ABSTRACT
Background: osteoarthritis [OA] is a progressive, age-associated disease that is characterized with cartilage destruction, subchondral bone remodeling and inflammation of the synovial membrane. Considering the complications of common treatments of OA, including non-steroidal anti-inflammatory drug [NSAIDs] and corticosteroids, investigating new treatments for this disorder is crucial. Recently, the role of matrix metalloproteinases [MMPs] expression in pathogenesis of OA has attracted attention
Objective: this study aimed to explore the effect of punicic acid [PA] in inhibition of MMPs gene expression in LPS-stimulated Bovine Fibroblast-like synoviocytes [BFLS] as a model of OA
Methods: in the first stage, the toxicity of PA was measured using MTT assay on BFLS cells. Afterward, the cells were stimulated by LPS [Lipopolysaccharide] and MMPs [Matrix Metalloproteinase] expression level in the BFLS cells were investigated using Real-Time PCR, in vitro Migration and Gelatin Zymography, Western Blot Analysis, ELISA Assay and Invasion Assay
Results: the results showed that PA significantly decreased MMP-9 expression levels in LPS stimulated BFLS cells; also, it suppressed migration and invasion of the mentioned cells. However, PA had no significant effect on MMP-1-2-3
Conclusion: based on our results PA could significantly reduce the activity and inflammatory effect of MMP-9 in OA, its potential role as a supplementary agent to common NSAIDs and corticosteroids was confirmed. Nonetheless, cellular modeling does not significantly confirm the beneficial effect of OA in patients
ABSTRACT
There are many techniques to knock out directed genes in bacteria, some of which have been described in Salmonella species. In this study, a combination of SOEing PCR method and the lamda Red disruption system were used to disrupt phoP gene in wild type and standard strains of Salmonella typhimurium. Three standards PCR and one fusion PCR reactions were performed to construct a linear DNA including upstream and downstream of phoP gene and Kanamycin cassette. As a template plasmid, we used pKD4 which carries kanamycin gene flanked by FRT [FLP recognition target] sites. The resulting construct was electroporated into prepared competent cells of S. typhimurium. The transformants colonies related to the standard strain appeared on the LB-Km-agar plates after incubation, but there was no colony on LB-Km-agar plates corresponding to the wild type strain. The failure in transformation of the wild type strain may be because of inflexibility of the lamda Red disruption system in this strain or its unique restriction-modification system. However, by this construct we are able to generate phoP mutant in many of the Salmonella species due to high homology of the phoP gene which exists in different species
ABSTRACT
Among all common techniques in site directed mutagenesis, Lambda Red recombinase system has been widely used to knock out chromosomal genes in bacteria. In this method, there is always the risk of DNA Linear digestion by host's restriction enzymes that leads to the low frequency of recombination. To overcome this, we constructed a recombinant vector to disrupt phoP gene in Salmonella typhimurium. The SOEing PCR method and restriction enzymes were used to construct the vector. The resulting plasmid, pTAAZ92, contains a Kanamycin cassette with two long homologous arms flanking of the phoP gene. After electrotransformation of the pTAAZ92 into the Salmonella typhimurium, the phoP gene is replaced by the Kanamycin cassette through homologous recombination. According to the high homology of the phoP gene in many of Salmonella species the pTAAZ92 can be used to disrupt the phoP gene in most of these species
Subject(s)
DNA, Recombinant , Genetic Vectors , MutagenesisABSTRACT
Clostridium difficile has been identified as a pathogen in antibiotic associated diarrhea [AAD], pseudomembranous colitis and also nosocomial diarrhea. The present study was performed to find the prevalence of toxigenic strains of C .difficile isolated from diarrhea patients hospitalized in Tehran hospitals. A total of 98 fecal samples obtained during July to December 2010 were studied. Samples were rapidly cultured on the CCFA medium and incubated at the anaerobic conditions. Then ELISA was done to detect toxin A and B in the stool. Molecular identification of C.difficile was done by cdd3 universal primer and toxin A gene [tcdA], toxin B gene [tcdB] and binary toxin profiles were determined by PCR method. From a total of 98 fecal samples, 15 samples [15.3%] were positive of which, 12 strains [21.2%] were A+B+, 2 strains [2%] were A+B-, and 1 strain [1%] was A-B+. This study showed that Clostridium difficile is an important pathogen in the development of nosocomial diarrhea. Therefore, routine detection of C.difficile in suspected cases is recommended
Subject(s)
Humans , Hospitalization , Diarrhea , Prevalence , Polymerase Chain ReactionABSTRACT
Photodynamic therapy is a treatment that uses photosensitizer and intense visible light. When photosensitizers get exposed to a specific light wavelength [preferentially in the red region], they produce reactive oxygen species that are toxic to cells. Recently, attention has been focused on porphyrins and their analogs as photosensitizers. Zn [II] tetrapyridinoporphyrazin complex is a water-soluble photosensitizer that has a good potential for application in photodynamic therapy. In this study, phototoxic effect of this complex on HeLa cancer cell line has been investigated. HeLa cell cultures were treated with different concentrations of Zn [II] tetrapyridinoporphyrazin. The cytotoxic effects were measured both in the presence and absence of light using the MTT assay. The light source was a 150W tungsten halogen lamp equipped with a red filter. Our data indicate that porphyrazine's photocytotoxicity is remarkably more significant than its cytotoxycity in the dark. Statistical analysis showed the effective dose [ED[50]] values in the dark and light conditions were 8.6 and 4.2 microM, respectively. In addition, the results imply that in the range of 0-12 microM, the increase in the complex concentration correlates with the increase in the cytotoxicity effect. However, the cytotoxicity decreases at the higher concentration [50microM], which is likely due to aggregation of the complex. Our results show that Zn [II] tetrapyridinoporphyrazin complex may be a promising photosensitizer for innovative photodynamic therapy and may have a high potential application in cancer treatment. Furthermore, it seems to have more benefits compared to other known photosensitizers
ABSTRACT
An immunosuppressed man was admitted to hospital with diarrhea and a history of urinary tract infection. He was subjected to treatment with antibiotics. The patient died of putative severe sepsis. The etiological agent was a carbapenemase producing isolate of Bacillus circulans with resistance to all prescribed antimicrobial agents
ABSTRACT
Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determination of subtypes of Cryptosporidium among children with diarrhea in Tehran by sequence analysis of the highly polymorphic 60-kDa glycoprotein [GP60] gene. Fecal samples were collected from 794 diarrheic children. Initial identification of Cryptosporidium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly. Out of 794 collected samples, 19 [2.40%] were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 [89.47%] of the positive isolates were Cryptosporidium parvum and 2 [10.52%] were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa [6/17] and IId [11/17]. The most common allele in all 17 isolates belonged to IId A20G1a [41.18%]. A22G1 [IF] subtype was detected in two C. hominis isolates of the children. The predominancy of C. parvum species [specially, IId A20G1a subtype] in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran