Co-production of hemagglutinin H9N2 influenza virus and fusion protein Newcastle virus in insect cell using baculovirus expression system.

Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.

Role of ESAT-6 in pathogenicity of Beijing and non-Beijing Mycobacterium tuberculosis isolates.

BACKGROUND: Mycobacterium tuberculosis Beijing genotype was associated with tuberculosis outbreaks and increased transmissibility. To understand the variation in virulence between Beijing and non-Beijing clinical isolates of M.tuberculosis genotypes, the esat-6 gene sequencing, and its expression was compared in the macrophage environment. MATERIALS & METHODS: Among 64 nonrepetitive, culture-positive M.tuberculosis, DNA extraction of 24 and 40 pure confirmed Beijing and non-Beijing isolates was accompanied by the boiling method. esat-6 gene PCR amplification and their sequencing were carried out by specific primers and its expression was performed on human macrophage cell line U937 after 6, 12, and 18 h of exposure to bacilli. The esat-6 mRNA transcription and expression in M. tuberculosis treated macrophage by Real-Time PCR and Western blot method. RESULTS: Data analysis based on sequencing of the east-6 gene PCR product showed that this gene exists in all isolates and there are no changes or single nucleotide variation between the Beijing and non-Beijing isolates. In Beijing strains, the esat-6 expression was increased during the study times, but it was constant in non-Beijing isolates. esat-6 gene expression in Beijing isolates reached to about 44.9 times more than non-Beijing isolates after 18 h incubation on the macrophages cell line. CONCLUSION: esat-6 is a conserved gene both in Beijing and non-Beijing isolates of M.tuberculosis. More expression of the east-6 gene in the macrophage model may indicate that this gene is likely to play a more important role in increasing the pathogenicity of Beijing strains.

Fusion-expressed CtxB-TcpA-C-CPE improves both systemic and mucosal humoral and T-cell responses against cholera in mice.

BACKGROUND: Development of an effective oral vaccine against Cholera, a life-threatening dehydrating diarrheal disease, proved to be a challenging task. To improve oral subunit vaccine immunogenicity and to prevent the state of oral tolerance, application of mucosal adjuvants might be a promising approach. In the present study, the CtxB-TcpA-C-CPE fusion was constructed in which CtxB and C-CPE were used as mucosal adjuvants and vaccine delivery system, respectively, to induce mucosal immune responses, and to improve the anti-toxin and anti-colonizing immunity against V. cholerae. MATERIALS & METHODS: The fusion construct was synthesized, sub-cloned in pQE30 and expressed in E. coli. The three antigen, making the fusion protein, were also separately expressed in E. coli. The recombinant proteins were purified by affinity chromatography using Ni-NTA agarose. Western blot analysis using anti-His antibody was applied to confirm identity of the purified proteins. BALB/c mice were subcutaneously immunized with CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE separately. The mice were orally immunized (in 3 boosts) by the same vaccine. Mucosal immune response stimulation was evaluated by measuring the levels of intestinal IgA. Systemic immune response was evaluated by measuring total serum IgG, IgG1, IgG2a, IgG2b subclasses, and also IL-4, IL-5, IL-10 and IFN-γ cytokines in spleen cell culture. RESULTS: The recombinant proteins CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE were expressed in E. coli and highly purified in a single step of chromatography. BALB/c mice immunized with the fusion protein had highest levels of intestinal IgA, serum IgG and IgG subclasses, compared to each of the three proteins making the fusion. Moreover, stimulated splenocytes of mice immunized with the fusion protein displayed significantly higher amounts of IL-5 and IFN-ɣ cytokines. Th2 dominance of the immune response was more evident in mice receiving the fusion protein. CONCLUSION: Inclusion of CtxB, as the mucosal adjuvant, and C-CPE, as the vaccine delivery system, in the fusion protein CtxB-TcpA-C-CPE significantly enhanced the elicited mucosal and systemic immune responses, compared to TcpA alone. Of note, significant production of intestinal IgA in mice immunized with the fusion protein is presumably capable of neutralizing TcpA, CtxB and C-CPE antigens, preventing V. cholera colonization, and toxic function of CtxB and C-CPE. Challenge infection of the immunized mice is required to evaluate protective potential of the fusion protein against V. cholera.

Downregulation of hepatitis C virus replication by miR-196a using lentiviral vectors.

Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis and hepatocellular carcinoma. Cellular microRNAs (miRNAs) directly modulate the viral infectivity and indirectly through targeting virus-related host factors. They play an essential role in the progression of different stages of HCV infection. The roles of miR-196 family in HCV infection and hepatocellular carcinoma progression remain poorly understood. Using ViTa databases, miR-196a as a high-score miRNA targeting the NS5 A region of HCV genome was selected. Using dual luciferase assay and an established cell-cultured HCV (HCVcc) system, the effect of miR-196a on HCV genome was assessed. In silico analysis demonstrated the significant role of miR-196a in the downregulation of HCV replication. Using dual luciferase assay, the liver-specific miR-196a and NS5 A gene binding was confirmed. To assess the experimental role of miR-196a, an HCVcc system was established in the Huh 7.5 cell lines. The HCV-RNA 1b derived from an infected patient was transfected into Huh 7.5 cells containing miR-196a lentiviral vectors (Huh 7.5/miR-196a), mocks (Huh 7.5/mock vector), and naïve Huh 7.5 cells. The rate of reduction of the HCV genome replication was assessed using relative real-time PCR assay. These results represent miR-196a overexpression and its roles in regulating HCV genome replication. However, miR-196a may inhibit HCV replication and accelerate the early stages of apoptosis. Overexpression of miR-196a in Huh 7.5 replicon cell is a potential new strategy to prevent hepatitis C infection. The results of this study suggest that miR-196a directly downregulates HCV replication and may serve as a new antiviral therapy.

Production of recombinant lethal factor of Bacillus anthracis in Bacillus subtilis.

Cancer is considered as a disease with high rates of mortality and morbidity. The limitations and side effects of common treatments have prompted the need for innovative cancer therapies. Furthermore, selectivity and targeting of cancer cells are crucial factors to successful treatment of cancer. One of these methods is the use of bacterial toxins including Bacillus anthracis toxin to aid cancer therapy. This toxin is composed of three polypeptides: protective factor (PA), lethal factor (LF), and edema factor (EF). PA can bind to various surface receptors of all types of human cells and it internalizes the lethal factor and edema factor subunits of the toxin in the cytosol. In the present study, we cloned and expressed the lef gene of B. anthracis as the lethal part of the toxin in Bacillus subtilis WB600 by a shuttle expression vector PHT4. The rLF made in B. subtilis is efficiently secreted by the host into the culture medium which facilitates downstream processing. The rLF can be used to study cancer treatment. Abbreviations: EF: edema factor; LF: lethal factor; PA: protective factor; rLF: recombinant lethal factor; rPAm: recombinant protective factor mutants; uPA: urokinase-type plasminogen activator; uPAR: urokinase-type plasminogen activator receptor.

Molecular Monitoring and Phylogenetic Analysis of Betanodavirus in Groupers (Epinephelus spp.) and Asian Sea bass (Lates calcarifer) of Iranian Northern Waters of the Persian Gulf.

The emergence of diseases has caused much health and economic damage. Viral Nervous Necrosis (VNN) is considered as one of the most important threats to aquatic ecosystems. VNN can cause severe mortality and economic loss in fish farms. The high water temperatures in southern Iran and the observed incidences of fish mortality in the Persian Gulf led to the hypothesis of the possible emergence of VNN. Therefore, this study aimed to monitor two species of fish susceptible to VNN using PCR, and Nested PCR methods and comparing the sensitivity of these methods to the identification of Betanodavirus infection in apparently healthy and symptomatic fish. About 850 Grouper (Epinephelus spp.) and Asian Sea bass (Lates calcarifer) fish of the Persian Gulf were collected randomly and examined. Molecular methods were used to identify NNV in visibly healthy and symptomatic fish of the Persian Gulf of Iran. The results of the PCR showed no positive cases, but the Nested PCR revealed some positive results. Then, the phylogenetic analysis of the virus sequence was performed. The nucleotide sequence of Nested PCR products revealed a 98-100% homology with Red Spotted Grouper Viral Nervous Necrosis (RGNNV). This is the first report on VNN tracing and detection as well as phylogenetic analysis of the virus from the Persian Gulf of Iran. Therefore, considering the importance of emerging viral diseases and the irreparable damage they cause, continuous monitoring and epidemiological studies of VNN were recommended by authorized organizations.

