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Posts Tagged ‘Epigenetics’


Deciphering the Epigenome

Curator: Larry H. Bernstein, MD, FCAP

 

UPDATED on 1/29/2016

 

RNA Epigenetics

DNA isn’t the only decorated nucleic acid in the cell. Modifications to RNA molecules are much more common and are critical for regulating diverse biological processes.

By Dan Dominissini, Chuan He and Gidi Rechavi | January 1, 2016

 

RNA SOUP: Newly transcribed messenger RNA exiting the nucleus via nuclear pores
© BENJAMIN CAMPILLO/SCIENCE SOURCE
http://www.the-scientist.com/January2016/feature2.jpg

For years, researchers described DNA and RNA as linear chains of four building blocks—the nucleotides A, G, C, and T for DNA; and A, G, C, and U for RNA. But these information molecules are much more than their core sequences. A variety of chemical modifications decorate the nucleic acids, increasing the alphabet of DNA to about a dozen known nucleotide variants. The alphabet of RNA is even more impressive, consisting of at least 140 alternative nucleotide forms. The different building blocks can affect the complementarity of the RNA molecules, alter their structure, and enable the binding of specific proteins that mediate various biochemical and cellular outcomes.

The large size of RNA’s vocabulary relative to that of DNA’s is not surprising. DNA is involved mainly with genetic information storage, while RNA molecules—mRNA, rRNA, tRNA, miRNA, and others—are engaged in diverse structural, catalytic, and regulatory activities, in addition to translating genes into proteins. RNA’s multitasking prowess, at the heart of the RNA World hypothesis implicating RNA as the first molecule of life, likely spurred the evolution of numerous modified nucleotides. This enabled the diversified complementarity and secondary structures that allow RNA species to specifically interact with other components of the cellular machinery such as DNA and proteins.

Methylating RNA

The nucleotide building blocks of RNA contain pyrimidine or purine rings, and each position of these rings can be chemically altered by the addition of various chemical groups. Most frequently, a methyl (–CH3) group is tacked on to the outside of the ring. Other chemical additions such as acetyl, isopentenyl, and threonylcarbamoyl are also found added to RNA bases.

Among the 140 modified RNA nucleotide variants identified, methylation of adenosine at the N6 position (m6A) is the most prevalent epigenetic mark in eukaryotic mRNA. Identified in bacterial rRNAs and tRNAs as early as the 1950s, this type of methylation was subsequently found in other RNA molecules, including mRNA, in animal and plant cells as well. In 1984, researchers identified a site that was specifically methylated—the 3′ untranslated region (UTR) of bovine prolactin mRNA.1 As more sites of m6A modification were identified, a consistent pattern emerged: the methylated A is preceded by A or G and followed by C (A/G—methylated A—C).

The alphabet of RNA consists of at least 140 alternative nucleotide forms.

Although the identification of m6A in RNA is 40 years old, until recently researchers lacked efficient molecular mapping and quantification methods to fully understand the functional implications of the modification. In 2012, we (D.D. and G.R.) combined the power of next-generation sequencing (NGS) with traditional antibody-mediated capture techniques to perform high-resolution transcriptome-wide mapping of m6A, an approach we termed m6A-seq.2 Briefly, the transcriptome is randomly fragmented and an anti-m6A antibody is used to fish out the methylated RNA fragments; the m6A-containing fragments are then sequenced and aligned to the genome, thus allowing us to locate the positions of methylation marks.

Analyzing the human transcriptome in this way, we identified more than 12,000 methylated sites in mRNA molecules derived from approximately 7,000 protein-coding genes. The transcripts of most expressed genes, in a variety of cell types, were shown to be methylated, indicating that m6A modifications are widespread. In addition, about 250 noncoding RNA sequences—including well-characterized long noncoding RNAs (lncRNAs), such as the XIST transcripts that have a key role in X-chromosome inactivation—are decorated by m6A. In almost all cases, the epigenetic mark was found on adenosines embedded in the predicted A/G—methylated A—C sequence. We found that this pattern was consistently preceded by an additional purine (A or G) and followed by a uracil (U), extending the known consensus sequence to A/G—A/G—methylated A—C—U.2

At the macro level, we found that m6A methylation sites were enriched at two distinct landmarks. The highest relative representation of m6A was found in the stop codon–3′ UTR segment of the RNA, with nearly a third of such methylation found in this sequence just beyond a gene’s coding region. Within the coding regions of the RNA molecules, m6A enrichment mapped to unusually long internal exons; 87 percent of the exonic methylation peaks were found in exons longer than 400 nucleotides. (The average human exon is only 145 nucleotides in length). This pattern of decoration of transcribed RNA suggests that m6A is involved in the mediation of splicing of long-exon transcripts. RNAs transcribed from single­-isoform genes were found to be relatively undermethylated, while transcripts that are known to have multiple isoforms, determined by alternative splicing patterns, were hypermethylated.2 Moreover, specific alternative splicing types, such as intron retention, exon skipping, and alternative first or last exon usage, were highly correlated with m6A decoration. And silencing the m6A methylating protein METTL3 affected global gene expression and alternative splicing patterns in both human and mouse cells.2

These findings clearly indicate the importance of m6A decoration in regulating the expression of diverse transcripts. Moreover, our parallel study of the human and mouse methylome by m6A-seq has uncovered a remarkable degree of conservation in both consensus sequence and areas of enrichment, further supporting the importance of m6A function.2 But research into understanding how m6A marks themselves are regulated, and how this affects various cellular processes, is only just beginning.

Writers, erasers, and readers

The accumulating findings regarding the cellular consequences of m6A transcriptome decoration led to the search for the mediators that enable m6A to exert its influence. Epigenetic marks are introduced by enzymes and cofactors known as “writers,” and m6A is no exception. This mark is added to RNA by a large protein complex that includes three well-characterized components: METTL3, METTL14, and WTAP.3,4 (See illustration on opposite page.)

The transcripts of most expressed genes, in a variety of cell types, were shown to be methylated.

The reverse process of RNA demethylation is performed by “erasers.” In 2011, one of us (C.H.) and an international group of colleagues identified the first m6A eraser: the fat mass and obesity–associated protein (FTO).5 Four years earlier, three independent studies had discovered that a single-nucleotide polymorphism in the first intron of Fto was strongly associated with body mass index and obesity risk, and studies of mouse models where Fto was deleted or overexpressed further demonstrated its link with altered body weight. The research from the C.H. group showed that silencing the Fto gene or protein increased total m6A levels, while overexpression decreased levels of the epigenetic mark.5 C.H.’s group later discovered that another protein from the same protein family as FTO, ALKBH5, behaves as an active m6A demethylase.6 In contrast to the ubiquitous expression of Fto in all tissues, the highest expression level of Alkbh5 was demonstrated in mouse testes. Indeed, Alkbh5-null male mice exhibit aberrant spermatogenesis, probably a result of m6A-mediated altered expression of spermatogenesis-related genes.6

 

RNA METHYLATION DYNAMICS: At least 140 alternative RNA nucleotide forms exist. On mRNA, the most common is the methylation of adenosine on the N6 position (m6A). This epigenetic mark is laid down by a “writer” protein complex that includes three well-characterized components: METTL3, METTL14, and WTAP. The reverse process of RNA demethylation is performed by “erasers,” such as the enzymes FTO and ALKBH5.

http://www.the-scientist.com/January2016/methylation2.jpg

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These writers and erasers facilitate the dynamic nature of m6A methylation, which was shown when we (D.D. and G.R.) demonstrated changes in response to environmental stimuli, such as UV irradiation, heat shock, and exposure to interferon gamma or hepatocyte growth factor.2 Once RNA epigenetic modifications are laid down, they are recognized by specific “reader” proteins that bind to the modified nucleotide and mediate enhancement or inhibition of gene expression. In 2012, the G.R. group used methylated and nonmethylated versions of synthetic RNA baits that include the m6A consensus sequence to identify such readers of m6A.2 By preferential binding to the methylated bait, we isolated several specific m6A-binding proteins, including members of the RNA-binding YTH domain family, whose function was previously unknown.2

The finding of the first m6A-binding reader proteins has accelerated the deciphering of the various molecular and cellular processes mediated by m6A marking. In 2014, for example, we (C.H. and colleagues) showed that the human YTH domain family 2 (YTHDF2) reader protein selectively recognizes m6A and mediates mRNA degradation.7 We identified more than 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs. Binding of YTHDF2 to m6A in mRNA results in the translocation of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies in the cytoplasm where mRNA turnover is regulated.

Recently, C.H. and colleagues identified another m6A reader protein, YTHDF1, with a very different function—stimulating protein synthesis by ramping up the efficiency of translation machinery.8 The dueling functions of YTHDF2 and YTHDF1 provide a mechanism by which cells can adjust gene expression promptly and precisely to environmental stimuli. Finally, G.R. and his group have identified an additional reader protein, the RNA-binding protein heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1),2 which directly binds a set of m6A decorated transcripts and mediates alternative splicing.9

Clearly, m6A plays diverse roles in regulating cellular function, starting with basic processes such as gene expression, translation, and alternative splicing. As work on this epigenetic mark continues, we will undoubtedly link m6A to numerous phenotypes, and its dysregulation may undergird various diseases and syndromes.

RNA epigenetics in action

Understanding the molecular mechanisms by which m6A regulation controls RNA stability, translation efficiency, and alternative splicing is helping researchers decipher the importance of this new epigenetic mark in physiological and pathological processes. For example, researchers recently showed that translation increases in stressed mice thanks to m6A decoration. In 2015, two studies from Cornell University and Weill Cornell Medical College found increased m6A methylation of specific 5′ UTR adenosines in newly transcribed mRNAs as a result of stress-induced nuclear localization of the m6A YTHDF2 reader. The researchers suggested that the nuclear YTHDF2 preserves the unique 5′ UTR m6A methylation of stress-induced transcripts by limiting the demethylation activity of the FTO eraser. Increased 5′ UTR m6A methylation in turn promotes translation of specific transcripts, such as those for the heat shock protein Hsp70. While conventional mRNA translation starts by binding of the ribosome components to a region of the 5′ UTR marked by the unusual nucleotide 7meG (the “cap”), under stress conditions initiation of translation can start farther downstream.10

DECIDING CELL FATE: Among its many roles in the cell, m6A methylation helps regulate the expression of RNA transcripts that mediate the transition from pluripotency to differentiation. The presence of m6A appears to decrease the stability of transcripts important for maintaining pluripotency, priming the cells for future differentiation. The loss of METTL3, an m6A methlyase component, in mouse embryonic stem cells leads to the cells’ inability to exit the pluripotent state, a lethal outcome in the early embryos.
http://www.the-scientist.com/January2016/feature2_21.jpg

See full infographic: WEB THE SCIENTIST STAFF

In a second study, Weill Cornell Medical College’s Samie Jaffrey, who collaborated on the previous study, led a team that showed m6A-methylated mRNAs can be translated in a cap-independent manner. The researchers showed that a specific 5′ UTR m6A binds the eukaryotic initiation factor 3 (eIF3), which recruits the ribosomal 43S complex and initiates cap-independent translation. This study also demonstrated increased m6A levels in the Hsp70 mRNA that enhanced its cap-independent translation following heat-shock stress.11

Other work has hinted at m6A’s role in the regulation of circadian rhythms. Researchers identified m6A sites on many transcripts of genes involved in the regulation of daily cycles. Inhibition of m6A methylation by silencing of the METTL3 writer led to circadian period elongation, with altered distribution and processing of the transcripts of the clock genes Per2 and Arntl.12

It’s quickly becoming clear that m6A decoration has diverse cellular and physiological functions. But perhaps the best illustration of its critical ability to precisely control processes at the cellular level is its involvement in early embryogenesis. Cell-fate decisions are coordinated by alterations in global gene expression, which are orchestrated by epigenetic regulation. Well-established epigenetic marks, such as DNA methylation and histone modifications, are known to mediate embryonic stem cell (ESC) cell-fate decisions, and it turns out that m6A modification is no different.

Dynamic m6A RNA markings, the new kid on the epigenetic block, herald the era of tripartite epigenetics where modifications of DNA, RNA, and proteins join hands to fine-tune gene expression and to execute prompt and precise responses to environmental stimuli and stresses.

We (G.R. and collaborators) and other groups recently demonstrated that the m6A writer METTL3 is also an essential regulator for termination of mouse embryonic stem cell pluripotency. Knocking out Mettl3 in preimplantation murine epiblasts and in undifferentiated ESCs led to depletion of m6A in mRNAs. Cell viability was not affected, suggesting that m6A decoration is not essential for the maintenance of the ESC naive state, but m6A marks were critical for early differentiation. The loss of this modification led to aberrant and restricted lineage priming at the post-implantation stage, resulting in early embryonic lethality.13 The presence of m6A also decreased mRNA stability, including in those transcripts important for maintaining pluripotency. These findings demonstrated, for the first time, an essential function for an mRNA modification in vivo.14

Beyond mRNA

While m6A methylation is most prevalent on mRNAs, this mark also decorates other RNA species. It is well established, for example, that m6A is abundant on rRNAs, tRNAs, and small nuclear RNAs (snRNAs), which mediate splicing and other RNA processing and protein synthesis reactions.

More recently, researchers found that the reader protein HNRNPA2B1 binds to m6A marks in a subset of primary microRNA (miRNA) transcripts, recruiting the miRNA-microprocessor complex and promoting primary miRNA processing that is essential for mature miRNA biogenesis.9 Not only is the biogenesis of miRNA regulated by m6A marking and recruitment of HNRNPA2B1, miRNAs themselves appear to play a role in the placement of the m6A epigenetic marks. MiRNAs regulate m6A modification in specific transcript sites using a sequence-pairing mechanism where the “seed” sequence of a specific miRNA binds a complementary target sequence in the 3′ UTR of mRNA and directs methylation.15 The interaction is bidirectional: manipulation of miRNA sequence or expression affects m6A modification also by reducing binding of the METTL3 writer to the target mRNA sites.

Similarly, m6A appears to be involved in structural alterations of mRNAs and lncRNAs to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant RNA-binding protein responsible for mRNA processing. This novel mechanism, termed m6A-switch, was shown to affect alternative splicing and abundance of multiple target mRNAs.16 Taken together, these results demonstrate that m6A is an important mark on diverse RNA species.

Dynamic m6A RNA markings, the new kid on the epigenetic block, herald the era of tripartite epigenetics where modifications of DNA, RNA, and proteins join hands to fine-tune gene expression and to execute prompt and precise responses to environmental stimuli and stresses. Indeed, m6A is just one of 140 modified RNA nucleotides that likely affect the function of the nucleic acid messenger and key cellular actor in diverse ways. Molecular approaches are paving the way for the study of additional RNA modifications.

As the list of RNA epigenetic marks continues to expand, researchers will gain a clearer picture of how diverse cellular processes are regulated. The extremely large repertoire of such modifications is expected to reveal various RNA marks analogous to the known DNA and histone epigenetic marks, and the various modifications of DNA, RNA, and proteins can enrich the language that allows the development, adaptation, and diversity of complex organisms.

Dan Dominissini is a postdoctoral fellow in Chuan He’s group at the University of Chicago. Gidi Rechavi is a pediatric hematologist-oncologist and a researcher in genetics and genomics at the Chaim Sheba Medical Center in Tel Hashomer, Israel, and a Professor of Hematology at the Sackler School of Medicine at Tel Aviv University. Sharon Moshitch-Moshkovitz, a senior researcher in RNA biology at the Chaim Sheba Medical Center, also contributed to this article.

References

  1. S. Horowitz et al., “Mapping of N6-methyladenosine residues in bovine prolactin mRNA,” PNAS, 81:5667-71, 1984.
  2. D. Dominissini et al., “Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq,” Nature, 485:201-06, 2012.
  3. Y. Fu et al., “Gene expression regulation mediated through reversible m6A RNA methylation,” Nat Rev Genet, 15:293-306, 2014.
  4. K.D. Meyer, S.R. Jaffrey, “The dynamic epitranscriptome: N6-methyladenosine and gene expression control,” Nat Rev Mol Cell Biol, 15:313-26, 2014.
  5. G. Jia et al., “N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO,” Nat Chem Biol, 7:885-87, 2011.
  6. G. Zheng et al., “ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility,” Mol Cell, 49:18-29, 2013.
  7. X. Wang et al., “N6-methyladenosine-dependent regulation of messenger RNA stability,” Nature, 505:117-20, 2014.
  8. X. Wang et al., “N6-methyladenosine modulates messenger RNA translation efficiency,” Cell, 161:1388-99, 2015.
  9. C.R. Alarcón et al., “HNRNPA2B1 is a mediator of m6A-dependent nuclear RNA processing events,”Cell, 162:1299-308, 2015.
  10. J. Zhou et al., “Dynamic m6A mRNA methylation directs translational control of heat shock response,” Nature, 526:591-94, 2015.
  11. K.D. Meyer et al. “5′ UTR m6A promotes cap-independent translation,” Cell, 163:999-1010, 2015.
  12. J.-M. Fustin et al., “RNA-methylation-dependent RNA processing controls the speed of the circadian clock,” Cell, 155:793-806, 2013.
  13. S. Geula et al., “m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation,” Science, 347:1002-06, 2015.
  14. P.J. Batista et al., “m6A RNA modification controls cell fate transition in mammalian embryonic stem cells,” Cell Stem Cell, 15:707-19, 2014.
  15. T. Chen et al., “m6A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency,” Cell Stem Cell, 16:289-301, 2015.
  16. N. Liu et al., “N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions,” Nature, 518:560-64, 2015.

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RNA methylationRNA epigeneticsrnamethylationepigenetics and epigenetic regulation

 

Telomerase Overdrive

Two mutations in a gene involved in telomere extension reverse the gene’s epigenetic silencing.

By Ashley P. Taylor | January 1, 2016

http://www.the-scientist.com//?articles.view/articleNo/44768/title/Telomerase-Overdrive/

EPIGENETIC ACTIVATION: A single base-pair mutation (lower allele) leads to epigenetic changes that promote expression of a telomerase gene.COURTESY OF JOSH STERN
http://www.the-scientist.com/January2016/shortlit2.jpg

EDITOR’S CHOICE IN GENETICS & GENOMICS

The paper
J.L. Stern et al., “Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERTexpression in multiple cancers,” Genes Dev, doi:10.1101/gad.269498, 2015.

The foundation
Chromosome ends are slightly shortened with each DNA replication. Terminal repetitive sequences called telomeres buffer coding DNA from this fate. In stem cells, telomerase extends the telomeres so that cell division can continue, perhaps indefinitely. In somatic cells, telomerase is inactive in part because the gene encoding telomerase’s catalytic sub­unit, telomerase reverse transcriptase (TERT), is epigenetically silenced. In most cancers, however, telomerase is again turned on and aids proliferation.

The mutations
In 2013, researchers found two mutations in the TERT promoter that occur frequently in cancer cell lines and are tied with TERT expression.

Regulation
To probe the mechanism of TERT activation, Josh Stern, a postdoctoral fellow in the lab of Thomas Cech at the University of Colorado Boulder, studied cancer cell lines that were heterozygous for one of these TERTmutations. Stern and his colleagues determined that the mutant TERT allele had histone methylation marks associated with gene activation and was transcribed, whereas the wild-type allele bore other histone methylation marks characteristic of gene silencing and was not transcribed.
“It’s very nice biochemical work to show that a single-base-pair mutation in the cancer genome activates the expression of the telomerase gene,” says Dana-Farber Cancer Institute’s Franklin Huang.

Application
“Telomerase is a fantastic therapeutic target for cancers because so many cancers are absolutely reliant on telomerase,” says Stern. “These TERT promoter mutations only occur in cancer, so if we can understand the mechanism, then we can potentially develop a highly specific cancer therapeutic.”

Tags

transcriptiontelomerestelomerasemutationliteraturegenetics & genomicsepigenetics and cancer

 

CRISPR Fixes Stem Cells Harboring Blindness-Causing Defect

http://www.genengnews.com/gen-news-highlights/crispr-fixes-stem-cells-harboring-blindness-causing-defect/81252293/

Marking yet another CRISPR-related first, scientists have replaced a defective gene associated with a sensory disease in stem cells that were derived from a patient’s tissue. The disease, retinitis pigmentosa (RP), is an inherited condition that degrades the retina and leads to blindness. A patient with the disease supplied a skin sample that was used to generate the stem cells, which were manipulated by means of the CRISPR/Cas9 gene-editing system.

CRISPR/Cas9, which zeroed in on a single disease-causing mutation in the RGPR gene, was able to make the necessary correction in 13% of the stem cells. This correction rate, according to the Columbia University and University of Iowa scientists who announced the results, is indicative of a practical approach—albeit one that still needs work. The Columbia/Iowa team added that they are working to show that their technique does not introduce any unintended genetic modifications in human cells, and that the corrected cells are safe for transplantation.

While the scientists freely acknowledge that their technique needs additional development before any cures are possible, they basked in the success of having accomplished a difficult genetic fix. The RGPR mutation that needed to be repaired sits in a highly repetitive sequence of the gene where it can be tricky to discriminate one region from another. In fact, it was not clear that CRISPR/Cas9 would be able to home in on and correct the point mutation.

The scientists described their work January 27 in the journal Scientific Reports in an article entitled “Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells.”

“Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce [induced pluripotent stem cells (iPSCs)] carrying the patient’s c.3070G > T mutation,” the authors wrote. “The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene’s repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele.”

The authors asserted that theirs was the first report of CRISPR/Cas9 being used to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This proof-of-concept finding, they added, supports the development of personalized iPSC-based transplantation therapies for retinal disease.

The authors also emphasized that because the corrections are made in cells derived from the patient’s own tissue, doctors can retransplant the cells with fewer fears of rejection by the immune system. Previous clinical trials have shown that generating retinal cells from embryonic stem cells and using them for transplantation is a safe and potentially effective procedure.

Recently, another group has used CRISPR to ablate a disease-causing mutation in rats with retinitis pigmentosa. Going forward, the first clinical use of CRISPR could be for treating an eye disease because compared to other body parts, the eye is easy to access for surgery, readily accepts new tissue, and can be noninvasively monitored.

 

 

Edited stem cells offer hope of precision therapy for blindness

http://www.rdmag.com/news/2016/01/edited-stem-cells-offer-hope-precision-therapy-blindness

http://www.rdmag.com/sites/rdmag.com/files/newsletter-ads/RD_stemcells.jpg

Skin cells from a patient with X-linked Retinitis Pigmentosa were transformed into induced pluripotent stem cells and the blindness-causing point mutation in the RPGR gene was corrected using CRISPR/Cas9. Credit: Vinit Mahajan, Univ.of Iowa Health Care

Using a new technology for repairing disease genes–the much-talked-about CRISPR/Cas9 gene editing–Univ. of Iowa researchers working together with Columbia Univ. Medical Center ophthalmologists have corrected a blindness-causing gene mutation in stem cells derived from a patient. The result offers hope that eye diseases might one day be treated by personalized, precision medicine in which patients’ own cells are used to grow replacement tissue.

With the aim of repairing the deteriorating retina in patients with an inherited blinding disease, X-linked Retinitis Pigmentosa (XLRP), Alexander Bassuk, MD, PhD, and Vinit Mahajan, MD, PhD, led a team of researchers who generated stem cells from patient skin cells and then repaired the damaged gene. The editing technique is so precise it corrected a single DNA change that had damaged the RPGR gene. More importantly, the corrected tissue had been derived from the patient’s own stem cells, and so could potentially be transplanted without the need for harmful drugs to prevent tissue rejection. The research was published Jan. 27 in the journal Scientific Reports.

“With CRISPR gene editing of human stem cells, we can theoretically transplant healthy new cells that come from the patient after having fixed their specific gene mutation, ” says Mahajan, clinical assistant professor of ophthalmology and visual sciences in the UI Carver College of Medicine. “And retinal diseases are a perfect model for stem cell therapy, because we have the advanced surgical techniques to implant cells exactly where they are needed.”

The study was a “proof-of-concept” experiment showing it is possible not only to repair a rare gene mutation, but that it can be done in patient stem cells. Use of stem cells is key because they can be re-programmed into retinal cells.

The CRISPR technology was able to correct the RPGR mutation in 13 percent of the stem cells, which is a practically workable correction rate.

Bassuk notes this result is particularly encouraging because the gene mutation sits in a highly repetitive sequence of the RPGR gene where it can be tricky to discriminate one region from another. In fact, initially determining the DNA sequence in this part of the gene was challenging. It was not clear that CRISPR/Cas9 would be able to home in on and correct the “point mutation.”

“We didn’t know before we started if we were going to be able to fix the mutation,” says Bassuk, associate professor in the Stead Family Department of Pediatrics at University of Iowa Children’s Hospital.

 

Epigenetics Research Reveals a Range of Clinical Possibilities

Advantageously Epigenetic Analyses Can Capture both Genetic Factors and Environmental Exposures

Richard A. Stein, M.D., Ph.D.

http://www.genengnews.com/gen-articles/epigenetics-research-reveals-a-range-of-clinical-possibilities/5650/

  • Over half a century ago, Conrad Hal Waddington introduced his model of the epigenetic landscape. He depicted a differentiating cell as a ball rolling down a landscape of bifurcating valleys and ridges, with each valley representing an alternative developmental path. Just as a ball may roll from valley to valley until it reaches the bottom of the landscape, a cell may progress from one developmental alternative to another until it reaches its fully differentiated state.

The model’s original purpose was to integrate concepts from genetics and developmental biology and to describe mechanisms that connect the genotype to the phenotype. Today, the model remains a compelling metaphor for epigenetics, which has developed into one of the most vibrant biomedical fields. Epigenetics has become indispensable for exploring development, differentiation, homeostasis, and diseases that span virtually every clinical discipline.

  • Analyzing Methylation Patterns

“Modern efforts toward explaining human disease purely based upon sequencing cannot possibly succeed in isolation,” says Andrew P. Feinberg, M.D., professor of medicine and director of the Center for Epigenetics at Johns Hopkins University School of Medicine. “At least half of human disease is caused by exposure to the environment.”

While the contribution of genetic factors to disease is more predictable and easier to study in the case of highly penetrant Mendelian disorders, most medical conditions involve multiple genes that may interact with one another and with environmental factors. Particularly for these conditions, capturing epigenetic changes becomes a crucial aspect of understanding pathogenesis and designing prophylactic and therapeutic interventions.

“In these cases,” notes Dr. Feinberg, “an approach not including epigenetics will be severely limited in what it can accomplish.”

In a recent study, Dr. Feinberg and colleagues reported that large blocks of the human genome are hypomethylated in the epidermis as a result of sun exposure, which together with aging represents a known risk factor for skin cancer. These hypomethylated regions overlap with regions that have methylation changes in patients with squamous cell carcinoma.

This overlap could explain the causal link between sun exposure and the increased risk of malignancy found in many epidemiological studies. Most of the methylation changes were observed in the epidermis, not in the dermis, pointing toward the combination between the genotype and exposure, acting on specific cell types, as a key factor in shaping disease.

“One of the advantages of epigenetic analyses is that they capture both genetic factors and environmental exposures,” explains Dr. Feinberg. In the study of complex diseases, the existence of many distinct genetic variants identified in different individuals makes it challenging to understand their roles in pathogenesis. “But if genetic variants converge on gene regulatory loci, then measuring methylation can still be informative about these variants,” continues Dr. Feinberg, “even if genetic changes are inconsistent across the patients.”

In combining data from genome-wide association analysis and epigenome-wide analysis, Dr. Feinberg and colleagues revealed that two single-nucleotide polymorphisms on human chromosome 11, located 100 kb apart and involved in different aspects of lipid metabolism, controlled DNA methylation at two CpG sites in a bidirectional promoter situated between two genes encoding the fatty acid desaturases FADS1 and FADS2. Genome-wide association studies alone would not capture the convergence of these two single-nucleotide polymorphisms as they regulate DNA methylation in the shared promoter region.

“Measuring DNA methylation,” concludes Dr. Feinberg, “can pick up the fact that these single nucleotide polymorphisms act through DNA methylation to regulate the genes.”

The image shows a cleavage-stage human embryo. This is around the same stage that DNA methylation is ‘set’ at metastable epialleles. [Instituto Bernabeu]

http://www.genengnews.com/Media/images/Article/Jan15_2016_RobertWaterland_CleavageStageHumanEmbryo3202193100.jpg

Identifying Metastable Epialleles

Over the years, genome-wide association studies provided opportunities to establish links between genetic variation and phenotypic changes. For these analyses, genetic material from any of an individual’s cells, such as a peripheral white blood cell, is informative about the individual’s genotype. However, for epigenetic changes, which vary across tissues and within the same tissue among different cells, it is much more challenging to examine associations with disease.

Robert A. Waterland, Ph.D., associate professor of pediatrics and molecular and human genetics at Baylor College of Medicine, thinks that identifying human metastable epialleles will help circumvent some of these challenges. “Getting investigators and the field interested in metastable epialleles is going to be an important first step in helping us understand how epigenetic dysregulation contributes to human disease,” says Dr. Waterland.

The term metastable epialleles refers to genomic loci with differential epigenetic regulation that are variably expressed in genetically identical individuals, and where the epigenetic state is established stochastically in the very early embryo, before gastrulation, and subsequently maintained. This leads to systemic (non-tissue-specific) interindividual epigenetic differences that are not genetically mediated.

