The DNA damage effector Chk1 kinase regulates Cdc14B nucleolar shuttling during cell cycle progression

Chk1 is a critical effector of DNA damage checkpoints necessary for the maintenance of chromosome integrity during cell cycle progression. Here we report, that Chk1 co-localized with the nucleolar marker, fibrillarin in response to radiation-induced DNA damage in human cells. Interestingly, in vitro studies using GST pull down assays identified the dual-specificity serine/threonine nucleolar phosphatase Cdc14B as a Chk1 substrate. Furthermore, Chk1, but not a kinase-dead Chk1 control, was shown to phosphorylate Cdc14B using an in vitro kinase assay. Co-immunoprecipitation experiments using exogenous Cdc14B transfected into human cells confirmed the interaction of Cdc14B and Chk1 during cell cycle. In addition, reduction of Chk1 levels via siRNA or UCN-01 treatment demonstrated that Chk1 activation following DNA damage was required for Cdc14B export from the nucleolus. these studies have revealed a novel interplay between Chk1 kinase and Cdc14B phosphatase involving radiation-induced nucleolar shuttling to facilitate error-free cell cycle progression and prevent genomic instability.Key words: Chk1, nucleoli, DNA damage, Cdc14B, genomic instabiliy, cell cycle     

Meraculous: de novo genome assembly with short paired-end reads

We describe a new algorithm, meraculous, for whole genome assembly of deep paired-end short reads, and apply it to the assembly of a dataset of paired 75-bp Illumina reads derived from the 15.4 megabase genome of the haploid yeast Pichia stipitis. More than 95% of the genome is recovered, with no errors; half the assembled sequence is in contigs longer than 101 kilobases and in scaffolds longer than 269 kilobases. Incorporating fosmid ends recovers entire chromosomes. Meraculous relies on an efficient and conservative traversal of the subgraph of the k-mer (deBruijn) graph of oligonucleotides with unique high quality extensions in the dataset, avoiding an explicit error correction step as used in other short-read assemblers. A novel memory-efficient hashing scheme is introduced. The resulting contigs are ordered and oriented using paired reads separated by ～280 bp or ～3.2 kbp, and many gaps between contigs can be closed using paired-end placements. Practical issues with the dataset are described, and prospects for assembling larger genomes are discussed.     

Genotype-Phenotype Correlations in CYP1B1-Associated Primary Congenital Glaucoma Patients Representing Two Large Cohorts from India and Brazil

BackgroundPrimary congenital glaucoma (PCG), occurs due to the developmental defects in the trabecular meshwork and anterior chamber angle in children. PCG exhibits genetic heterogeneity and the CYP1B1 gene has been widely implicated worldwide. Despite the diverse mutation spectra, the clinical implications of these mutations are yet unclear. The present study attempted to delineate the clinical profile of PCG in the background of CYP1B1 mutations from a large cohort of 901 subjects from India (n=601) and Brazil (n=300).MethodsGenotype-phenotype correlations was undertaken on clinically well characterized PCG cases from India (n=301) and Brazil (n=150) to assess the contributions of CYP1B1 mutation on a set of demographic and clinical parameters. The demographic (gender, and history of consanguinity) and quantitative clinical (presenting intraocular pressure [IOP] and corneal diameter [CD]) parameters were considered as binary and continuous variables, respectively, for PCG patients in the background of the overall mutation spectra and also with respect to the prevalent mutations in India (R368H) and Brazil (4340delG). All these variables were fitted in a multivariate logistic regression model using the Akaike Information Criterion (AIC) to estimate the adjusted odds ratio (OR) using the R software (version 2.14.1).ResultsThe overall mutation spectrum were similar across the Indian and Brazilian PCG cases, despite significantly higher number of homozygous mutations in the former (p=0.024) and compound heterozygous mutations in the later (p=0.012). A wide allelic heterogeneity was observed and only 6 mutations were infrequently shared between these two populations. The adjusted ORs for the binary (demographic) and continuous (clinical) variables did not indicate any susceptibility to the observed mutations (p>0.05).ConclusionsThe present study demonstrated a lack of genotype-phenotype correlation of the demographic and clinical traits to CYP1B1 mutations in PCG at presentation. However, the susceptibility of these mutations to the long-term progression of these traits are yet to be deciphered.     

Differential Effects of Human L1CAM Mutations on Complementing Guidance and Synaptic Defects in Drosophila melanogaster

A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.     

