The effect of Nrf2 deletion on the proteomic signature in a human colorectal cancer cell line.

BACKGROUND: Colorectal cancer is one of the most common cancer and the third leading cause of death worldwide. Increased generation of reactive oxygen species (ROS) is observed in many types of cancer cells. Several studies have reported that an increase in ROS production could affect the expression of proteins involved in ROS-scavenging, detoxification and drug resistance. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a known transcription factor for cellular response to oxidative stress. Several researches exhibited that Nrf2 could exert multiple functions and expected to be a promising therapeutic target in many cancers. Here, Nrf2 was knocked down in colorectal cancer cell line HT29 and changes that occurred in signaling pathways and survival mechanisms were evaluated. METHODS: The influence of chemotherapy drugs (doxorubicin and cisplatin), metastasis and cell viability were investigated. To explore the association between specific pathways and viability in HT29-Nrf2-, proteomic analysis, realtime PCR and western blotting were performed. RESULTS: In the absence of Nrf2 (Nrf2-), ROS scavenging and detoxification potential were dramatically faded and the HT29-Nrf2- cells became more susceptible to drugs. However, a severe decrease in viability was not observed. Bioinformatic analysis of proteomic data revealed that in Nrf2- cells, proteins involved in detoxification processes, respiratory electron transport chain and mitochondrial-related compartment were down regulated. Furthermore, proteins related to MAPKs, JNK and FOXO pathways were up regulated that possibly helped to overcome the detrimental effect of excessive ROS production. CONCLUSIONS: Our results revealed MAPKs, JNK and FOXO pathways connections in reducing the deleterious effect of Nrf2 deficiency, which can be considered in cancer therapy.

Analysis of FGFR3 and HRAS genes in patients with bladder cancer.

Background: Bladder cancer is the most frequent malignancy that affects the urinary tract. Studies have shown different types of FGFR3 and HRAS genes mutations in human bladder cancer, with a comprehensive range of mutation number in various populations. This study aimed to determine the specific point mutations of these 2 genes among Iranian patients with bladder cancer. Methods: In this study, 100 specimens of patients with transitional cell carcinoma were analyzed. All samples were examined for FGFR3 and HRAS mutations using PCR and direct DNA sequencing methods. Results: A total of 9 pathogenic mutations and 9 polymorphisms were found in 2 exons (7 and 15) of the FGFR3 genes in patients with bladder cancer (S249Y, I633I, L645L, D646E, Y647*, D628V, P250T, Q263H, Y305H). However, no mutation was found in exon 10 of FGFR3 and exon 1 of HRAS genes. Conclusion: In this study, 5 mutations were found in FGFR3 gene that have not been detected previously. There was no mutation in exon 10 of FGFR3 and exon1 of HRAS. The results of this study confirmed the association of ethnic-genetic factors in the occurrence of bladder cancer, so that these variables may not be present in all ethnic groups.

Analysis of KRT5 and KRT14 gene mutations and mode of inheritance in Iranian patients with clinical suspicion of Epidermolysis bullosa simplex.

Background: Epidermolysis bullosa simplex is a hereditary skin disorder caused by mutations in several genes such as KRT5 and KRT14 . Skin fragility in basal keratinocytes presence regions led to the cytolysis of epidermis and blistering. Aim of this study was to detect the molecular defects in KRT5 and KRT14 genes hot spots in patients with clinical suspicion of EBS and investigation of their probable genotype-phenotype correlations. Methods: Exons 1 and 6-7 of KRT5 and exons 1 and 4-7 of KRT14 amplification and mutation detection were performed by polymerase chain reaction and Sanger sequencing, respectively. Novel variants pathogenicity evaluated by bioinformatics tools. Results: Nine important variants detected in seven different patients within 6 Iranian families affected by Epidermolysis bullosa simplex, of which four variants were novel. Three patients had a mottled pigmentation phenotype [G96D (p.Gly96Asp) and F97I (p.Phe97Ile) in KRT5 ]. One of them showed a Dowling-Meara phenotype [A417P (p.Ala417Pro) and E477D (p.Glu477Asp) in KRT5 ] and another had a Koebner type phenotype [R397I (p.Arg397Ile) and Q444* (p.Gln444Ter) in KRT5 ]. A novel variant [G92E (p.Gly92Glu) in KRT5 ] in a double heterozygous state with a challenging variant [A413T (p.Ala413Thr) in KRT14 ] identified in one patient with Koebner type phenotype. Also, a previously reported mutation [I377T (p.Ile377Thr) in KRT14 gene] identified in this study. Conclusion: The results of molecular data analysis showed that the most severe phenotypes were associated with mutations in highly conserved regions. In some cases, different inheritance modes were observed.

