A Case-control Study of Urinary Tract Infection, 25‑hydroxyvitamin D Status and Associated Inflammatory and Regulatory Responses

Background: Vitamin D plays a key role in the modulation of numerous immune functions against infectious agents. We aimed to explore the association between serum 25‑hydroxyvitamin D (25[OH] D) levels and cytokine responses, along with hematological changes, in patients with urinary tract infection (UTI). Materials and Methods: Vitamin D level, cytokines (interferon [IFN]−γ, interleukin [IL]−4, IL−6, IL–10, IL−17A, tumor necrosis factor [TNF]−α, and transforming growth factor [TGF]−β), hematological indices (neutrophil-to-lymphocyte ratio [NLR], monocyte-to-lymphocyte ratio [MLR], neutrophil-to-monocyte ratio [NMR], platelet-to-lymphocyte ratio [PLR], and mean platelet volume [MPV]), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) were evaluated in a case-control human study included 65 patients and 45 controls. Results: Among the enhanced cytokine levels in patients, the cytokines IFN-γ, IL-17A, and IL-10 had a significant association with 25(OH)D, but not IL-6, TNF-α, and TGF-β. The IL-4 levels remained unchanged. By comparing hematological indices, we found the association of increased NLR and MLR with 25(OH)D and the cytokines IFN-γ and IL-17A, along with a decrease in the PLR without showing such an association. The NMR did not show any significant difference. The platelet count showed an association with IL-6, IL-17A, and TGF-β, but the association of MPV with 25(OH)D was significant. The ESR results exhibited statistically non-significant differences. CRP elevation was directly associated with IL-6 and IL-17A, but not with 25(OH)D. Conclusion: 25(OH)D-mediated inflammatory cytokine milieu might alter the proportion and function of peripheral blood cells in a regulated manner to support bacterial clearance which needs further studies to be validated.

Evaluating Cytotoxic Potential of the Fruit and the Leaf Extracts of Sambucus ebulus (L.) on MCF7 and AGs Cell Lines

Background: Breast and stomach cancers are the most common malignancies in Iranian females and males, respectively. Enriching with phytochemicals that have antioxidant and cytotoxic activities, extracts from dwarf elder (Sambucus ebulus L.) holds promises to be used for alternative medication. Materials and Methods: We investigated the cytotoxic and antiproliferative activities of the leaf and the fruit ethyl-acetate (EA), as well as the methanolic (MeOH) extracts of dwarf elder upon treatment of the MCF-7 and AGS cells. Twenty-seven concentration series ranging from 10 to 2000 μg/mL were administered to the cells, and their growth inhibitory potential was assessed using MTT assay. The potential anticancer compounds of the extracts were quantified applying an improved highperformance liquid chromatography (HPLC). Results: All extracts showed positive dose-dependent cytotoxic activities on both cell lines. The EA extracts demonstrated more cytotoxicity compared to those of the MeOH ones (P<0.0001). The leaf EA extract showed IC50 values of 65 and 50 μg/mL, while those of the fruit were estimated as 58 and 50 μg/mL on the MCF-7 and AGS cells, respectively. The AGS cell line showed more susceptibility to all extracts tested compared to the MCF-7. MeOH extracts caused only a maximum of ~20% reduction in cell viability at 2000 μg/mL concentration. According to the HPLC analysis, leaf extracts contained phenolic compounds, including p- coumaric acid (0.10 mg per g powder), rutin (0.07 mg per g powder), and quercetin (0.02 mg per g powder). Conclusion: EA extract of the fruit shows the highest cytotoxicity: reducing 35.3% viability of the AGS cells with 10 μg/mL concentration. It can be considered a potential chemopreventive agent for cancer therapies. However, MeOH extracts with far low cytotoxicity or non-cytotoxic at some concentrations would be an appropriate candidate for preventing tumor growth without affecting neighboring normal cells.

Cloning, Expression, and Purification of Recombinant Mouse Interferon-γ

Background: Interferon-gamma [IFN-γ) is the most important cytokine in the immune system. This protein has been expressed in bacterial cells. However, bacterial cloning is not an easy task. We aimed to clone, express, and purify recombinant mouse IFN-γ and overcome problems in favor of commercial purposes. Materials and Methods: To amplify the gene product for cloning, we primarily designed two specific primers for the target gene. Following PCR amplification, the amplicon was inserted into the pET-21b[+) vector. The E. coli BL21 [DE3) CodonPlus strain was chosen for the expression of the target gene. Finally, the expressed recombinant mouse IFN-γ was assessed through the western blotting method. Results: We performed a cloning process and produced recombinant mouse IFN-γ in an optimal condition. We also noticed that monomeric protein could be transformed to a homodimeric structure which can be observed using the SDS PAGE [SDS-polyacrylamide gel electrophoresis) and western blotting. Conclusion: Experimental conditions strongly affect the large-scale cloning procedures required to be optimized in each laboratory. The expressed recombinant mouse IFN-γ described here is appropriate for commercial purposes.

