Expression of F1L, a vaccinia virus H3L transmembrane protein analogue of orf virus, and its successful purification as a diagnostic antigen.

Orf or contagious ecthyma is a highly contagious, zoonotic, and economically important global viral disease of small ruminants and is endemic in India. Vaccination of susceptible goats/sheep along with suitable recombinant protein-based serological assay will be useful in the control of the infection. In this study, the full-length and truncated versions of F1L encoding gene (ORF 059) of orf virus were cloned into pFasBac HT A vector, transformed in DH10Bac cells, and expressed in insect cells. The full-length and truncated recombinant F1L proteins were expressed as a 6 × histidine-tagged fusion protein for ease of purification by Ni-NTA affinity chromatography under denaturing conditions. A protein with ~ 40 kDa and ~ 35 kDa for full-length and truncated F1L protein, respectively, were expressed and confirmed by SDS-PAGE and western blot. The protein reactivity evaluated by western blot analysis and indirect ELISA using ORFV hyperimmune serum was also found to be reactive. The results of the present study showed that the purified recombinant F1L protein can be used as a diagnostic antigen in sero-surveillance of ORFV infection in small ruminants. To the best of authors' knowledge, this is the first report on the expression of ORFV F1L in insect cells using a baculovirus vector and its successful purification to use as the potential diagnostic antigen in ELISA.

Prokaryotic expression, purification and evaluation of goatpox virus ORF117 protein as a diagnostic antigen in indirect ELISA to detect goatpox.

Goatpox is an economically significant transboundary viral disease of goats that is caused by goatpox virus (GTPV). This study describes the prokaryotic expression of the GTPV ORF117 protein, a homologue of vaccinia virus A27L, and evaluation of its diagnostic potential in ELISA. The GTPV ORF117 gene was cloned into the pET32a vector to express recombinant ORF117 protein (rA27L) in E. coli BL21-CodonPlus (DE3)-RIPL. The bacterial expression of the protein was confirmed by western blot analysis using anti-GTPV polyclonal antibodies that detected rA27L, which is ~ 35 kDa in size. rA27L was affinity purified under native conditions and used to assess the antibody response in an optimized indirect ELISA. The purified antigen specifically reacted with anti-GTPV and anti-SPPV serum in ELISA. A preliminary screening of random and purposive serum samples (n = 520) from sheep and goats using this optimized ELISA gave a positivity rate of 19.4 % with a diagnostic specificity of 88.7% and diagnostic sensitivity of 98.5% when compared to the gold standard serum neutralization test. Our results suggest that the indirect ELISA based on the rA27L protein has potential for serosurveillance and seromonitoring of GTPV in goats.

Ecosystem services in different agro-climatic zones in eastern India: impact of land use and land cover change.

Land use and land cover (LULC) change have considerable influence on ecosystem services. Assessing change in ecosystem services due to LULC change at different spatial and temporal scales will help to identify suitable management practices for sustaining ecosystem productivity and maintaining the ecological balance. The objective of this study was to investigate variations in ecosystem services in response to LULC change over 27 years in four agro-climatic zones (ACZ) of eastern India using satellite imagery for the year 1989, 1996, 2005, 2011 (Landsat TM) and 2016 (Landsat 8 OLI). The satellite images were classified into six LULC classes, agriculture land, forest, waterbody, wasteland, built-up, and mining area. During the study period (1989 to 2016), forest cover reduced by 5.2%, 13.7%, and 3.6% in Sambalpur, Keonjhar, and Kandhamal districts of Odisha, respectively. In Balasore, agricultural land reduced by 17.2% due to its conversion to built-up land. The value of ecosystem services per unit area followed the order of waterbodies > agricultural land > forests. A different set of indicators, e.g., by explicitly including diversity, could change the rank between these land uses, so the temporal trends within a land use are more important than the absolute values. Total ecosystem services increased by US$ 1296.4 × 105 (50.74%), US$ 1100.7 × 105 (98.52%), US$ 1867 × 105 (61.64%), and US$ 1242.6 × 105 (46.13%) for Sambalpur, Balasore, Kandhamal, and Keonjhar, respectively.