The fact that DNA methylation at metastable epialleles is particularly sensitive to environmental influences makes these loci valuable in mechanistically exploring the developmental origins hypothesis, the concept that environmental exposures during critical periods of prenatal and early postnatal development can have long-term implications in the risk of disease. Previous studies have implicated epigenetic modifications as a mechanism by which environmental changes during pregnancy may lead to epigenetic changes that influence health later in life.

In the most recent genome-wide screen meant to identify metastable epialleles in humans, Dr. Waterland teamed up with Dr. Andrew Prentice and colleagues at the London School of Hygiene and Tropical Medicine and used two independent and complementary experimental approaches to identify DNA methylation changes that occur in the cleavage-stage embryo (shortly after the time of conception). The first approach involved a genome-wide screen for DNA methylation in multiple tissues from two healthy Caucasian adults. In parallel, genome-wide DNA methylation profiling was performed in a rural population from The Gambia to examine the link between the season of conception (a proxy for maternal nutritional status) and DNA methylation in the offspring and sought to capture the effect of maternal nutritional status on the epigenetic profile of the offspring.

“We identified the same genomic locus as the top hit in both screens, suggesting that this is likely to be a key indicator of early environmental influences on the epigenome,” explains Dr. Waterland. Both approaches identified VTRNA2-1 as the lead candidate for an environmentally-responsive epiallele.

VTRNA2-1, a genomically imprinted small noncoding RNA and a putative tumor suppressor gene, is preferentially methylated on the maternally inherited allele, and loss of imprinting at this locus promises to link the early embryonic environment to epigenetic changes that shape disease risk later in life. Besides VTRNA2-1, over 100 metastable epialleles were identified in the study.

“At metastable epialleles such as VTRNA2-1, DNA methylation in peripheral blood or in any easily accessible tissue can give an indication about the epigenetic regulation throughout the body,” concludes Dr. Waterland. “That is what is really different.”

http://www.genengnews.com/Media/images/Article/thumb_Jan15_2016_DavidBazettJones_ElectronSpectroscopic1381311078.jpg

Electron spectroscopic image of a region of the nucleus of a mouse embryonic fibroblast. Phosphorus and nitrogen maps allow chromatin (yellow) to be distinguished from protein-based structures (cyan). The arrow indicates the nuclear envelope. The large structure in the middle of the field, a chromocentre, is an accumulation of pericentric heterochromatin. It is surrounded by dispersed chromatin fibers. The heterochromatin mark, trimethlated H3K9, is immunolabelled and visualized with gold tags (white foci). [David Bazett-Jones]

Mapping Heterochromatin Domains

Electron spectroscopic image of a region of the nucleus of a mouse embryonic fibroblast. Phosphorus and nitrogen maps allow chromatin (yellow) to be distinguished from protein-based structures (cyan). The arrow indicates the nuclear envelope. The large structure in the middle of the field, a chromocentre, is an accumulation of pericentric heterochromatin. It is surrounded by dispersed chromatin fibers. The heterochromatin mark, trimethlated H3K9, is immunolabelled and visualized with gold tags (white foci). [David Bazett-Jones]

“For the first time, we found that a histone chaperone is implicated in organizing chromatin at a large scale,” says David Bazett-Jones, Ph.D., professor of biochemistry at the University of Toronto and senior scientist at the Hospital for Sick Children. The discovery and characterization of histone variants has been a vital facet of understanding chromatin organization and dynamics.

One of the most extensively studied histone variants is H3.3. Although H3.3 is 96% identical at the amino acid level to histone H3.1, histones H3.3 and H3.1 are functionally distinct. Histone H3.3 is expressed throughout the cell cycle, and it is enriched in transcriptionally active chromatin and in certain types of post-translational modifications. The death domain-associated protein DAXX, one of the proteins associated with histone H3.3 deposition, was recently identified as its chaperone.

Dr. Bazett-Jones and colleagues, including his graduate student Lindsy Rapkin, revealed that the loss of DAXX led to a global structural change in the chromatin landscape, characterized by genomic regions enriched in the trimethylated H3K9 epigenetic mark that were juxtaposed to large chromatin domains devoid of this modification.

“These major changes probably occur because the boundaries between heterochromatin domains and other regions were not being respected, leading to the inappropriate insertions of histone H3.3, and this exerted quite profound effects,” explains Dr. Bazett-Jones. The loss of DAXX led to the uncoupling of the epigenetic marks from the global chromatin architecture. “This shows that a major global reorganization of the chromatin was taking place,” Dr. Bazett-Jones continues.

To visualize chromatin changes that result from the loss of DAXX, Dr. Bazett-Jones and colleagues used electron spectroscopic imaging, an experimental approach that is based on the principle of electron energy loss spectroscopy. When a biological specimen is targeted with electrons and its atoms become ionized, the ionization energy is equal to the energy that is lost by the incident electrons that generated the event.

The electron microscope technique generates nitrogen and phosphorus maps, which are used to discriminate between nucleic-acid-rich and protein-rich cellular structures. These maps offer high-contrast images of chromatin and its three-dimensional organization in intact cells.

Another component of the DAXX deletion phenotype included the loss of nucleolar structural integrity, resulting in an increased number of cells containing mini-nucleoli, and the dispersal of ribosomal DNA genes outside the nucleolus. Collectively, these findings pointed toward a novel role that DAXX plays in the subnuclear organization of chromatin and in maintaining nucleolar structural integrity.

“Historically, we thought that the well-known epigenetic modifications dictate the compact character of heterochromatin,” notes Dr. Bazett-Jones. “But our findings, and those from other groups, reveal that a heterochromatin domain epigenetically marked with H3K9 trimethylation, for example, can be found in a structurally ‘open’ state, similar to euchromatin.”

This indicates that the boundaries between heterochromatin and euchromatin are much more fluid than previously envisioned, a concept that is crucial for understanding factors that dynamically shape the three-dimensional interaction between epigenetic changes. A key implication of these findings is that the epigenetic marks at a specific genomic locus depend on both the local environment and the three-dimensional context.

“We need to look at what loci come together in specific regions of the nucleus in three dimensions and how they affect each other,” concludes Dr. Bazett-Jones. “This is on top of capturing epigenetic marks, which are on top of the genomic sequences that we need to explore.”

Identifying Druggable Epigenetic Processes

“There is a big gap in understanding the biology of epigenetics,” says Chris J. Burns, Ph.D., laboratory head, Division of Chemical Biology, Walter and Eliza Hall Institute of Medical Research, Melbourne. “And this goes hand in hand with the need to learn how to generate small molecule probes or drugs.”

When interrogating epigenetic processes, researchers find it useful to integrate biological and chemical perspectives. For example, researchers have generated a large body of literature demonstrating that many epigenetic processes involve highly complicated protein complexes.

Historically, genetics studies have typically relied on knocking down or knocking out a gene and its protein product to examine the resulting phenotype. “In contrast, knocking down a protein that is part of a protein complex fundamentally alters that complex, and the phenotype could be quite different from the one that can be seen with a small molecule inhibition of a catalytic component of the protein complex,” notes Dr. Burns. This opens an acute need to identify small molecules that can selectively impact just one particular aspect of these protein complexes.

A major effort in Dr. Burns’ lab is focusing on identifying therapeutic agents that could target epigenetic processes. “Epigenetics in terms of drug discovery and development is still in an early stage,” explains Dr. Burns. While several drugs that target epigenetic processes have become available in recent years—drugs such as HDAC inhibitors and DNA methyl transferase inhibitors—many other drugs are still at early stages of development.

“Some epigenetic processes have not yet been drugged,” Dr. Burns points out. “For some of them, there may not be any therapeutic agents that are particularly good.”

Dr. Burns’ lab has collaborated with investigators led by Carl Walkley, Ph.D., joint head, Stem Cell Regulation Unit, St. Vincent’s Institute of Medical Research, Melbourne. Together, the research teams revealed that several bromodomain inhibitors exert powerful antitumor activity in human osteosarcoma cell lines and in osteosarcoma primary cells from mouse models of the disease.

The researchers’ findings were surprising. JQ1, one the bromodomain inhibitors tested, exerted its antiproliferative activity by inducing apoptosis, and not by mediating cell cycle arrest, as expected. Moreover, even though previous studies identified MYC as an oncogenic driver in osteosarcoma, the activity of JQ1 was exerted independently of MYC downregulation.

At the same time, this work revealed that downregulation of FOSL1, a gene previously implicated in osteoblast differentiation, is an important contributor to the effects of JQ1, marking the first time when this gene was implicated in osteosarcoma.

“Because we used primary cell from animals, these findings reflect the disease process better than cell lines, which may take on a number of other mutations,” concludes Dr. Burns. “This explains why our findings are contrary to previous reports in the literature.”

“We have shown that epigenetic drugs may work not only on protein-coding genes but also on the noncoding part of the genome,” says Claes Wahlestedt, M.D., Ph.D., professor and associate dean for therapeutic innovation at the University of Miami Miller School of Medicine.

A therapeutically promising class of epigenetic compounds consists of bromodomain inhibitors. These compounds have received increasing attention in recent years, and several leads have entered clinical trials for malignancies, atherosclerosis, and type 2 diabetes.

”One of our interests is to see if bromodomain inhibitors could be used for diseases of the nervous system,” notes Dr. Wahlestedt.

Using in vitro and in vivo approaches, investigators in Dr. Wahlestedt’s group, in collaboration with investigators led by Nagi Ayad, Ph.D., found that BET bromodomain inhibitors can inhibit glioblastoma cell proliferation by inducing a cyclin-dependent kinase inhibitor. These findings set the stage for subsequent experiments that used single molecule sequencing to profile long noncoding RNAs (lncRNAs) differentially expressed in glioblastoma multiforme. This helped identify a set of transcripts that are specific for this malignancy and could be regulated by bromodomain inhibitors.

In glioblastoma multiforme cells, the I-BET151 bromodomain inhibitor localized to the promoter of HOTAIR, a tumor-promoting lncRNA that acts as an epigenetic silencer and has been implicated in several cancers, decreased its expression, and restored the expression of several lncRNA species that are downregulated in this malignancy.

In another collaborative endeavor, Dr. Wahlestedt and colleagues conducted a semi-high-throughput gene-expression-based screen to identify small molecules that could increase the expression of C9ORF72. A GGGGCC hexanucleotide repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic cause for amyotrophic lateral sclerosis. Individuals without this condition harbor 2 to 25 of these repeats, but their number can reach up to several hundreds in ALS patients, reducing C9ORF72 expression, which has been implicated in the pathogenesis of this condition.

The gene-expression-based screen identified, in fibroblasts from affected and unaffected individuals, small interfering RNAs against the BRD3 bromodomain protein and several small molecule bromodomain inhibitors that were able to increase C9ORF72 expression. This effect occurred without changes in promoter CpG hypermethylation and trimethylated H3K9 marks, which are heterochromatin markers of the expanded C9ORF72 alleles.

“The mechanism of action of these compounds is probably broader than we thought before,” concludes Dr. Wahlestedt.

 

CRISPR Works Well but Needs Upgrades

More Effective and Reliable CRISPR Tools Will Have To Be Developed

MaryAnn Labant

http://www.genengnews.com/gen-articles/crispr-works-well-but-needs-upgrades/5652/

http://www.genengnews.com/Media/images/Article/thumb_UnivIllinois_Cas9DSB1921455173.jpg

In this image, which comes from the University of Illinois at Urbana-Champaign, Cas9 (green) is shown cutting DNA (white and brown) at the target sequence specified by the single guide RNA (red). The image was created from the Protein Data Bank file 4un3.pdb using Pymol, and it was enhanced using Photoshop.

The gene-editing technology known as CRISPR-Cas9 went through a disruptive phase when it first took the research world by storm.

Now, thousands of research articles later, it is starting to raise expectations in the therapeutic realm. In fact, CRISPR-Cas9 and other CRISPR systems are moving so close to therapeutic uses that the technology’s ethical implications are starting to attract notice. For example, people worry that CRISPR could be used to alter human germline cells, introducing genomic changes that could impact future generations.

Before any of that can happen, however, CRISPR will have to overcome a number of practical obstacles. If CRISPR is to be harnessed effectively and leveraged to its full potential, it will have to be better understood. Also, more effective and reliable CRISPR tools will have to be developed.

For example, little progress has been made in the area of targeted integration. “We effectively have the tools to cut, yet we lack efficient tools to paste. How the cells repair the double-strand break created by the RNA-guided nucleases, or RGNs, depends almost exclusively on the cells themselves in that there is no control over the repair mechanism. In addition to the RGNs, we deliver a vector that can function as a repair template, and hope the cells will use it,” explained Pablo Perez-Pinera, M.D., Ph.D., assistant professor, department of bioengineering, University of Illinois at Urbana-Champaign.

 

 

Fun with Lego (molecules)

http://www.rdmag.com/news/2016/01/fun-lego-molecules

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Depending on the relative amounts of different building-block molecules, it is possible to create different sandwich and wheel topologies (shown above in micrographs and below as models). Credit: American Chemical Society. Copyright 2016

A great childhood pleasure is playing with Legos and marveling at the variety of structures you can create from a small number of basic elements. Such control and variety of superstructures is a goal of polymer chemists, but it is hard to regulate their specific size and how the pieces fit together. This week in ACS Central Science, researchers report a simple system to make different nano-architectures with precision.

Using a variety of highly efficient chemical transformations and other techniques to ensure high yields and purity, Stephen Z. D. Cheng, Yiwen Li, Wen-Bin Zhang and coworkers designed systems to create giant molecules with ‘orthogonal’ ends, meaning that they only fit together with a specific partner just like Legos. Depending on the relative amounts of different building-block molecules, these molecules come together in different superstructures — ranging from cubes to wheels and sandwiches. Eventually, they could be employed in device-creation, where it is crucial to have precise control over the positions of the components.

 

Protein Expression Systems Proliferate

Bioprocessing Assembly Lines Are Being Retooled, Often At the Genomic Scale

Angelo DePalma, Ph.D.

http://www.genengnews.com/Media/images/Article/thumb_iStock_32784234_eColi1930160883.jpg

Despite some bells and whistles, most E. coli production systems have been the same. Now, new systems are being introduced that purport to express proteins more efficiently. [iStock/Scharvik]

Biomanufacturers enjoy a host of tools to optimize the production of therapeutic proteins, including expression systems, media, feeds, and gene-editing tools. Suffice it to say that protein expression is a growth industry.

Industry research firm Future Market Insights (FMI) breaks down the protein expression market into four product areas: competent cells, expression vectors, instruments, and reagents serving demand for research-grade and therapeutic proteins.

FMI has identified noteworthy growth drivers: the rising significance of biologics; innovations in proteomics; and patent expirations among small-molecule drugs. “These demands will boost the overall protein expression market in the coming future,” FMI literature states. “However, [attempts to contain rising costs] in various R&D activities in the fields of biotechnology and pharmaceutical industry as well as market consolidation of a high degree are some restraining factors for this market.”

The largest market for protein expression is expected to emerge in North America, given this region’s “well-established healthcare infrastructure.” North America is followed by Europe, and the Asia-Pacific region shows the highest growth. This information was derived from an FMI report (“Protein Expression Market: Global Industry Analysis and Opportunity Assessment 2015–2025”) that was issued last December.

 

 

Landmark Year

Through the efforts of scientists at Thermo Fisher Scientific, 2015 was a landmark year for transient protein production in CHO cells. The company’s ExpiCHO™ transient expression system achieved multiple g/L levels of protein expression previously thought possible only in stable cell lines, according to Jonathan Zmuda, Ph.D., associate director of cell biology at Thermo Fisher Scientific’s Gibco business unit.

“ExpiCHO allows drug developers to obtain meaningful quantities of protein from CHO cells at the very earliest stages of biologics development,” Dr. Zmuda asserts. “It allows CHO-derived protein to be used from discovery day one through the transition to stable cell lines, bioproduction, clinical trials, and product licensing.”

This has had the effect of streamlining drug development by eliminating the risk of starting a program with HEK 293-derived drug candidates, while also providing an alternative high-expressing system for proteins that are difficult to express in HEK 293.

New E. Coli Expression System

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New England BioLabs says that its SHuffle T7 E. coli expression system is able to express non-di-sulfide bonded proteins more efficiently than wild-type E. coli. The actual SHuffle strain expressing GFP is shown here.

Since E. coli was recruited for service around 1950, hundreds of thousands of publications have sung the praises of this bedrock expression system. But Mehmet Berkmen, Ph.D., staff scientist at New England BioLabs, notes that no more than a dozen distinct protein production strains exist. When production strains are examined closely, all are found to belong to just two basic strains, E. coli K-12 and E. coli B.

“Some strains have ‘bells and whistles,’ but the basic platform is the same,” Dr. Berkmen points out. “People are still looking for engineered lines that express protein more efficiently.”

Most expression systems are based on E. coli B, but that strain is not engineered specifically for protein production. The B strain is somewhat less domesticated than K-12, which has gone through numerous generations of selection for DNA manipulation. “E. coli B is more wild and tends to make protein better,” Dr. Berkmen notes. “But if you ask people why that is the case, they can’t provide an answer.”

New England BioLabs claims that its SHuffle® T7 E. coli expression system represents a breakthrough for microbial fermentation. The bacteria, which are chemically competent E. coli K-12 cells engineered to form proteins containing disulfide bonds in the cytoplasm, are suitable for T7-promoter-driven protein expression. The company has recently produced full-length antibodies, complete with disulfide bonds, in SHuffle organisms, which Dr. Berkmen calls “a significant step toward engineering and developing novel antibody formats and tools.”

New England BioLabs manufactures more than 500 proteins, 98% of them in E. coli. Perhaps even more interesting is the SHuffle system’s ability to express non-disulfide-bonded proteins more efficiently than wild-type E. coli. “SHuffle,” insists Dr. Berkmen, “represents a new chassis for protein production.”

The E. coli bacterium does not form disulfide bonds in its cytoplasm because two reducing pathways maintain the cytoplasmic proteome in its reduced state. Dr. Berkmen’s group knocked out those pathways and inserted a gene for a disulfide bond isomerase that increases fidelity of disulfide bond formation.

In addition to benefits already mentioned, SHuffle has a greatly diminished reducing capacity, permitting the formation of disulfide bonds for proteins that require it for folding and activity. Additionally, the cells, which are under oxidative stress, produce chaperones that also improve folding. For example, the activity of green fluorescent protein (GFP) expressed in SHuffle is much higher than protein produced in wild-type E. coli B.

It should be noted that a lack of glycosylation machinery persists in SHuffle cells. This problem, however, can be circumvented, as demonstrated in a seminal study carried out by Dr. Berkmen and colleagues. This study, which appeared last year in Nature Communications, described how IgG could be produced in SHuffle cells. Specifically, the investigators introduced mutations into the Fc portion of IgG. This resulted in efficient binding of aglycosylated IgG to its cognate receptor FcγRI.

Even in the absence of such ingenuity, E. coli remains a valuable expression system. It can be used to produce diagnostic and reagent proteins, or proteins for which glycosylation is noncritical.

“A Matter of Trying”

The principal advantages of using E. coli. are time and cost. “It takes basically one day, more or less, to obtain enough protein to suit many applications,” says David Chereau, Ph.D., CSO at Biozilla, a biotechnology contract research organization. As previously noted, the main disadvantages are lack of glycosylation apparatus and inability to support disulfide bond formation.

Workaround strategies can achieve stable disulfide bonds for some proteins. One strategy involves the following steps: Express the protein as an inclusion body, in insoluble form. Isolate the insoluble fraction. Solubilize this fraction with urea or some other suitable agent. Refold the protein.

“The process is relatively straightforward,” observes Dr. Chereau. “It’s much more difficult to find refolding conditions, which are normally determined empirically.” Refolding requires just the right buffer, salt concentrations, and additives. Also, refolding must be done in an oxidizing environment if disulfide bonds are to be achieved or maintained.

Dr. Chereau is philosophical about CHO cells’ inability to glycosylate: “Lack of glycosylation can be seen as an advantage or an inconvenience, depending.” E. coli is definitely out where glycosylation is a sine qua non. “But for the many applications where glycosylation isn’t needed, E. coli can be advantageous,” comments Dr. Chereau. Diagnostics and reagents are two such products. Additionally, obtaining a crystal structure during protein characterization is easier with glycans absent.

As part of its proof-of-concept services, Biozilla performs rapid screens to determine if E. coli is the right expression system for a particular product. Screening resembles design-of-experiment for mammalian cells, varying plasmids and vectors, as well as expression conditions.

Due to the success of CHO cells, bioprocessors tend to dismiss microbial fermentation, particularly for large proteins. “A lot of people think that expressing large proteins in E coli is difficult,” Dr. Chereau states, “but it’s often just a matter of trying. We have recently expressed a protein of 215 kDa in E. coli, which most people will tell you cannot be done. And we achieved it in very high yield.”

Rapid Prototyping

In June 2015, Invenra, a preclinical stage biotech company specializing in next-generation antibodies and antibody derivatives, entered an agreement with Oxford BioTherapeutics (OBT) to identify and characterize fully human therapeutic monoclonal antibodies (mAbs) against a novel cancer target that OBT has identified.

Invenra’s protein expression platform, through which it is capable of producing hundreds of thousands of full-length antibodies, uses cell-free expression to multiplex up to 10,000 protein variants simultaneously.

“We think of our technology as a rapid prototyping tool for proteins,” says Bryan Glaser, Ph.D., Invenra’s R&D director. “Once we have DNA, we can get protein in less than a day.” Invenra’s expression platform is suitable mainly for discovery and rapid protein prototyping. Yields are quite good: up to 500 μg/mL.

Other firms, such as Sutro Biopharma, are working on cell-free expression at much larger scales. Sutro claims that its Express CF™ technology can produce g/L yields in eight hours.

Cell-free expression involves E. coli extracts, typically S30 (used by most cell-free expression systems) and S12. The numbers reflect centrifugation speed. “Our system is based on S12, which is spun at lower speed than S30,” informs Dr. Glaser. “Our extract also does not undergo dialysis. We think of it as a ‘whole grain’ version.”

In addition to E. coli extracts, additives contain varying quantities of supplemental energy sources, nucleotides, and other small molecules that facilitate in vitro transcription and translation. Every vendor has its own unique blend.

Invenra’s standard mix, which is similar to off-the-shelf products from most commercial sources, is optimized for less complex molecules that don’t require disulfide bonds. Another mix has been optimized to include chaperones formulated to help expression and folding of IgGs and IgG-like molecules.

The upshot: fully functional, correctly folded IgGs and some bispecific antibodies, scFvs, and Fabs. More complex molecules are also possible, but each must be investigated independently. It is possible those could be made, but they would need to be optimized structure by structure. Dr. Glaser says expression capability depends to a large extent on amino acid sequence.

“We can fine-tune expression and folding conditions better than is possible in E. coli,” Dr. Glaser asserts. “We have better control over redox environment to facilitate disulfide bond formation, and we can add chaperones that are not present in E. coli organisms.” Still, the more disulfides the more complex the structure, and the lower the yield.

Dr. Glaser adds that antibody frameworks that express well in E. coli express well in cell-free systems, and ones that don’t express well in bacteria or mammalian cells tend not to express well cell-free. “It could be a framework sequence dependency,” he speculates. “It could be how well that framework folds. Perhaps the best-expressing molecules are those that do not require as much assistance from various chaperones and isomerases.”

Invenra’s expression system lends itself well to large-scale parallelism. The company has developed a credit-card-sized nanowell platform that expresses up to 10,000 unique antibodies per nanowell array. Cell-free expression of IgG using the Invenra nanowell platform system enables the incorporation of functional screening very early into the discovery process.

The ability to screen in excess of 100,000 IgG molecules can reduce the antibody display selection steps and preserve a larger diversity of epitope coverage. In addition, large combinations of binding partners can be empirically tested in various bispecific formats with relevant functional assays to identify the best pair and format for activity.

Getting the Bugs Out

Interest is growing for insect cell expression systems transiently transfected through the baculovirus expression vector system (BEVS). More and more clinical candidates are being generated in insect cells, including development-stage products for respiratory syncytial virus, Ebola virus, and norovirus.

 

A good deal of BEVS’ success is the ability of insect cells to produce multivalent, multisubunit vaccines through virus-like particles. These proteins can be made at large scale with BEVS for structural studies or to elucidate protein function.

Additionally, insect cells are ideal for making proteins that are toxic to mammalian or E. coli expression systems. BEVS shows its flexibility by providing rapid development cycles for treatments like seasonal influenza or pandemic infection vaccines. Because it is a transient system, BEVS allows for rapid turnaround times compared with mammalian cells, from identification of vaccine candidates to production.

 

Progress toward Therapeutic Epigenetics    

Epigenetic Targets Are Plentiful but Well Camouflaged

Angelo DePalma, Ph.D.

GEN  Jan 15, 2016 (Vol. 36, No. 2)   http://www.genengnews.com/gen-articles/progress-toward-therapeutic-epigenetics/5664/

  • Epigenetics is poised to become a cornerstone of drug development in oncology, diabetes, inflammation, developmental and metabolic disorders, cardiovascular and autoimmune diseases, pain, and neurological disorders.

    According to citations from PubMed Epigenetics, 40% year-on-year increases in epigenetics-related scientific publications occurred during the last decade, accompanied by a substantial increase in research funding. Data from ClinicalTrials.gov indicate that more than 40 different epigenetics-related drugs are undergoing clinical trials. Epigenetics will also likely affect developments in animal, plant, and environmental health.

    Jim Corbett, president of the human health business at PerkinElmer, notes that epigenetics research is currently limited by the number and availability of fully validated targets and preclinical disease models. “Another limitation stems from the relative dearth of fully selective antibodies for some of the writer and eraser targets to elucidate these complex signaling and modification events,” he points out. “Epigenetics research also suffers from a lack of a translational continuum for specific applications and for solutions from bench to bedside.”

    Nevertheless, the field is characterized by a high level of optimism. Research by Mordor Intelligence (“North America and Europe Epigenetics Market Growth, Trends and Forecasts, 2014–2020”) estimates that epigenetics will grow in market reach from approximately $2.9 billion in 2012 to about $12 billion in 2018.

    “I anticipate the development of second-generation epigenetic inhibitors with increased selectivity and targeting potential, standardization of epigenetic assays, and the validation of preclinical disease models leading to an improved understanding of epigenetic targets and mechanisms,” Corbett ventures. “The emergence of selective genome-editing technologies such as CRISPR will also apply in epigenetics and epigenome editing. I envision the future the emergence of personalized epigenetic profiles in patients.”

  • Computer Analogy

    Randy L. Jirtle, Ph.D., professor of epigenetics at North Carolina State University, describes epigenetics as a type of biological software. He explains an embryo’s combination of paternal and maternal genetic information, and eventual differentiation into 200–300 cell types, on the basis of cells running different programs.

    “The cell can be thought of as a programmable computer where the hardware is DNA and the software is the epigenome,” says Dr. Jirtle. “Very shortly after fertilization, this computer tells the cell how to work. And as with actual computers, things can go wrong because of viruses or—in the case of cells—mutations.”

    Dr. Jirtle demonstrated in 2003 that epigenetic modifications in utero may determine adult disease susceptibility, a notion that was not welcomed enthusiastically. “If you think of life as hardware, no known mechanism would explain this [connection],” asserts Dr. Jirtle. “But when you consider the ‘software,’ it becomes understandable.”

    Epigenetics can bring about positive effects as well. Through a process known as hormesis, low doses of a toxic agent or low doses of radiation can be administered strategically to improve an organism’s subsequent health. For example, mice exposed to low levels of ionizing radiation experienced a positive adaptive effect, which flies in the face of prevailing “no safe dosage” logic.

    In one strain of an experimental mouse bred to develop human-like diseases, 1 cGy of exposure—about the dose received from five X-rays—resulted in a decidedly positive hypermethylation of the epigenome. Exposed mice developed obesity, diabetes, and cancer at significantly lower rates than nonexposed mice. Negative effects occur at significantly higher doses as expected.

    Similarly positive epigenetic effects have been observed in plants exposed to very low doses of herbicides.

    Dr. Jirtle believes that the characterization of the repertoire of genes imprinted in humans, and their regulatory elements, the imprintome, will guide epigenome-based therapies. Imprinting is the process by which one parental copy of a gene is silenced. Thus, depending on the effectiveness of silencing, one could have two copies of a gene or none, either of which could potentially be deadly. In some cancers, for example, the inability to silence one parental gene for IGF2, which influences apoptosis, allows cancer cells to grow out of control.

    “There are probably around 150–500 disease-influencing genes that are regulated this way,” Dr. Jirtle points out.

  • Implementation Hurdles

    The connection between dysregulated DNA methylation and cancer is well established. Keith Booher, Ph.D., epigenetic service projects manager at Zymo Research, believes that modifying how methylation patterns change could allow a reset. Essentially, cells destined to become cancerous could be returned to a normal state.

    But significant hurdles block straightforward implementation. For example, getting drugs into cells, particularly solid tumors, is not easy. “It’s no coincidence that DNA methylation inhibitors have proved most successful for blood-based cancers, which are easier to target,” Dr. Booher tells GEN.

    Another hurdle is drug resistance, an issue with nearly all oncology agents. Moreover, drugs that alter the activity of the ubiquitous DNA methyl transferase will have broad activity on normal as well as abnormal cell processes.

