Baby Hamster Kidney Cell Line Research Articles

Biosynthesis of Silver, Copper, and Their Bi-metallic Combination of Nanocomposites by Staphylococcus aureus: Their Antimicrobial, Anticancer Activity, and Cytotoxicity Effect

AbstractThe present study outlines an easy, cheap, and environmentally friendly way to make Staphylococcus aureus-mediated bimetallic silver-copper nanocomposites (Ag/Cu) that can fight cancer and germs. The gram-positive S. aureus synthesized Ag, Cu, and their bi-metallic nanocomposites extracellularly. We aimed to prepare the bimetallic nanocomposite in two different ways, and we compared them in terms of characterization and biological applications. The first one is a bimetallic nanocomposite (Ag/Cub) that was made by mixing Ag and Cu metal ions in equal amounts (50:50). Then, the whole mixture was reduced. The second is the after-reduction bimetallic nanocomposite (Ag/Cua), in which each metal ion was reduced separately, and then the nanocomposites were mixed (50:50%) during biological applications. Nanocomposites were characterized using UV–visible spectrophotometry, Fourier-transform infrared spectroscopy, dynamic light scattering, and transmission electron microscopy. The results demonstrated that surface plasmon bands were at 320 nm for Ag NPs and 525 nm for Cu NPs, and a shift from these peaks was observed at 290 nm in the Ag/Cub bimetallic nanocomposite. The synthesized nanocomposites were confirmed to be in the nanoscale with 20, 40, and 80 nm spherical crystals, respectively. Nanocomposites were assayed for their antimicrobial activity against the gram-negative Pseudomonas aeruginosa, the acid-fast Mycobacterium smegmatis, the gram-positive Bacillus cereus, and S. aureus, in addition to three fungal species, which were Aspergillus flavus, A. fumigatus, and Candida albicans. The minimum inhibitory concentration and minimum bactericidal concentration were determined. The Ag/Cua/Cuaetallic nanocomposite was the most potent antimicrobial compound. The anticancer activity of the tested compounds was assayed against the hepatocellular carcinoma cell line (HepG-2). Low cytotoxic activity was recorded in most assayed nanocomposites against the baby hamster kidney cell line (BHK).

Ciprofloxacin loaded PEG coated ZnO nanoparticles with enhanced antibacterial and wound healing effects

Antimicrobial resistance is a worldwide health problem that demands alternative antibacterial strategies. Modified nano-composites can be an effective strategy as compared to traditional medicine. The current study was designed to develop a biocompatible nano-drug delivery system with increased efficacy of current therapeutics for biomedical applications. Zinc oxide nanoparticles (ZnO-NPs) were synthesized by chemical and green methods by mediating with Moringa olifera root extract. The ZnO–NPs were further modified by drug conjugation and coating with PEG (CIP-PEG-ZnO-NPs) to enhance their therapeutic potential. PEGylated ZnO-ciprofloxacin nano-conjugates were characterized by Fourier Transform Infrared spectroscopy, X-ray diffractometry, and Scanning Electron Microscopy. During antibacterial screenings chemically and green synthesized CIP-PEG-ZnO-NPs revealed significant activity against clinically isolated Gram-positive and Gram-negative bacterial strains. The sustainable and prolonged release of antibiotics was noted from the CIP–PEG conjugated ZnO-NPs. The synthesized nanoparticles were found compatible with RBCs and Baby hamster kidney cell lines (BHK21) during hemolytic and MTT assays respectively. Based on initial findings a broad-spectrum nano-material was developed and tested for biomedical applications that eradicated Staphylococcus aureus from the infectious site and showed wound-healing effects during in vivo applications. ZnO-based nano-drug carrier can offer targeted drug delivery, and improved drug stability and efficacy resulting in better drug penetration.

Sir Michael George Parke Stoker. 4 July 1918—13 August 2013

Michael Stoker was a renowned virologist and cell biologist. He gained his interest in research while serving as a medical officer during the Second World War in India, studying infectious diseases such as typhus. He began to study viruses in the Department of Pathology in Cambridge and in 1958 was appointed to the first established UK chair of virology and as head of the Medical Research Council Virus Unit at the University of Glasgow. He pioneered studies of cancer-causing viruses using polyomavirus of mice, and developed the baby hamster kidney cell lines still used today for vaccine production. In 1968 he moved to London as director of research at the Imperial Cancer Research Fund (ICRF) Laboratories, now part of the Cancer Research UK funded laboratories at the Francis Crick Institute. He brought many virologists to ICRF, while he himself turned to cell biology, first of mesenchymal cells and then of normal and malignant epithelial cells, particularly breast epithelium. Returning to Cambridge in 1978, he discovered scatter factor (also known as hepatocyte growth factor), a paracrine cytokine that stimulates motility and proliferation of epithelial cells. Michael had broad-ranging interests and there are few virologists in the UK who have not been influenced either by him directly or by his protégés. Among the many activities beyond the institutions where he worked, he served from 1976 to 1981 as Foreign Secretary and Vice-President of the Royal Society.

The identification and genetic characteristics of Quang Binh virus from field-captured Culex tritaeniorhynchus (Diptera: Culicidae) from Guizhou Province, China

BackgroundMosquitoes carry a variety of viruses that can cause disease in humans, animals and livestock. Surveys for viruses carried by wild mosquitoes can significantly contribute to surveillance efforts and early detection systems. In addition to mosquito-borne viruses, mosquitoes harbor many insect-specific viruses (ISVs). Quang Binh virus (QBV) is one such example, categorized as an ISV within the Flavivirus genus (family Flaviviridae). QBV has been specifically documented in Vietnam and China, with reports limited to several mosquito species.MethodsThe homogenate obtained from female mosquitoes was cultured on C6/36 (Aedes albopictus) and BHK-21 (baby hamster kidney) cell lines. Positive cultures were identified by reverse transcription-polymerase chain reaction (RT‒PCR) with taxon- or species-specific primers. Next-generation sequencing was employed to sequence the complete genomes of the identified positive samples. Subsequently, phylogenetic, gene homology, molecular evolutionary and genetic variation analyses were conducted.ResultIn 2021, a total of 32,177 adult female mosquitoes were collected from 15 counties in Guizhou Province, China. The predominant mosquito species identified were Culex tritaeniorhynchus, Armigeres subalbatus and Anopheles sinensis. Among the collected mosquitoes, three positive cultures were obtained from Cx. tritaeniorhynchus pools, revealing the presence of Quang Binh virus (QBV) RNA sequences. Phylogenetic analysis indicated that the three Guizhou isolates, along with the prototype isolate from Vietnam, formed distinct branches. These branches were primarily closely related to other QBV isolates reported in China. Comparative analysis revealed a high degree of nucleotide and amino acid homology between the Guizhou isolates and both Vietnamese and other indigenous Chinese isolates. Additionally, nonsynonymous single-nucleotide variants (SNVs) were observed in these strains compared to the QBV prototype strain.ConclusionThis study represents the first report of QBV presences in Cx. tritaeniorhynchus mosquitoes in Guizhou Province, China. Phylogenetic tree analysis showed that the three Guizhou isolates were most closely related to the QBV genes found in China. In addition, the study of the genetic characteristics and variation of this virus provided a deeper understanding of QBV and enriched the baseline data of these insect-specific flaviviruses (ISFVs).Graphical

Circulating foot-and-mouth disease virus serotype A African-genotype IV in Egypt during 2022.

Foot-and-mouth disease (FMD) virus causes continuous outbreaks, leading to serious economic consequences that affect animal productivity and restrict trade movement. The potential influence of the disease was due to the emergence of new strains or re-emergence of local strains with major antigenic variations due to genetic mutations. This study aims to evaluate circulating virus in samples collected from infected animals during an outbreak using antigenic characterization and identify whether there is an emergence of a new strain or mutation. Reverse-transcription polymerase chain reaction (RT-PCR) was used to screen 86 samples. Viral protein 1 (VP1) codon sequencing was performed. The virus was isolated from the samples inoculated on the baby-hamster kidney cell line and Enzyme-linked immunosorbent assay was performed for serotyping and antigen detection. Based on the RT-PCR screening results, 10 positive samples were selected for sequencing. The sequences belonged to the FMD serotype A African topotype originating from the ancestor prototype Sudan/77, with which it shared 98.48% ± 1.2% similarity. The divergence with local isolates from 2020 was 9.3%. In addition, the sequences were 96.84% ± 1.01% and 95.84% ± 0.79% related to Egyptian-Damietta type 2016 and Sudanese-2018, respectively. Divergence with vaccinal strains ranged from 10% to 17%. Amino acid sequence analysis revealed that the isolates had variation in the most prominent antigenic regions (residues 35-75) and the immunogenic determinants of the G-H loop of VP1 (residues 100-146 and 161-175). The current isolates should be included in the locally produced vaccine to provide broader immunogenic coverage against serotype A African topotypes.

