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Introduction: Since its discovery in 1961, methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as a significant healthcare-associated pathogen (HA-MRSA) and a notorious 'superbug'. Typing is crucial for surveillance, epidemiology analysis, infection control of MRSA and sequencing of the spa gene is one of the most common methods used for determining the origin of this bacterium in humans and animals. This research aimed to determine the antibiotic resistance and spa type of S. aureus strains collected from outpatients in two hospitals in the Wasit province of Iraq. Material & Methods: The study analyzed 200 outpatient MRSA isolates by collecting nasal and sputum samples from patients. Standard biochemical and molecular methods based on the nuc gene were used to identify S. aureus bacteria and amplify the mecA and spa genes. The Kirby-Bauer disc diffusion method was employed to determine the antibiotic sensitivity of the isolates using penicillin, cefoxitin, vancomycin, gentamicin, erythromycin, tetracycline, imipenem, clindamycin, chloramphenicol and rifampicin. Results: Methods. The prevalence of MRSA was more common in women than in men. Antibiogram results showed that most of the isolates were resistant to penicillin (94.2%) and sensitive to imipenem (100%), clindamycin (100%), and chloramphenicol (100%). Of these 35 isolates, 30 (87.5%) and 26 strains (74.3%) were positive for the mecA and spa genes. Typing based on spa gene sequencing revealed four different patterns: t386, t3579, U0002 and U0234. Conclusion: Variations in the spa gene among different S. aureus isolates may he of clinical importance when treating staphylococcal infections. In this study, spa typing revealed four different patterns in Iraq, representing diagnostic and therapeutic implications. [ABSTRACT FROM AUTHOR]

Background and Aim: Entamoeba histolytica and Giardia intestinalis are two common parasites associated with gastrointestinal infections and diarrheal diseases worldwide. This study investigated the prevalence, molecular characterization, and phylogenetic relationships of E. histolytica and G. intestinalis among patients with diarrhea and intestinal disorders in hospitals across Iraq. Materials and Methods: Stool samples were collected from individuals presenting with gastrointestinal symptoms, and a microscopic examination was performed to screen for the presence of E. histolytica and G. intestinalis. DNA was extracted from positive samples, and PCR amplification targeting the 16S rRNA gene for E. histolytica and the triose phosphate isomerase (TPI) gene for G. intestinalis was carried out. The resulting PCR products were subjected to agarose gel electrophoresis, purified, and sequenced. Phylogenetic trees were constructed using the Maximum Likelihood method based on the Tamura-Nei model, and bootstrap analysis was performed. Results: Among the studied samples, 27.8% (n=85) were positive for E. histolytica, while 19.6% (n=60) were positive for G. intestinalis. Sequence analysis confirmed the presence of E. histolytica and G. intestinalis by matching the obtained sequences with those available in the NCBI gene bank. The phylogenetic analysis revealed the genetic diversity and relatedness of the E. histolytica and G. intestinalis isolates. Clustering patterns specific to the Iraqi population were observed, suggesting potential regional transmission dynamics. Conclusion: This study provides valuable insights into the prevalence, molecular characterization, and phylogenetic relationships of E. histolytica and G. intestinalis in Iraqi patients with diarrhea and intestinal disorders. [ABSTRACT FROM AUTHOR]

Introduction: Despite careful control of environmental factors, there is a wide variety of interpersonal muscle-induced muscle damage. Genetic changes can also be important in determining susceptibility to injury. Weightlifting is one of the most successful Iranian sports teams in Olympics; so, the aim of this study was the investigation of the relationship between ACE I/D gene polymorphism rs4646994 and incidence and severity of sport injuries in Iranian male elite weightlifters. Materials and Methods: The study was cross-sectional research that conducted in 2022. The statistical population consisted of male elite weightlifters from Isfahan consisting of 31 athletes who all were investigated as sample. A questionnaire was used to collect the injuries data. Saliva samples were taken from all subjects and DNA was extracted and genotyped by using real-time polymerase chain reaction (real time-PCR). Chi-square and regression tests were used to analyze the data. Significance level was also considered at P <0.05. Results: There was a significant difference in the incidence of injuries in different genotypes. ID genotype (59.4%) had the highest incidence of injury in compare with DD (23.2%) and II (17.4%) genotypes (χ2 = 21.47, P = 0.001). Also, there was a significant difference in the severity of injuries in different genotypes (χ2 =15.59, P = 0.049); but there was no relationship between genotype and incidence and the severity of sports injuries in selected Iranian elite male weightlifters (P= 0.715). Conclusion: The results showed differences in the incidence and severity of sport injuries in different genotypes, hence it would be important in the prediction injury risk in weightlifters. [ABSTRACT FROM AUTHOR]

