Assessment of the proteome profile of decellularized human amniotic membrane and its biocompatibility with umbilical cord-derived mesenchymal stem cells.

Extracellular matrix-based bio-scaffolds are useful for tissue engineering as they retain the unique structural, mechanical, and physiological microenvironment of the tissue thus facilitating cellular attachment and matrix activities. However, considering its potential, a comprehensive understanding of the protein profile remains elusive. Herein, we evaluate the impact of decellularization on the human amniotic membrane (hAM) based on its proteome profile, physicochemical features, as well as the attachment, viability, and proliferation of umbilical cord-derived mesenchymal stem cells (hUC-MSC). Proteome profiles of decellularized hAM (D-hAM) were compared with hAM, and gene ontology (GO) enrichment analysis was performed. Proteomic data revealed that D-hAM retained a total of 249 proteins, predominantly comprised of extracellular matrix proteins including collagens (collagen I, collagen IV, collagen VI, collagen VII, and collagen XII), proteoglycans (biglycan, decorin, lumican, mimecan, and versican), glycoproteins (dermatopontin, fibrinogen, fibrillin, laminin, and vitronectin), and growth factors including transforming growth factor beta (TGF-ß) and fibroblast growth factor (FGF) while eliminated most of the intracellular proteins. Scanning electron microscopy was used to analyze the epithelial and basal surfaces of D-hAM. The D-hAM displayed variability in fibril morphology and porosity as compared with hAM, showing loosely packed collagen fibers and prominent large pore areas on the basal side of D-hAM. Both sides of D-hAM supported the growth and proliferation of hUC-MSC. Comparative investigations, however, demonstrated that the basal side of D-hAM displayed higher hUC-MSC proliferation than the epithelial side. These findings highlight the importance of understanding the micro-environmental differences between the two sides of D-hAM while optimizing cell-based therapeutic applications.

Oxidative stress in polycystic ovary syndrome: a case-control study.

OBJECTIVE: To compare the serum levels of biochemical and oxidative stress markers i.e., malondialdehyde (MDA) and paraoxonase-1(PON1) in polycystic ovary syndrome (PCOS) patients and healthy female individuals of reproductive age group (18-40 years). METHODS: This case-control study was conducted in Dow University of Health Sciences (DUHS), Karachi from June 2019 to October 2020. Seventy Subjects including 35 PCOS patients that have primary subfertility problem (cases) and 35 healthy and fertile females (controls) were recruited. Serum samples were collected for analysis of insulin, sex hormone-binding globulin, testosterone, fasting blood glucose and lipid profile. PON 1 and MDA levels were estimated by ELISA. Comparison between the two groups was done using independent t-test. RESULTS: The patients had significantly increased mean body mass index (28.5+4.6 kg/m2 vs 25.7+4.5 kg/m2, p=0.014), systolic (129.6±13.9 mm of Hg vs 113±7.7 mm of Hg, p<0.001) and diastolic (78.7±8.8 mm of Hg vs 74.6±6.7 mm of Hg, p=0.032) blood pressures compared to controls. The high-density lipoprotein cholesterol levels were significantly lower in PCOS (42.2±8.6mg/dl) than controls (48.8±11.8mg/dl, p=0.009, p=0.009). Serum insulin (14.3±5.8 uIU/mL) vs (10.0±5.2 uIU/mL), p=0.002 and testosterone levels (1.3±0.9 nmol/L) vs (0.82±0.3 nmol/L), p<0.001 were significantly higher whereas sex hormone binding globulin (SHBG) levels (35.2±19.7nmol/L vs 58.8±31.0 nmol/L) were significantly lower in patients than healthy controls (p<0.001). Both oxidative stress markers, paraoxonase 1 (7.7±2.4 vs 6.4±2.6 µg/mL, p=0.04) and malondialdehyde (2.5±1.0 vs 1.9+0.51µg/mL, p=0.034) levels were significantly elevated in PCOS patients than controls. No significant correlation was found between dietary habits and life style between cases and controls. CONCLUSIONS: The study reported significantly elevated levels of oxidative stress markers in PCOS patients.

A Booster with a Genotype-Matched Inactivated Newcastle Disease Virus (NDV) Vaccine Candidate Provides Better Protection against a Virulent Genotype XIII.2 Virus.

Newcastle disease (ND) is endemic in Bangladesh. Locally produced or imported live Newcastle disease virus (NDV) vaccines based on lentogenic virus strains, locally produced live vaccines of the mesogenic Mukteswar strain, as well as imported inactivated vaccines of lentogenic strains, are being used in Bangladesh under different vaccination regimens. Despite these vaccinations, frequent outbreaks of ND are being reported in Bangladesh. Here we compared the efficacy of booster immunization with three different vaccines in chickens that had been primed with two doses of live LaSota vaccine. A total of 30 birds (Group A) were primed with two doses of live LaSota virus (genotype II) vaccine at days 7 and 28, while 20 birds (Group B) remained unvaccinated. At day 60, birds of Group A were divided into three sub-groups, which received booster immunizations with three different vaccines; A1: live LaSota vaccine, A2: inactivated LaSota vaccine, and A3: inactivated genotype XIII.2 vaccine (BD-C161/2010 strain from Bangladesh). Two weeks after booster vaccination (at day 74), all vaccinated birds (A1-A3) and half of the unvaccinated birds (B1) were challenged with a genotype XIII.2 virulent NDV (BD-C161/2010). A moderate antibody response was observed after the primary vaccination, which substantially increased after the booster vaccination in all groups. The mean HI titers induced by the inactivated LaSota vaccine (8.0 log2/5.0 log2 with LaSota/BD-C161/2010 HI antigen) and the inactivated BD-C161/2010 vaccine (6.7 log2/6.2 log2 with LaSota/BD-C161/2010 HI antigen) were significantly higher than those induced by the LaSota live booster vaccine (3.6 log2/2.6 log2 with LaSota/BD-C161/2010 HI antigen). Despite the differences in the antibody titers, all chickens (A1-A3) survived the virulent NDV challenge, while all the unvaccinated challenged birds died. Among the vaccinated groups, however, 50% of the chickens in Group A1 (live LaSota booster immunization) shed virus at 5- and 7-days post challenge (dpc), while 20% and 10% of the chickens in Group A2 (inactivated LaSota booster immunization) shed virus at 3 and 5 dpc, respectively, and only one chicken (10%) in Group A3 shed virus at 5 dpc. In conclusion, the genotype-matched inactivated NDV booster vaccine offers complete clinical protection and a significant reduction in virus shedding.

Poultry waste management practices in Bangladesh: Farmer's perceptions, and food and environmental hazards.

Objective: The poultry industry plays a key role in developing socio-economic and health sectors in Bangladesh. Poultry waste is a potential environmental threat as untreated poultry waste is used in vegetable gardens. The study aimed to investigate the current situation of small-scale poultry farms and their waste management practices in selected areas of Bangladesh and detect Escherichia coli and Salmonella in vegetables from farms using untreated poultry waste as fertilizer. Materials and Methods: A structured questionnaire-based survey was conducted in 86 small-scale poultry farms from different upazilas of Mymensingh and Khulna districts. 104 samples, including vegetables, poultry litter, water, and soil, were collected from vegetable gardens, ponds, fields, and wet markets in Mymensingh district to detect microbial contamination. Bacteria were identified based on their growth and colony morphology on selective media and motility tests. The presence of E. coli and Salmonella was confirmed by polymerase chain reaction (PCR) using a commercial PCR kit. Results: The survey revealed that mostly middle-aged males were involved in poultry farming. Most of the farmers had primary education and engaged in farming for about 5 years without training. In the study area, 37% of farmers collected droppings daily in the morning and used them as organic fertilizer. About 58% of farmers did not know the hygienic handlings of droppings and faced health problems. In PCR, either E. coli or Salmonella or both were confirmed in vegetables, litter, soil, and pond water. Conclusion: Appropriate poultry waste management practices can reduce the possible contamination of microbial agents in the human food chain.

Comparative pathogenicity of a genotype XXI.1.2 pigeon Newcastle disease virus isolate in pigeons and chickens.

