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Posts Tagged ‘PubMed’


An illustration of the central dogma of molecu...

An illustration of the central dogma of molecular biology annotated with the processes ncRNAs are involved in. (Photo credit: Wikipedia)

X-ray structure of the tRNA Phe from yeast. Da...

X-ray structure of the tRNA Phe from yeast. Data was obtained by PDB 1ehz and rendered with PyMOL. violet: acceptor stem wine red: D-loop blue: anticodon loop orange: variable loop green: TPsiC-loop yellow: CCA-3′ of the acceptor stem grey: anticodon (Photo credit: Wikipedia)

 Our genome must be packed tightly to fit into the nucleus. Genome is the blue print of a living organism whether made up off a single or multiple cell.   Recently, the genome seen as a functional network of physical contacts within (cis) and between (trans) chromosomes.  It became necessary to map these physical DNA contacts at high-resolution with technologies such as the “chromosome conformation capture” (3C) and other 3C-related methods including 3C-Carbon Copy (5C) and Hi-C.  Yet, we all know that in vivo conformation, gene to gene interactions from a long distance, histones and 3D have an impact on gene regulation and expression.  The game is not just a sequence but functional genomics with a correct translation of sequence for development so that proper molecular diagnostics can be applied not only for prevention but also for monitoring the efficacy of the intervention. Thus, we can provide a targeted therapy for personalized medicine.

On the other hand, we still know very little about genome organization at the molecular level, although spatial genome organization can critically affect gene expression.  It is important to recognize who is there to be present and who is there to create the functional impact for regulation in a specific tissue and time.  In addition, mediation of these chromatin contacts based on a specific tissue is quite essential.  For example, during long-range control mechanism specific enhancers and distal promoters needed to be invited to a close physical proximity to each other by transcription factors that has been found at other loci.  Furthermore, chromatin-binding proteins such as the CCCTC-binding factor (CTCF) and cohesin seem to have critical roles in genome organization and gene expression.  Let’s not forget about epigenetics, since there are so many methods to regulate chromatin interactions like cytosine methylation, maternal gene, gradient level, post-translational modifications and non-coding RNAs.

The non-coding RNAs (ncRNAs) are silent but they have the 99% power because ncRNAs are a broad class of transcripts consisting of structural (rRNAs, tRNAs, snRNAs, snoRNAs, etc.), regulatory (miRNAs, piRNAs, etc.), and of sense/antisense transcripts.  Among these an interesting class is the latter group.   This class includes transcriptional “features” (eRNAs, tiRNAs), and a very large number of long non-coding RNAs (lncRNAs), length from 200 nt to 100 kb.  The magnificent future of lncRNAs comes from their production, as they can be transcribed nearby known protein-coding genes or from their introns. As a result, because of their intergenical production they are also called as “lincRNAs (long intergenical non-coding RNAs).  They are abundant and specific as microRNAs.  Hence, their inclusion into the biomarker list and assuming their roles during targeted therapy don’t require us to be a wizard but a functional genomicist knowing evolution, development and molecular genetics and plus signaling.

lincRNA can both activate and repress the gene either cis or trans acting to effect gene regulation will be discussed next.

As a result, one gene expression regulation needs from twenty to several hundred genes. As they say raising a child needs a village.

References:

“Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs”.

Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY.  Cell. 2007 Jun 29; 129(7):1311-23.

“Long noncoding RNA as modular scaffold of histone modification complexes”

Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HYScience. 2010 Aug 6; 329(5992):689-93.

“Capturing Chromosome Conformation”.

Dekker J, Rippe K, Dekker M, Kleckner N.Science.2002;295:1306–1311.

“Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements”.

Dostie J, Richmond TA, Arnaout RA, Selzer RR, Lee WL, Honan TA, Rubio ED, Krumm A, Lamb J, Nusbaum C, Green RD, Dekker J.Genome Res. 2006;16:1299–1309.

“Chromosome conformation capture carbon copy technology”.

Dostie J, Zhan Y, Dekker J. Curr. Protoc. Mol. Biol. 2007 Chapter 21, Unit 21 14.

“Comprehensive mapping of long-range interactions reveals folding principles of the human genome”.

Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J.  Science. 2009;326:289–293.

“Chromatin conformation signatures: ideal human disease biomarkers?”

Crutchley JL, Wang XQ, Ferraiuolo MA, Dostie J.Biomark. Med. 2010;4:611–629.

“Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants”.

Folkersen L, Kyriakou T, Goel A, Peden J, Mälarstig A, Paulsson-Berne G, Hamsten A, Hugh Watkins, Franco-Cereceda A, Gabrielsen A, Eriksson P, PROCARDIS consortia

PLoS One. 2009 Nov 2; 4(11):e7677.

” A myelopoiesis-associated regulatory intergenic noncoding RNA transcript within the human HOXA cluster”.

Zhang X, Lian Z, Padden C, Gerstein MB, Rozowsky J, Snyder M, Gingeras TR, Kapranov P, Weissman SM, Newburger PE.  Blood. 2009 Mar 12; 113(11):2526-34.

Monk M.   Genes Dev. 1988 Aug; 2(8):921-5.

Hox genes specify vertebral types in the presomitic mesoderm

Marta Carapuço,1 Ana Nóvoa,1 Nicoletta Bobola,2 and Moisés Mallo1,3 .  Genes Dev. 2005 September 15; 19(18): 2116–2121.

