Apparent histological changes of adipocytes after treatment with CL 316,243, a β-3-adrenergic receptor agonist

Background and objectives: The objective of this experiment was to study the effect of CL 316,243 (CL) (a highly selective β3-adrenergic receptor agonist) on cellular changes occurring in retroperitoneal white adipose tissue (RWAT) of lean and obese rats.Methods: Ten-month-old lean and obese Zucker rats were implanted subcutaneously with osmotic mini-pumps, infusing either saline or CL (1 mg/kg body weight/day) for 4 weeks. Results: There was no effect of CL on food intake. However, the resting metabolic rate in lean and obese rats increased by 55 and 96 per rat, respectively. Total RWAT weight decreased in both lean and obese rats under influence of CL treatment by 65 and 38, respectively. Total body weight and body fat were lower in CL treated rats. Detection of uncoupling protein 1 (UCP1) in RWAT was confirmed qualitatively by both immunohistochemistry and immunofluorescence using a rabbit anti rat UCP1 antibody which showed the appearance of a marked increase of this protein in the adipose tissue. Stained semi-thin sections (0.5 µm) also demonstrated abundant nuclei in multilocular adipocytes, in endothelial cells associated with the vasculature, and in interstitial cells. In CL-treated obese rats, a clustering of several multilocular cells around the periphery of a white adipocyte was seen.Conclusion: These results indicate that treatment of both lean and obese Zucker rats with CL induces extensive remodeling of RWAT that includes shrinkage of white adipose tissue, appearance of abundant multilocular cells in RWAT together with the appearance of a marked increase of UCP, preferentially in lean rats. © 2015 Ghorbani et al

The inhibitory effect of a Lactobacillus acidophilus derived biosurfactant on Serratia marcescens biofilm formation

Objective(s): Serratia marcescens is one of the nosocomial pathogen. The ability to form biofilm is an important feature in the pathogenesis of S. marcescens. The aim of this study was to determine the anti�adhesive properties of a biosurfactant isolated from Lactobacillus acidophilus ATCC 4356, on S. marcescens strains. Materials and Methods: Lactobacillus acidophilus ATCC 4356, was selected as a probiotic strain to produce biosurfactant. Anti�adhesive activities was determined by pre�coating and co� incubating methods in 96�well culture plates. Results: The FTIR analysis of derived biosurfactant revealed the composition as protein component. Because of the release of such biosurfactants, L. acidophilus was able to interfere with the adhesion and biofilm formation of the S. marcescens strains. In co� incubation method this biosurfactant in 2.5 mg/ml concentration showed anti�adhesive activity against all tested strains of S. marcescens (P<0.05). Conclusion: Our results show anti�adhesive properties of L. acidophilus biosurfactant will be useful against microorganisms responsible for infections in the urinary, vaginal and gastrointestinal tracts, as well as skin, making it a suitable alternative to conventional antibiotics. © 2015, Mashhad University of Medical Sciences. All rights reserved

Transfection of an expressive construct including IgG1 and Fv1 genes in ovary cell line for infliximab expression

