Rad51-Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A(CaCse4) levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A(CaCse4) in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A(CaCse4) levels at the programmed stall sites of early replicating centromeres.

Effect of angle of incidence on mixed convective wake dynamics and heat transfer past a square cylinder in cross flow at Re=100

In this paper, a numerical investigation is performed to study the mixed convective flow and heat transfer characteristics past a square cylinder in cross flow at incidence. Utilizing air (Pr = 0.71) as an operating fluid, computations are carried out at a representative Reynolds number (Re) of 100. Angles of incidences are varied as, 0 degrees <= alpha <= 45 degrees. Effect of superimposed positive and negative cross-flow buoyancy is brought about by varying the Richardson number (RI) in the range -1.0 <= Ri <= 1.0. The detail features of flow topology and heat transport are analyzed critically for different angles of incidences. The thermo fluidic forces acting on the cylinder during mixed convection are captured in terms of the drag (C-D), lift (C-L), and moment (C-M) coefficients. The results show that the lateral width of the cylinder wake reduces with increasing alpha and the isotherms spread out far wide. In the range 0 degrees < alpha < 45 degrees, C-D reduces with increasing Ri. The functional dependence of C-M with Ri reveals a linear relationship. Thermal boundary layer thickness reduces with increasing angle of incidences. The global rate of heat transfer from the cylinder increases with increasing alpha. (C) 2014 Elsevier Ltd. All rights reserved.

Analysis of Entropy Generation During Mixed Convective Heat Transfer of Nanofluids Past a Rotating Circular Cylinder

The entropy generation due to mixed convective heat transfer of nanofluids past a rotating circular cylinder placed in a uniform cross stream is investigated via streamline upwind Petrov-Galerkin based finite element method. Nanosized copper (Cu) particles suspended in water are used with Prandtl number (Pr)=6.9. The computations are carried out at a representative Reynolds number (Re) of 100. The dimensionless cylinder rotation rate, a, is varied between 0 and 2. The range of nanoparticle volume fractions (phi) considered is 0 <= phi <= 5%. Effect of aiding buoyancy is brought about by considering two fixed values of the Richardson number (Ri) as 0.5 and 1.0. A new model for predicting the effective viscosity and thermal conductivity of dilute suspensions of nanoscale colloidal particles is presented. The model addresses the details of the agglomeration-deagglomeration in tune with the pertinent variations in the effective particulate dimensions, volume fractions, as well as the aggregate structure of the particulate system. The total entropy generation is found to decrease sharply with cylinder rotation rates and nanoparticle volume fractions. Increase in nanoparticle agglomeration shows decrease in heat transfer irreversibility. The Bejan number falls sharply with increase in alpha and phi.

Nitric oxide is the key mediator of death induced by fisetin in human acute monocytic leukemia cells

Nitric oxide ( NO) has been shown to be effective in cancer chemoprevention and therefore drugs that help generate NO would be preferable for combination chemotherapy or solo use. This study shows a new evidence of NO as a mediator of acute leukemia cell death induced by fisetin, a promising chemotherapeutic agent. Fisetin was able to kill THP-1 cells in vivo resulting in tumor shrinkage in the mouse xenograft model. Death induction in vitro was mediated by an increase in NO resulting in double strand DNA breaks and the activation of both the extrinsic and the intrinsic apoptotic pathways. Double strand DNA breaks could be reduced if NO inhibitor was present during fisetin treatment. Fisetin also inhibited the downstream components of the mTORC1 pathway through downregulation of levels of p70 S6 kinase and inducing hypo-phosphorylation of S6 Ri P kinase, eIF4B and eEF2K. NO inhibition restored phosphorylation of downstream effectors of mTORC1 and rescued cells from death. Fisetin induced Ca2+ entry through L-type Ca2+ channels and abrogation of Ca2+ influx reduced caspase activation and cell death. NO increase and increased Ca2+ were independent phenomenon. It was inferred that apoptotic death of acute monocytic leukemia cells was induced by fisetin through increased generation of NO and elevated Ca2+ entry activating the caspase dependent apoptotic pathways. Therefore, manipulation of NO production could be viewed as a potential strategy to increase efficacy of chemotherapy in acute monocytic leukemia.

Early postnatal exposure to lithium in vitro induces changes in AMPAR mEPSCs and vesicular recycling at hippocampal glutamatergic synapses

Lithium is an effective mood stabilizer but its use is associated with many side effects. Electrophysiological recordings of miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate receptor AMPA-subtype (AMPARs) in hippocampal pyramidal neurons revealed that CLi (therapeutic concentration of 1 mM lithium, from days in vitro 4-10) decreased the mean amplitude and mean rectification index (RI) of AMPAR mEPSCs. Lowered mean RI indicate that contribution of Ca2+-permeable AMPARs in synaptic events is higher in CLi neurons (supported by experiments sensitive to Ca2+-permeable AMPAR modulation). Co-inhibiting PKA, GSK-3 beta and glutamate reuptake was necessary to bring about changes in AMPAR mEPSCs similar to that seen in CLi neurons. FM1-43 experiments revealed that recycling pool size was affected in CLi cultures. Results from minimum loading, chlorpromazine treatment and hyperosmotic treatment experiments indicate that endocytosis in CLi is affected while not much difference is seen in modes of exocytosis. CLi cultures did not show the high KCl associated presynaptic potentiation observed in control cultures. This study, by calling attention to long-term lithium-exposure-induced synaptic changes, might have implications in understanding the side effects such as CNS complications occurring in perinatally exposed babies and cognitive dulling seen in patients on lithium treatment.