Krumlauf R.  Cell. 1994 Jul 29; 78(2):191-201.

“Noncoding RNA synthesis and loss of Polycomb group repression accompanies the colinear activation of the human HOXA cluster”.

Sessa L, Breiling A, Lavorgna G, Silvestri L, Casari G, Orlando V.  RNA. 2007 Feb; 13(2):223-39.

“Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs”.

Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY.  Cell. 2007 Jun 29; 129(7):1311-23.

“Long noncoding RNAs with enhancer-like function in human cells”.

Ørom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R

“Histone modifications at human enhancers reflect global cell-type-specific gene expression”.

Heintzman ND, Hon GC, Hawkins RD, Kheradpour P, Stark A, Harp LF, Ye Z, Lee LK, Stuart RK, Ching CW, Ching KA, Antosiewicz-Bourget JE, Liu H, Zhang X, Green RD, Lobanenkov VV, Stewart R, Thomson JA, Crawford GE, Kellis M, Ren B.   Nature. 2009 May 7; 459(7243):108-12.

“Tiny RNAs associated with transcription start sites in animals”.

Taft RJ, Glazov EA, Cloonan N, Simons C, Stephen S, Faulkner GJ, Lassmann T, Forrest AR, Grimmond SM, Schroder K, Irvine K, Arakawa T, Nakamura M, Kubosaki A, Hayashida K, Kawazu C, Murata M, Nishiyori H, Fukuda S, Kawai J, Daub CO, Hume DA, Suzuki H, Orlando V, Carninci P, Hayashizaki Y, Mattick JS.  Nat Genet. 2009 May; 41(5):572-8.

“Chromatin modifications and their function”.

Kouzarides T.   Cell. 2007 Feb 23; 128(4):693-705.

Tripathi V, Ellis JD, Shen Z, Song DY, Pan Q, Watt AT, Freier SM, Bennett CF, Sharma A, Bubulya PA, Blencowe BJ, Prasanth SG, Prasanth KV.   Mol Cell. 2010 Sep 24; 39(6):925-38.

Selected Further Reading

“Small and long non-coding RNAs in cardiac homeostasis and regeneration”

Ounzain, S.; Crippa, S.; Pedrazzini, T.  BBA – Molecular Cell Research vol. 1833 issue 4 April, 2013. p. 923-933

“Regulatory mechanisms of long noncoding RNAs in vertebrate central nervous system development and function.” 

Knauss, J.L.; Sun, T.  “Neuroscience vol. 235 April 3, 2013. p. 200-214

“Comparative genomics reveals ‘novel’ Fur regulated sRNAs and coding genes in diverse proteobacteria.”

Sridhar, J.; Sabarinathan, R.; Gunasekaran, P.; Sekar, K.   Gene vol. 516 issue 2 March 10, 2013. p. 335-344 DOI: 10.1016/j.gene.2012.12.057. ISSN: 0378-1119.

miRNAs Regulate Expression and Function of Extracellular Matrix Molecules”

Rutnam, Z.J.; Wight, T.N.; Yang,  B.B.Matrixixix Biology vol. 32 issue 2 March 11, 2013. p. 74-85 DOI: 10.1016/j.matbio.2012.11.003. ISSN: 0945-053X.

Transcript profiling of microRNAs during the early development of the maize brace root via Solexa sequencing

Liu, P.; Yan, K.; Lei, Y.x.; Xu, R.; Zhang, Y.m.; Yang, G.d.; Huang, J.g.; Wu, C.A.; Zheng, C.C.Genomics vol. 101 issue 2 February, 2013. p. 149-156 DOI: 10.1016/j.ygeno.2012.11.004. ISSN: 0888-7543.

Regulatory mechanisms of long noncoding RNAs in vertebrate central nervous system development and function

Knauss, J.L.; Sun, T.  Neuroscience vol. 235 April 3, 2013. p. 200-214 DOI: 10.1016/j.neuroscience.2013.01.022. ISSN: 0306-4522.

“The dynamic biliary epithelia: Molecules, pathways, and disease”

O’Hara, Steven P.; Tabibian, James H.; Splinter, Patrick L.; LaRusso, Nicholas F. Journal of Hepatology vol. 58 issue 3 March, 2013. p. 575-582 DOI: 10.1016/j.jhep.2012.10.011. ISSN: 0168-8278

ABBREVIATIONS

3C = Chromosome conformation capture

rRNAs = Ribosomal RNAs

tRNAs = Transfer RNAs

snRNAs = Small nuclear RNAs

snoRNAs = Small nucleolar RNAs

miRNAs = MicroRNAs

piRNAs = Piwi-interacting RNAs

eRNAs = Enhancer RNAs

tiRNAs = Transcription initiation RNAs

spliRNAs = Splice-site RNAs

lincRNAs = Long intergenic non-coding RNAs

lncRNPs = Long non-coding ribonucleoprotein complexes

Igf2r = Insulin-like growth factor II receptor

HMTs = Histone methyl transferases

TSSs = Transcriptional start sites

TFs = Transcription factors

RNAi = RNA interference

PTMs = Post-translational modifications

  • Patent. (postdocstreet.wordpress.com)

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Larry H Bernstein, MD, Reporter & Curator
https://pharmaceuticalintelligence.com/2013/06/21/Nrf2 Role in Blocking DNA Damage/lhbern

 

DNA damage has been a central focus of carcinogenesis.  The following is of great interest in this respect.

