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Lesson 1 & 2 Cell Signaling & Motility: Lessons, Curations and Articles of reference as supplemental information: #TUBiol3373

Curator: Stephen J. Williams, Ph.D.

UPDATED 2/05/2019

Syllabus for Cell Signaling & Motility for 2019

CELL SIGNALING AND MOTILITY (BIOL 3373)

SPRING 2017

Lectures:

Monday 5:00 PM – 8:00 PM

Biology Life Sciences, Room 342

Instructor:

Antonio Giordano, M.D., Ph.D.

Office hours: Biology Life Sciences Building, Room 431.

Friday: 12:00 noon – 2:00 PM. By appointment

(Phone: 215-2049520, or email: giordano@temple.edu).

Prerequisite:

BIO 3096, Cell Structure and Function (Minimum Grade of C- | May not be taken concurrently). 

Description:

The communication among cells is essential for the regulation of the development of an organism and for the control of its physiology and homeostasis. Aberrant cellular signaling events are often associated with human pathological conditions, such as cancer, neurological disorders, cardiovascular diseases and so on. The full characterization of cell signaling systems may provide useful insights into the pathogenesis of several human maladies.

Text:

Molecular Biology of the Cell 6th Edition, Alberts et al. Garland Science. This textbook is available at the Temple Bookstore.

Grading:

The final grade will be based on the score of four examinations that include both group and individuals assignment. Each exam accounts for 25% of the final grade. There will be no make-up tests during the course. If you have a documented medical excuse and you contact me as soon as possible after the emergency, I will arrange a make-up exam. Complaints regarding the grading will not be considered later than two weeks after the test is returned.

Blackboard:

Announcements will be readily posted on Blackboard. It is your responsibility to check Blackboard periodically.

Attendance: Lecture attendance is mandatory. In addition, punctuality is expected.

Disabilities: Students with documented disabilities who need particular accommodation should contact me privately as soon as possible.

Honesty and Civility:

Students must follow the Temple’s Code of Conduct (see http://www.temple.edu/assistance/udc/coc.htm). This Code of Conduct prohibits: 1. Academic dishonesty and impropriety, including plagiarism and cheating. 2. Interfering or attempting to interfere with or disrupting the conduct of classes or any other activity of the University.”

Academic Rights and Responsibilities:

The policy of the University that regulates Student and Faculty Academic Rights and Responsibilities (Policy # 03.70.02) is available at the following web link: http://policies.temple.edu/getdoc.asp?policy_no=03.70.02

This policy sets the parameters for freedom to learn and freedom to teach, which constitute the pillars of academia.

 

SCHEDULE

This schedule is a general outline, which may be eventually modified. Changes will be announced in advance. Please, always check Blackboard and your email.

Date Topic
Jan 14 Introduction (course overview  and discussion of syllabus). General concepts: Eukaryotic and prokaryotic cell; DNA, RNA  and proteins: Protein synthesis
Jan 21 Martin Luther King, Jr. Day (no classes held)
Jan 28 DNA analysis, RNA analysis; Proteins analysis; Microscopy.
Feb 4 Signaling: general concepts; Introduction to G-proteins; signaling via G-proteins (1)
Feb 11 Exam 1: In class presentation (group assignment)
Feb 18 Signaling via G-proteins (2); tyrosine kinase receptors signaling; Ras-MAPK pathway.
Feb 25 Exam 2: In class presentation (group assignment)
March 4- 10 Spring break
Mar 11

 

Cytoskeleton:  Intermediate filaments; actin
Mar 18 Cytoskeleton: actin binding proteins; microtubules
Mar 25

 

Cytoskeleton: microtubules
April 1

 

Exam 3: in class Multiple choice questions (individual assignment)
Apr 8 Extracellular matrix; cell adhesion; coordinated polarization.
Apr  15 Cell motility and Wnt Signal Signaling. 
Apr  22 Medical consequences of aberrant signaling pathways; production of small molecules for protein kinases In cancer therapy.
Study days
May 6 Exam 4: In class presentation (group assignment)

 

Below is Powerpoint presentations for Lesson 1 and Lesson 2.  Please check for UPDATES on this page for additional supplemental information for these Lessons including articles from this Online Access Journal

 

cell signaling and motility 1 lesson

 

cell signaling and motility 2 lesson

The following articles and curations discuss about the new paradigm how we now envision DNA, in particular how we now understand that the important parts of the genome are not just the exons which code for proteins but also the intronic DNA, which contains all the regulatory elements such as promoters, lnDNA, miRNA sequences etc.  These are good reads for your presentations.

The Search for the Genetic Code

Junk DNA codes for valuable miRNAs

 

And on How the Cell Creates Diversity post the Genetic Code by Use of Post Translational Modifications to Bring Diversity to Protein Structure/Function

Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

Also there is a link to a Blood article using FISH to detect gene amplifications after Gleevec resistance onset here

Novel Mechanisms of Resistance to Novel Agents

Other Articles related to the #TUBiol3373 course include:

Lesson 9 Cell Signaling: Curations and Articles of reference as supplemental information for lecture section on WNTs: #TUBioll3373

Curation of selected topics and articles on Role of G-Protein Coupled Receptors in Chronic Disease as supplemental information for #TUBiol3373

 

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The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

 

On 9/25/2017, Aviva Lev-Ari, PhD, RN commissioned Dr. Larry H. Bernstein to write a short article on the following topic reported on 9/22/2017 in sciencemission.com

 

We are publishing, below the new article created by Larry H. Bernstein, MD, FCAP.

 

Background

During the period between 9/2015  and 6/2017 the Team at Leaders in Pharmaceutical Business Intelligence (LPBI)  has launched an R&D effort lead by Aviva Lev-Ari, PhD, RN in conjunction with SBH Sciences, Inc. headed by Dr. Raphael Nir.

This effort, also known as, “DrugDiscovery @LPBI Group”  has yielded several publications on EXOSOMES on this Open Access Online Scientific Journal. Among them are included the following:

 

QIAGEN – International Leader in NGS and RNA Sequencing, 10/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

cell-free DNA (cfDNA) tests could become the ultimate “Molecular Stethoscope” that opens up a whole new way of practicing Medicine, 09/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

Detecting Multiple Types of Cancer With a Single Blood Test (Human Exomes Galore), 07/02/2017

Reporter and Curator: Irina Robu, PhD

 

Exosomes: Natural Carriers for siRNA Delivery, 04/24/2017

Reporter: Aviva Lev-Ari, PhD, RN

 

One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI, 01/05/2017

Curator: Marzan Khan, B.Sc

 

SBI’s Exosome Research Technologies, 12/29/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

A novel 5-gene pancreatic adenocarcinoma classifier: Meta-analysis of transcriptome data – Clinical Genomics Research @BIDMC, 12/28/2016

Curator: Tilda Barliya, PhD

 

Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute, Micro and Nanotechnology Lab, 12/28/2016

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN

 

Exosomes – History and Promise, 04/28/2016

Reporter: Aviva Lev-Ari, PhD, RN

 

Exosomes, 11/17/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Liquid Biopsy Assay May Predict Drug Resistance, 11/16/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Glypican-1 identifies cancer exosomes, 10/31/2015

Curator: Larry H. Bernstein, MD, FCAP

 

Circulating Biomarkers World Congress, March 23-24, 2015, Boston: Exosomes, Microvesicles, Circulating DNA, Circulating RNA, Circulating Tumor Cells, Sample Preparation, 03/24/2015

Reporter: Aviva Lev-Ari, PhD, RN

 

Cambridge Healthtech Institute’s Second Annual Exosomes and Microvesicles as Biomarkers and Diagnostics Conference, March 16-17, 2015 in Cambridge, MA, 03/17, 2015

Reporter: Aviva Lev-Ari, PhD, RN

 

The newly created think-piece on the relationship between regulatory functions of Exosomes and Metabolic processes is developed conceptually, below.