Alanine Aminotransferase and Total Bilirubin Concentration in Preeclampsia and Eclampsia.

Eclampsia is an important cause of maternal mortality in developing countries. This cross-sectional comparative study was conducted in the Departments of Clinical Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), and Obstetrics and Gynecology, BSMMU, and Dhaka Medical College Hospital, Dhaka, Bangladesh, from March 2012 to February 2013 and was designed to evaluate liver markers to predict preeclampsia. One hundred fifty (150) women with pregnancy of ≥28 weeks, 50 for each normal, preeclampsia and eclampsia group, were enrolled purposively. The protocol was approved by IRB of BSMMU. Consent was taken from each patient. Serum total Bilirubin and ALT were assessed. Data was collected in a questionnaire and analyzed by SPSS-16. Quantitative data were compared by ANOVA or 't' test and qualitative data by chi-square test. P value <0.05 was considered significant. The patients of all groups were similar in age and gravida. The mean±SD serum total bilirubin and ALT were significantly higher in preeclampsia and eclampsia groups than normal pregnancy. However they were similar in preeclampsia and eclampsia group.

Practically convenient and industrially-aligned methods for iridium-catalysed hydrogen isotope exchange processes.

The use of alternative solvents in the iridium-catalysed hydrogen isotope exchange reaction with developing phosphine/NHC Ir(I) complexes has identified reaction media which are more widely applicable and industrially acceptable than the commonly employed chlorinated solvent, dichloromethane. Deuterium incorporation into a variety of substrates has proceeded to deliver high levels of labelling (and regioselectivity) in the presence of low catalyst loadings and over short reaction times. The preparative outputs have been complemented by DFT studies to explore ligand orientation, as well as solvent and substrate binding energies within the catalyst system.

Isolation of bluetongue virus serotype 1 from aborted goat fetuses.

Abortions and stillbirths were noticed in pregnant goats on a farm in the state of Gujarat, India. About 50% of the pregnant goats aborted or gave birth to dead kids. Bluetongue virus (BTV) antibody in the sera of affected goats was detected using a competitive enzyme-linked immunosorbent assay (ELISA). Viral antigen in the blood of these goats and in the aborted fetal spleens was detected using a sandwich ELISA. Two viruses (SKN-9, SKN-10) were isolated in cell culture from aborted fetal spleens and were confirmed as Orbivirus by demonstration of ten bands in RNA polyacrylamide gel electrophoresis and identified as BTV-1 by sequencing of the VP2 gene. Sequence analyses revealed thatthese isolates were very closely related to a BTV-1 (strain SKN-8) isolated from Culicoides vectors captured on the same farm one month after the occurrence of abortion. Isolation of BTV-1 from fetuses is probably evidence of transplacental transmission of the wild-type strain, because attenuated or laboratory-adapted BTV-1 strains have never been used in this region. This may have important implications in the epidemiology of bluetongue, considering the presence of many BTV serotypes in India.

Physico-chemical characterization and biological response of Labeo rohita-derived hydroxyapatite scaffold.

The chemically treated Labeo rohita scale is used for synthesizing hydroxyapatite (HAp) biomaterials. Thermogravimetric and differential thermal analyses of fish scale materials reveal the different phase changes with temperature and find out the suitable calcination temperatures. The composition and structures of wet ball-milled calcined HAp powders are characterized by Fourier transform infrared spectroscopy, X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray analysis (EDX). The EDX as well as chemical analysis of fish scale-derived apatite materials confirms that the Ca/P ratio is 1.71. The compressive stress, hardness and porosity have been evaluated on sintered HAp biomaterials. The cell attachment on HAp surfaces, cytotoxicity evaluation and MTT assay, which are carried out in RAW macrophage-like cell line media demonstrate good biocompatibility. The histological analysis also supports the bioaffinity of processed HAp biomaterials in Wistar rat model for investigating the contact reaction and stability at the artificial or natural prosthesis interface.

