Nitric oxide and ascorbic acid confer cadmium (Cd) tolerance by improving plant terpenoid metabolism and epigenetically modifying DNA methylation.

This study investigated the efficacy of incorporating nitric oxide (NO; 10 µM) and ascorbic acid (Asc; 10 µM) into the culture medium to confer cadmium (Cd; 5 µM) tolerance in thyme (Zataria multiflora). The phytotoxicity of Cd resulted in a decrease in shoot biomass, which NO or Asc mitigated. Adding Asc and NO to the culture medium was associated with substantial DNA hypomethylation. The NO + Cd and Asc + Cd treatments were accompanied by an increase in the unmethylation percentages, about 3-fold higher than the control. The hemi-methylation percentages in the Asc-supplemented seedlings also displayed an upward trend. The transcriptional upregulation in the γ-terpinene synthase (TPS) gene resulted from the applied elicitors, especially NO. In response to the NO and Asc treatments, the transcription of two cytochrome P450 monooxygenase genes (CYP71D178 and CYP71D180) went up. Incorporating Asc or NO into the culture medium enhanced the concentrations of proline, carvacrol, and thymol metabolites. Employing NO or Asc mitigated the 43% decrease in protein content due to the Cd cytotoxicity. The NO and Asc applications improved the activity of the phenylalanine ammonia-lyase (PAL) enzyme. NO and Asc utilization increased the accumulation of flavonoids. NO and Asc also up-regulated the activities of two enzymatic antioxidants (catalase and peroxidase). Collectively, this study provided novel insight into how Asc or NO confers Cd tolerance by epigenetically remodeling DNA methylation, transcriptionally up-regulating terpenoid and phenylpropanoid metabolism, increasing proline concentration, and improving antioxidants.

Antibacterial and antibiofilm effect of Zinc Oxide nanoparticles on P. aeruginosa variants isolated from young patients with cystic fibrosis.

BACKGROUND: P. aeruginosa, a biofilm-forming bacteria, is the main cause of pulmonary infection in CF patients. We applied ZnO-np as a therapeutic agent for eradicating multi-drug resistance and biofilm-forming P. aeruginosa isolated from young CF patients. METHODS: A total of 73 throat and sputum samples taken from young CF patients were inquired. ZnO-np was synthesized and characterized in terms of size, shape, and structure for anti-bacterial activity. The antibiotic susceptibility of isolates before and after the addition of 16 µg/ml of ZnO was evaluated using disc diffusion and microtiter methods, respectively. The gene expression level of QS genes was assessed after treatment with 16 µg/ml ZnO-np. RESULTS: The optimum concentration of ZnO-np with a higher inhibitory zone was 16 µg/ml (MIC) and 32 µg/ml (MBC). All isolates were resistant to applied antibiotics, and about 45 % of isolates were strong biofilm-forming bacteria. After treatment with 16 µg/ml ZnO-np, all strains became susceptible to the applied antibiotic except for amikacin, which confers an intermediate pattern. About 63 % and 20 % of isolates were, respectively, non-biofilm and weak biofilm-forming bacteria following the addition of ZnO-np. Relative gene expression of gacA, lasR, and rhlR genes were downregulated significantly (P < 0.001). Although the retS did not have a significant reduction (P = 0.2) CONCLUSION: ZnO-np at a concentration of 16 µg/ml could significantly reduce the P. aeruginosa infection by altering the antibiotic susceptibility pattern and inhibiting biofilm formation. Due to their photocatalytic properties and their ability to penetrate the extracellular polysaccharide layer, ZnO nanoparticles can produce ROS, which increases their susceptibility to antibiotics. Nasal delivery of ZnO-np in the form of aerosol can be considered a potential strategy to decrease the mortality rate in CF patients at an early age.

Significance of genotypes and resistance status of Mycobacterium tuberculosis strains in gene expression of apoptosis cell death and inflammatory pathways in A549 lung epithelial cell line.