448 Establishing the efficacy of naturally occurring endocannabinoid-like substance in an in vitro model of Fragile X Tremor/Ataxia Syndrome (FXT/AS)

OBJECTIVES/GOALS: FXT/AS is a devastating, rare neurological syndrome that negatively impacts movement and cognition and is suspected to induce mitochondria dysfunction. Currently, no effective pharmacological treatments for FXTAS exist. The goal is to restore mitochondrial viability using endocannabinoid-like compounds in a cell culture model of FXTAS. METHODS/STUDY POPULATION: To establish a cell model of mitochondrial dysfunction, fibroblast baby hamster kidney (BHK-21) cell lines were treated with glucose oxidase (GluOx) at varying concentrations and times. Mitochondrial viability was assessed by the colorimetric Janus B Green Assay, which stains the mitochondria and enables assessment of cell numbers and the presence of oxygen in anchorage-dependent cell culture. Upon establishing this model of mitochondrial dysfunction, we next investigated the ability of three novel mitochondrial antioxidants (e.g., macamides) to protect mitochondrial viability. RESULTS/ANTICIPATED RESULTS: GluOx treatment of BHK-21 cells caused a dose- and time-dependent increase in oxidative stress. The data demonstrated significant disruption in the morphology of BHK-21 cells at a high glucose concentration, i.e., 40 nM, between 2 and 24 hours post-exposure. The morphology data were confirmed by the Janus B Green colorimetric assay. In examining the effects of glucose on mitochondrial viability, we demonstrated that at 15, 30, 35, and 40 nM, glucose significantly decreased mitochondria viability compared to the untreated, with 40 nM having the greatest effect. Under these conditions of mitochondrial dysfunction, co-incubation of the cells with the 0.5 uM MAM69 macamide attenuated the GluOx-induced increase in oxidative stress, with 0.5 uM MAM69 alone showing no effect on mitochondria viability. DISCUSSION/SIGNIFICANCE: This study illustrates the efficacy of macamides, natural occurring endocannabinoid like-compound, as novel prognostic and therapeutic candidates in the treatment of mitochondrial dysfunction that is associated with FXTAS. The use of BHK-21 fibroblast cells provides a rapid screening model to test for pharmacological therapeutic efficacy.

Immuno-affinity chromatography for purification of IgG from hyper-immune sera raised against 146S fraction of Foot and Mouth Disease Virus for diagnostic purposes.

Immunoaffinity chromatography (IAC) is a fundamental isolation and purification tool which is incorporated in a substantial range of therapeutic and diagnostic applications. This study has reappraised the usefulness of immunoaffinity chromatography for the purification of polyclonal antibodies. Protein A based IAC is a convenient and reliable method for purification of IgG, from hyperimmunesera (HIS) raised in experimental animals such as rabbits, guinea pigs and mice to be utilized in pharmaceutics and diagnostics. The 146S fraction of Foot and Mouth Disease virus (FMDV) TCID50=10 5.6 was cultured on a baby hamster kidney cell line 21 (BHK-21), concentrated using salt precipitation method using PEG 6000, purified by size exclusion chromatography (SEC) using Sepharose-30 at 254nm absorbance. Purification of 146S FMDV was analyzed using 12% SDS-PAGE which provided two bands of light and heavy chains. The alum-based vaccine, consisting of ≥10μg of 146S FMDV, was applied in 10 male rabbits and 10 male guinea pigs and two animals of each group were taken as a negative control. The titer of serum was calculated using virus neutralization test. A Protein-A kit (Thermo scientific- 44667, 0528.2) was used to purify HIS raised against 146S FMDV and validated using 12% SDS PAGE in reducing condition. The data demonstrate that protein-A affinity chromatography is an efficient tool for the purification of antibodies from hyper-immune sera raised against 146S FMDV and can be used for the production of diagnostic kits e.g. Enzyme linked immuno-sorbent assay (ELISA) and radioimmunoassay.