Stroke in Bangladeshi patients and risk factor.

To evaluate comprehensively the distribution of established risk factors of stroke among Bangladeshi patients. This is an observational study. It involved 8400 stroke patients from different hospitals in Bangladesh over a period of sixteen years. Common established risk factors of stroke e.g. age, sex, family history, hypertension, diabetes, ischemic heart disease, smoking, obesity, dyslipidaemia, alcoholism, use of oral contraceptive pill, lack of fresh fruit consumption etc. were evaluated in these patients through a preformed questionnaire and data were analyzed. Majority of the stroke events occurred after the age of forty (82.3%) and the ischemic stroke (72%) is the most common. Apart from non modifiable risk factors (advancing age, sex, Family history of stroke) hypertension was the most common modifiable risk factor found in stroke patients (57.6%) followed by smoking (44.6%), tobacco use (24.3%), OCP use in female (40% of female stroke), diabetes (23%), ischemic heart disease (17.1%), obesity (10.6%) and dyslipidaemia (5.3%). Lack of fresh fruit consumption and alcoholism were found in some of the patients. Stroke is common after the age of forty. Ischemic events are commonest type of stroke. Hypertension, smoking, tobacco use, diabetes and ischemic heart disease were five most common risk factors of stroke. Outlining the common stroke risk factors in our settings, may help the physicians and care givers in managing this disabling disease properly.

Effect of Duration of Hemostatic Compression on Radial Artery Occlusion after Transradial Percutaneous Coronary Intervention.

Radial artery occlusion (RAO) is now a major concern in transradial approach (TRA). RAO limits future radial artery use for further TRA, for as a conduit during CABG, for invasive hemodynamic monitoring and for creation of arteriovenous fistula for hemodialysis in Chronic Kidney Disease (CKD) patients through same vascular approach. The effect of duration of hemostatic compression of RAO is unknown in Bangladesh. This prospective observational study was conducted in the department of Cardiology, National Institute of Cardiovascular Diseases, Dhaka, Bangladesh (NICVD) from September 2018 to August 2019, to evaluate the effect of duration of hemostatic compression on the incidence of radial artery occlusion (RAO) after transradial percutaneous coronary intervention. A total of 140 patients underwent percutaneous coronary intervention (PCI) through TRA. RAO was defined as an absence of antegrade flow or monophasic flow or invert flow on Duplex study. In this study 70 patients (Group I) received 2 hours hemostatic compression after transradial PCI. Another 70 patients (Group II) received 6 hours hemostatic compression after transradial PCI. Radial arterial blood flow assessed at early (24 hours) and late (30 days) by color duplex study after the procedure in both groups. Early radial artery occlusion occurred in 4.3% of patients in Group I and 12.8% of patients in Group II, (p=0.04). Late radial artery occlusion occurred in 2.8% of patients in Group I and 11.4% of patients in Group II, the difference was statistically significant (p=0.04). From multivariate logistic regression analysis duration of hemostatic compression time 6 hours (p=0.01), post-procedural nitroglycerine use (p=0.03) and procedure time (p=0.03) were predictors of RAO. Shorter duration of hemostatic compression is associated with a lower incidence of early and late radial artery occlusion after transradial intervention.

Exploring the art of medical photography - an otolaryngologist's perspective.