Objectives: Tuberculosis (TB) has been a major health issue throughout history. As part of TB infection, host-Mycobacterium tuberculosis (Mtb) interactions are important. Through immune pathology and cell death control processes, Mtb infection facilitates intracellular growth. The relationship between apoptosis and inflammation in Mtb infection remains unclear. In this study, the levels of related apoptosis and inflammatory genes were assessed in A549 cells infected with a variety of Mtb strains. Materials and Methods: Mtb isolates with different phenotypes (sensitive, INHR, RifR, MDR, and XDR) were collected from the Pasteur Institute of Iran, during this study. Whole genome sequencing was previously performed on all strains, and the Beijing genotype was selected as sensitive. Also, for other resistant strains, the New-1 genotype was available and isolated for genotype comparison. A549 lung carcinoma cells were also grown and infected with selected Mtb strains. Genes involved in inflammation and apoptosis were detected using reverse transcription-PCR (RT-PCR). Results: All sensitive strains and resistant strains were found to significantly up-regulate anti-apoptotic (bcl2 and rb1), chemokine (IL-8 and MCP-1), and pro-inflammatory cytokine (TNF-α and IFN-γ) expression, while significant down-regulation was observed after 24 and 48 hr of infection in anti-inflammatory genes (IL-10) and pro-apoptotic genes (bad and bax). Besides resistance strains, Mtb genotypes also affected gene expression. The Beijing genotype (sensitive isolate) influences inflammatory and apoptotic genes more sharply than the New-1 genotype (INHR, RifR, MDR, and XDR). Conclusion: Gene expression differences related to apoptosis and inflammation examined in the current study may be attributed to genotypes rather than resistance status since the expression of most genes has been observed to be lower in resistant strains (INHR, RifR, MDR, and XDR belonging to the New-1 genotype) compared to sensitive strains (Beijing genotype).

Monophosphoryl lipid A as a co-adjuvant in methicillin-resistant Staphylococcus aureus vaccine development: improvement of immune responses in a mouse model of infection.

To increase the effectiveness of methicillin-resistant Staphylococcus aureus vaccines (MRSA), a new generation of immune system stimulating adjuvants is necessary, along with other adjuvants. In some vaccines, monophosphoryl lipid A (MPLA) as a toll-like receptor 4 agonist is currently used as an adjuvant or co-adjuvant. MPLA could increase the immune response and vaccine immunogenicity. The current investigation assessed the immunogenicity and anti-MRSA efficacy of recombinant autolysin formulated in MPLA and Alum as co-adjuvant/adjuvant. r-Autolysin was expressed and purified by Ni-NTA affinity chromatography and characterized by SDS-PAGE. Then, the vaccine candidate formulation in MPLAs and Alum was prepared. To investigate the immunogenic responses, total IgG, isotype (IgG1 and IgG2a) levels, and cytokines (IL-4, IL-12, TNF-α, and IFN-γ) profiles were evaluated by ELISA. Also, the bacterial burden in internal organs, opsonophagocytosis, survival rate, and pathobiology changes was compared among the groups. Results demonstrated that mice immunized with the r-Autolysin + Alum + MPLA Synthetic and r-Autolysin + Alum + MPLA Biologic led to increased levels of opsonic antibodies, IgG1, IgG2a isotype as well as increased levels of cytokines profiles, as compared with other experimental groups. More importantly, mice immunized with MPLA and r-Autolysin exhibited a decrease in mortality and bacterial burden, as compared with the control group. The highest level of survival was seen in the r-Autolysin + Alum + MPLA Synthetic group. We concluded that both MPLA forms, synthetic and biological, are reliable candidates for immune response improvement against MRSA infection.

Methyl jasmonate conferred Arsenic tolerance in Thymus kotschyanus by DNA hypomethylation, stimulating terpenoid metabolism, and upregulating two cytochrome P450 monooxygenases.

