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      Volume 40, Issue 5

      December 2015,   pages  829-968

    • What history tells us XXXIX. CRISPR-Cas : From a prokaryotic immune system to a universal genome editing tool

      Michel Morange

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    • Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro

      Swathi Kota Hari S Misra

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      Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase lb of D. radiodurans (DraTopolB) could change the electrophoretic mobility of fast migrating intramolecular rec𝐹-G4 DNA into the slow migrating species. DraTopolB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular rec𝐹-G4 DNA structures stabilized by K+. On the contrary, when DraTopolB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopolB. This implies that DraTopolB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopolB activity on intramolecular G4 DNA structures. These results suggested that DraTopolB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

    • Chromosomal context and replication properties of ARS plasmids in Schizosaccharomyces pombe

      Aditya S Pratihar Vishnu P Tripathi Mukesh P Yadav Dharani D Dubey

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      Short, specific DNA sequences called as Autonomously Replicating Sequence (ARS) elements function as plasmid as well as chromosomal replication origins in yeasts. As compared to ARSs, different chromosomal origins vary greatly in their efficiency and timing of replication probably due to their wider chromosomal context. The two Schizosaccharomyces pombe ARS elements, ars727 and ars2OO4, represent two extremities in their chromosomal origin activity - ars727 is inactive and late replicating, while ars2OO4 is a highly active, early-firing origin. To determine the effect of chromosomal context on the activity of these ARS elements, we have cloned them with their extended chromosomal context as well as in the context of each other in both orientations and analysed their replication efficiency by ARS and plasmid stability assays. We found that these ARS elements retain their origin activity in their extended/altered context. However, deletion of a 133-bp region of the previously reported ars727-associated late replication enforcing element (LRE) caused advancement in replication timing of the resulting plasmid. These results confirm the role of LRE in directing plasmid replication timing and suggest that the plasmid origin efficiency of ars2OO4 or ars727 remains unaltered by the extended chromosomal context.

    • Semaphorin 4A enhances lung fibrosis through activation of Akt via PlexinD1 receptor

      Hai-Ying Peng Wei Gao Fa-Rong Chong Hong-Yan Liu Ji Zhang

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      Semaphorin 4A plays a regulatory role in immune function and angiogenesis. However, its specific involvement in controlling lung fibrosis, a process that is closely related to angiogenesis and inflammation is still poorly understood. In the present study, we show that treatment of Sema4A on normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including a-smooth muscle actin (𝛼-SMA), ezrin, moesin, and paxillin. We confirm that Sema4A enhances the ability of lung fibroblasts to contract collagen gel. Sema4A treatment led to resistance to apoptosis in normal lung fibroblasts. Relative to normal lung fibroblasts, fibroblasts cultured from scars of patients with the flbrotic disease Systemic Sclerosis (SSc) showed elevated Sema4A secretion, enhanced 𝛼-SMA, ezrin, moesin, and paxillin expression, and high ability to induce collagen gel contraction. Using neutralizing antibody against Sema4A receptor, PlexinD1, we found that endogenous Sema4A signalling in SSc fibroblast was through PlexinD1 receptor. We then identified the signalling mechanism through which Sema4A-PlexinD1 promotes the ability of normal fibroblasts to contract a collagen gel matrix. Western blot analysis showed that Sema4A activated the Akt pathway in lung fibroblasts, and the specific inhibitor of Akt pathway, Akt inhibitor III, blocked the ability of Sema4A to promote the ability of lung fibroblasts to contract a collagen gel matrix. Thus, blocking Sema4A-PlexinD1-Akt cascades might be beneficial in reducing pulmonary fibrosis.

    • Interdependence of laforin and malin proteins for their stability and functions could underlie the molecular basis of locus heterogeneity in Lafora disease

      Shuchi Mittal Mamta Upadhyay Pankaj Kumar Singh Rashmi Parihar Subramaniam Ganesh

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      Lafora disease (LD), an autosomal recessive and fatal form of neurodegenerative disorder, is characterized by the presence of polyglucosan inclusions in the affected tissues including the brain. LD can be caused by defects either in the 𝐸𝑃𝑀2𝐴 gene coding for the laforin protein phosphatase or the 𝑁𝐻𝐿𝑅𝐶𝐼 gene coding for the malin ubiquitin ligase. Since the clinical symptoms of LD patients representing the two genetic groups are very similar and since malin is known to interact with laforin, we were curious to examine the possibility that the two proteins regulate each others function. Using cell biological assays we demonstrate here that

      1. malin promotes its own degradation via auto-ubiquitination,
      2. laforin prevents the auto-degradation of malin by presenting itself as a substrate and
      3. malin preferentially degrades the phosphatase-inactive laforin monomer. Our results that laforin and malin regulate each others stability and activity offers a novel and attractive model to explain the molecular basis of locus heterogeneityobserved in LD.
    • Regulatory single nucleotide polymorphisms at the beginning of intron 2 of the human 𝐾𝑅𝐴𝑆 gene

