Aflatoxin B1 (AFB1) is a well-established potent food mycotoxin. We previously evidenced that laccase could restrain AFB1's genetic and oxidative toxicity in vitro conditions; nevertheless, there is no report about the in vivo toxicity of the laccase-mediated metabolites of aflatoxin B1 (LM-AFB1). In this investigation, we evaluated the in vivo safety of LM-AFB1 in comparison with the parent toxin using serum liver function tests (LFTs), oxidative stress biomarkers, histopathological examinations, and CYP1A2 and GSTA3 gene expression. Adult male mice were divided into four groups: (1) Control; (2) 4 mg/kg AFB1; (3) 4 mg/kg LM-AFB1; (4) 4 mg/kg intermediate transformational metabolites of AFB1 (CA-AFB1). In this study, AFB1's liver toxicity was confirmed but LM-AFB1 did not significantly change the values. Gene expression analysis indicates that the utilization of laccase for bioremediation does not result in the suppression of glutathione S-transferase, in contrast to AFB1. Our research findings demonstrate that laccase has the potential to mitigate AFB1-induced liver toxicity in mice by averting oxidative stress caused by AFB1.

In this study, we used a simple green method for preparing tellurium nanoparticles and mainly evaluated their toxicological effects. The nanoparticles were synthesized using lactose and characterized with different instrumentation methods. The in vitro and in vivo cytotoxicity of tellurium nanoparticles and its effect on lipid profile were also evaluated. Hydroxyl-capped tellurium nanoparticles were successfully fabricated by lactose. The results showed spherical tellurium nanoparticles with a mean size of 89 nm. The toxicological study showed that the tellurium nanoparticles did not exhibit any toxicity on the primary cells. The LD50 values for the nanoparticles were 327 and 295 mg/kg for oral and intraperitoneal administrations, respectively. Also, the results showed a significant reduction in liver enzymes at the 16, 24, and 40 mg/kg doses. Hematological parameters indicated no significant suppressive changes between the animals that were administered tellurium nanoparticles and the control group. In addition, the effects of tellurium nanoparticles on hypercholesterolemic risk factors in mice fed with cholesterol demonstrated the depletion of triglyceride, cholesterol, and low-density lipoprotein. This study showed that the toxicity of tellurium nanoparticles was lower than tellurium ions. Furthermore, tellurium nanoparticles decreased the cholesterol and triglyceride levels in the animal model.

Regulation of immune responses is among the beneficial effects of probiotic bacteria on human health. In this study, we aim to investigate the effect of normal and heat-shocked Lactobacillus plantarum PTCC 1058 cell lysate on cytokine expression by human PBMCs. The mid-exponential phase L. plantarum (10 8 CFU/mL) were used to prepare cell lysate. Isolated PBMCs were stimulated with 100 µg/mL of each normal and heat-shocked L. plantarum cell lysate for 72 h. Non-stimulated PBMCs were also evaluated as negative control. The mRNA expression of IL-6, IL-10, IFN-ɣ, TNF-α, and TGF-β genes was determined by quantitative RT-PCR amplification of total RNA extracted from PBMCs. Both types of cell lysate were able to increase pro-inflammatory cytokines and decrease anti-inflammatory cytokines. However, this effect was significantly stronger in heat-shocked cell lysate–treated PBMCs. Moreover, comparison of IFN-ɣ/IL-10, IFN-ɣ/TGF-β, IL-6/IL-10, IL-6/TGF-β, and TNF-α/IL-10 ratios in both conditions demonstrated that in the heat-shocked group, all of the above ratios were significantly higher than normal lysate treatment ( p ˂0.001), suggesting that heat-shocked probiotics are a potent inducer of the immune system in comparison to intact probiotics. Regarding these results, it may be possible to develop a new postbiotic product for the stimulation of immune responses of cancer patients or individuals who suffer from an immune defect.