Arsenic (As) is a highly cytotoxic element impairing normal cellular functions, and its bioremediation has become one of the environmental concerns. This study explored the molecular and physiological responses of thyme (Thymus kotschyanus) seedlings to incorporating As (0 and 10 mgl-1) and methyl jasmonate (MJ; 0 and 10 µM) into the culture medium. The MJ treatment reinforced root system and mitigated the As cytotoxicity risk. MJ contributed to hypomethylation, a potential adaptation mechanism for conferring the As tolerance. Two cytochrome P450 monooxygenases, including CYP71D178 and CYP71D180 genes, were upregulated in response to As and MJ. The MJ treatment contributed to up-regulation in the γ-terpinene synthase (TPS) gene, a marker gene in the terpenoid metabolism. The As presence reduced photosynthetic pigments (chlorophylls and carotenoids), while the MJ utilization alleviated the As toxicity. The MJ supplementation increased proline accumulation and soluble phenols. The application of MJ declined the toxicity sign of As on the concentration of proteins. The activities of peroxidase, catalase, and phenylalanine ammonia-lyase (PAL) enzymes displayed an upward trend in response to As and MJ treatments. Taken collective, MJ can confer the As tolerance by triggering DNA hypomethylation, regulating CYPs, and stimulating primary and secondary metabolism, especially terpenoid.

Diversity of Bacteroidaceae family in gut microbiota of patients with chronic kidney disease and end stage renal disease.

Background: Human intestine microbiota are known to be directly and indirectly altered during some diseases such as kidney complications. Bacteroides is considered as the main and the most abundant phylum among human gut microbiota, which has been classified as enterotype 1. This study aimed to assess the abundance of Bacteroides spp. in fecal flora of end-stage renal disease (ESRD) and chronic kidney disease (CKD) patients and compare it with the Bacteroides composition among fecal flora of healthy individual. Methods: Fresh fecal samples were collected from 20 CKD/ESRD patients and 20 healthy individual without any kidney complications. The pure microbial DNA was extracted by QIAamp Stool Mini Kit from stool samples. MiSeq system was used to analyze the intestinal composition by next generation sequencing method. Results: A number of 651 bacterial strains were isolated and identified from 40 fecal samples of both patients and healthy groups. Bioinformatics analysis defined 18 different types of Bacteroides species which included 2.76% of all strains. Statistical analysis showed no significant difference between study groups (P>0.05). In both healthy and patient groups three species including B. dorei, B. uniformis, and B. ovatus have allocated the most abundance to themselves. The lowest abundance was related to B. eggerthii, A. furcosa and B. barnesiae among CKD/ESRD patients and A. furcosa, B. barnesiae, and B. coprocola had the lowest abundance among healthy people. Conclusion: This study indicates despite all previous evidence of Bacteroides role in gut microbiota, it had no different distribution between healthy persons and CKD/ESRD patients.

Core-shell inorganic NP@MOF nanostructures for targeted drug delivery and multimodal imaging-guided combination tumor treatment.

It is well known that metal-organic framework (MOF) nanostructures have unique characteristics such as high porosity, large surface areas and adjustable functionalities, so they are ideal candidates for developing drug delivery systems (DDSs) as well as theranostic platforms in cancer treatment. Despite the large number of MOF nanostructures that have been discovered, conventional MOF-derived nanosystems only have a single biofunctional MOF source with poor colloidal stability. Accordingly, developing core-shell MOF nanostructures with good colloidal stability is a useful method for generating efficient drug delivery, multimodal imaging and synergistic therapeutic systems. The preparation of core-shell MOF nanostructures has been done with a variety of materials, but inorganic nanoparticles (NPs) are highly effective for drug delivery and imaging-guided tumor treatment. Herein, we aimed to overview the synthesis of core-shell inorganic NP@MOF nanostructures followed by the application of core-shell MOFs derived from magnetic, quantum dots (QDs), gold (Au), and gadolinium (Gd) NPs in drug delivery and imaging-guided tumor treatment. Afterward, we surveyed different factors affecting prolonged drug delivery and cancer therapy, cellular uptake, biocompatibility, biodegradability, and enhanced permeation and retention (EPR) effect of core-shell MOFs. Last but not least, we discussed the challenges and the prospects of the field. We envision this article may hold great promise in providing valuable insights regarding the application of hybrid nanostructures as promising and potential candidates for multimodal imaging-guided combination cancer therapy.

