Exploring the potential of structural modeling and molecular docking for efficient siRNA screening: A promising approach to Combat viral mutants, with a focus on HIV-1.

RNA interference (RNAi) holds immense potential for sequence-specific downregulation of disease-related genes. Small interfering RNA (siRNA) therapy has made remarkable strides, with FDA approval for treating specific human diseases, showcasing its promising future in disease treatment. Designing highly efficient siRNAs is a critical step in this process. Previous studies have introduced various algorithms and parameters for siRNA design and scoring. However, these attempts have often fallen short of meeting all essential criteria or required modifications, resulting in variable and unclear effectiveness of screened siRNAs, particularly against viral mutants with non-conserved short sequences. In this study, we present a fully optimized siRNA screening system considering all necessary parameters. Notably, we highlight the critical role of molecular docking simulations between siRNA and two functional domains of the Argonaute protein (PAZ and PIWI) in identifying the most efficient siRNAs, since the appropriate interaction between the guide siRNA strand and the RISC complex is crucial. Through our stringent method, we designed approximately 50 potential siRNAs targeting the HIV-1 vpr gene. Evaluation through XTT, qRT-PCR, and flow cytometry analysis on RAW 264.7 macrophage stable cells revealed negligible cytotoxicity and exceptional gene-silencing efficiency at both the transcriptional and translational levels for the top-ranked screened siRNAs. Given the growing interest in siRNA-based therapeutics, we anticipate that the insights from this study will contribute to improving treatment strategies against mutant viruses, particularly HIV-1.

Gene expression and demographic analyses in women with the poor ovarian response: a computational approach.

PURPOSE: Poor response to ovarian stimulation (POR) typically is reflected as decreased follicular response and low estradiol (E2) levels following ovarian stimulation by FSH/HMG. Many genes are involved in oocyte maturation, and demographic features and lifestyle can affect the oocyte maturity and developmental competence. The present study was conducted to investigate the magnitude of gene expression and lifestyle habits in POR women as compared to healthy women, using different statistical and computational methods. METHODS: Fifty women in the two groups were studied. The study groups included POR women (n = 25) with 1-9 released oocytes, and the control group (normal women, n = 25) with 9-15 released oocytes. Quantitative PCR was used to estimate the expression of FIGLA, ZAR1, WNT4, LHX8, APC, H1FOO, MOS, and DMC1 genes in granulosa cells. RESULTS: The results showed no significant difference in the magnitude of the studied genes' expression and linear discriminant analysis did not differentiate the studied groups based on all the genes together. Redundancy analysis (RDA) and latent factor mixed model (LFMM) results produce no significant association between the genes' expression magnitude and the geographical variables of the patients' local habitat. Linear discriminant analysis (LDA) of the demographic features differentiated the two groups of women. CONCLUSION: Our results indicate that demographic features may have an effect on sample gene expression levels.

Induced genetic and chemical changes in medicinally important plant Catharanthus roseus (L.) G. Don: cold plasma and phytohormones.

BACKGROUND: Catharanthus roseus (L.) G. Donis a medicinal plant species belonging to the Apocynaceae family, which produces vinblastine and vincristine along with 100 other monoterpenoid indole alkaloids. The process of biosynthesis of C. roseus alkaloids is complex, in which many genes, enzymes, and regulators are involved. Induced mutations may be considered as a potential source for producing a higher amount of vinblastine and vincristine in this plant species. Therefore, the objective of the present study was to examine the effects of different treatments utilized on the induced genetic changes in C. roseus plants and enzyme activities. METHODS AND RESULTS: Spermine, jasmonic acid, methyjasmonate, putrescine, and cold plasma treatments were used for seed treatments. Different molecular markers, namely inter simple sequence repeat, inter retrotransposon amplified polymorphism, and retrotransposon microsatellite amplified polymorphism were employed to reveal the induced genetic changes. Antioxidant enzyme activities were also studied. The treated plants showed genetic variability and a significant increase in antioxidant enzyme activity compared to the control plants. The putrescine treatment resulted in the highest level of activity in superoxidase. A significant positive correlation occurred between the molecular markers data and antioxidant enzyme activities in treated plants. CONCLUSION: Our data revealed that the different phytohormones and cold plasma treatments could induce both genetic and chemical content changes in C. roseus plants.

