A novel mutation in GJC2 associated with hypomyelinating leukodystrophy type 2 disorder

Hypomyelinating leukodystrophy type 2 (HLD2), is an inherited genetic disease of the central nervous system caused by recessive mutations in the gap junction protein gamma 2 (GJC2/GJA12). HLD2 is characterized by nystagmus, developmental delay, motor impairments, ataxia, severe speech problem, and hypomyelination in the brain. The GJC2 sequence encodes connexin 47 protein (Cx47). Connexins are a group of membrane proteins that oligomerize to construct gap junctions protein. In the present study, a novel missense mutation gene c.760G>A (p.Val254Met) was identified in a patient with HLD2 by performing whole exome sequencing. Following the discovery of the new mutation in the proband, we used Sanger sequencing to analyze his affected sibling and parents. Sanger sequencing verified homozygosity of the mutation in the proband and his affected sibling. The autosomal recessive inheritance pattern was confirmed since Sanger sequencing revealed both healthy parents were heterozygous for the mutation. PolyPhen2, SIFT, PROVEAN, and CADD were used to evaluate the function prediction scores of detected mutations. Cx47 is essential for oligodendrocyte function, including adequate myelination and myelin maintenance in humans. Novel mutation p.Val254Met is located in the second extracellular domain of Cx47, both extracellular loops are highly conserved and probably induce intramolecular disulfide interactions. This novel mutation in the Cx47 gene causes oligodendrocyte dysfunction and HLD2 disorder.

OX40 gene and serum protein expression profiles in patients with Parkinson's disease

Objective: Inflammation of the immune system and the central nervous system has been known as an important predisposing factor for Parkinson's disease [PD]. Increased expression of OX40 protein [CD134] is a known factor for increased inflammation and initiation of NF-kappa-B signaling pathway in different diseases. We aimed to investigate the expression of OX40 at the transcript and serum protein levels

Materials and Methods: Twenty individuals with PD and 20 healthy individuals, as controls, were enrolled in this casecontrol study. Expression of OX40 at the transcript level and serum protein levels were measured by quantitative real-time polymerase chain reaction [qRT-PCR] and enzyme-linked immunosorbent assays respectively

Results: The mean expression level of OX40 was increased in patients but not at a significant level [P>0.05]. Consistently, the mean serum concentration of OX40 showed a mild, but non-significant, increase in the patients [P>0.05]

Conclusion: We conclude that OX40 expression at either the transcript or protein level has no diagnostic utility in asymptomatic PD. This shows the need for clinical, cellular and interventional research to detect new robust biomarkers

RORA and autism in Isfahan population: is there an epigenetic relationship

Objective: Autism is a neurodevelopmental disorder characterized by difficulty in verbal and non-verbal communication, impaired social interaction, and restricted and repetitive behavior. It has been recently introduced as a multigenic disorder with significant epigenetic effects on its pathology. Recently, epigenetic silencing of retinoic acid receptor-related orphan receptor alpha [ROR alpha] gene [which has an essential role in neural tissue development] was shown to have occurred in autistic children due to methylation of its promoter region. This may thus explain a significant part of the molecular pathogenesis of autism. Therefore, we aimed to confirm this finding by implementing a case-control [experimental] study in the population of Isfahan

Materials and Methods: The methylation status of a 136 bp sequence of a GpG island [encompassing 13 CpG sites] in the RORA promoter region [positions -200 to -64] as an experimental study was examined in the lymphocyte cells of 30 autistic children after sodium bisulfite treatment using the melting curve analysis-methylation [MCA-Meth] assay compared with normal children. Also, quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR] analysis was used to estimate the level of mRNA transcripts and to evaluate MCA-Meth analysis results

Results: This study revealed no methylation in the examined promoter regions in both autistic and normal children, with the melting curve of all studied samples being comparable to that of the non-methylated control. The results of MCA-Meth analysis were also consistent with qRT-PCR results. We therefore observed no significant difference in the levels of ROR alpha transcripts in the blood lymphocytes between autistic and healthy children

Conclusion: The methylation of the RORA promoter region may not be considered as a common epigenetic risk factor for autism in all populations. Hence, the molecular pathogenesis of autism remains unclear in the population investigated

Altered expression of KLC3 may affect semen parameters

Background: KLC3 protein as a member of the kinesin light-chain protein family plays an important role in spermatogenesis, during formation of mitochondrial sheath in the mid piece of the sperm tail

Objective:This study for the first time aims to compare the expression of the KLC3 gene between fertile and infertile individuals

Materials and Methods:Semen samples were collected from 19 fertile individuals who were selected from embryo-donor volunteers and 57 infertile individuals who had abnormal sperm parameters according to world health organization criteria. Sperm parameters using computer assisted sperm analysis and the quantitative KLC3-gene expression using the real-time PCR method were measured