    “Normal cells show low and high methylation levels,” Dr. Booher explains. “DNA methylation tends to limit gene expression, so you want to shut down those genes. And where DNA methylation is absent, genes tend to be expressed.

    “Methylation will change across the genome at different development stages, but in adult cells or developing blood cell you want methylation patterns to change in a regulated way. It’s difficult to limit the effect to diseased cells.”

    Finally, the way methylation inhibitors interact with DNA is in itself harmful. The original epigenetics-based drugs tested as broad chemotherapeutics, but their toxicology was high. It was only later, after the understanding the relationship between DNA methylation and carcinogenesis was better established, that the potential to use these agents at much lower doses became possible.

     

    Diagnostic Relevance

     

     

    This Circos plot from Swift Biosciences represents the methylation status of 1 Mb bins across chromo­somes 1–22 for Sample 8 (Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary).

    One of the most important advances in epigenetic research is the ability to obtain comprehensive, per-base methylation status of the methylome using next-generation sequencing (NGS). The significant drop in sequencing costs enables both whole-genome bisulfite sequencing and hybridization capture for targeted enrichment of the methylome.

    Initially, notes Laurie Kurihara, Ph.D., director of R&D at Swift Biosciences, these techniques were developed for microgram inputs of genomic DNA that undergo standard NGS library preparation followed by bisulfite conversion, a chemical process that converts nonmethylated cytosines to uracil. Subsequently, the polymerase chain reaction (PCR) process can be used to convert uracil to thymidine. “But the methylated cytosines are protected, thus demarcating methylation status when the DNA sequence is determined,” Dr. Kurihara observes. “The drawback is that bisulfite-induced DNA fragmentation destroys the bulk of the prepared NGS library. Hence the requirement for microgram DNA inputs.”

    To enable lower DNA inputs and improved methylome coverage and uniformity, Swift Biosciences has developed an NGS library preparation performed on bisulfite-converted DNA fragments. The underlying technology, Adaptase, is a proprietary NGS adapter attachment chemistry for single-stranded DNA.

    “By significantly improving sample recovery from bisulfite-converted DNA,” explains Dr. Kurihara, “more complete analysis of clinical samples is possible, particularly cell-free DNA from plasma that is limited to low-nanogram quantities of DNA.”

    Dr. Kurihara cites an example provided by Dennis Lo, M.D., Ph.D., professor of chemical pathology at the Chinese University of Hong Kong. Dr. Lo developed a noninvasive test for cancer by detection of genome-wide hypomethylation of cell-free DNA from patient plasma. Although this “liquid biopsy” does not uncover actionable cancer mutations, it may prove to be a sensitive blood test for early cancer detection as well as treatment monitoring.

    More recently, Dr. Lo’s group mapped the tissue of origin for cell-free plasma DNA using genome-wide bisulfite sequencing after mapping tissue-specific methylation patterns. Such noninvasive testing from blood may identify tissue- or organ-specific pathologies, including cancer, stroke, myocardial infarction, autoimmune disorders, and transplant rejection.

    “Given that advances in epigenetic technologies have enabled per-base methylation status from low DNA input clinical samples, proof of concept has been established that ‘liquid biopsy’ testing of patient blood may be a universal screen for a variety of diseases that may be pinpointed to individual organs or tissues,” Dr. Kurihara tells GEN. “Such universal testing could be particularly advantageous for early detection of cancer and other diseases where noninvasive screening has not previously been possible.”

  • NGS: An Enabling Technology

    The widespread adoption of next-generation genomic sequencing means that for the first time scientists can sequence large numbers of cancer patient genomes. Thus far, these studies have demonstrated that a large proportion of mutated cancer genes may be classified as epigenetic modifying factors.

    “Chromatin remodeling and modifying factors are involved in the regulation of gene expression,” says Ali Shilatifard, Ph.D., chairman of the department of biochemistry and molecular genetics at Northwestern University’s Feinberg School of Medicine. “The DNA methylation factors are highly mutated in most cancers characterized thus far.”

    Dr. Shilatifard provides the example of a family of mixed-lineage leukemia genes within the complexes known as COMPASS (complex proteins associated with Set1), which are highly mutated in a large number of cancers. “We’ve shown that MLL3/4, two members of the COMPASS family, modify regulatory elements known as enhancers,” notes Dr. Shilatifard. “The job of this COMPASS family member is to regulate these cis-regulatory elements during development.”

    It has been shown that MLL3/4 and another component of COMPASS, UTX, are some of the most mutated genes in cancer. “We propose that perhaps these mutations function through enhancer malfunction,” Dr. Shilatifard continues. “And enhancer malfunction through these family members could result in miscommunication of the regulatory elements and promoters and mis-regulation of the expression pattern, resulting in tissue-specific cancers. It’s now very clear that epigenetic regulation and enhancer malfunction are key events in cancer pathogenesis.”

    Dr. Shilatifard believes that over the next several years, academic labs and pharmaceutical companies will increasingly rely on agents that intervene epigenetically. For example, a recent study indicated that approximately 75% of patients with diffuse intrinsic pontine glioma (DIPG), a rare brain cancer in children, carried a single point mutation on histone H3, transforming lysine 27 into methionine. Many copies of histone H3 exist in these patients, but mutation in just one copy is sufficient to cause DIPG.

    After modeling this mutation in Drosophila, Dr. Shilatifard’s laboratory discovered that a single point mutation on one histone was associated with a global loss of histone methylation and an increase in histone acetylation.

    “Epigenetic regulators could be central for treating this disease,” comments Dr. Shilatifard. “Numerous examples in the literature suggest that inhibition of epigenetic regulators and interactors could be very important for treating cancer, and this may work for the treatment of DIPG through the inhibition of factors that bind to hyper-acetylated histones.”

 

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2.0 Genomics and Epigenetics: Genetic Errors and Methodologies – Cancer and Other Diseases

Writer and Curator: Larry H Bernstein, MD, FCAP

This is the second article in a series concerning genomic expression, The first of which was concerned with the expanded technologies in use for study of genomic expression.  This portion will also cover more of genetic errors as well as methodologies, but not all examples are in the realm of cancer.

I shall start with a New York Times editorial on July 24, 2015 by Angelina Jolie Pitt on her experience with BRCA1 gene and her family history.  It is very instructive on how she worked through her experience.

http://www.nytimes.com/2015/03/24/opinion/angelina-jolie-pitt-diary-of-a-surgery.html?

Two years ago she was found to have a positive test for BRCA1, carrying an 87 percent risk for breast cancer and a 50 percent risk for ovarian cancer.  At that time she had a preventive mastectomy.  The decision was not easy, but it also brought into consideration that her mother and grandmother both died of breast cancer.  She did not have an oophorectomy at that time because on considering the advice of medical experts, she would have been left with no estrogen support. She wanted to delay her early vegetative senescence.  She has reached the age of 39 years and on the advice of medical expert opinion, she proceeded with salpingo-oophorectomy, at age 39 years, a decade before  her  mother had developed cancer.  But her delay was to allow her to recover and adjust emotionally to her ongoing situation, with a remaining risk for ovarian cancer.

She tested negative for CA-1251-5 at this time prior to surgery. But the CA-125 test could well be negative with early onset ovarian cancer. It may be considered a better test for following treatment than for early diagnosis. Her choice was to sacrifice early menopause to the ability to live through her childrens’ childhood development.  This was a well thought out decision.  In addition, there were abnormal inflammatory markers that were not specific for cancer rsik, but were worth taking into account.  The procedure itself was simpler than the mastectomy.

23op-ed-thumbStandard

http://static01.nyt.com/images/2015/03/23/opinion/23op-ed/23op-ed-master315.jpg

2.1  CA-125 and Ovarian Cancer

2.1.1  lmmunoradiometric Assay of CA 125 in Effusions: Comparison with Carcinoembryonic Antigen

Marguerite M. Pinto, MD,‘ Larry H. Bernstein, MD,* Dennis A. Brogan, MPH, MT

and Elaine Criscuolo, CT(ASCP) CMIACS

The levels of CA 125 antigen were measured in 167 effusions from 150 patients using radioimmunoassay, and the results compared with the levels of carcinoembryonic antigen (CEA) in the fluids. The results indicate that an elevated fluid CA 125 level (>14,000 U/ml-68,000 U/ml) and a negative fluid CEA level (4 ng/ml) is suggestive of serous and endometrioid carcinoma of ovary, and adenocarcinoma of the endometrium and fallopian tube. Alternatively, an elevated fluid CEA level (14 ng/ml-600 ng/ml) and a negative CA 125 level (20-5000 U/ml) is seen in metastatic carcinomas of breast, lung, gastrointestinal tract, and mucinous ystadenocarcinoma. Lymphomas, melanomas, and benign effusions are negative for both antigens. The combined use of CEA and CA 125 antigen in fluids is useful in the differential diagnosis of adenocarcinoma of unknown primary. Cancer 59:218-222, 1987.

2.1.2 CA-125 in fine-needle aspirates of solid tumors: comparison with cytologic diagnosis and carcinoembryonic antigen (CEA) assay.

Marguerite M. Pinto, S Kotta

Diagnostic Cytopathology 03/1996; 14(2):121-5.
http://dx.doi.org:/10.1002/(SICI)1097-0339(199603)14:2<121::AID-DC4>3.0.CO;2-M

One hundred and twenty-two fine needle aspirates (FNA) from female patients were studied to determine whether CA-125 assay contributed to cytologic diagnosis and CEA assay. Cytologic examination was done on Papanicolaou-stained smears and cell blocks, CEA by EIA (Abbott Laboratory, > 5 ng/ml cutoff) and CA-125 by RIA (Abbott Laboratory, North Chicago, IL, > 66 mu/ml cutoff). Final diagnosis were correlated with histologic diagnosis when available, clinical, radiologic studies, and follow-up. Results: 29 benign, 93 malignant. Sensitivities and specificities: cytology, 91%, 100%; CEA: 59%, 86%; CA-125, 50%, 55%. CEA plus cytology sensitivity, 97%. CA-125 content was highest in endometrial/ovarian carcinoma (39,899 mu/ml) and < 5,000 mu/ml in other tumors and benign FNA in contrast to CEA which showed highest levels in carcinomas of colon, pancreas, and lung (> 280 ng/ml). While elevated CEA enhances the sensitivity of cytologic diagnosis of carcinomas of the colon, pancreas, and lung, low CEA and high CA-125 content supports an ovarian/endometrial primary.

2.1.3  Diagnostic efficiency of carcinoembryonic antigen and CA125 in the cytological evaluation of effusions.

Pinto MM, Bernstein LH, Rudolph RA, Brogan DA, Rosman M.
Arch Pathol Lab Med. 1992 Jun; 116(6):626-31.

In our previous study, the combination of the concentrations of carcinoembryonic antigen (CEA) and CA125 and the findings from cytological examination in 189 benign and malignant pleural and peritoneal effusions was useful in the diagnosis/classification of malignant effusions. Sensitivity of CEA (level, greater than 5 ng/mL) was 68%; specificity was 99% for the diagnosis of malignant effusions secondary to carcinoma of the lung, breast, gastrointestinal tract, and mucinous carcinoma of the ovary. Sensitivity of CA125 (level, greater than 5000 U/mL) was 85%; specificity was 96% for the diagnosis of malignant effusions in carcinoma of the ovary, fallopian tube, and endometrium. We now expanded the study to include 840 pleural and peritoneal effusions (benign, n = 520; malignant, n = 320) and analyzed the data by the statistical method of Rudolph and colleagues. Based on new cutoff values, ie, CEA level at 6.3 ng/mL and CA125 level at 3652 U/mL, the sensitivities for detection of malignant effusions secondary to carcinomas of the lung, breast, and gastrointestinal tract and mucinous carcinoma of the ovary varied between 75% and 100%; specificity was 98%. Sensitivity of CA125 for detection of malignant effusions from müllerian epithelial carcinoma was 71%; specificity was 99%. The elevated CEA fluid level alone helped to diagnose malignant effusions of the gastrointestinal tract in 54%, breast in 19%, and lung in 16%. The high CA125 fluid level was predictive of müllerian epithelial carcinoma. Adjunctive use of CEA and CA125 levels in fluid enhances the sensitivity of cytological diagnosis and may be predictive of the primary site in patients who present with carcinoma of an unknown primary source.

2.2 Carcinoembryonic antigen in diagnostics

2.2.1 Carcinoembryonic antigen content in fine needle aspirates of the lung. A diagnostic adjunct to cytology.

Pinto MM1, Ha DJ.
Acta Cytol. 1992 May-Jun; 36(3):277-82

Carcinoembryonic Antigen (CEA) was measured in 59 consecutive fine needle aspirates (FNAs) of the lung from 58 patients to determine if the CEA content would enhance the sensitivity of the cytologic diagnosis. Twenty-eight males and 30 females with tumors 1-40 cm in diameter were studied. Final diagnoses were correlated with the clinical history, radiologic studies, tissue (when available) and follow-up. Image-guided FNAs were performed by radiologists using a 22-gauge Chiba needle and 20-mL syringe with one to four passes per specimen. Cytologic examination included rapid assessment in the radiology suite and a final diagnosis in 24 hours. CEA was measured by enzyme immunoassay using monoclonal antibody. Nine benign aspirates and 50 malignant aspirates were diagnosed. The sensitivity of cytology was 86% and specificity, 100%. Using 5 ng/mL as the cutoff, the sensitivity of CEA for malignant aspirates was 50% and specificity, 90%. The combined sensitivity of CEA and cytology was 95%. The mean CEA in malignant aspirates was 131 ng/mL and in benign aspirates, 2.41. The highest mean CEA was seen in adenocarcinoma, 402.6 ng/mL. Lower CEA content was seen in epidermoid carcinoma (58.6 ng/mL), large cell carcinoma (8.09), oat cell carcinoma, metastatic carcinoma of the kidney and breast, thymoma and lymphoma (each less than 1 ng/mL). Elevated CEA alone was diagnostic in two aspirates of bronchioloalveolar carcinoma; carcinoma with an unknown primary source, three; and large cell carcinoma, one. The adjunctive use of CEA in FNAs of the lung enhances the sensitivity of the cytologic diagnosis.

2.2.2  Relationship between serum CA125 half life and survival in ovarian cancer

Table
Gupta and Lis Journal of Ovarian Research 2009 2:13
http://dx.doi.org:/10.1186/1757-2215-2-13

First Author, Year, Study Place Data Collection Study
Design
Sample
Size
RR/HR, (95% CI),
P-Value
Riedinger JM, 2006, France 1988 to
1996
R 553 2.04 (1.58-2.63), < 0.0001
Gadducci A, 2004, Italy 1996 to2002 R 71 3.11 (1.22-7.98), 0.0181
Munstedt K, 1997, Germany 1987 to1994 R 85 0.6184
Gadducci A, 1995, Italy 1986 to1992 R 225 2.13 (1.23-3.68), 0.0073
Rosman M, 1994, Connecticut 1985 to
1989
R 51 3.6 (1.8-7.4), < 0.001
Yedema C A, 1993, Netherlands 1984 to
1990
R 60 9.17 (1.49-56.3), 0.01
Hawkins RE, 1989, London NA P 29 3.7 (0.7-20.1), 0.001;27.8 (4.0-193), 0.001

1CA125 half-life was independent prognostic indicator for survival
2FIGO stage, tumor grade, residual disease, CA125
http://www.ovarianresearch.com/content/2/1/13/table/T6

3.3.0      DNA double strand breaks

2.3.1.  Collaboration and competition – DNA double-strand break repair pathways

Kass EM, Jasin M
FEBS Letters 2010; 584:3703-3708
http://dx.doi.org:/jfebslet.2010.07.057

DNA double-strand breaks occur in replication and exogenous sources pose risk to genome stability. There are two pathways to repair.  They are non-homologous end joining and homologous recombination. Both pathways cooperate and compete at double-strand break sites.

2.3.2 DNA Double-Strand Break Repair Inhibitors as Cancer Therapeutics

Srivastava M, Rashavan SC
Chem & Biol 2015 Jan; pp17-29
http://dx.doi.org:/10.1016/jchembiol.2014.11.013

Homologous recombination and non-homologous end joining are the two major repair pathways expressed in eukaryotes.  For double-strand breaks, and the DSB repair gene is vulnerable to chemotherapy and radiation therapy, accounting for treatment resistance. Therefore, targeting DSB repair is attractive. Blocking the residual repair using inhibitors can potentiate treatment.

2.3.3  Animation published in DNA Repair: Helleday T, Lo J, van Gent DC, Engelward BP. DNA double-strand break repair: From mechanistic understanding to cancer treatment. DNA Repair. (14 Mar 2007)
2.3.3.1 http://web.mit.edu/engelward-lab/animations/DSBR.html

2.3.3.2 https://www.youtube.com/watch?v=eg8rpYFsqCA

2.3.4 Homology-dependent double strand break repair. Oxford Academic (Oxford University Press)

https://www.youtube.com/watch?v=86JCMM5kb2A

2.4.0 Managing DNA data sets

2.4.1 Bionimbus –  a cloud for managing, analyzing and sharing large genomics datasets

The Bionimbus Protected Data Cloud (PDC) is a collaboration between the Open Science Data Cloud (OSDC) and the IGSB (IGSB,) the Center for Research Informatics (CRI), the Institute for Translational Medicine (ITM), and the University of Chicago Comprehensive Cancer Center (UCCCC). The PDC allows users authorized by NIH to compute over human genomic data from dbGaP in a secure compliant fashion. Currently, selected datasets from the The Cancer Genome Atlas (TCGA) are available in the PDC.

https://bionimbus-pdc.opensciencedatacloud.org/

2.4.1.2 Accounting for uncertainty in DNA sequencing data

O’Rawe JA, Ferson S, Lyon GJ
Trends in Genetics 2015 Feb; 31(2):61-66
http://dx.doi.org:/10.101/jtig.2014.12.002

This article reviews uncertainty in quantification in DNA sequency applications and sources of error propagation, and it proposes methods to account for errors and uncertainties.

2.5.0 Linking Traits to Mechanisms and UPR response/proteostasis

2.5.1 Stress-Independent Activation of XBP1s and/or ATF6 Reveals –Three Linking traits based on their shared molecular mechanisms

Shoulders MD, Ryno LM, Genereux JC,…Wiseman BL
Cell Reports 2013 Apr; 3, pp 1279-1292
http://dx.doi.org:/10.1016/j.celrep.2013.03.024

The unfolded protein response (UPR) maintains ER proteostasis through the transcription factors XP1s and ATF6. This study measured orthogonal small molecule-mediated activation of transcription factors nXP1s and/or ATF6 using transcriptomics and quantitative proteomics. The finding is that three ER proteostasis environmants differentially influence

  1. Folding
  2. Traffiking, and
  3. Degradation of destabilized ER client proteins

Without affecting endogenous proteome. The proteostasis network is remodeled with the potential for selective restoration of the aberrant ER proteostasis.

2.5.2 Biological and chemical approaches to diseases of proteostasis deficiency.

Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE
Annu Rev Biochem. 2009; 78:959-91.
http://dx.doi.org:/10.1146/annurev.biochem.052308.114844

Many diseases appear to be caused by the misregulation of protein maintenance. Such diseases of protein homeostasis, or “proteostasis,” include loss-of-function diseases (cystic fibrosis) and gain-of-toxic-function diseases (Alzheimer’s, Parkinson’s, and Huntington’s disease). Proteostasis is maintained by the proteostasis network, which comprises pathways that control protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. The decreased ability of the proteostasis network to cope with inherited misfolding-prone proteins, aging, and/or metabolic/environmental stress appears to trigger or exacerbate proteostasis diseases. Herein, we review recent evidence supporting the principle that proteostasis is influenced both by an adjustable proteostasis network capacity and protein folding energetics, which together determine the balance between folding efficiency, misfolding, protein degradation, and aggregation. We review how small molecules can enhance proteostasis by binding to and stabilizing specific proteins (pharmacologic chaperones) or by increasing the proteostasis network capacity (proteostasis regulators). We propose that such therapeutic strategies, including combination therapies, represent a new approach for treating a range of diverse human maladies.

2.5.3 Extracellular Chaperones and Proteostasis

Amy R. Wyatt, Justin J. Yerbury, Heath Ecroyd, and Mark R. Wilson
Annual Review of Biochemistry 2013 Jun; 82: 295-322
http://dx.doi.org:/10.1146/annurev-biochem-072711-163904

There exists a family of currently untreatable, serious human diseases that arise from the inappropriate misfolding and aggregation of extracellular proteins. At present our understanding of mechanisms that operate to maintain proteostasis in extracellular body fluids is limited, but it has significantly advanced with the discovery of a small but growing family of constitutively secreted extracellular chaperones. The available evidence strongly suggests that these chaperones act as both sensors and disposal mediators of misfolded proteins in extracellular fluids, thereby normally protecting us from disease pathologies. It is critically important to further increase our understanding of the mechanisms that operate to effect extracellular proteostasis, as this is essential knowledge upon which to base the development of effective therapies for some of the world’s most debilitating, costly, and intractable diseases.

http://www.proteostasis.com/our-technology/proteostasis-network.html

proteostasis model

http://www.proteostasis.com/images/stories/technology/illustration1.gif

2.6.0 Transcription

2.6.1 Looping Back to Leap Forward. Transcription Enters a New Era

Levine M, Cattoglio C, Tijan R
Cell 2014 Mar; 157: 13-22.
http://dx.doi.org:/10.1016/j.cell.2014.02.009

Organism complexity is not in gene number, but lies in gene regulation. The human genbome contains hundreds of thousands of enhancers, and genes are embedded in a milieu of enhancers . Proliferation of cis-regulatory DNAs is accompanied by complexity and functional diversity of transcription machinery recognizing distal enhancers and promotors, and high-order spatial organization. This article reviews the dynamic communication of remote enhancers with target promoters.

2.6.2 Activating gene expression in mammalian cells with promoter-targeted duplex RNAs.

Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, Corey DR.
Nat Chem Biol. 2007 Mar; 3(3):166-73
http://dx.doi.org:/10.1038/nchembio860

The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.
2.6.3 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

M Gossen and H Bujard
Proc Natl Acad Sci U S A. 1992 Jun 15; 89(12): 5547–5551.

Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of “on/off” situations for such genes in a reversible way.

Diagrams of two regulatable gene expression systems.

Diagrams of two regulatable gene expression systems.

http://www.intechopen.com/source/html/16788/media/image5.jpeg

schematic-representation-of-transgenic-mouse-breeding-scheme-h2b-gfp-mice-should-not-express-gfp-in-the-absence-of-a-tetracycline-regulatable-transactivator

schematic-representation-of-transgenic-mouse-breeding-scheme-h2b-gfp-mice-should-not-express-gfp-in-the-absence-of-a-tetracycline-regulatable-transactivator

http://openi.nlm.nih.gov/imgs/512/321/2408727/2408727_pone.0002357.g001.png

2.7.0 Epigenetics and Cancer

2.7.1 Epigenetics and cancer metabolism

Johnson C, Warmoes MO, Shen X, Locasale JW
Cancer Letters 2015;  356:309-314.
http://dx.doi.org:/10.1016/j.canlet.2013.09.043

Cancer is characterized by adaptive metabolic changes for proliferation and survival of the neoplastic cell, which is accompanied by dysfunctional metabolic enzyme changes in a specific nutrient supplied environment. The oncogenic change uses epigenetic level enzymes that catalyze posttranslational modifications of the DNA/histone expression with metabolites including cofactors and substrates for reactions. This interaction of epigenetics and metabolism provides new insights for anti-cancer therapy.

2.7.2 Cancer Epigenetics. From Mechanism to Therapy

Dawson MA, Konzarides T
Cell 2012 Jul; 150:12-27
http://dx.doi.org:/10.1016/j.cell.2012.06.013

Carcinogenesis requires all of the following:

  • DNA methylation
  • Histone modification
  • Nucleosome remodeling
  • RNA mediated targeting

This article reviews basic principles of epigenetic pathways that are dysregulated in carcinogenesis.

2.7.4 A subway review of cancer pathways

Hahn WC, Weinberg RA
Nature Reviews: Cancer
http://www.nature.com/nrc/poster/subpathways/index.html

Cancer arises from the stepwise accumulation of genetic changes that confer upon an incipient neoplastic cell the properties of unlimited, self-sufficient growth and resistance to normal homeostatic regulatory mechanisms. Advances in human genetics and molecular and cellular biology have identified a collection of cell phenotypes � the main destinations in the subway map below � that are required for malignant transformation1. Specific molecular pathways (subway lines) are responsible for programming these behaviours. Although the connections between cancer-cell wiring and function remain incompletely explored and specified � hence the many lines under construction � the broad outlines of the molecular circuitry of the cancer cell can now be sketched. Further advances in understanding these pathways and their interconnections will accelerate the development of molecularly targeted therapies that promise to change the practice of oncology.

cancer subway map

cancer subway map

http://www.nature.com/nrc/poster/subpathways/images/map.gif

Subway map designed by Claudia Bentley.

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Metabolomics Summary and Perspective


Metabolomics Summary and Perspective

Author and Curator: Larry H Bernstein, MD, FCAP 

 

This is the final article in a robust series on metabolism, metabolomics, and  the “-OMICS-“ biological synthesis that is creating a more holistic and interoperable view of natural sciences, including the biological disciplines, climate science, physics, chemistry, toxicology, pharmacology, and pathophysiology with as yet unforeseen consequences.

There have been impressive advances already in the research into developmental biology, plant sciences, microbiology, mycology, and human diseases, most notably, cancer, metabolic , and infectious, as well as neurodegenerative diseases.

Acknowledgements:

I write this article in honor of my first mentor, Harry Maisel, Professor and Emeritus Chairman of Anatomy, Wayne State University, Detroit, MI and to my stimulating mentors, students, fellows, and associates over many years:

Masahiro Chiga, MD, PhD, Averill A Liebow, MD, Nathan O Kaplan, PhD, Johannes Everse, PhD, Norio Shioura, PhD, Abraham Braude, MD, Percy J Russell, PhD, Debby Peters, Walter D Foster, PhD, Herschel Sidransky, MD, Sherman Bloom, MD, Matthew Grisham, PhD, Christos Tsokos, PhD,  IJ Good, PhD, Distinguished Professor, Raool Banagale, MD, Gustavo Reynoso, MD,Gustave Davis, MD, Marguerite M Pinto, MD, Walter Pleban, MD, Marion Feietelson-Winkler, RD, PhD,  John Adan,MD, Joseph Babb, MD, Stuart Zarich, MD,  Inder Mayall, MD, A Qamar, MD, Yves Ingenbleek, MD, PhD, Emeritus Professor, Bette Seamonds, PhD, Larry Kaplan, PhD, Pauline Y Lau, PhD, Gil David, PhD, Ronald Coifman, PhD, Emeritus Professor, Linda Brugler, RD, MBA, James Rucinski, MD, Gitta Pancer, Ester Engelman, Farhana Hoque, Mohammed Alam, Michael Zions, William Fleischman, MD, Salman Haq, MD, Jerard Kneifati-Hayek, Madeleine Schleffer, John F Heitner, MD, Arun Devakonda,MD, Liziamma George,MD, Suhail Raoof, MD, Charles Oribabor,MD, Anthony Tortolani, MD, Prof and Chairman, JRDS Rosalino, PhD, Aviva Lev Ari, PhD, RN, Rosser Rudolph, MD, PhD, Eugene Rypka, PhD, Jay Magidson, PhD, Izaak Mayzlin, PhD, Maurice Bernstein, PhD, Richard Bing, Eli Kaplan, PhD, Maurice Bernstein, PhD.

This article has EIGHT parts, as follows:

Part 1

Metabolomics Continues Auspicious Climb

Part 2

Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells

Part 3

Neuroscience

Part 4

Cancer Research

Part 5

Metabolic Syndrome

Part 6

Biomarkers

Part 7

Epigenetics and Drug Metabolism

Part 8

Pictorial

genome cartoon

genome cartoon

 iron metabolism

iron metabolism

personalized reference range within population range

personalized reference range within population range

Part 1.  MetabolomicsSurge

metagraph  _OMICS

metagraph _OMICS

Metabolomics Continues Auspicious Climb

Jeffery Herman, Ph.D.
GEN May 1, 2012 (Vol. 32, No. 9)

Aberrant biochemical and metabolite signaling plays an important role in

  • the development and progression of diseased tissue.

This concept has been studied by the science community for decades. However, with relatively

  1. recent advances in analytical technology and bioinformatics as well as
  2. the development of the Human Metabolome Database (HMDB),

metabolomics has become an invaluable field of research.

At the “International Conference and Exhibition on Metabolomics & Systems Biology” held recently in San Francisco, researchers and industry leaders discussed how

  • the underlying cellular biochemical/metabolite fingerprint in response to
  1. a specific disease state,
  2. toxin exposure, or
  3. pharmaceutical compound
  • is useful in clinical diagnosis and biomarker discovery and
  • in understanding disease development and progression.

Developed by BASF, MetaMap® Tox is

  • a database that helps identify in vivo systemic effects of a tested compound, including
  1. targeted organs,
  2. mechanism of action, and
  3. adverse events.

Based on 28-day systemic rat toxicity studies, MetaMap Tox is composed of

  • differential plasma metabolite profiles of rats
  • after exposure to a large variety of chemical toxins and pharmaceutical compounds.