Evaluation of the Function of a Rare Variant in the 3'-Untranslated Region of the ß-Globin Gene.

ß-Thalassemia (ß-thal) is an inherited genetic disease that occurs because of the absence or reduction of ß-globin chain synthesis. Genetic changes occur in different regions of the ß-globin gene, but these mutations are less reported in the 3' untranslated region (3'-UTR). The objective of the present investigation was to evaluate the functional effect of a rare variant in the 3'-UTR of the ß-globin gene. A variant at the first nucleotide of the 3'-UTR of the ß-globin gene (HBB: c.*1G > A) was identified by DNA sequencing in an individual with low hematological indices and a normal hemoglobin (Hb) electrophoresis pattern. To evaluate the functional effect of this variant, the normal and mutated 3'-UTR of the ß-globin gene was synthesized separately and sub cloned in the psiCHEK2 vector. Next, using the calcium phosphate method, the psiCHEK2 vectors containing normal and mutated 3'-UTR were transfected separately into the HEK293T cell line. Finally, the transfected cell line was analyzed by dual luciferase assay. The ratio of Renilla to firefly for the mutant sample was 1.26 ± 0.06, while for normal samples it was 1.12 ± 0.04. The results of the luciferase assay showed that there was no significant difference in the functional effect between the mutant and wild type construct. Therefore, it was concluded that this variant might not reduce the expression of the ß-globin gene. Future studies by globin chain synthesis or to evaluate the expression of the gene in erythroid cells, might be necessary to understand the regulatory function of this mutation.

Inhibitory effects of cold atmospheric plasma on the growth, virulence factors and HSP90 gene expression in Candida albicans.

In spite of the abundance of antifungal therapies, 75% of women in the world suffer from the second most common cause of vaginal infection named vulvovaginal candidiasis. This complication is characterized with overgrowth of Candida albicans. The low efficacy and side effects of current antifungal therapies have convinced the researchers to look for a non-antibiotic based treatment such as cold atmospheric plasmas (CAP). The aim of this research was to evaluate the effects of CAP on C. albicans growth, ergosterol and biofilm formation. In addition, antibiotic resistance, phospholipase and proteinase activity, and structural properties were examined with different exposure duration. Putative critical effect of CAP on the expression of HSP90 as a target of anti-fungal therapy was investigated. ROS production in C. albicans exposed to CAP was assessed. For this purpose, C. albicans subjected to 0, 90, 120, 150, 180 and 210 s of He/O2 (2%), and non-treated cells as control were examined in terms of the mentioned virulence factors. The results showed that CAP had a significant effect on inhibition of C. albicans growth, Inhibition of biofilm formation, ergosterol content, and fluconazole and amphotericin B antibiotic sensitivity were significant in 210 s treatment group. This effect was validated based on changes of the cell architecture and morphology given the microscopy imaging results. The expression of HSP90 in both C. albicans ATCC 10231 and C. albicans PFCC 9362 was inhibited in 210 s of exposition. CAP exposition induced intracellular ROS, which may cause membrane damage and cell death in C. albicans. Taken together, the potential of CAP for therapeutic purposes in C. albicans-induced fungal infections is supported.

Cold atmospheric plasma induced genotoxicity and cytotoxicity in esophageal cancer cells.

In this paper, we studied the functional effects of cold atmospheric plasma (CAP) on the esophageal cancer cell line (KYSE-30) by direct and indirect treatment and fibroblast cell lines as normal cells. KYSE-30 cells were treated with CAP at different time points of 60, 90, 120 and, 240 s for direct exposure and 90, 180, 240 and, 360 s for indirect exposure. Cell viability was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis induction in the treated cells was measured by Annexin-V/PI using flow cytometry. The expression of apoptotic related genes (BAX/BCL-2) was analyzed by real-time polymerase chain reaction. Moreover, the genotoxicity was analyzed by comet assay. Cell viability results showed that direct CAP treatment has a markedly cytotoxic impact on the reduction of KYSE-30 cells at 60 s (p = 0.000), while indirect exposure was less impactful (p > 0.05). The results of the Annexin-V/PI staining confirmed this analysis. Subsequently, the genotoxicity study of the direct CAP treatment demonstrated a longer tail-DNA length and caused increase in DNA damage in the cells (p < 0.00001) as well as shift BAX/BCL-2 toward apoptosis. The concentration of H2O2 and NO2- in direct CAP treatment was significantly higher than indirect (p > 0.05). Treatment with direct CAP showed genotoxicity in cancer cells. Collectively, our results pave a deeper understanding of CAP functions and the way for further investigations in the field of esophageal cancer treatment.

