Alpha amylase (α-1, 4-glucan-glucanhydrolase, EC 126.96.36.199), an extracellular enzyme, degrades α, 1–4 glucosidic linkages of starch and related substrates in an endo-fashion producing oligosaccharides including maltose, glucose and alpha limit dextrin (7). The present study deals with the production and comparative study of production of α-amylase from two strains of Bacillus licheniformis, MTCC 2617 and 2618, by using four different substrates, starch, rice, wheat and ragi powder as carbon source by submerged fermentation. The effect of varying pH and incubation temperature, activator, inhibitor, and substrate concentration was investigated on the activity of α-amylase produced by MTCC strain 2618. The results shows that the production of the α-amylase by the B.licheniformis strain MTCC 2618, using four different substrates were found to be maximum (Starch 3.64 IU/ml/minutes, Rice powder 2.93 IU/ml/minutes, Wheat powder 2.67 IU/ml/minutes, Ragi powder 2.36 IU/ml/minutes) on comparing the enzyme production of two strains. It was also observed that the maximum production was found on the 3rd day (i.e. 72 hr) and characterization of crude enzyme revealed that optimum activity was at pH 7 and 37°C.
amylase; Bacillus licheniformis; enzyme; extracellular; glucose
COX-2 has been implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) latency and pathogenesis (A. George Paul, N. Sharma-Walia, N. Kerur, C. White, and B. Chandran, Cancer Res. 70:3697-3708, 2010; P. P. Naranatt, H. H. Krishnan, S. R. Svojanovsky, C. Bloomer, S. Mathur, and B. Chandran, Cancer Res. 64:72-84, 2004; N. Sharma-Walia, A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog. 6:e1000777, 2010; N. Sharma-Walia, H. Raghu, S. Sadagopan, R. Sivakumar, M. V. Veettil, P. P. Naranatt, M. M. Smith, and B. Chandran, J. Virol. 80:6534-6552, 2006). However, the precise regulatory mechanisms involved in COX-2 induction during KSHV infection have never been explored. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 upon KSHV de novo infection. Promoter analysis using human COX-2 promoter deletion and mutation reporter constructs revealed that nuclear factor of activated T cells (NFAT) and the cyclic AMP (cAMP) response element (CRE) modulate KSHV-mediated transcriptional regulation of COX-2. Along with multiple KSHV-induced signaling pathways, infection-induced prostaglandin E2 (PGE2) also augmented COX-2 transcription. Infection of endothelial cells markedly induced COX-2 expression via a cyclosporine A-sensitive, calcineurin/NFAT-dependent pathway. KSHV infection increased intracellular cAMP levels and activated protein kinase A (PKA), which phosphorylated the CRE-binding protein (CREB) at serine 133, which probably led to interaction with CRE in the COX-2 promoter, thereby enhancing COX-2 transcription. PKA selective inhibitor H-89 pretreatment strongly inhibited CREB serine 133, indicating the involvement of a cAMP-PKA-CREB-CRE loop in COX-2 transcriptional regulation. In contrast to phosphatidylinositol 3-kinase and protein kinase C, inhibition of FAK and Src effectively reduced KSHV infection-induced COX-2 transcription and protein levels. Collectively, our study indicates that mediation of COX-2 transcription upon KSHV infection is a paradigm of a complex regulatory milieu involving the interplay of multiple signal cascades and transcription factors. Intervention at each step of COX-2/PGE2 induction can be used as a potential therapeutic target to treat KSHV-associated neoplasm and control inflammatory sequels of KSHV infection.
Glia, including astrocytes, are increasingly at the forefront of neurodegenerative research for their role in the modulation of neuronal function and survival. Improved understanding of underlying disease mechanisms, including the role of the cellular environment in neurodegeneration, is central to therapeutic development for these currently untreatable diseases. In these endeavours, experimental models that more closely reproduce the human condition have the potential to facilitate the transition between experimental studies in model organisms and patient trials. In this review we discuss the growing role of astrocytes in neurodegenerative diseases, and how astrocytes generated from human pluripotent stem cells represent a useful tool for analyzing astrocytic signalling and influence on neuronal function.
antioxidants; astrocytes; neuroprotection; Nrf2; oxidative stress; stem cells
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.