Cloning and expression of a novel synthetic gene containing VP1 and 3A in Bacillus subtilis as a vaccine candidate against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock animals and control of the disease based on vaccination against serotypes O, A and Asia 1 is important. VP1 (structural) protein and 3A (non-structural) protein is the important antigen in FMDV and they can be used to design recombinant vaccines. In this study the bioinformatics characteristics of VP1 [141-160 and 23-42] and 3A [21-35] of Iranian serotypes O, A and Asia 1 was obtained using on-line predicting software. Then the sequence VP1 [141-160]-GS-VP1 [23-42]-GS-3A [21-35]-GS were codon-optimized and cloned onpHT43shuttle vector and finally expressed in Bacillus subtilis WB600 strain. We could predict VP1 [141-160] as a B cell epitope, VP1 [23-42] as a CTL epitope and 3A [21-35] as a Th cell epitope. The 20KD recombinant protein expressed by Bacillus subtilis were detected by SDS-PAGE. The results showed that this recombinant protein had epitope characteristics and it could be useful as a vaccine candidate to control all serotypes of FMD in Iran.

Molecular surveillance of human rotaviruses in drinking water and investigation of the efficiency of their removal in Isfahan water treatment plant.

Enteric viruses, especially human rotaviruses present in aquatic environments, are microbial criteria in quality assessment of water resources. The present research aimed to investigate molecular monitoring of human rotavirus and efficacy evaluation of Isfahan water treatment plant (WTP) in the elimination of viruses. In total, 60 water samples were collected from different units of WTP. Zeta plus electropositive Virosorb cartridge filter and elution buffer was used for concentrating water samples. Enzyme-linked immunosorbent assay (ELISA) was used for detecting rotavirus antigen. Quantitative real-time reverse transcription PCR (qRT-PCR) with SYBR Green I fluorescent dye was performed for molecular detection of rotavirus. Multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) was used for rotavirus G genotyping. Total coliform count varies from 102-103 CFU/mL in the raw water resources. Rotavirus antigen was detected in 17 samples (28.33%) by ELISA, and 13 samples (21.67%) were found positive by RT-PCR. These included 41.18% (7 cases) of raw water influent, 29.41% (5 cases) after sedimentation, 23.52% (4 cases) after ozonation, and 5.88% (1 case) after filtration in ELISA method. The highest number of rotaviruses was detected by qRT-PCR in autumn (46.15% (6 cases)). The commonest circulating G type in the sampling points was the mixed types, which was identified in 6 samples (46.15%), followed by non-typeable (23.07%), G3 (15.38%), G1 (7.69%), and G8 (7.69%), respectively. Despite the presence of rotavirus in raw water, after clarification and ozonation, filtration and treated water did not show the presence of rotavirus. The results of this study showed that multi-stage treatment has a positive effect on virus removal in WTP.

The wide distribution of an extremely thermoacidophilic microorganism in the copper mine at ambient temperature and under acidic condition and its significance in bioleaching of a chalcopyrite concentrate.

Thermoacidophiles can exist in a state of dormancy both in moderate temperatures and even in cold conditions in heap leaching. Sulphide mineral ores such as chalcopyrite produce sulfuric acid when exposed to the air and water. The produced sulfuric acid leads to the decrease of pH and exothermic reactions in heap leaching causing the temperature to increase up to 55°C and the activation of thermoacidophilic microorganisms. The aim of the present study was to isolate indigenous extreme thermoacidophilic microorganisms at ambient temperature from Sarcheshmeh Copper Complex, to adapt them to the high pulp density of a chalcopyrite concentrate, and to determine their efficiency in chalcopyrite bioleaching in order to recover copper. In this study samples were collected at ambient temperature from Sarcheshmeh Copper Complex in Iran. Mixed samples were inoculated into the culture medium for enrichment of the microorganisms. Pure cultures from these enrichments were obtained by subculture of liquid culture to solid media. Morphological observation was performed under the scanning electron microscope. Isolates were adapted to 30% (w/v) pulp density. For the bioleaching test, the experiments were designed with DX7 software. Bioleaching experiments were carried out in Erlenmeyer flasks and a stirred tank reactor. The highest copper recovery in Erlenmeyer flasks was 39.46% with pulp 15%, inoculums 20%, size particle 90µm and 160rpm. The lowest recovery was 3.81% with pulp 20%, inoculums 20%, size particle 40µm and 140rpm after 28 days. In the reactor, copper recovery was 32.38%. Bioleaching residues were analyzed by the X-ray diffraction (XRD) method. The results showed no jarosite (KFe3(SO4)2(OH)6) had formed in the bioleaching experiments. It seems that the antagonistic reactions among various species and a great number of planktonic cells in Erlenmeyer flasks and the stirred tank reactor are the reasons for the low recovery of copper in our study.