Effect of memantine on expression of Bace1-as and Bace1 genes in STZ-induced Alzheimeric rats.

Recent studies have showed that the long non-coding RNAs (lncRNAs) expression is dysregulated in different neurodegenerative disorders like Alzheimer's disease (AD). In the present study, the effects of memantine on the level of Bace1-as and Bace1 genes' expression in streptozotocin (STZ)-induced Alzheimer's and memantine treated rats were investigated. The male Wistar rats were randomly divided into four groups: 1-Normal control, 2-Sham-operated control, 3- Alzheimer'scontrol rats (ICV-STZ), 4-Experimental group rats treated by memantine in a dose of 30 mg/kg/day for 28 days in ICV-STZ rats. The expression of Bace1-as and Bace1 genes was measured by quantitative-PCR in the brain and blood tissues. ELISA was used to analyze Bace1 and Aß proteins. Expression of Bace1-as was significantly increased in the brain and blood tissues of the experimental group (p = 0.032 and p = 0.034, respectively). The expression of Bace1 gene showed no significant changes in the brain. Furthermore, the ELISA analysis revealed that Bace1 protein was significantly increased in the plasma of the Alzheimer's control group (p = 0.000) and in the brain tissue of the experimental group (p = 0.000). Additionally, Aß levels had no significant changes between all groups studied. The Bace1 protein may be used as a prognostic biomarker in plasma, or before using memantine as a treatment. Furthermore, Bace1-as gene expression may play a role in monitoring the progression of AD.

The effects of UV-B radiation on genetic and biochemical changes of Pelargonium graveolens L'Her.

Ultraviolet radiation induces biochemical and genetic changes in plants. The aim of this study was to investigate the effects of UV-B radiation on genetic stability, phenolic compounds and antioxidant activity of Pelargonium graveolens L'Her. Plant cuttings were exposed to 0, 0.12. 0.26 and 0.38 W/m2 of UV-B radiation. Results indicated that by increasing the UV-B radiation intensity, total phenols, flavonoids and anthocyanin contents, Phenylalanine ammonia lyase activity and antioxidant capacity were increased. Analysis of four flavonols (quercetin, myricetin, kaempferol and rutin) contents of leaves extract by HPLC indicated that these four flavonols were enhanced in all treated plants and also the ratio of quercetin to kaempferol (Q/K) showed a significant increase (P ≤ 0.05) in UV-B treated plants in compare to control. To evaluate the genetic variation in treated plants, 10 ISSR primers were used. The highest level of percentage of polymorphism (P%), Shannon index (I), number of effective allele (Ne) and Nei' genetic diversity (He), were observed at the highest UV-B radiation (0.38 W/m2). The AMOVA analysis also showed a significant genetic differentiation (P ≥ 0.001) among the studied groups, and confirmed the differentiation of groups obtained by the cluster analysis of molecular data. Overall, these results showed that biochemical changes in different intensities of UV-B were in line with genetic variations, so that the highest biochemical and genetic variations were observed in 0.38 W/m2 treatment.

Carboxymethyl dextran-trimethyl chitosan coated superparamagnetic iron oxide nanoparticles: An effective siRNA delivery system for HIV-1 Nef.