Marburg virus (MARV) and Ebola virus (EBOV) are members of the family Filoviridae (‘filoviruses’) that cause severe hemorrhagic fever with human case fatality rates of up to 90%. Filovirus infection requires fusion of the host cell and virus membranes, a process that is mediated by the envelope glycoprotein (GP). GP contains two subunits, the surface subunit (GP1), which is responsible for cell attachment, and the transmembrane subunit (GP2), which catalyzes membrane fusion. The GP2 ectodomain contains two heptad repeat regions, N-terminal and C-terminal (NHR and CHR, respectively) that adopt a six-helix bundle during the fusion process. The refolding of this six-helix bundle provides the thermodynamic driving force to overcome barriers associated with membrane fusion. Here we report the crystal structure of the MARV GP2 core domain in its post-fusion (six-helix bundle) conformation at 1.9 Å resolution. The MARV GP2 core domain backbone conformation is virtually identical to that of EBOV GP2 (reported previously), and consists of a central NHR core trimeric coiled-coil packed against peripheral CHR α-helices and an intervening loop/helix-turn-helix segment. We previously reported that the stability of the MARV GP2 post-fusion structure is highly pH-dependent, with increasing stability at lower pH [Harrison, J.S.; Koellhoffer, J. K.; Chandran, K.; and Lai, J. R. Biochemistry, 2012, 51, 2515–2525]. We hypothesized that this pH-dependent stability provides a mechanism for conformational control such that the post-fusion six helix bundle is promoted in the environments of appropriately matured endosomes. In this report, a structural rationale for this pH-dependent stability is described, and involves a high-density array of core and surface acidic side chains at the midsection of the structure, termed the ‘anion stripe.’ In addition, many surface-exposed salt bridges likely contribute to stabilizing the post-fusion structure at low pH. These results provide structural insights into the mechanism of MARV GP2-mediated membrane fusion.
RNA splicing plays a critical role in the programming of neuronal differentiation and, consequently, normal human neurodevelopment, and its disruption may underlie neurodevelopmental and neuropsychiatric disorders. The RNA-binding protein, fox-1 homolog (RBFOX1; also termed A2BP1 or FOX1), is a neuron-specific splicing factor predicted to regulate neuronal splicing networks clinically implicated in neurodevelopmental disease, including autism spectrum disorder (ASD), but only a few targets have been experimentally identified. We used RNA sequencing to identify the RBFOX1 splicing network at a genome-wide level in primary human neural stem cells during differentiation. We observe that RBFOX1 regulates a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins. Downstream alterations in gene expression define an additional transcriptional network regulated by RBFOX1 involved in neurodevelopmental pathways remarkably parallel to those affected by splicing. Several of these differentially expressed genes are further implicated in ASD and related neurodevelopmental diseases. Weighted gene co-expression network analysis demonstrates a high degree of connectivity among these disease-related genes, highlighting RBFOX1 as a key factor coordinating the regulation of both neurodevelopmentally important alternative splicing events and clinically relevant neuronal transcriptional programs in the development of human neurons.
Morgagni-Larrey hernia is an uncommon entity. The majority of the literature describes hernia occurring mostly on the right side, a few on the left side and rarely bilateral. Retrospective chart review was done for the patients with the diagnosis of adult diaphragmatic hernia from January 1997 to December 2010. Post-operative course was evaluated for outcome, morbidity and mortality. Out of 20 patients, 13 (65 %) were males and 7 (35 %) were females. Their age ranged from 17 to 50 years (mean = 29.6). Abdominal discomfort was the most common presentation. Eight patients (40 %) were asymptomatic at presentation. Plain X-Ray chest was done for all. Ten patients (50 %) underwent suture repair, 6 (30 %) had mesh placement and the other 4 (25 %) underwent both: suture repair buttressed with mesh. Volvulus of stomach was noted in 5 (25 %) cases. All patients had left sided hernia. There was insignificant morbidity and no mortality. There was no recurrence in 16 patients followed up for a mean duration of 20 months (range = 8 to 32 months). In Morgagni-Larrey hernia, abdominal approach gives good accessibility to reduce the hernia and to undertake repair. When complicated with incarceration, perforation, gangrene or volvulus of the herniated bowel; this can be dealt with ease. Plain X Ray of the chest is fairly accurate in suggesting the diagnosis of Morgagni-Larrey hernia.
