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Dujardin G.,French Institute of Health and Medical Research | Buratti E.,ICGEB | Charlet-Berguerand N.,French Institute of Health and Medical Research | Mbopda A.,French Institute of Health and Medical Research | And 4 more authors.
Nucleic Acids Research | Year: 2010

Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF 65 and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain. © The Author(s) 2010.


Patra P.K.,Pt Jnm Medical College | Chauhan V.S.,ICGEB | Khodiar P.K.,Pt Jnm Medical College | Dalla A.R.,Red Cross | Serjeant G.R.,Sickle Cell Trust Jamaica
Journal of Community Genetics | Year: 2011

The aim of this study is to determine the feasibility of large-scale population screening for the sickle cell gene in high risk areas with limited resources. A programme designed to detect the sickle cell trait and sickle cell disease has screened 359,823 subjects among 2,087 (99.7%) of the villages in Raipur District, Chhattisgarh State, India between October 2007 and June 2010. Children aged 3-15 years were initially screened in the villages by solubility tests on fingerprick samples. Venipuncture was performed on subjects with positive solubility tests, and the samples were transferred to Raipur Medical College for alkaline haemoglobin electrophoresis. The sickle cell trait occurred in 33,467 (9.30%) and an SS phenotype in 747 (0.21%). The gene frequencies were not in Hardy- Weinberg equilibrium most likely due to a deficiency of the SS phenotype failing to enter the sampled population from either sickness or early death. Subjects with abnormal haemoglobin genotypes may factor this information into decisions regarding marriage and avoid the risks of having children with sickle cell disease. The techniques described may be a model for other developing societies with limited resources. © Springer-Verlag 2011.


News Article | October 26, 2016
Site: www.eurekalert.org

India suffers from the highest number of dengue infections in the world, but there are few studies, if any, to understand the immune cells involved in fighting the virus. A recent study published in the Journal of Virology, by joint efforts among scientists from Emory, India and Thailand, sheds novel insights on the properties of a class of immune cells known as CD8 T cells, which are involved in fighting dengue virus infection. Human infections with dengue virus often lead to debilitating illness, hemorrhage and death, especially among children. Currently one third of the global population is under the threat of dengue virus infection. The virus continues spreading globally, causing huge public health concern including the Americas. The study analyzed a large number of dengue-infected children from India. CD8 T cells expanded massively in the dengue patients, often reaching the levels seen in Ebola infected patients, the researchers say. The expansion was strikingly similar in dengue- infected children from different geographical regions across the continents. In addition to fighting the virus by killing infected cells, these CD8 T cells are notorious for releasing inflammatory cytokines, such as IFN-g. If all of these these massively expanding CD8 T cells produced these inflammatory cytokines, the patients would be expected to suffer from a dangerous "cytokine storm" like that seen in Ebola virus disease. Interestingly, the study shows that in the dengue patients, however, a vast majority of these massively expanding CD8 T cells kept their cytokine production under check while retaining the ability to kill virus-infected cells. According to Anmol Chandele, PhD, lead author of the study, harnessing this new knowledge for an understanding of how to maintain the balance between fighting the infection while inhibiting inflammation will be critical for developing more effective vaccines and therapeutics and could be a game changer in the fight against dengue. The study emphasizes the value of international collaboration and arises from a unique partnership established five years ago between Emory Vaccine Center and the International Centre for Genetic Engineering and Biotechnology (ICGEB) in New Delhi. Chandele is adjunct assistant professor of microbiology and immunology, and Murali Krishna Kaja, PhD, the study's senior author, is associate professor of pediatrics at Emory University School of Medicine and associate director for the ICGEB-Emory Vaccine Center partnership. The goal of the ICGEB-Emory Vaccine Center partnership is to bring state-of-the-art vaccine research to where it is needed most via international research collaborations and to increase India's capacity for vaccine and infectious disease research. The study was supported by a National Institutes of Health award for international collaboration in infectious disease research (ICIDR), and the Indian Government's Department of Biotechnology. This award to study dengue virus infection in India (principal investigators, Rafi Ahmed, Emory Vaccine Center and Navin Khanna, ICGEB) is the first ICIDR-funded project in India. In addition to scientists from the Emory Vaccine Center and ICGEB, scientists and physicians from Indian institutions such as All India Institute of Medical Sciences and the Translational Health Science and Technology Institute, and the Mahidol University of Thailand have played a critical role in this study. Journal of Virology: Characterization of human CD8 T cell responses in dengue virus infected patients from India


Kralovicova J.,University of Southampton | Lages A.,University of Southampton | Patel A.,University of Nottingham | Dhir A.,ICGEB | And 3 more authors.
Nucleic Acids Research | Year: 2014