“Using the reference data,

  • we have developed more than 110 patterns of metabolite changes, which are
  • specific and predictive for certain toxicological modes of action,”

said Hennicke Kamp, Ph.D., group leader, department of experimental toxicology and ecology at BASF.

With MetaMap Tox, a potential drug candidate

  • can be compared to a similar reference compound
  • using statistical correlation algorithms,
  • which allow for the creation of a toxicity and mechanism of action profile.

“MetaMap Tox, in the context of early pre-clinical safety enablement in pharmaceutical development,” continued Dr. Kamp,

  • has been independently validated “
  • by an industry consortium (Drug Safety Executive Council) of 12 leading biopharmaceutical companies.”

Dr. Kamp added that this technology may prove invaluable

  • allowing for quick and accurate decisions and
  • for high-throughput drug candidate screening, in evaluation
  1. on the safety and efficacy of compounds
  2. during early and preclinical toxicological studies,
  3. by comparing a lead compound to a variety of molecular derivatives, and
  • the rapid identification of the most optimal molecular structure
  • with the best efficacy and safety profiles might be streamlined.
Dynamic Construct of the –Omics

Dynamic Construct of the –Omics

Targeted Tandem Mass Spectrometry

Biocrates Life Sciences focuses on targeted metabolomics, an important approach for

  • the accurate quantification of known metabolites within a biological sample.

Originally used for the clinical screening of inherent metabolic disorders from dried blood-spots of newborn children, Biocrates has developed

  • a tandem mass spectrometry (MS/MS) platform, which allows for
  1. the identification,
  2. quantification, and
  3. mapping of more than 800 metabolites to specific cellular pathways.

It is based on flow injection analysis and high-performance liquid chromatography MS/MS.

Clarification of Pathway-Specific Inhibition by Fourier Transform Ion Cyclotron Resonance.Mass Spectrometry-Based Metabolic Phenotyping Studies F5.large

common drug targets

common drug targets

The MetaDisIDQ® Kit is a

  • “multiparamatic” diagnostic assay designed for the “comprehensive assessment of a person’s metabolic state” and
  • the early determination of pathophysiological events with regards to a specific disease.

MetaDisIDQ is designed to quantify

  • a diverse range of 181 metabolites involved in major metabolic pathways
  • from a small amount of human serum (10 µL) using isotopically labeled internal standards,

This kit has been demonstrated to detect changes in metabolites that are commonly associated with the development of

  • metabolic syndrome, type 2 diabetes, and diabetic nephropathy,

Dr. Dallman reports that data generated with the MetaDisIDQ kit correlates strongly with

  • routine chemical analyses of common metabolites including glucose and creatinine

Biocrates has also developed the MS/MS-based AbsoluteIDQ® kits, which are

  • an “easy-to-use” biomarker analysis tool for laboratory research.

The kit functions on MS machines from a variety of vendors, and allows for the quantification of 150-180 metabolites.

The SteroIDQ® kit is a high-throughput standardized MS/MS diagnostic assay,

  • validated in human serum, for the rapid and accurate clinical determination of 16 known steroids.

Initially focusing on the analysis of steroid ranges for use in hormone replacement therapy, the SteroIDQ Kit is expected to have a wide clinical application.

Hormone-Resistant Breast Cancer

Scientists at Georgetown University have shown that

  • breast cancer cells can functionally coordinate cell-survival and cell-proliferation mechanisms,
  • while maintaining a certain degree of cellular metabolism.

To grow, cells need energy, and energy is a product of cellular metabolism. For nearly a century, it was thought that

  1. the uncoupling of glycolysis from the mitochondria,
  2. leading to the inefficient but rapid metabolism of glucose and
  3. the formation of lactic acid (the Warburg effect), was

the major and only metabolism driving force for unchecked proliferation and tumorigenesis of cancer cells.

Other aspects of metabolism were often overlooked.

“.. we understand now that

  • cellular metabolism is a lot more than just metabolizing glucose,”

said Robert Clarke, Ph.D., professor of oncology and physiology and biophysics at Georgetown University. Dr. Clarke, in collaboration with the Waters Center for Innovation at Georgetown University (led by Albert J. Fornace, Jr., M.D.), obtained

  • the metabolomic profile of hormone-sensitive and -resistant breast cancer cells through the use of UPLC-MS.

They demonstrated that breast cancer cells, through a rather complex and not yet completely understood process,

  1. can functionally coordinate cell-survival and cell-proliferation mechanisms,
  2. while maintaining a certain degree of cellular metabolism.

This is at least partly accomplished through the upregulation of important pro-survival mechanisms; including

  • the unfolded protein response;
  • a regulator of endoplasmic reticulum stress and
  • initiator of autophagy.

Normally, during a stressful situation, a cell may

  • enter a state of quiescence and undergo autophagy,
  • a process by which a cell can recycle organelles
  • in order to maintain enough energy to survive during a stressful situation or,

if the stress is too great,

  • undergo apoptosis.

By integrating cell-survival mechanisms and cellular metabolism

  • advanced ER+ hormone-resistant breast cancer cells
  • can maintain a low level of autophagy
  • to adapt and resist hormone/chemotherapy treatment.

This adaptation allows cells

  • to reallocate important metabolites recovered from organelle degradation and
  • provide enough energy to also promote proliferation.

With further research, we can gain a better understanding of the underlying causes of hormone-resistant breast cancer, with

  • the overall goal of developing effective diagnostic, prognostic, and therapeutic tools.

NMR

Over the last two decades, NMR has established itself as a major tool for metabolomics analysis. It is especially adept at testing biological fluids. [Bruker BioSpin]

Historically, nuclear magnetic resonance spectroscopy (NMR) has been used for structural elucidation of pure molecular compounds. However, in the last two decades, NMR has established itself as a major tool for metabolomics analysis. Since

  • the integral of an NMR signal is directly proportional to
  • the molar concentration throughout the dynamic range of a sample,

“the simultaneous quantification of compounds is possible

  • without the need for specific reference standards or calibration curves,” according to Lea Heintz of Bruker BioSpin.

NMR is adept at testing biological fluids because of

  1.  high reproducibility,
  2. standardized protocols,
  3. low sample manipulation, and
  4. the production of a large subset of data,

Bruker BioSpin is presently involved in a project for the screening of inborn errors of metabolism in newborn children from Turkey, based on their urine NMR profiles. More than 20 clinics are participating to the project that is coordinated by INFAI, a specialist in the transfer of advanced analytical technology into medical diagnostics. The construction of statistical models are being developed

  • for the detection of deviations from normality, as well as
  • automatic quantification methods for indicative metabolites

Bruker BioSpin recently installed high-resolution magic angle spinning NMR (HRMAS-NMR) systems that can rapidly analyze tissue biopsies. The main objective for HRMAS-NMR is to establish a rapid and effective clinical method to assess tumor grade and other important aspects of cancer during surgery.

Combined NMR and Mass Spec

There is increasing interest in combining NMR and MS, two of the main analytical assays in metabolomic research, as a means

  • to improve data sensitivity and to
  • fully elucidate the complex metabolome within a given biological sample.
  •  to realize a potential for cancer biomarker discovery in the realms of diagnosis, prognosis, and treatment.

.

Using combined NMR and MS to measure the levels of nearly 250 separate metabolites in the patient’s blood, Dr. Weljie and other researchers at the University of Calgary were able to rapidly determine the malignancy of a  pancreatic lesion (in 10–15% of the cases, it is difficult to discern between benign and malignant), while avoiding unnecessary surgery in patients with benign lesions.

When performing NMR and MS on a single biological fluid, ultimately “we are,” noted Dr. Weljie,

  1. “splitting up information content, processing, and introducing a lot of background noise and error and
  2. then trying to reintegrate the data…
    It’s like taking a complex item, with multiple pieces, out of an IKEA box and trying to repackage it perfectly into another box.”

By improving the workflow between the initial splitting of the sample, they improved endpoint data integration, proving that

  • a streamlined approach to combined NMR/MS can be achieved,
  • leading to a very strong, robust and precise metabolomics toolset.

Metabolomics Research Picks Up Speed

Field Advances in Quest to Improve Disease Diagnosis and Predict Drug Response

John Morrow Jr., Ph.D.
GEN May 1, 2011 (Vol. 31, No. 9)

As an important discipline within systems biology, metabolomics is being explored by a number of laboratories for

  • its potential in pharmaceutical development.

Studying metabolites can offer insights into the relationships between genotype and phenotype, as well as between genotype and environment. In addition, there is plenty to work with—there are estimated to be some 2,900 detectable metabolites in the human body, of which

  1. 309 have been identified in cerebrospinal fluid,
  2. 1,122 in serum,
  3. 458 in urine, and
  4. roughly 300 in other compartments.

Guowang Xu, Ph.D., a researcher at the Dalian Institute of Chemical Physics.  is investigating the causes of death in China,

  • and how they have been changing over the years as the country has become a more industrialized nation.
  •  the increase in the incidence of metabolic disorders such as diabetes has grown to affect 9.7% of the Chinese population.

Dr. Xu,  collaborating with Rainer Lehman, Ph.D., of the University of Tübingen, Germany, compared urinary metabolites in samples from healthy individuals with samples taken from prediabetic, insulin-resistant subjects. Using mass spectrometry coupled with electrospray ionization in the positive mode, they observed striking dissimilarities in levels of various metabolites in the two groups.

“When we performed a comprehensive two-dimensional gas chromatography, time-of-flight mass spectrometry analysis of our samples, we observed several metabolites, including

  • 2-hydroxybutyric acid in plasma,
  •  as potential diabetes biomarkers,” Dr. Xu explains.

In other, unrelated studies, Dr. Xu and the German researchers used a metabolomics approach to investigate the changes in plasma metabolite profiles immediately after exercise and following a 3-hour and 24-hour period of recovery. They found that

  • medium-chain acylcarnitines were the most distinctive exercise biomarkers, and
  • they are released as intermediates of partial beta oxidation in human myotubes and mouse muscle tissue.

Dr. Xu says. “The traditional approach of assessment based on a singular biomarker is being superseded by the introduction of multiple marker profiles.”

Typical of the studies under way by Dr. Kaddurah-Daouk and her colleaguesat Duke University

  • is a recently published investigation highlighting the role of an SNP variant in
  • the glycine dehydrogenase gene on individual response to antidepressants.
  •  patients who do not respond to the selective serotonin uptake inhibitors citalopram and escitalopram
  • carried a particular single nucleotide polymorphism in the GD gene.

“These results allow us to pinpoint a possible

  • role for glycine in selective serotonin reuptake inhibitor response and
  • illustrate the use of pharmacometabolomics to inform pharmacogenomics.

These discoveries give us the tools for prognostics and diagnostics so that

  • we can predict what conditions will respond to treatment.

“This approach to defining health or disease in terms of metabolic states opens a whole new paradigm.

By screening hundreds of thousands of molecules, we can understand

  • the relationship between human genetic variability and the metabolome.”

Dr. Kaddurah-Daouk talks about statins as a current

  • model of metabolomics investigations.

It is now known that the statins  have widespread effects, altering a range of metabolites. To sort out these changes and develop recommendations for which individuals should be receiving statins will require substantial investments of energy and resources into defining the complex web of biochemical changes that these drugs initiate.
Furthermore, Dr. Kaddurah-Daouk asserts that,

  • “genetics only encodes part of the phenotypic response.

One needs to take into account the

  • net environment contribution in order to determine
  • how both factors guide the changes in our metabolic state that determine the phenotype.”

Interactive Metabolomics

Researchers at the University of Nottingham use diffusion-edited nuclear magnetic resonance spectroscopy to assess the effects of a biological matrix on metabolites. Diffusion-edited NMR experiments provide a way to

  • separate the different compounds in a mixture
  • based on the differing translational diffusion coefficients (which reflect the size and shape of the molecule).

The measurements are carried out by observing

  • the attenuation of the NMR signals during a pulsed field gradient experiment.

Clare Daykin, Ph.D., is a lecturer at the University of Nottingham, U.K. Her field of investigation encompasses “interactive metabolomics,”which she defines as

“the study of the interactions between low molecular weight biochemicals and macromolecules in biological samples ..

  • without preselection of the components of interest.

“Blood plasma is a heterogeneous mixture of molecules that

  1. undergo a variety of interactions including metal complexation,
  2. chemical exchange processes,
  3. micellar compartmentation,
  4. enzyme-mediated biotransformations, and
  5. small molecule–macromolecular binding.”

Many low molecular weight compounds can exist

  • freely in solution,
  • bound to proteins, or
  • within organized aggregates such as lipoprotein complexes.

Therefore, quantitative comparison of plasma composition from

  • diseased individuals compared to matched controls provides an incomplete insight to plasma metabolism.

“It is not simply the concentrations of metabolites that must be investigated,

  • but their interactions with the proteins and lipoproteins within this complex web.

Rather than targeting specific metabolites of interest, Dr. Daykin’s metabolite–protein binding studies aim to study

  • the interactions of all detectable metabolites within the macromolecular sample.

Such activities can be studied through the use of diffusion-edited nuclear magnetic resonance (NMR) spectroscopy, in which one can assess

  • the effects of the biological matrix on the metabolites.

“This can lead to a more relevant and exact interpretation

  • for systems where metabolite–macromolecule interactions occur.”

Diffusion-edited NMR experiments provide a way to separate the different compounds in a mixture based on

  • the differing translational diffusion coefficients (which reflect the size and shape of the molecule).

The measurements are carried out by observing

  • the attenuation of the NMR signals during a pulsed field gradient experiment.

Pushing the Limits

It is widely recognized that many drug candidates fail during development due to ancillary toxicity. Uwe Sauer, Ph.D., professor, and Nicola Zamboni, Ph.D., researcher, both at the Eidgenössische Technische Hochschule, Zürich (ETH Zürich), are applying

  • high-throughput intracellular metabolomics to understand
  • the basis of these unfortunate events and
  • head them off early in the course of drug discovery.

“Since metabolism is at the core of drug toxicity, we developed a platform for

  • measurement of 50–100 targeted metabolites by
  • a high-throughput system consisting of flow injection
  • coupled to tandem mass spectrometry.”

Using this approach, Dr. Sauer’s team focused on

  • the central metabolism of the yeast Saccharomyces cerevisiae, reasoning that
  • this core network would be most susceptible to potential drug toxicity.

Screening approximately 41 drugs that were administered at seven concentrations over three orders of magnitude, they observed changes in metabolome patterns at much lower drug concentrations without attendant physiological toxicity.

The group carried out statistical modeling of about

  • 60 metabolite profiles for each drug they evaluated.

This data allowed the construction of a “profile effect map” in which

  • the influence of each drug on metabolite levels can be followed, including off-target effects, which
  • provide an indirect measure of the possible side effects of the various drugs.

Dr. Sauer says.“We have found that this approach is

  • at least 100 times as fast as other omics screening platforms,”

“Some drugs, including many anticancer agents,

  • disrupt metabolism long before affecting growth.”
killing cancer cells

killing cancer cells

Furthermore, they used the principle of 13C-based flux analysis, in which

  • metabolites labeled with 13C are used to follow the utilization of metabolic pathways in the cell.

These 13C-determined intracellular responses of metabolic fluxes to drug treatment demonstrate

  • the functional performance of the network to be rather robust,
conformational changes leading to substrate efflux.

conformational changes leading to substrate efflux.

leading Dr. Sauer to the conclusion that

  • the phenotypic vigor he observes to drug challenges
  • is achieved by a flexible make up of the metabolome.

Dr. Sauer is confident that it will be possible to expand the scope of these investigations to hundreds of thousands of samples per study. This will allow answers to the questions of

  • how cells establish a stable functioning network in the face of inevitable concentration fluctuations.

Is Now the Hour?

There is great enthusiasm and agitation within the biotech community for

  • metabolomics approaches as a means of reversing the dismal record of drug discovery

that has accumulated in the last decade.

While the concept clearly makes sense and is being widely applied today, there are many reasons why drugs fail in development, and metabolomics will not be a panacea for resolving all of these questions. It is too early at this point to recognize a trend or a track record, and it will take some time to see how this approach can aid in drug discovery and shorten the timeline for the introduction of new pharmaceutical agents.

Degree of binding correlated with function

Degree of binding correlated with function

Diagram_of_a_two-photon_excitation_microscope_

Diagram_of_a_two-photon_excitation_microscope_

Part 2.  Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells

Biologists at UC San Diego have found

  • the “missing link” in the chemical system that
  • enables animal cells to produce ribosomes

—the thousands of protein “factories” contained within each cell that

  • manufacture all of the proteins needed to build tissue and sustain life.
‘Missing Link’

‘Missing Link’

Their discovery, detailed in the June 23 issue of the journal Genes & Development, will not only force

  • a revision of basic textbooks on molecular biology, but also
  • provide scientists with a better understanding of
  • how to limit uncontrolled cell growth, such as cancer,
  • that might be regulated by controlling the output of ribosomes.

Ribosomes are responsible for the production of the wide variety of proteins that include

  1. enzymes;
  2. structural molecules, such as hair,
  3. skin and bones;
  4. hormones like insulin; and
  5. components of our immune system such as antibodies.

Regarded as life’s most important molecular machine, ribosomes have been intensively studied by scientists (the 2009 Nobel Prize in Chemistry, for example, was awarded for studies of its structure and function). But until now researchers had not uncovered all of the details of how the proteins that are used to construct ribosomes are themselves produced.

In multicellular animals such as humans,

  • ribosomes are made up of about 80 different proteins
    (humans have 79 while some other animals have a slightly different number) as well as
  • four different kinds of RNA molecules.

In 1969, scientists discovered that

  • the synthesis of the ribosomal RNAs is carried out by specialized systems using two key enzymes:
  • RNA polymerase I and RNA polymerase III.

But until now, scientists were unsure if a complementary system was also responsible for

  • the production of the 80 proteins that make up the ribosome.

That’s essentially what the UC San Diego researchers headed by Jim Kadonaga, a professor of biology, set out to examine. What they found was the missing link—the specialized

  • system that allows ribosomal proteins themselves to be synthesized by the cell.

Kadonaga says that he and coworkers found that ribosomal proteins are synthesized via

  • a novel regulatory system with the enzyme RNA polymerase II and
  • a factor termed TRF2,”

“For the production of most proteins,

  1. RNA polymerase II functions with
  2. a factor termed TBP,
  3. but for the synthesis of ribosomal proteins, it uses TRF2.”
  •  this specialized TRF2-based system for ribosome biogenesis
  • provides a new avenue for the study of ribosomes and
  • its control of cell growth, and

“it should lead to a better understanding and potential treatment of diseases such as cancer.”

Coordination of the transcriptome and metabolome

Coordination of the transcriptome and metabolome

the potential advantages conferred by distal-site protein synthesis

the potential advantages conferred by distal-site protein synthesis

Other authors of the paper were UC San Diego biologists Yuan-Liang Wang, Sascha Duttke and George Kassavetis, and Kai Chen, Jeff Johnston, and Julia Zeitlinger of the Stowers Institute for Medical Research in Kansas City, Missouri. Their research was supported by two grants from the National Institutes of Health (1DP2OD004561-01 and R01 GM041249).

Turning Off a Powerful Cancer Protein

Scientists have discovered how to shut down a master regulatory transcription factor that is

  • key to the survival of a majority of aggressive lymphomas,
  • which arise from the B cells of the immune system.

The protein, Bcl6, has long been considered too complex to target with a drug since it is also crucial

  • to the healthy functioning of many immune cells in the body, not just B cells gone bad.

The researchers at Weill Cornell Medical College report that it is possible

  • to shut down Bcl6 in diffuse large B-cell lymphoma (DLBCL)
  • while not affecting its vital function in T cells and macrophages
  • that are needed to support a healthy immune system.

If Bcl6 is completely inhibited, patients might suffer from systemic inflammation and atherosclerosis. The team conducted this new study to help clarify possible risks, as well as to understand

  • how Bcl6 controls the various aspects of the immune system.

The findings in this study were inspired from

  • preclinical testing of two Bcl6-targeting agents that Dr. Melnick and his Weill Cornell colleagues have developed
  • to treat DLBCLs.

These experimental drugs are

  • RI-BPI, a peptide mimic, and
  • the small molecule agent 79-6.

“This means the drugs we have developed against Bcl6 are more likely to be

  • significantly less toxic and safer for patients with this cancer than we realized,”

says Ari Melnick, M.D., professor of hematology/oncology and a hematologist-oncologist at NewYork-Presbyterian Hospital/Weill Cornell Medical Center.

Dr. Melnick says the discovery that

  • a master regulatory transcription factor can be targeted
  • offers implications beyond just treating DLBCL.

Recent studies from Dr. Melnick and others have revealed that

  • Bcl6 plays a key role in the most aggressive forms of acute leukemia, as well as certain solid tumors.

Bcl6 can control the type of immune cell that develops in the bone marrow—playing many roles

  • in the development of B cells, T cells, macrophages, and other cells—including a primary and essential role in
  • enabling B-cells to generate specific antibodies against pathogens.

According to Dr. Melnick, “When cells lose control of Bcl6,

  • lymphomas develop in the immune system.

Lymphomas are ‘addicted’ to Bcl6, and therefore

  • Bcl6 inhibitors powerfully and quickly destroy lymphoma cells,” .

The big surprise in the current study is that rather than functioning as a single molecular machine,

  • Bcl6 functions like a Swiss Army knife,
  • using different tools to control different cell types.

This multifunction paradigm could represent a general model for the functioning of other master regulatory transcription factors.

“In this analogy, the Swiss Army knife, or transcription factor, keeps most of its tools folded,

  • opening only the one it needs in any given cell type,”

He makes the following analogy:

  • “For B cells, it might open and use the knife tool;
  • for T cells, the cork screw;
  • for macrophages, the scissors.”

“this means that you only need to prevent the master regulator from using certain tools to treat cancer. You don’t need to eliminate the whole knife,” . “In fact, we show that taking out the whole knife is harmful since

  • the transcription factor has many other vital functions that other cells in the body need.”

Prior to these study results, it was not known that a master regulator could separate its functions so precisely. Researchers hope this will be a major benefit to the treatment of DLBCL and perhaps other disorders that are influenced by Bcl6 and other master regulatory transcription factors.

The study is published in the journal Nature Immunology, in a paper titled “Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated by distinct biochemical mechanisms”.

Part 3. Neuroscience

Vesicles influence function of nerve cells 
Oct, 06 2014        source: http://feeds.sciencedaily.com

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Tiny vesicles containing protective substances

  • which they transmit to nerve cells apparently
  • play an important role in the functioning of neurons.

As cell biologists at Johannes Gutenberg University Mainz (JGU) have discovered,

  • nerve cells can enlist the aid of mini-vesicles of neighboring glial cells
  • to defend themselves against stress and other potentially detrimental factors.

These vesicles, called exosomes, appear to stimulate the neurons on various levels:

  • they influence electrical stimulus conduction,
  • biochemical signal transfer, and
  • gene regulation.

Exosomes are thus multifunctional signal emitters

  • that can have a significant effect in the brain.
Exosome

Exosome

The researchers in Mainz already observed in a previous study that

  • oligodendrocytes release exosomes on exposure to neuronal stimuli.
  • these are absorbed by the neurons and improve neuronal stress tolerance.

Oligodendrocytes, a type of glial cell, form an

  • insulating myelin sheath around the axons of neurons.

The exosomes transport protective proteins such as

  • heat shock proteins,
  • glycolytic enzymes, and
  • enzymes that reduce oxidative stress from one cell type to another,
  • but also transmit genetic information in the form of ribonucleic acids.

“As we have now discovered in cell cultures, exosomes seem to have a whole range of functions,” explained Dr. Eva-Maria Krmer-Albers. By means of their transmission activity, the small bubbles that are the vesicles

  • not only promote electrical activity in the nerve cells, but also
  • influence them on the biochemical and gene regulatory level.

“The extent of activities of the exosomes is impressive,” added Krmer-Albers. The researchers hope that the understanding of these processes will contribute to the development of new strategies for the treatment of neuronal diseases. Their next aim is to uncover how vesicles actually function in the brains of living organisms.

http://labroots.com/user/news/article/id/217438/title/vesicles-influence-function-of-nerve-cells

The above story is based on materials provided by Universitt Mainz.

Universitt Mainz. “Vesicles influence function of nerve cells.” ScienceDaily. ScienceDaily, 6 October 2014. www.sciencedaily.com/releases/2014/10/141006174214.htm

Neuroscientists use snail research to help explain “chemo brain”

10/08/2014
It is estimated that as many as half of patients taking cancer drugs experience a decrease in mental sharpness. While there have been many theories, what causes “chemo brain” has eluded scientists.

In an effort to solve this mystery, neuroscientists at The University of Texas Health Science Center at Houston (UTHealth) conducted an experiment in an animal memory model and their results point to a possible explanation. Findings appeared in The Journal of Neuroscience.

In the study involving a sea snail that shares many of the same memory mechanisms as humans and a drug used to treat a variety of cancers, the scientists identified

  • memory mechanisms blocked by the drug.

Then, they were able to counteract or

  • unblock the mechanisms by administering another agent.

“Our research has implications in the care of people given to cognitive deficits following drug treatment for cancer,” said John H. “Jack” Byrne, Ph.D., senior author, holder of the June and Virgil Waggoner Chair and Chairman of the Department of Neurobiology and Anatomy at the UTHealth Medical School. “There is no satisfactory treatment at this time.”

Byrne’s laboratory is known for its use of a large snail called Aplysia californica to further the understanding of the biochemical signaling among nerve cells (neurons).  The snails have large neurons that relay information much like those in humans.

When Byrne’s team compared cell cultures taken from normal snails to

  • those administered a dose of a cancer drug called doxorubicin,

the investigators pinpointed a neuronal pathway

  • that was no longer passing along information properly.

With the aid of an experimental drug,

  • the scientists were able to reopen the pathway.

Unfortunately, this drug would not be appropriate for humans, Byrne said. “We want to identify other drugs that can rescue these memory mechanisms,” he added.

According the American Cancer Society, some of the distressing mental changes cancer patients experience may last a short time or go on for years.

Byrne’s UT Health research team includes co-lead authors Rong-Yu Liu, Ph.D., and Yili Zhang, Ph.D., as well as Brittany Coughlin and Leonard J. Cleary, Ph.D. All are affiliated with the W.M. Keck Center for the Neurobiology of Learning and Memory.

Byrne and Cleary also are on the faculty of The University of Texas Graduate School of Biomedical Sciences at Houston. Coughlin is a student at the school, which is jointly operated by UT Health and The University of Texas MD Anderson Cancer Center.

The study titled “Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase” received support from National Institutes of Health grant (NS019895) and the Zilkha Family Discovery Fellowship.

Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase

Source: Univ. of Texas Health Science Center at Houston

http://www.rdmag.com/news/2014/10/neuroscientists-use-snail-research-help-explain-E2_9_Cchemo-brain

Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase

Rong-Yu Liu*,  Yili Zhang*,  Brittany L. Coughlin,  Leonard J. Cleary, and  John H. Byrne   +Show Affiliations
The Journal of Neuroscience, 1 Oct 2014, 34(40): 13289-13300;
http://dx.doi.org:/10.1523/JNEUROSCI.0538-14.2014

Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be

  • topoisomerase II inhibition, DNA cleavage, and free radical generation.

However, in non-neuronal cells, DOX also inhibits the expression of

  • dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby
  1. inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and
  2. p38 mitogen-activated protein kinase (p38 MAPK),
  3. two MAPK isoforms important for long-term memory (LTM) formation.

Activation of these kinases by DOX in neurons, if present,

  • could have secondary effects on cognitive functions, such as learning and memory.

The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia

  • to examine the effects of DOX on levels of phosphorylated ERK (pERK) and
  • phosphorylated p38 (p-p38) MAPK.

In addition, Aplysia neurons were used to examine the effects of DOX on

  • long-term enhanced excitability, long-term synaptic facilitation (LTF), and
  • long-term synaptic depression (LTD).

DOX treatment led to elevated levels of

  • pERK and p-p38 MAPK in SNs and cortical neurons.

In addition, it increased phosphorylation of

  • the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs.

DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to

  • overriding inhibitory effects of p-p38 MAPK, because
  • LTF was rescued in the presence of an inhibitor of p38 MAPK
    (SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) .

These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway.
Terms: Aplysia chemotherapy ERK  p38 MAPK serotonin synaptic plasticity

Technology that controls brain cells with radio waves earns early BRAIN grant

10/08/2014

bright spots = cells with increased calcium after treatment with radio waves,  allows neurons to fire

bright spots = cells with increased calcium after treatment with radio waves, allows neurons to fire

BRAIN control: The new technology uses radio waves to activate or silence cells remotely. The bright spots above represent cells with increased calcium after treatment with radio waves, a change that would allow neurons to fire.

A proposal to develop a new way to

  • remotely control brain cells

from Sarah Stanley, a research associate in Rockefeller University’s Laboratory of Molecular Genetics, headed by Jeffrey M. Friedman, is

  • among the first to receive funding from U.S. President Barack Obama’s BRAIN initiative.

The project will make use of a technique called

  • radiogenetics that combines the use of radio waves or magnetic fields with
  • nanoparticles to turn neurons on or off.

The National Institutes of Health is one of four federal agencies involved in the BRAIN (Brain Research through Advancing Innovative Neurotechnologies) initiative. Following in the ambitious footsteps of the Human Genome Project, the BRAIN initiative seeks

  • to create a dynamic map of the brain in action,

a goal that requires the development of new technologies. The BRAIN initiative working group, which outlined the broad scope of the ambitious project, was co-chaired by Rockefeller’s Cori Bargmann, head of the Laboratory of Neural Circuits and Behavior.