 

The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

We have had more than a half century of research into the genetic code and transcription leading to abundant work on RNA and proteomics. However, more recent work in the last two decades has identified RNA interference in siRNA. These molecules may be found in the circulation, but it has been a challenge to find their use in therapeutics. Exosomes were first discovered in the 1980s, but only recently there has been a huge amount of research into their origin, structure and function. Exosomes are 30–120 nm endocytic membrane-bound extracellular vesicles (EVs)(1-23) , and more specifically multiple vesicle bodies (MVBs) by a budding process from invagination of the outer cell membrane that carry microRNA (miRNA), and have structures composed of protein and lipids (1,23-27 ). EVs are the membrane vesicles secreted by eukaryotic cells for intracellular communication by transferring the proteins, lipids, and RNA under various physiologic conditions as well as during the disease stage. EVs also act as a signalosomes in many biological processes. Inward budding of the plasma membrane forms small vesicles that fuse. Intraluminal vesicles (ILVs) are formed by invagination of the limiting endosomal membrane during the maturation process of early endosome.

EVs are the MVBs secreted that serve in intracellular communication by transferring a cargo consisting of proteins, lipids, and RNA under various physiologic conditions (4, 23). Exosome-mediated miRNA transfer between cells is considered to be necessary for intercellular signaling and exosome-associated miRNAs in biofluids (23). Exosomes carry various molecular constituents of their cell of origin, including proteins, lipids, mRNAs, and microRNAs (miRNAs) (. They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, hydrothoracic fluid, and ascitic fluid, as well as in culture medium of most cell types.Exosomes have also been shown to be involved in noncoding RNA surveillance machinery in generating antibody diversity (24). There are also a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) that accumulate R-loop structures upon RNA exosome ablation, thereby, resolving deleterious DNA/RNA hybrids arising from active enhancers and distal divergent eRNA-expressing elements (lncRNA-CSR) engaged in long-range DNA interactions (25). RNA exosomes are large multimeric 3′-5′ exo- and endonucleases representing the central RNA 3′-end processing factor and are implicated in processing, quality control, and turnover of both coding and noncoding RNAs. They are large macromolecular cages that channel RNA to the ribonuclease sites (29). A major interest has been developed to characterize of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids (1). Moreover, exosomes play an active role in tumorigenesis, metastasis, and response to therapy through the transfer of oncogenes and onco-miRNAs between cancer cells and the tumor stroma. Blood cells and the vascular endothelium is also exosomal shedding, which has significance for cardiovascular,   neurologicological disorders, stroke, and antiphospholipid syndrome (1). Dysregulation of microRNAs and the affected pathways is seen in numerous pathologies their expression can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers (30).

Exosomes are secreted by many cells like B lymphocytes and dendritic cells of hematopoietic and non-hematopoietic origin viz. platelets, Schwann cells, neurons, mast cells, cytotoxic T cells, oligodendrocytes, intestinal epithelial cells were also found to be releasing exosomes (4). They are engaged in complex functions like persuading immune response as the exosomes secreted by antigen presenting cells activate T cells (4). They all have a common set of proteins e.g. Rab family of GTPases, Alix and ESCRT (required for transport) protein and they maintain their cytoskeleton dynamics and participate in membrane fusion. However, they are involved in retrovirus disease pathology as a result of recruitment of the host`s endosomal compartments in order to generate viral vesicles, and they can either spread or limit an infection based on the type of pathogen and its target cells (5).

Upon further consideration, it is understandable how this growing biological work on exosomes has enormous significance for laboratory diagnostics (1, 3, 5, 6, 11, 14, 15, 17-20, 23,30-41) . They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, thoracic and abdominal effusions, and ascitic fluid (1). The involvement of exosomes in disease is broad, and includes: cancer, autoimmune and infectious disease, hematologic disorders, neurodegenerative diseases, and cardiovascular disease. Proteins frequently identified in exosomes include membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), heat shock proteins (e.g., HSC70), tetraspanins (e.g., CD9, CD63, and CD81), MVB biogenesis proteins (e.g., alix and TSG101), and lipid-related proteins and phospholipases. The exosomal lipid composition has been thoroughly analyzed in exosomes secreted from several cell types including DCs and mast cells, reticulocytes, and B-lymphocytes (1). Dysregulation of microRNAs of pathways observed in numerous pathologies (5, 10, 12, 21, 27, 35, 37) including cancers (30), particularly, colon, pancreas, breast, liver, brain, lung (2, 6, 17-20, 30, 33-36, 38, 39). Following these considerations, it is important that we characterize the content of exosomal cargo to gain clues to their biogenesis, targeting, and cellular effects which may lead to identification of biomarkers for disease diagnosis, prognosis and response to treatment (42).

We might continue in pursuit of a particular noteworthy exosome, the NLRP3 inflammasome, which is activated by a variety of external or host-derived stimuli, thereby, initiating an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1b maturation and secretion (43).
Inflammasomes are multi-protein signaling complexes that activate the inflammatory caspases and the maturation of interleukin-1b. The NLRP3 inflammasome is linked with human autoinflammatory and autoimmune diseases (44). This makes the NLRP3 inflammasome a promising target for anti-inflammatory therapies. The NLRP3 inflammasome is activated in response to a variety of signals that indicate tissue damage, metabolic stress, and infection (45). Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Heritable and acquired inflammatory diseases are both characterized by dysregulation of NLRP3 inflammasome activation (45).
Receptors of innate immunity recognize conserved moieties associated with either cellular damage [danger-associated molecular patterns (DAMPs)] or invading organisms [pathogen-associated molecular patterns (PAMPs)](45). Either chronic stimulation or overwhelming tissue damage is injurious and responsible for the pathology seen in a number of autoinflammatory and autoimmune disorders, such as arthritis and diabetes. The nucleotide-binding domain leucine-rich repeat (LRR)-containing receptors (NLRs) are PRRs are found intracellularly and they share a unique domain architecture. It consists of a central nucleotide binding and oligomerization domain called the NACHT domain that is located between an N-terminal effector domain and a C-terminal LRR domain (45). The NLR family members NLRP1, NLRP3, and NLRC4 are capable of forming multiprotein complexes called inflammasomes when activated.

The (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease because the activation results, in pro-IL-1β processing and the secretion of the proinflammatory cytokine IL-1β (46). It has been proposed that Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma (46). First, NLRP3 interacts with the adaptor protein ASC by sensing microbial pathogens and self-danger signals. Then pro-caspase-1 is recruited and the large protein complex called the NLRP3 inflammasome is formed. This is followed by autocleavage and activation of caspase-1, after which pro-IL-1β and pro-IL-18 are converted into their mature forms. Ion fluxes disrupt membrane integrity, and also mitochondrial damage both play key roles in NLRP3 inflammasome activation (47). Depletion of mitochondria as well as inhibitors that block mitochondrial respiration and ROS production prevented NLRP3 inflammasome activation. Futhermore, genetic ablation of VDAC channels (namely VDAC1 and VDAC3) that are located on the mitochondrial outer membrane and that are responsible for exchanging ions and metabolites with the cytoplasm, leads to diminished mitochondrial (mt) ROS production and inhibition of NLRP3 inflammasome activation (47). Inflammasome activation not only occurs in immune cells, primarily macrophages and dendritic cells, but also in kidney cells, specifically the renal tubular epithelium. The NLRP3 inflammasome is probably involved in the pathogenesis of acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal-related nephropathy (48). The inflammasome also plays a role in autoimmune kidney disease. IL-1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and β-hydroxybutyrate, may prove to have value in the treatment of inflammasome-mediated conditions.

Autophagosomes derived from tumor cells are referred to as defective ribosomal products in blebs (DRibbles). DRibbles mediate tumor regression by stimulating potent T-cell responses and, thus, have been used as therapeutic cancer vaccines in multiple preclinical cancer models (49). It has been found that DRibbles could induce a rapid differentiation of monocytes and DC precursor (pre-DC) cells into functional APCs (49). Consequently, DRibbles could potentially induce strong innate immune responses via multiple pattern recognition receptors. This explains why DRibbles might be excellent antigen carriers to induce adaptive immune responses to both tumor cells and viruses. This suggests that isolated autophagosomes (DRibbles) from antigen donor cells activate inflammasomes by providing the necessary signals required for IL-1β production.