Background: The pro-inflammatory cytokine, interleukin 16 (IL-16), has been shown to be secreted in low levels in knee osteoarthritis (KOA). The aim of the study was to examine the relationship between IL-16 polymorphisms and the risk of KOA in the Egyptian population, as well as the clinical and radiographic severity of KOA. Results: IL16 rs11556218 thymidine triphosphate (T) T G (guanosine triphosphate), GG, TG + GG genotypes, and G allele (odd ratio (OR) = 0.315; 95% confidence interval (CI) = 0.191–0.518; P < 0.001; OR = 0.363; 95% CI = 0.162–0.815, P = 0.014; OR = 0.323; 95% CI = 0.202–0.519, P < 0.001; OR = 0.480; 95% CI = 0.338–0.683, P < 0.001 respectively); rs4778889 cytidine triphosphate (C) T,CC, TC + CC genotypes, and C allele (OR = 0.519, 95% CI = 0.319–0.844, P = 0.008; OR = 0.309, 95% CI = 0.105–0.916, P = 0.034; OR = 0.485, 95% CI = 0.304–0.775, P = 0.002; OR = 0.537, 95% CI = 0.365–0.791, P = 0.001 respectively); and rs4072111 CT, TT, CT + TT genotypes, and T allele (OR = 0.537, 95% CI = 0.323–0.893, P = 0.017, OR = 0.316, 95% CI = 0.096–0.843, P = 0.049, OR = 0.502, 95% CI = 0.309–0.816, P = 0.005; OR = 0.534, 95% CI = 0.353–0.809, P = 0.004 respectively) were associated with a decreased KOA risk, and they were significantly associated with decreased the Western Ontario and McMaster Universities Arthritis Index (WOMAC) and the Kellgren-Lawrence (K/L) scores. Neither IL-16 serum levels nor IL-16 polymorphisms were associated with the susceptibility to KOA. Low KOA risk was associated with the haplotypes GTC and TCT. Conclusion: There was no correlation between serum IL-16 levels and KOA susceptibility or IL-16 polymorphisms. GTC and TCT haplotypes were associated with low KOA risk. The variant alleles rs11556218GG, TG + GG; rs4778889 CC, TC + CC; and rs4072111 TT, CT + TT were associated with a reduced risk of KOA. [ABSTRACT FROM AUTHOR]

In most procedures that involve gene cloning, after the amplification of a target gene by PCR or by Real-time PCR, the purification of the trapped gene on agarose gel is a crucial stage. There are various methods for extracting genes from agarose gel by removing other contaminants. We isolated the amplified PqHMGR gene (derived from Ginseng (Panax quinquefolius)) from agarose gel by a quasi-electrophoresis device (similar to electro-elution technique). Moreover, the efficiency of this new approach was compared with that of the commercial kit 'Silica Bead DNA Gel Extraction' (Thermo Scientific American Company). Ligation to the PTG- 19 plasmid and cloning in E. coli bacteria were also done. The results showed successful isolations of targeted DNA, along with a high efficiency in producing recombinant DNA and in concluding a successful cloning procedure through this new device. The invented method provided a better purification ability than the commercial kit, but because of using the TAE 1X buffer as the purified gene storage solution, the plasmid and bacterial transformation rates were slower than the commercial kit method. It was found that using the new method for the purification of nucleotide sequences by electrophoresis and electrophoresis buffer is feasible, and that these purified fragments can be applied in cloning and sequencing. Using the TAE 1X buffer instead of distilled water did not cause problems in gene binding to PTG-19 plasmid. It also allowed a successful transformation of E. coli bacteria by the modified plasmid. Nonetheless, using TAE 1X buffer reduced the modification rate of the PTG-19 plasmid and decreased the rate of E. coli transformation by the modified plasmid. [ABSTRACT FROM AUTHOR]

Objective To establish a new indicator derived from reticulocyte hemoglobin (Ret-He) content and red blood cell (RBC) indices for screening for iron deficiency anemia (IDA) in an area in whch thalassemia is prevalent. Methods Blood specimens from 304 women aged between 18 and 30 years residing in northeast Thailand were collected and measured for RBC and reticulocyte parameters. Iron deficiency was diagnosed when a participant had a serum ferritin level of less than 15 ng per mL. Thalassemia genotypes were defined by hemoglobin (Hb) and DNA analyses. Results Of the total participants, 25% had iron deficiency (ID) and 50% carried the thalassemia gene. Various mathematical formulas were established and analyzed using the receiver operating characteristic (ROC) curve. The formula derived from Ret-He: (Ret-He/RDW-SD) × 10, was the best predictor for identifying ID among participants (area under the curve [AUC] = 0.812). Further testing of this indicator among individuals with positive thalassemia-screening results revealed stronger performance with an AUC of 0.874. Conclusions The findings indicate that the formula derived from Ret-He might be applicable for screening ID in areas in which thalassemia is prevalent. [ABSTRACT FROM AUTHOR]