Cloning and expression of nlpA gene as DNA vaccine candidate against Acinetobacter baumannii.

Acinetobacter baumannii is one of the highly antibiotic-resistant bacteria that cause infections with high rate of death. This bacterium is one the common causes of infection worldwide leading to endemic and epidemic nosocomial infections. Despite many efforts, there is no effective vaccine against A. baumannii. As NlpA is one of the important antigenic factors in biogenesis of outer membrane vesicles, and OMV-based reported vaccines in A. baumannii stimulated the immune responses, this study was aimed to clone and express nlpA gene in eukaryotic HDF cells and evaluate the induced immunization following the administration of resulting construct as DNA vaccine in BALB/c mice. The nlpA gene of A. baumannii was amplified using PCR. The PCR product was then cloned and subcloned into the pTZ57R/T and pEGFP-C2 vectors respectively. The cloning was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The pEGFP-C2-nlpA recombinant plasmid was transferred into the HDF cells using electroporation and the expression of target gene was validated by RT-PCR. The recombinant construct was injected to BALB/c mice through three IM injections and the levels of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 were determined using ELISA assay. The A. baumannii nlpA gene was amplified during PCR as 867 bp band which was successfully cloned in pEGFP-C2-nlpA vector. Obtained data from RT-PCR and presence of the 867 bp fragment in transformed HDF cells confirmed the nlpA gene expression. Following the injection of pEGFP-C2-nlpA showed the increased level of IgG, IgM, INF-γ, IL-2, IL-4, and IL-12 in serum of immunized mice. Overall, through this study recombinant pEGFP-C2-nlpA was generated and successfully expressed the A. baumannii nlpA gene in eukaryotic cells. Additionally, our in vivo study confirmed that the recombinant construct capable to induce the immune response in immunized mice. These findings suggest the pEGFP-C2-nlpA may be considered as DNA vaccine candidate against A. baumannii.

Cloning of ompA gene from Acinetobacter baumannii into the eukaryotic expression vector pBudCE4.1 as DNA vaccine.

Antibiotic resistant features of Acinetobacter baumannii is partly due to the decreased outer membrane proteins (OMPs) permeability. The OmpA is one of the most conserved proteins among A. baumannii with a considerable antigenic potential to stimulate the multidimensional immune system responses. The present study was aimed to clone the ompA gene into the eukaryotic expression vector with potential as DNA vaccine. The ompA gene of A. baumannii was amplified using polymerase chain reaction (PCR). The target DNA was cloned and sub-cloned into the pTZ57R/T and pBudCE4.1 vectors, respectively. The recombinant vectors containing ompA were then validated using colony PCR, vector sequencing and double-digestion strategies. The pBudCE4.1-ompA recombinant plasmid was transfected into the human dermal fibroblast cells (HDF) and presence of ompA transcript and protein was evaluated using reverse transcribed-PCR (RT-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Our finding from colony PCR, sequencing and enzyme double digestion result confirmed that target gene has been successfully inserted into the pTZ57RT and pBudCE4.1. The presence of an expected band (1112 bp) in RT-PCR as wells as a ~ 38 kDa band during SDS-PAGE showed that the recombinant pBudCE4.1-ompA construct was efficiently transfected into the HDF cells and expressed. Altogether, our observation demonstrated that the recombinant pBudCE4.1-ompA construct was successfully produced although further experiments are needed.

The chimeric UreB, FliD and Omp18 proteins for a sensitive and specific diagnosis of Helicobacter pylori infections.