Morgagni-Larrey hernia; Adult diaphragmatic hernia
Kaposi's sarcoma-associated herpesvirus (KSHV) infections of endothelial and B cells are etiologically linked with Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma (PEL), respectively. KS endothelial and PEL B cells carry multiple copies of the nuclear episomal latent KSHV genome and secrete a variety of inflammatory cytokines, including interleukin-1β (IL-1β) and IL-18. The maturation of IL-1β and IL-18 depends upon active caspase-1, which is regulated by a multiprotein inflammasome complex induced by sensing of danger signals. During primary KSHV infection of endothelial cells, acting as a nuclear pattern recognition receptor, gamma interferon-inducible protein 16 (IFI16) colocalized with the KSHV genome in the nuclei and interacted with ASC and procaspase-1 to form a functional inflammasome (Kerur N et al., Cell Host Microbe 9:363-375, 2011). Here, we demonstrate that endothelial telomerase-immortalized human umbilical cells (TIVE) supporting KSHV stable latency (TIVE-LTC cells) and PEL (cavity-based B-cell lymphoma 1 [BCBL-1]) cells show evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β and pro-IL-18. Interaction of ASC with IFI16 but not with AIM2 or NOD-like receptor P3 (NLRP3) was detected. The KSHV latency-associated viral FLIP (vFLIP) gene induced the expression of IL-1β, IL-18, and caspase-1 mRNAs in an NF-κB-dependent manner. IFI16 and cleaved IL-1β were detected in the exosomes released from BCBL-1 cells. Exosomal release could be a KSHV-mediated strategy to subvert IL-1β functions. In fluorescent in situ hybridization analyses, IFI16 colocalized with multiple copies of the KSHV genome in BCBL-1 cells. IFI16 colocalization with ASC was also detected in lung PEL sections from patients. Taken together, these findings demonstrated the constant sensing of the latent KSHV genome by IFI16-mediated innate defense and unraveled a potential mechanism of inflammation induction associated with KS and PEL lesions.
Zaire Ebola virus (EBOV) is a zoonotic pathogen that causes severe hemorrhagic fever in humans. A single viral glycoprotein (GP) mediates viral attachment and entry. Here, virus-like particle (VLP)-based entry assays demonstrate that a GP mutant, GP-F88A, which is defective for entry into a variety of human cell types, including antigen-presenting cells (APCs), such as macrophages and dendritic cells, can mediate viral entry into mouse CD11b+ APCs. Like that of wild-type GP (GP-wt), GP-F88A-mediated entry occurs via a macropinocytosis-related pathway and requires endosomal cysteine proteases and an intact fusion peptide. Several additional hydrophobic residues lie in close proximity to GP-F88, including L111, I113, L122, and F225. GP mutants in which these residues are mutated to alanine displayed preferential and often impaired entry into several cell types, although not in a species-specific manner. Niemann-Pick C1 (NPC1) protein is an essential filovirus receptor that binds directly to GP. Overexpression of NPC1 was recently demonstrated to rescue GP-F88A-mediated entry. A quantitative enzyme-linked immunosorbent assay (ELISA) demonstrated that while the F88A mutation impairs GP binding to human NPC1 by 10-fold, it has little impact on GP binding to mouse NPC1. Interestingly, not all mouse macrophage cell lines permit GP-F88A entry. The IC-21 cell line was permissive, whereas RAW 264.7 cells were not. Quantitative reverse transcription (RT)-PCR assays demonstrate higher NPC1 levels in GP-F88A permissive IC-21 cells and mouse peritoneal macrophages than in RAW 264.7 cells. Cumulatively, these studies suggest an important role for NPC1 in the differential entry of GP-F88A into mouse versus human APCs.
The simultaneous presentation of the Hodgkin lymphoma and multiple myeloma in the absence of prior chemotherapy or radiation is very rare. Here, we discuss a 72-year-old patient who initially presented with generalized pruritis. Workup led to a diagnosis of multiple myeloma which progressed and required treatment. As part of his pretreatment workup, an MRI was performed to evaluate skeletal lesions. This revealed diffuse and bulky adenopathy which was confirmed by PET. A biopsy of an axillary node was consistent with the nodular sclerosing type Hodgkin lymphoma. He was treated with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) chemotherapy × 6 resulting in complete resolution of his adenopathy and pruritis as well as improvement in his myeloma.