Stanley’s grant, for $1.26 million over three years, is one of 58 projects to get BRAIN grants, the NIH announced. The NIH’s plan for its part of this national project, which has been pitched as “America’s next moonshot,” calls for $4.5 billion in federal funds over 12 years.

The technology Stanley is developing would

  • enable researchers to manipulate the activity of neurons, as well as other cell types,
  • in freely moving animals in order to better understand what these cells do.

Other techniques for controlling selected groups of neurons exist, but her new nanoparticle-based technique has a

  • unique combination of features that may enable new types of experimentation.
  • it would allow researchers to rapidly activate or silence neurons within a small area of the brain or
  • dispersed across a larger region, including those in difficult-to-access locations.

Stanley also plans to explore the potential this method has for use treating patients.

“Francis Collins, director of the NIH, has discussed

  • the need for studying the circuitry of the brain,
  • which is formed by interconnected neurons.

Our remote-control technology may provide a tool with which researchers can ask new questions about the roles of complex circuits in regulating behavior,” Stanley says.
Rockefeller University’s Laboratory of Molecular Genetics
Source: Rockefeller Univ.

Part 4.  Cancer

Two Proteins Found to Block Cancer Metastasis

Why do some cancers spread while others don’t? Scientists have now demonstrated that

  • metastatic incompetent cancers actually “poison the soil”
  • by generating a micro-environment that blocks cancer cells
  • from settling and growing in distant organs.

The “seed and the soil” hypothesis proposed by Stephen Paget in 1889 is now widely accepted to explain how

  • cancer cells (seeds) are able to generate fertile soil (the micro-environment)
  • in distant organs that promotes cancer’s spread.

However, this concept had not explained why some tumors do not spread or metastasize.

The researchers, from Weill Cornell Medical College, found that

  • two key proteins involved in this process work by
  • dramatically suppressing cancer’s spread.

The study offers hope that a drug based on these

  • potentially therapeutic proteins, prosaposin and Thrombospondin 1 (Tsp-1),

might help keep human cancer at bay and from metastasizing.

Scientists don’t understand why some tumors wouldn’t “want” to spread. It goes against their “job description,” says the study’s senior investigator, Vivek Mittal, Ph.D., an associate professor of cell and developmental biology in cardiothoracic surgery and director of the Neuberger Berman Foundation Lung Cancer Laboratory at Weill Cornell Medical College. He theorizes that metastasis occurs when

  • the barriers that the body throws up to protect itself against cancer fail.

But there are some tumors in which some of the barriers may still be intact. “So that suggests

  • those primary tumors will continue to grow, but that
  • an innate protective barrier still exists that prevents them from spreading and invading other organs,”

The researchers found that, like typical tumors,

  • metastasis-incompetent tumors also send out signaling molecules
  • that establish what is known as the “premetastatic niche” in distant organs.

These niches composed of bone marrow cells and various growth factors have been described previously by others including Dr. Mittal as the fertile “soil” that the disseminated cancer cell “seeds” grow in.

Weill Cornell’s Raúl Catena, Ph.D., a postdoctoral fellow in Dr. Mittal’s laboratory, found an important difference between the tumor types. Metastatic-incompetent tumors

  • systemically increased expression of Tsp-1, a molecule known to fight cancer growth.
  • increased Tsp-1 production was found specifically in the bone marrow myeloid cells
  • that comprise the metastatic niche.

These results were striking, because for the first time Dr. Mittal says

  • the bone marrow-derived myeloid cells were implicated as
  • the main producers of Tsp-1,.

In addition, Weill Cornell and Harvard researchers found that

  • prosaposin secreted predominantly by the metastatic-incompetent tumors
  • increased expression of Tsp-1 in the premetastatic lungs.

Thus, Dr. Mittal posits that prosaposin works in combination with Tsp-1

  • to convert pro-metastatic bone marrow myeloid cells in the niche
  • into cells that are not hospitable to cancer cells that spread from a primary tumor.
  • “The very same myeloid cells in the niche that we know can promote metastasis
  • can also be induced under the command of the metastatic incompetent primary tumor to inhibit metastasis,”

The research team found that

  • the Tsp-1–inducing activity of prosaposin
  • was contained in only a 5-amino acid peptide region of the protein, and
  • this peptide alone induced Tsp-1 in the bone marrow cells and
  • effectively suppressed metastatic spread in the lungs
  • in mouse models of breast and prostate cancer.

This 5-amino acid peptide with Tsp-1–inducing activity

  • has the potential to be used as a therapeutic agent against metastatic cancer,

The scientists have begun to test prosaposin in other tumor types or metastatic sites.

Dr. Mittal says that “The clinical implications of the study are:

  • “Not only is it theoretically possible to design a prosaposin-based drug or drugs
  • that induce Tsp-1 to block cancer spread, but
  • you could potentially create noninvasive prognostic tests
  • to predict whether a cancer will metastasize.”

The study was reported in the April 30 issue of Cancer Discovery, in a paper titled “Bone Marrow-Derived Gr1+ Cells Can Generate a Metastasis-Resistant Microenvironment Via Induced Secretion of Thrombospondin-1”.

Disabling Enzyme Cripples Tumors, Cancer Cells

First Step of Metastasis

First Step of Metastasis

Published: Sep 05, 2013  http://www.technologynetworks.com/Metabolomics/news.aspx?id=157138

Knocking out a single enzyme dramatically cripples the ability of aggressive cancer cells to spread and grow tumors.

The paper, published in the journal Proceedings of the National Academy of Sciences, sheds new light on the importance of lipids, a group of molecules that includes fatty acids and cholesterol, in the development of cancer.

Researchers have long known that cancer cells metabolize lipids differently than normal cells. Levels of ether lipids – a class of lipids that are harder to break down – are particularly elevated in highly malignant tumors.

“Cancer cells make and use a lot of fat and lipids, and that makes sense because cancer cells divide and proliferate at an accelerated rate, and to do that,

  • they need lipids, which make up the membranes of the cell,”

said study principal investigator Daniel Nomura, assistant professor in UC Berkeley’s Department of Nutritional Sciences and Toxicology. “Lipids have a variety of uses for cellular structure, but what we’re showing with our study is that

  • lipids can send signals that fuel cancer growth.”

In the study, Nomura and his team tested the effects of reducing ether lipids on human skin cancer cells and primary breast tumors. They targeted an enzyme,

  • alkylglycerone phosphate synthase, or AGPS,
  • known to be critical to the formation of ether lipids.

The researchers confirmed that

  1. AGPS expression increased when normal cells turned cancerous.
  2. inactivating AGPS substantially reduced the aggressiveness of the cancer cells.

“The cancer cells were less able to move and invade,” said Nomura.

The researchers also compared the impact of

  • disabling the AGPS enzyme in mice that had been injected with cancer cells.

Nomura. observes -“Among the mice that had the AGPS enzyme inactivated,

  • the tumors were nonexistent,”

“The mice that did not have this enzyme

  • disabled rapidly developed tumors.”

The researchers determined that

  • inhibiting AGPS expression depleted the cancer cells of ether lipids.
  • AGPS altered levels of other types of lipids important to the ability of the cancer cells to survive and spread, including
    • prostaglandins and acyl phospholipids.

“What makes AGPS stand out as a treatment target is that the enzyme seems to simultaneously

  • regulate multiple aspects of lipid metabolism
  • important for tumor growth and malignancy.”

Future steps include the

  • development of AGPS inhibitors for use in cancer therapy,

“This study sheds considerable light on the important role that AGPS plays in ether lipid metabolism in cancer cells, and it suggests that

  • inhibitors of this enzyme could impair tumor formation,”

said Benjamin Cravatt, Professor and Chair of Chemical Physiology at The Scripps Research Institute, who is not part of the UC.

Agilent Technologies Thought Leader Award Supports Translational Research Program
Published: Mon, March 04, 2013

The award will support Dr DePinho’s research into

  • metabolic reprogramming in the earliest stages of cancer.

Agilent Technologies Inc. announces that Dr. Ronald A. DePinho, a world-renowned oncologist and researcher, has received an Agilent Thought Leader Award.

DePinho is president of the University of Texas MD Anderson Cancer Center. DePinho and his team hope to discover and characterize

  • alterations in metabolic flux during tumor initiation and maintenance, and to identify biomarkers for early detection of pancreatic cancer together with
  • novel therapeutic targets.

Researchers on his team will work with scientists from the university’s newly formed Institute of Applied Cancer Sciences.

The Agilent Thought Leader Award provides funds to support personnel as well as a state-of-the-art Agilent 6550 iFunnel Q-TOF LC/MS system.

“I am extremely pleased to receive this award for metabolomics research, as the survival rates for pancreatic cancer have not significantly improved over the past 20 years,” DePinho said. “This technology will allow us to

  • rapidly identify new targets that drive the formation, progression and maintenance of pancreatic cancer.

Discoveries from this research will also lead to

  • the development of effective early detection biomarkers and novel therapeutic interventions.”

“We are proud to support Dr. DePinho’s exciting translational research program, which will make use of

  • metabolomics and integrated biology workflows and solutions in biomarker discovery,”

said Patrick Kaltenbach, Agilent vice president, general manager of the Liquid Phase Division, and the executive sponsor of this award.

The Agilent Thought Leader Program promotes fundamental scientific advances by support of influential thought leaders in the life sciences and chemical analysis fields.

The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

Ping Xie, Minghua Zhang, Shan He, Kefeng Lu, Yuhan Chen, Guichun Xing, et al.
Nature Communications
  2014; 5(3733).  http://dx.doi.org:/10.1038/ncomms4733

Neddylation, the covalent attachment of ubiquitin-like protein Nedd8, of the Cullin-RING E3 ligase family

  • regulates their ubiquitylation activity.

However, regulation of HECT ligases by neddylation has not been reported to date. Here we show that

  • the C2-WW-HECT ligase Smurf1 is activated by neddylation.

Smurf1 physically interacts with

  1. Nedd8 and Ubc12,
  2. forms a Nedd8-thioester intermediate, and then
  3. catalyses its own neddylation on multiple lysine residues.

Intriguingly, this autoneddylation needs

  • an active site at C426 in the HECT N-lobe.

Neddylation of Smurf1 potently enhances

  • ubiquitin E2 recruitment and
  • augments the ubiquitin ligase activity of Smurf1.

The regulatory role of neddylation

  • is conserved in human Smurf1 and yeast Rsp5.

Furthermore, in human colorectal cancers,

  • the elevated expression of Smurf1, Nedd8, NAE1 and Ubc12
  • correlates with cancer progression and poor prognosis.

These findings provide evidence that

  • neddylation is important in HECT ubiquitin ligase activation and
  • shed new light on the tumour-promoting role of Smurf1.
 Swinging domains in HECT E3

Swinging domains in HECT E3

Subject terms: Biological sciences Cancer Cell biology

Figure 1: Smurf1 expression is elevated in colorectal cancer tissues.

Smurf1 expression is elevated in colorectal cancer tissues.

Smurf1 expression is elevated in colorectal cancer tissues.

(a) Smurf1 expression scores are shown as box plots, with the horizontal lines representing the median; the bottom and top of the boxes representing the 25th and 75th percentiles, respectively; and the vertical bars representing the ra

Figure 2: Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer.

Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer

Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer

(a) Representative images from immunohistochemical staining of Smurf1, Ubc12, NAE1 and Nedd8 in the same colorectal cancer tumour. Scale bars, 100 μm. (bd) The expression scores of Nedd8 (b, n=283 ), NAE1 (c, n=281) and Ubc12 (d, n=19…

Figure 3: Smurf1 interacts with Ubc12.

Smurf1 interacts with Ubc12

Smurf1 interacts with Ubc12

(a) GST pull-down assay of Smurf1 with Ubc12. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Smurf1 interacted with Ubc12 and UbcH5c, but not with Ubc9. (b) Mapping the regions…

Figure 4: Nedd8 is attached to Smurf1through C426-catalysed autoneddylation.

Nedd8 is attached to Smurf1through C426-catalysed autoneddylation

Nedd8 is attached to Smurf1through C426-catalysed autoneddylation

(a) Covalent neddylation of Smurf1 in vitro.Purified His-Smurf1-WT or C699A proteins were incubated with Nedd8 and Nedd8-E1/E2. Reactions were performed as described in the Methods section. Samples were analysed by western blotting wi…

Figure 5: Neddylation of Smurf1 activates its ubiquitin ligase activity.

Neddylation of Smurf1 activates its ubiquitin ligase activity.

Neddylation of Smurf1 activates its ubiquitin ligase activity.

(a) In vivo Smurf1 ubiquitylation assay. Nedd8 was co-expressed with Smurf1 WT or C699A in HCT116 cells (left panels). Twenty-four hours post transfection, cells were treated with MG132 (20 μM, 8 h). HCT116 cells were transfected with…

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The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response

M Simicek, S Lievens, M Laga, D Guzenko, VN. Aushev, et al.
Nature Cell Biology 2013; 15, 1220–1230    http://dx.doi.org:/10.1038/ncb2847

The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively

  • engaging two components of the exocyst complex, EXO84 and SEC5.
  1. RALB employs SEC5 to trigger innate immunity signalling, whereas
  2. RALB–EXO84 interaction induces autophagocytosis.

How this differential interaction is achieved molecularly by the RAL GTPase remains unknown.

We found that whereas GTP binding

  • turns on RALB activity,

ubiquitylation of RALB at Lys 47

  • tunes its activity towards a particular effector.

Specifically, ubiquitylation at Lys 47

  • sterically inhibits RALB binding to EXO84, while
  • facilitating its interaction with SEC5.

Double-stranded RNA promotes

  • RALB ubiquitylation and
  • SEC5–TBK1 complex formation.

In contrast, nutrient starvation

  • induces RALB deubiquitylation
  • by accumulation and relocalization of the deubiquitylase USP33
  • to RALB-positive vesicles.

Deubiquitylated RALB

  • promotes the assembly of the RALB–EXO84–beclin-1 complexes
  • driving autophagosome formation. Thus,
  • ubiquitylation within the effector-binding domain
  • provides the switch for the dual functions of RALB in
    • autophagy and innate immune responses.

Part 5. Metabolic Syndrome

Single Enzyme is Necessary for Development of Diabetes

Published: Aug 20, 2014 http://www.technologynetworks.com/Metabolomics/news.aspx?ID=169416

12-LO enzyme promotes the obesity-induced oxidative stress in the pancreatic cells.

An enzyme called 12-LO promotes the obesity-induced oxidative stress in the pancreatic cells that leads

  • to pre-diabetes, and diabetes.

12-LO’s enzymatic action is the last step in

  • the production of certain small molecules that harm the cell,

according to a team from Indiana University School of Medicine, Indianapolis.

The findings will enable the development of drugs that can interfere with this enzyme, preventing or even reversing diabetes. The research is published ahead of print in the journal Molecular and Cellular Biology.

In earlier studies, these researchers and their collaborators at Eastern Virginia Medical School showed that

  • 12-LO (which stands for 12-lipoxygenase) is present in these cells
  • only in people who become overweight.

The harmful small molecules resulting from 12-LO’s enzymatic action are known as HETEs, short for hydroxyeicosatetraenoic acid.

  1. HETEs harm the mitochondria, which then
  2. fail to produce sufficient energy to enable
  3. the pancreatic cells to manufacture the necessary quantities of insulin.

For the study, the investigators genetically engineered mice that

  • lacked the gene for 12-LO exclusively in their pancreas cells.

Mice were either fed a low-fat or high-fat diet.

Both the control mice and the knockout mice on the high fat diet

  • developed obesity and insulin resistance.

The investigators also examined the pancreatic beta cells of both knockout and control mice, using both microscopic studies and molecular analysis. Those from the knockout mice were intact and healthy, while

  • those from the control mice showed oxidative damage,
  • demonstrating that 12-LO and the resulting HETEs
  • caused the beta cell failure.

Mirmira notes that fatty diet used in the study was the Western Diet, which comprises mostly saturated-“bad”-fats. Based partly on a recent study of related metabolic pathways, he says that

  • the unsaturated and mono-unsaturated fats-which comprise most fats in the healthy,
  • relatively high fat Mediterranean diet-are unlikely to have the same effects.

“Our research is the first to show that 12-LO in the beta cell

  • is the culprit in the development of pre-diabetes, following high fat diets,” says Mirmira.

“Our work also lends important credence to the notion that

  • the beta cell is the primary defective cell in virtually all forms of diabetes and pre-diabetes.”

A New Player in Lipid Metabolism Discovered

Published: Aug18, 2014  http://www.technologynetworks.com/Metabolomics/news.aspx?ID=169356

Specially engineered mice gained no weight, and normal counterparts became obese

  • on the same high-fat, obesity-inducing Western diet.

Specially engineered mice that lacked a particular gene did not gain weight

  • when fed a typical high-fat, obesity-inducing Western diet.

Yet, these mice ate the same amount as their normal counterparts that became obese.

The mice were engineered with fat cells that lacked a gene called SEL1L,

  • known to be involved in the clearance of mis-folded proteins
  • in the cell’s protein making machinery called the endoplasmic reticulum (ER).

When mis-folded proteins are not cleared but accumulate,

  • they destroy the cell and contribute to such diseases as
  1. mad cow disease,
  2. Type 1 diabetes and
  3. cystic fibrosis.

“The million-dollar question is why don’t these mice gain weight? Is this related to its inability to clear mis-folded proteins in the ER?” said Ling Qi, associate professor of molecular and biochemical nutrition and senior author of the study published online July 24 in Cell Metabolism. Haibo Sha, a research associate in Qi’s lab, is the paper’s lead author.

Interestingly, the experimental mice developed a host of other problems, including

  • postprandial hypertriglyceridemia,
  • and fatty livers.

“Although we are yet to find out whether these conditions contribute to the lean phenotype, we found that

  • there was a lipid partitioning defect in the mice lacking SEL1L in fat cells,
  • where fat cells cannot store fat [lipids], and consequently
  • fat goes to the liver.

During the investigation of possible underlying mechanisms, we discovered

  • a novel function for SEL1L as a regulator of lipid metabolism,” said Qi.

Sha said “We were very excited to find that

  • SEL1L is required for the intracellular trafficking of
  • lipoprotein lipase (LPL), acting as a chaperone,” .

and added that “Using several tissue-specific knockout mouse models,

  • we showed that this is a general phenomenon,”

Without LPL, lipids remain in the circulation;

  • fat and muscle cells cannot absorb fat molecules for storage and energy combustion,

People with LPL mutations develop

  • postprandial hypertriglyceridemia similar to
  • conditions found in fat cell-specific SEL1L-deficient mice, said Qi.

Future work will investigate the

  • role of SEL1L in human patients carrying LPL mutations and
  • determine why fat cell-specific SEL1L-deficient mice remain lean under Western diets, said Sha.

Co-authors include researchers from Cedars-Sinai Medical Center in Los Angeles; Wageningen University in the Netherlands; Georgia State University; University of California, Los Angeles; and the Medical College of Soochow University in China.

The study was funded by the U.S. National Institutes of Health, the Netherlands Organization for Health Research and Development National Institutes of Health, the Cedars-Sinai Medical Center, Chinese National Science Foundation, the American Diabetes Association, Cornell’s Center for Vertebrate Genomics and the Howard Hughes Medical Institute.

Part 6. Biomarkers

Biomarkers Take Center Stage

Josh P. Roberts
GEN May 1, 2013 (Vol. 33, No. 9)  http://www.genengnews.com/

While work with biomarkers continues to grow, scientists are also grappling with research-related bottlenecks, such as

  1. affinity reagent development,
  2. platform reproducibility, and
  3. sensitivity.

Biomarkers by definition indicate some state or process that generally occurs

  • at a spatial or temporal distance from the marker itself, and

it would not be an exaggeration to say that biomedicine has become infatuated with them:

  1. where to find them,
  2. when they may appear,
  3. what form they may take, and
  4. how they can be used to diagnose a condition or
  5. predict whether a therapy may be successful.

Biomarkers are on the agenda of many if not most industry gatherings, and in cases such as Oxford Global’s recent “Biomarker Congress” and the GTC “Biomarker Summit”, they hold the naming rights. There, some basic principles were built upon, amended, and sometimes challenged.

In oncology, for example, biomarker discovery is often predicated on the premise that

  • proteins shed from a tumor will traverse to and persist in, and be detectable in, the circulation.

By quantifying these proteins—singularly or as part of a larger “signature”—the hope is

  1. to garner information about the molecular characteristics of the cancer
  2. that will help with cancer detection and
  3. personalization of the treatment strategy.

Yet this approach has not yet turned into the panacea that was hoped for. Bottlenecks exist in

  • affinity reagent development,
  • platform reproducibility, and
  • sensitivity.

There is also a dearth of understanding of some of the

  • fundamental principles of biomarker biology that we need to know the answers to,

said Parag Mallick, Ph.D., whose lab at Stanford University is “working on trying to understand where biomarkers come from.”

There are dogmas saying that

  • circulating biomarkers come solely from secreted proteins.

But Dr. Mallick’s studies indicate that fully

  • 50% of circulating proteins may come from intracellular sources or
  • proteins that are annotated as such.

“We don’t understand the processes governing

  • which tumor-derived proteins end up in the blood.”

Other questions include “how does the size of a tumor affect how much of a given protein will be in the blood?”—perhaps

  • the tumor is necrotic at the center, or
  • it’s hypervascular or hypovascular.

He points out “The problem is that these are highly nonlinear processes at work, and

  • there is a large number of factors that might affect the answer to that question,” .

Their research focuses on using

  1. mass spectrometry and
  2. computational analysis
  • to characterize the biophysical properties of the circulating proteome, and
  • relate these to measurements made of the tumor itself.

Furthermore, he said – “We’ve observed that the proteins that are likely to

  • first show up and persist in the circulation, ..
  • are more stable than proteins that don’t,”
  • “we can quantify how significant the effect is.”

The goal is ultimately to be able to

  1. build rigorous, formal mathematical models that will allow something measured in the blood
  2. to be tied back to the molecular biology taking place in the tumor.

And conversely, to use those models

  • to predict from a tumor what will be found in the circulation.

“Ultimately, the models will allow you to connect the dots between

  • what you measure in the blood and the biology of the tumor.”

Bound for Affinity Arrays

Affinity reagents are the main tools for large-scale protein biomarker discovery. And while this has tended to mean antibodies (or their derivatives), other affinity reagents are demanding a place in the toolbox.

Affimers, a type of affinity reagent being developed by Avacta, consist of

  1. a biologically inert, biophysically stable protein scaffold
  2. containing three variable regions into which
  3. distinct peptides are inserted.

The resulting three-dimensional surface formed by these peptides

  • interacts and binds to proteins and other molecules in solution,
  • much like the antigen-binding site of antibodies.

Unlike antibodies, Affimers are relatively small (13 KDa),

  • non-post-translationally modified proteins
  • that can readily be expressed in bacterial culture.

They may be made to bind surfaces through unique residues

  • engineered onto the opposite face of the Affimer,
  • allowing the binding site to be exposed to the target in solution.

“We don’t seem to see in what we’ve done so far

  • any real loss of activity or functionality of Affimers when bound to surfaces—

they’re very robust,” said CEO Alastair Smith, Ph.D.

Avacta is taking advantage of this stability and its large libraries of Affimers to develop

  • very large affinity microarrays for
  • drug and biomarker discovery.

To date they have printed arrays with around 20–25,000 features, and Dr. Smith is “sure that we can get toward about 50,000 on a slide,” he said. “There’s no real impediment to us doing that other than us expressing the proteins and getting on with it.”

Customers will be provided with these large, complex “naïve” discovery arrays, readable with standard equipment. The plan is for the company to then “support our customers by providing smaller arrays with

  • the Affimers that are binding targets of interest to them,” Dr. Smith foretold.

And since the intellectual property rights are unencumbered,

  • Affimers in those arrays can be licensed to the end users
  • to develop diagnostics that can be validated as time goes on.

Around 20,000-Affimer discovery arrays were recently tested by collaborator Professor Ann Morgan of the University of Leeds with pools of unfractionated serum from patients with symptoms of inflammatory disease. The arrays

  • “rediscovered” elevated C-reactive protein (CRP, the clinical gold standard marker)
  • as well as uncovered an additional 22 candidate biomarkers.
  • other candidates combined with CRP, appear able to distinguish between different diseases such as
  1. rheumatoid arthritis,
  2. psoriatic arthritis,
  3. SLE, or
  4. giant cell arteritis.

Epigenetic Biomarkers

Methylation of adenine

Sometimes biomarkers are used not to find disease but

  • to distinguish healthy human cell types, with
  •  examples being found in flow cytometry and immunohistochemistry.

These widespread applications, however, are difficult to standardize, being

  • subject to arbitrary or subjective gating protocols and other imprecise criteria.

Epiontis instead uses an epigenetic approach. “What we need is a unique marker that is

  • demethylated only in one cell type and
  • methylated in all the other cell types,”

Each cell of the right cell type will have

  • two demethylated copies of a certain gene locus,
  • allowing them to be enumerated by quantitative PCR.

The biggest challenge is finding that unique epigenetic marker. To do so they look through the literature for proteins and genes described as playing a role in the cell type’s biology, and then

  • look at the methylation patterns to see if one can be used as a marker,

They also “use customized Affymetrix chips to look at the

  • differential epigenetic status of different cell types on a genomewide scale.”

explained CBO and founder Ulrich Hoffmueller, Ph.D.

The company currently has a panel of 12 assays for 12 immune cell types. Among these is an assay for

  • regulatory T (Treg) cells that queries the Foxp3 gene—which is uniquely demethylated in Treg
  • even though it is transiently expressed in activated T cells of other subtypes.

Also assayed are Th17 cells, difficult to detect by flow cytometry because

  • “the cells have to be stimulated in vitro,” he pointed out.

Developing New Assays for Cancer Biomarkers

Researchers at Myriad RBM and the Cancer Prevention Research Institute of Texas are collaborating to develop

  • new assays for cancer biomarkers on the Myriad RBM Multi-Analyte Profile (MAP) platform.

The release of OncologyMAP 2.0 expanded Myriad RBM’s biomarker menu to over 250 analytes, which can be measured from a small single sample, according to the company. Using this menu, L. Stephen et al., published a poster, “Analysis of Protein Biomarkers in Prostate and Colorectal Tumor Lysates,” which showed the results of

  • a survey of proteins relevant to colorectal (CRC) and prostate (PC) tumors
  • to identify potential proteins of interest for cancer research.

The study looked at CRC and PC tumor lysates and found that 102 of the 115 proteins showed levels above the lower limit of quantification.

  • Four markers were significantly higher in PC and 10 were greater in CRC.

For most of the analytes, duplicate sections of the tumor were similar, although some analytes did show differences. In four of the CRC analytes, tumor number four showed differences for CEA and tumor number 2 for uPA.

Thirty analytes were shown to be

  • different in CRC tumor compared to its adjacent tissue.
  • Ten of the analytes were higher in adjacent tissue compared to CRC.
  • Eighteen of the markers examined demonstrated  —-

significant correlations of CRC tumor concentration to serum levels.

“This suggests.. that the Oncology MAP 2.0 platform “provides a good method for studying changes in tumor levels because many proteins can be assessed with a very small sample.”

Clinical Test Development with MALDI-ToF

While there have been many attempts to translate results from early discovery work on the serum proteome into clinical practice, few of these efforts have progressed past the discovery phase.

Matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry on unfractionated serum/plasma samples offers many practical advantages over alternative techniques, and does not require

  • a shift from discovery to development and commercialization platforms.

Biodesix claims it has been able to develop the technology into

  • a reproducible, high-throughput tool to
  • routinely measure protein abundance from serum/plasma samples.

“.. we improved data-analysis algorithms to

  • reproducibly obtain quantitative measurements of relative protein abundance from MALDI-ToF mass spectra.

Heinrich Röder, CTO points out that the MALDI-ToF measurements

  • are combined with clinical outcome data using
  • modern learning theory techniques
  • to define specific disease states
  • based on a patient’s serum protein content,”

The clinical utility of the identification of these disease states can be investigated through a retrospective analysis of differing sample sets. For example, Biodesix clinically validated its first commercialized serum proteomic test, VeriStrat®, in 85 different retrospective sample sets.

Röder adds that “It is becoming increasingly clear that

  • the patients whose serum is characterized as VeriStrat Poor show
  • consistently poor outcomes irrespective of
  1. tumor type,
  2. histology, or
  3. molecular tumor characteristics,”

MALDI-ToF mass spectrometry, in its standard implementation,

  • allows for the observation of around 100 mostly high-abundant serum proteins.

Further, “while this does not limit the usefulness of tests developed from differential expression of these proteins,

  • the discovery potential would be greatly enhanced
  • if we could probe deeper into the proteome
  • while not giving up the advantages of the MALDI-ToF approach,”

Biodesix reports that its new MALDI approach, Deep MALDI™, can perform

  • simultaneous quantitative measurement of more than 1,000 serum protein features (or peaks) from 10 µL of serum in a high-throughput manner.
  • it increases the observable signal noise ratio from a few hundred to over 50,000,
  • resulting in the observation of many lower-abundance serum proteins.

