Colloidal quantum dots initiating current bursts in lipid bilayers

The combination of inorganic semiconductor nanocrystals, also called quantum dots (QDs), with biological materials has recently attracted considerable interest since the QDs can be used as superior fluorescent labels. Here, we report on CdSe QD initiated current bursts in lipid bilayer membranes on application of a bias voltage. The current bursts observed resemble those produced by the peptaibol class of antibiotics such as alamethicin and trichorzins. The current fluctuations are dependent on the bias voltage and on the concentration of the QD applied to the membrane. Our data suggest that QDs with intrinsic dipole moments similar to alamethicin can be controlled by an external electric field, which creates a torque resulting in the insertion into the lipid membrane.     

IRREGULAR TRICHOME BRANCH1 in Arabidopsis encodes a plant homolog of the actin-related protein2/3 complex activator Scar/WAVE that regulates actin and microtubule organization

The dynamic actin cytoskeleton is important for a myriad of cellular functions, including intracellular transport, cell division, and cell shape. An important regulator of actin polymerization is the actin-related protein2/3 (Arp2/3) complex, which nucleates the polymerization of new actin filaments. In animals, Scar/WAVE family members activate Arp2/3 complex-dependent actin nucleation through interactions with Abi1, Nap1, PIR121, and HSCP300. Mutations in the Arabidopsis thaliana genes encoding homologs of Arp2/3 complex subunits PIR121 and NAP1 all show distorted trichomes as well as additional epidermal cell expansion defects, suggesting that a Scar/WAVE homolog functions in association with PIR121 and NAP1 to activate the Arp2/3 complex in Arabidopsis. In a screen for trichome branching defects, we isolated a mutant that showed irregularities in trichome branch positioning and expansion. We named this gene IRREGULAR TRICHOME BRANCH1 (ITB1). Positional cloning of the ITB1 gene showed that it encodes SCAR2, an Arabidopsis protein related to Scar/WAVE. Here, we show that itb1 mutants display cell expansion defects similar to those reported for the distorted class of trichome mutants, including disruption of actin and microtubule organization. In addition, we show that the scar homology domain (SHD) of ITB1/SCAR2 is necessary and sufficient for in vitro binding to Arabidopsis BRK1, the plant homolog of HSPC300. Overexpression of the SHD in transgenic plants causes a dominant negative phenotype. Our results extend the evidence that the Scar/WAVE pathway of Arp2/3 complex regulation exists in plants and plays an important role in regulating cell expansion.     

Next Generation Sequencing Reveals Regulation of Distinct Aedes microRNAs during Chikungunya Virus Development

Background

Application of genomics and Next Generation sequencing has led to the identification of new class of cellular functional molecules, namely, small RNAs. Of the several classes of ncRNAs (non-coding RNA), microRNAs have been demonstrated to exert determinative influence on various cellular processes. It is becoming abundantly clear that host/vector/pathogen encoded microRNAs impact eventual pathogenesis. In this context, the participation of vector based microRNAs in disease transmission and pathogen development is being investigated intensively. A few studies have highlighted the role of vector encoded microRNAs in pathogen infection. We conducted this study to evaluate the role of host miRNAs upon CHIKV (Chikungunya Virus) infection in an important vector, Aedes albopictus.

Findings

We identified 88 and 79 known miRNAs in uninfected and CHIKV infected Ae. albopictus Singh''s cell line respectively. We further identified nine novel miRNAs in Ae. albopictus. Comparison of the two libraries revealed differential expression of 77 common miRNAs between them. CHIKV infection specifically altered the miRNA profile of a specific set of eight miRNAs. Putative targets of these regulated miRNAs were identified and classified into their pathways.

Conclusions

In our study we have identified and described the profiles of various miRNAs upon CHIKV infection in Ae. albopictus. This investigation provides an insight about cellular modification by miRNAs during CHIKV infection and the results provide leads for identifying potential candidates for vector based antiviral strategies.     

Ca2+ signaling in human fetoplacental vasculature: effect of CGRP on umbilical vein smooth muscle cytosolic Ca2+ concentration

CGRP is a potent vasodilator with increased levels in fetoplacental circulation during late pregnancy. We have recently demonstrated that acute CGRP exposure to fetoplacental vessels in vitro induced vascular relaxation, but the signaling pathway of CGRP in fetoplacental vasculature remains unclear. We hypothesized that CGRP relaxes fetoplacental vasculature via regulating smooth muscle cytosolic Ca2+ concentrations. In the present study, by using human umbilical vein smooth muscle (HUVS) cells (HUVS-112D), we examined CGRP receptors, cAMP generation, and changes in cellular Ca2+ concentrations on CGRP treatment. These cells express mRNA for CGRP receptor components, calcitonin receptor-like receptor, and receptor activity-modifying protein-1. Direct saturation binding for 125I-labeled CGRP to HUVS cells and Scatchard analysis indicate specificity of the receptors for CGRP [dissociation constant (K(D)) = 67 nM, maximum binding capcity (Bmax) = 2.7 pmol/million cells]. Exposure of HUVS cells to CGRP leads to a dose-dependent increase in intracellular cAMP accumulation, and this increase is prevented by CGRP antagonist CGRP(8-37). Using fura-2-loaded HUVS cells, we monitored the effects of CGRP on intracellular Ca2+ concentration ([Ca2+]i). In the presence of extracellular Ca2+, bradykinin (10(-6) M), a fetoplacental vasoconstrictor, increases HUVS cells [Ca2+]i concentration. CGRP (10(-8) M) abolishes bradykinin-induced [Ca2+]i elevation. When the cells were pretreated with glibenclamide, an ATP-sensitive potassium channel blocker, the CGRP actions on bradykinin-induced Ca2+ influx were profoundly inhibited. In the absence of extracellular Ca2+, CGRP (10(-8) M) attenuated the increase of [Ca2+]i induced by a sarcoplasmic reticulum Ca2+ pump ATPase inhibitor thapsigargin (10(-5) M). Furthermore, Rp-cAMPS, a cAMP-dependent protein kinase A inhibitor, blocks CGRP actions on thapsigargin-induced Ca2+ release from sarcoplasmic reticulum. Our results suggested that CGRP relaxes human fetoplacental vessels by not only inhibiting the influx of extracellular Ca2+ but also attenuating the release of intracellular Ca2+ from the sarcoplasmic reticulum, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.     

Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin‐related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 ⁺ sectors, ACD2 functions cell autonomously, implicating a pro‐death ACD2 substrate as being cell non‐autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light‐dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro‐death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria.     

Sequential elimination of major-effect contributors identifies additional quantitative trait loci conditioning high-temperature growth in yeast

Several quantitative trait loci (QTL) mapping strategies can successfully identify major-effect loci, but often have poor success detecting loci with minor effects, potentially due to the confounding effects of major loci, epistasis, and limited sample sizes. To overcome such difficulties, we used a targeted backcross mapping strategy that genetically eliminated the effect of a previously identified major QTL underlying high-temperature growth (Htg) in yeast. This strategy facilitated the mapping of three novel QTL contributing to Htg of a clinically derived yeast strain. One QTL, which is linked to the previously identified major-effect QTL, was dissected, and NCS2 was identified as the causative gene. The interaction of the NCS2 QTL with the first major-effect QTL was background dependent, revealing a complex QTL architecture spanning these two linked loci. Such complex architecture suggests that more genes than can be predicted are likely to contribute to quantitative traits. The targeted backcrossing approach overcomes the difficulties posed by sample size, genetic linkage, and epistatic effects and facilitates identification of additional alleles with smaller contributions to complex traits.     

An electrophoretic mobility shift assay for the identification and kinetic analysis of acetyl transferase inhibitors

Histone acetyl transferases are important regulators of cellular homeostasis. This study describes a sensitive acetyl transferase electrophoretic mobility shift assay applicable both for kinetic analysis of acetyl transferase inhibitors and for high-throughput testing. Application of the assay for human GCN5L2 enabled dissection of inhibitor competition with respect to acetyl coenzyme A. Furthermore, we demonstrated that the assay can detect time-dependent inhibition of human GCN5L2 by reactive inhibitors.     

Tannins present in Cichorium intybus enhance glucose uptake and inhibit adipogenesis in 3T3-L1 adipocytes through PTP1B inhibition

Insulin resistance is a fundamental aspect for the etiology of non-insulin dependent diabetes mellitus (NIDDM) and has links with a wide array of secondary disorders including weight gain and obesity. The present study analyzes the effect of Cichorium intybus methanolic (CME) extract on glucose transport and adipocyte differentiation in 3T3-L1 cells by studying the radiolabelled glucose uptake and lipid accumulation assays, respectively. By performing detannification (CME/DT), the role of tannins present in CME on both the activities was evaluated. CME and CME/DT exhibited significant glucose uptake in 3T3-L1 adipocytes with a dose-dependent response. Glucose uptake profile in the presence of PI3K and IRTK inhibitors (Wortmannin and Genistein) substantiates the mechanism used by both the extracts. CME inhibited the differentiation of 3T3-L1 preadipocytes but failed to show glucose uptake in inhibitor treated cells. The activity exhibited by CME/DT is exactly vice versa to CME. Furthermore, the findings from PTP1B inhibition assay, mRNA and protein expression analysis revealed the unique behavior of CME and CME/DT. The duality exhibited by C. intybus through adipogenesis inhibition and PPARgamma up regulation is of interest. Current observation concludes that the activities possessed by C. intybus are highly desirable for the treatment of NIDDM because it reduces blood glucose levels without inducing adipogenesis in 3T3-L1 adipocytes.     

Thrombin induces tumor invasion through the induction and association of matrix metalloproteinase-9 and beta1-integrin on the cell surface

The procoagulatory serine protease, thrombin, is known to induce invasion and metastasis in various cancers, but the mechanisms by which it promotes tumorigenesis are poorly understood. Because the 92-kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion, we sought to determine whether and how thrombin regulates MMP-9. The thrombin receptor, PAR-1, and MMP-9 are expressed in osteosarcomas, as determined by immunohistochemistry. Stimulation of U2-OS osteosarcoma cells with thrombin and a thrombin receptor-activating peptide induced pro-MMP-9 secretion as well as cell surface-associated pro-MMP-9 expression and proteolytic activity. This was paralleled by an increase in MMP-9 mRNA and MMP-9 promoter activity. Thrombin-induced invasion of U2-OS cells through Matrigel was mediated by the phosphatidylinositol 3-kinase signaling pathway and could be inhibited with an MMP-9 antibody. The stimulation of MMP-9 by thrombin was paralleled by an increase in beta1-integrin mRNA and beta1-integrin expression on the cell surface, which was also mediated by phosphatidylinositol 3-kinase and was required for invasion. Thrombin activation induced and co-localized both beta1-integrin and pro-MMP-9 on the cell membrane, as evidenced by co-immunoprecipitation, confocal microscopy, and a protein binding assay. The thrombin-mediated association of these two proteins, as well as thrombin-mediated invasion of U2-OS cells, could be blocked with a cyclic peptide and with an antibody preventing binding of the MMP-9 hemopexin domain to beta1-integrin. These results suggest that thrombin induces expression and association of beta1-integrin with MMP-9 and that the cell surface localization of the protease by the integrin promotes tumor cell invasion.