Alum and a TLR7 agonist combined with built-in TLR4 and 5 agonists synergistically enhance immune responses against HPV RG1 epitope.

To relieve the limitations of the human papillomavirus (HPV) vaccines based on L1 capsid protein, vaccine formulations based on RG1 epitope of HPV L2 using various built-in adjuvants are under study. Herein, we describe design and construction of a rejoined peptide (RP) harboring HPV16 RG1 epitope fused to TLR4/5 agonists and a tetanus toxoid epitope, which were linked by the (GGGS)3 linker in tandem. In silico analyses indicated the proper physicochemical, immunogenic and safety profile of the RP. Docking analyses on predicted 3D model suggested the effective interaction of TLR4/5 agonists within RP with their corresponding TLRs. Expressing the 1206 bp RP-coding DNA in E. coli produced a 46 kDa protein, and immunization of mice by natively-purified RP in different adjuvant formulations indicated the crucial role of the built-in adjuvants for induction of anti-RG1 responses that could be further enhanced by combination of TLR7 agonist/alum adjuvants. While the TLR4/5 agonists contributed in the elicitation of the Th2-polarized immune responses, combination with TLR7 agonist changed the polarization to the balanced Th1/Th2 immune responses. Indeed, RP + TLR7 agonist/alum adjuvants induced the strongest immune responses that could efficiently neutralize the HPV pseudoviruses, and thus might be a promising formulation for an inexpensive and cross-reactive HPV vaccine.

Glucomannan as a polysaccharide adjuvant improved immune responses against Staphylococcus aureus: Potency and efficacy studies.

Staphylococcus aureus is a gram-positive bacterium, representing one of the most important nosocomial pathogens. The treatment of infections, caused by S. aureus, has become increasingly intricate due to the emergence of highly resistant strains. Therefore, it is obvious that an effective prevention strategy against this bacterium could significantly decrease such infections. In the present study, the protective efficacy and immunological properties of recombinant autolysin, formulated in Montanide ISA266 and Alum adjuvants with Glucomannan as a polysaccharide, were assessed in the systemic mouse model of infection. Mice were immunized with the purified recombinant protein in various formulations in different groups and, subsequently, mice were challenged with 5 × 108 CFU of bacteria for the evaluation of their survival and bacterial clearances in the internal organs. ELISA was performed to determine the type of induced immunity, cytokine secretion (IFN-γ, IL-4, IL-2, and IL-17), and isotyping (IgG1 and IgG2a). In addition, we measured the opsonophagocytic activities of the antibodies. Results showed that immunization with r-autolysin + Alum + Glucomannan and r-autolysin + MontanideISA266+Glucomannan formulations significantly increased total IgG and isotypes (IgG1 and IgG2a), as compared with other vaccinated and control groups. Furthermore, the formulation of r-autolysin in Alum and MontanideISA266 adjuvants with Glucomannan enhanced IFN-γ, IL-4, and IL-17 cytokine secretion as well as protectivity, following experimental challenge. We concluded that Glucomannan has the potential to induce immune responses and would be used as an adjuvant factor in vaccine formulation.

Synthetic selenium nanoparticles as co-adjuvant improved immune responses against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-acquired infections worldwide, which is resistant to many antibiotics, resulting in significant mortality in societies. Vaccination is a well-known approach to preventing disease. Autolysin, a surface-associated protein in S. aureus with multiple functions, is a suitable candidate for vaccine development. As a co-adjuvant, selenium nanoparticles (SeNPs) can increase the immune system, presumably resulting in increased vaccine efficacy. The present study evaluated the immunogenicity and defense of recombinant autolysin formulated in SeNPs and Alum adjuvants against MRSA. r-Autolysin was expressed and purified by the Ni-NTA affinity chromatography. SeNPs were synthetically obtained from sodium dioxide, followed by an assessment of shape and size using SEM and DLS. Balb/c mice were injected subcutaneously with 20 mg of r-autolysin formulated in Alum and SeNps adjuvants three times with the proper control group in 2 weeks intervals. Cytokine profile and isotyping ELISA were conducted to determine the type of induced immunity. Opsonophagocytosis tests assessed the functional activity of the vaccine, and the bacterial burden from the infected tissues was determined. Results showed that mice receiving SeNps and r-Autolysin had higher levels of total IgG and isotypes (IgG1 and IgG2a) and increased cytokine levels (IFN-γ, TNF-α, IL-12, and IL-4) as compared with those only receiving autolysin and PBS as a control. More importantly, mice immunized with SeNps and r-Autolysin exhibited a decrease in mortality and bacterial burden compared to the control group. We concluded that SeNps could stimulate immune responses and can be used as an adjuvant element in vaccine formulation.

Montanide ISA-720 and Naloxone in HBsAg Vaccine Formulation: Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses.

Objective: This study aimed to investigate the effects of Montanide ISA-720 and Naloxone (NLX) in Hepatitis B surface antigen (HBsAg) vaccine formulation on cytokine and long-lasting antibody responses. Methods: First, the HBsAg was formulated in Montanide ISA-720 adjuvant and Naloxone at 5 and 10 mg/kg. The experimental mice were immunized three times at a 2-week interval, and then IL-4, IL-2, TNF-α, and IFN-γ cytokines; long-lasting IgG antibody responses 220 days after the last shot; and IgG1/IgG2a isotypes were assessed by ELISA. Results: The HBsAg-Alum group exhibited the highest IL-4 cytokine response among the experimental groups, whereas NLX in HBsAg-MON720 vaccine formulation did not affect cytokine responses. In addition, NLX in Alum-based vaccine suppressed IL-4 cytokine response and increased the IL-2/IL-4 cytokine ratio. Moreover, HBsAg-MON720 was more potent than HBsAg-Alum in the induction of antibody responses, and NLX in Alum- and MON720-based vaccines induced long-lasting antibody responses. Conclusion: NLX in Alum-based vaccine decreased IL-4 cytokine response, increased IL-2/IL-4 cytokine ratio, and improved long-lasting humoral immune responses in both vaccine formulations. Therefore, the adjuvant activity of NLX in the vaccine formulation depends on the type of adjuvant and the nature of the antigen in the vaccine formulation.

Clostridium species diversity in gut microbiota of patients with renal failure.

The Pathology of digestive tract has long been known to be correlated with chronic kidney disease (CKD). The member of the major Firmicutes phylum especially Clostridium subcluster XIVa altered quantitatively and qualitatively in the gut microbiota of patients with End Stage Renal Disease (ESRD) and CKD. Therefore, in this study, the abundance of the species of Clostridium genus of Firmicutes phylum compared between intestine microbiota of patients with kidney failure and healthy individual. Fresh fecal specimens of 20 patients at different stages of CKD and 20 healthy individuals were collected. Bacterial DNA of samples were extracted to use for 16S ribosomal DNA sequencing targeting the V3-V4 region. Next generation sequencing (NGS) method at MiSeq system was used to find the diversity of gut microbiota composition. Totally, 11 (1.68%) of 651 bacterial strains which were isolated from forty fecal samples of both healthy volunteers and CKD/ESRD patients, were identified as Clostridium species. Eight genera of 11 Clostridium genera were related to Clostridium sensu stricto, and 3 other genera were as follows Vallitalea, Acidaminobacter and Caloramator. Among both group, the highest abundance was dedicated to Clostridium celatum genera. Sarcina maxima were not identified. The composition of Clostridium spp. showed the same frequency among CKD/ESRD and healthy groups (p < 0.05). The abundance of Clostridium spp. is virtually the same and not differs among healthy individuals and CKD/ESRD patients. Results of the present indicate despite of critical role of gut microbiota, some pathogens and their metabolites have no role on hemostasis and pathogenesis of kidney disorders.