The entry of Kaposi's sarcoma-associated herpesvirus (KSHV) into human dermal microvascular endothelial cells (HMVEC-d), natural in vivo target cells, via macropinocytosis is initiated through a multistep process involving the binding of KSHV envelope glycoproteins with cell surface α3β1, αVβ3, and αVβ5 integrin molecules and tyrosine kinase ephrin-A2 receptor, followed by the activation of preexisting integrin-associated signaling molecules such as focal adhesion kinase (FAK), Src, c-Cbl, phosphoinositide 3-kinase (PI-3K), and Rho-GTPases. Many viruses, including KSHV, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells; however, the role of ROS in early events of viral entry and the induction of signaling has not been elucidated. Here we show that KSHV induced ROS production very early during the infection of HMVEC-d cells and that ROS production was sustained over the observation period (24 h postinfection). ROS induction was dependent on the binding of KSHV to the target cells, since pretreatment of the virus with heparin abolished ROS induction. Pretreatment of HMVEC-d cells with the antioxidant N-acetylcysteine (NAC) significantly inhibited KSHV entry, and consequently gene expression, without affecting virus binding. In contrast, H2O2 treatment increased the levels of KSHV entry and infection. In addition, NAC inhibited KSHV infection-induced translocation of αVβ3 integrin into lipid rafts, actin-dependent membrane perturbations, such as blebs, observed during macropinocytosis, and activation of the signal molecules ephrin-A2 receptor, FAK, Src, and Rac1. In contrast, H2O2 treatment increased the activation of ephrin-A2, FAK, Src, and Rac1. These studies demonstrate that KSHV infection induces ROS very early during infection to amplify the signaling pathways necessary for its efficient entry into HMVEC-d cells via macropinocytosis.
Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2's tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.
KSHV is etiologically associated with Kaposi's sarcoma and primary effusion B-cell lymphoma. To initiate its in vitro infection of endothelial cells, KSHV interacts with cell surface heparan sulfate, integrins, and EphrinA2 (EphA2) molecules in the lipid raft (LR) regions, which induces the integrin-c-Cbl associated signaling and macropinocytic entry. In contrast, KSHV enters human foreskin fibroblast (HFF) cells via LR-independent clathrin mediated endocytosis. The present studies conducted to define the key molecules regulating KSHV entry in HFF cells demonstrate that KSHV induces the association of integrins (αVβ5, αVβ3 and α3β1) with EphA2 in the non-LR regions of HFF cells and activates EphA2, which in turn associates with c-Cbl, myosin IIA, FAK, Src, PI3-K, clathrin, AP2 and Epsin15. Loss of EphA2 function reduces the induction of these signals, virus entry and infection. c-Cbl knockdown also abolishes the EphA2 polyubiquitination and clathrin association with EphA2 and KSHV. These results reveal for the first time the role of EphA2 in clathrin mediated endocytosis of a virus and c-Cbl directed polyubiquitination of EphA2 regulating KSHV infection by coordinating signal induction and underscores EphA2 and c-Cbl as potential targets to intervene in KSHV entry and infection.
Can a wide range of complex biochemical behaviour arise from repeated applications of a highly reduced class of interactions? In particular, can the range of DNA manipulations achieved by protein enzymes be simulated via simple DNA hybridization chemistry? In this work, we develop a biochemical system which we call meta-DNA (abbreviated as mDNA), based on strands of DNA as the only component molecules. Various enzymatic manipulations of these mDNA molecules are simulated via toehold-mediated DNA strand displacement reactions. We provide a formal model to describe the required properties and operations of our mDNA, and show that our proposed DNA nanostructures and hybridization reactions provide these properties and functionality. Our meta-nucleotides are designed to form flexible linear assemblies (single-stranded mDNA (ssmDNA)) analogous to single-stranded DNA. We describe various isothermal hybridization reactions that manipulate our mDNA in powerful ways analogous to DNA–DNA reactions and the action of various enzymes on DNA. These operations on mDNA include (i) hybridization of ssmDNA into a double-stranded mDNA (dsmDNA) and heat denaturation of a dsmDNA into its component ssmDNA, (ii) strand displacement of one ssmDNA by another, (iii) restriction cuts on the backbones of ssmDNA and dsmDNA, (iv) polymerization reactions that extend ssmDNA on a template to form a complete dsmDNA, (v) synthesis of mDNA sequences via mDNA polymerase chain reaction, (vi) isothermal denaturation of a dsmDNA into its component ssmDNA, and (vii) an isothermal replicator reaction that exponentially amplifies ssmDNA strands and may be modified to allow for mutations.
