Alpha amylase (α-1, 4-glucan-glucanhydrolase, EC 188.8.131.52), 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
KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2's association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.
KSHV infection of endothelial cells in humans leads into the development of Kaposi's sarcoma (KS). Hence, understanding of in vitro KSHV entry in endothelial cells is critical to develop strategies to control KSHV infection and KS. The de novo KSHV infection of endothelial HMVEC-d cells is initiated by its attachment to cell surface integrins, activation of cellular signals, and interaction with the receptor tyrosine kinase EphrinA2. This results in plasma membrane protrusions (blebs) in the lipid raft regions that engulf and internalize the virus, a process known as macropinocytosis. However, the identity of the molecule(s) coordinating the macropinocytic KSHV entry is not completely known. The present study identifies calcium and integrin-binding protein-1 (CIB1) as a key effector molecule promoting EphA2 associated signal events. CIB1 depletion by shRNA significantly reduced KSHV-induced bleb formation, activation of EphA2, Src, and Erk1/2, virus entry by macropinocytosis, productive trafficking, and infection. Our results also demonstrated that CIB1 plays a role in scaffolding EphA2 with cytoskeletal myosin IIA and alpha-actinin 4 during KSHV entry. Together, these studies reveal for the first time the role of CIB1 as a potential adaptor molecule in virus macropinocytic entry and indicate CIB1 as an attractive target to block KSHV entry and infection.
Epstein-Barr virus (EBV), etiologically linked with human B-cell malignancies and nasopharyngeal carcinoma (NPC), establishes three types of latency that facilitate its episomal genome persistence and evasion of host immune responses. The innate inflammasome responses recognize the pathogen-associated molecular patterns which lead into the association of a cytoplasmic sensor such as NLRP3 and AIM2 proteins or nuclear interferon-inducible protein 16 (IFI16) with adaptor ASC protein (apoptosis-associated speck-like protein with a caspase recruitment domain) and effector procaspase-1, resulting in active caspase-1 formation which cleaves the proforms of inflammatory interleukin-1β (IL-1β), IL-18, and IL-33 cytokines. Whether inflammasome responses recognize and respond to EBV genome in the nuclei was not known. We observed evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β, -IL-18, and -IL-33, in EBV latency I Raji cells, latency II NPC C666-1 cells, and latency III lymphoblastoid cell lines (LCL). Interaction between ASC with IFI16 but not with AIM2 or NLRP3 was detected in all three latencies and during EBV infection of primary human B cells. IFI16 and cleaved caspase-1, IL-1β, IL-18, and IL-33 were detected in the exosomes from Raji cells and LCL. Though EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) are common to all forms of EBV latency, caspase-1 cleavage was not detected in cells expressing EBNA1 alone, and blocking EBER transcription did not inhibit caspase-1 cleavage. In fluorescence in situ hybridization (FISH) analysis, IFI16 colocalized with the EBV genome in LCL and Raji cell nuclei. These studies demonstrated that constant sensing of latent EBV genome by IFI16 in all types of latency results in the constitutive induction of the inflammasome and IL-1β, IL-18, and IL-33 maturation.
In January 2008, a national multifaceted road safety intervention program (IMESEVI) funded by the Bloomberg Philanthropies was launched in Mexico. Two years later in 2010, IMESEVI was refocused as part of a 10-country international consortium demonstration project (IMESEVI/RS10). We evaluate the initial effects of each phase of the road safety intervention project on numbers of RT crashes, injuries and deaths in Mexico and in the two main target cities of Guadalajara-Zapopan and León.
An interrupted time series analysis using autoregressive integrated moving average (ARIMA) modeling was performed using monthly data of rates of RT crashes and injuries (police data), as well as deaths (mortality system data) from 1999–2011 with dummy variables representing each intervention phase.
In the period following the first intervention phase at the country level and in the city of León, the rate of RT crashes decreased significantly (p<0.05). Notably, following the second intervention phase although there was no reduction at the country level, there has been a decrease in the RT crash rate in both Guadalajara-Zapopan (p = 0.029) and in León (p = 0.029). There were no significant differences in the RT injury or death rates following either intervention phase in either city.
These initial results suggest that a multi-faceted road safety intervention program appears to be effective in reducing road crashes in a middle-income country setting. Further analysis is needed to differentiate the effects of various interventions, and to determine what other economic and political factors might have affected this change.
The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.
Two rounds of integrated biological and behavioural assessment (IBBA) surveys were done among men who have sex with men (MSM) in Andhra Pradesh during 2006 and 2009. Avahan, the India AIDS initiative, funded by the Bill and Melinda Gates Foundation implemented HIV prevention interventions among MSM starting around the time of the first round of IBBA.
Data on socio-demographic, sex behaviour characteristics and HIV status of MSM from the two IBBA rounds were used. Changes in the rates of consistent condom use over the past one month by MSM with various types of partners between the two rounds were assessed. Multivariate analysis was performed to assess associations between various factors and inconsistent condom use for sex with regular partners as well as HIV in MSM.
A significant increase in consistent condom use by MSM was noted from 2006 to 2009 for paid male partners (19.5% to 93.8%), occasional male partners (13.2% to 86.2%), and paid female partners (25.9% to 94.2%). Consistent condom use with regular sex partners also increased but remained lower with regular male partner (75.8%) and very low with regular female partners (15.7%). MSM who used condoms inconsistently with their regular male partner were also more likely to use condoms inconsistently with their regular female partner. Multivariate analysis showed MSM who used condoms inconsistently with regular male partner had higher odds of HIV (odds ratio 1.8; 95% CI 1.2-2.7). MSM who received condoms from Avahan had the lowest odds (odds ratio 0.3; 95% CI 0.1-0.5) of inconsistent condom use with regular male partners.
Condom use by MSM increased markedly after implementation of Avahan, though a causal association cannot be assessed with the available data. The relatively lower condom use with regular partners of MSM suggests that additional programme effort is needed to address this aspect specifically.
Condom use; HIV; India; Men who have sex with men; Regular sex partner
Within the Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, we identified and analyzed novel enhancers that drive midline and/or tracheal expression and compared them to previously characterized enhancers. In addition, we tested expression of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea.
About 1–2% of the babies are born with bicuspid aortic valves instead of the normal aortic valve with three leaflets. A significant portion of the patients with the congenital bicuspid valve morphology suffer from aortic valve stenosis and/or ascending aortic dilatation and dissection thus requiring surgical intervention when they are young adults. Patients with bicuspid aortic valves (BAVs) have also been found to develop valvular stenosis earlier than those with the normal aortic valve. This paper overviews current knowledge of BAVs, where several studies have suggested that the mechanical stresses induced on the valve leaflets and the abnormal flow development in the ascending aorta may be an important factor in the diseases of the valve and the aortic root. The long-term goals of the studies being performed in our laboratory are aimed towards potential stratification of bicuspid valve patients who may be at risk for developing these pathologies based on analyzing the hemodynamic environment of these valves using fluid-structure interaction (FSI) modeling. Patient-specific geometry of the normal tri-cuspid and bicuspid valves are reconstructed from real-time 3D ultrasound images and the dynamic analyses performed in order to determine the potential effects of mechanical stresses on the valve leaflet and aortic root pathology. This paper describes the details of the computational tools and discusses challenges with patient-specific modeling.
Widespread use of human pluripotent stem cells (hPSCs) to study neuronal physiology and function is hindered by the ongoing need for specialist expertise in converting hPSCs to neural precursor cells (NPCs). Here, we describe a new methodology to generate cryo-preservable hPSC-derived NPCs that retain an anterior identity and are propagatable long-term prior to terminal differentiation, thus abrogating regular de novo neuralization. Key to achieving passagable NPCs without loss of identity is the combination of both absence of EGF and propagation in physiological levels (3%) of O2. NPCs generated in this way display a stable long-term anterior forebrain identity and importantly retain developmental competence to patterning signals. Moreover, compared to NPCs maintained at ambient O2 (21%), they exhibit enhanced uniformity and speed of functional maturation, yielding both deep and upper layer cortical excitatory neurons. These neurons display multiple attributes including the capability to form functional synapses and undergo activity-dependent gene regulation. The platform described achieves long-term maintenance of anterior neural precursors that can give rise to forebrain neurones in abundance, enabling standardised functional studies of neural stem cell maintenance, lineage choice and neuronal functional maturation for neurodevelopmental research and disease-modelling.
Peste des petits ruminants (PPR) is an economically important disease of small ruminants such as sheep and goats. The disease is characterized by severe pyrexia, oculo-nasal discharge, pneumonia, necrosis and ulceration of the mucous membrane and inflammation of the gastro-intestinal tract leading to severe diarrhea. A SYBR Green I based real time RT-PCR targeting the N gene of PPRV has not been established for PPRV detection. Thus, the objective of present study was to develop highly sensitive N gene target SYBR Green I real time RT-PCR for specific detection and quantification of PPRV in clinical samples. A set of primers was designed to detect the nucleocapsid (N) gene of PPRV.
The assay exhibited high specificity as all the viruses which have clinical and structural similarities to PPRV including Canine distemper virus (CDV), Measles virus (MV), Bluetongue virus (BTV) and Newcastle disease virus (NDV) failed to show an amplification signal. The lower detection limit of the assay was 5.11 copies/μl (Ct value of 33.67 ± 0.5) and 0.001 TCID50/ml (Ct value of 34.7 ± 0.5) based on plasmid copy number and tissue culture infectivity titre. The assay was 3-log more sensitive than the conventional RT-PCR. The coefficient of variation (CV) values for intra- and inter-assay variability were low, ranging from 0.32% - 2.31%, and 0.71% - 5.32%, respectively. To evaluate the performance of the newly developed assay, a total of 36 clinical samples suspected of PPR were screened for the presence of PPRV in parallel with conventional RT-PCR. The real time RT-PCR assay detected PPRV in 30 (83.3%) of clinical samples compared to 16 (44.4%) by conventional RT-PCR.
The two-step SYBR Green I based real time RT-PCR assay reported here is highly sensitive, specific, reproducible and rapid for detection and quantification of PPRV nucleic acids.
SYBR Green I; Real time RT-PCR; N gene; PPRV; Detection; Quantitation
The activity of the mammalian target of rapamycin (mTOR), an ubiquitously expressed serine/threonine kinase, is central to the regulation of translation initiation and, consequently protein synthesis required for long-term potentiation and new synaptic connections. Recent studies show that activation of the mTOR signaling pathway is required for the rapid antidepressant actions of glutamate N-methyl-D-aspartate (NMDA) receptor antagonists such as ketamine. Our prior work documented the first evidence of robust deficits in the mTOR signaling pathway in the prefrontal cortex (PFC) from subjects diagnosed with major depressive disorder (MDD). The goal of this study was to determine whether alterations in mTOR signaling can be observed in rats exposed to the chronic unpredictable stress (CUS) model of depression. In the present study, we examined the effect of CUS on the expression of phosphorylated mTOR and its downstream signaling components in the frontal cortex, hippocampus, amygdala, and dorsal raphe. We also examined the effect of CUS on the expression of kinases that phosphorylate mTOR such as extracellular signal-regulated kinase (ERK1/2) and protein kinase B/Akt (Akt1). In addition, we examined the effect of stress on the phosphorylation of GluR1 an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit. We found that eight-weeks of CUS exposure significantly decreased the phosphorylation levels of mTOR and its downstream signaling components in the amygdala. Reduced level of phospho-mTOR in the amygdala was accompanied by decreased phosphorylation of ERK-1/2, Akt-1, and GluR1. No significant changes were seen in the frontal cortex, hippocampus, or dorsal raphe. Our study demonstrates that long-term stress exposure results in brain region-specific abnormalities in signaling pathways previously linked to novel mechanisms for rapid antidepressant effects. These observations are in line with evidence showing that mTOR and its upstream and downstream signaling partners could be important targets for the development of novel antidepressants.
mTOR signaling pathway; chronic unpredictable stress; rats; frontal cortex; hippocampus; amygdala; dorsal raphe
Present study deals with the removal of Zn(II) ions from effluent using yeast biofilm formed on gravels.
The biofilm forming ability of Candida rugosa and Cryptococcus laurentii was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy (SEM), and Confocal laser scanning microscopy (CLSM). Copious amount of extracellular polymeric substances (EPS) produced by yeast species was quantified and characterized by Fourier transform infrared spectroscopy (FT-IR).
Yeast biofilm formed on gravels by C. rugosa and C. laurentii showed 88% and 74.2% removal of Zn(II) ions respectively in batch mode. In column mode, removal of Zn(II) ions from real effluent was found to be 95.29% by C. rugosa biofilm formed on gravels.
The results of the present study showed that there is a scope to develop a cost effective method for the efficient removal of Zn(II) from effluent using gravels coated with yeast biofilm.
Biofilm on gravels; CLSM; Electroplating effluent; Packed bed column; Yeast; Zn(II) removal
Little is known about whether public health (PH) enforcement of Ohio's 2007 Smoke Free Workplace Law (SFWPL) is associated with department (agency) characteristics, practice, or state reimbursement to local PH agencies for enforcement. We used mixed methods to determine practice patterns, perceptions, and opinions among the PH workforce involved in enforcement to identify agency and workforce associations.
Focus groups and phone interviews (n=13) provided comments and identified issues in developing an online survey targeting PH workers through e-mail recruitment (433 addresses).
A total of 171 PH workers responded to the survey. Of Ohio's 88 counties, 81 (43% rural and 57% urban) were represented. More urban than rural agencies agreed that SFWPL enforcement was worth the effort and cost (80% vs. 61%, p=0.021). The State Attorney General's collection of large outstanding fines was perceived as unreliable. An estimated 77% of agencies lose money on enforcement annually; 18% broke even, 56% attributed a financial loss to uncollected fines, and 63% occasionally or never fully recovered fines. About half of agency leaders (49%) felt that state reimbursements were inadequate to cover inspection costs. Rural agencies (59%) indicated they would be more likely than urban agencies (40%) to drop enforcement if reimbursements ended (p=0.0070). Prioritization of SFWPL vs. routine code enforcement differed between rural and urban agencies.
These findings demonstrate the importance of increasing state health department financial support of local enforcement activities and improving collection of fines for noncompliance. Otherwise, many PH agencies, especially rural ones, will opt out, thereby increasing the state's burden to enforce SFWPL and challenging widespread public support for the law.
The task of international expert groups is to recommend the classification and naming of viruses. The ICTV Filoviridae Study Group and other experts have recently established an almost consistent classification and nomenclature for filoviruses. Here, further guidelines are suggested to include their natural genetic variants. First, this term is defined. Second, a template for full-length virus names (such as “Ebola virus H.sapiens-tc/COD/1995/Kikwit-9510621”) is proposed. These names contain information on the identity of the virus (e.g., Ebola virus), isolation host (e.g., members of the species Homo sapiens), sampling location (e.g., Democratic Republic of the Congo (COD)), sampling year, genetic variant (e.g., Kikwit), and isolate (e.g., 9510621). Suffixes are proposed for individual names that clarify whether a given genetic variant has been characterized based on passage zero material (-wt), has been passaged in tissue/cell culture (-tc), is known from consensus sequence fragments only (-frag), or does (most likely) not exist anymore (-hist). We suggest that these comprehensive names are to be used specifically in the methods section of publications. Suitable abbreviations, also proposed here, could then be used throughout the text, while the full names could be used again in phylograms, tables, or figures if the contained information aids the interpretation of presented data. The proposed system is very similar to the well-known influenzavirus nomenclature and the nomenclature recently proposed for rotaviruses. If applied consistently, it would considerably simplify retrieval of sequence data from electronic databases and be a first important step toward a viral genome annotation standard as sought by the National Center for Biotechnology Information (NCBI). Furthermore, adoption of this nomenclature would increase the general understanding of filovirus-related publications and presentations and improve figures such as phylograms, alignments, and diagrams. Most importantly, it would counter the increasing confusion in genetic variant naming due to the identification of ever more sequences through technological breakthroughs in high-throughput sequencing and environmental sampling.
cuevavirus; Ebola; Ebola virus; ebolavirus; filovirid; Filoviridae; filovirus; genome annotation; ICTV; International Committee on Taxonomy of Viruses; Lloviu virus; Marburg virus; marburgvirus; mononegavirad; Mononegavirales; mononegavirus; virus classification; virus isolate; virus nomenclature; virus strain; virus taxonomy; virus variant
Overseas travel, as a risk factor for the acquisition of infections due to antimicrobial-resistant organisms, has recently been linked to carbapenemase-producing Gram-negative bacteria. Multiresistant Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii strains were isolated from a wound of a Canadian patient with a recent history of hospitalization in India. This resulted in the initiation of outbreak management that included surveillance cultures. Epidemiological and molecular investigations showed that NDM-1-producing K. pneumoniae ST16 and OXA-23-producing A. baumannii ST10 strains were transmitted to 5 other patients, resulting in the colonization of 4 patients and the death of 1 patient due to septic shock caused by the OXA-23-producing A. baumannii strain. The high rate of false positivity of the screening cultures resulted in additional workloads and increased costs for infection control and clinical laboratory work. We believe that this is the first report of an infection with carbapenemase-producing Gram-negative bacteria resulting in death attributed to a patient with recent foreign hospitalization. We recommend routine rectal and wound screening for colonization with multiresistant bacteria for patients who have recently been admitted to hospitals outside Canada.
HSV encephalitis (HSE) is the most prevalent sporadic viral encephalitis. Although safe and effective antiviral therapies and greatly improved noninvasive diagnostic procedures have significantly improved outcomes, mortality (~20%) and debilitating neurological sequelae in survivors remain unacceptably high. An encouraging new development is that the focus is now shifting away from the virus exclusively, to include consideration of the host immune response to infection in the pathology underlying development of HSE. In this article, the authors discuss results from recent studies in experimental mouse models, as well as clinical reports that demonstrate a role for exaggerated host inflammatory responses in the brain in the development of HSE that is motivating researchers and clinicians to consider new therapeutic approaches for treating HSE. The authors also discuss results from a few studies that have shown that immunomodulatory drugs can be highly protective against HSE, which supports a role for deleterious host inflammatory responses in HSE. The impressive outcomes of some immunomodulatory approaches in mouse models of HSE emphasize the urgent need for clinical trials to rigorously evaluate combination antiviral and immunomodulatory therapy in comparison with standard antiviral therapy for treatment of HSE, and support for such an initiative is gaining momentum.
acyclovir; encephalitis; herpesvirus; immune pathology; immunomodulation; inflammation; innate immunity; intravenous immunoglobulin; reactive oxygen species
Multiple Sclerosis has two clinical phases reflecting distinct but inter-related pathological processes: focal inflammation drives the relapse-remitting stage and neurodegeneration represents the principal substrate of secondary progression. In contrast to the increasing number of effective anti-inflammatory disease modifying treatments for relapse-remitting disease, the absence of therapies for progressive disease represents a major unmet clinical need. This raises the unanswered question of whether elimination of clinical relapses will prevent subsequent progression and if so how early in the disease course should treatment be initiated. Experimental autoimmune encephalomyelitis in the Biozzi ABH mouse recapitulates the clinical and pathological features of multiple sclerosis including relapse-remitting episodes with inflammatory mediated demyelination and progressive disability with neurodegeneration. To address the relationship between inflammation and neurodegeneration we used an auto-immune tolerance strategy to eliminate clinical relapses in EAE in a manner analogous to the clinical effect of disease modifying treatments.
By arresting clinical relapses in EAE at two distinct stages, early and late disease, we demonstrate that halting immune driven demyelination even after the first major clinical event is insufficient to prevent long-term neurodegeneration and associated gliosis. Nonetheless, early intervention is partially neuroprotective, whereas later interventions are not. Furthermore early tolerisation is also associated with increased remyelination.
These findings are consistent with both a partial uncoupling of inflammation and neurodegeneration and that the regenerative response of remyelination is negatively correlated with inflammation. These findings strongly support the need for early combinatorial treatment of immunomodulatory therapies and neuroprotective treatments to prevent long-term neurodegeneration in multiple sclerosis.
Multiple sclerosis; Experimental autoimmune encephalomyelitis; Neurodegeneration; Remyelination; Gliosis
Laboratory evidence suggests a plausible role for dietary fat in breast cancer pathophysiology. We conducted a systematic literature review to assess the epidemiological evidence on the impact of total dietary fat and fat subtypes, measured pre- and/or postcancer diagnosis, in relation to breast cancer–specific and all-cause mortality among breast cancer survivors. Studies were included if they were in English, had a sample size ≥200, and presented the hazard ratio/rate ratio for recurrence, diseasespecific mortality, or all-cause mortality (n = 18). Although the results are mixed, most studies suggested that higher saturated fat intake prediagnosis was associated with increased risk of breast cancer–specific and all-cause mortality. Postdiagnostic trans fat intake was associated with a 45% and 78% increased risk of all-cause mortality. Higher monounsaturated fat intake before and after diagnosis was generally associated with increased risk of all-cause and breast cancer–specific mortality, albeit the majority of the studies were statistically nonsignificant. Two studies evaluating omega-3 fat intake suggested an inverse association with all-cause mortality. Although there were too few studies on fat subtypes to draw definitive conclusions, high consumption of saturated fatmay exert a detrimental effect on breast cancer–specific and all-cause mortality, whereas omega-3 fat may be beneficial. The inconsistent and limited evidence warrants research to assess the impact of consumption of fat subtypes on breast cancer recurrence and mortality.
dietary fat; fat subtypes; cancer survival; cancer recurrence; breast cancer
Rabies in domestic and wild animals continues to be a major public health threat in India. Rapid and accurate diagnosis of rabies in animals is therefore of utmost importance as the individuals who were in contact with the rabid animals are at a greater risk. A significant amount of diagnostic tissue samples submitted to our laboratory are often autolysed and the WHO recommended direct fluorescent antibody test (FAT) for rabies diagnosis cannot be used in such samples. In this pilot study we have evaluated three different diagnostic primer sets for rapid sensitive and specific detection of rabies genome from the brain samples of different species of animals. We have validated a sensitive RT-PCR assay using brain tissue samples from different species of animals such as cat, cattle, dog, mouse and human, for routine diagnosis of rabies. Our results show the potential of this assay as a confirmatory test when the FAT results are unreliable and also as an alternative diagnostic test in circumstances when the diagnostic samples are unsuitable for use in FAT. Furthermore the nucleotide sequence of nucleoprotein gene amplified using this assay can also be used for the molecular epidemiological study of the rabies viruses in India.
Rabies; India; Diagnosis; FAT; RT-PCR
Kaposi sarcoma-associated herpesvirus (KSHV) stimulates proliferation, angiogenesis, and inflammation to promote Kaposi sarcoma (KS) tumor growth, which involves various growth factors and cytokines. Previously, we found that KSHV infection of human umbilical vein endothelial cells (HUVECs) induces a transcriptional induction of the proangiogenic and proinflammatory cytokine angiopoietin-2 (Ang-2). Here, we report that KSHV induces rapid release of Ang-2 that is presynthesized and stored in the Weibel-Palade bodies (WPB) of endothelial cells upon binding to its integrin receptors. Blocking viral binding to integrins inhibits Ang-2 release. KSHV binding activates the integrin tyrosine kinase receptor signaling pathways, leading to tyrosine phosphorylation of focal adhesion kinase (FAK), the tyrosine kinase Src, and the Calα2 subunit of the l-type calcium channel to trigger rapid calcium (Ca2+) influx. Pretreatment of endothelial cells with specific inhibitors of protein tyrosine kinases inhibits KSHV-induced Ca2+ influx and Ang-2 release. Inhibition of Ca2+ mobilization with calcium channel blockers also inhibits Ang-2 release. Thus, the interaction between KSHV and its integrin receptors plays a key role in regulating rapid Ang-2 release from endothelial cells. This finding highlights a novel mechanism of viral induction of angiogenesis and inflammation, which might play important roles in the early event of KS tumor development.
A male cattle calf was detected as subclinically and naturally infected with
Mycobacterium avium subspecies
paratuberculosis (MAP) by a series of antemortem and
postmortem tests. The MAP infection was identified by strong antibody and
cell-mediated immune (CMI) response by a commercial ELISA kit and an intradermal
Johnin test, respectively, in the initial antemortem examination. The antemortem
status of the calf was further confirmed by MAP-specific interferon gamma
(IFN-γ) response. For detection of IFN-γ response, MAP-specific IFN-γ release
assays (IGRAs): (a) immuno capture ELISA (IC-ELISA) and (b) ELISPOT was
employed. In addition, the presence of intracellular cytokine IFN-γ was detected
by flow cytometry. For all cytokine assays, MAP-specific recombinant antigens
HSP65 and 35 kDa were employed to overcome the poor sensitivity and specificity
resulting from the use of Johnin, the crude protein purified derivative of MAP.