Breast cancer, a disease now considered to be a collection of many complexes of symptoms and signatures—the dominant ones are labeled Luminal A, Luminal B, Her2, and Basal— which suggests different prognose, and

  • these labels are considered too simplistic for understanding and managing a woman’s cancer.

Studies published in the past year have looked at

  1. somatic mutations,
  2. gene copy number aberrations,
  3. gene expression abnormalities,
  4. protein and miRNA expression, and
  5. DNA methylation,

coming up with a list of significantly mutated genes—hot spots—in different categories of breast cancers. Targeting these will inevitably be the focus of much coming research.

“We’ve been taking these large trials and profiling these on a variety of array or sequence platforms. We think we’ll get

  1. prognostic drivers
  2. predictive markers for taxanes and
  3. monoclonal antibodies and
  4. tamoxifen and aromatase inhibitors,”
    explained Brian Leyland-Jones, Ph.D., director of Edith Sanford Breast Cancer Research. “We will end up with 20–40 different diseases, maybe more.”

Edith Sanford Breast Cancer Research is undertaking a pilot study in collaboration with The Scripps Research Institute, using a variety of tests on 25 patients to see how the information they provide complements each other, the overall flow, and the time required to get and compile results.

Laser-captured tumor samples will be subjected to low passage whole-genome, exome, and RNA sequencing (with targeted resequencing done in parallel), and reverse-phase protein and phosphorylation arrays, with circulating nucleic acids and circulating tumor cells being queried as well. “After that we hope to do a 100- or 150-patient trial when we have some idea of the best techniques,” he said.

Dr. Leyland-Jones predicted that ultimately most tumors will be found

  • to have multiple drivers,
  • with most patients receiving a combination of two, three, or perhaps four different targeted therapies.

Reduce to Practice

According to Randox, the evidence Investigator is a sophisticated semi-automated biochip sys­tem designed for research, clinical, forensic, and veterinary applications.

Once biomarkers that may have an impact on therapy are discovered, it is not always routine to get them into clinical practice. Leaving regulatory and financial, intellectual property and cultural issues aside, developing a diagnostic based on a biomarker often requires expertise or patience that its discoverer may not possess.

Andrew Gribben is a clinical assay and development scientist at Randox Laboratories, based in Northern Ireland, U.K. The company utilizes academic and industrial collaborators together with in-house discovery platforms to identify biomarkers that are

  • augmented or diminished in a particular pathology
  • relative to appropriate control populations.

Biomarkers can be developed to be run individually or

  • combined into panels of immunoassays on its multiplex biochip array technology.

Specificity can also be gained—or lost—by the affinity of reagents in an assay. The diagnostic potential of Heart-type fatty acid binding protein (H-FABP) abundantly expressed in human myocardial cells was recognized by Jan Glatz of Maastricht University, The Netherlands, back in 1988. Levels rise quickly within 30 minutes after a myocardial infarction, peaking at 6–8 hours and return to normal within 24–30 hours. Yet at the time it was not known that H-FABP was a member of a multiprotein family, with which the polyclonal antibodies being used in development of an assay were cross-reacting, Gribben related.

Randox developed monoclonal antibodies specific to H-FABP, funded trials investigating its use alone, and multiplexed with cardiac biomarker assays, and, more than 30 years after the biomarker was identified, in 2011, released a validated assay for H-FABP as a biomarker for early detection of acute myocardial infarction.

Ultrasensitive Immunoassays for Biomarker Development

Research has shown that detection and monitoring of biomarker concentrations can provide

  • insights into disease risk and progression.

Cytokines have become attractive biomarkers and candidates

  • for targeted therapies for a number of autoimmune diseases, including rheumatoid arthritis (RA), Crohn’s disease, and psoriasis, among others.

However, due to the low-abundance of circulating cytokines, such as IL-17A, obtaining robust measurements in clinical samples has been difficult.

Singulex reports that its digital single-molecule counting technology provides

  • increased precision and detection sensitivity over traditional ELISA techniques,
  • helping to shed light on biomarker verification and validation programs.

The company’s Erenna® immunoassay system, which includes optimized immunoassays, offers LLoQ to femtogram levels per mL resolution—even in healthy populations, at an improvement of 1-3 fold over standard ELISAs or any conventional technology and with a dynamic range of up to 4-logs, according to a Singulex official, who adds that

  • this sensitivity improvement helps minimize undetectable samples that
  • could otherwise delay or derail clinical studies.

The official also explains that the Singulex solution includes an array of products and services that are being applied to a number of programs and have enabled the development of clinically relevant biomarkers, allowing translation from discovery to the clinic.

In a poster entitled “Advanced Single Molecule Detection: Accelerating Biomarker Development Utilizing Cytokines through Ultrasensitive Immunoassays,” a case study was presented of work performed by Jeff Greenberg of NYU to show how the use of the Erenna system can provide insights toward

  • improving the clinical utility of biomarkers and
  • accelerating the development of novel therapies for treating inflammatory diseases.

A panel of inflammatory biomarkers was examined in DMARD (disease modifying antirheumatic drugs)-naïve RA (rheumatoid arthritis) vs. knee OA (osteoarthritis) patient cohorts. Markers that exhibited significant differences in plasma concentrations between the two cohorts included

  • CRP, IL-6R alpha, IL-6, IL-1 RA, VEGF, TNF-RII, and IL-17A, IL-17F, and IL-17A/F.

Among the three tested isoforms of IL-17,

  • the magnitude of elevation for IL-17F in RA patients was the highest.

“Singulex provides high-resolution monitoring of baseline IL-17A concentrations that are present at low levels,” concluded the researchers. “The technology also enabled quantification of other IL-17 isoforms in RA patients, which have not been well characterized before.”

The Singulex Erenna System has also been applied to cardiovascular disease research, for which its

  • cardiac troponin I (cTnI) digital assay can be used to measure circulating
  • levels of cTnI undetectable by other commercial assays.

Recently presented data from Brigham and Women’s Hospital and the TIMI-22 study showed that

  • using the Singulex test to serially monitor cTnI helps
  • stratify risk in post-acute coronary syndrome patients and
  • can identify patients with elevated cTnI
  • who have the most to gain from intensive vs. moderate-dose statin therapy,

according to the scientists involved in the research.

The study poster, “Prognostic Performance of Serial High Sensitivity Cardiac Troponin Determination in Stable Ischemic Heart Disease: Analysis From PROVE IT-TIMI 22,” was presented at the 2013 American College of Cardiology (ACC) Annual Scientific Session & Expo by R. O’Malley et al.

Biomarkers Changing Clinical Medicine

Better Diagnosis, Prognosis, and Drug Targeting Are among Potential Benefits

  1. John Morrow Jr., Ph.D.

Researchers at EMD Chemicals are developing biomarker immunoassays

  • to monitor drug-induced toxicity including kidney damage.

The pace of biomarker development is accelerating as investigators report new studies on cancer, diabetes, Alzheimer disease, and other conditions in which the evaluation and isolation of workable markers is prominently featured.

Wei Zheng, Ph.D., leader of the R&D immunoassay group at EMD Chemicals, is overseeing a program to develop biomarker immunoassays to

  • monitor drug-induced toxicity, including kidney damage.

“One of the principle reasons for drugs failing during development is because of organ toxicity,” says Dr. Zheng.
“proteins liberated into the serum and urine can serve as biomarkers of adverse response to drugs, as well as disease states.”

Through collaborative programs with Rules-Based Medicine (RBM), the EMD group has released panels for the profiling of human renal impairment and renal toxicity. These urinary biomarker based products fit the FDA and EMEA guidelines for assessment of drug-induced kidney damage in rats.

The group recently performed a screen for potential protein biomarkers in relation to

  • kidney toxicity/damage on a set of urine and plasma samples
  • from patients with documented renal damage.

Additionally, Dr. Zheng is directing efforts to move forward with the multiplexed analysis of

  • organ and cellular toxicity.

Diseases thought to involve compromised oxidative phosphorylation include

  • diabetes, Parkinson and Alzheimer diseases, cancer, and the aging process itself.

Good biomarkers allow Dr. Zheng to follow the mantra, “fail early, fail fast.” With robust, multiplexible biomarkers, EMD can detect bad drugs early and kill them before they move into costly large animal studies and clinical trials. “Recognizing the severe liability that toxicity presents, we can modify the structure of the candidate molecule and then rapidly reassess its performance.”

Scientists at Oncogene Science a division of Siemens Healthcare Diagnostics, are also focused on biomarkers. “We are working on a number of antibody-based tests for various cancers, including a test for the Ca-9 CAIX protein, also referred to as carbonic anhydrase,” Walter Carney, Ph.D., head of the division, states.

CAIX is a transmembrane protein that is

  • overexpressed in a number of cancers, and, like Herceptin and the Her-2 gene,
  • can serve as an effective and specific marker for both diagnostic and therapeutic purposes.
  • It is liberated into the circulation in proportion to the tumor burden.

Dr. Carney and his colleagues are evaluating patients after tumor removal for the presence of the Ca-9 CAIX protein. If

  • the levels of the protein in serum increase over time,
  • this suggests that not all the tumor cells were removed and the tumor has metastasized.

Dr. Carney and his team have developed both an immuno-histochemistry and an ELISA test that could be used as companion diagnostics in clinical trials of CAIX-targeted drugs.

The ELISA for the Ca-9 CAIX protein will be used in conjunction with Wilex’ Rencarex®, which is currently in a

  • Phase III trial as an adjuvant therapy for non-metastatic clear cell renal cancer.

Additionally, Oncogene Science has in its portfolio an FDA-approved test for the Her-2 marker. Originally approved for Her-2/Neu-positive breast cancer, its indications have been expanded over time, and was approved

  • for the treatment of gastric cancer last year.

It is normally present on breast cancer epithelia but

  • overexpressed in some breast cancer tumors.

“Our products are designed to be used in conjunction with targeted therapies,” says Dr. Carney. “We are working with companies that are developing technology around proteins that are

  • overexpressed in cancerous tissues and can be both diagnostic and therapeutic targets.”

The long-term goal of these studies is to develop individualized therapies, tailored for the patient. Since the therapies are expensive, accurate diagnostics are critical to avoid wasting resources on patients who clearly will not respond (or could be harmed) by the particular drug.

“At this time the rate of response to antibody-based therapies may be very poor, as

  • they are often employed late in the course of the disease, and patients are in such a debilitated state
  • that they lack the capacity to react positively to the treatment,” Dr. Carney explains.

Nanoscale Real-Time Proteomics

Stanford University School of Medicine researchers, working with Cell BioSciences, have developed a

  • nanofluidic proteomic immunoassay that measures protein charge,
  • similar to immunoblots, mass spectrometry, or flow cytometry.
  • unlike these platforms, this approach can measure the amount of individual isoforms,
  • specifically, phosphorylated molecules.

“We have developed a nanoscale device for protein measurement, which I believe could be useful for clinical analysis,” says Dean W. Felsher, M.D., Ph.D., associate professor at Stanford University School of Medicine.

Critical oncogenic transformations involving

  • the activation of the signal-related kinases ERK-1 and ERK-2 can now be followed with ease.

“The fact that we measure nanoquantities with accuracy means that

  • we can interrogate proteomic profiles in clinical patients,

by drawing tiny needle aspirates from tumors over the course of time,” he explains.

“This allows us to observe the evolution of tumor cells and

  • their response to therapy
  • from a baseline of the normal tissue as a standard of comparison.”

According to Dr. Felsher, 20 cells is a large enough sample to obtain a detailed description. The technology is easy to automate, which allows

  • the inclusion of hundreds of assays.

Contrasting this technology platform with proteomic analysis using microarrays, Dr. Felsher notes that the latter is not yet workable for revealing reliable markers.

Dr. Felsher and his group published a description of this technology in Nature Medicine. “We demonstrated that we could take a set of human lymphomas and distinguish them from both normal tissue and other tumor types. We can

  • quantify changes in total protein, protein activation, and relative abundance of specific phospho-isoforms
  • from leukemia and lymphoma patients receiving targeted therapy.

Even with very small numbers of cells, we are able to show that the results are consistent, and

  • our sample is a random profile of the tumor.”

Splice Variant Peptides

“Aberrations in alternative splicing may generate

  • much of the variation we see in cancer cells,”

says Gilbert Omenn, Ph.D., director of the center for computational medicine and bioinformatics at the University of Michigan School of Medicine. Dr. Omenn and his colleague, Rajasree Menon, are

  • using this variability as a key to new biomarker identification.

It is becoming evident that splice variants play a significant role in the properties of cancer cells, including

  • initiation, progression, cell motility, invasiveness, and metastasis.

Alternative splicing occurs through multiple mechanisms

  • when the exons or coding regions of the DNA transcribe mRNA,
  • generating initiation sites and connecting exons in protein products.

Their translation into protein can result in numerous protein isoforms, and

  • these isoforms may reflect a diseased or cancerous state.

Regulatory elements within the DNA are responsible for selecting different alternatives; thus

  • the splice variants are tempting targets for exploitation as biomarkers.
Analyses of the splice-site mutation

Analyses of the splice-site mutation

Despite the many questions raised by these observations, splice variation in tumor material has not been widely studied. Cancer cells are known for their tremendous variability, which allows them to

  • grow rapidly, metastasize, and develop resistance to anticancer drugs.

Dr. Omenn and his collaborators used

  • mass spec data to interrogate a custom-built database of all potential mRNA sequences
  • to find alternative splice variants.

When they compared normal and malignant mammary gland tissue from a mouse model of Her2/Neu human breast cancers, they identified a vast number (608) of splice variant proteins, of which

  • peptides from 216 were found only in the tumor sample.

“These novel and known alternative splice isoforms

  • are detectable both in tumor specimens and in plasma and
  • represent potential biomarker candidates,” Dr. Omenn adds.

Dr. Omenn’s observations and those of his colleague Lewis Cantley, Ph.D., have also

  • shed light on the origins of the classic Warburg effect,
  • the shift to anaerobic glycolysis in tumor cells.

The novel splice variant M2, of muscle pyruvate kinase,

  • is observed in embryonic and tumor tissue.

It is associated with this shift, the result of

  • the expression of a peptide splice variant sequence.

It is remarkable how many different areas of the life sciences are tied into the phenomenon of splice variation. The changes in the genetic material can be much greater than point mutations, which have been traditionally considered to be the prime source of genetic variability.

“We now have powerful methods available to uncover a whole new category of variation,” Dr. Omenn says. “High-throughput RNA sequencing and proteomics will be complementary in discovery studies of splice variants.”

Splice variation may play an important role in rapid evolutionary changes, of the sort discussed by Susumu Ohno and Stephen J. Gould decades ago. They, and other evolutionary biologists, argued that

  • gene duplication, combined with rapid variability, could fuel major evolutionary jumps.

At the time, the molecular mechanisms of variation were poorly understood, but today

  • the tools are available to rigorously evaluate the role of
  • splice variation and other contributors to evolutionary change.

“Biomarkers derived from studies of splice variants, could, in the future, be exploited

  • both for diagnosis and prognosis and
  • for drug targeting of biological networks,
  • in situations such as the Her-2/Neu breast cancers,” Dr. Omenn says.

Aminopeptidase Activities

“By correlating the proteolytic patterns with disease groups and controls, we have shown that

  • exopeptidase activities contribute to the generation of not only cancer-specific
  • but also cancer type specific serum peptides.

according to Paul Tempst, Ph.D., professor and director of the Protein Center at the Memorial Sloan-Kettering Cancer Center.

So there is a direct link between peptide marker profiles of disease and differential protease activity.” For this reason Dr. Tempst argues that “the patterns we describe may have value as surrogate markers for detection and classification of cancer.”

To investigate this avenue, Dr. Tempst and his colleagues have followed

  • the relationship between exopeptidase activities and metastatic disease.

“We monitored controlled, de novo peptide breakdown in large numbers of biological samples using mass spectrometry, with relative quantitation of the metabolites,” Dr. Tempst explains. This entailed the use of magnetic, reverse-phase beads for analyte capture and a MALDI-TOF MS read-out.

“In biomarker discovery programs, functional proteomics is usually not pursued,” says Dr. Tempst. “For putative biomarkers, one may observe no difference in quantitative levels of proteins, while at the same time, there may be substantial differences in enzymatic activity.”

In a preliminary prostate cancer study, the team found a significant difference

  • in activity levels of exopeptidases in serum from patients with metastatic prostate cancer
  • as compared to primary tumor-bearing individuals and normal healthy controls.

However, there were no differences in amounts of the target protein, and this potential biomarker would have been missed if quantitative levels of protein had been the only criterion of selection.

It is frequently stated that “practical fusion energy is 30 years in the future and always will be.” The same might be said of functional, practical biomarkers that can pass muster with the FDA. But splice variation represents a new handle on this vexing problem. It appears that we are seeing the emergence of a new approach that may finally yield definitive diagnostic tests, detectable in serum and urine samples.

Part 7. Epigenetics and Drug Metabolism

DNA Methylation Rules: Studying Epigenetics with New Tools

The tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.

Patricia Fitzpatrick Dimond, Ph.D.

http://www.genengnews.com/media/images/AnalysisAndInsight/Feb7_2013_24454248_GreenPurpleDNA_EpigeneticsToolsII3576166141.jpg

New tools may help move the field of epigenetic analysis forward and potentially unveil novel biomarkers for cellular development, differentiation, and disease.

DNA sequencing has had the power of technology behind it as novel platforms to produce more sequencing faster and at lower cost have been introduced. But the tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.

Among these mechanisms, DNA methylation, or the enzymatically mediated addition of a methyl group to cytosine or adenine dinucleotides,

  • serves as an inherited epigenetic modification that
  • stably modifies gene expression in dividing cells.

The unique methylomes are largely maintained in differentiated cell types, making them critical to understanding the differentiation potential of the cell.

In the DNA methylation process, cytosine residues in the genome are enzymatically modified to 5-methylcytosine,

  • which participates in transcriptional repression of genes during development and disease progression.

5-methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. DNA methylation affects gene transcription by physically

  • interfering with the binding of proteins involved in gene transcription.

Methylated DNA may be bound by methyl-CpG-binding domain proteins (MBDs) that can

  • then recruit additional proteins. Some of these include histone deacetylases and other chromatin remodeling proteins that modify histones, thereby
  • forming compact, inactive chromatin, or heterochromatin.

While DNA methylation doesn’t change the genetic code,

  • it influences chromosomal stability and gene expression.

Epigenetics and Cancer Biomarkers

multistage chemical carcinogenesis

multistage chemical carcinogenesis

And because of the increasing recognition that DNA methylation changes are involved in human cancers, scientists have suggested that these epigenetic markers may provide biological markers for cancer cells, and eventually point toward new diagnostic and therapeutic targets. Cancer cell genomes display genome-wide abnormalities in DNA methylation patterns,

  • some of which are oncogenic and contribute to genome instability.

In particular, de novo methylation of tumor suppressor gene promoters

  • occurs frequently in cancers, thereby silencing them and promoting transformation.

Cytosine hydroxymethylation (5-hydroxymethylcytosine, or 5hmC), the aforementioned DNA modification resulting from the enzymatic conversion of 5mC into 5-hydroxymethylcytosine by the TET family of oxygenases, has been identified

  • as another key epigenetic modification marking genes important for
  • pluripotency in embryonic stem cells (ES), as well as in cancer cells.

The base 5-hydroxymethylcytosine was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. In 2011, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2′-deoxycytidine (5hmdC) and 5-methyl-2′-deoxycytidine (5mdC) in genomic DNA, scientists at the Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California investigated

  • whether levels of 5hmC can distinguish normal tissue from tumor tissue.

They showed that in squamous cell lung cancers, levels of 5hmdC showed

  • up to five-fold reduction compared with normal lung tissue.

In brain tumors,5hmdC showed an even more drastic reduction

  • with levels up to more than 30-fold lower than in normal brain,
  • but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1, the enzyme that converts 5hmC to 5hmdC.

Immunohistochemical analysis indicated that 5hmC is “remarkably depleted” in many types of human cancer.

  • there was an inverse relationship between 5hmC levels and cell proliferation with lack of 5hmC in proliferating cells.

Their data suggest that 5hmdC is strongly depleted in human malignant tumors,

  • a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue.

In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.

Enzymatic Mapping

But according to New England Biolabs’ Sriharsa Pradhan, Ph.D., methods for distinguishing 5mC from 5hmC and analyzing and quantitating the cell’s entire “methylome” and “hydroxymethylome” remain less than optimal.

The protocol for bisulphite conversion to detect methylation remains the “gold standard” for DNA methylation analysis. This method is generally followed by PCR analysis for single nucleotide resolution to determine methylation across the DNA molecule. According to Dr. Pradhan, “.. bisulphite conversion does not distinguish 5mC and 5hmC,”

Recently we found an enzyme, a unique DNA modification-dependent restriction endonuclease, AbaSI, which can

  • decode the hydryoxmethylome of the mammalian genome.

You easily can find out where the hydroxymethyl regions are.”

AbaSI, recognizes 5-glucosylatedmethylcytosine (5gmC) with high specificity when compared to 5mC and 5hmC, and

  • cleaves at narrow range of distances away from the recognized modified cytosine.

By mapping the cleaved ends, the exact 5hmC location can, the investigators reported, be determined.

Dr. Pradhan and his colleagues at NEB; the Department of Biochemistry, Emory University School of Medicine, Atlanta; and the New England Biolabs Shanghai R&D Center described use of this technique in a paper published in Cell Reports this month, in which they described high-resolution enzymatic mapping of genomic hydroxymethylcytosine in mouse ES cells.

In the current report, the authors used the enzyme technology for the genome-wide high-resolution hydroxymethylome, describing simple library construction even with a low amount of input DNA (50 ng) and the ability to readily detect 5hmC sites with low occupancy.

As a result of their studies, they propose that

factors affecting the local 5mC accessibility to TET enzymes play important roles in the 5hmC deposition

  • including include chromatin compaction, nucleosome positioning, or TF binding.
  •  the regularly oscillating 5hmC profile around the CTCF-binding sites, suggests 5hmC ‘‘writers’’ may be sensitive to the nucleosomal environment.
  • some transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.

“We were able to do complete mapping in mouse embryonic cells and are pleased about what this enzyme can do and how it works,” Dr. Pradhan said.

And the availability of novel tools that make analysis of the methylome and hypomethylome more accessible will move the field of epigenetic analysis forward and potentially novel biomarkers for cellular development, differentiation, and disease.

Patricia Fitzpatrick Dimond, Ph.D. (pdimond@genengnews.com), is technical editor at Genetic Engineering & Biotechnology News.

Epigenetic Regulation of ADME-Related Genes: Focus on Drug Metabolism and Transport

Published: Sep 23, 2013

Epigenetic regulation of gene expression refers to heritable factors that are functionally relevant genomic modifications but that do not involve changes in DNA sequence.

Examples of such modifications include

  • DNA methylation, histone modifications, noncoding RNAs, and chromatin architecture.

Epigenetic modifications are crucial for

packaging and interpreting the genome, and they have fundamental functions in regulating gene expression and activity under the influence of physiologic and environmental factors.

In this issue of Drug Metabolism and Disposition, a series of articles is presented to demonstrate the role of epigenetic factors in regulating

  • the expression of genes involved in drug absorption, distribution, metabolism, and excretion in organ development, tissue-specific gene expression, sexual dimorphism, and in the adaptive response to xenobiotic exposure, both therapeutic and toxic.

The articles also demonstrate that, in addition to genetic polymorphisms, epigenetics may also contribute to wide inter-individual variations in drug metabolism and transport. Identification of functionally relevant epigenetic biomarkers in human specimens has the potential to improve prediction of drug responses based on patient’s epigenetic profiles.

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=157804

This study is published online in Drug Metabolism and Disposition

Part 8.  Pictorial Maps

 Prediction of intracellular metabolic states from extracellular metabolomic data

MK Aurich, G Paglia, Ottar Rolfsson, S Hrafnsdottir, M Magnusdottir, MM Stefaniak, BØ Palsson, RMT Fleming &

Ines Thiele

Metabolomics Aug 14, 2014;

http://dx.doi.org:/10.1007/s11306-014-0721-3

http://link.springer.com/article/10.1007/s11306-014-0721-3/fulltext.html#Sec1

http://link.springer.com/static-content/images/404/art%253A10.1007%252Fs11306-014-0721-3/MediaObjects/11306_2014_721_Fig1_HTML.gif

Metabolic models can provide a mechanistic framework

  • to analyze information-rich omics data sets, and are
  • increasingly being used to investigate metabolic alternations in human diseases.

An expression of the altered metabolic pathway utilization is the selection of metabolites consumed and released by cells. However, methods for the

  • inference of intracellular metabolic states from extracellular measurements in the context of metabolic models remain underdeveloped compared to methods for other omics data.

Herein, we describe a workflow for such an integrative analysis

  • emphasizing on extracellular metabolomics data.

We demonstrate,

  • using the lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM,

how our methods can reveal differences in cell metabolism. Our models explain metabolite uptake and secretion by predicting

  • a more glycolytic phenotype for the CCRF-CEM model and
  • a more oxidative phenotype for the Molt-4 model,
  • which was supported by our experimental data.

Gene expression analysis revealed altered expression of gene products at

  • key regulatory steps in those central metabolic pathways, and

literature query emphasized the role of these genes in cancer metabolism.

Moreover, in silico gene knock-outs identified unique

  •  control points for each cell line model, e.g., phosphoglycerate dehydrogenase for the Molt-4 model.

Thus, our workflow is well suited to the characterization of cellular metabolic traits based on

  • -extracellular metabolomic data, and it allows the integration of multiple omics data sets
  • into a cohesive picture based on a defined model context.

Keywords Constraint-based modeling _ Metabolomics _ Multi-omics _ Metabolic network _ Transcriptomics

1 Introduction

Modern high-throughput techniques have increased the pace of biological data generation. Also referred to as the ‘‘omics avalanche’’, this wealth of data provides great opportunities for metabolic discovery. Omics data sets

  • contain a snapshot of almost the entire repertoire of mRNA, protein, or metabolites at a given time point or

under a particular set of experimental conditions. Because of the high complexity of the data sets,

  • computational modeling is essential for their integrative analysis.

Currently, such data analysis is a bottleneck in the research process and methods are needed to facilitate the use of these data sets, e.g., through meta-analysis of data available in public databases [e.g., the human protein atlas (Uhlen et al. 2010) or the gene expression omnibus (Barrett et al.  2011)], and to increase the accessibility of valuable information for the biomedical research community.

Constraint-based modeling and analysis (COBRA) is

  • a computational approach that has been successfully used to
  • investigate and engineer microbial metabolism through the prediction of steady-states (Durot et al.2009).

The basis of COBRA is network reconstruction: networks are assembled in a bottom-up fashion based on

  • genomic data and extensive
  • organism-specific information from the literature.

Metabolic reconstructions capture information on the

  • known biochemical transformations taking place in a target organism
  • to generate a biochemical, genetic and genomic knowledge base (Reed et al. 2006).

Once assembled, a

  • metabolic reconstruction can be converted into a mathematical model (Thiele and Palsson 2010), and
  • model properties can be interrogated using a great variety of methods (Schellenberger et al. 2011).

The ability of COBRA models

  • to represent genotype–phenotype and environment–phenotype relationships arises
  • through the imposition of constraints, which
  • limit the system to a subset of possible network states (Lewis et al. 2012).

Currently, COBRA models exist for more than 100 organisms, including humans (Duarte et al. 2007; Thiele et al. 2013).

Since the first human metabolic reconstruction was described [Recon 1 (Duarte et al. 2007)],

  • biomedical applications of COBRA have increased (Bordbar and Palsson 2012).

One way to contextualize networks is to

  • define their system boundaries according to the metabolic states of the system, e.g., disease or dietary regimes.

The consequences of the applied constraints can

  • then be assessed for the entire network (Sahoo and Thiele 2013).

Additionally, omics data sets have frequently been used

  • to generate cell-type or condition-specific metabolic models.

Models exist for specific cell types, such as

  1. enterocytes (Sahoo and Thiele2013),
  2. macrophages (Bordbar et al. 2010),
  3. adipocytes (Mardinoglu et al. 2013),
  4. even multi-cell assemblies that represent the interactions of brain cells (Lewis et al. 2010).

All of these cell type specific models, except the enterocyte reconstruction

  • were generated based on omics data sets.

Cell-type-specific models have been used to study

  • diverse human disease conditions.

For example, an adipocyte model was generated using

  • transcriptomic, proteomic, and metabolomics data.

This model was subsequently used to investigate metabolic alternations in adipocytes

  • that would allow for the stratification of obese patients (Mardinoglu et al. 2013).

The biomedical applications of COBRA have been

  1. cancer metabolism (Jerby and Ruppin, 2012).
  2. predicting drug targets (Folger et al. 2011; Jerby et al. 2012).

A cancer model was generated using

  • multiple gene expression data sets and subsequently used
  • to predict synthetic lethal gene pairs as potential drug targets
  • selective for the cancer model, but non-toxic to the global model (Recon 1),

a consequence of the reduced redundancy in the cancer specific model (Folger et al. 2011).

In a follow up study, lethal synergy between FH and enzymes of the heme metabolic pathway

  • were experimentally validated and resolved the mechanism by which FH deficient cells,
    e.g., in renal-cell cancer cells survive a non-functional TCA cycle (Frezza et al. 2011).