DNA self-assembly; synthetic biology; DNA nanostructures
Tissue engineered heart valves (TEHV) have been observed to respond to mechanical conditioning in vitro by expression of activated myofibroblast phenotypes followed by improvements in tissue maturation. In separate studies, cyclic flexure, stretch, and flow (FSF) have been demonstrated to exhibit both independent and coupled stimulatory effects. Synthesis of these observations into a rational framework for TEHV mechanical conditioning has been limited, however, due to the functional complexity of trileaflet valves and the inherent differences of separate bioreactor systems. Toward quantifying the effects of individual mechanical stimuli similar to those that occur during normal valve function, a novel bioreactor was developed in which FSF mechanical stimuli can be applied to engineered heart valve tissues independently or in combination. The FSF bioreactor consists of two identically equipped chambers, each having the capacity to hold up to 12 rectangular tissue specimens (25 × 7.5 × 1 mm) via a novel “spiral-bound” technique. Specimens can be subjected to changes-in-curvature up to 50 mm−1 and uniaxial tensile strains up to 75%. Steady laminar flow can be applied by a magnetically coupled paddlewheel system. Computational fluid dynamic (CFD) simulations were conducted and experimentally validated by particle image velocimetry (PIV). Tissue specimen wall shear stress profiles were predicted as a function of paddlewheel speed, culture medium viscosity, and the quasi-static state of specimen deformation (i.e., either undeformed or completely flexed). Velocity profiles predicted by 2D CFD simulations of the paddlewheel mechanism compared well with PIV measurements, and were used to determine boundary conditions in localized 3D simulations. For undeformed specimens, predicted inter-specimen variations in wall shear stress were on average ±7%, with an average wall shear stress of 1.145 dyne/cm2 predicted at a paddlewheel speed of 2000 rpm and standard culture conditions. In contrast, while the average wall shear stress predicted for specimens in the quasi-static flexed state was ~59% higher (1.821 dyne/cm2), flexed specimens exhibited a broad intra-specimen wall shear stress distribution between the convex and concave sides that correlated with specimen curvature, with peak wall shear stresses of ~10 dyne/cm2. This result suggests that by utilizing simple flexed geometric configurations, the present system can also be used to study the effects of spatially varying shear stresses. We conclude that the present design provides a robust tool for the study of mechanical stimuli on in vitro engineered heart valve tissue formation.
Tissue engineering; Bioreactor; Heart valve; Flexure; Tension; Flow; Fluid shear stress
This cross-sectional case control study included subjects aged between 18 and 65 years with diagnosis of myasthenia gravis (MG) in Osserman's Stage I and Stage IIa and those in remission with positive and negative acetylcholine receptor antibody (AChRAb). They were evaluated for heart rate variability (HRV) and other conventional autonomic functions. Patients with co-morbidities that can affect autonomic nervous system were excluded. Repetitive nerve stimulation test (RNST), nerve conduction test, AChRAb assay, and computerized tomography (CT) of chest were done in all the patients. All patients of MG who fulfilled the inclusion criteria had a minimum drug-free period of 6 h which was followed by HRV and other conventional tests. Thirty subjects fulfilling study criteria and an equal number of age and gender-matched healthy subjects were enrolled as controls. Autonomic function tests revealed significant changes in HRV (both time and frequency domain) parameters suggestive of parasympathetic deficiency as well as shifting of sympathovagal balance towards raised sympathetic tone. With regards to conventional autonomic function tests, there was statistically significant decrease in values of heart rate-based tests as well as blood pressure-based test (isometric handgrip test) in study group compared with controls, again indicative of significant parasympathetic deficiency and minimal sympathetic deficiency. We conclude that in MG, cholinergic transmission is affected more diffusely than previously thought.
Blood pressure changes; cholinergic function; heart rate variability; myasthenia gravis; receptors
While the role of nutrition, physical activity, and body size on breast cancer risk has been extensively investigated, most of these studies were conducted in Caucasian populations. However, there are well known differences in tumor biology and the prevalence of these factors between African American and Caucasian women. The objective of this paper was to conduct a review on the role of dietary factors, anthropometry, and physical activity on breast cancer risk in African American women.
