Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

Temperature-based phenology model for predicting the present and future establishment and distribution of recently invasive Spodoptera frugiperda (J. E. Smith) in India.

Fall armyworm, Spodoptera frugiperda (J. E. Smith) is a polyphagous and highly destructive invasive insect pest of many crops. It was recently introduced into India and widely reported in almost all parts of India. Development of a temperature-based phenology model for predicting its rate of development and distribution will help in understanding the establishment and further spread of introduced invasive insect pests. Development, survival and reproduction parameters of S. frugiperda at six constant temperature conditions (15, 20, 25, 27, 30 and 35°C) were investigated and further validated with data generated under fluctuating temperature conditions. The estimated lower developmental threshold temperatures were 12.1°C for eggs, 11°C for larvae, 12.2°C for pupae, 15.13°C for males and 12.66°C for females. Degree-day (DD) requirements for the development of the different stages of S. frugiperda were 50, 250 and 200 DD for egg, larva and pupa, respectively. The best-fitted functions were compiled for each life stage to yield a phenology model, which was stochastically simulated to estimate the life table parameters. The developed phenology model predicted temperature ranges between 27 and 30°C as favourable for S. frugiperda development, survival and reproduction. The results revealed that maximum net reproductive rate (215.66 females/female/generation) and total fecundity (981.08 individuals/female/generation) were attained at 30°C constant temperature. The mean length of generations decreased from 74.29 days at 15°C to 38.74 days at 30°C. The maximum intrinsic rate of increase (0.138 females/female/day) and shortest doubling time (4.9 days) were also observed at 30°C. Results of simulated life table parameters showed high temperature-dependent development of S. frugiperda and complete development within all the tested constant temperature ranges (15-35°C). Simulated life table parameters for predicting risk indices of S. frugiperda in India indicated a significant increase in activity indices and establishment risk indices with a higher number of generations during future (2050 and 2070) climatic change scenarios compared to present conditions. Our results indicate that India will be highly suitable for the establishment and survival of S. frugiperda in future time periods.

Heavy Metals Scavenging Potential of Trichoderma asperellum and Hypocrea nigricans Isolated from Acid Soil of Jharkhand.

Trichoderma asperellum (NAIMCC-F-03167) and Hypocrea nigricans (NAIMCC-F-03168) were isolated from the acidic soil of the vicinity of Litchi orchard, Ranchi, Jharkhand and were characterized on the basis of morphological, molecular and biochemical features. Both strains are fast growing, light to dark green, highly sporulative and have ability to cover 90 mm Petri dish within 96 h of inoculation. Biochemcial estimation of both isolates indicated significant cellulase and phosphate solubilisation activity. Highest cellulase activity was observed in T. asperellum (5.63 cm) followed by H. nigricans (5.10 cm) and phosphate solubilisation index was observed maximum in T. asperellum (1.93) followed by H. nigricans (1.39). Moreover, these isolates were molecularly identified on the basis of ribosomal DNA based sequences database and phylogenetic analysis in NCBI GenBank as T. asperellum (NCBI-KM 438015) and H. nigricans (NCBI-KJ910335). Negetive effect on sporulation of Lead (Pb) and Cadmium (Cd) was observed while in heavy metal scavenging potential, T. asperellum (88.9% Cd) showed highest scavenging potential followed by H. nigricans (87.2% Cd) while in Pb scavenging potential, H. nigricans (88% Pb) followed highest scavenging potential followed by T. asperellum (81.30% Pb) after 21 days of inoculation from 30 µg/ml heavy metals concentrated broth medium. If both potential bioagents can apply in Cd and Pb affected soil/water will be helpful in scavenging of heavy metals as well as management of phosphorus deficiency and soilborne fungal diseases.

Using PCR-PIRA based genotyping for identifying complex vertebral malformation allele in Frieswal young bulls in India.