Contextualized models, which contain only the subset of reactions active in a particular tissue (or cell-) type,

  • can be generated in different ways (Becker and Palsson, 2008; Jerby et al. 2010).

However, the existing algorithms mainly consider

  • gene expression and proteomic data
  • to define the reaction sets that comprise the contextualized metabolic models.

These subset of reactions are usually defined

  • based on the expression or absence of expression of the genes or proteins (present and absent calls),
  • or inferred from expression values or differential gene expression.

Comprehensive reviews of the methods are available (Blazier and Papin, 2012; Hyduke et al. 2013). Only the compilation of a large set of omics data sets

  • can result in a tissue (or cell-type) specific metabolic model, whereas

the representation of one particular experimental condition is achieved

  • through the integration of omics data set generated from one experiment only (condition-specific cell line model).

Recently, metabolomic data sets have become more comprehensive and

  • using these data sets allow direct determination of the metabolic network components (the metabolites).

Additionally, metabolomics has proven to be stable, relatively inexpensive, and highly reproducible (Antonucci et al. 2012). These factors make metabolomic data sets particularly valuable for

  • interrogation of metabolic phenotypes.

Thus, the integration of these data sets is now an active field of research (Li et al. 2013; Mo et al. 2009; Paglia et al. 2012b; Schmidt et al. 2013).

Generally, metabolomic data can be incorporated into metabolic networks as

  • qualitative, quantitative, and thermodynamic constraints (Fleming et al. 2009; Mo et al. 2009).

Mo et al. used metabolites detected in the

  • spent medium of yeast cells to determine intracellular flux states through a sampling analysis (Mo et al. 2009),
  • which allowed unbiased interrogation of the possible network states (Schellenberger and Palsson 2009) and
  • prediction of internal pathway use.
Modes of transcriptional regulation during the YMC

Modes of transcriptional regulation during the YMC

Such analyses have also been used to reveal the effects of

  1. enzymopathies on red blood cells (Price et al. 2004),
  2. to study effects of diet on diabetes (Thiele et al. 2005) and
  3. to define macrophage metabolic states (Bordbar et al. 2010).

This type of analysis is available as a function in the COBRA toolbox (Schellenberger et al. 2011).

In this study, we established a workflow

  • for the generation and analysis of condition-specific metabolic cell line models
  • that can facilitate the interpretation of metabolomic data.

Our modeling yields meaningful predictions regarding

  • metabolic differences between two lymphoblastic leukemia cell lines (Fig. 1A).

Fig. 1

metabol leukem cell lines11306_2014_721_Fig1_HTML

metabol leukem cell lines11306_2014_721_Fig1_HTML

A Combined experimental and computational pipeline to study human metabolism.

  1. Experimental work and omics data analysis steps precede computational modeling.
  2. Model predictions are validated based on targeted experimental data.
  3. Metabolomic and transcriptomic data are used for model refinement and submodel extraction.
  4. Functional analysis methods are used to characterize the metabolism of the cell-line models and compare it to additional experimental data.
  5. The validated models are subsequently used for the prediction of drug targets.

B Uptake and secretion pattern of model metabolites. All metabolite uptakes and secretions that were mapped during model generation are shown.

  • Metabolite uptakes are depicted on the left, and
  • secreted metabolites are shown on the right.
  1. A number of metabolite exchanges mapped to the model were unique to one cell line.
  2. Differences between cell lines were used to set quantitative constraints for the sampling analysis.

C Statistics about the cell line-specific network generation.

D Quantitative constraints.

For the sampling analysis, an additional set of constraints was imposed on the cell line specific models,

  • emphasizing the differences in metabolite uptake and secretion between cell lines.

Higher uptake of a metabolite was allowed

  • in the model of the cell line that consumed more of the metabolite in vitro, whereas
  • the supply was restricted for the model with lower in vitro uptake.

This was done by establishing the same ratio between the models bounds as detected in vitro.

X denotes the factor (slope ratio) that distinguishes the bounds, and

  • which was individual for each metabolite.

(a) The uptake of a metabolite could be x times higher in CCRF-CEM cells,

(b) the metabolite uptake could be x times higher in Molt-4,

(c) metabolite secretion could be x times higher in CCRF-CEM, or

(d) metabolite secretion could be x times higher in Molt-4 cells.LOD limit of detection.

The consequence of the adjustment was, in case of uptake, that one model was constrained to a lower metabolite uptake (A, B), and the difference depended on the ratio detected in vitro. In case of secretion, one model

  • had to secrete more of the metabolite, and again
  • the difference depended on the experimental difference detected between the cell lines

2 Results

We set up a pipeline that could be used to infer intracellular metabolic states

  • from semi-quantitative data regarding metabolites exchanged between cells and their environment.

Our pipeline combined the following four steps:

  1. data acquisition,
  2. data analysis,
  3. metabolic modeling and
  4. experimental validation of the model predictions (Fig. 1A).

We demonstrated the pipeline and the predictive potential to predict metabolic alternations in diseases such as cancer based on

^two lymphoblastic leukemia cell lines.

The resulting Molt-4 and CCRF-CEM condition-specific cell line models could explain

^  metabolite uptake and secretion
^  by predicting the distinct utilization of central metabolic pathways by the two cell lines.
^  the CCRF-CEM model resembled more a glycolytic, commonly referred to as ‘Warburg’ phenotype,
^  our model predicted a more respiratory phenotype for the Molt-4 model.

We found these predictions to be in agreement with measured gene expression differences

  • at key regulatory steps in the central metabolic pathways, and they were also
  • consistent with additional experimental data regarding the energy and redox states of the cells.

After a brief discussion of the data generation and analysis steps, the results derived from model generation and analysis will be described in detail.

2.1 Pipeline for generation of condition-specific metabolic cell line models

integration of exometabolomic (EM) data

integration of exometabolomic (EM) data

2.1.1 Generation of experimental data

We monitored the growth and viability of lymphoblastic leukemia cell lines in serum-free medium (File S2, Fig. S1). Multiple omics data sets were derived from these cells.Extracellular metabolomics (exo-metabolomic) data,

integration of exometabolomic (EM) data

integration of exometabolomic (EM) data

^  comprising measurements of the metabolites in the spent medium of the cell cultures (Paglia et al. 2012a),
^ were collected along with transcriptomic data, and these data sets were used to construct the models.

2.1.4 Condition-specific models for CCRF-CEM and Molt-4 cells

To determine whether we had obtained two distinct models, we evaluated the reactions, metabolites, and genes of the two models. Both the Molt-4 and CCRF-CEM models contained approximately half of the reactions and metabolites present in the global model (Fig. 1C). They were very similar to each other in terms of their reactions, metabolites, and genes (File S1, Table S5A–C).

(1) The Molt-4 model contained seven reactions that were not present in the CCRF-CEM model (Co-A biosynthesis pathway and exchange reactions).
(2) The CCRF-CEM contained 31 unique reactions (arginine and proline metabolism, vitamin B6 metabolism, fatty acid activation, transport, and exchange reactions).
(3) There were 2 and 15 unique metabolites in the Molt-4 and CCRF-CEM models, respectively (File S1, Table S5B).
(4) Approximately three quarters of the global model genes remained in the condition-specific cell line models (Fig. 1C).
(5) The Molt-4 model contained 15 unique genes, and the CCRF-CEM model had 4 unique genes (File S1, Table S5C).
(6) Both models lacked NADH dehydrogenase (complex I of the electron transport chain—ETC), which was determined by the absence of expression of a mandatory subunit (NDUFB3, Entrez gene ID 4709).

Rather, the ETC was fueled by FADH2 originating from succinate dehydrogenase and from fatty acid oxidation, which through flavoprotein electron transfer

FADH2

FADH2

  • could contribute to the same ubiquinone pool as complex I and complex II (succinate dehydrogenase).

Despite their different in vitro growth rates (which differed by 11 %, see File S2, Fig. S1) and
^^^ differences in exo-metabolomic data (Fig. 1B) and transcriptomic data,
^^^ the internal networks were largely conserved in the two condition-specific cell line models.

2.1.5 Condition-specific cell line models predict distinct metabolic strategies

Despite the overall similarity of the metabolic models, differences in their cellular uptake and secretion patterns suggested distinct metabolic states in the two cell lines (Fig. 1B and see “Materials and methods” section for more detail). To interrogate the metabolic differences, we sampled the solution space of each model using an Artificial Centering Hit-and-Run (ACHR) sampler (Thiele et al. 2005). For this analysis, additional constraints were applied, emphasizing the quantitative differences in commonly uptaken and secreted metabolites. The maximum possible uptake and maximum possible secretion flux rates were reduced
^^^ according to the measured relative differences between the cell lines (Fig. 1D, see “Materials and methods” section).

We plotted the number of sample points containing a particular flux rate for each reaction. The resulting binned histograms can be understood as representing the probability that a particular reaction can have a certain flux value.

A comparison of the sample points obtained for the Molt-4 and CCRF-CEM models revealed

  • a considerable shift in the distributions, suggesting a higher utilization of glycolysis by the CCRF-CEM model
    (File S2, Fig. S2).

This result was further supported by differences in medians calculated from sampling points (File S1, Table S6).
The shift persisted throughout all reactions of the pathway and was induced by the higher glucose uptake (34 %) from the extracellular medium in CCRF-CEM cells.

The sampling median for glucose uptake was 34 % higher in the CCRF-CEM model than in Molt-4 model (File S2, Fig. S2).

The usage of the TCA cycle was also distinct in the two condition-specific cell-line models (Fig. 2). Interestingly,
the models used succinate dehydrogenase differently (Figs. 2, 3).

TCA_reactions

TCA_reactions

The Molt-4 model utilized an associated reaction to generate FADH2, whereas

  • in the CCRF-CEM model, the histogram was shifted in the opposite direction,
  • toward the generation of succinate.

Additionally, there was a higher efflux of citrate toward amino acid and lipid metabolism in the CCRF-CEM model (Fig. 2). There was higher flux through anaplerotic and cataplerotic reactions in the CCRF-CEM model than in the Molt-4 model (Fig. 2); these reactions include

(1) the efflux of citrate through ATP-citrate lyase,
(2) uptake of glutamine,
(3) generation of glutamate from glutamine,
(4) transamination of pyruvate and glutamate to alanine and to 2-oxoglutarate,
(5) secretion of nitrogen, and
(6) secretion of alanine.

energetics-of-cellular-respiration

energetics-of-cellular-respiration

The Molt-4 model showed higher utilization of oxidative phosphorylation (Fig. 3), again supported by
elevated median flux through ATP synthase (36 %) and other enzymes, which contributed to higher oxidative metabolism. The sampling analysis therefore revealed different usage of central metabolic pathways by the condition-specific models.

Fig. 2

Differences in the use of  the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

The table provides the median values of the sampling results. Negative values in histograms and in the table describe reversible reactions with flux in the reverse direction. There are multiple reversible reactions for the transformation of isocitrate and α-ketoglutarate, malate and fumarate, and succinyl-CoA and succinate. These reactions are unbounded, and therefore histograms are not shown. The details of participating cofactors have been removed.

Figure 3.

Molt-4 has higher median flux through ETC reactions II–IV 11306_2014_721_Fig3_HTML

Molt-4 has higher median flux through ETC reactions II–IV 11306_2014_721_Fig3_HTML

Atp ATP, cit citrate, adp ADP, pi phosphate, oaa oxaloacetate, accoa acetyl-CoA, coa coenzyme-A, icit isocitrate, αkg α-ketoglutarate, succ-coa succinyl-CoA, succ succinate, fumfumarate, mal malate, oxa oxaloacetate,
pyr pyruvate, lac lactate, ala alanine, gln glutamine, ETC electron transport chain

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

metabolic pathways 1476-4598-10-70-1

metabolic pathways 1476-4598-10-70-1

Metabolic Systems Research Team fig2

Metabolic Systems Research Team fig2

Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001

Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001

Metabolome Informatics Research fig1

Metabolome Informatics Research fig1

Modelling of Central Metabolism network3

Modelling of Central Metabolism network3

N. gaditana metabolic pathway map ncomms1688-f4

N. gaditana metabolic pathway map ncomms1688-f4

protein changes in biological mechanisms

protein changes in biological mechanisms

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Writer and curator: Larry H. Bernstein, MD, FCAP and
Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013-01-23/larryhbern/Regulation-of-somatic-stem-cell-function/

There is an explosion of work-in-progress in applications to regenerative medicine using inducible pluripotent stem cells in both endothelial and cardiomyocyte postischemic repair, and also in post bone marrow radiation restoration, with benefits and hazards.  The following article is quite novel in that it deals with stem cell regulation by DNA methylation.  Therefore, it deals with the essentiality of methylation of DNA in epigenetic regulation.

This is the fourth discussion of a several part series leading from the genome, to protein synthesis (1), posttranslational modification of proteins (2), examples of protein effects on metabolism and signaling pathways (3), and leading to disruption of signaling pathways in disease (4), and effects leading to mutagenesis.

1.  A Primer on DNAand DNA Replication

2.  Overview of translational medicine

3.  Genes, proteomes, and their interaction

4. Regulation of somatic stem cell Function

5.  Proteomics – The Pathway to Understanding and Decision-making in Medicine

6.  Genomics, Proteomics and standards

7.  Long Non-coding RNAs Can Encode Proteins After All

8.  Proteins and cellular adaptation to stress

9.  Loss of normal growth regulation

 

Posttranslational modification is a step in protein biosynthesis. Proteins are created by ribosomes translating mRNA into polypeptide chains. These polypeptide chains undergo
PTM before becoming the mature protein product.

Regulation of somatic stem cell Function by DNA Methylation and Genomic Imprinting

Mo Li1, Na Young Kim1, Shigeo Masuda1 and Juan Carlos izpisua Belmonte1,2 1Salk institute for Biological Studies, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA. 2Center of Regenerative Medicine in Barcelona, Dr Aiguader, 88, 08003 Barcelona, Spain. Corresponding author email: mli@salk.edu

Cell & Tissue Transplantation & Therapy 2013:5 19–23
http://dx.doi.org/10.4137/CTTT.S12142
This article is available from http://www.la-press.com

Abstract:

Epigenetic regulation is essential for self-renewal and differentiation of somatic stem cells, including

  • hematopoietic stem cells (HSCs) and
  • neural stem cells (NSCs).

The role of DNA methylation, a key epigenetic pathway,

  • in regulating somatic stem cell function
    • under physiological conditions and during aging

has been intensively investigated.

Accumulating evidence highlights the dynamic nature of

  • the DNAmethylome
    • during lineage commitment of somatic stem cells and
  • the pivotal role of DNAmethyltransferases in
    • stem cell self-renewal and differentiation.

Recent studies on genomic imprinting have shed light on

  • the imprinted gene network (IGN) in somatic stem cells,
  1. where a subset of imprinted genes remain expressed and
  2. are important for maintaining self-renewal of these cells.

Together with emerging technologies, elucidation of the epigenetic mechanisms regulating somatic stem cells with normal or pathological functions may contribute to the development of regenerative medicine.

Keywords: somatic stem cells, epigenetics, DNA methylation, genomic imprinting, hematopoietic stem cells, neural stem cells

Introduction

In adult animals, somatic stem cells (also known as adult stem cells) are responsible for maintaining tissue homeostasis and participate in tissue regeneration under injury conditions. Self-renewal and differentiation are two important aspects of somatic stem cell function. Epigenetic mechanisms underlying these processes have been intensively investigated. With the increasing ability

  • to identify and manipulate somatic stem cell populations from diverse tissues,
  • it is possible to dissect the epigenetic pathways that are
  1. either unique for a specific tissue or
  2. universally important in regulating stemness and differentiation.

Epigenetic control of somatic stem cell function exists at various levels, including

  • DNA methylation,
  • histone modification, and
  • higher-order chromatin structure dynamics.

Here, we focus on recent progress in our understanding of how

  • DNA methylation regulates somatic stem cell function.

DNA Methylation and stem cell Function

The role of DNA methylation in somatic stem cell compartments has gained increasing attention. Recent  evidence has shown that

  • DNA methylation is dynamically regulated during somatic stem cell differentiation and aging.1

A study of methylomes of human hematopoietic stem cells (HSCs) and two mature hematopoietic lineages,

  • including B cells and neutrophils, showed that
    • hypomethylated regions of lineage-specific genes often become methylated in opposing lineages, and that
    • progenitors display an intermediate methylation pattern

that is poised for lineage-specific resolution.2

Another study compared genome-wide promoter DNA methylation in human cord blood hematopoietic progenitor cells (HPCs) with

  • that in mobilized peripheral blood HPCs from aged individuals.

It was found that aged HPCs lose DNA methylation in a subset of genes that are hypomethylated in differentiated myeloid cells and

  • gain de novo DNA methylation at polycomb repressive complex 2 (PRC2) target sites.3

It was hypothesized that such epigenetic changes contribute to age-related loss of HSC function, such as a bias toward myeloid lineages. Recently, Beerman et al. studied the global DNA methylation landscape of HSCs in the context of

  • age-associated decline of HSC function.4

Over- all, the DNA methylation landscape remains stable during HSC ontogeny. However, HSCs isolated from old mice display higher global DNA methylation. Interestingly, they observed

  • localized DNA methylation changes in genomic regions associated with hematopoietic lineage differentiation.

These methylation changes preferentially map to genes

  • that are expressed in downstream progenitor and effector cells.

For example, genes that are important for the lymphoid and erythroid lineages

  • become methylated in “old” HSCs,

which is consistent with

  • the decline of lymphopoiesis and erythropoiesis during aging.

Additionally, inducing HSC proliferation by 5-fluorouracil treatment or

  • by limiting the number of transplantedHSCs
    • recapitulates the functional decline and DNA methylation changes during physiological aging.

A closer examination of the overlapping genes with significant DNA methylation changes during aging or enforced proliferation showed

  • an enrichment of DNA hypermethylation at PRC2 target loci,

echoing the observation by Bocker et al. in human HSCs.

Interestingly, a recent report showed that epigenetic alterations such as DNA hypermethylation that are accrued during aging,

  • can be fully reset by somatic reprogramming,

raising an interesting possibility that these aging-related epigenetic defects may be reserved by small molecules.5

Methylation of cytosines at CpG dinucleotides is catalyzed by three key enzymes.

DNA (cytosine-5)- methyltransferase 1 (DNMT1) is responsible for maintaining DNA methylation patterns during DNA replication

  • by methylating the newly synthesized hemi-methylated DNA.

The other two DNA methyltransferases, DNMT3a and DNMT3b,

  • are not DNA replication-dependent and can methylate fully unmethylated DNA de novo.

They are responsible for establishing new DNA methylation patterns during development.

DNMT3a, a gene required for neurogenesis,

  • is expressed in postnatal neural stem cells (NSCs).

In NSCs, DNMT3a methylates non-proximal promoter regions, such as gene bodies and intergenic regions. Surprisingly, rather than silencing gene expression,

DNMT3a-mediated DNA methylation in gene bodies antagonizes Polycomb-dependent repression and

  • facilitates the expression of neurogenic genes.6

The role of DNMT3a in HSCs has also been investigated. Both Dnmt3a and Dnmt3b are expressed in HSCs. An earlier study did not identify any defects in HSC function when Dnmt3a or Dnmt3b was removed.  However,

  • HSCs lackingboth of these de novomethyltransferases
    • fail to self-renew, yet retain the capacity to differentiate.7

A more recent study re-examined

  • the consequences of Dnmt3a loss in HSCs and
  • uncovered a progressive defect in differentiation that is only manifested during serial transplantation.8

At the molecular level, while Dnmt3a loss results in the expected hypomethylation at some loci,

  • it counterintuitively causes hypermethylation in even more regions.8

This seemingly paradoxical result echoes the  unconventional role of Dnmt3a in transcriptional  activation in NSCs (as discussed above). Both cases suggest a more complex regulatory function of DNMT3a that is

  • beyond simply methylating DNA.

In contrast, the loss of Dnmt1 produces more dramatic and immediate phenotypes in HSCs, manifested

  • in premature HSC exhaustion and
  • block of lymphoid differentiation,

highlighting the distinct requirements for different DNA methyltransferases in HSCs.9,10

Genomic Imprinting and stemness

DNA methylation also underlies genomic imprinting, which is an

  • evolutionarily conserved epigenetic mechanism of ensuring appropriate gene dosage during development.

One allele of the imprinted genes is

  • epigenetically marked by DNA methylation to be silenced according to the parental origin.

The pattern of imprinting

  • is established in germ cells and maintained in somatic cells.

Imprinted genes are thought to play critical roles in organismal growth and are relatively downregulated after birth.11 Recently, a series of reports demonstrated that

  • a subset of imprinted genes belonging to the purported imprinted gene network (IGN)12
  • remain expressed in somatic stem cells and
  • are important for maintaining self-renewal of these cells.

Through gene expression profiling, one group identified that several members of the IGN are expressed in

  1. murine muscle,
  2. epidermal, and
  3. long-term hematopoietic stem cells
  4. as well as in human epidermal and hematopoietic stem cells.13

In particular, the paternally expressed gene 3 (Peg3) gene was shown by another group

  • to mark cycling and quiescent stem cells in a wide variety of mouse tissues.14

The role of imprinted genes in regulating somatic stem cell function has been examined in two types of tissues.

In bronchioalveolar stem cells (BASCs), a lung epithelial stem cell population,

  • expression of IGN members is required for their self-renewal.

Bmi1, a polycomb repressive  complex 1 (PRC1) subunit,

  • is essential for controlling the expression of imprinted genes in BASCs without affecting their imprinting status.15

In Bmi1 mutant BASCs,  many members of the IGN become derepressed,

  • including p57, H19, Dlk1, Peg3, Ndn, Mest, Gtl2, Grb10, Plagl1, and Igf2.

Knockdown of p57, which is the most differentially expressed imprinted gene between normal and mutant BASCs,

  • partially rescues the self-renewal defect of lung stem cells.

Interestingly, insufficient levels of p57 also inhibit self-renewal of lung stem cells. Because p57 expression

  • remains monoallelic in Bmi1 knockdown cells,
  • Bmi1 is thought to maintain an appropriate level of expression from the expressed allele of p57.15

Another IGN member- delta-like homologue 1 (Dlk1) has been shown to be important for postnatal neurogenesis. Interestingly, in this context,

  • Dlk1 loses its imprinting in postnatal neural stem cells and niche astrocytes.16

These studies suggest that modulating IGN may represent another

  • epigenetic mechanism for balancing self-renewal and differentiation in somatic stem cells.

Thus, somatic stem cells either co-opt or remodel these developmental pathways involving the IGN

  • to fulfill the needs of tissue homeostasis during the adult stage.

In summary, several factors participate in regulating the epigenome of somatic stem cells.

Perturbations in the epigenome of somatic stem cells,

  • either during organismal aging or under pathological conditions,

will tip the balance between self-renewal and differentiation of somatic stem cells (Fig. 1). A detailed understanding of the mechanisms underlying these changes will likely result in novel therapeutic approaches targeting somatic stem cells.

Figure 1. The epigenome of somatic stem cells is regulated by diverse factors.

Future perspectives The epigenetic mechanisms governing self-renewal and differentiation of somatic stem cells are likely to be complex because of the diverse needs of different tissues. It would be interesting to determine whether a common mechanism, such as the IGN, exists across different somatic stem cells. Additionally, study- ing epigenetic pathways that are specific to one type of somatic stem cell requires the isolation of these cells and their differentiated progeny, which is more practical in model organisms than in humans. Along these lines, developing robust in vitro culture methods for human somatic stem cells and protocols for differentiating these cells into specific lineages are critical for uncovering epigenetic pathways that are unique to human somatic stem cells. In recent years, the field has seen a great improvement in methods of directed differentiation of human embryonic stem cells and induced pluripotent stem cells (iPSCs). For example, it is relatively straightforward to produce high-purity cell populations that resemble neural stem cells or mesenchymal stem cells from iPSCs.17

These methodologies not only are useful for studying the normal function of somatic stem cells, but also provide an exciting opportunity for understanding the role of somatic stem cells in disease pathology and a platform to screen for drugs. A recent study under- scored the usefulness of this approach. Liu et al. studied neural stem cells derived from Parkinson’s disease human iPSCs and uncovered previously unknown defects in nuclear morphology and epigenetic regulation in these derived NSCs.18 The cellular defects only menifest in “aged” neural stem cells, which is consistent with the fact that Parkinson’s disease pri- marily manifests in old age. More  importantly, this study identified neural stem cell as a potential target of therapeutic intervention for Parkinson’s disease.

Targeted modification of the human genome is  another technological advancement that is on the horizon to greatly facilitate the dissection of epige- netic pathways in somatic stem cells. Although gene targeting in somatic stem cells has been historically challenging, there have been encouraging successful reports following development of new genome-e diting technologies, such as Helper-dependent adenovi- ral vectors, TALENs, and CAS9/CRISPR. With the development of these new technologies, it seems that the stage has been set for a new wave of discoveries in epigenetic mechanisms of somatic stem cells.

References

1. Li M, Liu GH, Izpisua Belmonte JC. Navigating the epigenetic landscape of pluripotent stem cells. Nat Rev Mol Cell Biol. 2012;13(8):524–535.

2. Hodges E, Molaro A, Dos Santos CO, et al. Directional DNA methylation changes and complex intermediate states accompany lineage specificity in the adult hematopoietic compartment. Mol Cell. 2011;44(1):17–28.

3. Bocker MT, Hellwig I, Breiling A, Eckstein V, Ho AD, Lyko F. Genome- wide promoter DNA methylation dynamics of human hematopoietic progen- itor cells during differentiation and aging. Blood. 2011;117(19):e182–e189.

4. Beerman I, Bock C, Garrison BS, et al. Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging. Cell Stem Cell. 2013;12(4):413–425.

5. Wahlestedt M, Norddahl GL, Sten G, et al. An epigenetic component of hematopoietic stem cell aging amenable to reprogramming into a young state. Blood. 2013;121(21):4257–4264.

6. Wu H, Coskun V, Tao J, et al. Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes. Science. 2010; 329(5990):444–448.

7. Tadokoro Y, Ema H, Okano M, Li E, Nakauchi H. De novo DNA meth- yltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells. J Exp Med. 2007;204(4):715–722.

8. Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet. 2011;44(1):23–31.

9. Broske AM, Vockentanz L, Kharazi S, et al. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Nat Genet. 2009;41(11):1207–1215.

10. Trowbridge JJ, Snow JW, Kim J, Orkin SH. DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells. Cell Stem Cell. 2009;5(4):442–449.

11. Wood AJ, Oakey RJ. Genomic imprinting in mammals: emerging themes and established theories. PLoS Genet. 2006;2(11):e147.

12. Lui JC, Finkielstain GP, Barnes KM, Baron J. An imprinted gene network that controls mammalian somatic growth is down-regulated during postna- tal growth deceleration in multiple organs. Am J Physiol Regul Integr Comp Physiol. 2008;295(1):R189–R196.

13. Berg JS, Lin KK, Sonnet C, et al. Imprinted genes that regulate early mam- malian growth are coexpressed in somatic stem cells. PLoS One. 2011; 6(10):e26410.

14. Besson V, Smeriglio P, Wegener A, et al. PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell popu- lations. Proc Natl Acad Sci U S A. 2011;108(28):11470–11475.

15. Zacharek SJ, Fillmore CM, Lau AN, et al. Lung stem cell self-renewal relies on BMI1-dependent control of expression at imprinted loci. Cell Stem Cell. 2011;9(3):272–281.

16. Ferron SR, Charalambous M, Radford E, et al. Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis. Nature. 2011;475(7356):381–385.

17. Li W, Sun W, Zhang Y, et al. Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proc Natl Acad Sci U S A. 2011;108(20):8299–8304.

18. Liu GH, Qu J, Suzuki K, et al. Progressive degeneration of human neural stem cells caused by pathogenic LRRK2. Nature. 2012;491(7425):603–607.

 

Additional References in Leaders in Pharmaceutical Intelligence

Proteomics and Biomarker Discovery

https://pharmaceuticalintelligence.com/2012/08/21/proteomics-and-biomarker-discovery/

Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets

https://pharmaceuticalintelligence.com/2013/12/08/developments-in-the-genomics-and-proteomics-of-type-2-diabetes-mellitus-and-treatment-targets/

Immune activation, immunity, antibacterial activity

https://pharmaceuticalintelligence.com/2014/07/06/immune-activation-immunity-antibacterial-activity/

Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis/

Research on inflammasomes opens therapeutic ways for treatment of rheumatoid arthritis

https://pharmaceuticalintelligence.com/2014/07/12/research-on-inflammasomes-opens-therapeutic-ways-for-treatment-of-rheumatoid-arthritis/

Update on mitochondrial function, respiration, and associated disorders

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-disorders/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified

https://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-in-the-mitochondrial-matrix-identified/

Mitochondrial Damage and Repair under Oxidative Stress

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Bzzz! Are fruitflies like us?

https://pharmaceuticalintelligence.com/2014/07/07/bzzz-are-fruitflies-like-us/

Discovery of Imigliptin, a Novel Selective DPP-4 Inhibitor for the Treatment of Type 2 Diabetes

https://pharmaceuticalintelligence.com/2014/06/25/discovery-of-imigliptin-a-novel-selective-dpp-4-inhibitor-for-the-treatment-of-type-2-diabetes/

Molecular biology mystery unravelled

https://pharmaceuticalintelligence.com/2014/06/22/molecular-biology-mystery-unravelled/

Gene Switch Takes Blood Cells to Leukemia and Back Again

https://pharmaceuticalintelligence.com/2014/06/20/gene-switch-takes-blood-cells-to-leukemia-and-back-again/

Wound-healing role for microRNAs in colon offer new insight to inflammatory bowel diseases

https://pharmaceuticalintelligence.com/2014/06/19/wound-healing-role-for-micrornas-in-colon-offer-new-insight-to-inflammatory-bowel-diseases/

Targeting a key driver of cancer

https://pharmaceuticalintelligence.com/2014/06/20/targeting-a-key-driver-of-cancer/

Tang Prize for 2014: Immunity and Cancer

https://pharmaceuticalintelligence.com/2014/06/20/tang-prize-for-2014-immunity-and-cancer/

Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Hemeostasis of Immune Responses for Good and Bad                             Demet Sag, PhD

https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/

3:45 – 4:15, 2014, Scott Lowe “Tumor suppressor and tumor maintenance genes”

12:00 – 12:30, 6/13/2014, John Maraganore “Progress in advancement of RNAi therapeutics”

9:30 – 10:00, 6/13/2014, David Bartel “MicroRNAs, poly(A) tails and post-transcriptional gene regulation.”