PURPOSE:Sepsis originates from the host inflammatory response, especially to bacterial infections, and is considered one of the main causes of death in intensive care units. Various agents have been developed to inhibit mediators of the inflammatory response; one prospective agent is β-sitosterol (βS), a phytosterol with a structure similar to cholesterol. This study is aimed at evaluating the effects of βS on the biomarkers of inflammation and liver function in cecal ligation and puncture- (CLP-) induced septic rats.METHODS:Thirty male Wistar rats were divided equally into six groups as follows: sham, CLP, CLP+dexamethasone (DX, 0.2 mg/kg), CLP+βS (1 mg/kg), CLP+imipenem (IMI, 20 mg/kg), and CLP+IMI (20 mg/kg)+βS (1 mg/kg). Serum levels of IL-1β, IL-6, IL-10, AST, ALT, and liver glutathione (GSH) were assessed by ELISA. Liver expression levels of TNF-α and NF-κBi mRNAs were evaluated by RT-qPCR.RESULTS:Serum concentrations of IL-1β, IL-6, IL-10, ALT, and AST and mRNA levels of TNF-α and NF-κBi were all significantly higher in septic rats than in normal rats (p < 0.05). Liver GSH content was markedly lower in the CLP group than that in the sham group. βS-treated rats had remarkably lower levels of IL-1β, IL-6, IL-10, TNF-α, NF-κBi, AST, and ALT (51.79%, 62.63%, 41.46%, 54.35%, 94.37%, 95.30%, 34.87%, and 46.53% lower, respectively) and greater liver GSH content (35.71% greater) compared to the CLP group (p < 0.05).CONCLUSION:βS may play a protective role in the septic process by mitigating inflammation. This effect is at least partly mediated by inhibition of the NF-κB signaling pathway. Thus, βS can be considered as a supplementary treatment in septic patients.

BACKGROUND AND PURPOSE:Granulocyte colony-stimulating factor (G-CSF) is routinely used in combination with chemotherapy to battle neutropenia. However, studies suggest that this chemokine may increase the risk of metastasis and malignancy in many cancers. To counteract the adverse effects of G-CSF in cancer, antibodies have been used to block its action. However, antibodies are large and complex molecules which makes their production expensive. Thus in this study, we aim to construct different structure variants of the G-CSF receptor containing different domains and select the best variant that prevents the adverse actions of this chemokine. These novel structures are smaller than antibodies and easier to produce.EXPERIMENTAL APPROACH:Different domains of the G-CSF receptor were designed and cloned into the pET28a expression vector. These recombinant receptor subunits were then expressed in Escherichia coli and purified using standard affinity chromatography techniques. Interaction of recombinant receptor subunits with G-CSF was assessed using enzyme-linked immunosorbent assay and NFS60 cells.FINDINGS / RESULTS:Two recombinant receptor subunits containing D1 + D2 + D3 domains and D2 domain showed the strongest inhibitory activity to G-CSF.CONCLUSION AND IMPLICATIONS:These novel recombinant receptor variants could be candidates for further studies in the development of novel therapeutics.

Despite the existence of vaccination, antibiotic therapy, and antibody therapies, infectious diseases still remain as one of the biggest challenges to human health all over the world. Among the different methods for treatment and prevention of infectious diseases, antibodies are well known but poorly developed. There is a new subclass of antibodies calledheavy-chain antibodies that belong to the IgG isotype. However, they are low in molecular weight and lost the first constant domain (CH1). Their single-domain antigen-binding fragments, identified as nanobodies, have unique characteristics, which make them superior in comparison with the conventional antibodies. Low molecular weight and small size, high stability and solubility, ease of expression, good tissue penetration, and low-cost production make nanobodies an appropriate alternative to use against infectious disease. In this research, we review the properties of nanobodies and their potential applications in controlling human infections and inflammations.