Development and Evaluation of Prussian Blue Mediated Glucose Sensor for Reverse Iontophoresis Application

The screen printed electrochemical glucose sensor is developed suitable for revere iontophoresis (RI) application. Glucose oxidase is immobilized on screen printed sensor using crosslinking method. Electrochemical and material characterization studies are conducted on the developed sensor and the obtained results confirm the suitability of the developed sensor for RI application. The developed sensor is validated by conducting clinical investigations on 10 human subjects through RI. A correlation is established between the blood glucose and extracted glucose, and correlation is found to be 0.73. (C) 2015 The Electrochemical Society. All rights reserved.

Effect of Channel Confinement on Mixed Convective Flow Past an Equilateral Triangular Cylinder

The present work investigates the mixed convective flow and heat transfer characteristics past a triangular cylinder placed symmetrically in a vertical channel. At a representative Reynolds number, Re = 100, simulations are carried out for the blockage ratios beta = 1/3; 1/4; and 1/6. Effect of aiding and opposing buoyancy is brought about by varying the Richardson number in the range -1.0 <= Ri <= 1.0. At a blockage ratio of 1/3, suppression of vortex shedding is found at Ri = 1, whereas von Karman vortex street is seen both at beta = 1/4 and 1/6, respectively. This is the first time that such behavior of blockage ratio past a triangular cylinder in the present flow configuration is reported. Drag coefficient increases progressively with increasing Ri and a slightly higher value is noticed at beta = 1/3. For all b, heat transfer increases with increasing Ri. Flattening of Nu(avg)-Ri curve beyond Ri > 0: 75 is observed at beta = 1/3.

Fully 3D refraction correction dosimetry system

The irradiation of selective regions in a polymer gel dosimeter results in an increase in optical density and refractive index (RI) at those regions. An optical tomography-based dosimeter depends on rayline path through the dosimeter to estimate and reconstruct the dose distribution. The refraction of light passing through a dose region results in artefacts in the reconstructed images. These refraction errors are dependant on the scanning geometry and collection optics. We developed a fully 3D image reconstruction algorithm, algebraic reconstruction technique-refraction correction (ART-rc) that corrects for the refractive index mismatches present in a gel dosimeter scanner not only at the boundary, but also for any rayline refraction due to multiple dose regions inside the dosimeter. In this study, simulation and experimental studies have been carried out to reconstruct a 3D dose volume using 2D CCD measurements taken for various views. The study also focuses on the effectiveness of using different refractive-index matching media surrounding the gel dosimeter. Since the optical density is assumed to be low for a dosimeter, the filtered backprojection is routinely used for reconstruction. We carry out the reconstructions using conventional algebraic reconstruction (ART) and refractive index corrected ART (ART-rc) algorithms. The reconstructions based on FDK algorithm for cone-beam tomography has also been carried out for comparison. Line scanners and point detectors, are used to obtain reconstructions plane by plane. The rays passing through dose region with a RI mismatch does not reach the detector in the same plane depending on the angle of incidence and RI. In the fully 3D scanning setup using 2D array detectors, light rays that undergo refraction are still collected and hence can still be accounted for in the reconstruction algorithm. It is found that, for the central region of the dosimeter, the usable radius using ART-rc algorithm with water as RI matched medium is 71.8%, an increase of 6.4% compared to that achieved using conventional ART algorithm. Smaller diameter dosimeters are scanned with dry air scanning by using a wide-angle lens that collects refracted light. The images reconstructed using cone beam geometry is seen to deteriorate in some planes as those regions are not scanned. Refraction correction is important and needs to be taken in to consideration to achieve quantitatively accurate dose reconstructions. Refraction modeling is crucial in array based scanners as it is not possible to identify refracted rays in the sinogram space.

Effect of potassium present in stratum corneum during non-invasive measurement of potassium in human subjects using reverse iontophoresis

Purpose: Reverse iontophoresis (RI) is one of the potential techniques used to monitor the concentration of various analytes in body fluids non -invasively. Transdermal extraction of potassium is investigated using RI. In the present work, the effect of potassium on stratum corneum (SC) during RI, feasibility of RI for continuous monitoring of potassium, and use of potassium as internal standard in RI, are investigated. Methods: Tape stripping experiment is carried out to find potassium concentration in SC. RI is carried out continuously for 180 min without passive diffusion and after passive diffusion for 60 min. Skin impedance measurements are done at 20 Hz and 20 kHz. Results: Potassium is found to be in the range 300-650 nmol/cm(2) on SC by tape stripping experiment. Correlation coefficient between blood potassium and extracted potassium through RI after passive diffusion (R-2 = 0.5870) is more than without passive diffusion (R-2 = 0.5117). The skin impedance measurement shows that RI has more effect on SC than superficial layer of SC during RI. Conclusion: The present investigations conclude that it is possible to monitor potassium continuously through RI and using potassium as internal standard in RI.