Cellulase stimulation during biodegradation of lignocellulosic residues at increased biomass loading

Microbial degradation of lignocellulosic biomass is primarily affected by the composition and structure of biomass, as well as enzyme activities that are influenced by the presence of in-process degradation products. This study focuses on the latter, and demonstrates that cellulase activity of Neurospora discreta is stimulated in the presence of in-process soluble lignin degradation products. Two types of biomass - cocopeat and sugarcane bagasse, with contrasting lignin content and cellulose structure were tested at two biomass loadings each. At the higher biomass loading, cocopeat showed the highest amount of hydrolyzed cellulose and cellulase activity, despite its low cellulose content and recalcitrant cellulose structure. A strong positive correlation was revealed between the amount of in-process degraded lignin and cellulase activity, indicating a stimulatory effect on cellulase, which contradicts most previous literature. Furthermore, the causal relationship between the amount of degraded lignin and cellulase activity was established in a model system of commercial cellulase and standard soluble lignin. This work could pave the way for using biomass loading as a process lever to enhance cellulose hydrolysis in microbial conversion of lignocellulosic biomass.     

E-cadherin dis-engagement activates the Rap1 GTPase

E-cadherin based adherens junctions are finely regulated by multiple cellular signaling events. Here we show that the Ras-related Rap1 GTPase is enriched in regions of nascent cell-cell contacts and strengthens E-cadherin junctions: constitutively active Rap1 expressing MDCK cells exhibit increased junctional contact and resisted calcium depletion-induced cell-cell junction disruption. E-cadherin disengagement activated Rap1 and this correlated with E-cadherin association with the Rap GEFs, C3G and PDZ-GEF I. PDZ-GEF I associated with E-cadherin and beta-catenin whereas C3G interaction with E-cadherin did not involve beta-catenin. Knockdown of PDZ-GEF I in MDCK cells decreased Rap1 activity following E-cadherin junction disruption. We hereby show that Rap1 plays a role in the maintenance and repair of E-cadherin junctions and is activated via an "outside-in" signaling pathway initiated by E-cadherin and mediated at least in part by PDZ-GEF I.     

Exposure to titanium dioxide and other metallic oxide nanoparticles induces cytotoxicity on human neural cells and fibroblasts

The use of titanium dioxide (TiO₂) in various industrial applications (eg, production of paper, plastics, cosmetics, and paints) has been expanding thereby increasing the occupational and other environmental exposure of these nanoparticles to humans and other species. However, the health effects of exposure to TiO₂ nanoparticles have not been systematically assessed even though recent studies suggest that such exposure induces inflammatory responses in lung tissue and cells. Because the effects of such nanoparticles on human neural cells are unknown, we have determined the putative cytotoxic effects of these nanoparticles on human astrocytes-like astrocytoma U87 cells and compared their effects on normal human fibroblasts. We found that TiO₂ micro- and nanoparticles induced cell death on both human cell types in a concentration-related manner. We further noted that zinc oxide (ZnO) nanoparticles were the most effective, TiO₂ nanoparticles the second most effective, and magnesium oxide (MgO) nanoparticles the least effective in inducing cell death in U87 cells. The cell death mechanisms underlying the effects of TiO₂ micro- and nanoparticles on U87 cells include apoptosis, necrosis, and possibly apoptosis-like and necrosis-like cell death types. Thus, our findings may have toxicological and other pathophysiological implications on exposure of humans and other mammalian species to metallic oxide nanoparticles.     

Calmodulin-like protein and the phospholipids of Mycobacterium smegmatis

When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed. Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above). Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium. Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM. Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium. Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.     

Quorum sensing involvement in response surface methodology for optimisation of sclerotiorin production by Penicillium sclerotiorum in shaken flasks and bioreactors

Sclerotiorin, an azaphilone produced by some filamentous fungi including Penicillium sclerotiorum, is a pigment with variety of biological activities including lipoxygenase inhibition, reduction of cholesterol levels, and anti-cancer properties. Sclerotiorin has potential use in pharmaceutical as well as food industries. In this context, the purpose of this study was to provide a simple and robust procedure for optimised production of sclerotiorin by P. sclerotiorum using a central composite design developed through response surface methodology (RSM) and to identify the molecule(s) involved in the signalling mechanism in P. sclerotiorum. The optimisation of sclerotiorin production was carried out using RSM in shaken flasks and the obtained results were then replicated using a 2-L stirred tank bioreactor. Penicillium sclerotiorum ethyl acetate culture extract was analysed using thin layer chromatography (TLC) and potential signalling molecules were identified using Gas chromatography-mass spectrometry (GC-MS). The experimental studies suggested an increase in the sclerotiorin production by 2.1-fold and 2.2-fold in shaken flasks and stirred tank bioreactors respectively. Further analysis of P. sclerotiorum ethyl acetate culture extract reported the presence of ricinoleic acid, an oxylipin, belonging to a family of signalling molecules tentatively involved in the enhancement of sclerotiorin production. This paper has highlighted the positive effect of the optimal supplementation of P. sclerotiorum culture extracts for enhanced production of sclerotiorin. It has also examined potential molecules involved in the signalling mechanism in P. sclerotiorum culture extract for the overproduction of sclerotiorin.