Gene therapy, including small interfering RNA (siRNA) technology, is one of the leading strategies that help to improve the outcomes of the current therapeutic systems against HIV-1 infection. The successful therapeutic application of siRNAs requires their safe and efficient delivery to specific cells. Here, we introduce a superparamagnetic iron oxide nanoparticle (SPION) for delivering siRNA against HIV-1 nef (anti-nef siRNA) into two cell lines, HEK293 and macrophage RAW 264.7. SPIONs were coated with trimethyl chitosan (TMC), and thereafter, different concentrations of SPION-TMC were coated with different ratios of a carboxymethyl dextran (CMD) to modify the physicochemical properties and improve the biological properties of the nanocarriers. The nanoparticles exhibited a spherical shape with an average size of 112 nm. The obtained results showed that the designed delivery route enhanced the uptake of siRNA into both HEK293 and RAW 264.7 cells compared with control groups. Moreover, CMD-TMC-SPIONs containing anti-nef siRNA significantly reduced the expression of HIV-1 nef in HEK293 stable cells. The modified siRNA-loaded SPIONs also displayed no toxicity or apoptosis-inducing effects on the cells. The CMD-TMC-SPIONs are suggested as potential nanocarriers for siRNA delivery in gene therapy of HIV-1 infection.

ANRIL and ANRASSF1 long noncoding RNAs are upregulated in gastric cancer.

Gastric cancer (GC) is the fifth most frequent cancer and the third-leading cause of cancer-related death worldwide. It is a highly heterogeneous disease regarding the morphological and molecular viewpoints. Since it is curable in primary stages, early detection could improve the survival rate. Long noncoding RNAs contribute to a variety of cellular mechanisms, and their dysregulation is reported in various diseases such as cancer. Thus, they have a great potential to be used as diagnostic and prognostic biomarkers and therapeutic targets as well. In the current study, ANRIL and ANRASSF1 expression levels were compared between GC tumors and the adjacent normal tissues collected from 39 Iranian patients using the quantitative real-time polymerase chain reaction method. Correlation between ANRIL and ANRASSF1 expression levels and other clinical parameters was also evaluated. ANRIL and ANRASSF1 were significantly overexpressed in GC tumors compared with adjacent tissues ( P < 0.0001 and P = 0.001, respectively). No significant correlation between ANRIL and ANRASSF1 expression levels and demographic information was found. This study suggests that ANRIL and ANRASSF1 may play a critical role in GC progression and can be considered as a potential diagnostic or therapeutics biomarkers.

The effects of 6-Gingerol on reproductive improvement, liver functioning and Cyclooxygenase-2 gene expression in estradiol valerate - Induced polycystic ovary syndrome in Wistar rats.

6-Gingerol is the major pungent ingredient of ginger with anti-inflammatory and antioxidant properties. In this study, we evaluate the effects of 6-gingerol on the biochemical parameters and ovarian histological improvements in estradiol valerate (EV) induced PCOS rats. Thirty six female Wistar rats were divided into 4 groups: control, received normal diet, PCOS control, received 4 mg/kg EV injection for 28 days and two experimental groups, received an EV injection for 28 days and followed by 6-gingerol (200 µg/kg and 400 µg/kg) for 14 days. The administration of EV led to increase body and ovarian weights, abnormality in serum sex steroid profile, decrease in antioxidant activity and increase in COX-2 gene expression. 6-gingerol treatments, particularly the 400 µg/kg dose, markedly attenuated these alterations. 6-gingerol showed beneficial effects in the EV induced PCOS rats via decreased expression of COX-2, restored biochemical parameters to normal and decreased of cysts in the ovaries.

Development a rapid and accurate multiplex real time PCR method for the detection Chlamydia trachomatis and Mycoplasma hominis.

BACKGROUND: Sexually transmitted diseases easily spread among sexually active people and often have no symptoms. Rapid and accurate method for detecting these infections are necessary in early stages. The traditional detection methods of them are difficult and time-consuming. METHODS: In this study, multiplex real time PCR was optimized for rapid identification of Chlamydia trachomatis and Mycoplasma hominis in a single tube and was performed with our designed primers. The sensitivity test was carried out to designed primers with diluted genomic DNA. To defined the specificity, non STD bacteria were used as DNA template. RESULTS: This study indicated that the developed multiplex real time PCR can be an effective alternative procedure to the conventional methods for rapid and accurate identification of C Chlamydia trachomatis and Mycoplasma hominis. Multiplex real-time PCR Results of them were checked with melting curves. The sensitivity of our designed primer by multiplex real time PCR for Chlamydia trachomatis and Mycoplasma hominis were 4.78×1010 and 8.35×1010 , respectively, Which the primers did not amplify any product from a non-STD species. CONCLUSIONS: Multiplex real time PCR by our new primers and analysis of melting curves were successfully usable for rapid and accurate detection of Chlamydia trachomatis and Mycoplasma hominis. This assay instead of traditional culture method, has considerable potential to be rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous and direct detection.