Keratins and epidermolysis bullosa simplex.

Keratin intermediate filaments play an important role in maintaining the integrity of the skin structure. Understanding the importance of this subject is possible with the investigation of keratin defects in epidermolysis bullosa simplex (EBS). Nowadays, in addition to clinical criteria, new molecular diagnostic methods, such as next generation sequencing, can help to distinguish the subgroups of EBS more precisely. Because the most important and most commonly occurring molecular defects in these patients are the defects of keratins 5 and14 (KRT5 and KRT14), comprehending the nature structure of these proteins and their involved processes can be very effective in understanding the pathophysiology of this disease and providing new and effective therapeutic platforms to treat it. Here, we summarized the various aspects of the presence of KRT5 and KRT14 in the epidermis, their relation to the incidence and severity of EBS phenotypes, and the processes with which these proteins can affect them.

Mutation analysis of Phenylalanine hydroxylase gene in Iranian patients with Phenylketonuria.

Background: Phenylketonuria as the most common genetic metabolic disorder is the result of disruption of the phenylalanine hydroxylase gene. This study was carried out to explore the phenylalanine hydroxylase gene mutation status of Iranian phenylketonuria patients. Methods: Blood samples were collected from 30 patients, and hot spot areas of the phenylalanine hydroxylase gene, including exons 6, 7, 8, 11, and 12 were studied through polymerase chain reaction and sequencing techniques. Results: Eight different mutations, including 5 missense mutations, 1 splice mutation, 1 nonsense mutation, and 1 Silent/Splice mutation were detected. These mutations were R243X, R261Q, R261X, P281L, R241C, V399V, E280K, and IVS11+1G>C. V399V and R241C were reported for the first time in Iranian population. Three polymorphisms including Q232Q, V245V and L385L and 3 novel intronic variants including IVS10-15A>C, IVS6+44T>G, and IVS6+36 T>G were also detected in this study. Conclusion: The results of this study prove the heterogeneous status of phenylalanine hydroxylase gene mutations in the Iranian population, which can be useful in carrier testing and genetic counseling.

Distribution of KRAS, DDR2, and TP53 gene mutations in lung cancer: An analysis of Iranian patients.

PURPOSE: Lung cancer is the deadliest known cancer in the world, with the highest number of mutations in proto-oncogenes and tumor suppressor genes. Therefore, this study was conducted to determine the status of hotspot regions in DDR2 and KRAS genes for the first time, as well as in TP53 gene, in lung cancer patients within the Iranian population. EXPERIMENTAL DESIGN: The mutations in exon 2 of KRAS, exon 18 of DDR2, and exons 5-6 of TP53 genes were screened in lung cancer samples, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) using PCR and sequencing techniques. RESULTS: Analysis of the KRAS gene showed only a G12C variation in one large cell carcinoma (LCC) patient, whereas variants were not found in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) cases. The Q808H variation in the DDR2 gene was detected in one SCC sample, while no variant was seen in the ADC and LCC subtypes. Variations in the TP53 gene were seen in all NSCLC subtypes, including six ADC (13.63%), seven SCC (15.9%) and two LCC (4.54%). Forty-eight variants were found in the TP53 gene. Of these, 15 variants were found in coding regions V147A, V157F, Q167Q, D186G, H193R, T211T, F212L and P222P, 33 variants in intronic regions rs1625895 (HGVS: c.672+62A>G), rs766856111 (HGVS: c.672+6G>A) and two new variants (c.560-12A>G and c.672+86T>C). CONCLUSIONS: In conclusion, KRAS, DDR2, and TP53 variants were detected in 2%, 2.17% and 79.54% of all cases, respectively. The frequency of DDR2 mutation is nearly close to other studies, while KRAS and TP53 mutation frequencies are lower and higher than other populations, respectively. Three new putative pathogenic variants, for the first time, have been detected in Iranian patients with lung cancer, including Q808H in DDR2, F212L, and D186G in coding regions of TP53. In addition, we observed five novel benign variants, including Q167Q, P222P and T211T in coding sequence, and c.560-12A>G and c.672+86T>C, in intronic region of TP53. Mutations of KRAS and DDR2 were found in LCC and SCC subtypes, respectively, whereas mutations of TP53 were seen in SCC and ADC subtypes with higher frequencies and LCC subtype with lower frequency. Therefore, Iranian lung cancer patients can benefit from mutational analysis before starting the conventional treatment. A better understanding of the biology of these genes and their mutations will be critical for developing future targeted therapies.

Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity.

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.

Activity, stability and structure of laccase in betaine based natural deep eutectic solvents.

Natural deep eutectic solvents (NADES) play a role as alternative media to water in living organisms. They are formed by mixing two or more natural compounds in certain ratios producing a liquid having a lower melting point than those of the individual NADES components. Although, the eutectics medium bring several advantages as enhanced solubility of non-polar substrates and/or products, however, these advantages would often be limited by a lower stability of biocatalysts in these systems. To examine this matter, biochemical characterization, thermal stability and tertiary structure of laccase from Bacillus HR03 was investigated as a model in betaine and choline based NADES. In eutectics containing choline, a sudden drop in enzyme activity and stability was observed. Betaine based eutectics exhibited a better media for the laccase stability in comparison with the aqueous buffer and choline chloride eutectics. The enzyme highest activity was observed in 20% (v/v) glycerol:betaine (2:1). Among betaine based eutectics, the enzyme exhibited its highest stability in sorbitol:betaine:water (1:1:1) and glycerol:betaine (2:1) compared to the aqueous buffer at 80 and 90°C. Associated conformational changes caused by solvents were monitored using fluorescence technique. Finally, the effects of NADES on the enzyme activity and stability were discussed.

Genetic Screening of Iranian Patients with 46,XY Disorders of Sex Development.

BACKGROUND: Disorders of sex development (DSDs) belong to uncommon pathologies and result from abnormalities during gonadal determination and differentiation. Various gene mutations involved in gonadal determination and differentiation have been associated with gonadal dysgenesis. Despite advances in exploration of genes and mechanisms involved in sex disorders, most children with severe 46,XY DSDs have no definitive etiological diagnoses; therefore, the possibility that other genes or loci might play important roles in these disorders needs to be explored. METHODS: Patients (37) clinically suspicious for 46,XY gonadal dysgenesis (46,XY GD) of unknown etiology were studied. SRY, encoding the sex-determining region Y protein, NR5A1, encoding a transcription factor called steroidogenic factor 1, and DHH, encoding the desert hedgehog protein, were directly sequenced. Multiplex ligation-dependent probe amplification (MLPA) was used to detect deletions in NR0B1, encoding the DAX1 protein, and WNT4, encoding the WNT4 protein, and real-time PCR (qPCR) confirmed the MLPA data. Other potential loci have been investigated in the complete genome using Array-Comparative Genomic Hybridization, (Array CGH). RESULTS: The SRY deletion was found in five patients. One each of previously described NR5A1, DHH, and AR (androgen receptor) allelic variants were identified. A pathogenic c.2522G>A AR mutation was found in two affected brothers. A heterozygous partial deletion was found in NR5A1 and heterozygous partial duplications were found in WNT4. These deletions and duplications (del/dup) were confirmed by qPCR. The Array CGH result demonstrated one partial deletion in SOX2-OT, which encodes a member of the SOX family of transcription factors, and the exact region of the rearrangements. CONCLUSION: According to our study, del/dup mutations could be checked prior to point mutations, SOX2-OT has a potential role in gonadal dysgenesis, and Array CGH has a prominent role in gonadal dysgenesis diagnosis.

CTNS molecular genetics profile in a Persian nephropathic cystinosis population / Perfil genético molecular del gen CTNS en una población persa con cistinosis nefropática