Background: Numerous molecular changes are involved in the development and progression of bladder cancer. Regular follow-up of patients is crucial due to the high recurrence rate of bladder cancer. The aim of this study is to determine the role of B-Raf proto-oncogene, serine/threonine kinase and ZEB2 expressions in onset and progression of bladder cancer. We have also investigated their relationships to pathological characteristics. Methods: We conducted this case-control study on bladder cancer and its healthy adjacent tissue, and normal bladder tissue from patients with benign prostatic hyperplasia. After extraction of total RNA and cDNA synthesis, quantitative expression analysis was performed in duplicate using real-time PCR. Changes in the gene expression were calculated according to the 2(-ΔΔCt) equation. The products were confirmed by 1% agarose gel electrophoresis and sequenced by Bioneer Company. Data was analyzed using the SPSS software (version 16). Results: There was significantly greater B-Raf proto-oncogene, serine/threonine kinase expression in 82% of bladder tumor samples compared to the adjacent tissues. In 91.1% of tumor samples, the gene expression was also significantly higher than healthy bladder tissues from patients with benign prostatic hyperplasia. We observed overexpression of B-Raf proto-oncogene, serine/threonine kinase in 61.7% of the healthy margin tissue samples compared to healthy bladder tissues of patients with benign prostatic hyperplasia (P<0.001). Expression of ZEB2 in 52.9% of the bladder tumor samples was significantly higher than healthy peripheral tissues. This increase was observed in 94.1% of tumor samples compared to healthy bladder tissues of patients with benign prostatic hyperplasia (P<0.001). Pearson correlation coefficient showed a positive relationship between B-Raf proto-oncogene, serine/threonine kinase and ZEB2 in cancerous samples (r = 0.75) and healthy margin tissue samples (r = 0.49). Conclusion: During the carcinogenesis process, molecular changes are seen in healthy margin tissue. These molecular changes may be the reason for the high recurrence rate of bladder cancer. B-Raf proto-oncogene, serine/threonine kinase can potentially be a target cancer therapy in antisense technologies. [ABSTRACT FROM AUTHOR]

Crime scene investigation is an important step in the entire criminal investigation process because this is where evidence is gathered. Blood from the perpetrator or victim of a crime can be left at crime scenes or transferred to other materials such as clothing, knives and guns. Most often, this body fluid is contaminated with soil at outdoor crime scenes but this might be the only or the most important evidence in solving a crime. This work aimed at identifying the most appropriate method of storing crime scene soil-blood mixed sample prior to analysis at the laboratory. Human blood was mixed with soil and stored at three different storage conditions (i.e., Room temperature/25°C, 4°C and -20°C). Samples stored at room temperature saw significant reduction in DNA concentration as storage time increased (P=0.001). Samples stored at 4°C saw a drastic decrease in DNA concentration just after two weeks of storage. By the eighth week of storage at 4°C, there was no detectable DNA (P=0.000). Samples stored at -20°C recorded no specific pattern in decrease or increase in DNA concentration for the entire 12week storage (P=0.324). There were full STR Profiles generated for room temperature stored samples and -20°C stored samples throughout the study. There were full, partial and null Profiles generated for 4°C stored samples depending on the sample storage duration. In conclusion, -20°C was identified as the best storage condition for soil-blood mixed sample followed by room temperature and 4°C, respectively. [ABSTRACT FROM AUTHOR]

Background: Cytochrome P450 3A enzymes exhibit a variety of physiological roles and have been reported to be the most predominant enzymes involved in drugs metabolism. Single nucleotide polymorphisms (SNPs) in the genes that code for these enzymes may result in functional changes that affect enzyme activity. CYP3A4 is an important enzyme in the metabolism of many important drugs used in the treatment of breast cancer. Methods: A total of 94 post-menopausal breast cancer patients were recruited for the study and their DNA was isolated for polymerase chain reaction (PCR). The primers were designed using Primer3 software with primer specificities checked via the Basic Local Alignment Tool (BLAST) database. The primer specificity, functionality and annealing temperature were first investigated using uniplex PCR protocols, followed by a single multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The digested amplification fragments were analysed by gel electrophoresis and subsequently validated by sequencing. Results: A multiplex PCR-RFLP method was successfully developed for simultaneous detection of CYP3A4*4, CYP3A4*18B and CYP3A4*22 in a population of post-menopausal breast cancer patients. Conclusion: The technique is simple, cost-effective, time-saving and can be routinely applied in the identification of SNPs and determination of allelic and genotypic frequencies of CYP3A4*4, CYP3A4*18B and CYP3A4*22. [ABSTRACT FROM AUTHOR]