 

 

 

Nrf2 as a novel molecular target for chemoprevention.

 

Lee JS, Surh YJ.

 

Cancer Lett. 2005 Jun 28;224(2):171-84. Epub 2004 Nov 11.

 

Source

 

National Research Laboratory of Molecular Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku, Seoul 151-742, South Korea.

 

 

Abstract

 

One of the rational and effective strategies for chemoprevention is the blockade of DNA damage caused by carcinogenic insult. This can be achieved either

 

  • by reducing the formation of reactive carcinogenic species
  • or stimulating their detoxification.

 

A wide spectrum of xenobiotic metabolizing enzymes catalyze both phase I (oxidation and reduction) and phase II biotransformation (conjugation) reactions involved in carcinogen activation and/or deactivation. Several antioxidant-response element (ARE)-regulated gene products such as

 

  • glutathione S-transferase,
  • NAD(P)H:quinone oxidoreductase 1,
  • UDP-glucuronosyltransferase,
  • gamma-glutamate cysteine ligase, and
  • hemeoxygenase-1

 

are known to mediate detoxification and/or to exert antioxidant functions thereby protecting cells from genotoxic damage.

 

The transcription of ARE-driven genes is regulated, at least in part,

 

  • by nuclear transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2),
  • which is sequestered in cytoplasm by Kelch-like ECH-associated protein 1 (Keap1).

 

Exposure of cells to ARE inducers results in

 

  1. the dissociation of Nrf2 from Keap1 and
  2. facilitates translocation of Nrf2 to the nucleus,
  3. where it heterodimerizes with small Maf protein, and
  4. binds to ARE,

 

eventually resulting in the transcriptional regulation of target genes.

 

The Nrf2-Keap1-ARE signaling pathway can be modulated by several upstream kinases including

 

  • phosphatidylinositol 3-kinase,
  • protein kinase C, and
  • mitogen-activated protein kinases.

 

Selected Nrf2-Keap1-ARE activators, such as

 

  • oltipraz,
  • anethole dithiolethione,
  • sulforaphane,
  • 6-methylsulphinylhexyl isothiocyanate,
  • curcumin,
  • caffeic acid phenethyl ester,
  • 4′-bromoflavone, etc.

 

are potential chemopreventive agents. This mini-review will focus on a chemopreventive strategy directed towards

 

  • protection of DNA and other important cellular molecules by
  • inducing de novo synthesis of phase II detoxifying or antioxidant genes via the Nrf2-ARE core signaling pathway.

 

 

PMID: 15914268 [PubMed – indexed for MEDLINE]

 

 

 

 

 

English: Graph of Nrf2 publications (pubmed se...

English: Graph of Nrf2 publications (pubmed search) by year (Photo credit: Wikipedia)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Most lifespan influencing genes affect the rat...

Most lifespan influencing genes affect the rate of DNA damage (Photo credit: Wikipedia)

 

Single-strand and double-strand DNA damage

Single-strand and double-strand DNA damage (Photo credit: Wikipedia)

 

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Zebrafish—Susceptible to Cancer

Reporter: Larry H Bernstein, MD, FCAP

 

 

Models of Transparency

Researchers are taking advantage of small, transparent zebrafish embryos and larvae—and a special strain of see-through adults—to understand the development and spread of cancer.
By Joan K. Heath, David Langenau, Kirsten C. Sadler, and Richard White | April 1, 2013
LOOKING INSIDE DISEASE: The wild-type zebrafish larva on the left is stained for the two neuronal proteins (green) and membrane-trafficking proteins expressed near synapses (blue). On the right, the neurons of a transgenic zebrafish larva produce the dementia-associated Tau protein (red), a disease-specific form of which is stained in blue. Tubulin is stained in green.
COURTESY OF DOMINIK PAQUET, THE ROCKEFELLER UNIVERSITY, NEW YORK, USA
From frogs to dogs and people, cancer wreaks havoc across the animal kingdom—and fish are no exception. Coral trout, for example, develop melanoma from overexposure to sun, just as humans do. Rainbow trout develop liver cancer in response to environmental toxins. And zebrafish—small, striped fish indigenous to the rivers of India and a widely used model organism—are susceptible to both malignant and benign tumors of the brain, nervous system, blood, liver, pancreas, skin, muscle, and intestine.
Importantly, tumors that arise in the same organs in humans and fish look and behave alike, and the cancers often share common genetic underpinnings. As a result, most researchers believe that the basic mechanisms underlying tumor formation are conserved across species, allowing them to study the formation, expansion, and spread of tumors in animal models with the hope of eventually finding new insights into cancer in people.
Zebrafish are an increasingly popular choice among cancer biologists. Between 1995 and 2012, there was a 10-fold increase in the number of yearly PubMed citations of cancer studies in the species, with more than 200 research papers published last year.  Although dwarfed by cancer studies using human tissue and mouse models, the optical transparency of zebrafish embryos and larvae—and now, adult fish of a recently created strain—allows researchers to track tumors in a way that is not possible in other vertebrate models. Furthermore, their small size—embryos are small enough to be reared in 96-well plates—make them a more practical laboratory system than other cancer models. Indeed, researchers are now using these fish to identify druggable oncogenic drivers of specific tumor types, to tease apart the complex network of cancer genes that cooperate in tumor formation and progression, to probe the interplay between the genes that govern embryonic development and those that cause cancer, and to uncover how tumors metastasize and kill their host. The zebrafish model offers a major opportunity to discover important pathways underlying cancer and to identify novel therapies in high-throughput drug screens in a way that mice never could.