Study of single nucleotide polymorphism (rs28368082) in SPO11 gene and its association with male infertility.

PURPOSE: The present study is a case-control analysis of a SNP (rs28368082) in exon 7 of the SPO11 gene and its possible association with male infertility in three provinces of Iran. We also searched for genetic differences among populations. METHODS: Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, we genotyped 113 infertile men and 50 fertile controls. Then, samples consisting SNP, as determined by PCR-RFLP, were genotyped by sequencing. The differences in genotype distributions between cases and fertile controls were examined using Chi-squared analysis. The genetic difference between individuals with mutated nucleotide was investigated by phylogenetic trees. Genetic difference among populations (provinces) was analyzed through ANOVA test, and homogeneity was investigated using STRUCTURE and K-means clustering analysis. RESULTS: According to the statistical analysis, the SNP was significantly associated with male infertility in all populations except oligozoospermic cases of the Center region. The phylogenetic trees showed partial genetic variation among the individuals, although ANOVA test showed no significant genetic difference between populations (provinces) for both azoospermic, and oligozoospermic cases. Eventually, we affirmed that individuals in the inclusive populations had genetic difference, but it was not statistically significant for dividing underlying populations to separate groups, so each population was homogenous. CONCLUSION: Our study indicates that the mentioned polymorphism in SPO11 gene may be linked to the susceptibility of azoospermia and oligozoospermia male infertility in three provinces of Iran. Further studies are required to support obtained results. It finally should be noted that the possible association between a particular SNP and a specific disease completely depends on the underlying population.

Whole Exome Sequencing of Non-Syndromic Hearing Loss Patients.

Background: Hearing loss is the second most common disease after mental retardation in Iran. Autosomal recessive non-syndromic hearing loss (ARNSHL) is an extreme and highly heterogeneous disease, for which more than 70 genes have been identified. Considering the frequency of family marriage as well as the importance of ARNSHL in Iran, we evaluated the genetic factors involved in this type of deafness. Methods: We performed the whole exome sequencing (WES) of eight Iranian subjects with severe nonsyndromic hearing loss selected from 110 well-characterized subjects with non-syndromic hearing loss from 2017-2019. The patients with mutated GJB2 and GJB6 genes were excluded from the study. Results: The use of the whole exome sequencing method revealed 10 different mutations in 7 genes, including SLC26A4 (c.1234G>T), FGF3 (c.45DelC, c.466T>C), ADGRV1 (c.12528-2A>C, c.16226-16227insAGTC), OTOG (c.7454delG), OTOF (c.3570+2T>C), ESPN (c.992G>A), OTOA (c.2359G>T, c.2353A>C). Seven new variants were observed in seven families including SLC26A4 (c.1234G>T), FGF3 (c.45DelC), ADGRV1 (c.12528-2A>C), OTOG (c.7454delG), ADGRV1 (c.16226-16227insAGTC), OTOF (c.3570+2T>C). Conclusion: The causal mutation of ARNSHL was found in all patients using the WES. Meta-analysis studies can help to identify common mutations causing deafness in any population to facilitate identification of carriers and subjects with deafness.

Interference of Bisphenol A on Cumulus Cells Development and Number of Retrieved Mature Oocytes in Unexpected Poor Ovarian Response Women: A Prospective Cohort Study.