Cytotoxicity and Genotoxicity Evaluation of Some Stored Grain Insects and Their Infested Flour Using the BHK-21 Cell Line in an In Vitro Experimental Model

Globally, stored grain is vulnerable to pest infestation, resulting in significant economic losses for some crops. Wheat is one of the most important crops in the world. Many sucking, piercing insects infect wheat in the form of grains or flour and may produce toxic residues that are harmful to human health. The current study aimed to estimate the safe use of four stored grain insects by evaluating the potential genotoxic effects and cytotoxicity of crushed insects (T. granarium, S. oryzae, R. dominica, and T. castaneum) and their flour residues. MTT and comet assays were conducted to assess the effects of six concentrations of insect flour residues (0, 6.5, 12.5, 25, 50, and 100%) on the baby hamster kidney cell line (BHK-21). The lowest BHK-21 cell viability was noted against T. granarium (LC50% 36.42 μg/ml) followed by T. castaneum flour (LC50% 46.73 μg/ml) compared to the control (LC50% 808.2 μg/ml). Significantly high DNA comet (%) was observed in the treatments of T. castaneum flour (18.8%), S. oryzae wheat (15.6%), T. granarium (15.4%), T. castaneum (13.6%), and T. granarium wheat (13.1%). FTIR spectra of stored grain insects and their flour residues identified various functional metabolite groups, including alkynes and phenols, which could enhance cell apoptosis and genotoxicity. T. granarium, T. castaneum, and their flour residues had the highest cytotoxic and genotoxic effects on the BHK-21 cell line. The current study concludes that insect residues in flour may have cytotoxic and genotoxic effects on living cells, potentially affecting public health, particularly after consuming T. granarium and T. castaneum-infested flour. Therefore, good storage of stored grains and their products is recommended.

Packaging, Purification, and Titration of Replication-Deficient Semliki Forest Virus-Derived Particles as a Self-Amplifying mRNA Vaccine Vector

Background:Self-amplifying mRNA is the next-generation vaccine platform with the potential advantages in efficacy and speed of development against infectious diseases and cancer. The main aim was to present optimized and rapid methods for SFV-PD SAM preparation, its packaging, and titer determination. These protocols are provided for producing and harvesting the high yields of VRP-packaged SAM for vaccine studies.Methods:pSFV-PD-EGFP plasmid was linearized and subjected to in vitro transcription. Different concentrations of SFV-PD SAM were first transfected into HEK-293 and BHK-21 cell lines, and EGFP expression at different time points was evaluated by fluorescent microscopy. Replicon particle packaging was achieved by co-transfection of SFV-PD SAM and pSFV-Helper2 RNA into BHK-21 cells. The VRPs were concentrated using ultrafiltration with 100 kDa cut-off. The titers of replicon particles were determined by RT-qPCR. Results: In vitro transcribed SAM encoding EGFP was successfully transfected and expressed in HEK-293 and BHK-21 cell lines. Higher levels of EGFP expression was observed in BHK-21 compared to HEK-293 cells showing more stable protein overexpression and VRP packaging. Using ultrafiltration, the high yields of purified SFV-PD-EGFP particles were rapidly obtained with only minor loss of replicon particles. Accurate and rapid titer determination of replication-deficient particles was achieved by RT-qPCR. Conclusion:Using optimized methods for SAM transfection, VRP packaging, and concentration, high yields of SFV-PD VRPs could be produced and purified. The RT-qPCR demonstrated to be an accurate and rapid method for titer determination of replication deficient VRPs.