Background and Objectives: Helicobacter pylori is known as the main cause of gastrointestinal diseases including gastritis, gastric ulcer and stomach cancer. Serodiagnosis of H. pylori infection is a noninvasive and rapid method but the efficiency of this method is highly dependent to the antigens used. This study evaluated the efficacy of recombinant UreB-Omp18 and FliD for serodiagnosis of H. pylori infection. Materials and Methods: The genes encoding for fliD, ureB, and omp18 was amplified by PCR and cloned into pET-22b and pET-28a vectors. The constructs were expressed in E. coli BL21 and purified by affinity chromatography. The antigenic properties and diagnostic potential of the recombinant proteins were analysed by immunoblotting and ELISA, respectively. Results: The recombinant UreB-Omp18 and FliD with molecular weights of 48 kDa and 25 kDa were observed on SDS-PAGE and purified by the Ni-NTA column. The ELISA results showed that the sensitivity and specificity of recombinant UreB-Omp18 protein in serodiagnosis of H. pylori infection were 89% and 83%, respectively. Also, the sensitivity and specificity of the recombinant FliD protein were calculated to be 91% and 76%, respectively. Conclusion: The results indicated that the recombinant UreB-Omp18 and FliD could diagnose H. pylori infection with high sensitivity and specificity.

Lag Plot: A Novel Method to Determine Viable Bacterial Cell Number via a Single OD Measurement.

Bactericidal activity is a valuable parameter which is considered and measured for antimicrobial compounds. The available standard protocol to evaluate bactericidal activity is based on the direct colony count. Colony counting requires serial dilution, plating, overnight incubation, and direct counting, which is time-and labor-intensive. In regard to eliminate direct plate count, novel techniques were developed based on the real-time growth monitoring which can come with some limitations and drawbacks. These drawbacks encourage us to develop a novel technique with simple procedure to determine viable bacterial cell count. In this procedure, real-time growth monitoring is not required. In fact, after an incubation time, the number of viable bacteria can be determined with a single OD measurement and through an equation. In this regard, four standard bacterial strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 23857), were cultured with descending inoculum densities (1.5 × 107 to 15 CFU/mL) and growth curves were drawn. As expected, growth in the samples with lower inoculum densities recorded with longer lag phase. Also, a direct relation was observed between the recorded turbidities and initial cell counts. A logarithmic curve (lag plot) was obtained by plotting the OD, after 12-18 h incubation, against initial cell counts. In all examined strains, R2 was calculated in the range of 0.96-0.99 which is acceptable value for coefficient of determination. Equation corresponding to the lag plot was obtained and called lag equation. This equation is applicable to calculate the number of viable cells in unknown samples simply by inoculation, incubation, and single OD measurement. Developed technique can be introduced as a simple substitution for labor- and time-intensive direct colony counting.

Effect of Crocin and Crocetin Compared to Cyclophosphamide on the Expression Level of miRNA-16-1 in a B Cell Transformed with EBV Virus Cell Line.

INTRODUCTION: Crocin and Crocetin are compounds that have shown promising therapeutic potentials in various medical contexts. To date, the effect of crocin and crocetin on the expression level of miRNA-16-1 in Epstein Barr Virus (EBV)-induced lymphoma has not been investigated. This research delved into a comparative analysis of the cytotoxic effects of crocin and crocetin compared to cyclophosphamide, the main drug used in the treatment of lymphoma and PTLD, on B-cell lymphoma infected with EBV (cell line CO 88BV59-1). Additionally, the study examines the changes in miRNA-16-1 expression following these treatments in this cell line. MATERIALS AND METHODS: CO 88BV59-1 LCL cells were treated with crocin, crocetin (0.2 to 200 µM), and cyclophosphamide (0.05 to 50 µM) for 72 hours. Cell viability and apoptosis were assessed using resazurin and Annexin V/PI techniques, respectively. Additionally, the expression of miRNA-16-1-3p and miRNA-16-1-5p was determined using the Real-Time PCR method. The data were analyzed using one-way analysis of variance (ANOVA) with Tukey's multiple comparisons post-hoc test. RESULTS: Crocin and crocetin inhibited the proliferation and apoptosis caused by EBV-infected cells in a dose- and time-dependent manner (P<0.05). The expression levels of miRNA-16-1-3p and miRNA-16-1-5p remained unchanged in cells treated with crocin and crocetin. CONCLUSION: The study found that the cytotoxic effect of Crocin, Crocetin, and Cyclophosphamide on CO 88BV59-1 LCL is independent of the expression level of miRNA-16-1. The results showed a reduction in cell survival and an increase in cell death.