AIMS:Granulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is overexpressed in several malignant tumors and blocking its binding to the receptor can lead to significant decrease in tumor growth, vascularization and metastasis. Furthermore, targeting G-CSF receptor has shown therapeutic benefit in other diseases such as rheumatoid arthritis, progressive neurodegenerative disorder and uveitis. Camelid single-chain antibodies (nanobodies) have exceptional properties making them appropriate for tumor imaging and therapeutic application. In this study we aim to use the rational design approach to engineer a previously described G-CSF-R targeting nanobody (VHH1), to improve its affinity toward G-CSF-R.MAIN METHODS:We redesigned the complementary determining region 3 (CDR3) domain of the VHH1 nanobody to mimic G-CSF interaction to its receptor and developed five new engineered nanobodies. Binding affinity of the engineered nanobodies was evaluated by ELISA (Enzyme-linked immunosorbent assay) on NFS60 cells.KEY FINDINGS:Enzyme-linked immunosorbent assay (ELISA) confirmed the specificity of the engineered nanobodies and ELISA-based determination of affinity revealed that two of the engineered nanobodies (1c and 5a) bind to G-CSF-R on the surface of NFS60 cells in a dose-dependent manner and with a higher potency compared to the parental nanobody.SIGNIFICANCE:Additional studies are required to better characterize these nanobodies and assess their interaction with G-CSF-R in vitro and in vivo. These newly developed nanobodies could be beneficial in tumor imaging and therapy and make a basis for development of additional engineered nanobodies.

BACKGROUND:Circumsporozoite protein (CSP) is one of the most important surface sporozoite antigens in malaria, recently considered as a candidate for vaccination. Considering the importance of CSP, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax Circumsporozoite Protein (Pvcsp) in the southeastern region of Iran during 2015-2016.METHODS:To investigate polymorphism and genetic diversity, 20 blood samples were collected from patients with P. vivax, then DNA was extracted and amplified using partial sequence of CSP gene. Polymerase chain reaction (PCR) products were sequenced and compared to sequences from genomic databases using BLAST. Genetic evaluation and phylogenic analysis were performed using MEGA7 and DnaSP5 software's on 38 sequences include 20 sequences of our study and 18 sequences of Gene Bank.RESULTS:Eleven isolates were VK210 genotype and 9 isolates contained VK247. The result of variable segregation nucleotide site indicated that the differentiation of sequences in CSP were 25.67% in our 20 samples which are less than the 38 samples with a value of 26.67%. Comparing the ratio of dN/dS regions in the CSP gene indicates that the CSP varies more synonymously and amino acid has lower variation. Out of 38 samples, 35 unique haplotypes were identified based on 1042 nucleotide sequences in CSP, showing a variation percentage of 99.4%.CONCLUSION:The Tajima D analyses showed that CSP gene in P. vivax had a positive number in the total analyzed sequences, which means that the P. vivax mutations are in order to select positive evolution.

Treponema (T.) denticola is one of the key etiological agents in the development of periodontitis. The major outer sheath protein (Msp) of T. denticola has been shown to mediate pathogenesis and to facilitate adhesion of T. denticola to mucosal surfaces. This study aimed to find short polypeptides in the amino acid sequence of Msp which may be immunogenic and might elicit protective antisera against T. denticola. The complete msp sequence was divided into six fragments and the corresponding genes were cloned and expressed. Antisera against the polypeptides were raised in rabbits and fragment 3 (F3), hereinafter called PerioVax3 was the most potent fragment of the Msp in terms of yielding high titer antiserum. An adhesion assay was done to examine the inhibitory effects of antisera on the attachment of T. denticola to human gingival fibroblasts (HGFs) and human fibronectin. Antiserum against PerioVax3 significantly inhibited attachment of T. denticola to the substratum. Also, antiserum against PerioVax3 inhibited detachment of HGFs upon T. denticola exposure. To begin examining the clinical relevance of this work, blood samples from 12 sever periodontitis patients were collected and the sera were used in western blotting against the recombinant polypeptides. Periodontitis patient antisera exclusively detected PerioVax3 in western blotting. The data suggest that PerioVax3 carries epitopes that may trigger humoral immunity against T. denticola, which may protect against its adhesion functions. The complexity of periodontitis suggests that PerioVax3 may be considered for testing as a component of an experimental multivalent periodontal vaccine in further preclinical and clinical studies.

The Lactococcus lactis is known as a probiotic bacterium and also as a producer of nisin. Nisin has been approved by related legal agencies to be used as an antimicrobial peptide in food preservation. In fact, the L. lactis is present in different food products along with other micro-organisms especially pathogenic bacteria. So, it is important to predict the behavior of nisin-producer strain in contact with other pathogens. In this regard, nisin gene expression and the level of secreted biologically active form of nisin by L. lactis subsp. lactis in modified MRS broth and whey solution in co-culture with Listeria monocytogenes or Salmonella enterica were studied. The nisin concentration was determined by microbiological assay method and the transcription level of nisin gene was assayed through quantitative reverse transcription PCR (RT-qPCR). According to our results, the highest concentration of nisin and its gene transcription level were detected in mono- and co-cultures after 16 h of incubation, concurrent with the end of L. lactis exponential phase of growth. The nisin mRNA copies in co-cultures were higher than mono-cultures only at 16 h of incubation. But, differences between nisin concentrations in mono- and co-cultures were significant at 16, 24 h and at 12, 16, 24 h of incubation in the modified MRS medium and whey solution, respectively. This incompatibility could be related to the low availability of components required for nisin precursor modification, transportation and processing in mono-cultures. Overall, the L. lactis produced more mature and active nisin when it was in contact with pathogenic bacteria.

BACKGROUND:Invasive candidiasis management through the rapid initiation of appropriate antifungal therapy has been shown to be associated with the better prognosis, improved clinical outcome and reduced mortality in critically ill patients. Therefore, selection of  an appropriate antifungal therapy should be based on the distribution of candida species and the pattern of antifungal resistance. This study aimed to assess the prevalence of candidemia and associated subtypes following severe sepsis in non-neutropenic critically ill patients.METHODS:This study was a cross-sectional study that was conducted on severe sepsis patients stayed at least seven days in intensive care unit. Patients less than 18 years old, pregnant and breastfeeding patients, immunocompromised patients, neutropenic patients, patients with concurrent use of antifungal medicines and cytotoxic agents were excluded.To asses the candidemia, one mililiter of patients' blood sample was collected. Sample analysis was performed by Real-Time PCR and high resolution melting curve analysis method.RESULTS:Thirty-one critically ill patients were recruited in this study over 12-month period. Candidemia with a detection limit of 100 pg per 0.2 ml blood sample was not recognized in any of the included patients.CONCLUSION:The present result indicates low incidence of candidemia in the targeted intensive care units, but other factors such as small sample size, exclusion of patients with compromised immune system and the low fungal load at the time of sampling may also account for our observation.

The use of the methylotrophic yeast, Pichia pastoris , as one of the most effective and versatile systems for the expression of heterologous proteins in biopharmaceutical manufacturing has become increasingly popular in recent years. The impurity caused by residual host cell DNA is one of the major concerns in production of recombinant therapeutics. The aim of the present study was to develop a semi-quantitative, multiplex PCR method to determine the level of impurity in biopharmaceuticals produced in Pichia pastoris as the host. Primers were designed based on the rDNA repeat region and optimized to achieve the limit of detection in a multiplex PCR following by analyzing with MYImageAnalysis (Thermo Fisher Scientific, USA) software to quantify the concentration of Pichia pastoris genomic DNA in pertinent controls and drug samples. The multiplex PCR were able to detect up to 1 femtogram (fg) of genomic DNA under optimized conditions. Moreover, achieved concentration of DNA in controls and samples through relevant standard curve indicates the feasibility of this method in the presence of inhibitory effects. In comparison with other methods such as real-time PCR and Threshold assay, the assay shows acceptable sensitivity, precision and linearity along with ease of use, low equipment costs and analyte flexibility. We thus propose this method to be considered as a useful tool to estimate host cell residual DNA in biopharmaceuticals produced in Pichia pastoris . Highlights The impurity of residual host cell DNA is an important concern in production of biopharmaceuticals. Pichia pastoris is an effective and versatile system for the expression of recombinant proteins Quantitative Polymerase Chain Reaction could be used for quantifying residual host-cell DNA We designed a sensitive and valid PCR method for detection and quantification of Pichia residual DNA

It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models.

Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T1/2, respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p < 0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations.

