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      Volume 40, Issue 3

      September 2015,   pages  473-666

    • What history tells us XXXVIII. Resurrection of a transient forgotten model of gene action

      Michel Morange

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    • Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin

      Qizhuang Lv Kangkang Guo Tao Wang Chengcheng Zhang Yanming Zhang

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      Porcine circovirus type 2 (PCV2) is the primary infectious agent of PCV-associated disease (PCVAD) in swine. ORF4 protein is a newly identified viral protein of PCV2 and is involved in virus-induced apoptosis. However, the molecular mechanisms of ORF4 protein regulation of apoptosis remain unclear, especially given there is no information regarding any cellular partners of the ORF4 protein. Here, we have utilized the yeast two-hybrid assay and identified four host proteins (FHC, SNRPN, COX8A and Lamin C) interacting with the ORF4 protein. Specially, FHC was chosen for further characterization due to its important role in apoptosis. GST pull-down, subcellular co-location and co-immunoprecipitation assays confirmed that the PCV2 ORF4 protein indeed interacted with the heavy-chain ferritin, which is an interesting clue that will allow us to determine the role of the ORF4 protein in apoptosis.

    • A rapidly progressing, deadly disease of Actias selene (Indianmoonmoth) larvae associated with a mixed bacterial and baculoviral infection

      Marta A Skowron Beata Guzow-Krzemińska Sylwia Barańska Paulina Jędrak Grzegorz Węgrzyn

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      The outbreak of an infectious disease in captive-bred Lepidoptera can cause death of all the caterpillars within days. A mixed baculoviral–bacterial infection observed among Actias selene (Hübner 1807), the Indian moon moth (Insecta: Lepidoptera: Saturniidae), larvae was characterized and followed by a photographic documentation of the disease progression. The etiological agents were determined using mass spectrometry and polymerase chain reaction (PCR). It appeared that the disease was caused by a mixed infection of larvae with a baculovirus and Morganella morganii. A molecular phylogenetic analysis of the virus and microbiological description of the pathogenic bacterium are presented.

    • Potential gene regulatory role for cyclin D3 in muscle cells

      Fathima Athar Veena K Parnaik

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      Cyclin D3 is important for muscle development and regeneration, and is involved in post-mitotic arrest of muscle cells. Cyclin D3 also has cell-cycle-independent functions such as regulation of specific genes in other tissues. Ectopic expression of cyclin D3 in myoblasts, where it is normally undetectable, promotes muscle gene expression and faster differentiation kinetics upon serum depletion. In the present study, we investigated the mechanistic role of cyclin D3 in muscle gene regulation. We initially showed by mutational analysis that a stable and functional cyclin D3 was required for promoting muscle differentiation. Using chromatin immunoprecipitation assays, we demonstrated that expression of cyclin D3 in undifferentiated myoblasts altered histone epigenetic marks at promoters of muscle-specific genes like MyoD, Pax7, myogenin and muscle creatine kinase but not non-muscle genes. Cyclin D3 expression also reduced the mRNA levels of certain epigenetic modifier genes. Our data suggest that epigenetic modulation of muscle-specific genes in cyclin-D3-expressing myoblasts may be responsible for faster differentiation kinetics upon serum depletion. Our results have implications for a regulatory role for cyclin D3 in muscle-specific gene activation.

    • Bifidogenic effect of grain larvae extract on serum lipid, glucose and intestinal microflora in rats