OBJECTIVE: This study aimed to investigate the relationship between follicular fluid Bisphenol A (BPA) concentrations with alterations in the expressions of NOTCH1-3, CASPASE 3/7, HLA-G, and ICAM-1 genes and the number of retrieved mature oocytes (MII oocyte) in the cumulus cells of infertile poor ovarian response stimulates women. MATERIALS AND METHODS: In this prospective cohort study, 80 infertile unexpected poor ovarian response (POR) subjects were selected on the basis of subgroup 1a of the POSEIDON classification. They were divided into two groups: group 1 consisted of 40 women, each with a higher number of metaphase II (MII) oocytes (G1, 3-4 oocytes retrieved), while group 2 comprised of 40 women, each with a lower number of MII oocytes (G2, ≤2 oocytes retrieved). The expressions of the studied genes were evaluated by quantitative-real time polymerase chain reaction (PCR). The concentration of BPA in follicular fluid was measured with HPLC. RESULTS: The expression levels of NOTCH1-3, HLA-G, and ICAM-1 genes were significantly lower in G2 than G1 (P<0.05). Meanwhile, CASPASE 3/7 expression levels were higher in unexpected POR patients in G2 compared to G1 (P<0.05). There was a significant direct correlation between the levels of NOTCH1-3, HLA-G and ICAM-1 gene expressions and there was also a significant inverse correlation (P<0.05) between the levels of CASPASE 3/7, with the number of MII oocytes and embryo development between the two groups. The concentration of BPA in the follicular fluids of G2 was higher compared to G1 (P<0.05). CONCLUSION: A higher concentration of BPA was associated with a lower number of mature oocytes and oocyte quality in these patients. Also, alterations of NOTCH1-3, CASPASE 3/7, HLA-G, and ICAM-1 transcript levels in unexpected POR women were associated with BPA concentration.

A novel EGFR-specific recombinant ricin-panitumumab (scFv) immunotoxin against breast and colorectal cancer cell lines; in silico and in vitro analyses.

The Epidermal Growth Factor Receptor (EGFR) has been of high importance as it is over expressed in a wide diversity of epithelial cancers, promoting cell proliferation and survival pathways. Recombinant immunotoxins (ITs) have emerged as a promising targeted therapy for cancer treatment. In this study, we aimed to investigate the antitumor activity of a novel recombinant immunotoxin designed against EGFR. Using an in silico approach, we confirmed the stability of the RTA-scFv fusion protein. The immunotoxin was successfully cloned and expressed in the pET32a vector, and the purified protein was analyzed by electrophoresis and western blotting. In vitro evaluations were conducted to assess the biological activities of the recombinant proteins (RTA-scFv, RTA, scFv). The novel immunotoxin demonstrated significant anti-proliferative and pro-apoptotic effects against cancer cell lines. The MTT cytotoxicity assay revealed a decrease in cell viability in the treated cancer cell lines. Additionally, Annexin V/Propidium iodide staining followed by flow cytometry analysis showed a significant induction of apoptosis in the cancer cell lines, with half maximal inhibitory concentration (IC50) values of 81.71 nM for MDA-MB-468 and 145.2 nM for HCT116 cells (P < 0.05). Furthermore, the EGFR-specific immunotoxin exhibited non-allergenic properties. The recombinant protein demonstrated high affinity binding to EGFR. Overall, this study presents a promising strategy for the development of recombinant immunotoxins as potential candidates for the treatment of EGFR-expressing cancers.

Evaluation of the Immunogenicity of Recombinant Espb, Espc Proteins from Mycobacterium Tuberculosis and the Fusion Espc/Espb Protein in BALB/C Mice.

Background: Two newly identified proteins, EspB and EspC are involved in the pathogenesis of Mycobacterium tuberculosis. The objective of the present study was to evaluate the immunogenicity of recombinant EspC, EspB, and EspC/EspB fusion proteins in mice. Methods: BALB/c mice were immunized subcutaneously with recombinant EspC, EspB, and fusion EspC/EspB proteins, three times with along with Quil-A as an adjuvant. The cellular and humoral immune responses were evaluated by quantifying IFN-γ, IL-4, IgG, IgG1, and IgG2a antibodies against the antigens. Results: The results showed that the mice immunized with recombinant EspC, EspB, and EspC/EspB proteins did not produce IL-4, whereas IFN-γ was secreted in response to all three proteins. EspC/EspB group produced significant amounts of IFN-γ in response to stimulation with all the three recombinant proteins (P<0.001). In mice immunized with EspC, high levels of IFN-γ were detected in response to EspC/EspB, and EspC (P<0.0001); while mice immunized with EspB produced lower levels of IFN-γ in response to EspC/EspB, and EspB (P<0.05).Mice immunized with recombinant EspC, EspB, and EspC/EspB proteins exhibited significantly high levels of IgG and IgG2a/IgG1 ratio (P< 0.001). Moreover, high levels of IgG and IgG2a were detected in the sera of mice immunized with EspC/EspB fusion protein. Conclusions: All the three recombinant proteins induced Th1-type immune responses in mice against EspB and EspC; however, EspC/EspB protein is more desirable due to the presence of epitopes from both EspC and EspB proteins and the production of immune responses against both.