Complex vertebral malformation (CVM) has considerable economic impact on dairy cattle breeding due to extensive use of artificial insemination (AI). Identifying the carrier is an important factor to reduce the incidence of the genetic disorder. The study was conducted to identify the carriers of CVM in Frieswal cattle by polymerase chain reaction-primer-introduced restriction analysis (PCR-PIRA) method, which was further confirmed by sequencing. Carrier prevalence of 1% was observed in the Frieswal cattle. The results of the study clearly demonstrated the existence of carriers of CVM among Frieswal bull calves. Due to the widespread use of AI it is recommended to screen young bulls at early stages for this defective allele in order to avoid its rapid spread within the population.

Population genetic structure of cotton pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) using mitochondrial cytochrome oxidase I (COI) gene sequences from India.

Pink bollworm (PBW), Pectinophora gossypiella is one of the most destructive pest's globally inflicting huge economic losses in cotton even during later stages of crop growth. In the present investigation, the population genetic structure, distribution, and genetic diversity of P. gossypiella in cotton growing zones of India using partial mitochondrial DNA cytochrome oxidase-I (COI) gene was addressed. The overall haplotype (Hd), number of nucleotide differences (K), and nucleotide diversity (π) were 0.3028, 0.327, and 0.00047, respectively which suggest that entire population exhibited low level of genetic diversity. Zone-wise clustering of population revealed that central zone recorded low level of Hd (0.2730) as compared to north (0.3619) and south (0.3028) zones. The most common haplotype (H1) reported in all 19 locations could be proposed as ancestral/original haplotype. This haplotype with one mutational step formed star-like phylogeny connected with 11 other haplotypes. The phylogenetic relationship studies revealed that most haplotypes of populations are closely related to each other. Haplotype 5 was exclusively present in Dharwad (South zone) shared with populations of Hanumangarh and Bathinda (North zone). The result indicated that there is no isolation by distance effect among the Indian populations of PBW. The present study reports a low genetic diversity among PBW populations of India and H1, as ancestral haplotype from which other haplotypes have evolved suggests that the migration and dispersal over long distance and invasiveness are major factors.

SynGAP isoforms exert opposing effects on synaptic strength.

Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5' rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing.

Exploring pharmacological significance of chalcone scaffold: a review.

Chalcones (1,3-diaryl-2-propen-1-ones) and their heterocyclic analogues, belong to the flavonoid family, which possess a number of interesting biological properties such as antioxidant, cytotoxic, anticancer, antimicrobial, antiprotozoal, antiulcer, antihistaminic and anti-inflammatory activities. Several pure chalcones have been approved for clinical use or tested in humans. Clinical trials have shown that these compounds reached reasonable plasma concentration and are well-tolerated. For this reason they are an object of continuously growing interest amongst the scientists. However, much of the pharmacological potential of chalcones is still not utilized. The purpose of this review is to provide an overview of the pharmacological activity of naturally occurring and synthetic chalcones. This review highlights more recent pharmacological screening of these compounds, their mechanisms of action and relevant structure-activity relationships.

De novo CNV analysis implicates specific abnormalities of postsynaptic signalling complexes in the pathogenesis of schizophrenia.

A small number of rare, recurrent genomic copy number variants (CNVs) are known to substantially increase susceptibility to schizophrenia. As a consequence of the low fecundity in people with schizophrenia and other neurodevelopmental phenotypes to which these CNVs contribute, CNVs with large effects on risk are likely to be rapidly removed from the population by natural selection. Accordingly, such CNVs must frequently occur as recurrent de novo mutations. In a sample of 662 schizophrenia proband-parent trios, we found that rare de novo CNV mutations were significantly more frequent in cases (5.1% all cases, 5.5% family history negative) compared with 2.2% among 2623 controls, confirming the involvement of de novo CNVs in the pathogenesis of schizophrenia. Eight de novo CNVs occurred at four known schizophrenia loci (3q29, 15q11.2, 15q13.3 and 16p11.2). De novo CNVs of known pathogenic significance in other genomic disorders were also observed, including deletion at the TAR (thrombocytopenia absent radius) region on 1q21.1 and duplication at the WBS (Williams-Beuren syndrome) region at 7q11.23. Multiple de novos spanned genes encoding members of the DLG (discs large) family of membrane-associated guanylate kinases (MAGUKs) that are components of the postsynaptic density (PSD). Two de novos also affected EHMT1, a histone methyl transferase known to directly regulate DLG family members. Using a systems biology approach and merging novel CNV and proteomics data sets, systematic analysis of synaptic protein complexes showed that, compared with control CNVs, case de novos were significantly enriched for the PSD proteome (P=1.72 × 10⁻6. This was largely explained by enrichment for members of the N-methyl-D-aspartate receptor (NMDAR) (P=4.24 × 10⁻6) and neuronal activity-regulated cytoskeleton-associated protein (ARC) (P=3.78 × 10⁻8) postsynaptic signalling complexes. In an analysis of 18 492 subjects (7907 cases and 10 585 controls), case CNVs were enriched for members of the NMDAR complex (P=0.0015) but not ARC (P=0.14). Our data indicate that defects in NMDAR postsynaptic signalling and, possibly, ARC complexes, which are known to be important in synaptic plasticity and cognition, play a significant role in the pathogenesis of schizophrenia.