      Sang-Oh Park Byung-Sung Park

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      The main objective of this study was to investigate whether orally administered Korean grain larvae ethanol extract (GLE) had a bifidogenic effect in normal rats. Male Sprague–Dawley rats were divided into a negative control group (CO) and GLE orally administered (5.0, 7.0 and 9.0 mg/100 g body weight) groups. Thymus and spleen weights dosedependently increased by 128.58% and 128.58%, respectively, but abdominal fat decreased by 19.18% after GLE administration compared with that in the CO group (𝑝<0.05). Serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, and glucose decreased by 30.26%, 7.33%, 27.20%, and 6.96%, respectively, whereas highdensity lipoprotein cholesterol increased by 129.93% in the GLE groups compared with those in the CO group (𝑝<0.05). IgG, IgM, IgA in the GLE groups increased 203.68%, 181.41%, and 238.25%, respectively, compared to that in the CO group (𝑝<0.05). Bifidobacteria and Lactobacillus increased by 115.74% and 144.28%, whereas Bacteroides, Clostridium, Escherichia, and Streptococcus decreased by 17.37%, 17.46%, 21.25%, and 19.16%, respectively, in the GLE groups compared with those in the CO group (𝑝<0.05). Total organic acids, acetic acid, and propionic acid increased by 151.40%, 188.09%, and 150.17%, whereas butyric acid and valeric acid decreased by 40.65% and 49.24%, respectively, in the GLE groups as compared with those in the CO group (𝑝<0.05). These results suggest that Korean GLE improves the bifidogenic effect by increasing cecal organic acids and modulating gut microflora via a selective increase in Bifidobacterium in normal rats.

    • Role of leptin G-2548A polymorphism in age- and gender-specific development of obesity

      Adeela Shahid Sobia Rana Saqib Mahmood Shahid Saeed

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      Leptin is involved in the regulation of food intake and energy expenditure, and therefore, is central to adipositysensing pathway. We examined the relationship of the leptin G-2548A polymorphism with obesity and obesityrelated anthropometric and metabolic parameters in a total of 394 (239 obese and 155 non-obese) subjects between 5 and 45 years of age. Body weight, height, waist circumference (WC), hip circumference (HC) and blood pressure (BP) were measured. Body mass index (BMI) and waist-to-hip ratio (WHR) were calculated. Levels of fasting blood glucose (FBG), insulin, leptin and leptin receptor were determined, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The LEP G-2548A polymorphism showed association with obesity in children and adolescents (≤18 years of age) but not in adults. However, analysis by gender stratification revealed association with obesity in girls only. In addition, G-2548A polymorphism showed association with BMI, WC, HC, fasting blood glucose and serum leptin levels. This suggests that G-2548A polymorphism may influence the susceptibility to metabolic disturbances and obesity at an early life. Further investigation with a larger sample size is required to validate the effect of LEP G-2548A polymorphism in obese Pakistani girls.

    • The potential role of myostatin and neurotransmission genes in elite sport performances

      L Filonzi N Franchini M Vaghi S Chiesa F Nonnis Marzano

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      Elite athletes are those who represent their sport at such major competition as the Olympic Games or World contests. The most outstanding athletes appear to emerge as a result of endogenous biologic characteristics interacting with exogenous influences of the environment, often described as a `Nature and Nurture’ struggle. In this work, we assessed the contribution given by 4 genes involved in muscles development (MSTN) and behavioural insights (5HTT, DAT and MAOA) to athletic performances. As for neurotransmission, 5HTT, DAT and MAOA genes have been considered as directly involved in the management of aggressiveness and anxiety.

      Genotypes and allelic frequencies of 5HTTLPR, MAOA-u VNTR, DAT VNTR and MSTN K153R were determined in 50 elite athletes and compared with 100 control athletes.

      In this work we found a significant correlation between the dopamine transporter genotype 9/9 and allele 9 and elite sport performances. On the contrary, no association was found between muscle development regulation or serotonin pathway and elite performances. Our data, for the first time, suggest a strong role of dopamine neurotransmitter in determining sport success, highlighting the role of emotional control and psycological management to reach high-level performances.

    • Diversity of sickle cell trait in Jharkhand state in India: Is it the zone of contact between two geographically and ethnically distinct populations in India?