Background: Infeliximab is a form of chimeric antibody which neutralizes the most important inflammatory cytokine, TNF-α, in inflammatory disorders. The aim of current study was to pilot expression of chimeric infliximab in Chinese Hamster ovary (CHO) cells. Methods: In this research study, pVITRO2-neo-mcs vector that consist of infliximab light chain and heavy chain was used to transform into the E.coli by CaCl2 method. The plasmid was then purified and transfected to cultured CHO cells by Lipofectamine 2000® (Invitrogen GmbH, Germany). Transfected cells were selected upon G-418 treatment after 2 weeks and the level of expression, based on standard curve, was measured using IgG ELISA kit after 48 hours for each clone. High level expressed clone was then cultured in roller bottles and recombinant chimeric product was purified by protein A affinity chromatography. The purity of the product was analyzed by 10 gel SDS-PAGE from eluted samples. The efficacy of the purification was analyzed by ELISA before and after purification step. This article is a master's student thesis from February 2015 to August 2016 in pharmaceutical technology development center, Tehran University of Medical Sciences, Tehran, Iran. Results: The purified plasmid was analyzed on 2 agarose gel. After selective pressure of G-418, 10 stable transfect clones were assessed for infliximab secretion by IgG ELISA kit at 450 nm. The maximum and minimum expression which detected by ELISA were 23 ng/ml and 6 ng/ml, respectively. The band width of infliximab fraction during purification procedure was observed at 0.7-0.8 min. The efficiency of the purification by ELISA was 70. On SDS-PAGE analysis, two bands, 25 and 50 kDa, respect to light and heavy chains of Infliximab, was confirmed the expression of recombinant protein. Conclusion: In the current study, the construct for infliximab monoclonal antibody production was designed using genetic engineering techniques and the expression was confirmed by conventional molecular biology methods. The high yield production was carried out in semi-industrial scale using roller bottles with a 70 percentage of purification efficiency. © 2016, Tehran University of Medical Sciences. All rights reserved

The Founder Effect? -FXIII Deficiency in Southeast Iran: A Molecular Study Report

Background: Congenital factor XIII (FXIII) deficiency is an extremely rare bleeding disorder (RBD) with different clinical coagulation disorders and great impacts on the perioperative patient outcome. Its prevalence in Southeast Iran is approximately 4,000 times higher than the worldwide prevalence, with Trp187Arg (c.559T&gt; C as the only causative mutation of FXIIID there. We investigated the founder effect of rs1742924, rs4960181, rs3778360 and rs4142290 using haplotype analysis to define the genetic phenomenon in this geographic region. Materials and Methods: In a case-control study, 10 patients with FXIIID and 10 healthy individuals were assessed. Initially, Trp187Arg (c.559T&gt; C) mutation was assessed in all study populations using a PCR-RFLP technique, then haplotype analysis was performed by assessing rs1742924, rs4960181, rs3778360 and rs4142290 polymorphisms. Data were analyzed using a two-proportion z-test. Results: All patients were homozygote for Trp187Arg (c.559T&gt;C), and this mutation was not observed in any form of homozygote or heterozygote in the control group. Polymorphisms in rs1742924, rs4960181, and rs377836 were homozygote (TT, GG, GG, respectively) and T, G, and G alleles distribution in cases and controls with significant difference (P&lt;0.001, P&lt;0.001, and P=0.01 respectively). Rs4142290 polymorphism showed no significant difference between patients and controls (P=0.3). Two types of haplotypes were observed in the case group, and haplotype number 1* was observed among 90% of them, while not observed in the control group. Conclusion: It seems that founder effectors of haplotype number *1 have more antiquity versus other haplotypes, and probably founder effect is responsible for this high prevalence of FXIIID in the southeast of Iran

Hereditary ataxia with a novel mutation in the Senataxin gene: A case report

Hereditary ataxias (HA) are a group of inherited neurological disorders caused by changes in genes. At least 115 different mutations in the senataxin (SETX) gene causing ataxia have been identified. There are no reports of any SETX gene mutation among the Iranian population. Here we report on two cases with homozygous and heterozygous mutations in which one patient was affected by HA with oculomotor apraxia type 2, and the other was a carrier of the disorder. In 2016, the affected patient was referred to the Biogene Medical and Genetic Laboratory (Tehran, Iran) suffering from imbalance and tremor of both head and body. The coding regions of 18 genes, including the SETX gene, were screened. The target regions were captured using the NimbleGen chip followed by next-generation sequencing (NGS) technology on the Illumina Hiseq2500 platform. NGS, a DNA sequencing technology, has greatly increased the ability to identify new causes of ataxia; a useful tool for the prevention of primary manifestations and treatment of affected patients. In the present study, a novel mutation in the SETX gene has been identified. © 2019, Shiraz University of Medical Sciences. All rights reserved

Effects of oral probiotic feeding on toll-like receptor gene expression of the chicken's cecal tonsil