Twenty-six research articles that presented risk estimates on these factors in African American women and five articles involving non-US Black women were included in this review.
Racial disparities in the impact of anthropometric and nutritional factors on breast cancer risk
African American and non-US Black women
Based on the few studies that presented findings in African American women, an inverse association with physical activity was found for pre and postmenopausal African American women, while the association for anthropometric and other dietary factors, such as alcohol, was unclear. Studies assessing the effect by molecular subtypes in African American women were too few and based on sample sizes too small to provide definitive conclusions.
The effect of certain nutrition and lifestyle factors on breast cancer in African American is not starkly distinct from those observed in White women. However, there is an enormous need for further research on this minority group to obtain more confirmatory findings.
breast cancer; African American; nutrition; obesity; physical activity
Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca2+) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca2+ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca2+-ATPase (SERCA) which is involved in the regulation of the intracellular Ca2+ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes.
To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of three genome-wide association studies (GWAS) and two independent datasets genotyped on the Immunochip, involving 10,588 cases and 22,806 controls in total. We identified 15 new disease susceptibility regions, increasing the number of psoriasis-associated loci to 36 for Caucasians. Conditional analyses identified five independent signals within previously known loci. The newly identified shared disease regions encompassed a number of genes whose products regulate T-cell function (e.g. RUNX3, TAGAP and STAT3). The new psoriasis-specific regions were notable for candidate genes whose products are involved in innate host defense, encoding proteins with roles in interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C), and NF-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense.
Recruitment of controls remains a challenge in case–control studies and particularly in studies involving minority populations.
We compared characteristics of controls recruited through random digit dialing (RDD) to those of community controls enrolled through churches, health events and other outreach sources among women of African ancestry (AA) participating in the Women’s Circle of Health Study, a case–control study of breast cancer. Odds ratios and 95% confidence intervals were also computed using unconditional logistic regression to evaluate the impact of including the community controls for selected variables relevant to breast cancer and for which there were significant differences in distribution between the two control groups.
Compared to community controls (n=347), RDD controls (n=207) had more years of education and higher income, lower body mass index, were more likely to have private insurance, and less likely to be single. While the percentage of nulliparous women in the two groups was similar, community controls tended to have more children, have their first child at a younger age, and were less likely to breastfeed their children. Dietary intake was similar in the two groups. Compared to census data, the combination of RDD and community controls seems to be more representative of the general population than RDD controls alone. Furthermore, the inclusion of the community group had little impact on the magnitude of risk estimates for most variables, while enhancing statistical power.
Community-based recruitment was found to be an efficient and feasible method to recruit AA controls.
African American; Case–control studies; Controls; Community controls; Minorities; Random digit dialing; Recruitment
Several studies have documented a significant association between vasomotor symptoms (VMS) and a decrement in health outcomes among postmenopausal women, but these studies have mostly focused on the US. The aim of the current study was to broaden this investigation by examining the burden of VMS symptoms in the European Union with respect to both humanistic and economic outcomes.
All women aged 40–75 years who completed the 2010 5EU (France, Germany, Italy, Spain, and the UK) National Health and Wellness Survey were identified as potential respondents and invited to participate in an additional cross-sectional, Internet-based survey. Only postmenopausal women from 5EU were included in the current analyses (n = 3801). VMS was assessed using the Menopausal Rating Scale, and was used in multiple regression models as the primary predictor of health status (EQ-5D-3L), work productivity loss, and the number of physician visits due to menopause.
Over half (50.3%) of postmenopausal women experienced either mild (24.6%), moderate (17.6%), or severe (8.1%) VMS. Controlling for confounding variables, mild (b = −0.03, P < 0.05), moderate (b = −0.07, P < 0.05), and severe VMS (b = −0.17, P < 0.05) were each associated with worse health utilities relative to women without VMS. Similarly, increased resource use (b = 1.04–2.39, all P < 0.05), overall work impairment (b = 8.71–19.69, all P < 0.05), and activity impairment (b = 11.22–24.54, all P < 0.05) were also observed as VMS severity increased (with each b representing the difference between each level of severity and the reference category).