Here, we performed molecular and pathogenic characterization of a Newcastle disease virus (NDV) isolate from pigeons in Bangladesh. Molecular phylogenetic analysis based on the complete fusion gene sequences classified the three study isolates into genotype XXI (sub-genotype XXI.1.2) together with recent NDV isolates obtained from pigeons in Pakistan (2014-2018). The Bayesian Markov Chain Monte Carlo analysis revealed that the ancestor of Bangladeshi pigeon NDVs and the viruses from sub-genotype XXI.1.2 existed in the late 1990s. Pathogenicity testing using mean embryo death time pathotyped the viruses as mesogenic, while all isolates carried multiple basic amino acid residues at the fusion protein cleavage site. Experimental infection of chickens and pigeons revealed no or minimum clinical signs in chickens, while a relatively high morbidity (70%) and mortality (60%) were observed in pigeons. The infected pigeons showed extensive and systemic lesions including hemorrhagic and/or vascular changes in the conjunctiva, respiratory and digestive system and brain, and atrophy in the spleen, while only mild congestion in the lungs was noticed in the inoculated chickens. Histologically, consolidation in the lungs with collapsed alveoli and edema around the blood vessels, hemorrhages in the trachea, severe hemorrhages and congestion, focal aggregation of mononuclear cells, and single hepatocellular necrosis in the liver, severe congestion, multifocal tubular degeneration, and necrosis, as well as mononuclear cell infiltration in the renal parenchyma, encephalomalacia with severe neuronal necrosis with neuronophagia were noticed in the brain in infected pigeons. In contrast, only slight congestion was found in lungs of the infected chickens. qRT-PCR revealed the replication of the virus in both pigeons and chickens; however, higher viral RNA loads were observed in oropharyngeal and cloacal swabs, respiratory tissues, and spleen of infected pigeons than the chickens. In conclusion, genotype XXI.1.2 NDVs are circulating in the pigeon population of Bangladesh since 1990s, produce high mortality in pigeons with pneumonia, hepatocellular necrosis, renal tubular degeneration, and neuronal necrosis in pigeons, and may infect chickens without overt signs of clinical disease and are likely to shed viruses via the oral or cloacal routes.

Experimental Pathogenicity of H9N2 Avian Influenza Viruses Harboring a Tri-Basic Hemagglutinin Cleavage Site in Sonali and Broiler Chickens.

Low-pathogenic avian influenza (LPAI) H9N2 virus is endemic in Bangladesh, causing huge economic losses in the poultry industry. Although a considerable number of Bangladeshi LPAI H9N2 viruses have been molecularly characterized, there is inadequate information on the pathogenicity of H9N2 viruses in commercial poultry. In this study, circulating LPAI H9N2 viruses from recent field outbreaks were characterized, and their pathogenicity in commercial Sonali (crossbred) and broiler chickens was assessed. Phylogenetic analysis of currently circulating field viruses based on the hemagglutinin (HA) and neuraminidase (NA) gene sequences revealed continuous circulation of G1 lineages containing the tri-basic hemagglutinin cleavage site (HACS) motif (PAKSKR*GLF) at the HA protein. Both the LPAI susceptible Sonali and broiler chickens were infected with selected H9N2 isolates A/chicken/Bangladesh/2458-LT2/2020 or A/chicken/Bangladesh/2465-LT56/2021 using intranasal (100 µL) and intraocular (100 µL) routes with a dose of 106 EID50/mL. Infected groups (LT_2-So1 and LT_56-So2; LT_2-Br1 and LT_56-Br2) revealed no mortality or clinical signs. However, at gross and histopathological investigation, the trachea, lungs, and intestine of the LT_2-So1 and LT_56-So2 groups displayed mild to moderate hemorrhages, congestion, and inflammation at different dpi. The LT 2-Br1 and LT 56-Br2 broiler groups showed nearly identical changes in the trachea, lungs, and intestine at various dpi, indicating no influence on pathogenicity in the two commercial bird species under study. Overall, the prominent lesions were observed up to 7 dpi and started to disappear at 10 dpi. The H9N2 viruses predominantly replicated in the respiratory tract, and higher titers of virus were shed through the oropharyngeal route than the cloacal route. Finally, this study demonstrated the continuous evolution of tri-basic HACS containing H9N2 viruses in Bangladesh with a low-pathogenic phenotype causing mild to moderate tracheitis, pneumonia, and enteritis in Sonali and commercial broiler chickens.

The role of epigenetic modifiers in the hepatic differentiation of human umbilical cord derived mesenchymal stem cells.

Human umbilical cord (hUC) derived mesenchymal stem cells (MSCs) can be progressively differentiated into multiple lineages including hepatic lineages, and thus provide an excellent in vitro model system for the study of hepatic differentiation. At present, hepatic differentiation protocols are based on the use of soluble chemicals in the culture medium and provide immature hepatic like cells. Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) are two important epigenetic modifiers that regulate stem cell differentiation. Therefore, this study aimed to investigate the role of HDACi, valproic acid (VPA) and DNMTi,5-azacytidine (5-aza) along with a hepatic inducer in the hepatic differentiation of hUC-MSCs. hUC-MSCs were characterized via immunocytochemistry and flow cytometry. The final concentrations of VPA and 5-aza were optimized via MTT cytotoxicity assay. All treated groups were assessed for the presence of hepatic genes and proteins through qPCR and immunocytochemistry, respectively. The results showed that the pretreatment of epigenetic modifiers not only increased the hepatic genes but also increased the expression of the hepatic proteins. VPA induces hepatic differentiation in hUC-MSCs with significant gene expression of hepatic markers i.e., FOXA2 and CK8. Moreover, VPA pretreatment enhanced the expression of hepatic proteins AFP and TAT. The pretreatment of 5-aza shows significant gene expression of hepatic marker LDL-R. However, 5-aza treatment failed to induce hepatic protein expression. The results of the current study highlighted the effectiveness of epigenetic modifiers in the hepatic differentiation of hUC-MSCs. These differentiated cells can be employed in cell-based therapeutics for hepatic diseases in future.