Background: The prognostic significance of free cancer cells detected in peritoneal fluid at the time of rectal surgery remains unclear. A substantial number of patients will develop metastatic disease even with successful local treatment. This prospective non-randomized study investigated the prognostic value of intraperitoneal free cancer cells harvested in peritoneal lavage after surgery for rectal cancer. Mutational hotspots in mitochondrial DNA were examined as potential molecular signatures to detect circulating intraperitoneal free cancer cells when present in primary tumor and in lavage.Methods: Point mutations in mitochondrial DNA amplifications were determined in primary tumors and corresponding exfoliated intraperitoneal free cancer cells in lavage from 191 patients with locally advanced rectal cancer scheduled for radical treatment. Mitochondrial DNA target sequences were amplified by polymerase chain reaction and base substitutions were detected by denaturant, cycling temperature capillary electrophoresis. Detection of intraperitoneal free cancer cells was correlated to survival.Results: Of 191patients analyzed, 138 (72%) were identified with somatic mitochondrial point mutations in rectal cancer tumors. From this fraction, 45 patients (33%) had positive lavage fluid with corresponding somatic mtDNA point mutations in lavage representing circulating intraperitoneal free cancer cells. There was no significant survival difference between patients identified with or without somatic mitochondrial DNA point mutations in the corresponding lavage.Conclusion: Somatic mitochondrial DNA point mutations identified in primary rectal tumors enable detection of circulating intraperitoneal free cancer cells in lavage fluid. Intraperitoneal free cancer cells harvested from lavage immediately after surgery for rectal cancer does not represent an independent prognostic factor on survival. [ABSTRACT FROM AUTHOR]

Outdoor microalgae cultivation process is threatened by many issues, such as pest pollution and complex, changeable weather. Therefore, it is difficult to have identical growth rate for the microalgae cells and to keep their continuous growth. Outdoor cultivation requires the algae strains not only to have a strong ability to accumulate oil, but also to adapt to the complicated external environment. Using 18S rRNA technology, one wild strain Scenedesmus sp. FS was isolated and identified from the culture of Chlorella zofingiensis. Upon contamination by Scenedesmus sp., the species could quickly replace Chlorella zofingiensis G1 and occupy ecological niche in the outdoor column photobioreactors. The results indicated that Scenedesmus sp. FS showed high alkali resistance. It also showed that even under the condition of a low inoculum rate (OD680, 0.08), Scenedesmus sp. FS could still grow in the outdoor raceway pond under a high alkaline environment. Even under unoptimized conditions, the oil content of Scenedesmus sp. FS could reach more than 22% and C16–C18 content could reach up to 79.68%, showing that this species has the potential for the biodiesel production in the near future. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the contribution of SHISA3 promoter methylation to laryngeal squamous cell carcinoma (LSCC). SHISA3 promoter methylation status and expression were determined using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (qRT-PCR) in 93 paired LSCC and adjacent normal tissues, respectively. Furthermore, the regulatory function of the SHISA3 promoter fragment was analyzed using a luciferase reporter assay. The results reveal that there is a significant increase in SHISA3 methylation in LSCC tissues compared with corresponding nontumor tissues P=4.58E-12. The qRT-PCR results show a significant association between SHISA3 methylation and expression in LSCC P=1.67E-03. In addition, the area under the receiver operating characteristic curve was 0.91. Consequently, a log-rank test and multivariate Cox analysis suggest that SHISA3 promoter hypermethylation is a predictor of poor overall survival for LSCC (log-rank P = 0.024; HR = 2.71; 95% CI = 1.024–7.177; P = 0.047). The results indicate that SHISA3 promoter hypermethylation might increase the risk of LSCC through regulation of gene expression and is a potential diagnostic and prognostic biomarker for LSCC. [ABSTRACT FROM AUTHOR]

GPR1 is a G protein-coupled receptor that plays critical roles in eukaryotic cells: typically, response to glucose stimulation, lipid accumulation, and transmitting nutrition signals to cAMP pathway. However, the alternative splicing of the GPR1 gene and its expression pattern in chicken tissues and ovarian follicles were unknown. In our current study, we used RACE-PCR to identify three GPR1 variants, including the full-length variant (GPR1-va1) and two alternatively spliced variants (GPR1-va2, GPR1-vb). Quantitative real-time PCR examined the expression pattern of GPR1 mRNA in chicken tissues and ovarian follicles. The result reveals that the coding sequence of the three variants cDNA is 1053, 1053, and 627 bp in length, encoding 350, 350, and 208 amino acids, respectively. The three variants of GPR1 show similar tissue distributions; GPR1 expression was abundant in the abdominal fat, lung, and heart. With the follicular development, the expression of GPR1 gene gradually increased, and GPR1-va1 and GPR1-va2 spliced variants expression in F2 were significantly higher than in F5, F4, and prehierarchical follicles (P<0.05). Taken together, we found three novel variants of GPR1, and the results of GPR1 expression profiling in adipose tissues and ovarian follicles suggest that GPR1 may play a significant role in the lipid accumulation and progression of follicular development. [ABSTRACT FROM AUTHOR]

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori, by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana, the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm. [ABSTRACT FROM AUTHOR]

