Role of endoplasmic reticulum aminopeptidase-1 gene polymorphism (rs13167972) in occurrence susceptibility of ankylosing spondylitis in a sample of Iraqi male patients.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic and systemic seronegative inflammatory spondyloarthropathy, an autoimmune disease that has been associated with impaired Endoplasmic Reticulum Aminopeptidase (ERAP)-1 activity, which is involved in priming antigenic peptides. The purpose of this study is to investigate the association of 3-UTR of ERAP1 gene polymorphism (rs13167972) with the AS occurrence susceptibility in a sample of Iraqi male patients. METHODS: The AS patients were diagnosed clinically and by magnetic resonance imaging (MRI) and other clinical and laboratory criteria like symptoms, increased C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). The blood grouping and Body Mass Index (BMI) were also investigated to be associated with AS occurrence. The genotyping of the 3-UTR region of the ERAP1 gene (rs13167972) was done by Sanger sequencing. RESULTS: The results revealed that the AS occurred significantly in the age group of 20-35 years (p = 0.013). The BMI shows that the AS patients were overweighted males (p = 0.013) and the most predominant blood group in AS patients was O- (p = 0.002). The ESR and serum level of CRP were significantly raised in AS patient sera (< 0.001). The results of the receiver-operating characteristics curve analysis (ROC) revealed that the CRP (AUC: 0.995, cut-off: 2.48 mg/L, had 95% %sensitivity, 100% specificity, p < 0.001) is more discriminative than BMI (AUC: 0.300, cut-off: 46.91 kg, had 0% sensitivity, 100% specificity, p = 0.001), and ESR (AUC: 0.808, cut-off: 7.50 mm/hr, had 60% sensitivity, 88% specificity, p < 0.001) in distinguishing between AS patients and control group. The genotyping of the 3-UTR region of ERAP1 gene (rs13167972) result shows that the AG and GG genotypes are significantly occurring in AS patients (70%, OR: 2.33, 95%CI: 1.02-5.36, p = 0.04). The G allele is significantly occurring in AS patients (47%, OR: 2.07, 95CI%: 1.15-3.71, p = 0.01). CONCLUSION: The AS occurred in young overweight males with blood group O-. The AG and GG genotypes are risk factors for AS development while the G allele is a risk factor that increases the chances for disease incidence.

The C/A functional polymorphism of TGF-ß2 gene (rs991967) in primary open angle glaucoma patients.

BACKGROUND: Primary Open-angle Glaucoma (POAG) is a functional disease that.leads to blindness globally. The aims of this study are estimation the importance.of transforming growth factor-beta 2 (TGF-ß2) in the pathogenicity of POAG and.to evaluate the effect of the C/A SNP of the TGF-ß2 gene (rs991967) on POAG development. METHODS: Blood samples and some topographic data were collected from POAG.patients and the controls. The serum level of TGF-ß2 was estimated by ELISA.and the C/A SNP of the TGF-ß2 gene (rs991967) was determined by RFLP-PCR. RESULTS: The males are more susceptible to having POAG (p = 0.0201). The serum.TGF-ß2 is higher in POAG patients as compared with the control (p < 0.0001). The.AA (reference) genotype was the most common in the patients (61.7%). While..CC genotype (45.0%, OR: 0.136, 95%CI: 0.05-0.36, P < 0.0001) and AC..genotypes (41.7%, OR: 0.051, 95%CI: 0.01-0.16, P < 0.001) were most common..in the control group. Moreover, the TGF-ß2 C allele is protective (OR: 0.25,..95%, CI: 0.15-0.44, P < 0.0001). Patients with AA, CC, and AC genotypes have..significantly high levels of TGF-ß2 (P < 0.001) than the control. CONCLUSIONS: The males were more susceptible to acquiring POAG than females,.. especially the elderly. The TGF-ß2 plays important role in the pathogenesis of POAG. The CC and AC genotypes are common in the control and the C allele is a protective factor.

Evidence of anti- K. pneumoniae biofilm activity of novel Entrococcus faecalis enterocin GLHM.