Effect of tamoxifen treatment on motility related proteins in rat spermatozoa.

The study was undertaken to identify the effect of tamoxifen on the expression and phosphorylation of motility related proteins in the adult male rats. For this purpose, tamoxifen, at a dose of 0.4 mg/kg/day, was administered per os to the male rats for a period of 60 days. Cauda sperms, epididymal fluid and tissue proteins were extracted and analyzed by electrophoresis. Testicular tissues fixed in paraffin wax were analyzed for changes in the immunoexpression of interstitial tissue estrogen receptor alpha. Phosphorylation pattern of sperm proteins was studied in vitro after incubating with 32P-ATP. The expression of dynein and tubulin in sperms, and estrogen receptors in epididymis were analyzed by immunoblotting. Tamoxifen treatment did not alter the protein profile in the cauda sperms, epididymal fluid and tissues. Endogenous phosphorylation pattern of sperm proteins in vitro was also not affected, though it is possible that 32P incorporation observed in the 66 kDa protein could be estrogen receptor. Expression of sperm dynein, tubulin and epididymal estrogen receptors was unchanged as was the expression of testicular estrogen receptors. It was concluded that tamoxifen administration alters forward motility pattern characteristic of cauda sperm without any demonstrable change in the expression or activation of motility related proteins and the phosphorylation of the sperm estrogen receptors may be involved in the regulation of sperm motility.

Antifertility effects of fluphenazine in adult male rats.

The underlying mechanisms in human infertility associated with hyperprolactinemia have yet to be established. Hyperprolactinemia is a known side-effect of fluphenazine, a broad spectrum, long-acting phenothiazine known to be D2 dopamine receptor antagonist. Dose-related effects of fluphenazine decanoate were ascertained on the fertility of 60-day treated, adult male rats. Significant increase in the serum levels of prolactin and decrease in the levels of LH and FSH were seen at doses of 1-3 mg/kg/day. No effect was evident on the serum testosterone (T) and estradiol. The tissue levels of Inhibins were not affected. The weights of testes, epididymides, seminal vesicles, ventral prostate, adrenal and pituitary glands were not affected. Testicular histology showed sloughing indicating the sensitivity of this parameter to FSH deficiency. Mating occurred within 10 days of cohabitation in the control and 1-2 mg/kg/day treated groups but delayed in the 3 mg/kg/day treated group with a significant effect on potency. Implantation sites, litter size and fertility index were significantly reduced at 2-3 mg/kg/day doses of fluphenazine. No effects however were seen on sperm counts or motility whereas morphological changes were apparent in the acrosome. Chromatin decondensation in vitro was enhanced and sperm chromatin structure assay revealed DNA denaturation. Hypothalamic tyrosine hydroxylase levels were increased in 1-3 mg/kg/day dose range. Hyperprolactinemic males sired fewer pups as compared to controls. Hypothalamic tyrosine hydroxylase was upregulated at all the doses. The antifertility effects of fluphenazine-induced hyperprolactinemia appeared to be unrelated to testosterone (T). In addition, FSH decrease might have affected the intrinsic sperm quality and thereby reduced litter size.