The potent in vitro skin permeation of archaeosome made from lipids extracted of Sulfolobus acidocaldarius.

Archaeosomes are a new generation of liposomes that exhibit higher stabilities under different conditions, such as high temperatures, alkaline or acidic pH, and presence of bile salts in comparison with liposomes, and can be used in biotechnology including drug, gene, and vaccine delivery. The objective of this study was to prepare archaeosomes using lipid extracted from Sulfolobus acidocaldarius and evaluate their physicochemical properties. The lipids were extracted from S. acidocaldarius and assayed by High Performance Thin-Layer Chromatography (HPTLC). Archaeosomes were prepared using film method and methylene blue was used as drug model. They were characterized for their vesicle size and Differential Scanning Calorimetry (DSC) was used to investigate changes in their thermal behavior. The released amount of methylene blue was determined using a dialysis membrane and rat skin. HPTLC analysis of the extracted lipids showed that glycerol ether may be the major lipid with more than 78 percent probability. Results of particle size determination showed a mean size of 158.33 nm and the results of DSC indicated the possible interaction of methylene blue with lipids during the preparation of archaeosome. The addition of cholesterol significantly improved the encapsulation of methylene blue in the archaeosome so that the encapsulation efficiency was 61.66 ± 2.88%. The result of in vitro skin permeation showed that methylene blue could pass through skin model according to Peppas model and there was about 41.66% release after 6 h, whereas no release was observed through dialysis membrane. According to the results of the study, it is concluded that archaeosome may be successfully used as drug delivery system.

Effect of plasma-activated water on the biofilm-forming ability of Salmonella enterica serovar Enteritidis and expression of the related genes.

In recent years, microbial decontamination with plasma-activated water (PAW) has attracted a lot of research attention in the field of food industry. Despite several studies showing that PAW effectively inactivates planktonic bacteria, few studies have been conducted on biofilms. The present study was, therefore, designed to evaluate the effect of PAW on the biofilm formation characteristics of Salmonella Enteritidis. Comparing the expression patterns of biofilm-related genes in PAW-treated and non-treated planktonic and biofilm cells provided insight into how PAW regulates this process. The results showed that a 30-minute exposure to PAW at room temperature significantly reduced S. enteritidis planktonic cells. This exposure resulted in a decreased expression of the genes involved in the early stages of biofilm formation (csgD, agfA, fimA, lpfE, and rpoS), and an increased expression of the csrA gene in S. enteritidis planktonic cells. These results indicated the inhibitory effect of PAW on the biofilm formation process in S. enteritidis. Results of the initial attachment assay confirmed these findings, where, after 6 h, the number of PAW-treated cells attached to the stainless steel surfaces were significantly lower than non-treated ones. Furthermore, biofilm development assay revealed that the number of PAW-treated biofilm cells were significantly lower than non-treated ones after 24 h incubation at 37 °C. These findings were confirmed by measurements of the major components of biofilm i.e., extracellular DNA (eDNA), protein and carbohydrate. The amount of these components in 24-hour biofilms produced by PAW-treated S. enteritidis cells was significantly lower than that of non-treated cells. PAW's treatment on preformed 24-hour biofilms for 30 min led to a decrease in the expression of genes involved in quorum sensing and cellulose synthesis (csgD, bapA, adrA, luxS and sdiA) and an increase in the expression of the csrA gene. This treatment also reduced the number and metabolic activity of biofilm cells compared to non-treated biofilm cells. In total, the present study demonstrated that PAW has an inhibitory effect on the process of biofilm formation in S. enteritidis and hence, the food industry should pay special attention to PAW as a promising treatment to eliminate bacterial biofilms.

Productivity Loss of Temporary Work Absenteeism Due to COVID-19 and Its Determinant Factors in Northeastern Iran.

OBJECTIVE: This study aimed to estimate the lost productivity cost of temporary work absenteeism due to COVID-19. METHODS: This study conducted on all hospitalized patients with COVID-19 in northeastern Iran between February 2020 and March 2022 (10,406 cases). Data were collected from the Hospital Information System. Indirect costs were estimated using the human capital approach. Data were analyzed with the STATA version 17. RESULTS: The total indirect cost of work absenteeism due to COVID-19 was estimated at $513,688. There was a statistically significant relationship between the mean lost productivity cost and COVID-19 peak, sex, insurance type, age, and hospitalization. CONCLUSIONS: Because the absenteeism costs of COVID-19 had increased in the second peak, which coincided with the summer holidays, the country's crisis management headquarters should pay more attention to formulating and implementing appropriate preventive programs in future epidemics.