Background: It was proposed that probiotics may influence immune system through direct or indirect exposure. Direct exposure is mostly mediated by surface receptors. Toll-like receptors (TLRs) are conserved molecular sensors which could be triggered via some pathogen associated structures, hence, modulate the immune responses. This study was conducted to elucidate the impact of lactobacillus acidophilus as a common probiotic on the expression level of TLRs in the chicken's cecal tonsil. Methods: Thirty one-day-old chicken were selected and separated into three groups as probiotic-fed, dairy-fed and control. In addition to commercial powder supply, each chicken in the probiotic-fed group received 109 CFU/Kg of L. acidophilus daily. While, chickens in the dairy-fed group were provided with commercial powder feed and sterile dairy milk. After 14 and 21 days of oral feeding the cecal tonsil was removed and the expression of TLR2, TLR4 and TLR5 were examined by real-time PCR. Results: At the age of 14-day, there was a slight upregulation in the expression levels of TLR2 (118.9), TLR4 (129.6) and TLR5 (123.7) of the cecal tonsil in the probiotic-fed group; however, these alterations were not statistically significant. At the age of 21-day, a non-significant downregulation was observed in TLR expression level of both dairy-fed (TLR2, 85; TLR4, 79.5; and TLR5, 86.5) and probiotic-fed (TLR2, 88.8; TLR4, 81; and TLR5, 87.2) groups in comparison to controls. Conclusions: The findings revealed that although the probiotic supplementation could be useful but it did not significantly affect innate immunity state through alteration of TLRs

Effects of oral probiotic feeding on toll-like receptor gene expression of the chicken's cecal tonsil

Background: It was proposed that probiotics may influence immune system through direct or indirect exposure. Direct exposure is mostly mediated by surface receptors. Toll-like receptors (TLRs) are conserved molecular sensors which could be triggered via some pathogen associated structures, hence, modulate the immune responses. This study was conducted to elucidate the impact of lactobacillus acidophilus as a common probiotic on the expression level of TLRs in the chicken's cecal tonsil. Methods: Thirty one-day-old chicken were selected and separated into three groups as probiotic-fed, dairy-fed and control. In addition to commercial powder supply, each chicken in the probiotic-fed group received 109 CFU/Kg of L. acidophilus daily. While, chickens in the dairy-fed group were provided with commercial powder feed and sterile dairy milk. After 14 and 21 days of oral feeding the cecal tonsil was removed and the expression of TLR2, TLR4 and TLR5 were examined by real-time PCR. Results: At the age of 14-day, there was a slight upregulation in the expression levels of TLR2 (118.9), TLR4 (129.6) and TLR5 (123.7) of the cecal tonsil in the probiotic-fed group; however, these alterations were not statistically significant. At the age of 21-day, a non-significant downregulation was observed in TLR expression level of both dairy-fed (TLR2, 85; TLR4, 79.5; and TLR5, 86.5) and probiotic-fed (TLR2, 88.8; TLR4, 81; and TLR5, 87.2) groups in comparison to controls. Conclusions: The findings revealed that although the probiotic supplementation could be useful but it did not significantly affect innate immunity state through alteration of TLRs