The First Report of a Virulent Newcastle Disease Virus of Genotype VII.2 Causing Outbreaks in Chickens in Bangladesh.

Newcastle disease (ND) is endemic in poultry in Bangladesh. We performed genotypic and pathotypic characterization of four ND virus (NDV) isolates from recent outbreaks in broiler chickens in Bangladesh during the period of 2020-2021. Phylogenetic analysis based on the complete fusion protein gene coding sequences classified the viruses into NDV class II genotype VII.2 together with viruses from Indonesia isolated between 2014 and 2021 and a single 2020 Indian isolate. Pathogenicity testing using the intracerebral pathogenicity index in day-old chickens and mean embryo death time in embryonating chicken eggs revealed that the Bangladeshi isolates are velogenic. Inoculation of 35-day-old chickens with two NDV isolates (LT67 and N5) resulted in 100% morbidity by 3 days post inoculation (DPI), and all birds succumbed to infection by 7 DPI. Massive hemorrhages, congestion and necrotic lesions were observed in different visceral organs, which were typical for infection with a velogenic viscerotropic pathotype of NDV. At microscopic examination, tracheitis, severe pneumonia, focal proventriculitis, transmural enteritis, focal myocarditis, severe congestion and necrosis in kidneys, and lymphoid depletion in lymphoid tissues were found. Our study reports the first outbreak of the panzootic genotype VII.2 NDV in poultry in Bangladesh and documents a possible recent re-introduction of this NDV genotype from Southeast or East Asia. This study further provides viral distribution and epidemiological data that can facilitate the effective control of NDV.

Investigation of respiratory disease outbreaks of poultry in Bangladesh using two real-time PCR-based simultaneous detection assays.

For rapid and sensitive pathogen screening from field outbreaks, molecular techniques such as qPCR-based simultaneous detections are efficient. Respiratory diseases are the most detrimental diseases to the poultry industry and need to be addressed because of their major economic losses. In the current study, we have applied two different detection assays: one for simultaneous detection of avian influenza virus (AIV; M gene) and subtyping (H5, N1, H9, N2) using TaqMan probe chemistry (TaqMan multitarget) and another for simultaneous detection of Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV) using SYBR Green chemistry (SYBR Green multitarget). Two individual qPCRs were conducted for the detection of four pathogens. Surveillance of tissue (n = 158) and oropharyngeal swab (206) samples from multiple poultry flocks during the years April 2020-July 2022 applying the TaqMan and SYBR Green multitarget qPCRs revealed that 48.9% of samples were positive for respiratory infections, of which 17.2% were positive for NDV, 25.5% were positive for AIV, 9.9% were positive for IBV, and only a single positive (0.3%) for ILTV. Among the AIV, 35% were highly pathogenic subtype H5N1 and 65% were low pathogenic subtype H9N2. Co-infections of 2-3 respiratory viruses were also accurately detected. Respiratory viral pathogens are quite common in Bangladeshi poultry and can be successfully detected using multitarget simultaneous real-time quantitative polymerase chain reaction (RT-qPCR) assays like those adopted in the current study. Increased mass surveillance, along with the molecular characterization of the circulating respiratory viruses, is crucial to control the epidemic and subsequently save the Bangladeshi poultry industry.

Clinical Epidemiology, Pathology, and Molecular Investigation of Lumpy Skin Disease Outbreaks in Bangladesh during 2020-2021 Indicate the Re-Emergence of an Old African Strain.