The zebrafish toolbox

Zebrafish (Danio rerio) have fast made their way from pet stores and home aquaria into research laboratories worldwide. Their weekly matings produce 100 to 200 embryos that rapidly and synchronously march through embryonic development, so that within 5 days of fertilization, they are mature, feeding larvae. Zebrafish are small and inexpensive to maintain in high numbers, facilitating large-scale experimentation and cheap in vivo drug screens. Famously, the fish are transparent during early larval stages, allowing investigators to directly observe internal development and making the fish a favorite of developmental biologists since the 1960s. But in recent years, the utility of zebrafish has been proven beyond developmental fields, and they are now being found in more and more laboratories studying behavior, diabetes, heart disease, regeneration, stem cell biology—and cancer.
Critically, zebrafish can be used to identify the important pathways and processes that cause cancer in people. Common organ systems and cell types are shared between human and zebrafish, and whether induced by transgenesis or carcinogens, cancers arising from the blood (leukemia and lymphoma), pigmented cells of the skin (melanoma), and the cells that line the bile ducts (cholangiocarcinoma) have microscopic features that are essentially indistinguishable between humans and zebrafish.
The zebrafish model offers a major opportunity to discover important pathways underlying cancer and to identify novel therapies in high-throughput drug screens, in a way that mice never could.
Comparing gene-expression profiles of tumors across various species provides a powerful mechanism for identifying genes that likely represent core functions of cancer. For example, microarray gene-expression analyses have compared the gene signatures of fish hepatocellular carcinoma to that of human liver, gastric, prostate, and lung tumors. Remarkably, this analysis revealed that fish and human liver tumors are more similar to each other than either tumor type is to human tumors derived from different tissues. Moreover, comparative studies can often be used to pinpoint pathways that are active in human disease. This is illustrated by work on a zebrafish model of rhabdomyosarcoma (RMS), a cancer of skeletal muscle, which revealed a gene signature that is also commonly found in human RMS, highlighting the importance of the RAS signaling pathway in the genesis of human RMS.1

A window into cancer

In 2003, the laboratory of A. Thomas Look at Children’s Hospital Boston was the first to realize the long-held dream of following the behavior of cancer cells as they initiate tumor growth and invade structures within live animals. Specifically, the researchers engineered leukemia-afflicted T cells to express green fluorescent protein (GFP) and visualized cancer onset within the zebrafish thymus.2 Moreover, these GFP-positive tumor cells were transplantable into recipient fish, a hallmark of the malignant cell type. Following up on this work, several researchers have now begun transplanting fluorescently labeled human cancer cells into zebrafish larvae to visualize tumor growth and spread in a manner not achievable in more common mouse xenograft models.
Capitalizing on the lack of an acquired immune system during larval stages and the ability to rear zebrafish at temperatures that mimic the human core temperature, Stefania Nicoli of the University of Brescia in Italy and colleagues implanted human cancer cells expressing high levels of vascular endothelial growth factor (VEGF) into zebrafish larvae with GFP-labeled blood vessels.3 VEGF is a factor commonly produced by growing cancers and is responsible for coaxing blood vessels to invade the developing tumor. Nicoli’s work allowed the direct visualization of vasculature remodeling and new vessel formation—and showed that it could be blocked by the addition of VEGF-inhibitory drugs to the water in which the larvae lived. Using similar approaches, many laboratories have successfully engrafted human cancer cells from a range of tumor types into zebrafish embryos.
Researchers have also used zebrafish to visualize the role of tumor heterogeneity within cancer over time. Work from one of our labs (David Langenau’s) has utilized a model of RAS-induced RMS to fluorescently label tumor cells based on differentiation status. This allowed the team to watch the never-before-visualized birth of cancer—the acquisition of invasive properties by normal muscle stem cells and the breakdown of normal muscle architecture, clearing the way for continued tumor expansion. The researchers also characterized two molecularly distinct cell populations, one that is responsible for tumor growth and another that drives cancer spread or metastasis. (See photos below—Imaging Blood Cancers; Solid Tumor Development.)
SEE-THROUGH SUBJECTS: Zebrafish embryos (bottom right), shown here at 28 hours, are naturally transparent, as are zebrafish larvae (bottom left) until around 3 weeks of age. A new strain of zebrafish, called casper, maintains its transparency into adulthood (top right), allowing researchers to observe cancer formation in adult fish. A wall of tanks (top left) at the Zebrafish Resource Center, Karlsruhe Institute of Technology.
CLOCKWISE FROM TOP: © MARTIN LOBER; COURTESY OF RICHARD WHITE; COURTESY OF GRAHAM SCOTT; COURTESY OF KIRSTEN EDEPLI
T-cell leukemia and RMS are pediatric diseases, favoring cancer development in early larval stages of these zebrafish models. However, cancer is predominantly a disease that affects adults, and zebrafish lose their transparency at around 3 weeks of age. To visualize tumor formation in older zebrafish, one of us (Richard White) and Len Zon of Children’s Hospital Boston have developed a strain of zebrafish called casper, which lacks pigment and is optically clear into adulthood.4 (See photo here.) Using these animals, investigators have implanted pigmented melanomas and witnessed local spread and metastasis over time. First described in 2008, casper is now the zebrafish strain of choice for imaging studies in the field.
Zebrafish have truly proven to be ideal organisms for visualizing cancer; there is no other animal system that allows researchers to literally watch tumors grow and spread. Because we can follow cells as they escape from the primary tumor, migrate, and form metastases in a variety of organs, zebrafish provide an unsurpassed model to describe the distinct steps of cancer progression, and researchers using this model are already contributing much to our understanding of cancer.

