Evolution, Expression, and Function of Non-neuronal Ligand-Gated Chloride Channels in Drosophila melanogaster

Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927 and CG11340, are highly expressed in non-neuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing and in situ hybridization to examine cell-type and tissue specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including salivary gland, ejaculatory duct, malpighian tubules and early midgut. CG11340 is found in malpighian tubules, and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific non-neuronal role for this subunit in D. melanogaster. Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets.

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Histone H1 Limits DNA Methylation in Neurospora crassa

Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stochiometery of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in wildtype and the hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the hH1 mutant. Together, these data suggest that H1 acts as a non-specific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.

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Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs

Drosophila larval crawling is an attractive system to study patterned motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors including head casts, turning, and feeding. It is likely that some neurons are used in all these behaviors (e.g. motor neurons), but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines--chosen for sparse neuronal expression--to express the warmth-inducible neuronal activator TrpA1 and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°C). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program.

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Identifying Genetic Signatures of Natural Selection Using Pooled Population Sequencing in Picea abies

The joint inference of selection and past demography remain a costly and demanding task. We used next generation sequencing of two pools of 48 Norway spruce mother trees, one corresponding to the Fennoscandian domain and the other to the Alpine domain, to assess nucleotide polymorphism at 88 nuclear genes. These genes are candidate genes for phenological traits and most belong to the photoperiod pathway. Estimates of population genetic summary statistics from the pooled data are similar to previous estimates suggesting that pooled sequencing is reliable. The non-synonymous SNPs tended to have both lower frequency differences and lower F_ST values between the two domains than silent ones. These results suggest the presence of purifying selection. The divergence between the two domains based on synonymous changes was around 5 million years, a time similar to a recent phylogenetic estimate of 6 million years, but much larger than earlier estimates based on isozymes. Two approaches, one of them novel and that considers both F_ST and difference in allele frequencies between the two domains, were used to identify SNPs potentially under diversifying selection. SNPs from around 20 genes were detected, including genes previously identified as main target for selection such as PaPRR3 and PaGI.

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Budding Yeast SLX4 Contributes to the Appropriate Distribution of Crossovers and Meiotic Double-strand Break Formation on Bivalents during Meiosis

The number and distribution of meiosis crossover (CO) events on each bivalent are strictly controlled by multiple mechanisms to assure proper chromosome segregation during the first meiotic division. In Saccharomyces cerevisiae, Slx4 is a multi-functional scaffold protein for structure-selective endonucleases, such as Slx1 and Rad1 (which involved in DNA damage repair) and also is a negative regulator of the Rad9-dependent signaling pathway with Rtt107. Slx4 has been believed to facilitate only a minor role in meiotic recombination. Here we report that Slx4 is involved in proper intra-chromosomal distribution of meiotic CO formation, especially in the region near centromeres. We observed an increase in uncontrolled CO formation only in the region near the centromere in the slx4 mutant. Interestingly, this phenomenon was not observed in the slx1, rad1, or rtt107 mutants. In addition, we observed a reduced number of DNA double-strand breaks (DSBs) and altered meiotic DSB distribution on chromosomes in the slx4 mutant. This suggests that multi-functional Slx4 is required for proper CO formation and meiotic DSB formation.

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Partial Functional Diversification of Drosophila melanogaster Septins Sep2 and Sep5

The septin family of hetero-oligomeric-complex-forming proteins can be divided into subgroups, and subgroup members are interchangeable at specific positions in the septin complex. Drosophila melanogaster has five septin genes, including the two SEPT6 subgroup members Sep2 and Sep5. We previously found that Sep2 has a unique function in oogenesis, which is not performed by Sep5. Here, we find Sep2 is uniquely required for follicle cell encapsulation of female germline cysts, and that Sep2 and Sep5 are redundant for follicle cell proliferation. The five D. melanogaster septins localize similarly in oogenesis, including as rings flanking the germline ring canals. Pnut fails to localize in Sep5; Sep2 double mutant follicle cells, indicating that septin complexes fail to form in the absence of both Sep2 and Sep5. We also find that mutations in septins enhance the mutant phenotype of bazooka, a key component in the establishment of cell polarity, suggesting a link between septin function and cell polarity. Overall, this work suggests Sep5 has undergone partial loss of ancestral protein function, and demonstrates redundant and unique functions of septins.

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CORRELATION OF COAGULATION PROFILE IN LIVER DISEASE PATIENTS IN A TERTIARY CARE HOSPITAL

2016-05-06T23-58-19Z
Source: International Journal of Current Research and Review
G. K. Tripathi, Ankita A. Katara, Shweta V. Awasthi, Shashank V. Srivastava.
Objective: To determine the coagulation abnormalities among liver disease patients admitted in a tertiary care hospital of Hind Institute of Medical Sciences, Barabanki, Uttar Pradesh (India) for a period of 1 year (January 2015 to December 2015). Methods: Patients were labeled as liver disease patients on the basis of Liver Function Tests (LFT) done in the hospital at the time of admission. Patients blood were collected in sodium citrate and plain vaccutainer for coagulation tests [prothrombin time (PT), activated partial thromboplastin time (aPTT)] and liver function tests respectively. The prothrombin time ratio was calculated and then used to calculate the international normalized ratio. Following parameters were included in Liver Function Test, serum bilirubin, SGOT, SGPT, ALP, Total Protein and serum albumin. Results: Total number of cases studied in a period of 1 year (January 2015 to December 2015) were 311. In 284(91.3%) cases both LFT and coagulation profile were done. In 261 (83.9%) cases (out of total 311 cases) coagulation tests were normal and in 50 (16.1%) cases coagulation tests were found to be deranged. Out of these 50 cases, PT was raised in 44(100%) cases, APTT was raised in 21(95.4%) cases and both PT and APTT were raised in 15(93.75%) cases. Conclusion: Coagulation abnormalities were profound patients with chronic liver diseases. In case of acute liver disease ,usually PT( Prothrombin Time) is increased, but aPTT (activated partial thromboplastin time)is found to be normal. aPTT (activated partial thromboplastin time)is usually increased in chronic liver diseases, but the PT(Prothrombin Time) prolongation is usually not seen in initial stages of chronic liver disease until the stage of liver fibrosis and cirrhosis is reached. Nevertheless, these parameters (PT and aPTT) were still widely used as prognostic markers in liver disease patients..


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