In vitro apoptotic, anti-proliferative and antioxidant activities of therapeutic plant Datura metel

Natural bioactive compounds with apoptotic action might be a promising new anti-cancer drug source. The purpose of the present study was to assess the apoptotic, anti-proliferative, antioxidative activities of a therapeutic plant Datura metel in liver hepatocellular carcinoma, HepG2 cell lines, as well as in normal baby hamster kidney (BHK) cell lines as controls. Ethanol and n-hexane solvents were used to extract Datura metel leaves extract. Standard techniques for identifying components were used to conduct phytochemical analysis. Cell death and viability in all sets of the cells were assessed using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), trypan blue and crystal violet tests. For the estimate of apoptosis and cell death in all groups ELISA of Annexin-V was used. In addition, antioxidant enzyme activity assays were also conducted to estimate H2O2, nitric oxide, superoxide and DPPH radical scavenging activities. The outcomes revealed that when cancer cells from the HepG2 cell lines were treated with Datura metel extracts, they demonstrated decreased viability, proliferation, and enhanced apoptosis as compared to normal BHK cells and untreated control cells. Anti-oxidative scavenging activities were higher in cancer cells treated with Datura metel extract than in untreated ones. It was concluded that the Datura metel leaves extract induces apoptosis, enhance antioxidant status, decrease proliferation in HepG2 cells.

Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire

Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell lines and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.

Optimization of a GDNF production method based on Semliki Forest virus vector

Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson's disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature-dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33°C to 37°C during the “shut-off period”, produced a significant improvement in cell survival and hGDNF expression. In consonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a “recovery period” at 37°C before cells are exposed at 33°C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF.

Epigallocatechin-3-gallate exhibits antiviral effects against the duck Tembusu virus via blocking virus entry and upregulating type I interferons

The duck Tembusu virus (DTMUV) is a novel mosquito-borne Flavivirus which caused huge economic losses for poultry industries in Southeast Asia and China. Currently, no effective antiviral drugs against this virus have been reported. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in green tea, has recently been demonstrated to have an antiviral activity for many viruses; however, whether EGCG can inhibit DTMUV infection remains unknown. Here, we tried to explore the anti-DTMUV effects and mechanisms of EGCG both in vitro and in vivo. Several EGCG treatment regimens were used to study the comprehensive antiviral activity of EGCG in DTMUV-infected baby hamster kidney cell line (BHK-21). The DTMUV titers of mock- and EGCG-treated infected cell cultures were determined using the tissue culture infective dose assay and the DTMUV mRNA copy number as determined using quantitative Real Time PCR. Moreover, the therapeutic efficacy of EGCG against DTMUV was assessed in DTMUV-infected ducklings. Our results suggested that EGCG significantly reduced the viral infection in BHK-21 cells in a dose-dependent manner, as reflected by the reduction of virus titers, virus copy number, and the expressions of viral E protein. We also observed that EGCG exhibited direct virucidal abilities against DTMUV. Notably, a significant reduction in virus binding ability was also observed, indicating that EGCG possesses excellent inhibitory effects on the viral adsorption step. In addition, DTMUV replication was also suppressed in BHK-21 cells treated with EGCG after viral entry, likely because of upregulation of the levels of interferon alfa and interferon beta. Finally, we also proved that EGCG exhibited anti-DTMUV efficacy in a duckling infection model because the survival rate was significantly improved. This is the first study to demonstrate the protective effect of EGCG against DTMUV, suggesting its potential use as an antiviral drug for DTMUV infection.

Antiviral effect of honey extract Camelyn against SARS-CoV-2

This study aimed to evaluate the potential antiviral effects of honey extract “Camelyn” against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). The baby hamster kidney cell line 21 (BHK‐21), bone marrow-derived hematopoietic stem cells (HSCs), and splenic cells were used for Camelyn cytotoxicity assay. After the isolation procedures, cell viability was assessed by trypan blue dye exclusion under a microscope using a hemocytometer. The in vitro cell growth rate was carried out using the cell counting Kit 8 (CCK-8) assay. The cells were seeded in growth media with various Camelyn concentrations (35 μg, 50 μg, 70 μg, 100 μg, 150 μg, and 200 μg). The absorbance at 450 nm was determined by the multiplate reader. The antiviral effect was assessed by plaque reduction assay for the determination of drug susceptibility against SARS-CoV-2. Serial dilution of the selected compounds was pre-incubated with 40 to 100 plaque-forming units (PFUs) of SARS-CoV-2. The pre-incubated mix of Camelyn and SARS-CoV-2 was then added to the confluent Vero E6 cells after incubation cells were fixed and stained and the number of PFUs was counted under an inverted microscope and plotted against the logarithm of antiviral concentrations. Our study showed that Camelyn is not cytotoxic, has a stimulatory effect on cell proliferation, and has an inhibitory effect against SARS-CoV-2 with EC50 (half-maximal effective concentration) from 85.7 μg/mL to 192.4 μg/mL depending on product concentration and viral plaque per cell. © 2021 Global Research Online. All rights reserved.