Lumpy skin disease (LSD) emerged in Bangladesh in mid-2019, leading to great economic losses for cattle farmers. This study describes the recent occurrence of the LSDV in Bangladesh and examines the clinical manifestation of the disease in local cattle breeds, characteristic epidemiological features, and pathological findings in affected animals. In addition, a full-genome sequencing of two local LSDV isolates was carried out. A total of 565 animals from 88 households were investigated, and 165 samples (skin lesions, saliva, nasal discharge, feces, and milk) were collected for virus detection. Pathology and immunohistochemistry were performed on nodule biopsies. Fever, nodular skin lesions, and swelling of the joints were the most common clinical manifestations. Skin lesions had a higher concentration of viral DNA compared to other sample types and were therefore selected for virus isolation and characterization. Pathology of the LSD skin nodules comprised a granulomatous reaction in the dermis and hypodermis that extended to the surrounding tissues. Development of the skin lesions started with swelling of keratinocytes with cytoplasmic vacuolation, vasculitis, panniculitis, thrombosis, and infarction. Altogether, the LSDV produced transmural, hemorrhagic, necrotizing, proliferative and ulcerative dermatitis. The LSD viral antigen was detected occasionally in the macrophages, epithelial cells, and vascular smooth muscle cells. The complete genome sequence analysis revealed that the two Bangladeshi field strains (BD-V392.1 and BD-V395.1) were distinct from the contemporary field strains and were closely related to the ancestral African Neethling strain. The findings of this study will improve the diagnosis, monitoring, and control of LSD in Bangladesh.

Differentiation of CD117+ Amniotic Fluid Stem Cells towards Nephron Progenitors.

Objective: The study aimed at isolation of CD117+ stem cells from amniotic fluid samples followed by their invitro differentiation towards nephron progenitors that can be potentially used for regenerative medicine studies and to further understand pathways involved in renal pathogenesis. Methods: This experimental study was conducted at Dow Research Institute of Biotechnology and Biomedical Sciences (DRIBBS), Dow University of Health Sciences, OJHA Campus Karachi from November 2019 to December 2020. After patient consent, a Pfannenstiel incision was performed by the gynecologist through abdominal and uterine muscles without cutting into Amniotic Membrane. Using a needle of 5CC syringe connected to sterile Redivac bottle, a blunt end insertion was passed through the membrane and the amniotic fluid was aseptically sucked into Redivac bottle, the ice bag was used for transporting amniotic fluid from hospital to the lab and samples were processed within 60 minutes after collection. Amniotic fluid was centrifuged at 4º C for 20 minutes at 1400xg. After centrifugation the cell pellet was treated for analysis of CD117+ cells using flowcytometry, once small percentage of CD117+ cells were identified the cells were prepared for differentiation and that was carried out using specific growth factors including BMP4, BMP7, FGF2, and retinoic acid, providing the niche to the stem cells for differentiation towards nephron progenitors which was confirmed by protein expression of Wilms Tumor-1 (WT1) using immunofluorescence analysis. The sample size for this invitro work was n=3. Results: We successfully isolated small percentage of CD117+ cells in amniotic fluid followed by in vitro expansion and differentiation towards nephron progenitor cells (NPCs) using well defined media and growth factors, initially differentiated cells were spindle shaped and showed fibroblastic appearance later at stage of nephron progenitors it attained the shape of rounded big clusters, differentiated cells stained positive for WT1 and negative for cluster of differentiation (CD117). Therefore, confirming the successful isolation and differentiation of amniotic fluid stem cells towards nephron progenitors. Conclusion: To the best of our knowledge this is the first study from the country on the use of Amniotic fluid stem cells and their differentiation towards nephron progenitors that can be used as substitution source of cell therapy for exploration of renal diseases at cellular and molecular level and potential regenerative medicine applications.

Peste des petits ruminants virus antibodies in domestic large ruminants in Bangladesh.

INTRODUCTION: Peste des petits ruminants (PPR) is an important transboundary animal disease of small ruminants which causes serious damage to the livelihood and food security of millions of small-scale farmers. PPR is endemic in goats in Bangladesh since 1993. The aim of this study was to determine the seroprevalence of PPR in sheep, cattle, and buffaloes in Bangladesh. METHODOLOGY: A total of 434 blood samples from sheep (n = 100), cattle (n = 190) and buffalo (n = 144) were collected aseptically. Sera were separated and antibody titer was determined using a commercially available c-ELISA kit. RESULTS: The overall seroprevalence was 16% and 3.68% in sheep and cattle, respectively, while buffaloes had a considerably higher seroprevalence of 42.36%. The study suggests that buffaloes are more prone to the PPR virus (PPRV) infection and cattle. CONCLUSIONS: This study provides serological evidence of PPRV infection in cattle and buffaloes. These results may warrant further studies to find out the role of large ruminants in transmitting PPRV infection to small ruminants and vice versa and inclusion of all domestic and wild ruminants for regular surveillance program.

