ATM- and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair

The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G(2) phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G(2)/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.

EFFECT OF CORE-SHELL STRUCTURE OF HYDROGEL BEADS ON THE THRESHOLD CONCENTRATION OF WATER FOR SWELLING AND ITS PH SENSITIVITY

In this paper we investigate the effect of core-shell structure of Sodium Alginate based hydrogel beads and their size on certain activation threshold concentration of water for applications in swelling and pH sensing. This type of hydrogel experiences diffusive pressure due to transport of certain free charges across its interface with a solvent or electrolyte. This process is essentially a dynamic equilibrium of the electric force field, stress in the polymeric network with cage like structure and molecular diffusion including phase transformation due to pressure imbalance between the hydrogel and its surroundings. The effect of pH of the solvant on the swelling rate of these beads has been studied experimentally. A mathematical model of the swelling process has been developed by considering Nernst-Planck equation representing the migration of mobile ions and Er ions, Poisson equation representing the equilibrium of the electric field and mechanical field equation representing swelling of the gel. An attempt has been made to predict the experimentally observed phenomena using these numerical simulations. It is observed experimentally that certain minimum concentration called activation threshold concentration of the water molecules must be present in the hydrogel in order to activate the swelling process. For the required activation threshold concentration of water in the beads, the pH induced change in the rate of swelling is also investigated. This effect is analyzed for various different core-shell structures of the beads.

Reduced graphene oxide induced phase miscibility in polystyrene-poly(vinyl methyl ether) blends

Graphene oxide and reduced graphene oxide (r-GO) were synthesized by wet chemistry and the effect of r-GO in PS-PVME blends was investigated here with respect to phase miscibility, intermolecular cooperativity in the glass transition region and concentration fluctuation variance by shear rheology and dielectric spectroscopy. The spinodal decomposition temperature (T-s) and correlation length were evaluated from isochronal temperature scans in shear rheology. The r-GO is shown to induce miscibility in the blends, which may lead to increased local heterogeneity in the blends, though the length of cooperatively re-arranged regions (xi) at T-g is more or less unaltered. The evolution of the phase morphology as a function of temperature was assessed using polarized optical microscopy (POM). In the case of the 60/40 PS-PVME blends with 0.25 wt% r-GO, apart from significant refinement in the morphology, retention of the interconnected ligaments of PVME was observed, even in the late stages of phase separation suggesting that the coarsening of the phase morphology has been slowed down in the presence of r-GO. This phenomenon was also supported by AFM. Surface enrichment of PVME, owing to its lower surface tension, in the demixed samples was supported by XPS scans. The interconnected network of PVME has resulted in significantly higher permittivity in the bi-phasic blends, although the concentration of r-GO is below the percolation threshold.

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 mu M) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading Delta psi m dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.

SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae

Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1. cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1. sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration

The epsilon 4 isoform of apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We created a transgenic model of neurodegenerative disorder by expressing epsilon 3 and epsilon 4 isoforms of human ApoE in the Drosophila melanogaster. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. Moreover, acute oxidative damage in the hApoE transgenic flies triggered a neuroprotective response of hApoE3 while chronic induction of oxidative damage accelerated the rate of neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity. (C) 2015 Elsevier B.V. All rights reserved.

Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration

The epsilon 4 isoform of apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We created a transgenic model of neurodegenerative disorder by expressing epsilon 3 and epsilon 4 isoforms of human ApoE in the Drosophila melanogaster. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. Moreover, acute oxidative damage in the hApoE transgenic flies triggered a neuroprotective response of hApoE3 while chronic induction of oxidative damage accelerated the rate of neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity. (C) 2015 Elsevier B.V. All rights reserved.