The Hsp90 system is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function (50). The co-chaperones enable Hsp90 to chaperone structurally and functionally diverse client proteins. Sahasrabudhe et al. (50) show that the nature of the client protein dictates the contribution of a co-chaperone to its maturation. The study reveals the general importance of the cochaperone Sgt1 (50). In addition to Hsp90, we have to consider Hsp60. Adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins (21).

A new Protein Organic Solvent Precipitation (PROSPR) method efficiently isolates the EV repertoire from human biological samples. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes. In addition lipidomic characterization of enriched CNS vesicles identified previously reported EV-specific lipid families and novel lipid isoforms not previously detected in human EVs. The characterization of these structures from central nervous system (CNS) tissues is relevant to current neuroscience, especially to advance the understanding of neurodegeneration in amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD)(15). In addition, study of EVs in brain will enable characterization of the degenerative posttranslational modifications (DPMs) occurring in those proteins.
Neurodegenerative disease is characterized by dysregulation because of NLRP3 inflammasome activation. Alzheimer’s disease (AD) and Parkinson’s disease (PD), both neurodegenerative diseases are associated with the NLRP3 inflammasome. PD is characterized by accumulation of Lewy bodies (LB) formed by a-synuclein (aSyn) aggregation. A recent study revealed that aSyn induces synthesis of pro-IL-1b by an interaction with TLR2 and activates NLRP3 inflammasome resulting in caspase-1 activation and IL-1b maturation in human primary monocytes (43). In addition mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals. It is notable that in this aberrant activation mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals (43).

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  46. Hirota JA, Im H, Rahman MM, Rumzhum NN, Manetsch M, Pascoe CD, Bunge K, Alkhouri H, Oliver BG, Ammit AJ. The nucleotide-binding domain and leucine-rich repeat protein-3 inflammasome is not activated in airway smooth muscle upon toll-like receptor-2 ligation. Am J Respir Cell Mol Biol. 2013 Oct; 49(4):517-24. doi: 10.1165/rcmb.2013-0047OC.
  47. Zhong Z, Sanchez-Lopez E, Karin M. Autophagy, NLRP3 inflammasome and auto-inflammatory immune diseases. Clin Exp Rheumatol. 2016 Jul-Aug; 34(4 Suppl 98):12-6. Epub 2016 Jul 21.
  48. Hutton HL, Ooi JD, Holdsworth SR, Kitching AR. The NLRP3 inflammasome in kidney disease and autoimmunity. Nephrology (Carlton). 2016 Sep; 21(9):736-44. doi: 10.1111/nep.12785
  49. Xing Y, Cao R and Hu H-M. TLR and NLRP3 inflammasome-dependent innate immune responses to tumor-derived autophagosomes (DRibbles). Cell Death and Disease (2016) 7, e2322; doi:10.1038/cddis.2016.206
  50. Sahasrabudhe P, Rohrberg J, Biebl MM, Rutz DA, Buchner J. The Plasticity of the Hsp90 Co-chaperone System. Molecular Cell 2017 Sept; 67:947–961. http://dx.doi.org/10.1016/j.molcel.2017.08.004

 

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RNA Testing

Curator: Larry H. Bernstein, MD, FCAP

LPBI

 

RNA Testing: A Fast, Accurate Tool for Diagnosing Autoimmune Disease

05/17/2016 –   Dr. Chase Spurlock, IQuity CEO and Dr. Thomas Aune, IQuity Chief Science Officer
http://www.dddmag.com/articles/2016/05/rna-testing-fast-accurate-tool-diagnosing-autoimmune-disease

Diagnosing complex autoimmune diseases and related conditions is a challenging endeavor, as these multifaceted conditions often present with non-specific symptoms across several organ systems. Historically, physicians have diagnosed autoimmune disease through detailed physical examination, collection of medical and family history, multiple laboratory tests and monitoring symptoms over time. Current diagnostic algorithms can lead to a protracted and costly diagnostic process of elimination.

However, emerging RNA-based diagnostic technologies can dramatically reduce the time to diagnosis of complex diseases, including autoimmune disease. With the completion of the Human Genome Project and basic research in the field of RNA biology over the past two decades, it is now accepted that the majority of the human genome is transcribed into unique patterns of information that exhibit cell type specificity and are consistent in a variety of chronic conditions, including autoimmune disease. With the invention of new technologies, including next-generation RNA sequencing, we are able to assess global changes in gene expression.

As such, RNA-based tools are now poised to provide physicians with actionable information early in the diagnostic process. While RNA-based testing is a relatively new innovation in the clinical setting and widespread adoption efforts are still in their infancy, the science behind these techniques makes them a reliable method for helping physicians make fast, accurate decisions.

The science behind RNA

Analysis of RNA expression paints a molecular portrait of what’s going on in an individual’s cells at a given point in time. This can provide a picture of disease manifestation. At a high level, current RNA testing involves collecting a patient’s blood sample via standard venipuncture, isolating the RNA and then detecting RNA expression patterns in real time using polymerase chain reaction (PCR). The turning on or off of these expression patterns can serve as an indicator of the presence or absence of disease.

In contrast, DNA tests examine changes in nucleotide sequence to establish a patient’s risk of developing a particular condition. These genotype associations do not always reveal information about disease manifestation. A DNA test, for example, might indicate that a patient is at high risk for an autoimmune disorder, such as rheumatoid arthritis (RA) or multiple sclerosis (MS), but the patient may never develop the condition. Just because the patient has a particular DNA risk marker does not mean he or she actually has—or will have—the disease. Thus, a major limitation with DNA methodology is its inability to reliably forecast active disease. RNA tests are more specific in this regard.

Other types of testing, such as pharmacologic and serologic testing, have also been used to detect autoimmune disorders. However, due to the complexity of these diseases, a single test can only put a physician one small step closer to diagnosis and additional tests are often required. For instance, to accurately identify RA, a clinician may need to perform approximately 10 to 15 different molecular tests over what is often a lengthy time period. If the doctor concludes that a portion of these tests point to the presence of RA, then he or she may be able to make a diagnosis of RA. In some cases, it can take years for the patient to receive a definitive diagnosis. In comparison, new RNA-based diagnostic testing for the same condition offers an accuracy of greater than 90 percent, enabling a higher degree of certainty for establishing the presence or absence of the disease.

The impact on patients, providers and the scientific community

As RNA testing becomes more widespread, its impact on patients, providers and other stakeholders promises to be significant. Once widely adopted, this kind of testing could cut down on the number of tests and amount of the time needed to confirm diagnosis. This, in turn, would enhance the patient experience, as individuals would no longer have to undergo repeated or invasive testing to determine if they have a disease.

Current autoimmune therapies are reasonably effective at slowing disease advancement.  Across many autoimmune diseases, the best outcomes are achieved when therapies are initiated early in the disease process. Providing physicians with actionable diagnostic information enables the provider to place patients on the appropriate treatments faster, limiting disease progression and supporting better long-term health outcomes.

RNA testing can improve outcomes for providers by helping to get patients on the path to suitable treatment earlier. This not only facilitates optimal medication performance, but also lessens the cost of diagnosis and management of disease.  In turn, this ensures a better quality of life for patients.

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RNA in synthetic biology

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

RNA May Surpass DNA in Precision Medicine

http://www.genengnews.com/gen-news-highlights/rna-may-surpass-dna-in-precision-medicine/81252507/

 

Scientists based at the Translational Genomics Research Institute have published a review heralding the promise of RNA sequencing (RNA-seq) for precision medicine. The scientists also note that progress will be needed on analytical, bioinformatics, and regulatory fronts, particularly in light of the transcriptome’s variety, dynamism, and wealth of detail. In this image, one aspect of RNA-seq is shown, the alignment with intron-split short reads. It reflects the alignment of mRNA sequence obtained via high-throughput sequencing and the expected behavior of the alignment to the reference genome when the read falls in an exon–exon junction. [Rgocs, Wikipedia]
http://www.genengnews.com/Media/images/GENHighlight/thumb_Mar22_2016_Rgocs_RNASeqAlignment1872484040.jpg

 

It’s not an either/or situation. Both DNA sequencing and RNA sequencing hold clinical promise—diagnostically, prognostically, and therapeutically. It must be said, however, that RNA sequencing reflects the dynamic nature of gene expression, shifting with the vagaries of health and disease. Also, RNA sequencing captures more biochemical complexity, in the sense that it allows for the detection of a wide variety of RNA species, including mRNA, noncoding RNA, pathogen RNA, chimeric gene fusions, transcript isoforms, and splice variants, and provides the capability to quantify known, predefined RNA species and rare RNA transcript variants within a sample.