Sero-prevalence of anti-SARS-CoV-2 antibodies in Chattogram Metropolitan Area, Bangladesh

BackgroundSeroprevalence studies of coronavirus disease 2019 (COVID-19) assess the degree of undetected transmission in the community. Different groups, such as healthcare workers (HCWs), garment workers, and others, are deemed vulnerable due to their workplace hazards and immense responsibility. PurposeThe present study was conducted to estimate the seroprevalence of anti-SARS-CoV-2 antibody (IgG) and its association with different explanatory variables. Further, the antibody was quantified to assess the increasing or decreasing trend over different intervention periods and according to other factors. MethodologyThis cross-sectional study observed health workers - doctor, nurse, hospital staff, etc. in and outpatients (non-COVID-19) and garments workers of Chattogram metropolitan area (CMA, N=748) from randomly selected six government and private hospitals and two garment factories. Study subjects were included upon written consent, fulfilling specific inclusion criteria. Venous blood was collected following standard aseptic methods. Qualitative and quantitative ELISA was used to identify and quantify antibodies (IgG) in serum samples. Descriptive, univariable, and multivariable statistical analysis was performed. ResultsOverall seroprevalence was estimated as 66.99% (95% CI: 63.40%-70.40%). Seroprevalence among HCWs, in and outpatients, and garments workers were 68.99 % (95% CI: 63.8%-73.7%), 81.37 % (95% CI: 74.7%-86.7%), and 50.56 % (95% CI: 43.5%-57.5%), respectively. Seroprevalence was 44.47 % (95% CI: 38.6%-50.4%) in the non-vaccinated population while it was significantly (p <0.001) higher in the population receiving the first dose (61.66 %, 95% CI: 54.8%-68.0%) and both (first and second) doses of vaccine (100%, 95% CI: 98.4%-100%). The mean titer of the antibody was estimated as 255.46 DU/ml and 159.08 DU/ml in the population with both doses and one dose of vaccine, respectively, compared to 53.71 DU/ml of the unvaccinated population. A decreasing trend in the titer of antibodies with increasing time after vaccination was observed. ConclusionsSeroprevalence and mean antibody titer varied according to different factors in this study. The second dose of vaccine significantly increased the seroprevalence and titer, which decreased to a certain level over time. Although antibody was produced following natural infection, the mean titer was relatively low compared to antibody after vaccination. This study emphasizes the role of the vaccine in antibody production. Based on the findings, interventions like continuing extensive mass vaccination of the leftover unvaccinated population and bringing the mass population with a second dose under a third dose campaign might be planned.

Significance of peritoneal washing cytology in the accurate staging of malignant ovarian tumors.

OBJECTIVES: To identify the percentage of ovarian cancers with positive peritoneal cytology and to correlate the positive cytology with the prognostic factors. METHODS: This retrospective, cross-sectional study, evaluated the data of surgical specimens of malignant ovarian tumors, received in the Department of Pathology, Dow University of Health Sciences over a period of three years. The peritoneal cytology was correlated with these prognostic parameters: the size of the tumor, stage, capsular invasion, omental, and lymph node metastasis. RESULTS: Eighty malignant ovarian tumors were diagnosed. Serous carcinoma was the most common ovarian tumor, diagnosed in 24(30.0%) cases, followed by endometrioid carcinoma in 17(21.25%) and Granulosa cell tumor in 11 (13.75%) cases. The mean age of the patients was 41.91 years (range 7-71 years). The mean size of the tumors was 10.03 cm (SD 5.62 cm). The ovarian capsular invasion was present in 27(33.75%) tumors. Peritoneal cytology was positive in 10/24 cases, with a detection rate of 41.66%. Omentum was involved in 12/34(35.29%) cases. Lymph node dissection was performed in three cases, two were reported as positive for metastasis. Peritoneal cytology significantly correlated with the tumor size (p=0.045), and with ovarian capsular invasion (p=0.054) and omental metastasis (p=0.052). Most of the tumors were staged as FIGO stage IA. CONCLUSION: Peritoneal cytology correlates with the tumor size, stage, and omental metastasis of the malignant ovarian tumors. It should be routinely performed at the time of surgery for the optimal staging of the patients.

Biomedical Applications of Carbohydrate-based Polyurethane: From Biosynthesis to Degradation.