Effect of Rhamnolipids on Pathogenicity Characteristics of Microorganisms in Organic Compost

Background: Rhamnolipids are hydrophilic glycolipids, often classified as biosurfactants. They are produced by different bacterial species. Rhamnolipids are extensively studied in biological research because of their interesting features like antimicrobial, antifungal, and antiviral activities. Materials and Methods: To study the antimicrobial effect of rhamnolipid, we conducted a crosssectional study on the eight different pathogenic bacterial strains from November 2019 to June 2020. These bacterial strains were isolated from the organic compost. Both disk diffusion and broth microdilution methods were used to evaluate the inhibitory effects of rhamnolipids on these pathogenic bacteria. Also, protease and amylase enzyme activities were evaluated in these eight bacterial isolates. Results: For Bacillus stearothermophilus, within the area of 31.5 mm, no growth was observed, hence proving the inhibitory effect of rhamnolipid. After calculating the minimum inhibitory (MIC) and minimum lethal concentrations (MLC) for each bacterial strain, it has been found that the studied bacteria were more susceptible to rhamnolipids than most of the antibiotics. Strains were also quantified for their enzymatic activity of proteases and amylases. The bacterial strains of Bacillus stearothermophilus, Brevibacillus brevis, Bacillus licheniformis, and Bordetella petrii showed maximum protease activity. Whereas Escherichia coli, Enterococcus faecalis, Enterobacter aerogenes, and Pseudomonas aeruginosa showed high amylase activity. Conclusion: Rhamnolipids can be used as a potential antimicrobial agent for treatment of infections.

Spata19 Inactivation as a Cause of Oligospermia

Background: Spermatogenesis associated 19 (Spata19) was introduced as a testis-specific gene that was probably involved in spermatogenesis cell apoptosis. Therefore, this study aimed to investigate the effect of Spata19 inactivation on sperm count. Materials and Methods: We generated global Spata19 knockout mice by CRISPR/Cas9 nickase technology. Disability was validated in three levels of DNA, RNA, and protein using PCR, RT-PCR, and immunohistochemistry. Histological studies were performed for testis. Sperm characteristics were also assessed with CASA software. Results: Spata19 knockout mice had a 43 nucleotides deletion in exon 4 of this gene. The presence and absence of Spata19 were confirmed in normal and knockout mice, respectively. The presence of Spata19 in normal NMRI mice was detected in the brain, heart, and thymus by semi-nested RT-PCR and in Leydig cells by immunohistochemistry. Histological studies revealed a decrease in sperm count in knockout mice. Also, CASA parameters were significantly reduced (P<0.05). Conclusion: These data indicate that Spata19 inactivation is a cause of oligospermia, and its role could be beyond an adhesive molecule.

Susceptible Single Nucleotide Polymorphisms in Exon 10 and Intron 9 of Complement Factor H Gene in Patients With Age-related Macular Degeneration

Background: Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly. Although it has been shown that Y402H polymorphism in the CFH gene was strongly associated with AMD in the Iranian population, there were no data on other single nucleotide polymorphisms (SNPs), which have the most significant association with AMD. This study aimed to investigate hot point regions in exon 10 and intron 9. Materials and Methods: One hundred and sixty-six AMD patients and 69 controls were recruited. Their blood was collected in the tubes containing EDTA. Then, DNA was extracted from the blood, and its quality was evaluated. Primers were designed for intron 9 and exon10 sequencing. A viral polymorphisms analysis software named CEQ was used for the analysis of putative polymorphisms. Results: We noticed three polymorphisms in study cases: rs7535263 and C66379A in intron 9 and rs2274700 in exon 10. Based on the McNamara’s test (rs7535263 and rs2274700) and the Phi and Cramer’s test (C66379A), a significant difference was found between the control and patient groups regarding rs7535263 and rs2274700 polymorphisms. Conclusion: We found a synonymous or silent mutation, A473A, rs2274700 in exon 10 in 85% of patients. From two intronic SNPs, just rs7535263 showed association with the disease in studied patients living in Gilan Province, Iran. Although no significant relationship was found between controls and patients regarding the C66379A allele, it would be important that no other sources have reported C66379A polymorphism in AMD yet.

Comparing the Effect of Grape Fermentative Product and Fresh Red Grape Juice on Antioxidant Biomarkers of Liver Mitochondria Isolated From Rats in Vitro

Background: Mitochondria are a source of reactive oxygen species (ROS), and several natural compounds are used as antioxidant agents. This study aimed to investigate and compare the effects of fresh grape juice red wine on oxidative stress biomarkers in rat liver mitochondria. Materials and Methods: In this regard, mitochondria were isolated from the liver of 27 male Wistar rats (220-250 g). The isolated mitochondria were cultured in different doses of red wine and fresh red grape juice for 24, 48, and 72 h. After treatment, total antioxidant capacity, lipid peroxidation, total thiol groups, and catalase activity were determined in the isolated mitochondria of the rat liver. Results: The results confirmed the oxidant/antioxidant effects of red wine and fresh red grape juice at different times. Conclusion: According to the results, the fresh red grape juice showed higher antioxidant properties than red wine in the liver mitochondrial samples.