All these potential advantages were cited in a paper that appeared March 21 in Nature Reviews Genetics, in an article entitled, “Translating RNA Sequencing into Clinical Diagnostics: Opportunities and Challenges.” The paper, contributed by scientists based at the Translational Genomics Research Institute (TGen), was definitely optimistic about the clinical utility of RNA sequencing, but it also highlighted the advances that would have to occur if RNA sequencing is to achieve its promise.

In general, the very things that make RNA sequencing so interesting are the same things that make it so challenging. RNA sequencing would take the measure of a world—the transcriptome—that is incredibly rich. To capture all the relevant subtleties of the transcriptome, scientists will have to develop sensitive, precise, and trustworthy analytical techniques. What’s more, scientists will need to find efficient and reliable means of processing and interpreting all of the transcriptome data they will collect. Finally, they will need to continue integrating RNA-based knowledge with DNA-based knowledge. That is, RNA sequencing results can be used to guide the interpretation of DNA sequencing results.

In their Nature Reviews Genetics paper, the TGen scientists review the state of RNA sequencing and offer specific recommendations to enhance its clinical utility. The TGen scientists make a special point about the promise held by extracellular RNA (exRNA). Because exRNA can be monitored by simply taking a blood sample, as opposed to taking a tumor biopsy, it could serve as a noninvasive diagnostic indicator of disease.

“Detection of gene fusions and differential expression of known disease-causing transcripts by RNA-seq represent some of the most immediate opportunities,” wrote the authors. “However, it is the diversity of RNA species detected through RNA-seq that holds new promise for the multi-faceted clinical applicability of RNA-based measures, including the potential of extracellular RNAs as non-invasive diagnostic indicators of disease.”

The first test measuring exRNA was released earlier this year, the paper said, for use measuring specific exRNAs in lung cancer patients. And, the potential for using RNA-seq in cancer is expanding rapidly. Commercial RNA-seq tests are now available, and they provide the opportunity for clinicians to profile cancer more comprehensively and use this information to guide treatment selection for their patients.

In addition, the authors reported on several recent applications for RNA-seq in the diagnosis and management of infectious diseases, such as monitoring for drug-resistant populations during therapy and tracking the origin and spread of the Ebola virus.

Despite these advances, the authors also sounded a few cautionary notes. “There are currently few agreed upon methods for isolation or quantitative measurements and a current lack of quality controls that can be used to test platform accuracy and sample preparation quality,” they wrote. “Analytical, bioinformatics, and regulatory challenges exist, and ongoing efforts toward the establishment of benchmark standards, assay optimization for clinical conditions and demonstration of assay reproducibility are required to expand the clinical utility of RNA-seq.”

Overall, the authors remain hopeful that precision medicine will embrace RNA sequencing. For example, lead author Sara Byron, research assistant professor in TGen’s Center for Translational Innovation, said, “RNA is a dynamic and diverse biomolecule with an essential role in numerous biological processes. From a molecular diagnostic standpoint, RNA-based measurements have the potential for broad application across diverse areas of human health, including disease diagnosis, prognosis, and therapeutic selection.”

 

RNA Bacteriophages May Open New Path to Fighting Antibiotic-Resistant Infections

http://www.genengnews.com/gen-news-highlights/rna-bacteriophages-may-open-new-path-to-fighting-antibiotic-resistant-infections/81252521/

http://www.genengnews.com/Media/images/GENHighlight/thumb_Mar25_2016_Wikimedia_RNABacteriophages2091791481.jpg

Micrograph image of RNA bacteriophages attached to part of the bacterium E. coli. A new study at Washington University School of Medicine in St. Louis suggests that bacteriophages made of RNA, a close chemical cousin of DNA, likely play a much larger role in shaping the bacterial makeup of worldwide habitats than previously recognized. [Graham Beards/Wikimedia]

Scientists at Washington University School of Medicine in St. Louis report that bacteriophages made of RNA likely play a much larger role in shaping the bacterial makeup of worldwide habitats than previously recognized. Their study (“Hyperexpansion of RNA Bacteriophage Diversity”), published in PLOS Biology, identified 122 new types of RNA bacteriophages in diverse ecological niches, providing an opportunity for scientists to define their contributions to ecology and potentially to exploit them as novel tools to fight bacterial infections, particularly those that are resistant to antibiotics.

“Lots of DNA bacteriophages have been identified, but there’s an incredible lack of understanding about RNA bacteriophages,” explained senior author David Wang, Ph.D., associate professor of molecular microbiology. “They have been largely ignored—relatively few were known to exist, and for the most part, scientists haven’t bothered to look for them. This study puts RNA bacteriophages on the map and opens many new avenues of exploration.”

Dr. Wang estimates that of the more than 1500 bacteriophages that have been identified, 99% of them have DNA genomes. The advent of large-scale genome sequencing has helped scientists identify DNA bacteriophages in the human gut, skin, and blood, as well as in the environment, but few researchers have looked for RNA bacteriophages in those samples (doing so requires that RNA be isolated from the samples and then converted back to DNA before sequencing).

As part of the new study, first author and graduate student Siddharth Krishnamurthy, and the team, including Dan Barouch, M.D., Ph.D., of Beth Israel Deaconess Medical Center and Harvard Medical School, identified RNA bacteriophages by analyzing data from samples taken from the environment, such as oceans, sewage, and soils, and from aquatic invertebrates including crabs, sponges, and barnacles, as well as insects, mice, and rhesus macaques.

RNA bacteriophages have been shown to infect Gram-negative bacteria, which have become increasingly resistant to antibiotics and are the source of many infections in health care settings. But the researchers also showed for the first time that these bacteriophages also may infect Gram-positive bacteria, which are responsible for strep and staph infections as well as MRSA (methicillin-resistant Staphylococcus aureus).

“What we know about RNA bacteriophages in any environment is limited,” Dr. Wang said. “But you can think of bacteriophages and bacteria as having a predator–prey relationship. We need to understand the dynamics of that relationship. Eventually, we’d like to manipulate that dynamic to use phages to selectively kill particular bacteria.”

 

Hyperexpansion of RNA Bacteriophage Diversity

Siddharth R. Krishnamurthy , Andrew B. Janowski , Guoyan Zhao , Dan Barouch
24 Mar 2016 | PLOS Biology   
   http://dx.doi.org:/10.1371/journal.pbio.1002409

Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent.

Bacteriophages (viruses that infect bacteria) can alter biological processes in numerous ecosystems. While there are numerous studies describing the role of bacteriophages with DNA genomes in these processes, the role of bacteriophages with RNA genomes (RNA bacteriophages) is poorly understood. This gap in knowledge is in part because of the limited diversity of known RNA bacteriophages. Here, we begin to address the question by identifying 122 novel RNA bacteriophage partial genome sequences present in metagenomic datasets that are highly divergent from each other and previously described RNA bacteriophages. Additionally, many of these sequences contained novel properties, including novel genes, segmentation, and host range, expanding the frontiers of RNA bacteriophage genomics, evolution, and tropism. These novel RNA bacteriophage sequences were globally distributed from numerous ecological niches, including animal-associated and environmental habitats. These findings will facilitate our understanding of the role of the RNA bacteriophage in microbial communities. Furthermore, there are likely many more unrecognized RNA bacteriophages that remain to be discovered.

 

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RNA Modification

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New RNA Modification Added to Epitranscriptomic Library   

GEN News Highlights  Feb 17, 2016    http://www.genengnews.com/gen-news-highlights/new-rna-modification-added-to-epitranscriptomic-library/81252376/

 

http://www.genengnews.com/Media/images/GENHighlight/109124_web3461772372.jpg

 

In 1956, Francis Crick—co-discoverer of DNA’s helical structure—postulated what is now considered to be a central doctrine of the biological sciences stating that “The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.” What Crick was suggesting was that DNA makes RNA and, in turn, RNA makes protein.