Introduction. Infections due to extended spectrum β-lactamase producing Enterobacteriaceae are on the rise. They pose serious public health problems due to their resistance to large number of antibiotics. However, little is known about the genotypes of ESBL from Nepal. Therefore, the study presents results of phenotypic and molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. isolated from various clinical specimens in a tertiary care teaching hospital of Nepal. Methods. A total of 172 Enterobacteriaceae clinical isolates recovered from various clinical specimens were analyzed for their antibiotic susceptibility test. Detection of ESBLs was carried out using combination disk test and multiplex PCR for their genotypes (CTX-M, SHV, and TEM). Results. Out of 172 clinical isolates, 70 (40.6%) of them were found ESBL producers. The major source of ESBL producers was urinary tract samples and the highest ESBL production was observed in Escherichia coli (46.5%). Among ESBL genotypes, CTX-M (91.4%) was most predominant, followed by TEM (65.7%) and SHV (11.4%) in both of the isolates. Conclusions. High level of drug resistance and ESBL production was observed among the clinical isolates. There is a need for longitudinal and nationwide surveillance for drug resistance in clinical isolates and antimicrobial stewardship is necessary to guide the appropriate and judicious antibiotic use. [ABSTRACT FROM AUTHOR]

The detection of Toxoplasma gondii in free-range chickens is a good indicator of possible risk to human beings. The aim of this study was to investigate the occurrence of T. gondii in free-range chicken using polymerase chain reaction (PCR). Brain samples from 105 free-range chickens from three administrative areas in Thika region, Kenya, were collected, DNA-extracted, and analyzed using PCR to detect presence of T. gondii. The overall prevalence of T. gondii in all the three areas was 79.0% (95% CI: 70.0–86.4%) and the prevalence across the three areas was not significantly different (P=0.5088; χ2=1.354). Female chickens had higher (79.4%) prevalence than males (78.6%), although the difference was not significant (P=0.922, χ2 = 0.01). However, chickens that were more than 2 years old had significantly (P=0.003; χ2 = 11.87) higher prevalence compared to younger ones. The study indicates that there was a high occurrence of T. gondii infection in free-range chickens from Thika region and that the infection rate is age dependent. Further studies should be carried out to determine the possible role of roaming chickens in the epidemiology of the disease among humans in the area. [ABSTRACT FROM AUTHOR]

The identification of snails of the genus Biomphalaria can be done using morphological characteristics which depends on the size of the snails and skill and knowledge of researcher. These methods sometimes are not adequate for identification of species. The PCR-RFLP, using the ITS region of the rDNA, has been used to identify Brazilian species of the genus Biomphalaria. Nevertheless, there is a lack of information about snails from other Latin American countries. In addition, some snails may be infected by Schistosoma mansoni and when submitted to PCR-RFLP they show molecular profiles different from those previously standardized for the other mollusc species. In this work the molecular profiles of 15 species and the subspecies were established by PCR-RFLP of ITS-rDNA with the enzyme DdeI. Moreover, the molecular profiles of host species, B. glabrata, B. straminea, B. tenagophila, and B. prona, infected by S. mansoni were also established. The molluscs were dissected to permit morphological identification. These results contribute to a correct identification of snails of the genus Biomphalaria and detection of these snails infected by S. mansoni. [ABSTRACT FROM AUTHOR]

Pulmonary diseases caused by nontuberculous mycobacteria (NTM) are increasing in incidence and prevalence worldwide. In this study, we identified NTM species of the clinical isolates from 8 provinces in China, in order to preliminarily provide some basic scientific data in the different species and distribution of NTM related to pulmonary disease in China. A total of 523 clinical isolates from patients with tuberculosis (TB) diagnosed clinically from 2005 to 2012 were identified to the species using conventional and molecular methods, including multilocus PCR, rpoB and hsp65 PCR-PRA, hsp65, rpoB, and 16S-23S internal transcribed spacer region sequencing. The isolates were identified into 3 bacterium genera, including NTM, Gordonia bronchialis, and Nocardia farcinica, and, for the 488 NTM isolates, 27 species were identified. For all the 27 species of NTM which were found to cause pulmonary infections in humans, the most prevalent species was M. intracellulare, followed by M. avium and M. abscessus. And seven other species were for the first time identified in patients with TB in China. NTM species identification is very important for distinguishing between tuberculosis and NTM pulmonary diseases, and the species diversity drives the creation of diverse and integrated identification methods with higher accuracy and efficacy. [ABSTRACT FROM AUTHOR]

A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg++, Fe++, Zn++, Cu++, and Pb++) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries. [ABSTRACT FROM AUTHOR]