Phloridzin reduces blood glucose levels and improves lipids metabolism in streptozotocin-induced diabetic rats.

Phloridzin is the specific and competitive inhibition of sodium/glucose cotransporters in the intestine (SGLT1) and kidney (SGLT2). This property which could be useful in the management of postprandial hyperglycemia in diabetes and related disorders. Phloridzin is one of the dihydrochalcones typically contained in apples and in apple-derived products. The effect of phloridzin orally doses 5, 10, 20 and 40 mg/kg body weight on diabetes was tested in a streptozotocin-induced rat model of diabetes type 1. From beneficial effect of this compound is significant reduction of blood glucose levels and improve dyslipidemia in diabetic rats. As a well-known consequence of becoming diabetic, urine volume and water intake were significantly increased. Administration of phloridzin reduced urine volume and water intake in a dose-dependent manner. Phloretin decreases of food consumption, as well as a marked lowering in the weight. In conclusion, this compound could be proposed as an antihyperglycemic and antihyperlipidemic agent in diabetes and potential therapeutic in obesity.

Designing of novel chimeric PvpA-pMGA protein of Mycoplasma gallisepticum, applicable for indirect ELISA.

BACKGROUND: Mycoplasma gallisepticum is the primary agent of chronic respiratory disease in chickens creating important economic losses in poultry industry. pMGA and pvpA genes encode major surface proteins in M. gallisepticum containing pathogenic, antigenic, and immune evasion characteristics. The objective of the present study was to design, express, and purify the recombinant chimeric PvpA-pMGA protein from M.gallisepticum for using in serological diagnostic test. METHODS: Antigenic regions of PvpA and pMGA proteins were predicted for designing chimeric pvpA-pMGA gene construct. The codon optimized sequence was cloned into the expression vector pET32a+ and transformed into the Escherichia coli strain BL21 (DE3). The pET32a-PvpA-pMGA recombinant plasmid was expressed and confirmed by SDS-PAGE and immunoblotting. PvpA-pMGA recombinant protein (20µg and 50µg), ts-11 vaccine strain, and S6 strain that formulated by montanide adjuvant and two control groups (PBS and adjuvant) were injected subcutaneously to six groups of chickens. RESULTS: High yield of protein was purified amount 138 mg/L by affinity batch formation method. Indirect ELISA showed the levels of antibodies in rPvpA-pMGA was significantly higher than ts-11 and S6 groups (p<0.05). The results indicated that antigen-specific response was successfully elicited by the rpMGA-PvpA in chickens. The result of the ELISA with sera collected from ts-11 and S6 groups showed that indirect PvpA-pMGA-ELISA is appropriate candidate for detection of specific antibodies against M. gallisepticum with 100% sensitivity and specificity. CONCLUSIONS: The rPvpA-pMGA is a highly candidate immunogenic protein which induced high amount of humoral immune response. Novel rPvpA-pMGA protein could be useful for evaluation of antibody level in vaccinated poultry flocks.

Detection and evaluation of rotavirus surveillance methods as viral indicator in the aquatic environments.

Group A rotaviruses (RVAs) have been introduced as the most important causative agents of acute gastroenteritis in the young children. One of every 260 children born globally will die due to rotavirus (RV) before 5 years old. The RV is widely known as a viral indicator for health (fecal contamination) because this pathogen has a high treatment resistance nature, which has been listed as a relevant waterborne pathogen by the World Health Organization (WHO). Therefore, monitoring of environmental is important, and RV is one of the best-known indicators for monitoring. It has been proved that common standards for microbiological water quality do not guarantee the absence of viruses. On the other hand, in order to recover and determine RV quantity within water, standard methods are scarce. Therefore, dependable prediction of RV quantities in water sample is crucial to be able to improve supervision efficiency of the treatment procedure, precise quantitative evaluation of the microbial risks as well as microbiological water safety. Hence, this study aimed to introduce approaches to detecting and controlling RV in environmental waters, and discussed the challenges faced to enable a clear perception on the ubiquity of the RV within different types of water across the world.