 Screening for drugs

In terms of drug discovery, zebrafish have emerged as the only vertebrate organism amenable to high-throughput and high-content chemical screening in vivo. The small size of freshly hatched zebrafish embryos means that up to 20 embryos can be dispensed into the individual wells of a 96-well plate, thereby providing a platform for the robotic delivery and testing of hundreds or thousands of compounds in living animals. Because of the parallels between embryonic development and cancer, compounds producing changes in the growth or proliferation of developing organs may also be relevant to cancer. However, to more directly search for small molecules capable of putting the brakes on cancerous growth, researchers are turning to several established tumor-prone zebrafish lines.
One prominent success from such endeavors is the identification of a drug to treat melanoma. Like human melanoma skin lesions, zebrafish melanomas exhibit a gene-expression signature characteristic of the embryonic neural crest, a multipotent group of stem cells that give rise to dozens of cell types, including pigmented skin cells called melanocytes. The genes in this signature, including sox10 and mitf, were hypothesized to be important for melanoma growth, so in 2011 White and Zon performed an in vivo screen to identify small molecules that suppressed the expression of these neural crest genes in developing embryos.5 After screening 2,000 molecules, they identified leflunomide, an approved treatment for rheumatoid arthritis. Importantly, leflunomide was then found to inhibit the growth of both zebrafish and human melanoma xenografts in vivo, and the drug moved from discovery to Phase 1/2 trials in only 4 years, demonstrating just how quickly discoveries in zebrafish can have clinical impact. Similar efforts to discover novel modulators of leukemia growth have recently been reported to work in both fish and humans, suggesting that this approach will be broadly applicable to a wide range of solid and liquid tumors.
Probing the cancer genome
The genesis of cancer generally depends on the inactivation of one or more tumor suppressor genes in conjunction with signaling from oncogenes. Indeed, rapid advances in sequencing technologies and efforts such as The Cancer Genome Atlas (TCGA) have revealed surprisingly few “driver” mutations capable of causing cancer alone. Instead, TCGA and other sequencing studies have identified vast genetic heterogeneity both across and within tumor types, with mutations extending well beyond genes likely to represent classical oncogenes or tumor suppressor genes. How these mutations influence tumor growth remains a major unanswered question in cancer biology.
IMAGING BLOOD CANCERS: Developing T lymphocytes in the thymus of a transgenic zebrafish (top right) express green fluorescent protein (GFP). A transgenic zebrafish (bottom right) that coexpresses the Myc oncogene with GFP shows signs of prominent leukemia, which has spread well beyond the boundaries of the thymus (T).
COURTESY OF DAVID LANGENAU
Enter zebrafish, and a range of high-throughput reverse-genetic techniques for cancer gene discovery. By transiently overexpressing each of 30 candidate genes in zebrafish larvae, for example, Craig Ceol and Yariv Houvras in Zon’s group identified a single cooperating oncogene, SETDB1, as a new player in melanoma.6 In this study, the researchers created and analyzed more than 3,000 transgenic animals. Because they used a transposon-based transgenic approach that leads to high-level, uniform expression, they could directly assess how the injected genes affected tumor onset without going through the lengthy process of germline transgenics, a major bottleneck in mouse genetics. This rapid screening approach is a prime example of how the mountains of data generated by TCGA can be quickly assessed for biological function, and zebrafish are the only in vivo whole-animal vertebrate system that enables researchers to rapidly sift through these data to understand which mutations drive cancer.
Other new approaches are advancing loss-of-function analyses. Until recently, such studies relied on TiLLING (targeting induced local lesions in genomes), in which a chemical carcinogen, ethylnitrosourea (ENU), introduces point mutations throughout the genome and high-throughput methods then look for mutations in genes of interest. This method yielded several valuable strains of tumor-prone zebrafish harboring clinically relevant mutations in the well-known tumor-suppressor genes p53, apc, and pten, and these have been pivotal to the development of multiple zebrafish cancer models. However, the unbiased nature of ENU mutagenesis makes TiLLING a labor-intensive and impractical business in most laboratory settings. Instead, precision editing of the genome has emerged as the method of choice for the systematic creation of knockout and mutant animals. Specifically, homology-based editing, using TALENs (transcription activator–like effector nucleases) and, more recently, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems, has revolutionized the field.7,8 Using relatively simple procedures, virtually any gene can now be mutated in zebrafish, allowing for very large-scale, in vivo assessments of novel cancer genes and the analysis of interacting mutations—one of the greatest challenges facing the cancer field in the coming decade.
SOLID TUMOR DEVELOPMENT: A transgenic zebrafish (bottom left) with fluorescent-labeled RAS-induced rhabdomyosacoma. Green fluorescent protein is expressed in the tumor-propagating cells, which drive continued tumor growth. Red fluorescent protein is localized to the nucleus and expressed in myoblast-like cells, while blue fluorescent protein is confined to terminally differentiated cancer cells that express myosin (magnified image, bottom right). Live-cell imaging permits dynamic visualization of the birth of cancer and the functional consequences of tumor cell heterogeneity within established tumors.
COURTESY OF DAVID LANGENAU
To complement approaches that directly inactivate genes within the genome, strategies to achieve interference RNA-mediated gene silencing in zebrafish have come of age as well. Expression of short hairpin RNAs, for example, have produced stable and tissue-specific knockdown in cancer-related genes such as chordin and wnt5b.9,10 Because of the ease of manipulating the genome as well as the large number of well-characterized zebrafish gene promoters, such strategies immediately afford the opportunity to knockdown known gene functions in a tissue-specific fashion, and it is likely that temporal control will be readily achievable as well.
In all, combining the descriptive data from TCGA with zebrafish transgenesis, high-throughput overexpression and knockout techniques, and unbiased genetic screens offers an unprecedented opportunity to functionally probe the cancer genome.