      Elena V Antontseva Marina Yu Matveeva Natalia P Bondar Elena V Kashina Elena Yu Leberfarb Leonid O Bryzgalov Polina A Gervas Anastasia A Ponomareva Nadezhda V Cherdyntseva Yury L Orlov Tatiana I Merkulova

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      There are two regulatory single nucleotide polymorphisms (rSNPs) at the beginning of the second intron of the mouse 𝐾-𝑟𝑎𝑠 gene that are strongly associated with lung cancer susceptibility. We performed functional analysis of three SNPs (rs12228277: T>A, rs12226937: G>A, and rs61761074: T>G) located in the same region of human 𝐾𝑅𝐴𝑆. We found that rs12228277 and rs61761074 result in differential binding patterns of lung nuclear proteins to oligonucleotide probes corresponding two alternative alleles; in both cases, the transcription factor NF-Y is involved. G>A substitution (rs12226937) had no effect on the binding of lung nuclear proteins. However, all the nucleotide substitutions under study showed functional effects in a luciferase reporter assay. Among them, rs61761074 demonstrated a significant correlation with allele frequency in non-small-cell lung cancer (NSCLC). Taken together, the results of our study suggest that a T>G substitution at nucleotide position 615 in the second intron of the KRAS gene (rs61761074) may represent a promising genetic marker of NSCLC.

    • Signatures of positive selection at hemopexin (PEX) domain of matrix metalloproteinase-9 (MMP-9) gene

      Yang Liu Yang Zhao Chunlei Lu Maobin Fu Tonghai Dou Xiaoming Tan

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      Matrix metalloproteinases-9 (MMP-9) is an important cancer-associated, zinc-dependent endopeptidase. To investigate the natural selection hypothesis of MMP-9, the orthologous sequences from 12 vertebrates were compared and a molecular evolution analysis was performed. Results suggest that amino acid residues present in the middle region of the protein are more selectively constrained, whereas amino acid residues in the C-terminal region of the MM~P-9 protein including exon 13 showed lowest conservation level in non-primate species, suggesting that it is an exon with fast evolving rate compared to the others analyzed. InterProScan analysis shows that exon 13 was located in hemopexin (PEX) domain of MM~P-9. Positive selection was detected in PEX domain of MMP-9 protein between human and other species, which indicates that selective pressure may play a role in shaping the function of MM~P-9 in the course of evolution.

    • Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus

      Puli Chandramouli Reddy Ishani Sinha Ashwin Kelkar Farhat Habib Saurabh J Pradhan Raman Sukumar Sanjeev Galande

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      The Asian elephant Elephas maximus and the African elephant Loxodonta africana that diverged 5-7 million years ago exhibit differences in their physiology, behaviour and morphology. A comparative genomics approach would be useful and necessary for evolutionary and functional genetic studies of elephants. We performed sequencing of E. maximus and map to L. africana at ∼ 15X coverage. Through comparative sequence analyses, we have identified Asian elephant specific homozygous, non-synonymous single nucleotide variants (SNVs) that map to 1514 protein coding genes, many of which are involved in olfaction. We also present the first report of a high-coverage transcriptome sequence in E. maximus from peripheral blood lymphocytes. We have identified 103 novel protein coding transcripts and 66-long non-coding (Inc)RNAs. We also report the presence of 181 protein domains unique to elephants when compared to other Afrotheria species. Each of these findings can be further investigated to gain a better understanding of functional differences unique to elephant species, as well as those unique to elephantids in comparison with other mammals. This work therefore provides a valuable resource to explore the immense research potential of comparative analyses of transcriptome and genome sequences in the Asian elephant.

    • Differential neural activation for camouflage detection task in Field-independent and Field-Dependent individuals: Evidence from fMRI

      Janani Rajagopalan Shilpi Modi Pawan Kumar Subash Khushu Manas K Mandal

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      It is not clearly known as to why some people identify camouflaged objects with ease compared with others. The literature suggests that Field-Independent individuals detect camouflaged object better than their Field-Dependent counterparts, without having evidence at the neural activation level. A paradigm was designed to obtain neural correlates of camouflage detection, with real-life photographs, using functional magnetic resonance imaging. Twenty-three healthy human subjects were stratified as Field-Independent (Fl) and Field-Dependent (FD), with Witkins Embedded Figure Test. FIs performed better than FDs (marginal significance; 𝑝=0.054) during camouflage detection task. fMRI revealed differential activation pattern between Fl and FD subjects for this task. One sample T-test showed greater activation in terms of cluster size in FDs, whereas FIs showed additional areas for the same task. On direct comparison of the two groups, Fl subjects showed additional activation in parts of primary visual cortex, thalamus, cerebellum, inferior and middle frontal gyrus. Conversely, FDs showed greater activation in inferior frontal gyms, precentral gyms, putamen, caudate nucleus and superior parietal lobule as compared to FIs. The results give preliminary evidence to the differential neural activation underlying the variances in cognitive styles of the two groups.