Many enterocins were produced from the lactic acid bacteria, Enterococcus faecalis, they belonged to different types of bacteriocins and have different characteristics. The present study aimed to search for another enterocin and test its ability to inhibit Klebsiella pneumoniae biofilm as compared with the most effective antibacterial agent. E. faecalis isolates were isolated from stools of breastfeeding babies. Klebsiella pneumoniae isolates were from urinary tract infections and urinary catheters. K. pneumoniae isolates showed biofilm formation potential and multidrug resistance phenotype but amikacin was the most effective one. Enterocin production by gene harboring E. faecalis was screened, then enterocin was purified, characterized, and antibacterial activity and MIC of enterocin were determined. Produced enterocin has characteristics differ from other discovered enterocins. Furthermore, the crude and purified enterocin of E. faecalis possess significant antibacterial activity against K. pneumoniae isolates as compare with control (p < 0.05), and antibiofilm activity of enterocin was stronger than the antibiofilm activity of amikacin (P < 0.05), as well as the enterocin, was potent than the amikacin in preventing the formation of biofilm on the catheter. In conclusion, a novel enterocin was produced from Enterococcus faecalis (enterocin GLHM) is proteinous bacteriocin, relatively heat-stable and have full activity at neutral pH and was belong to type II bacteriocin. Enterocin GLHM have anti-K. pneumoniae and anti-K. pneumoniae biofilm significantly better than amikacin.

L-asparaginase from human breast milk Lactobacillus reuteri induces apoptosis using therapeutic targets Caspase-8 and Caspase-9 in breast cancer cell line.

BACKGROUND: Breast cancer is the most fatal type of cancer in women worldwide. Many chemotherapeutics targeted breast cancer however, they have frightening side effects. One method of controlling cancer cell growth is targeting apoptosis. OBJECTIVE: This study aimed to induce apoptosis in breast cancer cells by purifying L-asparaginase from human breast milk Lactobacillus reuteri isolates via inhibition of Caspases 8 and 9. METHODS: The best L. reuteri isolates producing L-asparagine with the highest enzyme activity were identified from human breast milk and chosen for L-asparaginase purification. The MTT cell viability assay used for measure the toxicity of the enzyme. Breast cancer cell line was used to study the effect of the enzyme on the caspase 8 and caspase 9 gene expression. RESULTS: The MTT cell viability assay showed the inhibition rates ranged between 30% and 80%, of cell death, occurred when 3.125, 6.25, 12.5, 25, 50, and 100 µg/ml of the enzyme used and IC50 was 4.305 µg/ml. The breast cell lines were treated with the enzyme at a concentration of IC50 value. The Cas8 and Cas9 genes expression in L-asparagine treated breast cancer cell line at a concentration of IC50 value were upregulated (the fold of gene expression are 2.071 and 1.197 respectively). CONCLUSIONS: Breast milk L. reuteri L-asparaginase induces apoptosis via Cas8 and Cas9 upregulation in the breast cancer cell line. L. reuteri L-asparaginase treatment may be the hopeful approach for the management of breast cancer. Furthermore, the results may highlight the fact that the presence of L-asparaginase-producing L. reuteri isolates in human breast milk may aid in breast cancer improvement or even prevention.

Anti-biofilm and anti-virulence effects of silica oxide nanoparticle-conjugation of lectin purified from Pseudomonas aeruginosa.

Pseudomonas aeruginosa lectin is purified and nanoparticle-conjugated in an attempt to inhibit biofilm formation. Thirteen (23.6%) P. aeruginosa isolates are obtained from chicken meat samples, of which 30.8% are biofilm producers and 69.2% are lectin producers. Lectin is purified 36.8-fold to final specific activity of 506.9 U/mg. Four nanoparticle types are prepared via laser ablation: platinum (Pt), gold (Au), silica oxide (SiO2 ), and tin oxide (SnO2 ). The four types are characterised, and pulse feeding is used to conjugate the lectin and nanoparticles. Pt, Au, SiO2, and SnO2 nanoparticles inhibit biofilm formation, especially SiO2 nanoparticles, which have higher effectiveness when conjugated with purified lectin. SiO2 -conjugated lectin significantly (p < 0.05) inhibits biofilm formation more effectively than control and other nanoparticle-conjugated lectins. Au-, Pt nanoparticle-, and SnO2 -conjugated lectins inhibit biofilm significantly compared with control (p < 0.05), and rhlR gene expression is decreased in the presence of SiO2 -conjugated lectin. Furthermore, lectin and Pt, Au, SiO2 and SnO2 nanoparticles separately, and their conjugated lectins, are effective biofilm inhibitors. Of these, SiO2 -conjugated lectin was most significant as an anti-biofilm. Moreover, virulence factors regulon and RhlR were reduced by SiO2 -conjugated lectin, indicating that this conjugation may also decrease the virulence of P. aeruginosa.