In the time since the initial proposal of the central dogma, scientists have come to understand that there are not only instances of reverse information flow from RNA to DNA, but chemical alterations to RNA structures that can have a profound effect on gene regulation. The discovery of these alterations has added a critical dimension to how scientists view the genetic code and recently spawned an entirely new field of study within molecular biology: the epitranscriptome.

Now, a recent study by scientists at the University of Chicago and Tel Aviv University has revealed evidence that provides a promising new lever in the control of gene expression. The researchers describe a small chemical modification to RNA that can significantly boost the conversion of genes to proteins.

The findings from this study were published recently in Nature through an article entitled “The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA.”

“Epigenetics, the regulation of gene expression beyond the primary information encoded by DNA, was thought until recently to be mediated by modifications of proteins and DNA,” explained co-senior study author Gidi Rechavi, Ph.D., chair in oncology at Tel Aviv University’s Sackler Faculty of Medicine and head of the Cancer Research Center at Sheba Medical Center. “The new findings bring RNA to a central position in epigenetics.”

“This discovery further opens the window on a whole new world of biology for us to explore,” added co-senior study author Chuan He, Ph.D., professor in the department of chemistry and investigator within the Howard Hughes Medical Institute at the University of Chicago. “These modifications have a major impact on almost every biological process.”

Previously, Dr. He’s laboratory discovered the first RNA demethylase that reverses the most prevalent mRNA methylation N6-methyladenosine (m6A), implying that the addition and removal of the methyl group could dramatically affect these messengers and the outcome of gene expression—as also seen for DNA and histones—which subsequent research found to be true.

In the current study, the investigators described a second functional mRNA methylation, N1-methyladenosine (m1A). Like m6A, the small modification is evolutionarily conserved and common, present in humans, rodents, and yeast. However, its location and effect on gene expression reflect a new form of epitranscriptome control.

“The discovery of m1A is extremely important, not only because of its own potential in affecting biological processes but also because it validates the hypothesis that there is not just one functional modification,” Dr. He stated. “There could be multiple modifications at different sites where each may carry a distinct message to control the fate and function of mRNA.”

From their findings, the research team estimates that that m1A may be present on transcripts of more than one out of three expressed human genes—suggesting that m1A, like m6A, may be a mechanism by which cells rapidly boost the expression of hundreds or thousands of specific genes.

“mRNA is the perfect place to regulate gene expression because they can code information from transcription and directly impact translation—you can add a consensus sequence to a group of genes and use a modification of the sequence to readily control several hundred transcripts simultaneously,” Dr. He said. “If you want to rapidly change the expression of several hundred or a thousand genes, this offers the best way.”

The scientists were excited by their findings and have plans for future studies that will examine the role of m1A methylation in human development, for diseases such as diabetes and cancer, and its potential as a target for therapeutic uses.

“This study represents a breakthrough discovery in the exciting, nascent field of the ‘epitranscriptome,’ which is how RNAs are regulated, akin to the genome and the epigenome,” commented Christopher Mason, Ph.D., associate professor at Weill Cornell Medicine, who was not affiliated with the study. “What is important about this work is that m6A was recently found to enrich at the ends of genes, and now we know that m1A is what is helping regulate the beginning of genes, and this opens up many questions about revealing the ‘epitranscriptome code’ just like the histone code or the genetic code.”

 

The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA

Dan DominissiniSigrid NachtergaeleSharon Moshitch-MoshkovitzNitzan Kol, et al.
Nature(2016 10 Feb )      http://dx.doi.org:/10.1038/nature16998      http://www.nature.com/nature/journal/vaop/ncurrent/full/nature16998.html

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N6-methyladenosine (m6A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N1-methyladenosine (m1A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m1A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m1A in promoting translation of methylated mRNA.

 

Figure 1: Development of m1A-seq to map a newly identified constituent of mammalian mRNA.

Development of m1A-seq to map a newly identified constituent of mammalian mRNA.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16998-f1.jpg

a, Chemical structures of m1A and m6A. Methyl groups (-CH3) are in red and the positive charge (+) on m1A is in blue. b, LC-MS/MS quantitation of m1A, m6A and Ψ in human and mouse mRNA isolated from the indicated cell types. …

 

Figure 3: m1A occurs in GC-rich sequence contexts and in genes with structured 5′ UTRs.

m1A occurs in GC-rich sequence contexts and in genes with structured 5′ UTRs.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16998-f3.jpg

a, Sequence frequency logo for a set of 192 adenosines in peak areas that have a higher mismatch rate in immunoprecipitation relative to input (FC ≥ 6) in HepG2 demonstrates the GC-rich context of m1A. b, Length-adjusted minimum free energy…

 

Figure 5: m1A in mRNA is a dynamic modification that responds to changing physiological and stress conditions, and varies between tissues.

m1A in mRNA is a dynamic modification that responds to changing physiological and stress conditions, and varies between tissues.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16998-f5.jpg

a, LC-MS/MS quantification of m1A (left, grey) and m6A (right, black) in mRNA of untreated and glucose-starved (upper panels) or heat shock-treated (lower panels) HepG2 cells, presented as percentage of unmodified A. Mean values ± s.e.m…

 

RNA modification discovery suggests new code for control of gene expression

A new cellular signal discovered by a team of scientists at the University of Chicago and Tel Aviv University provides a promising new lever in the control of gene expression.    Gene expression study

The study, published online Feb. 10 in the journal Nature, describes a small chemical modification that can significantly boost the conversion of genes to proteins. Together with other recent findings, the discovery enriches a critical new dimension to the “Central Dogma” of molecular biology: the epitranscriptome.

“This discovery further opens the window on a whole new world of biology for us to explore,” said Chuan He, the John T. Wilson Distinguished Service Professor in Chemistry, Howard Hughes Medical Institute investigator and senior author of the study. “These modifications have a major impact on almost every biological process.”

The central dogma of molecular biology describes the cellular pathway where genetic information from DNA is copied into temporary RNA “transcripts,” which provide the recipe for the production of proteins. Since Francis Crick first postulated the theory in 1956, scientists have discovered a multitude of modifications to DNA and proteins that regulate this process.

Only recently, however, have scientists focused on investigating dynamic modifications that specifically target the RNA step. In 2011, He’s group discovered the first RNA demethylase that reverses the most prevalent mRNA methylation N6-methyladenosine, or m6A, implying that the addition and removal of the methyl group could dramatically affect these messengers and impact the outcome of gene expression, as also seen for DNA and histones. Subsequently, scientists discovered that the dynamic and reversible methylation of m6A dramatically controlled the metabolism and function of most cellular messenger RNA, and thus, the production of proteins.

In the new Nature study, researchers from UChicago and Tel Aviv University describe a second functional mRNA methylation, N1-methyladenosine, or m1A. Like m6A, the small modification is evolutionarily conserved and common, and present in humans, rodents and yeast, the authors found. But its location and effect on gene expression reflect a new form of epitranscriptome control and suggest an even larger cellular “control panel.”

“The discovery of m1A is extremely important, not only because of its own potential in affecting biological processes, but also because it validates the hypothesis that there is not just one functional modification,” He said. “There could be multiple modifications at different sites where each may carry a distinct message to control the fate and function of mRNA.”

The researchers estimated that m1A was present on transcripts of more than one out of three expressed human genes. Methylated genes exhibited enhanced translation compared to unmethlyated genes, producing protein levels nearly twice as high in all cell types. This increase suggests that m1A, like m6A, may be a mechanism by which cells rapidly boost the expression of hundreds or thousands of specific genes, perhaps during important processes such as cell division, differentiation or under stress.

“mRNA is the perfect place to regulate gene expression, because they can code information from transcription and directly impact translation; you can add a consensus sequence to a group of genes and use a modification of the sequence to readily control several hundred transcripts simultaneously,” He said. “If you want to rapidly change the expression of several hundred or a thousand genes, this offers the best way.”

However, despite their complementary effects, m1A and m6A exert their influence on mRNA through different pathways. While studies have found that m6A localizes predominantly to the tail of messenger RNA molecules, increasing their translation and turnover rate, m1A was found largely near the start codon of mRNA transcripts, where protein translation begins. The different mechanisms could allow for finer tuning of post-transcriptional gene expression, or the selective activation of particular genes in different physiological situations.