The future of the field

Given its power for imaging, transplantation, small-molecule screens, and high-throughput transgenesis, the zebrafish model should become a major platform for deeply interrogating cancer biology in vivo over the next decade. One major area where zebrafish are particularly valuable is in teasing apart the extreme complexity of cancer. Because combinations of genetic pathways can be assessed simultaneously, potentially dozens of genomic alterations found in human cancer could be tested for their effects in the fish, allowing us to sort biologically meaningful alterations from neutral ones. These techniques will also allow us to understand how numerous small changes, which on their own have little phenotypic effect, can combine to cause cancer.
The success of the field will depend upon improved funding for zebrafish cancer research, however. Currently, only a small fraction of National Institutes of Health RO1 grants for cancer research are awarded for zebrafish studies, with the vast majority going to work in mice, humans, and human cells. Consortium efforts analogous to the Mouse Model Consortium will be necessary to develop more faithful zebrafish models of human cancer, which can then be used as the basis for further screens. Whereas mouse models of cancer have delivered great insights into the biological mechanisms underlying human malignancy, we view zebrafish models as a springboard for the rapid launch of unbiased genetic and chemical screens.
With any cancer model, bridging the gap between the animals and human patients is the ultimate proof of its utility. For the zebrafish, this can occur not only through bringing drugs to the clinic, but also in the development of novel biomarkers and early detection methods. The next 10 years will be an exciting time, and we have great confidence that the zebrafish will contribute major discoveries to the treatment of human cancers.
Joan K. Heath is an associate professor in the ACRF Chemical Biology Division at the Walter and Eliza Hall Institute of Medical Research and the Department of Medical Biology at the University of  Melbourne, Australia, where her laboratory is studying the genetic regulation of  intestinal organogenesis and colorectal cancer.
David Langenau, an assistant professor of pathology at Harvard Medical School, studies the mechanisms that drive pediatric cancer relapse within the Molecular Pathology Unit and the Cancer Center at Massachusetts General Hospital.
Kirsten C. Sadler is an assistant professor in the Division of Liver Diseases/Department of Medicine and in Developmental and Regenerative Biology at the Icahn School of Medicine at Mount Sinai in New York City, where she studies the mechanisms of liver development, regeneration, and cancer.
Richard White is an assistant professor at the Memorial Sloan-Kettering Cancer Center and Weill Cornell Medical College in New York City. His laboratory studies the evolutionary mechanisms by which tumors develop the capacity for metastasis.

References

D.M. Langenau et al., “Effects of RAS on the genesis of embryonal rhabdomyosarcoma,” Genes Dev, 21:1382-95, 2007.
D.M. Langenau et al., “Myc-induced T cell leukemia in transgenic zebrafish,” Science, 299:887-90, 2003.
S. Nicoli et al., “Mammalian tumor xenografts induce neovascularization in zebrafish embryos,” Cancer Res, 67:2927-31, 2007.
R.M. White et al., “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell, 2:183-89, 2008.
R.M. White et al., “DHODH modulates transcriptional elongation in the neural crest and melanoma,” Nature, 471:518-22, 2011.
V.M. Bedell et al., “In vivo genome editing using a high-efficiency TALEN system,” Nature, 491:114-18, 2012.
W.Y. Hwang et al., “Efficient genome editing in zebrafish using a CRISPR-Cas system,” Nat Biotechnol, doi: 10.1038/nbt.2501, 2013.
M. Dong et al., “Heritable and lineage-specific gene knockdown in zebrafish embryo,” PLOS ONE, 4:e6125, 2009.
G. De Rienzo et al., “Efficient shRNA-mediated inhibition of gene expression in zebrafish,” Zebrafish, 9:97-107, 2012.
Tags
zebrafish, model organisms, cancer therapetics, cancer research, cancer genomics, cancer gene expression, cancer and animal models
Danio rerio, better known as the zebrafish

Danio rerio, better known as the zebrafish (Photo credit: Wikipedia)

Zebrafish embryo development

Zebrafish embryo development (Photo credit: Carl Zeiss Microscopy)

English: zebrafish (Danio rerio) ovary oocyte ...

English: zebrafish (Danio rerio) ovary oocyte maturation (Photo credit: Wikipedia)

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Reporter: Aviva Lev-Ari, PhD, RN

 

‘We Have a Problem in Science’

October 02, 2012

A recent study in the Proceedings of the National Academy of Sciences found that more than two-thirds of 2,000 retractions in the life science literature were attributable to some form of misconduct, including fraud, duplicate publication, and plagiarism.