Establishing novel roles of bifidocin LHA, antibacterial, antibiofilm and immunomodulator against Pseudomonas aeruginosa corneal infection model.

Bifidocin LHA, a novel bacteriocin, was extracted from bee honey B. adolescentis and purified. Bifidocin LHA was characterized as a protein in nature, without lipid or carbohydrate moieties, the molecular weight was 16,000 Da protein, heat-stable and active at a wide range of pH values, bactericidal effect, detergent, and solvents did not affect bifidocin activity and can be classified as type II bacteriocin. In vitro, the antibacterial activity of purified bifidocin LHA was significantly higher than crude bifidocin LHA (P < 0.05) against Pseudomonas aeruginosa (P. aeruginosa). The antibiofilm activity of bifidocin LHA was significantly higher than the antibiofilm activity of Amikacin (P < 0.05). In vivo, bifidocin LHA demonstrates a significant decreased in the number of P. aeruginosa in the eye, while complete clearance of P. aeruginosa comparing with the control (P < 0.05) when treating with Bifidobacterium adolescentis and bifidocin LHA together. Bifidobacterium adolescentis and bifidocin LHA treatment together induced substantial elevation of IL10 and IL-12 concentrations (P < 0.01) that helped to prevent damage caused by the inflammatory response. Succeeded to eradicate P. aeruginosa infection improved by histological patterns of the eye tissues. This study indicated Bifidobacterium adolescentis and bifidocin LHA consider as crucial strategies for the practical treatment of eye infection in the future.

Antibacterial immunomodulatory and antibiofilm triple effect of Salivaricin LHM against Pseudomonas aeruginosa urinary tract infection model.

The study sought to purify and characterize a novel bacteriocin from oral L. salivarius and studying the effect of L. salivarius and its bacteriocin against multidrug-resistant (MDR) P. aeruginosa in vivo and in vitro. Saliva Lactobacillus salivarius bacteriocin was prepared and purified. The molecular weight of purified L. salivarius bacteriocin was 13,500Da protein. The antibacterial activity of purified salivaricin LHM was higher than crude (P<0.05) and was active at a wide range of pH values, thermostable and has no lipid or carbohydrate moiety. The antibiofilm activity of salivaricin LHM was observed. In vivo, Lactobacillus salivarius and salivaricin LHM significantly decrease the effect of bacteria in the kidney and bladder, while there is an improvement of P. aeruginosa infection in ureter salivaricin LHM-treated groups (P<0.05). Analysis of serum IL-10 and IL-4 levels revealed salivaricin LHM has prophylaxis effect. In conclusion, salivaricin LHM is protein in nature, without lipid or carbohydrate moieties, heat-stable and active at a wide range of pH values and can be classified as type II bacteriocin. Lactobacillus salivarius and salivaricin LHM has anti-pseudomonas activity, immunomodulatory by increasing pro-inflammatory cytokines and antibiofilm against P. aeruginosa urinary tract infection model in vivo and in vitro.

A Novel Genetic Determination of a Lectin Gene in Iraqi Acinetobacter baumannii Isolates and Use of Purified Lectin as an Antibiofilm Agent.