    • Asymmetric cell division and its role in cell fate determination in the green alga Tetraselmis indica

      Mani Arora Arga Chandrashekar Anil Karl Burgess Jane Delany Ehsan Mesbahi

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      The prasinophytes (early diverging Chlorophyta), consisting of simple unicellular green algae, occupy a critical position at the base of the green algal tree of life, with some of its representatives viewed as the cell form most similar to the first green alga, the `ancestral green flagellate'. Relatively large-celled unicellular eukaryotic phytoflagellates (such as Tetraselmis and Scherffelia), traditionally placed in Prasinophyceae but now considered as members of Chlorodendrophyceae (core Chlorophyta), have retained some primitive characteristics of prasinophytes. These organisms share several ultrastructural features with the other core chlorophytes (Trebouxiophyceae, Ulvophyceae and Chlorophyceae). However, the role of Chlorodendrophycean algae as the evolutionary link between cellular individuality and cellular cooperation has been largely unstudied. Here, we show that clonal populations of a unicellular chlorophyte, Tetraselmis indica, consist of morphologically and ultrastructurally variant cells which arise through asymmetric cell division. These cells also differ in their physiological properties. The structural and physiological differences in the clonal cell population correlate to a certain extent with the longevity and function of cells.

    • Structural studies on a non toxic homologue of type II RIPs from bitter gourd: Molecular basis of non toxicity, conformational selection and glycan structure

      Thyageshwar Chandran Alok Sharma M Vijayan

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      The structures of nine independent crystals of bitter gourd seed lectin (BGSL), a non-toxic homologue of type II RIPS, and its sugar complexes have been determined. The four-chain, two-fold symmetric, protein is made up of two identical two-chain modules, each consisting of a catalytic chain and a lectin chain, connected by a disulphide bridge. The lectin chain is made up of two domains. Each domain carries a carbohydrate binding site in type II RIPS of known structure. BGSL has a sugar binding site only on one domain, thus impairing its interaction at the cell surface. The adenine binding site in the catalytic chain is defective. Thus, defects in sugar binding as well as adenine binding appear to contribute to the non-toxicity of the lectin. The plasticity of the molecule is mainly caused by the presence of two possible well defined conformations of a surface loop in the lectin chain. One of them is chosen in the sugar complexes, in a case of conformational selection, as the chosen conformation facilitates an additional interaction with the sugar, involving an arginyl residue in the loop. The 𝑁-glycosylation of the lectin involves a plant-specific glycan while that in toxic type H RIPS of known structure involves a glycan which is animal as well as plant specific.

    • Occurrence, detection and detoxification of mycotoxins

      Visenuo Aiko Alka Mehta

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      Mycotoxins have been identified as important toxins affecting animal species and humans ever since the discovery of aHatoxin Bl in 1960. Mycotoxigenic fungi are ubiquitous in nature and are held responsible for economic loss as they decrease crop yield and quality of food. The presence of fungi and their mycotoxins are reported not only in food grains but also in medicinal herbs and processed foods. Since prevention is not always possible, detoxification of mycotoxins have been attempted using several means; however, only few have been accepted for practical use, e.g. ammonia in the com industry. Organizations such as the World Health Organization, US Food and Drug Adminis-tration and European Union have set regulations and safety limits of important mycotoxins, viz. aHatoxins, fusarium toxins, ochratoxin, patulin zearalenone, etc., to ensure the safety of the consumers. This review article is a brief and up-to-date account of the occurrence, detection and detoxification of mycotoxins for those interested in and considering research in this area.

    • In search of principles for a Theory of Organisms

      Giuseppe Longo Maël Montévil Carlos Sonnenschein Ana M Soto

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      Lacking an operational theory to explain the organization and behaviour of matter in unicellular and multicellular organisms hinders progress in biology. Such a theory should address life cycles from ontogenesis to death. This theory would complement the theory of evolution that addresses phylogenesis, and would posit the oretical extensions to accepted physical principles and default states in order to grasp the living state of matter and define proper biological observables. Thus, we fanout adopting the default state implicit in Darwins theory, namely, cell proliferation with variation plus motility, and a framing principle, namely, life phenomena manifest themselves as non-identical iterations of morphogenetic processes. From this perspective, organisms become a consequence of the inherent variability generated by proliferation, motility and self-organization. Morphogenesis would then be the result of the default state plus physical constraints, like gravity, and those present in living organisms, like muscular tension.

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