The foremost common natural polymers are carbohydrate-based polymers or polysaccharides, having a long chain of monosaccharide or disaccharide units linked together via glycosidic linkage to form a complex structure. There are several uses of carbohydrate-based polymers in the biomedical sector due to their attractive features, including less toxicity, biocompatibility, biodegradability, high reactivity, availability, and relative inexpensiveness. The aim of our study was to explore the synthetic approaches for the preparation of numerous carbohydrate-based polyurethanes (PUs) and their wide range of pharmaceutical and biomedical applications. The data summarized in this study show that the addition of carbohydrates in the structural skeleton of PUs not only improves their suitability but also affects their applicability for use in biological applications. Carbohydrate- based units are incorporated into the PUs, which is the most convenient method for the synthesis of novel biocompatible and biodegradable carbohydrate-based PUs for use in various biomedical applications.

Molecular insights into peste des petits ruminants virus identified in Bangladesh between 2008 and 2020.

An in-depth knowledge of the molecular evolution of the peste des petits ruminants virus (PPRV) is critical for the success of the current global eradication program. For this reason, a molecular evolutionary analysis of PPRVs circulating in Bangladesh over a decade (2008-2020) was performed. The complete genome sequencing of three PPRV isolates from 2008 (BD2), 2015 (BD12) and 2017 (BD17) as well as full length nucleocapsid (N), matrix (M) and fusion (F) gene sequencing of seven more samples from 2015 to 2020 was performed. Phylogenetic analysis classified all ten PPRVs from Bangladesh as members of lineage IV and showed that they were closely related to PPRV strains detected in China and Tibet during 2007-2008, and India during 2014-2018. Time scale Bayesian Maximum Clade Credibility (MCC) phylogenetic analysis of the three complete genomes revealed a mean Time to Most Recent Common Ancestor (TMRCA) of 2000. Comparative deduced amino acid residue analysis at various functional motifs of PPRVs related to virus structure and function, virulence and host adaptation, receptor binding sites and polymerase activity revealed conserved residues among the PPRVs from Bangladesh. In total sixteen epitopes were predicted from four immunogenic proteins i.e. N, M, F and haemagglutinin (H). Interestingly, the predicted epitopes from the N and M proteins shared conserved epitopes with two vaccine strains currently being used, indicating that the strains from Bangladesh could be potentially used as alternative local vaccines.

Circulation of three genotypes and identification of unique mutations in neutralizing epitopes of infectious bronchitis virus in chickens in Bangladesh.

Infectious bronchitis (IB) is a highly contagious respiratory disease caused by a gammacoronavirus that has been circulating for many years in chickens in Bangladesh, resulting in significant economic losses. The aim of this study was to detect and characterize infectious bronchitis virus (IBV) from clinical outbreaks and surveillance samples. Real-time RT-PCR was used to detect IBV in pooled lung and tracheal tissue samples (n = 78), oropharyngeal swabs (n = 19), and pooled fecal samples (n = 13) from live-bird markets. Both respiratory and nephropathogenic forms of IB were suspected at necropsy (n = 7) from clinical outbreaks. Sequencing of hypervariable regions (HVR1-2 and HVR3) of the region of the spike gene (S) encoding the S1 subunit of five isolates revealed circulation of the Mass-like, QX-like, and 4/91-like genotypes of IBV in Bangladesh. Each genotype was extremely variable, as shown by separate clustering of the viruses in a phylogenetic tree and high nucleotide (nt) sequence divergence (38.8-41.2% and 25.7-37.4% in the HVR1-2 and HVR3 sequence, respectively). The unique mutation G65E was observed in each Mass-like isolate, and Y328S was observed in each 4/91-like Bangladeshi isolate. Three neutralizing epitope sites were predicted within the HVRs that differed significantly among the three genotypes. In addition, one Bangladeshi isolate carried fixed mutations at 294F and 306Y, like other pathogenic QX-like IBVs, which could affect epitopes involved in neutralization, facilitating virus circulation among vaccinated flocks. Therefore, continuous screening and genotype characterization will be necessary to track the epidemiology of IBV and control IB infection in Bangladesh.

Molecular Analysis of SARS-CoV-2 Circulating in Bangladesh during 2020 Revealed Lineage Diversity and Potential Mutations.