Splice-switching oligonucleotides (SSOs) have been widely used to inhibit exon usage but antisense strategies that promote removal of entire introns to increase splicing-mediated gene expression have not been developed. Here we show reduction of INS intron 1 retention by SSOs that bind transcripts derived from a human haplotype expressing low levels of proinsulin. This haplotype is tagged by a polypyrimidine tract variant rs689 that decreases the efficiency of intron 1 splicing and increases the relative abundance of mRNAs with extended 5' untranslated region (5' UTR), which curtails translation. Co-expression of haplotype-specific reporter constructs with SSOs bound to splicing regulatory motifs and decoy splice sites in primary transcripts revealed a motif that significantly reduced intron 1-containing mRNAs. Using an antisense microwalk at a single nucleotide resolution, the optimal target was mapped to a splicing silencer containing two pseudoacceptor sites sandwiched between predicted RNA guanine (G) quadruplex structures. Circular dichroism spectroscopy and nuclear magnetic resonance of synthetic G-rich oligoribonucleotide tracts derived from this region showed formation of a stable parallel 2-quartet G-quadruplex on the 3' side of the antisense retention target and an equilibrium between quadruplexes and stable hairpin-loop structures bound by optimal SSOs. This region interacts with heterogeneous nuclear ribonucleoproteins F and H that may interfere with conformational transitions involving the antisense target. The SSO-assisted promotion of weak intron removal from the 5' UTR through competing noncanonical and canonical RNA structures may facilitate development of novel strategies to enhance gene expression. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.


Buratti E.,ICGEB | Baralle D.,University of Southampton
Methods in Molecular Biology | Year: 2012

Defects at the level of pre-mRNA splicing represent a common source of disease mutations in almost all known diseases with a genetic aetiology. In general, it is commonly accepted that 15% of all pathogenic mutations are caused by splicing defects. However, this is probably a conservative estimate since clinical practice has only recently begun to routinely assess for this types of abnormalities. Therefore, it is expected that many currently unclassified or apparently harmless genetic variants will really turn out to be splicing-affecting defects. It is also well known that some genes are more susceptible than others to alterations in their splicing processes. Among these genes, one of the most representative is the NF-1 gene. In this gene, almost 50% of all reported disease-causing mutations can be directly attributed to alterations of the pre-mRNA process. In this chapter, we review the splicing process of the NF-1 gene and the most commonly used methods to identify splicing alterations. In particular, we provide practical notes on how to perform this analysis to maximize the chance of correctly identifying aberrant pre-mRNA splicing events in this gene. © 2012 Springer Science+Business Media, LLC.


Skoko N.,ICGEB | Baralle M.,ICGEB | Baralle F.E.,ICGEB
EMBO Journal | Year: 2016

It is well established that TDP-43 accumulates in degenerating neurons in patients with ALS/FTLD, which might affect normal TDP-43 function. In this issue of The EMBO Journal Xia et al () show a novel connection between TDP-43 loss of function and autophagy failure. Using knockdown models of TDP-43, they observed enhanced autophagosome and lysosome biogenesis through mTORC1 activity inhibition and TFEB activation. Impaired autophagosome-lysosome fusion was also observed, however in an mTORC1-independent manner. The data identify dysfunctions at multiple stages of the autophagic pathway following TDP-43 depletion that might represent possible targets of future therapeutic interventions. The ALS-associated RNA binding protein TDP-43 regulates autophagy and lysosome gene expression as well as dynactin 1-mediated autophagosome fusion. Abnormal regulation of autophagy contributes to TDP-43-associated neurotoxicity. © 2015 The Authors.


Negro R.,ICGEB | Gobessi S.,ICGEB | Longo P.G.,ICGEB | He Y.,Indiana University | And 3 more authors.
Blood | Year: 2012

A polymorphic variant of the phosphatase PTPN22 has been associated with increased risk for multiple autoimmune diseases. The risk allele is thought to function by diminishing antigen-receptor signals responsible for negative selection of autoreactive lymphocytes. We now show that PTPN22 is markedly overexpressed in chronic lymphocytic leukemia (CLL), a common malignancy of autoreactive B lymphocytes. We also show that overexpression of PTPN22 significantly inhibits antigen-induced apoptosis of primary CLL cells by blocking B-cell receptor (BCR) signaling pathways that negatively regulate lymphocyte survival. More importantly, we show that PTPN22 positively regulates the antiapoptotic AKT kinase, which provides a powerful survival signal to antigen-stimulated CLL cells. This selective uncoupling of AKT from other downstream BCR signaling pathways is a result of inhibition of a negative regulatory circuit involving LYN, CD22, and SHIP. Finally, we show that PTPN22 can be effectively down-regulated by the PKC inhibitors ruboxistaurin and sotrastaurin, resulting in enhanced killing of CLL cells exposed to proapoptotic BCR stimuli. Collectively, these data suggest that PTPN22 overexpression represents a protective mechanism that allows autoantigen-activated CLL cells to escape from negative selection and indicate that this mechanism could be exploited for therapeutic purposes by targeting PTPN22 with PKC inhibitors. © 2012 by The American Society of Hematology.