      Rachana Nagar Rajiva Raman

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      Incidence of sickle cell trait in India is high in peninsular south, south-eastern, central and south-western India, while in north and north-eastern India, it is absent. Unicentric origin of SCD in the tribals of nilgiri hills in southern India has been proposed. The present study on the frequency of HbS trait and 𝛽-globin gene haplotypes was conducted in the tribal-rich states of Chhattisgarh and Jharkhand to get an insight into the uneven distribution of HbS in India. Jharkhand borders with the HbS-high Odisha and Chhattisgarh, and HbS-low UP, Bihar and Bengal. Cellulose acetate gel electrophoresis was performed on the collected blood samples, to detect sickle haemoglobin (HbS) followed by DNA analysis. HbS associated 𝛽-gene haplotype was constructed for the samples positive for HbS and all the tribals by PCR-RFLP. Out of 805 (Chhattisgarh – 261, Jharkhand – 544; >36% tribals) samples analysed HbS frequency was 13% in Chhattisgarh and 3.3% in Jharkhand. Within Jharkhand, frequencies varied considerably from 10% in Tatanagar to nil in Sahibganj. The Arab-India (AI) haplotype of 𝛽-globin cluster occurred in low frequency, confined mainly to Chhattisgarh. The most abundant haplotype in all the populations was the East Asian, + − − − − − +, rare in HbS, mainly in Sahibganj in east Jharkhand, which lacked AI. Our results indicate that besides the heterozygote advantage againstmalaria, the uneven regional distribution of HbS trait is because of restricted movement of two different populations, Dravidian from the south and Tibeto-Burman from the east into the Indianmainland which failed tomeet, we conjecture, due to severe climatic conditions (deserts and heat) prevailing through parts of central India. Apparently, Jharkhand became a zone of contact between them in recent times.

    • Actin filaments as the fast pathways for calcium ions involved in auditory processes

      Miljko V Sataric Dalibor L Sekulic Bogdan M Sataric

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      We investigated the polyelectrolyte properties of actin filaments which are in interaction with myosin motors, basic participants in mechano-electrical transduction in the stereocilia of the inner ear. Here, we elaborated a model in which actin filaments play the role of guides or pathways for localized flow of calcium ions. It is well recognized that calcium ions are implicated in tuning of actin-myosin cross-bridge interaction, which controls the mechanical property of hair bundle. Actin filaments enable much more efficient delivery of calcium ions and faster mechanism for their distribution within the stereocilia. With this model we were able to semiquantitatively explain experimental evidences regarding the way of how calcium ions tune the mechanosensitivity of hair cells.

    • Strain- and context-based 50 kHz ultrasonic vocalizations and anxiety behaviour in the Wistar-Kyoto rat

      Rashmi Madhava Rao Monika Sadananda

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      Rodent ultrasonic vocalizations (USVs) are influenced by immediate, prior contexts and have emerged as important indicators that monitor an individual’s ‘state’. They also index direct reflections of inherent ‘trait’ and are suggested to constitute non-invasive read-outs of pathological conditions. Analysis of USVs emitted under particular contexts could help discern strain-specific differences and existence of individual USV profiles. USVs of the Wistar-Kyoto (WKY) strain, a putative model of depression, could indicate social communication deficits. In the cage, USV emission was significantly reduced in WKYs. An elevated plus maze exposure led to no change in USV emission in WKYs, while it significantly reduced USVs in Wistars. Re-exposure induced strain-specific differences in behaviour and total calling time. Sonographic patterns indicated that the predominant USV subtype were flat 50 kHz USVs. EPMexposure induced a reduction in peak amplitude in WKY USVs and in USV length in both strains. USV peak frequency and amplitude, genetically determined spectral features, were strain-specific, while bandwidth and temporal features such as total calling time and USV duration were context-dependent. WKY USVs demonstrated characteristic spectral structures such as increased call length and reduced peak frequency while other parameters were not quantitatively different, reflecting the shared phylogeny between Wistars and WKYs.