Virus evolution and mutation analyses are crucial for tracing virus transmission, the potential variants, and other pathogenic determinants. Despite continuing circulation of the SARS-CoV-2, very limited studies have been conducted on genetic evolutionary analysis of the virus in Bangladesh. In this study, a total of 791 complete genome sequences of SARS-CoV-2 from Bangladesh deposited in the GISAID database during March 2020 to January 2021 were analyzed. Phylogenetic analysis revealed circulation of seven GISAID clades G, GH, GR, GRY, L, O, and S or five Nextstrain clades 20A, 20B, 20C, 19A, and 19B in the country during the study period. The GISAID clade GR or the Nextstrain clade 20B or lineage B.1.1.25 is predominant in Bangladesh and closely related to the sequences from India, USA, Canada, UK, and Italy. The GR clade or B.1.1.25 lineage is likely to be responsible for the widespread community transmission of SARS-CoV-2 in the country during the first wave of infection. Significant amino acid diversity was observed among Bangladeshi SARS-CoV-2 isolates, where a total of 1023 mutations were detected. In particular, the D614G mutation in the spike protein (S_D614G) was found in 97% of the sequences. However, the introduction of lineage B.1.1.7 (UK variant/S_N501Y) and S_E484K mutation in lineage B.1.1.25 in a few sequences reported in late December 2020 is of particular concern. The wide genomic diversity indicated multiple introductions of SARS-CoV-2 into Bangladesh through various routes. Therefore, a continuous and extensive genome sequence analysis would be necessary to understand the genomic epidemiology of SARS-CoV-2 in Bangladesh.

Genetic reprogramming of cord blood derived endothelial colony forming cells towards human induced pluripotent stem cells using episomal plasmids.

OBJECTIVE: This study aimed to isolate human umbilical cord blood derived endothelial colony forming cells (ECFCs) followed by their integration free reprogramming towards induced pluripotent stem cells (iPSCs) and molecular characterization of both cell types using multicolour flowcytometery and immunofluorescence respectively. METHODS: The cord blood was collected from 37-39 weeks of gestational ages after C-section ex-utero from Dow University Hospital. The ECFCs isolated after ficoll based separation of cord blood mononuclear cells (CBMNCs) which on emergence characterized through flow cytometry and reprogrammed towards induced pluripotent stem cells (iPSCs) using episomal vectors, the iPSCs were characterized using immunofluorescence. The study was conducted at Stem Cells and Regenerative lab, Dow Research Institute of Biotechnology and Biomedical Sciences, Dow University of health sciences OJHA campus. The study time duration was about one year (October 2017-October 2018); study design was in vitro experimental. The sample size of the study was n=3. RESULTS: The isolated ECFCs were evaluated using flowcytometery which showed positive expression for CD31, CD34, CD146 cell surface markers and negative for CD90. The successful reprogramming of ECFCs towards iPSCs was confirmed by immunofluorescence using OCT-4 which is considered to be a master regulator of pluripotency. CONCLUSIONS: To the best of our knowledge this study was the first attempt to integration free reprogramming of cord blood derived endothelial colony forming cells towards induced pluripotent stem using episomal plasmids. Cells that have been isolated from cord blood and those that have been reprogrammed both have potential therapeutic applications in regenerative medicine.

Antiproliferative Activity and Apoptotic Efficiency of Syzygium cumini Bark Methanolic Extract against EAC Cells In Vivo.

BACKGROUND: Syzygium cumini is one of the evidence-based traditional medicinal plant used in the treatment of various ailments. OBJECTIVES: Herein, the antioxidant property and anticancer property of Syzygium cumini against Ehrlich Ascites Carcinoma (EAC) cells were examined to find effective chemotherapeutics. METHODS: In vitro assays, and phytochemical and chromatographic analyses were used to determine antioxidant properties and chemical constituents of Syzygium cummini Bark Methanolic Extract (SCBME). Functional assays were used to measure the anticancer activity of SCBME. Fluorescence microscopy and RT-PCR were used to examine morphological and molecular changes of EAC cells followed by SCBME treatment. RESULTS: Phytochemical and GC-MS analyses confirmed the presence of compounds with antioxidant and anticancer activities. Accordingly, we have noted a strong antioxidant activity of SCBME with an IC50 value of ~10µg/ml. Importantly, SCBME exerted a dose-dependent anticancer activity with significant inhibition of EAC cell growth (71.08±3.53%; p<0.001), reduction of tumor burden (69.50%; p<0.01) and increase of life span (73.13%; p<0.001) of EAC-bearing mice at 75mg/kg/day. Besides, SCBME restored the blood toxicity towards normal in EAC-bearing mice (p<0.05). DISCUSSION: SCBME treated EAC cells showed apoptotic features under a fluorescence microscope and fragmented DNA in DNA laddering assay. Moreover, up-regulation of the tumor suppressor p53 and pro-apoptotic Bax and down-regulation of NF-κB and anti-apoptotic Bcl-2 genes implied induction of apoptosis followed by SCBME treatment. CONCLUSION: The antiproliferative activity of SCBME against EAC cells is likely due to apoptosis, mediated by regulation of p53 and NF-κB signaling. Thus, SCBME can be considered as a useful resource in cancer chemotherapy.