BACKGROUND: Modern day otolaryngology has expanded beyond the ear, nose and throat to include head and neck surgery and aesthetic facial procedures. Photographic documentation is important within this expanded horizon. The spectrum of clinical photography includes photomicrographs, endoscopic photographs, peri-operative photography and medical social photography. METHOD: This article aimed to review the standard guidelines essential to obtain, store and disseminate photographs and looked at setting up a small clinic with minimal gadgets to suit clinical photography requirements. Elaboration of basic photography techniques in otolaryngology was reviewed, with examples of photographs taken in a clinic by a clinician. Advances and innovation in clinical photography, in the form of smartphone photography, artificial intelligence, device editing and newer hardware and software in otorhinolaryngology was reviewed. CONCLUSION: Having a professional photographer to aid a clinician is a luxury. Simple knowledge and regular practice of basic photography guidelines by a clinician is imperative.

Vapor Pressure versus Temperature Relations of Common Elements.

The vapor pressure values of common elements are available in the literature over a limited temperature range and the accuracy and reliability of the reported data are not generally available. We evaluate the reliability and uncertainty of the available vapor pressure versus temperature data of fifty common pure elements and recommend vapor pressure versus temperature relations. By synthesizing the vapor pressure values from measurements reported in the literature with the values computed using the Clausius Clapeyron relation beyond the boiling point, we extend the vapor pressure range from 10-8 atm to 10 atm. We use a genetic algorithm to optimize the fitting of the vapor pressure data as a function of temperature over the extended vapor pressure range for each element. The recommended vapor pressure values are compared with the corresponding literature values to examine the reliability of the recommended values.

Propagation of vaccine strain of duck enteritis virus in a cell line of duck origin as an alternative production system to propagation in embryonated egg.

Duck virus enteritis (DVE) also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with a chicken embryo-adapted live virus that is known to be poorly immunogenic and affords partial protection. Further, the risk of harboring other infectious agents in the embryo particularly the deadly and zoonotic avian influenza virus is also high. In this paper, we report propagation of a chicken embryo-adapted vaccine strain of duck enteritis virus in duck embryo fibroblast (DEF) cell line. Thirty serial passages were done in DEF cell that made the vaccine virus further attenuated which was tested in ducks. The growth behaviors of the virus in DEF cells were studied and at 30th passage level the virus titre was found to be 10(6.8) TCID(50)/ml. Ducks were immunized with this virus and challenged after 21 days with high dose of virulent DEV. All the immunized ducks withstood challenge with no clinical symptoms in any of the ducks while all the control ducks died. DEF cell which is free from other infectious agents appears to be a good system for cultivation of duck enteritis virus vaccine strain.

Evidence of mixed infection of peste des petits ruminants virus and bluetongue virus in a flock of goats as confirmed by detection of antigen, antibody and nucleic acid of both the viruses.

A mixed infection with peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) occurred in goats which exhibited symptoms characteristic of PPR. A number of samples were collected from ailing or dead goats for labrotory diagnosis. Antibody to BTV and PPRV was detected in sera samples by competitive ELISA. No PPRV antigen was detected in tissue samples like lung and spleen, however, presence of PPRV antigen in some sera samples was confirmed by sandwich ELISA. All the blood samples collected from the ailing animals were found positive for BTV antigen by a sandwich ELISA. BTV- and PPRV nucleic acids were amplified from the pooled blood and tissue samples respectively by RT-PCR assays. The identity of the amplicons was confirmed by cloning and sequencing. All these tests confirm that the goats were infected with PPRV and BTV simultaneously. Isolation of viruses from the clinical samples is underway.

A novel non-enzymatic zinc oxide thin film based electrochemical recyclable strip with device interface for quantitative detection of catechol in water.