    • FASTR: A novel data format for concomitant representation of RNA sequence and secondary structure information

      Tungadri Bose Anirban Dutta Mohammed Mh Hemang Gandhi Sharmila S Mande

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      Given the importance of RNA secondary structures in defining their biological role, it would be convenient for researchers seeking RNA data if both sequence and structural information pertaining to RNA molecules are made available together. Current nucleotide data repositories archive only RNA sequence data. Furthermore, storage formats which can frugally represent RNA sequence as well as structure data in a single file, are currently unavailable. This article proposes a novel storage format, `FASTR’, for concomitant representation of RNA sequence and structure. The storage efficiency of the proposed FASTR format has been evaluated using RNA data from various microorganisms. Results indicate that the size of FASTR formatted files (containing both RNA sequence as well as structure information) are equivalent to that of FASTA-format files, which contain only RNA sequence information. RNA secondary structure is typically represented using a combination of a string of nucleotide characters along with the corresponding dot-bracket notation indicating structural attributes. `FASTR’ – the novel storage format proposed in the present study enables a frugal representation of both RNA sequence and structural information in the form of a single string. In spite of having a relatively smaller storage footprint, the resultant `fastr’ string(s) retain all sequence as well as secondary structural information that could be stored using a dot-bracket notation. An implementation of the `FASTR’ methodology is available for download at http://metagenomics.atc.tcs.com/compression/fastr.

    • The emerging roles of inositol pyrophosphates in eukaryotic cell physiology

      Swarna Gowri Thota Rashna Bhandari

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      Inositol pyrophosphates are water soluble derivatives of inositol that contain pyrophosphate or diphosphate moieties in addition to monophosphates. The best characterised inositol pyrophosphates, are IP7 (diphosphoinositol pentakisphosphate or PP-IP5), and IP8 (bisdiphosphoinositol tetrakisphosphate or (PP)2-IP4). These energy-rich small molecules are present in all eukaryotic cells, from yeast to mammals, and are involved in a wide range of cellular functions including apoptosis, vesicle trafficking, DNA repair, osmoregulation, phosphate homeostasis, insulin sensitivity, immune signalling, cell cycle regulation, and ribosome synthesis. Identified more than 20 years ago, there is still only a rudimentary understanding of the mechanisms by which inositol pyrophosphates participate in these myriad pathways governing cell physiology and homeostasis. The unique stereochemical and bioenergetic properties these molecules possess as a consequence of the presence of one or two pyrophosphate moieties in the vicinity of densely packed monophosphates are likely to form the molecular basis for their participation in multiple signalling and metabolic pathways. The aim of this review is to provide first time researchers in this area with an introduction to inositol pyrophosphates and a comprehensive overview on their cellular functions.

    • Approaches for targeted proteomics and its potential applications in neuroscience

      Sumit Sethi Dipti Chourasia Ishwar S Parhar

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      An extensive guide on practicable and significant quantitative proteomic approaches in neuroscience research is important not only because of the existing overwhelming limitations but also for gaining valuable understanding into brain function and deciphering proteomics from the workbench to the bedside. Early methodologies to understand the functioning of biological systems are now improving with high-throughput technologies, which allow analysis of various samples concurrently, or of thousand of analytes in a particular sample. Quantitative proteomic approaches include both gel-based and non-gel-based methods that can be further divided into different labelling approaches. This review will emphasize the role of existing technologies, their advantages and disadvantages, as well as their applications in neuroscience. This review will also discuss advanced approaches for targeted proteomics using isotope-coded affinity tag (ICAT) coupled with laser capture microdissection (LCM) followed by liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis. This technology can further be extended to single cell proteomics in other areas of biological sciences and can be combined with other ‘omics’ approaches to reveal the mechanism of a cellular alterations. This approach may lead to further investigation in basic biology, disease analysis and surveillance, as well as drug discovery. Although numerous challenges still exist, we are confident that this approach will increase the understanding of pathological mechanisms involved in neuroendocrinology, neuropsychiatric and neurodegenerative disorders by delivering protein biomarker signatures for brain dysfunction.