Gastric cancer (GC) is one of the most common types of cancer worldwide. Owing to the distinct molecular pathology and the increasing progression rate of GC in Asia, suppression subtractive hybridization (SSH) was used as a high throughput gene expression analysis method to find genes associated with GC pathogenesis. Total RNA was extracted from the clinical samples, and mRNA was isolated and used in SSH method. The subtracted library was subjected to cloning and the randomly selected clones were sequenced. qRT-PCR was used for expression analysis. The overexpression of vesicle amine transport protein-1 (VAT-1) gene was observed and its expression was analyzed by qRT-PCR in clinical tissue samples. According to the potential oncogenic activity of VAT-1 and its probable surface-occurrence in GC cells, it might be involved in GC pathogenesis and invasion, and it is suggested to be investigated in the diagnosis and therapeutics of GC.

•Both wild-type and truncated forms, coding for lipA from Serratia marcescen were successfully cloned and expressed.•Deletion of in the second β-roll domain at the C-terminal region of SML does not deleteriously affect enzyme catalysis.•The truncated form of SML with strong catalytic efficiency could be employed potential to industrial applications.

Gastric cancer (GC) is one of the most common and life-threatening types of malignancies. Identification of the differentially expressed genes in GC is one of the best approaches for establishing new diagnostic and therapeutic targets. Furthermore, these investigations could advance our knowledge about molecular biology and the carcinogenesis of this cancer. To screen for the overexpressed genes in gastric adenocarcinoma, we performed suppression subtractive hybridization (SSH) on gastric adenocarcinoma tissue and the corresponding normal gastric tissue, and eight genes were found to be overexpressed in the tumor compared with those of the normal tissue. The genes were ribosomal protein L18A, RNase H2 subunit B, SEC13, eukaryotic translation initiation factor 4A1, tetraspanin 8, cytochrome c oxidase subunit 2, NADH dehydrogenase subunit 4, and mitochondrially encoded ATP synthase 6. The common functions among the identified genes include involvement in protein synthesis, involvement in genomic stability maintenance, metastasis, metabolic improvement, cell signaling pathways, and chemoresistance. Our results provide new insights into the molecular biology of GC and drug discovery: each of the identified genes could be further investigated as targets for prognosis evaluation, diagnosis, treatment, evaluation of the response to new anticancer drugs, and determination of the molecular pathogenesis of GC.

In this paper, the enzymatic detoxification of aflatoxin B1 (AFB1) by laccase was studied, and the prooxidant properties and mutagenicity of the detoxification products were compared with those of AFB1. The optimal enzymatic reaction occurred in 0.1M of citrate buffer containing 20% DMSO at 35 °C, a pH of 4.5, and a laccase activity of 30 U mL(-1). After 2 d, sixty-seven percent of the toxic substrate was removed. The prooxidative properties of the detoxified products (27% versus 86%) and the mutagenicity were significantly decreased in comparison with the parent toxin. Unlike AFB1, which promoted metabolism-dependent genetic mutations by base-pair substitution, the detoxified products did not induce genotoxicity. Comparison of the Km values for AFB1 and riboflavin, a valuable food nutrient, indicated that laccase showed greater affinity for the toxin than for riboflavin.

Hypercholesterolemia is an important risk factor contributing to atherosclerosis and coronary heart disease. Lactic acid bacteria have attracted much attention regarding their promising effect on serum cholesterol levels. Tellurium (Te) is a rare element that has also gained considerable interest for its biological effects. There have been some recent in vivo reports on the reduction effect of Te on cholesterol content. In this study, Lactobacillus plantarum PTCC 1058 was employed for the intracellular biosynthesis of Te NPs. The UV-visible spectrum of purified NPs showed a peak at 214 nm related to the surface plasmon resonance of the Te NPs. Transmission electron microscopy showed that spherical nanoparticles without aggregation had the average size of 45.7 nm as determined by the laser scattering method. The energy dispersive X-ray pattern confirmed the presence of Te atoms without any impurities. A significant reduction was observed in group which received L. plantarum with or without Te NPs during propylthiouracil and cholesterol diet in compare with the control group which received just propylthiouracil and cholesterol. The levels of triglycerides also remarkably decrease (p<0.05) in mice given L. plantarum with intracellular Te NPs.