Catechol, one of the major effluents released by various chemical and metal processing industries, causes severe pollution of groundwater. Monitoring of catechol in water using cost-effective, handheld sensor is demanding for the safety of the environment. In this work, non-enzymatic zinc oxide thin film based electrochemical strip sensor is developed on conducting glass substrate for detection of catechol. The preparation of strip without employing standard Pt or Ag/AgCl electrodes and simply depositing ZnO through wet chemical process represents a cost-effective innovative technique. The ZnO thin film is characterized using field emission scanning electron microscope (FESEM), energy dispersive X-ray spectroscopy (EDS), atomic force microscopy (AFM) and grazing incidence X-ray diffractometer (GIXRD). Catechol is electrochemically detected by means of cyclic voltammetry and amperometry. A prominent redox peak of the developed strip attributed to the detection of catechol is observed at -0.26 V in cyclic voltammetry. The strip is integrated with readout meter and an algorithm is built based on the experimentally observed linear variation of amperometric current with catechol concentration. The quantitative detection performance is demonstrated by testing 0.1-12 ppm catechol solutions.

Prokaryotic expression of truncated VP7 of bluetongue virus (BTV) and reactivity of the purified recombinant protein with all BTV type-specific sera.

Purification of bluetongue virus (BTV) group-specific VP7 protein, expressed in prokaryotic system as histidine-tagged fusion protein is described in the present study. The major antigenic portion of VP7 gene of BTV 23 was amplified from the extracted RNA by reverse transcription polymerase chain reaction and cloned. The recombinant expression construct (pET-VP7) was identified by the polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion truncated VP7 protein with a molecular mass of 36 kDa was determined by Western blot analysis using anti-His antibody. The expressed VP7 was purified to near homogeneity by chromatography on nickel-agarose column as judged by sodium dodesyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP7 protein was recognized by antibody to BTV in Western blot analysis. The capability of the recombinant VP7 protein to differentiate hyperimmune serum of rabbit to BTV from normal rabbit serum was evident in the enzyme-linked immunosorbent assay (ELISA). The purified VP7 reacted well with the 24 BTV serotype-specific sera obtained from OIE Reference Laboratory on bluetongue. Our results indicated that the expressed VP7 protein could be used as antigen for development of antibody-capture ELISA for detection BTV group-specific antibodies. This recombinant protein may also be used as antigen in competitive ELISA format.

Use of shredded tire chips and tire crumbs as packing media in trickling filter systems for landfill leachate treatment.

Scrap tire stockpiles are breeding grounds for pests, mosquitoes and west Nile viruses and, thereby, become a potential health risk. This experimental study was carried out in six stages to determine the suitability of shredded tire materials in a trickling filter system to treat landfill leachate. Biochemical oxygen demand (BOD5), chemical oxygen demand (COD) and NH3-N removals were obtained in the range of 81 to 96%, 76 to 90% and 15 to 68%, respectively. The removal of organics appears to be largely related to total dissolved solids reduction in leachate. A sudden increase, from time to time, in organic content of effluent could be attributed to biomass sloughing and clogging in the trickling filters. However, tire crumbs exhibited more consistent organics removal throughout the experimental program. Due to the high surface area of shredded tire chips and crumbs, a layer of biomass, 1-2 mm thick, was attached to them and was sloughed off at an interval of 21 days. Apart from that, as shredded tires are comparatively cheaper than any other usable packing material, tire chips and tire crumbs appeared to be quite promising as packing media in trickling filters for landfill leachate treatment.

Integrative functional genomic analysis identifies epigenetically regulated fibromodulin as an essential gene for glioma cell migration.