10:00 – 10:30, 6/13/2014, Joshua Mendell “Novel microRNA functions in mammalian physiology and cancer”

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/06/04/koch-institute-for-integrative-cancer-research-mit-summer-symposium-2014-rna-biology-cancer-and-therapeutic-implications-june-13-2014-830am-430pm-kresge-auditorium-mit/

Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases          Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/06/04/targeted-genome-editing-by-lentiviral-protein-transduction-of-zinc-finger-and-tal-effector-nucleases/

Illana Gozes discovered Novel Protein Fragments that have proven Protective Properties for Cognitive Functioning

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/06/03/prof-illana-gozes-discovered-novel-protein-fragments-that-have-proven-protective-properties-for-cognitive-functioning/

 

 

 

 

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Larry H Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013-12-07/larryhbern/Epigenomics and Companion Diagnostics

The development of epigenomics has reached a new stage.  Therapeutics has long been hampered from making important inroads to personalized medicine by the inability to differentiate patients with the same disorder having subvariants that react favorably and others that fail a treatment with an expected response.  These developments in diagnostics have been needed and rely specifically on laboratory-based diagnostics.  As these developments unfold, it will ba a very significant challenge to the clinical laboratory industry in the education and staffing of technologists.  This is already at a time that laboratory post-bacchalaureate education and staffing and accreditation of laboratories has not yet caught up to the need.

Epigenetics: Diagnostic and Therapeutic Applications

DDD Dec07, 2013

RiboMed and NuvOx Pharma to Collaborate on Brain Cancer Drug and Companion Diagnostic Test

RiboMed Biotechnologies, Inc. and NuvOx Pharma today jointly announced that they have entered into a collaborative agreement that will utilize RiboMed’s epigenetic biomarker test, GliomaSTRAT™, to characterize tumors from brain cancer patients and correlate response to NuvOx’s new drug, NVX-108 in the treatment of Glioblastoma multiforme (GBM).
“Our goal at RiboMed is to develop Companion Diagnostic tests that will help to prevent the treatment of patients with drugs to which they will not respond,” said Dr. Michelle Hanna, CEO and Scientific Director at RiboMed.
Carlsbad, CA and Tucson, AZ (PRWEB) December 05, 2013
RiboMed Biotechnologies, Inc. and NuvOx Pharma today jointly announced that they have entered into a collaborative agreement that will utilize
  • RiboMed’s epigenetic biomarker test, GliomaSTRAT™, to characterize tumors from brain cancer patients and
  • correlate response to NuvOx’s new drug, NVX-108 in the treatment of Glioblastoma multiforme (GBM).
NVX-108 is an intravenously delivered drug that, in animal models, increases the concentration of oxygen in tumors and consequently increases tumor sensitivity to radiation treatment. In animals implanted with human tumors which are resistant to radiation because they are low in oxygen, NVX-108 increased tumor oxygen levels by 400% and prolonged survival of the animals with tumors that were treated with radiation.
“The Phase 1B will begin early in Q1 2014 to evaluate the safety and efficacy of NVX-108 in combination with the current standard of care. We anticipate that incorporating RiboMed’s GliomaSTRAT into our Phase 1B clinical protocol will improve our ability to further stratify the resulting clinical data to ascertain optimal dosing and corresponding benefit of NVX-108 to patients with this disease, for which there are no good therapies,” noted NuvOx’s Chief Business Officer David Wilson.
The standard of care for patients with Glioblastoma brain cancer is surgery, followed by a 6-week course of radiation therapy and treatment with temozolomide. The patient’s response to treatment depends upon their tumor’s sensitivity to both the radiation and to the drug. RiboMed’s GliomaSTRAT is a DNA methylation based test that stratifies brain tumors into 4 groups, by
  • both grade (low grade vs high grade) and
  • response to certain chemotherapeutic drugs, including temozolomide (Temodar®).
“Our goal at RiboMed is to develop Companion Diagnostic tests that will help to prevent the treatment of patients with drugs to which they will not respond,” said Dr. Michelle Hanna, CEO and Scientific Director at RiboMed. “Given the differential response of high grade and low grade tumors to radiation, this stratification step could identify the patients that are most likely to benefit from treatment with NVX-108.”
RiboMed’s technology for epigenetic testing provides up to 100-fold greater sensitivity than competing methods. Tests utilize RiboMed’s bisulfite-free, methylated DNA enrichment process, MethylMagnet®, and their proprietary biomarker detection technology, Abscription®, which together in MethylMeter® provide superior sensitivity and specificity for the detection of DNA methylation in clinical samples. MethylMeter®
  • allows quantitative analysis of DNA methylation,
  • even with small samples containing damaged DNA, including formalin fixed paraffin embedded (FFPE) tissues.

About RiboMed

RiboMed Biotechnologies, Inc. (http://www.ribomed.com) is a College of American Pathology (CAP) accredited and CLIA-certified molecular diagnostic clinical laboratory and Contract Research Organization. The RiboMed Clinical Services Laboratory offers DNA methylation based tests for cancer and drug response related biomarkers to physicians and for use in clinical trials, as well as development services to research institutions and Pharma. Research Use Only (RUO) kits, reagents, and technology licensing are also available. More information can be requested at info(at)  http://ribomed.com.

About NuvOx

NuvOx was founded in 2008, after in-licensing NVX-108 for therapeutic applications, after it was demonstrated to be safe and effective as an ultrasound contrast imaging agent in more than 2200 patients. Serving as an oxygen therapeutic, previously demonstrated to be safe in humans, testing in animal models has demonstrated potential therapeutic application for sensitization to radiation treatment in many cancers, the mitigation of brain damage from stroke, heart damage from heart attack and general tissue destruction resulting from hemorrhagic shock. For additional information, please contact David Wilson at dwilson(at)nuvoxpharma(dot)com or call +1 (520) 624-6688 x1004.  http://www.nuvoxpharma.com.

Disclosure Regarding Forward-Looking Statements

Except for historical information contained herein, the matters set forth in this press release, including statements regarding the Company’s expectations, are forward-looking statements within the meaning of the “safe harbor” provisions of the Private Securities Litigation Reform Act of 1995. These forward-looking statements are subject to risks and uncertainties that may cause actual results to differ materially, including the risks and uncertainties associated with market demand for and acceptance and use of technology and tests such as the GliomaSTRAT test and NVX-108, separately or in combination, reliance upon the collaborative efforts of other parties including without limitation NuvOx, RiboMed or third parties obtaining or maintaining regulatory approvals that impact the Company’s business, government regulation particularly with respect to diagnostic products and laboratory developed tests, the Company’s ability to develop and commercialize technologies and products, particularly new technologies such as laboratory developed tests and genetic analysis platforms, the Company’s financial position, the Company’s ability to manage its existing cash resources or raise additional cash resources, competition, intellectual property protection and intellectual property rights of others, litigation involving the Company, and other risks. These forward-looking statements are based on current information that may change and you are cautioned not to place undue reliance on these forward-looking statements, which speak only as of the date of this press release. All forward-looking statements are qualified in their entirety by this cautionary statement, and the Company undertakes no obligation to revise or update any forward-looking statement to reflect events or circumstances after the issuance of this press release.
SOURCE: RiboMed Biotechnologies, Inc. and NuvOx Pharma, LLC

Epigenomics FDA Advisory Panel Date to Review Epi proColon®

Jordan deVos Marketing Specialist at Epigenomics AG

Epigenomics has announced that the Company was informed by the U.S. Food and Drug Administration (FDA) via the premarket approval (PMA) review process for Epi proColon® that the Meeting of the Molecular and Clinical Genetics Panel of the Medical Devices Advisory Committee has been tentatively scheduled for Tuesday, March 25th, 2014. Epi proColon® is Epigenomics’ blood-based screening test for colorectal cancer.
Epigenomics AG Announces FDA Advisory Committee Meeting to Review Epi… epigenomics.com
Berlin, Germany, and U.S.A., November 27, 2013 – Epigenomics AG (Frankfurt Prime Standard: ECX, OTC: EPGNY), the German-American cancer molecular diagnostics company, today announced that the Company was informed by the U.S. Food and Drug…

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Under Nutrition Early in Life may lead to Obesity

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

With the growing worldwide obesity epidemic, including huge populations in developing countries, such as China, India, Mexico and Brazil, the causes of this health and economic catastrophe have been increasingly studied. It is well known that metabolic syndrome and obesity exhibit a high correlation with low or absent physical exercise practices and the consumption of calorie-rich diets in developing countries; however, although the inhabitants may actually experience a nutrition transition, high levels of overweight and obese individuals could not be justified solely by diet and physical inactivity, other hallmarks, such as metabolic programming by the under nutrition early in life and epigenetic modification could also be underlining the obesity onset.

In addition to the pathophysiological aspects that have emerged from studies on metabolic programming caused by environmental insults during fetal life, another interesting point that is relevant to this issue is the role of epigenetic changes in the increased risk of developing metabolic diseases, such as type 2 diabetes and obesity, later in life. Epigenetic mechanisms, such as DNA methylation and/or nucleoprotein acetylation/methylation, are crucial to the normal/physiological development of several tissues in mammals, and they involve several mechanisms to guarantee fluctuations of enzymes and other proteins that regulate the metabolism. As previously reviewed, the intrauterine phase of development is particularly important for the genomic processes related to genes associated with metabolic pathways. Therefore, this phase of life may be particularly important for nutritional disturbance. In humans who experienced the Dutch famine Winter in 1944–1945 and in rats that were deprived of food in utero, epigenetic modifications were detected in the insulin-like growth factor 2 (IGF2) and pancreatic and duodenal home box 1 (Pdx1), which are the major factors involved in pancreas development and pancreatic β-cell maturation. Although it is known that the pancreas and the pancreatic β-cells develop/maturate during the embryonic phase, the postnatal life is also crucial for the maintenance processes that control the β-cell mass, such as proliferation, neogenesis and apoptosis. Nevertheless, no data on metabolic programming as the result of protein-restricted diet during lactation only have yet been reported, and no direct association with epigenetic modifications has been observed; on the other hand, because stressor insults during the milk suckling phase can lead to disturbances in glucose metabolism, hypothalamic neurons, ANS activity and β-cell mass/function of the pancreatic β-cells in rodents, further studies are needed on this topic.

Two decades ago, it was observed that low birth weight was related to adult chronic, non-transmissible diseases, such as type 2 diabetes, cardiovascular disease and obesity. It has been speculated that a nutritional injury during perinatal growth, including uterine and early postnatal life, may contribute to adapting the adult metabolism toward nutritional restriction. However, if an abundant diet is offered to people who have been undernourished during the perinatal life, this opportunity induces a metabolic shift toward the storage of energy and high fat tissue accumulation, thus leading to high risks of the onset of metabolic/coronary diseases onset. These observations led to the introduction of the term DOHaD (Developmental Origins of Health and Disease) previously known as the Barker thrifty phenotype hypothesis. Currently, the concept of DOHaD is extended to any other insults during perinatal life, pregnancy and/or lactation, such as underweight, overweight, diabetic or hyperplasic mothers. This concept also includes any type of stressful situations that may predispose babies or pups to develop metabolic disorders when they reach adulthood.

Source References:

 

http://www.nutritionandmetabolism.com/content/9/1/80

 

http://www.ncbi.nlm.nih.gov/pubmed/19955786?dopt=Abstract&holding=f1000,f1000m,isrctn

 

http://www.ncbi.nlm.nih.gov/pubmed/12886432?dopt=Abstract&holding=f1000,f1000m,isrctn

 

http://www.ncbi.nlm.nih.gov/pubmed/8733829?dopt=Abstract&holding=f1000,f1000m,isrctn

 

http://www.ncbi.nlm.nih.gov/pubmed/9478036?dopt=Abstract&holding=f1000,f1000m,isrctn

 

 

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Epigenetic mechanisms

Epigenetic mechanisms

Image Source:http://nihroadmap.nih.gov/EPIGENOMICS/images/epigeneticmechanisms.jpg |Author=National Institute of Health |Date=2005

The Underappreciated EpiGenome

Author:  Demet Sag, PhD

Early 1990’s Kavai group developed a method called Restriction Landmark Genomic Scanning using Methylation-sensitive endonucleases (RLGS-M) to identify differential methylation during development based on CpG islands. In their study they showed that the appearance and disappearance of the spots were specific to tissue and affecting gene regulation.

English: Revised definition of gene and flow o...

Revised definition of gene and flow of genetic information (adapted from Mattick JS (2003). Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms. BioEssays 25:930. doi:10.1002/bies.10332).

Epigenetics is getting a big attention recently to understand genomics and provide better results. However, this field is studied for many years under functional genomics and developmental biology for cellular and molecular biology. Stem cells have a free drive that we have not figured out yet. So genomics must be studied essentially with people training in developmental biology and comparative molecular genetics knowledge to make heads and tail for translational medicine.

There are three main routes of epigenetic modifications one

In 1993, Kavai group showed brain development assays of mice showed that only 0.7% genome has tissue and cellular specificity, and 1.7% of genome was able to turn on and off. This conclusion is relevant to genome sequencing data. Also, previous studies in genome and RNA biology presented that RNA directed DNA modifications lead into splicing and transcriptional silencing for gene regulation in Arapsidosis, mice, and Drosophila. (Borge, F. and. Martiensse, R.A. 2013; Di Croce L, Raker VA, Corsaro M, et al. 2002; Piferrer, F, 2013; Jun Kawai1 et al. 1993)

Comparative developmental biology studies and genetics in organisms give away clues that can be applied or open the door for a new discovery in human disease models.  Plants do utilize methylation and transposomes, which are viral particles that can affect the genome structure and present in all organisms including human, for their gene regulation and development extensively (The EMBO Journal, (22 March 2013) | doi:10.1038/emboj.2013.49). Thus, Arapsidosis is a good model.

The environment and gene expression define inheritable materials at transcriptional levels. He group (Cui-Jun Zhang et al, 2013, doi:10.1038/emboj.2013.49) suggested splicing machinery affecting RdDM and transcriptional silencing to control gene regulation during development since an RNA-directed DNA methylation (RdDM) pathway directs de novo methylation. Furthermore, these differences are highly regulated at RNA level with silencing, splicing, transposon activation/inactivation and modulation at epigenetic level.

In fruit fly with more than five hundreds years of accumulated data, the sex determination pathways, soma or germline, share common genes but they act differently in each pathway. Sxl (sex-lethal), which is key gene of somatic sex determination and is an RNA binding protein, regulates the genes through splicing in one its mechanisms (DOI: 10.1002/dvdy.23924). Yet, in germline ovo, which is a DNA binding protein, regulates the expression and development with different sets of rules yet these two paths always communicate to make the final outcome. The effects of RNA on epigenetics and gene regulation during development will be another topic to discuss. However, the three major epigenetic factors do not run in order necessarily, but always have three targeted outcome: initiate, differentiate and maintain. Thus, from environment to phenotype there are places/parts scientists can modulate or reprogram but there parts must be kept intact.

Yang et al, 2012 presented a study on PHD Finger Protein 7 (PHF7), which is an important factor for male germline sexual identity in Drosophila, and called this gene as a “epigenetic reader’ since the expression of this gene in XX soma started a female germline development. This type of epigenetic readers may also alter the outcome to balance cell metabolism towards desired phenotype in stem cells.

Adenine methylation

Adenine methylation (Photo credit: Allen Gathman)

The environment creates the epigenerators including temperature, differentiation signals and metabolites that trigger the cell membrane proteins for development of signal transduction within the cell to activate gene(s) and to create cellular response.  These changes can be modulated but they are not necessary for modulation. The second step involves epigenetic initiators that require precise coordination to recognize specific sequences on a chromatin in response to epigenerator signals. These molecules are

After they are involved they are on for life and controlled by autoregulatory mechanisms, like Sxl (sex lethal) RNA binding protein in somatic sex determination and ovo DNA binding protein in germline sex determination of fruit fly. Both have autoregulation mechanisms, cross talks, differential signals and cross reacting genes since after the final update made the soma has to maintain the decision to stay healthy and develop correctly.  Then, this brings the third level mechanism called epigenetic maintainers that are DNA methylating enzymes, histone modifying enzymes and histone variants.  The good news is they can be reversed. As a result the phonotype establishes either a

  • short term phenotype, transient for transcription,
  • DNA replication and repair or
  • long term phenotype outcomes that are chromatin conformation and heritable markers.

Early in development things are short term and stop after the development seized but be able to maintain the short term phonotype during wound healing, coagulation, trauma, disease and immune responses. Some cells will loose their ability to differentiate to very low levels. Yet, in life everything is possible even with less than 1% chance because nothing is accidental.

X-chromosome has fascinating characteristics simply because they present unusual mechanisms among female and male differentiation in fruit flies and mammals but their distinct characteristics in evolution marry with surprising parallel mechanisms in regulation. These features are the importance of noncoding RNAs, and epigenetic spreading of chromatin-modifying activities, and at the end of the actions most part of the Y chromosome is lost and one of the X-chromosome is downregulated in the big picture.

Figure 2: DNA methylation analysis methods not...

DNA methylation analysis methods not based on methylation-specific PCR. Following bisulfite conversion, the genomic DNA is amplified with PCR that does not discriminate between methylated and non-methylated sequences. The numerous methods available are then used to make the discrimination based on the changes within the amplicon as a result of bisulfite conversion. (Photo credit: Wikipedia)

Revisiting RNA directed DNA methylation study once again shows that unread sequence has the word on gene expression; it can still create the diversity that may help rebirth of stem cells with a correct program and develop tools for unmet human diseases. This will be the next topic to discuss.

Personal Impression

While I was listening Dr. Ecker, I remembered these studies. The question becomes what we know then what we know now. “The Underappreciated Epigenome: Methylation of Brain” by Joseph Ecker, Ph.D. of Salk Institute gave a talk on differential expression in adult vs. fetal brain development at Future of Genomics VI Medicine on March 7, 2013.

I like to give snapshot of his talk, and relating to the third wheel of the epigenetics: non coding RNAs for epigenetics, stem cell biology and development. He also reconnecting the dots and demonstrated that there is a linear relation between gene regulation region and methylation type.  As a result the plasticity of development takes place with the extensive mutation reconfigurations during early post natal stages up to two years at synaptogenesis. 

Ecker’s Study

The study focused onto inheritability of methylation in different organisms and comparative expression pattern. The data from chip sequencing for

  • histone modifications,
  • whole genome bisulfide sequencing for DNA modifications and
  • methylome profiling

projected a differential expression pattern between

  • adult and
  • fetal brain

for 5 hydroxymethyl cytosine hmC and 5 methyl cytosine mC.

Completion of base resolution of human methylation and aberrant epigenomic reprogramming in induced stem cells showed that the density of genic mCH is positively correlated with gene expression.

  • There was an increased mCH and an elevated gene expression pattern, unlike mCG that the gene is silenced at stem cell differentiation.
  • mCH expression was not only tissue specific but also cell specific based on comparative expression study.
  • There was no mCH expression in fetal frontal cortex unlike adult frontal cortex with accumulation of mCH during synaptogenesis. Also
  • deserts of methylation can be counted as heterochromatic regions and protective transfactors between mCG and mCH methylation.
  • In DNMT pattern showed neurons enriched with mCH but glia was depleted.  Furthermore, these
  • sites are not randomly but occurring with correlation.

Ecker group also published (Lister et al, 2009) the first genome-wide study in a mammalian genome, from both

  • human embryonic stem cells and
  • fetal fibroblasts, along with
  • comparative analysis of messenger RNA and
  • small RNA components of the transcriptome, several
  • histone modifications, and
  • sites of DNA–protein interaction for several key regulatory factors.

Like the related review paper by Spivakov and Fisher pointed out the search for molecular signatures of ‘stemness’ and pluripotency is becoming important for cell therapy. Thus, there is a huge effort on transcription machinery of key genes during early development and understanding of stem cells, but working on the epigenetic profiles and their interaction with transcription machinery is equally important. This poised but activable factors under the stem cell genome may open new doors for diagnostics and therapies.  As a result, “restricted” human diversity will open doors for a personalized medicine and delivery mechanisms.

Until human genome was sequenced the expected number of genes was high but only 1% of genome is read producing about 25000 genes. That brings up three modules to be concerned:

1. Use the RNA wisely as the ancestor of transferable genetic material that viruses used, even human has embedded over 90% natural viral in their genome;

2. Apply epigenetics with all three types from a scratch;

3. Inheritance.

RNA is regulating the methylation on genome through transposons and silencing the genes at transcriptional level to create intergenerational or transgenerational reprograming. This makes sense since after the decision is made there are two intentions passing onto next generation and maintaining the decision consistently.  If

  • in soma by mitosis to daughter cells, or
  • in germline by meiosis

the characters are transferred to the next generation. However, we also need to mind after the fact because no one is choosing what they get let alone not being able to choose their parents. Choosing the healthy tolerance levels in genome for future medicine is the key.

REFERENCES

Borge, F. and Martiense, R.A. “Establishing epigenetic variation during genome reprogramming” RNA Biology, Volume 10, Issue 4, April 2013,  doi: org/10.4161/rna.24085

Dalakouras,A. and Wassenegger, M. “Revisiting RNA-directed DNA methylation” RNA Biology, Volume 10, Issue 3 March 2013 Pages 453 – 455 http://dx.doi.org/10.4161/rna.23542

Piferrer, F. “Epigenetics of sex determination and gonadogenesis” Developmental Dynamics 8 FEB 2013 DOI: 10.1002/dvdy.23924

Yang SY, Baxter EM, Van Doren M. “Phf7 controls male sex determination in the Drosophila germline” Dev Cell. 2012 May 15;22(5):1041-51. doi: 10.1016/j.devcel.2012.04.013.

Yongkyu Park,  Mitzi I. KurodaEpigenetic Aspects of X-Chromosome Dosage Compensation” Science 10 August 2001: Vol. 293 no. 5532 pp. 1083-1085  DOI: 10.1126/science.1063073

Okano M, Xie S, Li E (July 1998). “Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases” Nat Genet 19 (3): 219–20. doi:10.1038/890    

Di Croce L, Raker VA, Corsaro M, et al. (2002). “Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor” Science 295 (5557): 1079–82. doi:10.1126/science.1065173

Jun Kawai1,+, Shinji Hirotsune2,3,Kenji Hirose1,3,+,Shinji Fushiki4, Sachihiko Watanabe1,+ and Yoshihide Hayashizaki2,3, “Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method” Nucl. Acids Res. (1993) 21 (24): 5604-5608. doi: 10.1093/nar/21.24.5604

Cui-Jun Zhang, Jin-Xing Zhou, Jun Liu, Ze-Yang Ma, Su-Wei Zhang, Kun Dou, Huan-Wei Huang, Tao Cai, Renyi Liu, Jian-Kang Zhu and Xin-Jian He. “The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis” The EMBO Journal , (22 March 2013) | doi:10.1038/emboj.2013.49

Ryan Lister1,9, Mattia Pelizzola1,9, Robert H. Dowen1, R. David Hawkins2, Gary Hon2, Julian Tonti-Filippini4, Joseph R. Nery1, Leonard Lee2, Zhen Ye2, Que-Minh Ngo2, Lee Edsall2, Jessica Antosiewicz-Bourget5,6, Ron Stewart5,6, Victor Ruotti5,6, A. Harvey Millar4, James A. Thomson5,6,7,8, Bing Ren2,3 & Joseph R. Ecker1 “Differential methylation between stem cells and adult stem cellsNature 462, 315-322 (19 November 2009) | doi:10.1038/nature08514

Mikhail Spivakov & Amanda G. Fisher “Epigenetic signatures of stem-cell identity” Nature Reviews Genetics 8, 263-271 (April 2007) | doi:10.1038/nrg2046

Louise C Laurent1,2,3,15, Caroline M Nievergelt4,15, Candace Lynch2,3, Eyitayo Fakunle2,3, Julie V Harness5, Uli Schmidt6, Vasiliy Galat7,8, Andrew L Laslett9,10,11, Timo Otonkoski12,13, Hans S Keirstead5, Andrew Schork4, Hyun-Sook Park14 & Jeanne F Loring2  “Restricted human ethnic diversity in human stem cell lines.” Nature Methods 7, 6 – 7 (2010) doi:10.1038/nmeth0110-06

Other related articles on this topic were published on this Open Access Online Scientific Journal, including the following:

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition

SJ Williams, PhD

https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-transition-in-prostate-cancer-cells/

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

SJ Williams, PhD

https://pharmaceuticalintelligence.com/2012/10/31/how-mobile-elements-in-junk-dna-prote-cacner-part1-transposon-mediated-tumorigenesis/

Diagnosing Diseases & Gene Therapy: Precision Genome Editing and Cost-effective microRNA Profiling 

Aviva Lev-Ari, PhD, RN, March 28, 2013
https://pharmaceuticalintelligence.com/2013/03/28/diagnosing-diseases-gene-therapy-precision-genome-editing-and-cost-effective-microrna-profiling/

Genomics-based cure for diabetes on-the-way 

Ritu Saxena, PhD, March 4, 2013
https://pharmaceuticalintelligence.com/2013/03/04/genomics-based-cure-for-diabetes-on-the-way/

How Genes Function

Larry H Bernstein, MD, FACP, March 4, 2013 

https://pharmaceuticalintelligence.com/2013/03/04/how-genes-function/

Long noncoding RNA: UCSF Researchers have Uncovered its role in Brain Development and in Neurological Diseases

Aviva Lev-Ari, PhD, RN, April 17, 2013 

https://pharmaceuticalintelligence.com/2013/04/17/long-noncoding-rna-ucsf-researchers-have-uncovered-its-role-in-brain-development-and-in-neurological-diseases/

Bibliographies on Genomics by Subject Matter

Genomics and Genetics Articles on this Open Access Online Scientific Journal 2/2012 — 1/2013

Aviva Lev-Ari, PhD, RN, 2/25/2013

https://pharmaceuticalintelligence.com/biomed-e-books/genomics-orientations-for-personalized-medicine/bibliographies-on-genomics/

The Initiation and Growth of Molecular Biology and Genomics – Part I

Larry H Bernstein, MD, FACP, 2/8/2013

https://pharmaceuticalintelligence.com/2013/02/08/the-initiation-and-growth-of-molecular-biology-and-genomics/

CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

Larry H Bernstein, MD, FACP, February 14, 2013

https://pharmaceuticalintelligence.com/2013/02/14/cracking-the-code-of-human-life-recent-advances-in-genomic-analysis-and-disease/

Genomic Endocrinology and its Future

Sudipta Saha, December 27, 2012
https://pharmaceuticalintelligence.com/2012/12/27/genomic-endocrinology-and-its-future-2/

Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes
Sudipta Saha, PhD, December 18, 2012
https://pharmaceuticalintelligence.com/2012/12/18/exome-sequencing-of-serous-endometrial-tumors-shows-recurrent-somatic-mutations-in-chromatin-remodeling-and-ubiquitin-ligase-complex-genes

Pancreatic Cancer: Genetics, Genomics and Immunotherapy
Tilda Barlyia, PhD, April 11, 2013 
https://pharmaceuticalintelligence.com/2013/04/11/update-on-pancreatic-cancer/

Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes
Sudipta Saha, December 18, 2012
https://pharmaceuticalintelligence.com/2012/12/18/exome-sequencing-of-serous-endometrial-tumors-shows-recurrent-somatic-mutations-in-chromatin-remodeling-and-ubiquitin-ligase-complex-genes/

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics
3/2010 – 3/2013 

Aviva Lev-Ari, PhD, RN and Larry H Bernstein, MD, FACP, March 7, 2013

https://pharmaceuticalintelligence.com/2013/03/07/genomics-genetics-of-cardiovascular-disease-diagnoses-a-literature-survey-of-ahas-circulation-cardiovascular-genetics-32010-32013/

What is the Future for Genomics in Clinical Medicine?

Larry H Bernstein, MD, FACP, February 17, 2013
https://pharmaceuticalintelligence.com/2013/02/17/what-is-the-future-for-genomics-in-clinical-medicine/

CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

Larry H Bernstein, MD, FACP, February 14, 2013
https://pharmaceuticalintelligence.com/2013/02/14/cracking-the-code-of-human-life-recent-advances-in-genomic-analysis-and-disease/

 

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