Supplementation of Freezing Media with Cyclic Adenosine Monophosphate Analog and Isobutylmethylxanthine on Sperm Quality

Background: This study aimed to explore whether the addition of a cyclic adenosine monophosphate (cAMP) analog and isobutylmethylxanthine (IBMX) in freezing media improved sperm quality and what role cAMP has in this recovery. Materials and methods: ach semen sample was cryopreserved into four groups: fresh semen sample, as a control group, freezing medium + 2.5 mM cAMP analog and 0.2 mM IBMX, freezing medium + 12.5 mM cAMP analog and 0.2 mM IBMX, and freezing medium + 25 mM cAMP analog and 0.2 mM IBMX. Sperm parameters after post-thaw were analyzed according to WHO instruction (2010). Viability, acrosome reaction, and DNA damage levels of the samples were evaluated. Results: Our results indicated that the effective concentrations of 12.5 and 25 mM cAMP analog and 0.2 mM IBMX significantly improved the total motility, progressive motility, and viability of the frozen-thawed (P<0.05). However, non-progressive motility and immotile were significantly reduced in the 12.5 and 25 mM cAMP analogs and 0.2 mM IBMX groups after thawing (P<0.05). During freezing the spermatozoa, the high concentration of the cAMP analog increased acrosome reaction after thawing in the 25 mM and 0.2 mM IBMX treated samples (P<0.05). DNA fragmentation in 25 mM cAMP analog and 0.2 mM (IBMX) supplementation was significantly lower compared to the other groups (P<0.05). Conclusions: Our findings revealed that in vitro cAMP analog and IBMX supplementation in freezing media play an important role in preventing cryodamage by maintaining the sperm functional parameters.

Isolation and Characterization of Lactic Acid Bacteria From Milk and Their Effects on the Pathogenic Bacteria

Background: Probiotics are “live microbial cells” that are beneficial for human and animal health. Lactobacilli are such a diverse group of bacteria with similar metabolic and physiological characteristics, and constitute important and beneficial gut microflora. During carbohydrate fermentation, lactobacilli produce lactic acid as an end product in metabolism. Hence, lactobacilli have high significance to be used as probiotics in the food industry, because of their acidifying properties. Also, lactobacilli are considered “safe”, owing to their ubiquitous presence in the food. Many researchers provided evidence for the presence of lactobacilli in milk sources. Thus, the present study aimed to isolate and characterize different lactobacilli strains from milk sources and analyze their “probiotic potential”. Materials and methods: Forty-one lactobacilli isolates were obtained from raw cow milk. Then, the strains were characterized by morphological identification and biochemical tests. Besides, probiotic potentials were evaluated with the bile tolerance test, antibiotic susceptibility test, and determining suitable pH for the optimal growth of lactobacilli. The lactobacilli isolates were also analyzed for their probiotic characteristics and the release of antimicrobial substances. Their antimicrobial activities against pathogenic strains were assessed by determining the minimum inhibitory concentration, with the help of agar diffusion methods. Results: From 50 milk samples, 41 lactobacilli isolates were obtained, out of which five lactobacilli strains were identified as Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus acidophilus, and Lactobacillus lactis. Moreover, 35 isolates showed an inhibitory effect. These strains were able to survive and grow in 0.5% to 2.5% bile salt concentrations. Lactic acid bacteria were susceptible to antibiotics, and 35 isolates obtained from raw milk showed an inhibitory effect against pathogenic bacteria. The observed minimum inhibitory concentration ranged from 50 to 100 µL and varied between the different pathogens. Conclusion: Out of 41 Lactobacillus isolates obtained from cow milk samples, 35 were identified with probiotic characteristics. Hence, this study highlighted the novel probiotic bacteria and validated the antimicrobial properties of the Lactobacillus spp against pathogenic bacteria.

Transcription Factor Assay of Peripheral Blood T cells in Different Groups of Rheumatoid Arthritis Patients

Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and other tissues and organs of the body. Previous reports have demonstrated the imbalance of T helper (Th) subsets and Treg activity in the development, progression, and remission of RA. Here, we investigated the mRNA expression of four major transcription factors T-bet (Th1), GATA (Th2), RORc (Th17), and Foxp3 (Treg) in peripheral blood of different groups of RA patients. Materials and methods: In this case-control study, 60 patients with RA, including 20 newly diagnosed, 20 under treatment, and 20 in remission, as well as 20 patients with osteoarthritis, and 20 age- and the sex-matched healthy individual were enrolled. Diagnosis and classification of patients were done according to the American College of Rheumatology criteria. The relative mRNA expression of transcription factors, including T-bet, GATA, RORc, and Foxp3, was measured using qRT-PCR. Results: The relative expression of T-bet in RA patients was significantly increased in healthy controls (P = 0.002), while the relative expression of Foxp3 in RA patients was significantly decreased in healthy controls (P < 0.0001). There was no significant difference in the expression of GATA3 or RORc among RA patients, healthy controls, and osteoarthritis group. Conclusions: The results indicate the importance of Th1 and Treg cells in RA; however, the role of Th17 cells appear to be of little importance in these patients. It seems that Th2 cells do not interfere with RA development.