Aims: The increasing usage of antibiotics can cause resistance to the treatment of infections, which can caused by bacteria, e.g. Pseudomonas aeruginosa. The aim of this study was to trace the class I, II and III integrons in isolates of P. aeruginosa of nosocomial infection and determining the antibiotic resistance pattern of the bacteria. Instrument & Methods: In this cross-sectional study, 100 Pseudomonas aeruginosa clinical isolates of infected wounds, bedsores, burns, urinary tract infections and respiratory tract infections were collected from patients of 3 Isfahan City hospitals, Iran (Al Zahra, Kashani, Shariati) in 2015. After identification tests and antibiogram, integrons class I, II and III were detected by M-PCR method. Data analysis were performed in SPSS 16 software using Chi-square and Fisher exact tests and the relationship between the presence of class III, II, I was calculated by M-PCR test. Findings: All isolates had multiple antibiotic resistances. The highest antibiotic resistance was to Tetracycline (85%) and the lowest to Norfloxacin (12.5%). There were significant differences between class I and the two other classes of integrons (p=0.036). There was a statistically significant difference between the presence of blaTEM gene in Pseudomonas aeruginosa with other coding genes for antibiotic resistance (p=0.029). Conclusion: Pseudomonas aeruginosa isolates are multi-drug resistant and almost all isolates from clinical infections have class I, II and III Integrons. [ABSTRACT FROM AUTHOR]

In Errachidia province, the incidence of cutaneous leishmaniasis (CL) has increased over the past decade and it was higher in 2010 (860.34 per 100,000 inhabitants), with 3445 cases. The number of cases declined sharply and decreased from 3445 cases in 2010 to 8 cases in 2014 following the control action plan interventions. The total of patients was diagnosed only on clinical basis and the lesions were considered caused by L. major. The epidemiological study was conducted between 2001 and 2014 and the molecular detection of CL was studied to identify the circulating parasite species in this province by using the ITS1-PCR-RFLP methods. In 2010, the molecular identification of 11 samples revealed the presence of L. major in the most affected circles: Goulmima, Er-Rissani, and Errachidia. In 2014 the molecular characterization of 7 among 8 cases reported in this year showed the presence of L. tropica in Errachidia circle. This is the first molecular identification of L. tropica in Errachidia province. The detection of this species after the intensified control measures strategies suggests that it was probably dissipated through the dominance of L. major. [ABSTRACT FROM AUTHOR]

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder. Considering that XP patients have a defect of the nucleotide excision repair (NER) pathway which enables them to repair DNA damage caused by UV light, they have an increased risk of developing skin and eyes cancers. In the present study, we investigated the involvement of the prevalent XPA and XPC genes mutations—nonsense mutation (c.682C>T, p.Arg228X) and a two-base-pair (2 bp) deletion (c.1643_1644delTG or p.Val548Ala fsX25), respectively—in 19 index cases from 19 unrelated families in the West of Algeria. For the genetic diagnosis of XPA gene, we proceeded to PCR-RFLP. For the XPC gene, we validated a routine analysis which includes a specific amplification of a short region surrounding the 2 bp deletion using a fluorescent primer and fragment sizing (GeneScan size) on a sequencing gel. Among the 19 index cases, there were 17 homozygous patients for the 2 bp deletion in the XPC gene and 2 homozygous patients carrying the nonsense XPA mutation. Finally, XPC appears to be the major disease-causing gene concerning xeroderma pigmentosum in North Africa. The use of fragment sizing is the simplest method to analyze this 2 bp deletion for the DNA samples coming from countries where the mutation c.1643_1644delTG of XPC gene is prevalent. [ABSTRACT FROM AUTHOR]

Diagnosis of the endemic cutaneous leishmaniasis (CL) in Jordan relies on patient clinical presentation and microscopic identification. Studies toward improved identification of the causative Leishmania species, especially in regions where multiple species exist, and the introduction of these techniques into medical diagnosis is paramount. This study looked at the current epidemiology of CL in Jordan. Clinically diagnosed 41 patients with CL were tested for the presence of Leishmania parasite using both Giemsa staining from skin scraps on glass slides and ITS1-PCR from samples blotted onto storage cards (NucleoCards®). Microscopically, 28 out of the 41 (68.3%) collected samples were positive for amastigotes, whereas the molecular ITS1-PCR amplification successfully identified 30 of the 41 samples (73.2%). Furthermore, PCR-RFLP analysis allowed species identification which is impossible microscopically. Of the 30 PCR positive samples, 28 were Leishmania major positive and the other two samples were Leishmania tropica. This indicates that L. major is the most prevalent species in Jordan and the two L. tropica cases originated from Syria indicating possible future L. tropica outbreaks. Diagnosis of CL based on clinical presentation only may falsely increase its prevalence. Although PCR is more sensitive, it is still not available in our medical laboratories in Jordan. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the intra- and intergenetic diversities of eight different goat populations in Turkey including Hair, Angora, Kilis, Yayladag, Shami, Honamli, Saanen, and Alpine. A total of 244 DNA samples were genotyped using 11 microsatellites loci. The genetic differentiation between breeds was considerable as a result of the statistically significant (P<0.001) pairwise FST values of each pair of breeds. Exceptionally, FST values calculated for Honamli and Hair breeds were statistically nonsignificant (P>0.05). Heterozygosity values ranged between 0.62 and 0.73. According to the structure and assignment test, Angora and Yayladag goats were assigned to the breed they belong to, while other breeds were assigned to two or more different groups. Because this study for the first time presented genetic data on the Yayladag goat, results of structure analysis and assigned test suggest that further analyses are needed using additional and different molecular markers. [ABSTRACT FROM AUTHOR]