Serum overexpression of miR-301a and miR-23a in patients with colorectal cancer

BACKGROUND: Extracellular vesicles (EVs) are a heterogeneous group of membrane-bound vesicles with complex cargoes including proteins, lipids, and nucleic acids. EVs have received significant attention due to their specific features including stability under harsh conditions and involvement in cell-to-cell communication. Circulating EVs and the molecules associated with them are important in the diagnosis and prognosis of cancers. MicroRNAs (miRNAs) are a group of small noncoding RNAs that have a role in regulating gene expression. Current literature shows that circulating miRNAs can be used as noninvasive biomarkers for early detection of cancers. The present study was set to investigate the potential role of serum exosomal miRNA expression levels in colorectal cancer (CRC) patients and evaluate their correlation with clinicopathologic features. METHODS: Exosome-enriched fractions were isolated from the serum of 25 CRC patients and 13 age- and sex-matched healthy controls using a polymer-based precipitation method. During the pilot phase, real-time polymerase chain reaction (RT-PCR) was carried out on 12 CRC patients and eight healthy participants to evaluate the expression difference of 11 candidate miRNAs between CRC patients and tumor free subjects. Finally, the results were validated in a separate group, which was similar in size to the pilot group. The clinicopathologic data were also collected and the relationship between aberrant miRNA expression and clinicopathological parameters were investigated. RESULTS: There were high expressions of exosomal miR-23a and miR-301a in serum samples of CRC patients compared to normal controls in training and validation phases; these differences were not significantly correlated with clinicopathologic features. Receiver operating characteristic curve analysis showed that miR-301a and miR-23a were able to discriminate CRC patients from normal subjects. CONCLUSION: The findings provide evidence on the roles of miR-301a and miR-23a in CRC development and their potential roles as noninvasive biomarkers for early detection of CRC

Antagonistic activities of some probiotic lactobacilli culture supernatant on serratia marcescens swarming motility and antibiotic resistance

Background and Objectives: Serratia marcescens, a potentially pathogenic bacterium, benefits from its swarming motility and resistance to antibiotic as two important virulence factors. Inappropriate use of antibiotics often results in drug resistance phenomenon in bacterial population. Use of probiotic bacteria has been recommended as partial replacement. In this study, we investigated the effects of some lactobacilli culture supernatant on swarming, motility and antibiotic resistance of S. marcescens. Materials and Methods: Antimicrobial activity of lactobacilli supernatant and susceptibility testing carried out on S. marcescens isolates. Pretreatment effect of lactobacilli culture supernatant on antibiotic - resistance pattern in S. marcescens was determined by comparison of the MIC of bacteria before and after the treatment. Results: Our results showed that pretreatment with L. acidophilus ATCC 4356 supernatant can affect the resistance of Serratia strains against ceftriaxone, but it had no effect on the resistance to other antibiotics. Furthermore, culture supernatant of lactobacilli with concentrations greater than 2, had an effect on the swarming ability of S. marcescens ATCC 13880 and inhibited it. Conclusion: Probiotic bacteria and their metabolites have the ability to inhibit virulence factors such as antibiotic resistance and swarming motility and can be used as alternatives to antibiotics. © 2018, Tehran University of Medical Science. All rights reserved

Mutation characterization and heterodimer analysis of patients with leukocyte adhesion deficiency: Including one novel mutation

Abstract Background and aim Leukocyte adhesion deficiency type 1 (LAD-I) is a rare, autosomal recessive disorder of neutrophil migration, characterized by severe, recurrent bacterial infections, inadequate pus formation and impaired wound healing. The ITGB2 gene encodes the β2 integrin subunit (CD18) of the leukocyte adhesion cell molecules, and mutations in this gene cause LAD-I. The aim of the current study was to investigate the mutations in patients diagnosed with LAD-I and functional studies of the impact of two previously reported and a novel mutation on the expression of the CD18/CD11a heterodimer. Materials and methods Blood samples were taken from three patients who had signed the consent form. Genomic DNA was extracted and ITGB2 exons and flanking intronic regions were amplified by polymerase chain reaction. Mutation screening was performed after Sanger sequencing of PCR products. For functional studies, COS-7 cells were co-transfected with an expression vector containing cDNA encoding mutant CD18 proteins and normal CD11a. Flow cytometry analysis of CD18/CD11a expression was assessed by dimer-specific IB4 monoclonal antibody. Results Two previously reported mutations and one novel mutation,p. Cys562Tyr, were found. All mutations reduced CD18/CD11 heterodimer expression. Conclusion Our strategy recognized the p.Cys562Tyr mutation as a pathogenic alteration that does not support CD18 heterodimer formation. Therefore, it can be put into a panel of carrier and prenatal diagnosis programs. Keywords Leukocyte adhesion deficiency type I Immunodeficiency Mutation Genetic testing CD1