Postmortem examination of the MAP-infected/suspected cattle calf did not reveal
any pathognomonic gross lesions in the gastro-intestinal tract.
Histopathological examination of multiple organs showed the presence of
epithelioid cells/macrophages and edematous lesions in the mesenteric lymph
nodes suggestive of MAP; however, no granulomas were observed in the intestinal
tract. The necropsy samples of rectum and mesenteric lymph nodes were positive
for isolation of MAP by culture in the BACTEC™ MGIT™ 960 system, and acid fast
bacilli were demonstrated by fluorescence microscopy confirming the infection.
Due to differential and complex expression patterns of MAP antigens reported in
literature, a combination of assays such as those based on IGRAs and antibody
detection is essential. Therefore, the current experimental evidence confirms
the efficacy of the approach adopted. However, further studies will be needed to
understand the optimal combination MAP-specific antigens for use in IGRAs or
antibody assays that can be used for detecting MAP infection in every stage of
bovine IFN-γ; ELISA; ELISPOT; flow cytometry; histopathology; MAP culture; MAP diagnosis; paratuberculosis
The present work discusses and compares the toluene sensing behavior of polyaniline (PANI) and graphene/polyaniline nanocomposite (C-PANI) films. The graphene–PANI ratio in the nanocomposite polymer film is optimized at 1:2. For this, N-methyl-2-pyrrolidone (NMP) solvent is used to prepare PANI-NMP solution as well as graphene-PANI-NMP solution. The films are later annealed at 230 °C, characterized using scanning electron microscopy (SEM) as well Fourier transform infrared spectroscopy (FTIR) and tested for their sensing behavior towards toluene. The sensing behaviors of the films are analyzed at different temperatures (30, 50 and 100 °C) for 100 ppm toluene in air. The nanocomposite C-PANI films have exhibited better overall toluene sensing behavior in terms of sensor response, response and recovery time as well as repeatability. Although the sensor response of PANI (12.6 at 30 °C, 38.4 at 100 °C) is comparatively higher than that of C-PANI (8.4 at 30 °C, 35.5 at 100 °C), response and recovery time of PANI and C-PANI varies with operating temperature. C-PANI at 50 °C seems to have better toluene sensing behavior in terms of response time and recovery time.
graphene; PANI; nanocomposite polymer; toluene; sensing
Ebola Virus (EBOV) is a highly pathogenic member of the family Filoviridae of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann-Pick C1 (NPC1) and may induce conformational changes in GP leading to membrane fusion. However, the precise details of the structural changes in GP associated with proteolysis and the role of these changes in viral entry have not been established. Here, we have employed synthetic antibody technology to identify antibodies targeting EBOV GP prior to and following proteolysis (i.e. in the “uncleaved” [GPUNCL] and “cleaved” [GPCL] forms). We identified antibodies with distinct recognition profiles: FabCL bound preferentially to GPCL (EC50 = 1.7 nM), whereas FabUNCL bound specifically to GPUNCL (EC50 = 75 nM). Neutralization assays with GP-containing pseudotyped viruses indicated that these antibodies inhibited GPCL or GPUNCL mediated viral entry with specificity matching their recognition profiles (IC50: 87 nM for IgGCL; 1 μM for FabUNCL). Competition ELISAs indicate that FabCL binds an epitope distinct from that of KZ52, a well-characterized EBOV GP antibody, and from that of the luminal domain of NPC1. The binding epitope of FabUNCL was also distinct from that of KZ52, suggesting that FabUNCL binds a novel neutralization epitope on GPUNCL. Furthermore, the neutralizing ability of FabCL suggests that there are targets on GPCL available for neutralization. This work showcases the applicability of synthetic antibody technology to the study of viral membrane fusion, and provides new tools for dissecting intermediates of EBOV entry.
Ebola Virus; Filovirus; Viral Membrane Fusion; Synthetic Antibodies; Antibody Engineering; Phage Display