The prevalence of Salmonella spp. was investigated in 109 wild birds poached in the illegal wildlife trade in Rio de Janeiro; most of them are passerines from Thraupidae family and three from Psittacidae. One strain of Salmonella ser. Typhimurium and two strains of Salmonella ser. Panama were isolated from passerine species and all of them showed resistance to multiple antimicrobial drugs, like ampicillin, ceftriaxone, ceftiofur, tetracycline, gentamicin, nalidixic acid, ciprofloxacin, and enrofloxacin. PFGE showed 100% similarity among the Salmonella ser. Typhimurium strain isolated from a Temminck’s seedeater (Sporophila falcirostris) and the strains isolated from a human outbreak, in southern Brazil. The two Salmonella ser. Panama strains isolated from two chestnut-capped blackbirds (Chrysomus ruficapillus) present in the same catch showed the same clonal origin and have never been associated with epizooties and human outbreaks. Potential for dissemination of resistant Salmonella through situations offered by captive management and the isolation of the same strain from wild birds and human sources may become a problem for the conservation of natural populations and to public health. [ABSTRACT FROM AUTHOR]

Identifying human remains often begins with cleaning and imaging the material. Hot water maceration is used to remove adherent soft tissue from bone and radiographs are taken to better visualize osseous details. Heat and radiation are known to have harmful effects on DNA, but their ability to degrade DNA when used for cleaning and imaging has not been well studied. To better understand their individual and combined effects on the recoverability of DNA from bone, skeletal samples were subjected to (1) hot water maceration (62 °C for 45 min); (2) CT scanning (0.6mm slices, 120 kV, 10.4s); (3) X-ray (50 kVp, 150 mA, 0.03 s, 40 in); and (4) all 3 treatments combined. Forty-eight DNA samples were extracted, quantified and amplified with the AmpFLSTR(®) Identifiler(®) system. Nearly all of the processed samples had reduced RFU values relative to the unprocessed samples, indicating some amount of genetic loss. This loss did not always translate into loss of profile completeness, since only a few samples had a reduction in the number of loci detected after processing. DNA yields were not significantly reduced by any one of the processing methods, however the results indicate that the damaging effects are additive. It is possible that processing may reduce a bone's DNA reservoir and as more procedures are preformed, the pool of available genetic information might be diminished. Many intrinsic and extrinsic factors can affect the recoverability of DNA from bone. Collecting a DNA sample prior to processing avoids the negative effects from hot water maceration and radiological imaging. [ABSTRACT FROM AUTHOR]

In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market and to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owing to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g., loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g., PCR capillary gel electrophoresis, microarray, and Luminex), more accurate quantification of GM targets (e.g., digital PCR), or characterization of partially known (e.g., DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g., NGS) GMO. The benefits and drawbacks of these methods are discussed in this review. [ABSTRACT FROM AUTHOR]

Background: Multiple sclerosis (MS) is a chronic, demyelinating, autoimmune and also complex disease of the central nervous system the etiology of which is not completely defined; but both genetic and environmental factors are regarded as main factors in its susceptibility. HLA-DQB1*0602 is considered as one of the most important genetic factors in MS predisposition but contradictory results have been reported in different populations worldwide. Since there are no data with respect to the correlation of HLA-DQB1*0602 and multiple sclerosis in Khuzestan province, and because of ethnic diversity in Khuzestan province, the aim was to examine the association of this aliele with multiple sclerosis in Khuzestan. Methods: This is a case-control study that evaluated 200 MS patients from Khuzestan and 200 healthy individuals from the same geographical region. DNA extraction was performed by salting out method; in addition, HLA typing was carried out by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP) method. The present study also considered probable association among HLA-DQB1*0602 with sex, ethnicity, and type of disease. Results: Results revealed that distribution of mentioned allele was not statistically different among cases and controls (61.5% vs. 64%, P = 0.605); furthermore, no association was shown between this allele and gender, ethnicity or type of disease. Conclusion: On the whole, our result is consistent with most of the other studies in Iran; but contrasts with most of the studies in European populations. [ABSTRACT FROM AUTHOR]