The study, led by Arturo Casadevall of Albert Einstein College of Medicine, estimates that the percentage of scientific papers retracted because of fraud has increased more than 10-fold since 1975.

Carl Zimmer notes in The New York Times that previous studies have concluded that most retractions were attributable to “honest errors,” but the new study “challenges that comforting assumption.”

The authors compiled more than 2,000 retraction notices published before May 3, 2012, and then dug into the reasons behind each retraction. Some reasons were cited by the journals, but the authors also found that the retraction notices for some papers did not cite fraud as the reason for the retraction.

The rise in fraudulent papers “is a sign of a winner-take-all culture in which getting a paper published in a major journal can be the difference between heading a lab and facing unemployment,” Zimmer says.

According to Casadevall, the fact that “some fraction of people are starting to cheat” should not be taken lightly, even if the overall number of fraudulent papers is relatively low. “It convinces me more that we have a problem in science,” he says.

 Source:

 http://www.genomeweb.com//node/1133746?hq_e=el&hq_m=1361468&hq_l=1&hq_v=09187c3305

Misconduct accounts for the majority of retracted scientific publications

  1. Ferric C. Fanga,b,1,
  2. R. Grant Steenc,1, and
  3. Arturo Casadevalld,1,2

+Author Affiliations


  1. Departments of aLaboratory Medicine and

  2. bMicrobiology, University of Washington School of Medicine, Seattle, WA 98195;

  3. cMediCC! Medical Communications Consultants, Chapel Hill, NC 27517; and

  4. dDepartment of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
  1. Edited by Thomas Shenk, Princeton University, Princeton, NJ, and approved September 6, 2012 (received for review July 18, 2012)

Abstract

A detailed review of all 2,047 biomedical and life-science research articles indexed by PubMed as retracted on May 3, 2012 revealed that only 21.3% of retractions were attributable to error. In contrast, 67.4% of retractions were attributable to misconduct, including fraud or suspected fraud (43.4%), duplicate publication (14.2%), and plagiarism (9.8%). Incomplete, uninformative or misleading retraction announcements have led to a previous underestimation of the role of fraud in the ongoing retraction epidemic. The percentage of scientific articles retracted because of fraud has increased ∼10-fold since 1975. Retractions exhibit distinctive temporal and geographic patterns that may reveal underlying causes.

Footnotes

  • Author contributions: F.C.F., R.G.S., and A.C. designed research, performed research, analyzed data, and wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online atwww.pnas.org/lookup/suppl/doi:10.1073/pnas.1212247109/-/DCSupplemental.

    Source:

    http://www.pnas.org/content/early/2012/09/27/1212247109.abstract

     

    Misconduct Widespread in Retracted Science Papers, Study Finds

    By CARL ZIMMER
    Published: October 1, 2012

    Last year the journal Nature reported an alarming increase in the number of retractions of scientific papers — a tenfold rise in the previous decade, to more than 300 a year across the scientific literature.

    Other studies have suggested that most of these retractions resulted from honest errors. But a deeper analysis of retractions, being published this week, challenges that comforting assumption.

    In the new study, published in the Proceedings of the National Academy of Sciences, two scientists and a medical communications consultant analyzed 2,047 retracted papers in the biomedical and life sciences. They found that misconduct was the reason for three-quarters of the retractions for which they could determine the cause.

    “We found that the problem was a lot worse than we thought,” said an author of the study, Dr. Arturo Casadevall of Albert Einstein College of Medicine in the Bronx.

    Dr. Casadevall and another author, Dr. Ferric C. Fang of the University of Washington, have been outspoken critics of the current culture of science. To them, the rising rate of retractions reflects perverse incentives that drive scientists to make sloppy mistakes or even knowingly publish false data.

    “We realized we would really like more hard data for what the reasons were for retractions,” Dr. Fang said.

    They began collaborating with R. Grant Steen, a medical communications consultant in Chapel Hill, N.C., who had already published a study on 10 years of retractions. Together they gathered all the retraction notices published before May 2012 by searching PubMed, a database of scientific literature maintained by the National Library of Medicine.

    “I guess our O.C.D. kicked in and we started trying to look at every paper we could look at,” Dr. Fang said.

    The researchers analyzed the reasons for retractions cited by the scientific journals. But they also looked beyond the journals for the full story.

    In the mid-2000s, for example, Boris Cheskis, then a senior scientist at Wyeth Research, and his colleagues published two papers on estrogen. Later, the scientists retracted both papers, explaining that some of the data in them were “unreliable.” In 2010, the Office of Research Integrity at the federal Department of Health and Human Services ruled that Dr. Cheskis had engaged in misconduct, having falsified the figures.

    Dr. Cheskis settled with the government. Although he neither accepted nor denied the charges, he agreed not to serve on any advisory boards for the United States Public Health Service and agreed to be supervised on any Public Health Service-financed research for two years.

    Neither of the notices for the two retracted papers has been updated to reflect the finding of fraud. Dr. Cheskis could not be reached for comment.

    Dr. Fang and his colleagues dug through other reports from the Office of Research Integrity, as well as newspaper articles and the blog Retraction Watch. All told, they reclassified 158 papers as fraudulent based on their extra research.