EVALUATION OF SUITABILITY OF METHODS FOR TESTING BLOOD PROTEIN HYDROLYSATES FOR THEIR BIOLOGICAL ACTIVITY AND TOXICITY

The use of high quality nutrient media comprising protein hydrolysates as the main components for maintaining their nutrient value is a critical prerequisite for appropriate fnal product preparation in FMD vaccine biotechnology. The incoming control of raw materials, in particular testing blood protein hydrolysates of different batches for their quality, is of considerable importance. Currently, there are different methods for the determination of hydrolysate efcacy using cell cultures and unicellular organisms as test systems. We used suspended baby hamster kidney (ВНК-21) cell line as a model for testing blood protein hydrolysates in the raw materials intended for FMD vaccine production for their biological activity and toxicity. The validation of the proposed method was carried out to confrm the reliability of the test results obtained. The following main validation characteristics were determined through testing 560 blood protein hydrolysate samples: trueness, specifcity, sensitivity, as well as precision under repeatability and reproducibility conditions. The validation results for the proposed method demonstrated high reliability (Fisher’s criterion – 0.021; ≤0.050) and full compliance with acceptance criteria.

Characterization of liver specific promoters in a foamy viral vector pMD09.

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.

Lyophilization as an alternative method for preservation of some continuous cell cultures

This work is a trial to provide lyophilization as a simple method for preservation of continuous cell lines instead of the freezing process in liquid nitrogen. Comparative evaluation of lyophilized African green monkey kidney (VERO), baby hamster kidney (BHK) and Madin Darby bovine kidney (MDBK) cell lines and those preserved by freezing in liquid nitrogen was carried out. Such evaluation on 6 months intervals revealed that both of lyophilized cell cultures showed delayed cell adhesion to the culture surface extending to 2-3 days post culturing while propagated and frozen cells adhered to the culture surface within few hours. However there were an increased number of adhered viable cells in case of loading of cells using trehalose and sucrose on days post culturing. In addition cultured lyophilized cells which loaded by trehalose or sucrose exhibited abnormal shape (showing cell rounding) in comparison to the other cultures. Also cell dispersing of confluent sheets of cultured lyophilized cells were found to take longer time (one hour) than that required to other cultures (few minutes) even on using EDTA and incubation at 37 °C. Further studies are needed to investigate the biological behavior or cell changes which may be occurred through the lyophilization process in addition to study the susceptibility of such cells to virus infection. Depending on these facts it could be said that lyophilization is not valid for VERO, BHK and MDBK which usually used for preparation of virus vaccine in stationary or roller systems. It also could be suggested that preservation of cell lines by lyophilization may be of value in cell culturing suspension system.

In vitro biocompatibility evaluation of canasite-calcium phosphate glass-ceramics

This research introduces a new bioactive glass-ceramic material based on canasite and calcium phosphate with different molar ratios in the range from 2 to 0.5. The glass-ceramics were obtained by controlled two-stage heat-treatment of the parent glass (encoded G) at different temperatures (700, 800, 900 and 1000 °C, encoded C700, C800, C900 and C1000, respectively). The glass and glass-ceramics were characterized by DTA, XRD, FTIR and SEM. The four point bending strength was also measured. In vitro bioactivity of the selected samples was investigated in SBF, and the cytotoxicity test was performed by culturing the samples with normal human lung fibroblast cells (WI-38 cells) and baby hamster kidney cell line (BHK cells). The results of XRD analyses showed that crystalline phases of frankamenite/canasite-A and fluorapatite were detected, and xonotlite crystal phase was formed at high heat-treatment temperature. The bending strength was significantly affected by the heat-treatment temperature. It increased with increasing crystallization temperature. SEM micrographs showed a homogeneous microstructure of interlocked needle- and rod-like crystals. Moreover, immersion of the samples in SBF demonstrated that the samples G and C900 showed a relatively better ability than other two samples. The results of the cytotoxicity test showed that cytocompatibility behavior of the samples was varied with the type of cell line. G sample showed a significant higher viability than C900 sample with the WI-38 cell line for all sample concentrations, while, this significant viability differences were not observed at low and high sample concentrations cultured with BHK cells.

Dual function of active constituents from bark of Ficus racemosa L in wound healing

BackgroundDifferent parts including the latex of Ficus racemosa L. has been used as a medicine for wound healing in the Ayurveda and in the indigenous system of medicine in Sri Lanka. This plant has been evaluated for its wound healing potential using animal models. The aim of this study was to obtain an insight into the wound healing process and identify the potential wound healing active substance/s present in F. racemosa L. bark using scratch wound assay (SWA) as the in-vitro assay method.MethodStem bark extracts of F. racemosa were evaluated using scratch wound assay (SWA) on Baby Hamster Kidney (BHK 21) and Madin-Darby Canine Kidney (MDCK) cell lines and Kirby Bauer disc diffusion assay on common bacteria and fungi for cell migration enhancing ability and antimicrobial activity respectively. Dichloromethane and hexanes extracts which showed cell migration enhancement activity on SWA were subjected to bioactivity directed fractionation using column chromatography followed by preparative thin layer chromatography to identify the compounds responsible for the cell migration enhancement activity.ResultsDichloromethane and hexanes extracts showed cell migration enhancement activity on both cell lines, while EtOAc and MeOH extracts showed antibacterial activity against Staphylococcus and Bacillus species and antifungal activity against Saccharomyces spp. and Candida albicans. Lupeol (1) and β-sitosterol (2) were isolated as the potential wound healing active compounds which exhibited significant cell migration enhancement activity on BHK 21 and MDCK cell lines (> 80%) in par with the positive control, asiaticoside at a concentration of 25 μM. The optimum concentration of each compound required for the maximum wound healing has been determined as 30 μM and 35 μM for 1 and 2 respectively on both cell lines. It is also established that lupeol acetate (3) isolated from the hexanes extract act as a pro-drug by undergoing hydrolysis into lupeol in the vicinity of cells.ConclusionDifferent chemical constituents present in stem bark of Ficus racemosa L show enhancement of cell migration (which corresponds to the cell proliferation) as well as antimicrobial activity. This dual action of F. racemosa stem bark provides scientific support for its traditional use in wound healing.

Pigsties near dwellings as a potential risk factor for the prevalence of Japanese encephalitis virus in adult in Shanxi, China

BackgroundThe increasing trend of adult cases of Japanese encephalitis (JE) in China, particularly in northern China, has become an important public health issue. We conducted an epidemiological investigation in the south of Shanxi Province to examine the relationships between mosquitoes, Japanese encephalitis virus (JEV), and adult JE cases.MethodsMosquito specimens were collected from the courtyards of farmers’ households and pig farms in Shanxi Province. Mosquitoes were pooled, homogenized, and centrifuged. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mosquito-borne arbovirus genes in homogenates. Specimens positive for these genes were inoculated into the baby hamster kidney cell line (BHK-21) to isolate virus. Minimum infection rate was calculated and phylogenetic analyses were performed.ResultsA total of 7 943 mosquitoes belonging to six species in four genera were collected; Culex tritaeniorhynchus accounted for 73.08% (5 805/7 943), C. pipiens pallens for 24.75% (1 966/7 943), and the remaining 3% (104/ 7943) consisted of Anopheles sinensis, Aedes vexans, Ae. dorsalis, and Armigeres subalbatus. Sixteen pools were positive for JEV based on RT-PCR using JEV pre-membrane gene nested primers. Phylogenetic analyses showed that all JEVs belonged to genotype I; two pools were positive using Getah Virus (GETV) gene primers. In addition, one JEV strain (SXYC1523) was isolated from C. pipiens pallens specimens. These results indicate that the minimum infection rate of JEV in mosquito specimens collected from the courtyards of farmers’ households with pigsties was 7.39/1 000; the rate for pig farms was 2.68/1 000; and the rate for farmers’ courtyards without pigsties was zero.ConclusionsThe high-prevalence regions of adult JE investigated in this study are still the natural epidemic focus of JEV. Having pigsties near dwellings is a potential risk factor contributing to the prevalence of adult JE. To prevent the occurrence of local adult JE cases, a recommendation was raised that, besides continuing to implement the Expanded Program on Immunization for children, the government should urge local farmers to cease raising pigs in their own courtyards to reduce the probability of infection with JEV.