These results suggest a high prevalence of VMS in Western Europe. These symptoms are also associated with both humanistic and economic outcomes. Improved management of VMS may be able to increase the health status and ability to work productively as well as reduce societal direct costs.
menopause; vasomotor symptoms; health status; work productivity
•Human embryonic stem cell derived neurons develop NMDA and AMPA receptors in culture.•Enriched HESC-derived neurons develop functional glutamate responses in vitro.•MK 801 confers a protective effect on glutamate-induced excitotoxic neuronal death.•Studies using human pluripotent stem cells may aid the discovery of novel therapeutic targets.
Thanks to the development of efficient differentiation strategies, human pluripotent stem cells (HPSC) offer the opportunity for modelling neuronal injury and dysfunction in human neurons in vitro. Critically, the effective use of HPSC-derived neural cells in disease-modelling and potentially cell replacement therapies hinges on an understanding of the biology of these cells, specifically their development, subtype specification and responses to neurotoxic signalling mediators. Here, we generated neurons from human embryonic stem cells and characterised the development of vulnerability to glutamate excitotoxicity, a key contributor to neuronal injury in several acute and chronic neurodegenerative disorders. Over two months of differentiation we observed a gradual increase in responsiveness of neurons to glutamate-induced Ca2+ influx, attributable to NMDA receptor activity. This increase was concomitant with an increase in expression of mRNA encoding NMDA and AMPA receptor subunits. Differentiated neurons were vulnerable to glutamate excitotoxicity in a dose-dependent manner, which was reduced by NMDA receptor antagonists.
aCSF, artificial cerebrospinal fluid; AMPA, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid; AMPAR, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptor; D-APV, aminophosphonopentanoic acid; HESC, human embryonic stem cell; HPSC, human pluripotent stem cell; iPSC, induced pluripotent stem cell; NPC, neural precursor cell; NMDA, N-methyl-d-aspartic acid; NMDAR, N-methyl-d-aspartic acid receptor; Human pluripotent stem cells; MK 801; Excitotoxicity; Glutamate; Disease-modelling
The Nod-like receptor, Nlrp3, has been linked to inflammatory diseases and adjuvant-mediated immune responses. A wide array of structurally diverse agents does not interact directly with Nlrp3, but is thought to activate the Nlrp3 inflammasome by inducing a common upstream signal, such as lysosome rupture. To test the connection between lysosome integrity and Nlrp3 signaling, we analyzed inflammasome activation following stimulation of murine macrophages with lysosome-destabilizing agents and pyroptosis inducers. Here we provide evidence that lysosomal rupture and the corresponding release of lysosomal hydrolases is an early event in macrophages exposed to the lysosome-destabilizing adjuvants LLOMe and alum. Lysosome rupture preceded cell death induction mediated by these agents and was associated with the degradation of low-molecular weight proteins, including the inflammasome component caspase-1. Proteolysis of caspase-1 was controlled by specific cathepsins, but was independent of autocatalytic processes and Nlrp3 signaling. Consistent with these findings, lysosome-disrupting agents triggered only minimal caspase-1 activation and failed to cause caspase-1-dependent cell death (pyroptosis), generally associated with Nlrp3 signaling. In contrast, lysosome rupture was a late event in macrophages exposed to prototypical pyroptosis inducers. These agents triggered extensive Nlrp3 signaling prior to lysosome rupture with only minimal impact on the cellular proteome. Taken together, our findings suggest that lysosome impairment triggers a cascade of events culminating in cell death but is not crucial for Nlrp3 signaling. The significant differences observed between lysosome-disrupting agents and pyroptosis inducers might explain the distinct immunologic responses associated with these compounds.
Nlrp3 inflammasome; caspase-1; lysosome rupture; necrosis; pyroptosis
Hypertension is a risk factor for several cardiovascular diseases and oxidative stress suggested to be involved in the pathophysiology. Antihypertensive drug Clonidine action in ameliorating oxidative stress was not well studied. Therefore, this study investigate the effect of Clonidine on oxidative stress markers and nitric oxide (NO) in SHR and nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) administered SHR. Male rats were divided into four groups [SHR, SHR+Clonidine (SHR-C), SHR+L-NAME, SHR+Clonidine+L-NAME(SHRC+L-NAME)]. Rats (SHRC) were administered with Clonidine (0.5 mg kg−1 day−1) from 4 weeks to 28 weeks in drinking water and L-NAME (25 mg kg−1 day−1) from 16 weeks to 28 weeks to SHRC+L-NAME. Systolic blood pressure (SBP) was measured. At the end of 28 weeks, all rats were sacrificed and in their heart homogenate, oxidative stress parameters and NO was assessed. Clonidine treatment significantly enhanced the total antioxidant status (TAS) (P < 0.001) and reduced the thibarbituric acid reactive substances (TBARS) (P < 0.001) and protein carbonyl content (PCO) (P < 0.05). These data suggest that oxidative stress is involved in the hypertensive organ damage and Clonidine not only lowers the SBP but also ameliorated the oxidative stress in the heart of SHR and SHR+L-NAME.