Results: Our results revealed a significant correlations between sperm concentration with relative expression of KLC3 only in infertile groups [r=0.45, p=0.00]. A significant correlation was not found between KLC3 expression and sperm motility; however, the relative expression of KLC3 was significantly higher in asthenozoospermic compared to non-asthenozoospermic individuals

Conclusion: Low expression of KLC3 may result in improper function of midpiece, which has important function in sperm motility. The results of this study show that aberrant expression of KLC3 might be associated with phenomena like oligozoospermia and asthenozoospermia. This article is extracted from student's thesis

effect of plasma exchange on theexpression of FOXP3 and rorc2 inrelapsed multiple sclerosis patients

Background: Lack of sufficient information on the mechanism of plasma exchange[PE] therapy in multiple sclerosis [MS], has limited this treatment to individual patientswith severe relapses who have been refractory to other treatments. This is while PE isused very successfully as a first-line standard treatment in many other neuro-immunedisorders. Recent data suggest that Treg/Th17 counterbalance may indicate theboundaries between promotion and regulation of inflammatory responses in MS andTreg/Th17 ratio may be useful as a marker for monitoring the efficiency of MStherapies

Objective: To evaluate the effect of PE on the frequency and ratio ofTreg/Th17 cells through concomitant measurement of the expression levels of Treg andTh17 lineage specific transcription factors, FOXP3 and RORC2, respectively

Methods: Peripheral blood mononuclear cells of 8 relapsed MS patients were obtainedbefore and after a complete course of PE therapy and the FOXP3 and RORC2 mRNAlevels were assayed using real-time PCR approach

Results: No significant change inthe expression levels of individual transcription factors existed, but a significantincrease in FOXP3/RORC2 ratio [p=0.036] was observed

Conclusions: Our resultssuggest that PE therapy influences Treg/Th17 ratio and this maybe a mechanism bywhich this procedure exerts its improving effects in MS disease

P-glycoprotein a gene expression in glucantime-resistant and sensitive leishmania major [MRHO/IR/75/ER]

Leishmaniasis is a parasitic disease caused by different species of Leishmania parasites with a wide range of clinical manifestations. Antimonial compounds such as meglumine antimoniate [glucantime] are the first line drugs for the treat-ment of leishmaniasis. However, according to reports of the drug resistance of parasites, the efficacy of antimonial compounds is low. The ATP-binding cassette [ABC] proteins are present in all organisms and mediate the transport of vital elements through biological membranes. One of the important mechanisms of resistance in Leishmania parasites is the overexpression of ABC efflux pumps. P-glycoprotein A [pgpA] is a related gene for ABC transporter in Leishmania species. The aim of this study was to compare the pgpA expression in laboratory-induced resistant L. major [MRHO/IR/75/ER] and sensitive parasites. RNA extraction of promastigotes of sensitive and resistant clones was performed and total RNA was reverse transcribed. The real-time quantitative polymerase chain reaction [PCR] was used to assess RNA expression profiles and the expression levels were calculated using 2-deltaCt method. The mean expression level of pgpA mRNA was 2.70 +/- 0.51 in in sensitive Leishmania clone and 6.08 +/- 1.50 in resistant Leishmania clone [P = 0.021]. The expression of pgpA gene in resistant strains of L. major was almost fivefold higher than those in susceptible strains. Therefore, this can be used in field isolates, i.e. overexpression of the gene can prove resistance in wild type field isolates

Optimization of human immunodeficiency virus type one replication assay using single cycle replicable virions

HIV virions with replication capacity are needed for HIV researches, like investigating for new anti HIV agents. Here HIV-1 replication assay was optimized with HIV-1 single cycle replicable [SCR] virions to improve biological safety condition. pSPAX2, pmzNL4-3 and pMD2G plasmids were co-transfected to the HEK293T by using polyfect reagent to produce the SCR HIV-1 virions. Virions were quantified using capture ELISA P24. Different MOI of SCR virions were used for infecting of Target cells [HEK] and the load of the supernatant P24 was monitored days after infection. Single cycle replication assay [SCRA] was developed using kinetic studies data. The P24 load of the infected cells supernatant has linear relation to the beginning infectious MOI. 24 hours post infection with HIV-1 SCR virions the viral particle production was detectable. The highest load of P24 in infected cells supernatant was detected 48 hours after infection. Using this developed method, the 50 and 95 percent inhibitory concentration of [IC[95] and IC[50]] Indinavir and Nevirapine were calculated as 25nM and 50nM. In this study, using SCR HIV-1 virions the SCRA was developed. SCR HIV-1 virions are replicable only for one cycle and this improves the safety of developed assays. The accuracy of assay was examined by quantifying the anti HIV-1 potential of two commercial anti-AIDS drugs and the calculated activity for test agents was equal to previously known amounts