Molecular Detection of gyrA Mutation in Clinical Strains of Klebsiella pneumoniae.

Background: Multi-drug resistant (MDR) Klebsiella pneumoniae strains cause the majority of community acquired and life-threatening infections. We aimed to detect the gyrA mutations in the clinical strains of nalidixic acid and ciprofloxacin resistant K. pneumoniae isolated from the patients with urinary tract infections. Methods: Bacterial strains were isolated from the patients with urinary tract infections admitted to a major hospital in Tehran, Iran (2017-2018). Bacterial identification was done according to standard microbiological tests. Antimicrobial susceptibility testing for quinolones and fluoroquinolone antibiotics was done using both disc diffusion and minimal inhibitory concentrations (MICs) methods. PCR-RFLP was used to detect the probable mutation in the gyrA gene in nalidixic acid and ciprofloxacin resistant strains. Finally sequencing was performed to detect point mutations in isolated K. pneumoniae strains. Results: One hundred K. pneumonia isolates were recovered from the urine samples of the clinical cases. Antibiotic resistance testing showed that among all K. pneumoniae isolates, 26% and 19% of the strains were resistant to nalidixic acid and ciprofloxacin respectively. MIC value was ≥4 µg/ml for ciprofloxacin resistant isolates. The results of RFLP on gyrA PCR amplicons using HinfI restriction enzyme showed point mutation in this gene in 46% of nalidixic acid and ciprofloxacin resistant K. pneumonia. The data obtained from the sequencing confirmed the RFLP results and indicated the presence of point mutations in codons 83 and 87 in the gyrA gene which leads to the substitution of different amino acids in gyrA protein. Conclusion: Our findings indicated a relative increased rate of resistance against quinolones and fluoroquinolone antibiotics that raised a concern about extensive dissemination of clinical strains of nalidixic acid and ciprofloxacin resistant K. pneumonia. Point mutation of gyrA gene was responsible for the resistance in our strains however to gain more insight into the molecular characterization of quinolone-resistant isolates, other possible mechanisms of the resistance should also be investigated.

A new insight on genetic diversity of sweet oranges: CAPs-SSR and SSR markers.

BACKGROUND: Citrus species are among the most important and widely consumed fruit trees in the world and are subjected to increasing global cultivation. Sweet orange (Citrus sinensis L. Osbeck) is one of 30 species of citrus which is cultivated in different regions of Iran. In this study, 80 trees of 13 sweet orange cultivars of Mazandaran province were studied for genetic diversity and fingerprinting by five short simple repeat (SSR) marker. RESULTS: The studied cultivars showed a high degree of genetic variability with an average genetic polymorphism of 98.46%. Behshahr and Jadeh Ghadim2 genotypes had the highest and lowest values in Nei genetic diversity, number of effective alleles, and Shannon index, respectively. Based on k-means clustering, the studied genotypes were divided into two main different groups. The high magnitude of genetic similarity between replicates of different cultivars indicated a potential case of homonymy or synonymy. DAPC analysis showed genetic admixture among some of the cultivars. The heatmap plot illustrated the alleles involved in cultivar differentiation. The CAPs analysis of monomorphic alleles of SSR loci indicated that these alleles differ in their sequences which add up to the genetic variability of citrus germplasm. CONCLUSION: In general, SSR markers, due to their codominant nature and abundance in genome, are a good indicator for cultivar fingerprinting and hybrid prediction in orange cultivars. The present results showed the high diversity of sweet orange trees in different cultivars in the north of the country.