Angiogenin, a 14-kDa multi-functional pro-angiogenic growth factor, is up-regulated in several types of cancers. Anti-angiogenin monoclonal antibodies used as antagonists inhibited the establishment, progression, and metastasis of human cancer cells in athymic mice (Olson et al. 1994). Silencing angiogenin and inhibition of angiogenin’s nuclear translocation blocked cell survival and induced cell death in B-lymphoma and endothelial cells latently infected with Kaposi sarcoma associated herpesvirus (KSHV) (Sadagopan et al. 2009) suggesting that actively proliferating cancer cells could be inducing angiogenin for inhibiting apoptotic pathways. However, the mechanism of cell survival and apoptosis regulation by angiogenin and their functional significance in cancer is not known. We demonstrate that angiogenin interacts with p53 and colocalizes in the nucleus. Silencing endogenous angiogenin induced p53 promoter activation and p53 target gene (p53, p21 and Bax) expression, down-regulated anti-apoptotic Bcl-2 gene expression and increased p53 mediated cell death. In contrast, angiogenin expression blocked pro-apoptotic Bax and p21 expression, induced Bcl-2 and blocked cell death. Angiogenin also co-immunoprecipitated with p53 regulator protein Mdm2. Angiogenin expression resulted in the inhibition of p53 phosphorylation, increased p53-Mdm2 interaction, and consequently increased ubiquitination of p53. Taken together these studies demonstrate that angiogenin promotes the inhibition of p53 function to mediate anti-apoptosis and cell survival. Our results reveal for the first time a novel p53 interacting function of angiogenin in anti-apoptosis and survival of cancer cells and suggest that targeting angiogenin could be an effective therapy for several cancers.
Angiogenin; p53; Mdm2; KSHV; cancer; anti-apoptosis; p53 ubiquitination
Commensal Escherichia coli are a prominent reservoir of genes coding for antibiotic resistance and also responsible for endogenous infections in pregnant women. We studied the factors in pregnant women associated with carriage of multi-drug resistant (MDR) E. coli and genetic determinants of antibiotic resistance in them.
Women attending to Obstetric and Gynaecology department outpatient clinics for routine antenatal check-up were administered a questionnaire. Peri-anal swabs were collected for culture isolation and identification of E.coil. Antibiotic sensitivity was done using the Kirby-Bauer disc diffusion method as recommended by the CLSI guidelines. MICs for quinolones and third generation cephalosporins were done using the agar dilution method. Genes coding for production of beta lactamses and for the quinolone resistance determinant were screened by polymerase chain reaction. Rep-PCR was done on MDR isolates for detecting possible genetic similarity. Multiple logistic regression models were used to determine the independent factors associated with carriage of MDR isolates.
A total of 710 isolates of E. coli from 710 women (mean age 26 years) were included in the study. Resistance to at least one antibiotic tested was detected in 94% of the E. coli isolates. A total of 109 isolates were ESBL producing and 35 isolates were MDR. In the MDR isolates MIC50 and MIC90 for quinolones and third generation cephalosporins were high for those isolates that carried blaTEM gene (26 isolates) and blaCTX-M gene (24 isolates). Both blaTEM and blaCTX-M genes were detected in 19 isolates. The commonest Plasmid Mediated Quinolone Resistance (PMQR) gene identified was aac(6′)-Ib-cr (n = 23/25). All isolates carrying the PMQR genes were also positive for blaCTX-M and blaTEM gene. Mutations in gyr A and par C genes were present in all 35 MDR isolates. The statistically significant risk factors for carriage of MDR E. coli were graduate or post-graduate education, a self-employed status, a family size of more than 10 members, antibiotic usage in last four weeks, and history of hospitalization in the last four weeks.
The presence of genes coding for extended spectrum of beta lactamases and plasmid mediated quinolone resistance in commensal E. coli is disconcerting. The study provides strong basis good antibiotic stewardship.