“This study represents a breakthrough discovery in the exciting, nascent field of the ‘epitranscriptome,’ which is how RNAs are regulated, akin to the genome and the epigenome,” said Christopher Mason, associate professor at Weill Cornell Medicine, who was not affiliated with the study. “What is important about this work is that m6A was recently found to enrich at the ends of genes, and now we know that m1A is what is helping regulate the beginning of genes, and this opens up many questions about revealing the ‘epitranscriptome code’ just like the histone code or the genetic code.”

Future studies will examine the role of m1A methylation in human development, diseases such as diabetes and cancer, and its potential as a target for therapeutic uses.


Citation: “The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA,” Nature, Feb. 10, 2016, by Chuan He, Dan Dominissini, Sigrid Nachtergaele, Qing Dai, Dali Han, Wesley Clark, Guanqun Zheng, Tao Pan and Louis Dore from the University of Chicago, and Sharon Moshitch-Moshkovitz, Eyal Peer, Nitkan Kol, Moshe Shay Ben-Haim, Ayelet Di Segni, Mali Salmon-Divon, Oz Solomon, Eran Eyal, Vera Hershkovitz, Ninette Amariglio and Gideon Rechavi from Tel Aviv University. DOI: 10.1038/nature16998

Funding: National Institutes of Health, Howard Hughes Medical Institute, Flight Attendant Medical Research Institute, Israel Science Foundation, Israeli Centers of Excellence Program, Ernest and Bonnie Beutler Research Program, Chicago Biomedical Consortium, Damon Runyon Cancer Research Foundation and Kahn Family Foundation.

– See more at: http://news.uchicago.edu/article/2016/02/16/rna-modification-discovery-suggests-new-code-control-gene-expression#sthash.HX6wUgKW.dpuf

RNA modifications and epitranscriptomics conference   
University of Chicago, Chicago, Illinois, US   September 8-9, 2016
The meeting is aimed at bringing in students and postdocs as well as faculty involved in RNA modification and epitranscriptome research.  In addition to talks, there will be a poster session and reception.

Topics

  • M6A mRNA methylation
  • Biological functions of m6A RNA methylation
  • Dynamic RNA modifications

Registration will open on March 1, 2016

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From GEN News Highlights

Reposted from GEN News

Nov 18, 2015

RNA-Based Drugs Turn CRISPR/Cas9 On and Off

  • This image depicts a conventional CRISPR-Cas9 system. The Cas9 enzyme acts like a wrench, and specific RNA guides act as different socket heads. Conventional CRISPR-Cas9 systems act continuously, raising the risk of off-target effects. But CRISPR-Cas9 systems that incorporate specially engineered RNAs could act transiently, potentially reducing unwanted changes. [Ernesto del Aguila III, NHGRI]

    By removing parts of the CRISPR/Cas9 gene-editing system, and replacing them with specially engineered molecules, researchers at the University of California, San Diego (UCSD) and Isis Pharmaceutical hope to limit the CRISPR/Cas9 system’s propensity for off-target effects. The researchers say that CRISPR/Cas9 needn’t remain continuously active. Instead, it could be transiently activated and deactivated. Such on/off control could prevent residual gene-editing activity that might go awry. Also, such control could be exploited for therapeutic purposes.

    The key, report the scientists, is the introduction of RNA-based drugs that can replace the guide RNA that usually serves to guide the Cas9 enzyme to a particular DNA sequence. When Cas9 is guided by a synthetic RNA-based drug, its cutting action can be suspended whenever the RNA-based drug is cleared. The Cas9’s cutting action can be stopped even more quickly if a second, chemically modified RNA drug is added, provided that it is engineered to direct inactivation of the gene encoding the Cas9 enzyme.

    Details about temporarily activated CRISPR/Cas9 systems appeared November 16 in the Proceedings of the National Academy of Sciences, in a paper entitled, “Synthetic CRISPR RNA-Cas9–guided genome editing in human cells.” The paper’s senior author, the USCD’s Don Cleveland, Ph.D., noted that the RNA-based drugs described in the study “provide many advantages over the current CRISPR/Cas9 system,” such as increased editing efficiency and potential selectivity.

    “Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells,” wrote the authors of the PNAS paper. “Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA.”

    Not only did the synthetic RNA functionally replace the natural crRNA, it produced enhanced cleavage activity at a target DNA site with apparently reduced off-target cleavage. These findings, Dr. Cleveland explained, could provide a platform for multiple therapeutic applications, especially for nervous system diseases, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 activity. “In addition,” he said, “[these designer RNAs] can be synthesized efficiently, on an industrial scale and in a commercially feasible manner today.”

 

 

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BioMEMS The Market aspects of Oligonucleotide-Chips, Products and Applications, Competition, January 21, 2016

Curator: Gérard LOISEAU, ESQ

 

BioMEMS

The Market aspects of Oligonucleotide-Chips, Products, Applications, Competition 

January 21, 2016

2015-2020

The oligonucleotide synthesis market is expected to reach USD 1.918.6Billion at a CAGR of 10.1% by 2020 from USD 1.078.1Billion in 2015.

SOURCE

MARKETSANDMARKETS marketsandmarkets.com/

 

PLAYERS

  • Agilent Technologies Inc.
  • BioAutomation Corp.
  • Biosearch Technologies
  • Gen9 Inc.
  • GenScript Inc.
  • Illumina Inc.
  • Integrated DNA Technologies
  • New England Biolabs Inc.
  • Nitto Denko Avecia Inc.
  • OriGene Technologies Inc.
  • Sigma-Aldrich Corporation
  • Thermo Fisher Scientific Inc.
  • TriLink Biotechnologies

 

Agilent Technologies
 CA NYSE :A


http://www.agilent.com/

  • Agilent was created as a spin off from Hewlett-Packard Company in 1999.
  • Agilent Technologies Inc. is engaged in the life sciences, diagnostics and applied chemical markets. The Company provides application focused solutions that include instruments, software, services and consumables for the entire laboratory workflow. The Company has three business segments:

the life sciences and applied markets business,

the diagnostics and genomics business, and

the Agilent Cross Lab business

  • The Company’s life sciences and applied markets business segment brings together the Company’s analytical laboratory instrumentation and informatics.
  • The Company’s diagnostics and genomics business segment consists of three businesses: the Dako business, the genomics business and the nucleic acid solutions business.
  • The Company’s Agilent Cross Lab business segment combines its analytical laboratory services and consumables business

SOURCE

http://reuters.com/

PRODUCTS AND SERVICES

https://www.agilent.com/en-us/default#collapse-0

  • October 09, 2015 03:21 PM Eastern Daylight Time
  • CARPINTERIA, Calif.–(BUSINESS WIRE)–Dako, an Agilent Technologies company and a worldwide provider of cancer diagnostics, today announced the U.S. Food and Drug Administration has approved a new test that can identify PD-L1 expression levels on the surface of non-small cell lung cancer tumor cells and provide information on the survival benefit with OPDIVO® (nivolumab) for patients with non-squamous NSCLC.

SOURCE

BUSINESS WIRE busibesswire.com/

 

BioAutomation Corp.