Recombinant PBP2a/autolysin conjugate as PLGA-based nanovaccine induced humoral responses with opsonophagocytosis activity, and protection versus methicillin-resistant Staphylococcus aureus infection.

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) reasons extreme infections, can resist various conventional antimicrobial agents, and cause morbidity and mortality worldwide. Vaccination seems to help modulate MRSA infections. Nanovaccine is considered a novel strategy in vaccine technology. The primary purpose of the present study was to develop a conjugate vaccine based on recombinant PBP2a and MRSA autolysin formulated in PLGA as a nanoparticle capable of enhancing protective responses against MRSA in the murine model. Materials and Methods: Recombinant PBP2a and autolysin have been expressed and purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column and characterized by SDS-PAGE and western blot. PLGA was bound to recombinant proteins by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and adipic acid dihydrazide (ADH) as a linker and spacer, respectively. Conjugation of recombinant proteins to PLGA was confirmed by the AFM assay, zeta potential, and size distribution, and its efficacy was evaluated in mice. Total IgG, IgG1, IgG2a, IgG2b, and IgM titers were analyzed to assess immune responses. Lastly, the bioactivity of antibodies was tested by using the opsonophagocytosis assay. Results: Mice immunized with the r-PBP2a-r-autolysin-PLGA nanovaccine led to increased levels of opsonic antibodies and IgG1, IgG2a, IgG2b, and IgM when compared with other experimental groups. Our results confirmed that vaccination with nanovaccine could reduce the mortality rate against the sub-lethal dose of MRSA challenge. Furthermore, the nanovaccine could eliminate MRSA from the kidney of infected mice. Conclusion: This study may provide valuable insights into the protective power of the r-PBP2a-r-autolysin-PLGA conjugate vaccine against MRSA infection.

Investigation of Curcumin-Loaded OA400 Nanoparticle's Effect on the Expression of E6 and E7 Human Papilloma-Virus Oncogenes and P53 and Rb Factors in HeLa Cell Line.

Background: Curcumin, a compound derived from the root of the Curcuma longa, has been confirmed as an anticancer, chemoprotective, and gene/protein regulatory agent. Nanoformulation of curcumin has been developed to increase its targeting efficiency, solubility, controlled release, and physical and chemical stability. Objectives: This study investigated the effect of new nano-type curcumin, oleic acid-derived dendrosome (OA400 nanoparticles), on the expression of E6 and E7 human papillomavirus oncogenes and P53 and Rb factors in the HeLa cell line. After preparing nano-curcumin by mixing OA400 nano-carrier and curcumin, its effect was considered on the human cervical cancer cell line (HeLa cell line RRID: CVCL_003) and normal fibroblast cells. Methods: MTT assay and flow cytometry were used to evaluate cell viability and apoptosis. Furthermore, real-time RT-PCR and western blot analyses assessed RNA and protein expression of E6, E7, P53, and Rb. Statistical analyses were performed by GraphPad Prism 7 software. Results: The nanoformulation of curcumin could reduce the expression of E6 and E7 oncogenes and increase P53 and Rb tumor suppressors in HeLa cancerous cells at 15 µM concentration; however, it had no significant effect on the viability of normal fibroblast cells. On the other hand, curcumin altered the expression of these genes at a 50-µM concentration. Gene and protein expression analysis indicated the up-regulation of P53 and Rb factors and the down-regulation of E6 and E7 under the influence of nano-curcumin treatment more than curcumin. Conclusions: These data indicate the potential of curcumin-loaded OA400 nanoparticles to be considered as a treatment option in cervical cancer investigations.