Purpose: In this report, we document the CTNS gene mutations of 28 Iranian patients with nephropathic cystinosis age 1-17 years. All presented initially with severe failure to thrive, polyuria, and polydipsia. Methods: Cystinosis was primarily diagnosed by a pediatric nephrologist and then referred to the Iran University of Medical Sciences genetics clinic for consultation and molecular analysis, which involved polymerase chain reaction (PCR) amplification to determine the presence or absence of the 57-kb founder deletion in CTNS, followed by direct sequencing of the coding exons of CTNS. Results: The common 57-kb deletion was not observed in any of the 28 Iranian patients. In 14 of 28 patients (50%), mutations were observed in exons 6 and 7. No mutation was detected in exon 5, and only one (3.6%) patient with cystinosis showed a previously reported 4-bp deletion in exon 3 of CTNS. Four patients (14.3%) had a previously reported mutation (c.969C>A; p.N323K) in exon 11, and five (18%) had novel homozygous deletions in exon 6 leading to premature truncation of the protein. These deletions included c.323delA; p.Q108RfsX10 in three individuals and c.257-258delCT; p.S86FfsX37 in two cases. Other frame-shift mutations were all novel homozygous single base pair deletion/insertions including one in CTNSexon 9 (c.661insT; p.V221CfsX6), and four (14.3%) in exon 4, i.e., c.92insG; p.V31GfsX28 in two and c.120delC; p.T40TfsX10 in two. In total, we identified eight previously reported mutations and eight novel mutations in our patients. The only detected splice site mutation (IVS3-2A>C) was associated with the insertion mutation in the exon 9. Conclusion: This study, the first molecular genetic analysis of non-ethnic-specific Iranian nephropathic cystinosis patients, may provide guidance for molecular diagnostics of cystinosis in Iran (AU)

Objetivo: En este informe, documentamos las mutaciones del gen CTNS de 28 pacientes iraníes con cistinosis nefropática y una edad de 1-17 años. En un principio, todos presentaron retraso del desarrollo, poliuria y polidipsia. Métodos: En primer lugar, un nefrólogo pediátrico diagnosticó la cistinosis y luego los pacientes fueron trasladados a la clínica genética de la Universidad de Ciencias Médicas de Irán para consulta y análisis molecular, que incluía la multiplicación por reacción en cadena de la polimerasa (PCR), para determinar la existencia o ausencia de la deleción del fundador del 57kb en el CTNS, seguida por la secuenciación directa de los exones de codificación del CTNS. Resultados: La deleción frecuente del 57kb no se observó en ninguno de los 28 pacientes iraníes. En 14 de los 28 pacientes (50%) se observaron mutaciones en los exones 6 y 7. No se detectó ninguna mutación en el exón 5 y solo un paciente (3,6%) con cistinosis mostró una deleción del 4pb, anteriormente comunicada, en el exón 3 del CTNS. De ellos, 4 pacientes (14,3%) tenían una mutación anteriormente comunicada (c.969C > A; p.N323K) en el exón 11 y 5 (18%) tenían nuevas deleciones homocigóticas en el exón 6 que produjeron el vaciamiento prematuro de la proteína. Entre estas deleciones se puede citar c.323delA; p.Q108RfsX10 en 3 personas y c.257-258delCT; p.S86FfsX37 en 2 casos. Otras mutaciones con desplazamiento del marco de lectura fueron todas nuevas deleciones/inserciones de un par de bases únicas homocigóticas, incluyendo una en el exón 9 del CTNS(c.661insT; p.V221CfsX6) y 4 (14,3%) en el exón 4, es decir, c.92insG; p.V31GfsX28 en 2 y c.120delC; p.T40TfsX10 en 2. En total, en nuestros pacientes se identificaron 8 mutaciones anteriormente comunicadas y 8 mutaciones nuevas. La única mutación del sitio de empalme detectada (IVS3-2A>C) estaba asociada con la mutación de inserción en el exón 9. Conclusión: Este estudio, el primer análisis genético molecular de pacientes iraníes con cistinosis nefropática de carácter no específicamente étnico, puede servir como guía para el diagnóstico molecular de la cistinosis en Irán (AU)

CTNS molecular genetics profile in a Persian nephropathic cystinosis population.