 TX


 

http://bioautomation.com/

          PRODUCTS AND SERVICES

  • DNA and RNA synthesis reagents for the MerMades

 

Note: The MerMade 192E Oligonucleotide synthesizer is designed to synthesize DNA, RNA & LNA oligonucleotides in a column format

          PARTNERSHIPS

  • HONGENE BIOTECH : BIOAUTOMATION is the exclusive distributor for the Americas
  • EMD MILLIPORE
  • BIOSEARCH TECHNOLOGIES

 

DISTRIBUTORS

  • LINK TECHNOLOGIES : UK
  • AME BIOSCIENCE : UK
  • BOSUNG SCIENCE : KOREA
  • DNA CHEM : CHINA
  • WAKO : JAPAN
  • ACE PROBE : INDIA

SOURCE

bioautomation.com/

 

Biosearch Technologies
 CA


http://biosearchtech.com/

          PRODUCTS

  • qPCR & SNP Genotyping
  • Custom Oligonucleotides
  • – highly sophisticated oligonucleotides
  • – simple PCR primers
  • Oligos in Plates
  • RNA FISH
  • Synthesis Reagents
  • Immunochemicals
  • Primers
  • Probes
  • Large-Scale Synthesis Oligos
  • Intermediate-Scale Synthesis Oligos

          SERVICES

  • GMP & Commercial Services
  • OEM & Kit Manufacturing
  • qPCR Design Collaborations

          DISTRIBUTORS

Argentina | Australia | Austria | Brazil | Canada |Chile | China | Colombia | Czech Republic | Denmark | Ecuador | Finland | Germany |Hong Kong | Israel | Italy | Japan | Korea | Malaysia | Mexico | New Zealand | Norway | Paraguay | Peru| Philippines | Poland | Romania | Singapore | South Africa | Spain | Sweden |Switzerland | Taiwan ROC | Thailand | Turkey | United Kingdom | Uruguay | Vietnam

SOURCE

biosearchtech.com/

 

Gen9 Inc.
 MA 


http://www.gen9bio.com/

          PRODUCTS

Gen9 is building on advances in synthetic biology to power a scalable fabrication capability that will significantly increase the world’s capacity to produce DNA content. The privately held company’s next-generation gene synthesis technology allows for the high-throughput, automated production of DNA constructs at lower cost and higher accuracy than previous methods on the market. Founded by world leaders in synthetic biology, Gen9 aims to ensure the constructive application of synthetic biology in industries ranging from enzyme and chemical production to pharmaceuticals and biofuels.

          SERVICES

  • Synthetic Biology
  • Gene Synthesis Services
  • Variant Libraries
  • Gene Sequence Design Services

         INVESTORS

  • Agilent Technologies : Private Equity
  • CAMBRIDGE, Mass. and SANTA CLARA, Calif. — April 24, 2013 —Gen9 Receives $21 Million Strategic Investment from Agilent Technologies

SOURCE

gen9bio.com/

 

GenScript Inc.
 NJ 


http://www.genscript.com/

  • GenScript is the largest gene synthesis provider in the USA
  • GenScript Corporation, a biology contract research organization, provides biological research and drug discovery services to pharmaceutical companies, biotech firms, and research institutions in the United States, Europe, and Japan. It offers bio-reagent, custom molecular biology, custom peptide, protein production, custom antibody production, drug candidates testing, assay development and screening, lead optimization, antibody drug development, gene synthesis, and assay-ready cell line production services.
  • The company also offers molecular biology, peptide, protein, immunoassay, chemicals, and cell biology products. It offers its products through distributors in Tokyo, Japan; and Seoul, Korea. GenScript Corporation has a strategic partnership with Immunologix, Inc. The company was founded in 2002 and is based in Piscataway, New Jersey. It has subsidiaries in France, Japan, and China.

 

Note: As of October 24, 2011, Immunologix, Inc. was acquired by Intrexon Corporation. Immunologix, Inc. develops and produces antibody-based therapeutics for various biological targets. It produces human monoclonal antibodies against viral, bacterial, and tumor antigens, as well as human auto antigens.

Intrexon Corporation, founded in 1998, is a leader in synthetic biology focused on collaborating with companies in Health, Food, Energy, Environment and Consumer sectors to create biologically based products that improve quality of life and the health of the planet.

 

 

             PRODUCTS AND SERVICES

  • Gene synthesis
  • Antibody services
  • Protein Services
  • Peptide services

 

               INVESTORS


Note: The Balloch Group (‘TBG’) was established in 2001 by Howard Balloch (Canada‘s ambassador to China from 1996 to 2001). TBG has since grown from a market-entry consultancy working with North American clients in China to a leading advisory and merchant banking firm serving both domestic Chinese companies and multinational corporations. TBG was ranked as the number one boutique investment bank in China by ChinaVenture in 2008.

Kleiner, Perkins, Caufield and Byers

 

Illumina
Inc. CA


http://illumina.com/

 

Monica Heger : SAN FRANCISCO (GenomeWeb) – Illumina today announced two new next-generation sequencing platforms, a targeted sequencing system called MiniSeq and a semiconductor sequencer that is still under development.

Illumina disclosed the initiatives during a presentation at the JP Morgan Healthcare conference held here today. During the presentation, Illumina CEO Jay Flatley also announced a new genotyping array called Infinium XT; a partnership with Bio-Rad to develop a single-cell sequencing workflow; preliminary estimates of its fourth-quarter 2015 revenues; and an update on existing products. The presentation followed the company’s announcement on Sunday that it has launched a new company called Grail to develop a next-generation sequencing test for early cancer detection from patient blood samples.

The MiniSeq system, which is based on Illumina’s current sequencing technology, will begin shipping early this quarter and has a list price of $49,500. It can perform a variety of targeted DNA and RNA applications, from single-gene to pathway sequencing, and promises “all-in” prices, including library prep and sequencing, of $200 to $300 per sample, Flatley said during the JP Morgan presentation.

SOURCES

https://www.genomeweb.com/sequencing-technology/illumina-unveils-mini-targeted-sequencer-semiconductor-sequencing-project-jp

http://investor.biospace.com/biospace/quote?Symbol=ILMN

 

              PRODUCTS AND SERVICES

  •               Mid to large scale manufacturing assets
  •               Analytical Labs
  •               Pre-clinical
  •               Clinical
  •               Launched products

 

              COMPETITORS

https://finance.yahoo.com/q/co?s=ILMN+Competitors Tue, Feb 2, 2016, 2:16pm EST – US Markets

ILMN PVT1 AFFX LMNX Industry
Market Cap: 22.75B N/A 1.13B 835.66M 134.14M
Employees: 3,700 10,000 1,200 745 45.00
Qtrly Rev Growth (yoy): 0.14 N/A -0.01 0.07 0.18
Revenue (ttm): 2.14B 3.80B1 357.74M 235.37M 8.47M
Gross Margin (ttm): 0.73 N/A 0.63 0.71 0.58
EBITDA (ttm): 770.84M N/A 46.64M 52.99M -12.31M
Operating Margin (ttm): 0.30 N/A 0.08 0.17 -1.62
Net Income (ttm): 510.36M 430.90M1 11.22M 39.29M N/A
EPS (ttm): 3.42 N/A 0.13 0.93 -0.34
P/E (ttm): 45.43 N/A 104.40 20.91 25.33
PEG (5 yr expected): 2.68 N/A 4.66 0.55 N/A
P/S (ttm): 10.87 N/A 3.13 3.45 13.65

 

Pvt1 = Life Technologies Corporation (privately held)

AFFX = Affymetrix Inc.

LMNX = Luminex Corporation

 

 

Integrated DNA Technologies (IDT)
IOWA + CA

http://www.com/

 

Integrated DNA Technologies, Inc. (IDT), the global leader in nucleic acid synthesis, serving all areas of life sciences research and development, offers products for a broad range of genomics applications. IDT’s primary business is the production of custom, synthetic nucleic acids for molecular biology applications, including qPCR, sequencing, synthetic biology, and functional genomics. The company manufactures and ships an average of 44,000 custom nucleic acids per day to more than 82,000 customers worldwide. For more information, visit idtdna.com.

 

               PRODUCTS AND SERVICES

               https://eu.idtdna.com/site

  • DNA & RNA Synthesis
  • Custom DNA Oligos 96- & 384-Well Plates Ultramer Oligos Custom RNA Oligos SameDay Oligos HotPlates ReadyMade Primers Oligo Modifications Freedom
  • Dyes GMP for Molecular Diagnostics Large Scale Oligo Synthesis

 

Note : Skokie, IL – December 1, 2015. Integrated DNA Technologies Inc. (“IDT”), the global leader in custom nucleic acid synthesis, has entered into a definitive agreement to acquire the oligonucleotide synthesis business of AITbiotech Pte. Ltd. in Singapore (“AITbiotech”). With this acquisition, IDT expands its customer base across Southeast Asia making it possible for these additional customers to now have access to its broad range of products for genomic applications. AITbiotech will continue operations in its other core business areas.