Background: Diet-induced hepatic steatosis is highly associated with nonalcoholic fatty liver disease, which is related to the development of metabolic syndrome. While advanced stage nonalcoholic hepatic steatosis and steatohepatitis (NASH) result ultimately in fibrosis and cirrhosis, the molecular basis for lipid droplet formation is poorly understood. Common pathways underlie the pathology of mammalian obesity and the zebrafish diet-induced obesity model (DIO-zebrafish) used in this study. Methods: Our analysis involved a combination of transcriptome (DNA microarray) and proteome (two-dimensional electrophoresis) methods using liver tissue from DIO-zebrafish to find candidate genes involved in hepatic steatosis. We conducted intraperitoneal injection (i.p.) of morpholino antisense oligonucleotides (MOs) for each gene into DIO-zebrafish. We also conducted in vitro overexpression in human cells. Additionally, we examined gene expression during feeding experiments involving anti-obesity compounds, creatine and anserine. Results: We found that fatty acid binding protein 3 (fabp3) and E2F transcription factors were upregulated in hepatic steatosis. E2f8 MO i.p. suppressed fabp3 expression in liver, and ameliorated hepatic steatosis. In human cells (HepG2), E2F8 overexpression promoted FABP3 expression. Additionally, co-administration of creatine and anserine suppressed obesity associated phenotypes including hepatic steatosis as indicated by e2f8 and fabp3 down regulation. Conclusion: We discovered that the e2f8--fabp3 axis is important in the promotion of hepatic steatosis in DIO-zebrafish. The combination of transcriptome and proteome analyses using the disease model zebrafish allow identification of novel pathways involved in human diseases. [ABSTRACT FROM AUTHOR]

Background: It has been recognized that infectious agents, such as different bacteria and viruses, may play a role in the developing of rheumatoid arthritis (RA). Recently, the mycoplasma species has been implicated in the pathogenesis of RA. Aim: The aim of this study was to design a multiplex PCR for rapid and simultaneous detection of Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma arthritidis in the synovial fluid of patients with rheumatoid arthritis (RA). Methods: A total of 131 synovial fluid (SF) samples from patients with RA were assayed. Mycoplasma pneumoniae (ATCC: 29342), M. hominis (native strain), and the synthetic complete genome of M. arthritidis mitogen (MAM) superantigen were used as controls. All SF samples were subjected to DNA extraction separately and multiplex PCR was performed. The PCR products were confirmed by sequencing. Results: The designed multiplex PCR was able to detect M. pneumoniae, M. hominis, and M. arthritidis in the SF of patients with RA with a frequency of 30 (22.9%), 23 (17.5%) and 13 (9.9%), respectively. Conclusion: In this study, the overall detection of the Mycoplasma species in RA patients was 53.4%; thus, we recommend the application of multiplex PCR assays when searching for a specific anti mycoplasma treatment for RA patients. [ABSTRACT FROM AUTHOR]

Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR) loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections. [ABSTRACT FROM AUTHOR]

In the New World, the leishmaniases are primarily transmitted to humans through the bites of Leishmania-infected Lutzomyia (Diptera: Psychodidae) phlebotomine sand flies. Any or both of two basic clinical forms of these diseases are endemic to several cities in Brazil—the American cutaneous leishmaniasis (ACL) and the American visceral leishmaniasis (AVL). The present study was conducted in the urban area of a small-sized Brazilian municipality (Jaboticatubas), in which three cases of AVL and nine of ACL have been reported in the last five years. Jaboticatubas is an important tourism hub, as it includes a major part of the Serra do Cipó National Park. Currently, no local data is available on the entomological fauna or circulating Leishmania. During the one-year period of this study, we captured 3,104 phlebotomine sand flies belonging to sixteen Lutzomyia species. In addition to identifying incriminated or suspected vectors of ACL with DNA of the etiological agent of AVL and vice versa, we also detected Leishmania DNA in unexpected Lutzomyia species. The expressive presence of vectors and natural Leishmania infection indicates favorable conditions for the spreading of leishmaniases in the vicinity of the Serra do Cipó National Park. [ABSTRACT FROM AUTHOR]

Evidence of dried blood is very valuable in forensic science. Since the discovery of luminescence with Luminol and dried blood spots (DBSs) in 1928, interest and research on blood have continued to date. One of the most important factor that DBSs have is genes. However, the current use of distilled water (DDW) to collect and extract blood samples has disadvantages related to DNA stability. Therefore, this study aimed to develop an extraction reagent that is most suitable for gene extraction from DBSs. Blood was collected from 45 healthy adult men and women in vacuum blood containers without coagulants or anticoagulants. The collected blood was dried in various settings to check the performance of the extraction reagent. Extraction with Tris-EDTA (TE) and phosphate-buffered saline (PBS) was found more suitable in terms of gene interference effects compared with DDW; their performance was also compared with those of the newly developed extraction reagents. Upon comparing the results of polymerase chain reaction for human genomic DNA samples using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as the target, the performance of the newly developed extraction reagents, modified TE and PBS, was found to be relatively good. To determine the optimal composition of the developed extraction reagents, 12 new extraction reagents were developed with different pH and sodium concentrations. Among them, the best results were found when the DNA was extracted using extraction reagent No. 3 with pH 8.0 and containing 1 M NaCl. Next, the four extraction reagents, DDW, TE, PBS, and No. 3 were compared under nine different temperature and humidity conditions. Similarly, under various environmental conditions, extraction reagent No. 3 performed better than other reagents. It is proposed that modified TE and PBS mixed extraction reagents are the most suitable for collecting and preserving crime site samples. The proposed composition for a DNA extraction reagent can contribute greatly to crime scene reconstruction. [ABSTRACT FROM AUTHOR]