BACKGROUND: Lectin was initially called hemagglutinin or agglutinin because of its capacity to agglutinate human as well as human erythrocytes. They are a heterogeneous group of proteins or glycoproteins of nonimmune origin. Because of their chemical properties, they have become a useful tool in several fields such as immunology, cell biology, molecular biology, membrane structure, pharmacology, cancer research, clinical chemistry, and genetic engineering. OBJECTIVE: The wide applications of lectins users urged the need to isolate lectins from a new strain of bacteria can produce new and high yield of lectin because the current production of lectin from Pseudomonas spp. is very expensive. The goal of this study was to screen the ability of Acinetobacter baumannii isolates to produce lectin and detection of its phenotypic and genotypic profiles and detection of lectin ability to inhibit ofbiofilm formation. METHODS: Fifty-one isolates from different sources were collected and detected genetically by using the recA gene. Phenotypic detection of lectin by using semi-quantitative analysis and quantitative analysis in microtiter plate. Genotypic detection of lectin by designed lec gene and used PCR technique. The lectin was extracted by using glass beads and purified by chromatographyic technique followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for determination the molecular size of lectin and finally detection the spectrum of biofilm inhibition by the purified lectin toward biofilm producers. RESULTS: Of 51 A. baumannii isolates, 17 (33.3%) have been found to produce lectin. Ten of 17 were sequenced, of which 2 were submitted and tested by the gene bank National Center for Biotechnology Information (NCBI), and accession numbers (KX766405.1 and KX766406.1) were obtained. These 17 isolates were phenotypically and genotypically positive for lectin and showed different lec gene expression in semi-quantitative and quantitative analysis. The activities ranged between 4-128 U/mL. Lectin purified by ammonium sulfate precipitation was used to inhibit biofilm formation. We found reduction at three different types of bacteria ranging from 26% for Klebsiella pneumonia, 46.7% for P. stutzeri and 53% for A. baumannii. These results suggested that lectin has a promising application as an antibiofilm agent to combat the growing number of multidrug-resistant pathogen-associated infections. CONCLUSIONS: Lectin has been detected recently in A. baumannii, but the genetic property of this lectin has not yet been fully studied. In our study, we determined the presence of the lectin gene (lec gene) in A. baumannii by using PCR technique, and lec PCR products were identified with various source of isolation and sequenced to screening for epidemiology and submitted to the gene bank NCBI under accession number (KX766405.1 and KX766406.1). HIGHLIGHTS: A. baumannii has an ability to produce lectin protein; Lec gene was detected in A. baumannii, and the sequence was recorded under accession number KX766405.1 and KX766406.1.; Lectin was extracted by glass beads and purified by chromatographyic technique; Lectin had strong effect against biofilm formation.

RAPD- and ERIC-Based Typing of Clinical and Environmental Pseudomonas aeruginosa Isolates.

Pseudomonas aeruginosa is a major cause of nosocomial infection in children and adults, resulting in significant morbidity and mortality due to its ability to acquire drug resistance. The ability of P. aeruginosa in the environment to cause infection in individuals has been reported previously; henceforth, surveillance of the emergence and transmission of P. aeruginosa strains among patients is important for infection control in a clinical setup. Various gene-typing methods have been used for epidemiological typing of P. aeruginosa isolates for the purpose of surveillance. In this work, the suitability and comparability of two typing methods, enterobacterial repetitive intergenic consensus (ERIC)-PCR and random amplification of polymorphic DNA (RAPD)-PCR fingerprinting, were studied to characterize P. aeruginosa strains isolated from clinical and environmental sources. Forty-four clinical and environmental bacterial isolates of P. aeruginosa were collected between October 2015 and January 2016. DNA extraction, ERIC-PCR and RAPD-PCR, agarose gel electrophoresis, and phylogenetic analyses were carried using the unweighted pair-group method with mean. RAPD typing revealed less clonality among clinical isolates, whereas the ERIC method showed greater similarity in comparison with RAPD. Environmental isolates, however, showed greater similarity using RAPD compared with ERIC typing. With only a few exceptions, most clinical isolates were distinct from environmental isolates, irrespective of the typing method. In conclusion, both the RAPD and ERIC typing methods proved to be good tools in understanding clonal diversity. The results also suggest that there is no relationship between clinical and environmental isolates. The absence of clonality among the clinical isolates may indicate that most P. aeruginosa infection cases could be endemic and not epidemic and that endemic infections may be due to nonclonal strains of P. aeruginosa.