An integrative functional genomics study of multiple forms of data are vital for discovering molecular drivers of cancer development and progression. Here, we present an integrated genomic strategy utilizing DNA methylation and transcriptome profile data to discover epigenetically regulated genes implicated in cancer development and invasive progression. More specifically, this analysis identified fibromodulin (FMOD) as a glioblastoma (GBM) upregulated gene because of the loss of promoter methylation. Secreted FMOD promotes glioma cell migration through its ability to induce filamentous actin stress fiber formation. Treatment with cytochalasin D, an actin polymerization inhibitor, significantly reduced the FMOD-induced glioma cell migration. Small interfering RNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on integrin-FAK-Src-Rho-ROCK signaling pathway. FMOD lacking C-terminus LRR11 domain (ΔFMOD), which does not bind collagen type I, failed to induce integrin and promote glioma cell migration. Further, FMOD-induced integrin activation and migration was abrogated by a 9-mer wild-type peptide from the FMOD C-terminus. However, the same peptide with mutation in two residues essential for FMOD interaction with collagen type I failed to compete with FMOD, thus signifying the importance of collagen type I-FMOD interaction in integrin activation. Chromatin immunoprecipitation-PCR experiments revealed that transforming growth factor beta-1 (TGF-ß1) regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but enrichment of Sma- and Mad-related protein-2 (SMAD2) and CBP. FMOD silencing inhibited the TGF-ß1-mediated glioma cell migration significantly. In univariate and multivariate Cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, this study identified several epigenetically regulated alterations responsible for cancer development and progression. Specifically, we found that secreted FMOD as an important regulator of glioma cell migration downstream of TGF-ß1 pathway and forms a potential basis for therapeutic intervention in GBM.

e19a2 BCR-ABL fusion transcript in typical chronic myeloid leukaemia: a report of two cases.

This report describes two patients with chronic myeloid leukaemia (CML): one of them developed accelerated phase CML and died 8 years after diagnosis and the other is at the chronic phase. Sequence analysis of reverse transcription-polymerase chain reaction products showed the presence of BCR-ABL fusion transcript e19a2. This finding suggests that CML carrying mu-BCR breakpoint may exhibit a clinical course similar to typical CML.

A novel biocompatible conducting polyvinyl alcohol (PVA)-polyvinylpyrrolidone (PVP)-hydroxyapatite (HAP) composite scaffolds for probable biological application.

We have prepared biocompatible composites of 80wt% polyvinyl alcohol (PVA)-(20wt%) polyvinylpyrrolidone (PVP) blend with different concentrations of bioactive nanohydroxyapatite, Ca10(PO4)6(HO)2 (HAP). The composite films demonstrated maximum effective conductivity (σ∼1.64×10(-4)S/m) and effective dielectric constant (ε∼290) at percolation threshold concentration (∼10wt% HAP) at room temperature. These values of σ and ε are much higher than those of PVA, PVP or HAP. Our preliminary observation indicated excellent biocompatibility of the electrospun fibrous meshes of two of these composites with different HAP contents (8.5 and 5wt% within percolation threshold concentration) using NIH 3T3 fibroblast cell line. Cells viability on the well characterized composite fibrous scaffolds was determined by MTT [3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay analysis. Enhancement of σ, due to HAP addition, was found to show increased biocompatibility of the fibrous scaffold. Enhanced σ value of the PVA/PVP-HAP composite provided supporting cues for the increased cell viability and biocompatibility of the composite fibrous meshes. Excellent biocompatibility these electrospun composite scaffolds made them to plausible potential candidates for tissue engineering or other biomedical applications.

Isolation of bluetongue virus-1 from cattle in India and phylogenetic analysis of the complete coding sequence of the segment-2 gene.

The isolation of BTV-1 serotype from cattle in India and its phylogenetic relationship based on VP2 gene has been reported. Virus (JBP42/12/Ind) is isolated in BHK-21 cell line from blood sample tested positive for BTV antigen in sandwich ELISA from asymptomatic cattle. Full length VP2 gene of cattle isolate was amplified, cloned and sequenced. On BLAST analysis virus isolate was identified as BTV-1 serotype. Phylogenetic tree based on complete VP2 coding region segregated Indian isolates, Australian isolates and African/European isolates in three distinct clusters. Segregation of Indian BTV-1 isolates at close proximity to the monophyletic cluster of Australian BTV-1 isolates indicates the present isolate as "eastern topotype' of BTV. Multiple alignments of VP2 gene nucleotide sequences suggest that, Indian BTV-1 isolate is more closely related to Australian BTV-1 isolates; where 14.1% to 14.4% and 6.8% to 7.4% divergence was observed at nucleotide and deduced amino acid sequence level respectively.