Distribution of HLA-B*27 alleles in patients with ankylosing spondylitis in Iran

HLA-B*27 is strongly associated with ankylosing spondylitis [AS]. It represents a family of alleles that differ among ethnic groups. The aim of this study was to determine the distribution of HLA-B*27 alleles in AS patients and healthy controls in Isfahan [Iran]. Sixty AS patients and 430 healthy blood donors were selected. All subjects were HLA-B*27 positive by flow cytometry. HLA-B*27 subtypes were determined by PCR-SSP. Forty patients [66.7%] and 17 controls [3.95%] were HLA-B*27 positive. Subtypes detected by PCR-SSP were B*2705, B*2702, B*2704 and B*2707. One patient was B*2702/B*2710. No significant difference was found in the distribution of these alleles between AS patients and controls. Although Caucasian subtypes are predominant among Iranians, this population is characterized by a combination of both specific Caucasian and Oriental subtypes. However such results should be interpreted carefully because of the small sample size in our investigation and definitive conclusion awaits more ethnic-group studies

Emergence of cefepime resistance in gram-negative induced nosocomial infections

The rapid emergence of antibiotic resistance, especially broad-spectrum antibiotics, resulted in the avid use of new potent antibiotics. Ceftriaxone and ceftazidime, two third-generation cephalosporin, are usually used to manage complicated and uncomplicated infections. The use of cefepime in resistant infections is increasing gradually, which put this potent antibiotic at risk of resistance. During an 18-month period, a total of 220 gram-negative bacteria including Pseudomonas spp, Serratia spp, Acinetobacter spp, Proteus spp, E-coli and Kiebsiella spp. have been isolated by standard microbiological methods from nosocomial surgical site, abscess, blood stream and urinary tract infections. MIC of antibiotics on isolated bacteria was determined by gradient concentration method. Totally, 29.4%, 19.5% and 23.3% of isolated bacteria with MIC /= 256micro g/ml to cefepime, cefiriaxone and ceftazidime was also observed in 47.1%, 70.8% and 62.5% of cases, respectively [p<0.05]. High level resistance to cefepime were more commonly observed for pseudomonas [73.1%] and Klebsiella spp. [73.5%], respectively [p<0.05]. According to CLSI criteria, 47.1% of isolated bacteria in this study showed high level of resistance [MIC >/= 256micro g/ml] to cefepime. Therefore application of cefepime, as a drug of choice, for gram-negative organisms is not reasonable. Our result demonstrated that this potent antibiotic should not be used as a choice for empiric antibiotic therapy, in the cases of nosocomial infections caused by gram-negative organisms

Detection of Toxoplasma Parasitemia by PCR: Does it correlate with IgG and IgM antibody titers?

Toxoplasmosis is a zoonotic disease with high seroprevalence worldwide. Several immunological methods have been described for diagnosis of toxoplasmosis. To determine the parasitemia priod in patients infected with toxoplasma using PCR and comparing serological data with molecular results. In this study 154 serum samples from patients with toxoplasmosis were examined. Presence of parasite DNA was evaluated using PCR method. IgG and IgM antibody titers were measured using IFA test. Of 154 studied samples, 28 were positive for IgM and 60 were positive for IgG with titers higher than 1/400. PCR was performed on those samples having either IgG or IgM titers. Samples with IgM titers lower than 1/800 and higher than 1/3200 had no detectable level of parazite DNA. Parsetemia was detected in cases with IgG titer of 1/100 to 1/200. All samples with no detectable IgM and with IgG titers higher than 1/400 were negative when tested by PCR. IgM specific antibody titer between 1/800 and 1/3200 represents a window opportunity in treatment of patients with toxoplasmosis. Absence of parasite's DNA in patient with higher IgM antibody titer is explained by the effector mechanism of antibody for clearance of the parasite

Detection of HBV DNA in HBsAg negative normal blood donors

The risk of infection by transfusion-transmitted viruses has been reduced remarkably. However, a zero-risk blood supply is still desirable. The screening for antibody to HBc [anti-HBc] has been shown as an alternative test for the detection of HBV infection. The main aim of this study was to evaluate HBV infection markers and the potential value of anti-HBc testing of blood donors to detect HBV infection. In this descriptive cross-sectional study, 545 blood samples were collected and tested for HbsAg using ELISA method. Then all HBsAg negative samples were tested for anti-HBc by the same method. To detect HBV infection, all HBsAg negative and anti-HBc positive samples were tested by PCR for HBV DNA. All blood samples were HBsAg negative of which, 43 [8%] were anti-HBc positive. From those which were positive for anti-HBc, five samples were also positive for HBV DNA. Occult HBV infection is a clinical form of HBV infection in which HBsAg is not expressed by HBV and blood samples cannot be screened by ELISA method, therefore more sensitive techniques are needed. Our results demonstrate that a complementary test such as PCR, for detecting HBV DNA, is essential to ensure safety of blood samples