 

New England Biolabs Inc.
 MA 


http://www.neb.com/

 

                PRODUCTS AND SERVICES

  •                 Restriction Endonucleases
  •                 PCR, Polymerases & Amplification Technologies
  •                 DNA Modifying Enzymes
  •                 Library Preparation for Next Generation Sequencing
  •                 Nucleic Acid Purification
  •                 Markers & Ladders
  •                 RNA Reagents
  •                 Gene Expression
  •                 Cellular Analysis

SOURCE

neb.com/

 

Nitto Denko Avecia Inc.
 MA


http://avecia.com/

 

With over 20 years of experience in oligonucleotide development and production, and over 1000 sequences manufactured, Avecia has played an integral role in the advancing oligo therapeutic market. Our mission is to continue to build value for our customers, as they progress through drug development into commercialization. And as a member of the Nitto Denko Corporation (nitto.com), Avecia is committed to the future of the oligonucleotide market. We are driven by innovative ideas and flexible solutions, designed to provide our customers with the best in service, quality, and technology.

 

SOURCE

http://avecia.com/

 

Note : 1918 Nitto Electric Industrial Co., Ltd. forms in Ohsaki, Tokyo, to produce electrical insulating materials in Japan.

2011 Acquires Avecia Biotechnology Inc. in the U.S.A.

 

 

OriGene Technologies Inc.
 CA

http://www.com/

 

OriGene Technologies, Inc. develops, manufactures, and sells genome wide research and diagnostic products for pharmaceutical, biotechnology, and academic research applications. The company offers cDNA clones, including TrueORF cDNA, viral ORF, destination vectors, TrueClones (human), TrueClones (mouse), organelle marker plasmids, MicroRNA tools, mutant and variant clones, plasmid purification kits, transfection reagents, and gene synthesis service; and HuSH shRNA, siRNA, miRNA, qPCR reagents, plasmid purification products, transfection reagents, PolyA+ and total RNA products, first-strand cDNA synthesis, and CRISPR/Cas9 genome products. It also provides proteins and lysates, such as purified human proteins, over-expression cell lysates, mass spectrometry standard proteins, and protein purification reagents; UltraMAB IHC antibodies, TrueMAB primary antibodies, anti-tag and fluorescent proteins, ELISA antibodies, luminex antibodies, secondary antibodies, and controls and others; and anatomic pathology products, including IHC antibodies, detection systems, and IHC accessories

The company offers luminex and ELISA antibody pairs, autoantibody profiling arrays, ELISA kits, cell assay kits, assay reagents, custom development, and fluorogenic cell assays; TissueFocus search tools; tissue sections; tissue microarrays, cancer protein lysate arrays, TissueScan cDNA arrays, tissue blocks, and quality control products, as well as tissue RNA, DNA, and protein lysates; and lab essentials. Its research areas include cancer biomarker research, RNAi, pathology IHC, stem cell research, ion channels, and protein kinase products. The company provides gene synthesis and molecular biology services, genome editing, custom cloning, custom shRNA, purified protein, monoclonal antibody development, and assay development. It sells its products through distributors worldwide, as well as online. OriGene Technologies, Inc. was incorporated in 1995 and is based in Rockville, Maryland.

SOURCE

http://BLOOMBERG.com

               PRODUCTS AND SERVICES

  •                cDNA Clones
Human, mouse, rat
Expression validated
  •                RNAi
shRNA, siRNA
microRNA & 3’UTR clones
  •                Gene Synthesis
Codon optimization
Variant libraries
  •                Real-time PCR
Primer pairs, panels
SYBR green reagents
  •                Lab Essentials
DNA/RNA purification kits
Transfection reagents
  •                Anatomic Pathology
UltraMAB antibodies
Specificity validated
  •                Recombinant Proteins
10,000 human proteins
from mammalian system
  •                Antibodies
TrueMAB primary antibodies
Anti-tag antibodies
  •                Assays and Kits
ELISA & Luminex antibodies
Autoantibody Profiling Array
  •                Cancer & Normal Tissues
Pathologist verified
gDNA, RNA, sections, arrays

SOURCE

origene.com/

 

Sigma-Aldrich Corporation 
MI 


http://www.sigmaaldrich.com/

Louis, MO – November 18, 2015 Merck KGaA, Darmstadt, Germany, Completes Sigma-Aldrich Acquisition

Merck KGaA today announced the completion of its $17 billion acquisition of Sigma-Aldrich, creating one of the leaders in the $130 billion global industry to help solve the toughest problems in life science.

Press Release: 18-Nov-2015

Letter to our Life Science Customers from Dr. Udit Batra

The life science business of Merck KGaA, Darmstadt, Germany brings together the world-class products and services, innovative capabilities and exceptional talent of EMD Millipore and Sigma-Aldrich to create a global leader in the life science industry.

Everything we do starts with our shared purpose – to solve the toughest problems in life science by collaborating with the global scientific community. 

This combination is built on complementary strengths, which will enable us to serve you even better as one organization than either company could alone.

This means providing a broader portfolio with a catalog of more than 300,000 products, including many of the most respected brands in the industry, greater geographic reach, and an unmatched combination of industry-leading capabilities.

                PRODUCTS AND SERVICES

                http://www.sigmaaldrich.com/configurator/servlet/DesignCenter?btnOpen_0.x=1

                http://www.sigmaaldrich.com/content/dam/sigma-aldrich/common/quality-products.jpg

 

Thermo Fisher Scientific Inc.
 MA 
NYSE :TMO


http://thermofisher.com/

Thermo Fisher Scientific Inc. is a provider of analytical instruments, equipment, reagents and consumables, software and services for research, manufacturing, analysis, discovery and diagnostics. The company operates through four segments: Life Sciences Solutions, provides reagents, instruments and consumables used in biological and medical research, discovery and production of new drugs and vaccines as well as diagnosis of disease; Analytical Instruments, provides instruments, consumables, software and services that are used in the laboratory; Specialty Diagnostics, offers diagnostic test kits, reagents, culture media, instruments and associated products, and Laboratory Products and Services, offers self-manufactured and sourced products for the laboratory.

SOURCE

http://REUTERS.com

 

                PRODUCTS AND SERVICES

  •                 Oligos Value – Standard – Plate
  •                 Primers
  •                 Probes
  •                 Nucleotides

 

                BRANDS

  1.                THERMO SCIENTIFIC
  2.                 APPLIED BIOSYSTEMS
  3.                 INVITROGEN
  4.                 FISHER SCIENTIFIC
  5.                 UNITY LAB SERVICES

 

                 PARTNERSHIPS

AFFYMETRIX : NASDAQ : AFFX : affymetrix.com/

WALTHAM, Mass. & SANTA CLARA, Calif.–(BUSINESS WIRE)–Jan. 8, 2016– Thermo Fisher Scientific Inc. (NYSE:TMO), the world leader in serving science, and Affymetrix Inc. (NASDAQ:AFFX), a leading provider of cellular and genetic analysis products, today announced that their boards of directors have unanimously approved Thermo Fisher’s acquisition of Affymetrix for $14.00 per share in cash. The transaction represents a purchase price of approximately $1.3 billion.

SOURCE

http://BUSINESSWIRE.com

 

TriLink Biotechnologies
 CA 


http://www.com/

 

              PRODUCTS

              Oligonucleotides

  •               DNA Oligos
  •               RNA Oligos
  •               Modified Oligos
  •               Specialty Oligos

              Nucleotides

  •               NTPs (Nucleoside Triphosphates)
  •               Biphosphates
  •               Monophosphates

 

              SERVICES

  •              Custom Chemistry
  •              Reagents
  •              Aptamers

 

             PARTNERSHIPS

  • LIFE TECHNOLOGIES,
  • TERMO FISHER SCIENTIFIC since July 2015 thermofisher.com/
  • GENMARK genmarkdx.com/

SOURCE

http://trilinkbiotech.com/

 

Other related articles published in this Open Access Online Scientific Journal include the following:

Gene Editing: The Role of Oligonucleotide Chips

https://pharmaceuticalintelligence.com/2016/01/07/gene-editing-the-role-of-oligonucleotide-chips/

Gene Editing for Exon 51: Why CRISPR Snipping might be better than Exon Skipping for DMD

https://pharmaceuticalintelligence.com/2016/01/23/gene-editing-for-exon-51-why-crispr-snipping-might-be-better-than-exon-skipping-for-dmd/

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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