    “We haven’t seen this level of analysis before,” said Dr. Ivan Oransky, an author of Retraction Watch and the executive editor at Reuters Health. “It confirms what we suspected.”

    Dr. Oransky said he expected the rise to continue in the near future. He and his co-author, Adam Marcus, have been scrambling to keep up with new cases of fraud.

    In July, for example, the Japanese Society of Anesthesiologists reported that Dr. Yoshitaka Fujii had falsified data in 172 papers. Most of those papers have yet to be officially retracted. “They’re headed for the fraud pile,” Dr. Oransky said.

    Dr. Benjamin G. Druss, a professor of health policy of Emory University, said he found the statistics in the paper to be sound but added that they “need to be kept in perspective.” Only about one in 10,000 papers in PubMed have been officially retracted, he noted. By contrast, 112,908 papers have had published corrections.

    Dr. Casadevall disagreed. “It convinces me more that we have a problem in science,” he said.

    While the fraudulent papers may be relatively few, he went on, their rapid increase is a sign of a winner-take-all culture in which getting a paper published in a major journal can be the difference between heading a lab and facing unemployment. “Some fraction of people are starting to cheat,” he said.

    Better policing techniques, like plagiarism-detecting software, might help slow the rise in misconduct, Dr. Casadevall said, but the most important thing the scientific community can do is change its culture.

    “I don’t think this problem is going to go away as long as you have this disproportionate system of rewards,” he said.

    <nyt_correction_bottom>

    This article has been revised to reflect the following correction:

    Correction: October 1, 2012

     

    An earlier version of this story misstated the federal agency housing the Office of Research Integrity. It is the Department of Health and Human Services, not the National Institutes of Health. The earlier version also misstated the reason cited in the study for three-quarters of the retractions for which researchers could determine the cause. It was misconduct, not fraud. (Fraud or suspected fraud accounted for 41.3 percent of retractions; other forms of misconduct made up the rest.)

    Source:

    http://www.nytimes.com/2012/10/02/science/study-finds-fraud-is-widespread-in-retracted-scientific-papers.html?_r=1 

 

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Molecular pathogen identification comes to the bedside

Reporter:  Larry H Bernstein, MD, FCAP

The developments in molecular diagnostics have been proceeding at a rapid pace.  Naturally it is not surprising that it would reach into clinical microbiology early.  Microbiology and virology have many methods for validation of type of pathogen, and the identification of new pathogens can require delay because of use of a State laboratory.  This will be less an issue with the consolidation of regional facilities and associated laboratories.

I present an example of point-of-care technology from the University of California, Davis developed by Gerald Kost and colleagues with UC Lawrence Livermore National Point-of-Care Technologies Center .

Tran NK, Wisner DH, Albertson TE, Cohen S, et al.  Multiplex polymerase chain reaction pathogen detection in patients with suspected septicemia after trauma, emergency, and burn surgery. Surgery 2012 Mar;151(3):456-63. Epub 2011 Oct 5.  nktran@ucdavis.edu

The goal of the study:  to determine the clinical value of multiplex polymerase chain reaction (PCR) study for enhancing pathogen detection in patients with suspected septicemia after trauma, emergency, and burn surgery.

Finding: PCR-based pathogen detection quickly reveals occult bloodstream infections in these high-risk patients and may accelerate the initiation of targeted antimicrobial therapy.

Type study: a prospective observational study

Population:  30 trauma and emergency surgery patients compared to 20 burn patients.

Method:  Whole- routine blood cultures (BCs) were tested using a new multiplex, PCR-based, pathogen detection system. PCR results were compared to culture data.

Arbitrated Case Review

Arbitrated case review was performed by a medical intensivist, 3 trauma surgeons, 3 burn surgeons, 1 microbiologist, and an infectious disease physician to determine antimicrobial adequacy based on paired PCR/BC results. The arbitrated case review process is adapted from a previous study. Physicians were first presented cases with only BC results. Cases were then represented with PCR results included.

Results:

  • PCR detected rapidly more pathogens than culture methods.
  • Acute Physiology and Chronic Health Evaluation II (APACHE II), Sequential Organ Failure Assessment (SOFA), and Multiple Organ Dysfunction (MODS) scores were greater in PCR-positive versus PCR-negative trauma and emergency surgery patients (P ≤ .033).
  • Negative PCR results (odds ratio, 0.194; 95% confidence interval, 0.045-0.840; P = .028) acted as an independent predictor of survival for the combined surgical patient population.

CONCLUSION:

  • PCR results were reported faster than blood culture results.
  • Severity scores were significantly greater in PCR-positive trauma and emergency surgery patients.
  • The lack of pathogen DNA as determined by PCR served as a significant predictor of survival in the combined patient population.
  • PCR testing independent of traditional prompts for culturing may have clinical value in burn patients.

NK Tran, et al.  Multiplex Polymerase Chain Reaction Pathogen Detection in Trauma, Emergency, and Burn Surgery Patients with Suspected Septicemia.  Surgery. 2012 March; 151(3): 456–463. PMID: 21975287 [PubMed – indexed for MEDLINE] PMCID: PMC3304499 On-line 2011 October 5.
doi:  10.1016/j.surg.2011.07.030
PMCID: PMC3304499.  NIHMSID: NIHMS288960

Plymerase chain reaction, PCR

Plymerase chain reaction, PCR (Photo credit: Wikipedia)

 

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