Exploring the interaction of quercetin-3-O-sophoroside with SARS-CoV-2 main proteins by theoretical studies: A probable prelude to control some variants of coronavirus including Delta.

The aim of this study was to investigate the mechanism of interaction between quercetin-3-O-sophoroside and different SARS-CoV-2's proteins which can bring some useful details about the control of different variants of coronavirus including the recent case, Delta. The chemical structure of the quercetin-3-O-sophoroside was first optimized. Docking studies were performed by CoV disease-2019 (COVID-19) Docking Server. Afterwards, the molecular dynamic study was done using High Throughput Molecular Dynamics (HTMD) tool. The results showed a remarkable stability of the quercetin-3-O-sophoroside based on the calculated parameters. Docking outcomes revealed that the highest affinity of quercetin-3-O-sophoroside was related to the RdRp with RNA. Molecular dynamic studies showed that the target E protein tends to be destabilized in the presence of quercetin-3-O-sophoroside. Based on these results, quercetin-3-O-sophoroside can show promising inhibitory effects on the binding site of the different receptors and may be considered as effective inhibitor of the entry and proliferation of the SARS-CoV-2 and its different variants. Finally, it should be noted, although this paper does not directly deal with the exploring the interaction of main proteins of SARS-CoV-2 Delta variant with quercetin-3-O-sophoroside, at the time of writing, no direct theoretical investigation was reported on the interaction of ligands with the main proteins of Delta variant. Therefore, the present data may provide useful information for designing some theoretical studies in the future for studying the control of SARS-CoV-2 variants due to possible structural similarity between proteins of different variants.

Improvement of hepatitis B vaccine to induce IFN-γ cytokine response: A new formulation.

Hepatitis B virus (HBV) infection is limited through vaccination against HBsAg formulated in the Alum adjuvant. However, this alum-formulated vaccine fails to be preventive in some cases, also known as non-responders. Recent studies have shown the immunomodulatory effect of α-tocopherol in various models. Here, we developed a new formulation for HBsAg using α-tocopherol, followed by assessment of immune responses. Experimental BALB/c mice were immunized with a commercial alum-based vaccine or the one formulated in α-tocopherol at different doses. Mice were immunized subcutaneously with 5 µg of HBsAg with different formulations three times with 2-week intervals. Specific total IgG, IgG1, and IgG2a isotypes of antibodies were measured by ELISA. Immunologic cytokines, such as IFN-γ, IL-4, IL-2, and TNF-α, were also evaluated through commercial ELISA kits. Our results showed that the new α-tocopherol-formulated vaccine had the ability to reinforce specific total IgG responses. Moreover, α-tocopherol in the HBsAg vaccine increased IFN-γ, IL-2, and TNF-α cytokines at higher concentrations; however, the vaccine suppressed IL-4 cytokine release. At a lower concentration of α-tocopherol, the IL-4 cytokine response increased without a positive effect on IFN-γ and TNF-α cytokine response. It seems that α-tocopherol can change the immune responses against HBsAg; however, the type of response depends on the dose of α-tocopherol used in the vaccine formulation.

Clonal Relationship and Resistance Profiles Among ESBL-Producing Escherichia coli.