    • Microirradiation techniques in radiobiological research

      Guido A Drexler Miguel J Ruiz-Gómez

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      The aim of this work is to review the uses of laser microirradiation and ion microbeam techniques within the scope of radiobiological research. Laser microirradiation techniques can be used for many different purposes. In a specific condition, through the use of pulsed lasers, cell lysis can be produced for subsequent separation of different analytes. Microsurgery allows for the identification and isolation of tissue sections, single cells and subcellular components, using different types of lasers. The generation of different types of DNA damage, via this type of microirradiation, allows for the investigation of DNA dynamics. Ion microbeams are important tools in radiobiological research. There are only a limited number of facilities worldwide where radiobiological experiments can be performed. In the beginning, research was mostly focused on the bystander effect. Nowadays, with more sophisticated molecular and cellular biological techniques, ion microirradiation is used to unravel molecular processes in the field of radiobiology. These include DNA repair protein kinetics or chromatin modifications at the site of DNA damage. With the increasing relevance of charged particles in tumour therapy and new concepts on how to generate them, ion microbeam facilities are able to address unresolved questions concerning particle tumour therapy.

    • Genome engineering and parthenocloning in the silkworm, Bombyx mori

      Valeriya Zabelina Vyacheslav Klymenko Toshiki Tamura Karina Doroshenko Haoyuan Liang Hideki Sezutsu František Sehnal

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      Genetic engineering of the silkworm, Bombyx mori, opens door to the production of new kinds of silk and to the use of silkworms as proteosynthetic bioreactors. The insertion of foreign genes into silkworm genome and the control of their expression by diverse promoters have become possible but are not yet efficient enough for commercial use. Several methods of gene targeting are being developed to minimize position effect on transgene expression and facilitate cloning. Parthenocloning can be exploited to conserve genetic traits and improve selection and amplification of clones containing genes of interest. Some silkworm clones have been bred for decades as genetically stable female stocks whose unfertilized eggs are induced to develop by heat-shock treatment. Any exclusively female generation contains exact copies of the maternal clone-founder genome. Ovaries transplanted in either direction between the standard and the parthenogenetic genotypes yield eggs capable of parthenocloning. In addition, use ofmale larvae as ovary recipients eliminates diapause in eggs produced in the implants. Unfertilized eggs of some silkworm clones respond also to the cold-shock treatment by producing homozygous fertile sons; cloned females can be crossed with their parthenogenetic sons to obtain progeny homozygous for the transgene in both sexes. Rational exploitation of available parthenozygous pools and the use of parthenocloning methods enable rapid fixation and maintenance of the desired genotypes.

    • Cancer research in need of a scientific revolution: Using `paradigm shift’ as a method of investigation

      Didier Wion Florence Appaix Meriwether Burruss François Berger Boudewijn Van Der Sanden

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      Despite important human and financial resources and considerable accumulation of scientific publications, patents, and clinical trials, cancer research has been slow in achieving a therapeutic revolution similar to the one that occurred in the last century for infectious diseases. It has been proposed that science proceeds not only by accumulating data but also through paradigm shifts. Here, we propose to use the concept of `paradigm shift’ as a method of investigation when dominant paradigms fail to achieve their promises. The first step in using the `paradigm shift’ method in cancer research requires identifying its founding paradigms. In this review, two of these founding paradigms will be discussed:

      1. the reification of cancer as a tumour mass and
      2. the translation of the concepts issued from infectious disease in cancer research.

      We show how these founding paradigms can generate biases that lead to over-diagnosis and over-treatment and also hamper the development of curative cancer therapies. We apply the `paradigm shift’ method to produce perspective reversals consistent with current experimental evidence. The `paradigm shift’ method enlightens the existence of a tumour physiologic–prophylactic–pathologic continuum. It integrates the target/antitarget concept and that cancer is also an extracellular disease. The `paradigm shift’ method has immediate implications for cancer prevention and therapy. It could be a general method of investigation for other diseases awaiting therapy.

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