Estrogen receptor, calcium mobilization and rat sperm motility.

Oral treatment with 0.4 mg/kg/day of tamoxifen citrate, an antiestrogen, has been reported to reduce the fertility of adult male rat, presumably through estrogen receptors expressed throughout the male reproductive tract. During the course of these studies, tamoxifen was observed to gradually alter the pattern of sperm motility in the cauda epididymides without reducing sperm counts. Studies were carried out to understand the mechanism involved in tamoxifen induced change in the sperm motility pattern. In order to study the direct effects of tamoxifen on motility, biochemical levels/activities of sperm calcium, cAMP, phosphodiesterase and dynein ATPase, normally implicated in sperm motility were studied In view of the fact that tamoxifen is a ligand of estrogen receptor, estrogen receptor alpha protein and transcript were localized on rat sperm membrane and the effect of tamoxifen studied. The present study demonstrated presence of estrogen receptor protein and mRNA in the rat sperm by immunofluorescence, western blotting and in situ hybridization respectively. Specificity of sperm estrogen receptors was confirmed by conventional binding studies using [3H]-estradiol. There was no effect of tamoxifen treatment on estrogen receptors in rat sperms. Biochemical analysis of the sperms from tamoxifen treated cauda epididymides revealed a significant increase in the levels of calcium and cAMP. A significant reduction was also apparent in the activity of dynein ATPase. Tamoxifen treatment did not alter phosphodiesterase activity. Estrogen receptors could be identified both in the control as well as tamoxifen treated rat sperms. It was concluded that tamoxifen treatment mobilized calcium from the intra- or extra-cellular pools with a concomitant increase in cAMP and presumably activation of PKA (protein kinase A). Tamoxifen altered the pattern of sperm motility through a calcium induced block in the activity of dynein ATPase, presumably through the activation of sperm phosphatase. The putative estrogen receptor mediated signal transduction pathway appears to be directly affected in the tamoxifen treated, sub-motile rat sperm.

Interrogating the human genome using uninterpreted mass spectrometry data.

The public availability of a draft assembly of the human genome has enabled us to demonstrate, for the first time, the feasibility of searching a complete, unmasked eukaryotic genome using uninterpreted mass spectrometry data. A complex LC-MS/MS data set, containing peptides from at least 22 human proteins, was searched against a comprehensive, nonidentical protein database, an expressed sequence tag (EST) database, and the International Human Genome Project draft assembly of the human genome. The results from the three searches are compared in detail, and the merits of the different databases for this application are discussed. In the case of the EST database, the UniGene index provided a method of simplifying and summarising the search results. In the case of the genomic DNA, the presence of introns prevented matching of roughly one quarter of the spectra, but the technique can provide primary experimental verification of predicted coding sequences, and has the potential to identify novel coding sequences.

Proteomics characterization of abundant Golgi membrane proteins.

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Matching peptide mass spectra to EST and genomic DNA databases.

The use of mass spectrometry data to search molecular sequence databases is a well-established method for protein identification. The technique can be extended to searching raw genomic sequences, providing experimental confirmation or correction of predicted coding sequences, and has the potential to identify novel genes and elucidate splicing patterns.

Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

Applications of protein mass spectrometry in cell biology.

Advances in mass spectrometry combined with accelerated progress in genome sequencing projects have facilitated the rapid identification of proteins by enzymatic digestion, mass analysis, and sequence database searching. Applications for this technology range from the surveillance of protein expression in cells, tissues, and whole organisms, to the identification of proteins and posttranslational modifications. Here we consider practical aspects of the application of mass spectrometry in cell biology and illustrate these with examples from our own laboratories.

E. C. T. And drug induced parkinsonism.

A cross-sectional evaluation of the presence of drug induced parkinsonian symptoms in hospitalized patients was done. Patients who had received two or more ECTs had lower scores of parkinsonism when compared to those who were net receiving ECT. Since the patient groups were comparable on parameters which would influence the occurrence and development of drug induced parkinsonism, the lowered scores in one group could be attributed to the effect of ECT.