Suljagic M.,ICGEB | Laurenti L.,Catholic University Hospital melli | Tarnani M.,Catholic University Hospital melli | Alam M.,University of Michigan | And 2 more authors.
Leukemia | Year: 2010

The PI3K/Akt pathway is activated in response to various microenvironmental stimuli that regulate the survival and proliferation of chronic lymphocytic leukemia (CLL) B-cells, including triggering of the B-cell receptor (BCR). Although this pathway is frequently targeted in cancer, no significant alterations have yet been identified in CLL. We now show that the phosphatase PH domain leucin-rich repeat protein phosphatase (PHLPP1), a recently identified tumor suppressor and negative regulator of the Akt kinase, is absent or expressed at substantially reduced levels in CLL B-cells. To determine what the consequences of PHLPP1 loss on BCR signaling are, we downregulated or re-expressed PHLPP1 in lymphoma cell lines and primary CLL B-cells, respectively. Downregulation of PHLPP1 increased BCR-induced phosphorylation and activation of the Akt, GSK3 and ERK kinases, whereas re-expression had the opposite effect. Importantly, re-expression of PHLPP1 in primary CLL cells prevented upregulation of Mcl-1 and inhibited the increase in leukemic cell viability induced by sustained BCR engagement. Enforced expression of PHLPP1 also affected the response to other microenvironmental stimuli, particularly in terms of ERK phosphorylation. Collectively, these data show that CLL cells lack an important negative regulator of the Akt and ERK pathways, which could confer them a growth advantage by facilitating the propagation of crucial microenvironment-derived stimuli. © 2010 Macmillan Publishers Limited All rights reserved.


PubMed | Health Science University, International School for Advanced Studies, University of Udine, Third University of Rome and 3 more.
Type: Journal Article | Journal: Cell death & disease | Year: 2017

Hemoglobin (Hb) is the major protein in erythrocytes and carries oxygen (O


Genetic engineering is the science for cloning or manipulating DNAs or genes to create new characteristic in organism or produce protein or hormone. Genetic engineering is mostly used for generating recombinant DNA and is done by inserting desired trait gene or by deletion or mutating gene in cell or virus known as vector. Genetically engineered plants, animals or microbes are called as genetically modified organisms. Increased productivity due to genetically modified plants and government subsidy for development of genetically modified organisms are factors driving the growth of genetically modified organism market. Furthermore, strong inclination of R&D in genetic research and expanding biotech and pharmaceuticals industries triggers the growth in the market. Also affordable food, long shelf life, resistance to disease and increased flavors are some aspects that help in development of market. Yet, strict regulatory policy issues, moral concern of human health and development of superweeds are prime restraints affecting the growth of genetically modified organisms market. Nevertheless, funding from government and private institutions for research and developing of genetically modified organism is likely to open new market avenues in the near future. The report covers forecast and analysis for the genetically modified organisms market on a global and regional level. The study provides significant data of 2014 along with estimated from 2014 to 2020 based on revenue (USD Million). The report also offers detailed competitive landscape of the global genetically modified organisms market. It includes company market share analysis, product portfolio of the major industry participants. The report provides detailed segmentation of the genetically modified organisms market based on application segment, end users segment and region. Know more before buying this report @ http://www.syndicatemarketresearch.com/market-analysis/genetically-modified-organisms-market-global-industry-analysis-size.html#inquiry-for-buying The genetically modified organisms market is segmented on the basis of application, end user and region. Modified plants, animals and cell line are three types of applications. Modified plants are also known as transgenic plants. GM rice, GM cotton, GM soybeans, GM maize and others are some of the modified plants. Further, dolly the sheep, ruppy the fluorescent dog, glofish, vacanti mouse and others are types of genetically modified animals. Modified plants accounted for major market share due to widespread applications and advantages. Biotech and pharmaceuticals companies and research centers, clinical trial centers are the different end users of the genetically modified organisms market. The U.S., India, Brazil, China, Argentina and Canada are witnessing an unprecedented boom in GM crops in recent times. There was sharp rise in demand of GM crops in South Africa, Paraguay and Australia in 2006. Europe has shown exceptional growth due to appreciable production of GM maize in Spain owing to high cultivation rate. In Germany, Portugal, Czech Republic and France, transgenic crops continue to gain traction in the years to come. Some top research centers in genetically modified organisms markets are NIAS, and ICGEB among others. The report segments of the genetically modified organisms market into:

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