AmpC ß-lactamases hydrolyze all ß-lactams except cefepime and carbapenems. The study of AmpC-producing E. coli has high priority for the infection control committee. This research is aimed to investigate the resistant urinary AmpC-generating E. coli isolates and identify their genetic variety. Some 230 E. coli isolates from patients suffering urinary tract infection symptoms were studied in 2017-2018 to assess their susceptibility toward antimicrobial agents. AmpC gene was evaluated by PCR and molecular typing using the 10-loci MLVA method. MLVA images were examined by BioNumerics 6.6 software through the use of the UPGMA algorithms. Thirty-eight AmpC-generating E. coli isolates were detected. The most abundant determinant was blaCIT and blaEBC , blaFOX , and blaDHA had the next ranks, respectively. Six major clusters and a singleton were identified by MLVA. AmpC beta-lactamases in urinary isolates of E. coli in the hospital under study and high rate of additional resistance to gentamicin, cotrimoxazole and ciprofloxacin. The most frequent gene determinant of AmpC beta-lactamase was blaCIT and vary depending on time and geographical location.

Subtyping ß-lactamase-producing Escherichia coli strains isolated from patients with UTI by MLVA and PFGE methods.

OBJECTIVES: Strain subtyping is an important epidemiological tool to trace contamination, determine clonal relationships between different strains, and the cause of outbreaks. Current subtyping methods, however, yield less than optimal subtype discrimination. Pulsed-field gel electrophoresis is the gold standard method for Escherichia coli and Multiple-Locus Variable-number tandem repeat Analysis is a rapid PCR-based method. The purpose of this study was to evaluate MLVA and PFGE methods for subtyping ß -lactamase-producing E. coli strains isolated from urinary tract infections. MATERIALS AND METHODS: Overall, 230 E. coli isolates from patients with urinary tract infections were examined for antimicrobial susceptibility testing. 10-loci and 7-loci MLVA and PFGE methods were used for molecular typing of ß -lactamase-producing E. coli isolates. RESULTS: Out of 230 isolates, 130 (56.5%) ß -lactamase-producing E. coli isolates were found in this study. The diversity indices of the VNTR loci showed an average diversity of 0.48 and 0.54 for 7-loci and 10-loci MLVA, respectively. The discriminatory power of PFGE showed a value of 0.87. The discordance between the methods was high. CONCLUSION: Our study showed that PFGE is more discriminatory than MVLA. MLVA is a PCR- based method and can generate unmistakable data, in contrast to PFGE. Optimization of polymorphic VNTR is essential to improve the discriminatory power of MLVA based on geographical region.

Molecular cloning and immunogenicity evaluation of IsdE protein of methicillin resistant Staphylococcus aureus as vaccine candidates.

Methicillin resistant Staphylococcus aureus is one of the most common causes of nosocomial infections. Current therapeutic approaches are not always effective in treatment of nosocomial infections, thus, there is a global demand for the development of novel therapeutic strategies. Staphylococcus aureus possesses various systems to uptake iron. One of the most important of them is iron regulated surface determinant (Isd) which can be an excellent candidate for immunization. Here, following the preparation of recombinant IsdE protein, 20 µg of r-IsdE prepared in various formulations were subcutaneously injected in different groups of mice. Two booster vaccinations were administered in two-week intervals, then, blood samples were collected two weeks after each injection. ELISA was used for the evaluation of total IgG and its isotypes (IgG1 and IgG2a) as well as quantity of IFN-γ, IL-4, IL-17, IL-2 and TNF-α cytokines on the serum samples. Meanwhile, the immunized mice were intraperitoneally inoculated with 5 × 108 CFU of bacteria then, their mortality rate and bacterial load were assessed. Our results showed that immunization with the r-IsdE in various formulations raised total IgG and isotypes (IgG1 and IgG2a) compared with the control groups. Moreover, r-IsdE formulation with MF59 and Freund adjuvants raised production of IFN-γ, IL-4, IL-17, IL-2 and TNF-α cytokines and provided an acceptable protection against Staphylococcus aureus infections. Results of present study suggest that r-IsdE which can easily be expressed by Escherichia coli BL21 system shows a great potential to develop a protective immunity against infections caused by Methicillin resistant Staphylococcus aureus.