PURPOSE: In this report, we document the CTNS gene mutations of 28 Iranian patients with nephropathic cystinosis age 1-17 years. All presented initially with severe failure to thrive, polyuria, and polydipsia. METHODS: Cystinosis was primarily diagnosed by a pediatric nephrologist and then referred to the Iran University of Medical Sciences genetics clinic for consultation and molecular analysis, which involved polymerase chain reaction (PCR) amplification to determine the presence or absence of the 57-kb founder deletion in CTNS, followed by direct sequencing of the coding exons of CTNS. RESULTS: The common 57-kb deletion was not observed in any of the 28 Iranian patients. In 14 of 28 patients (50%), mutations were observed in exons 6 and 7. No mutation was detected in exon 5, and only one (3.6%) patient with cystinosis showed a previously reported 4-bp deletion in exon 3 of CTNS. Four patients (14.3%) had a previously reported mutation (c.969C>A; p.N323K) in exon 11, and five (18%) had novel homozygous deletions in exon 6 leading to premature truncation of the protein. These deletions included c.323delA; p.Q108RfsX10 in three individuals and c.257-258delCT; p.S86FfsX37 in two cases. Other frame-shift mutations were all novel homozygous single base pair deletion/insertions including one in CTNS exon 9 (c.661insT; p.V221CfsX6), and four (14.3%) in exon 4, i.e., c.92insG; p.V31GfsX28 in two and c.120delC; p.T40TfsX10 in two. In total, we identified eight previously reported mutations and eight novel mutations in our patients. The only detected splice site mutation (IVS3-2A>C) was associated with the insertion mutation in the exon 9. CONCLUSION: This study, the first molecular genetic analysis of non-ethnic-specific Iranian nephropathic cystinosis patients, may provide guidance for molecular diagnostics of cystinosis in Iran.

MicroRNAs as biomarkers associated with bladder cancer.

Bladder cancer is the fifth most common cancer with significant morbidity and mortality. Recently, numerous studies demonstrated that microRNAs are emerging as diagnostic biomarkers for bladder cancer. Specific miRNA profiles have been identified for several samples from patients with bladder cancer. MicroRNAs are noncoding RNA molecules of approximately 23 nucleotides that play important roles in multiple steps during the progression of bladder cancer. Here, we review the expression profiles of miRNAs and their biological functions, regulation, and clinical implications in bladder cancer. Either downregulation or upregulation of miRNAs occurs in bladder cancer through epigenetic changes or defects of the biogenesis apparatus. Deregulation of miRNAs is involved in cell cycle arrest, apoptosis, proliferation, metastasis, drug resistance, and other functions in bladder cancer. A number of miRNAs, have been associated with tumor type, stage, or patient survival, and miRNAs might be developed as diagnostic or prognostic markers. A better understanding of the roles of miRNAs in bladder cancer will shed light on the molecular mechanisms of bladder cancer.

A semi-rational approach to obtain an ionic liquid tolerant bacterial laccase through π-type interactions.

Laccases are particularly promising enzymes for biotechnology and bioremediation purposes. They are among the most effective enzymes capable of catalyzing the degradation of phenolic compounds with poor water solubility. The technological utility of lacasses can be enhanced greatly by their use in ionic liquids rather than in conventional organic solvents or in their natural aqueous reaction media. In the current study, a laccase from Bacillus HR03 has been engineered through a semi rational method. By screening a library of 450 clones, Glu188Tyr and Glu188Phe showed a distinct improvement in thermal stability and ionic liquid tolerance. In comparison with the wild type, selected mutants exhibited higher kcat/Km against ABTS in the imidazolium based ionic liquids, (1-ethyl-3-methyl imidazolium chloride [EMIm][Cl], butyl-3-methyl imidazolium chloride [BMIm][Cl] and hexyl-3-methyl imidazolium chloride [HMIm][Cl]). Glu188Tyr had a catalytic efficiency, two times greater when compared to the wild type in [HMIm][Cl]. Far-UV circular dichroism (CD) exhibited no significant changes in the secondary structure of the mutants and wild type. Glu188Tyr revealed a more compact structure using Near-UV CD and fluorescence spectroscopy that could account for its high thermal stability. According to bioinformatic analysis, π-π and anion-π interactions played the dominant role in stabilizing both variants.

Molecular detection of human parechovirus type 1 in stool samples from children with diarrhea.

BACKGROUND: Human parechovirus type 1 (HPeV-1) appears to be associated with gastrointestinal and respiratory infections, and occasionally with central nervous system symptoms. Since there is no accurate information about the incidence and epidemiology of HPeV-1 in Iran, this study was designed to describe the frequency of HPeV-1 in stool samples from children under 4 years of age with gastroenteritis. METHODS: RNA was isolated from stool suspensions and cDNA was prepared and amplified using a specific nested reverse transcription PCR (RT-PCR). We also compared this RT-PCR method with a cell culture procedure. RESULTS: Out of 472 samples, 112 (23.7%) were HPeV-1-positive. The incidence and prevalence of this virus was highest in children under 1 year old with diarrhea (p=0.036), and was higher in the spring and autumn than in summer and winter (p<0.001). More males than females were HPeV-1-positive (p<0.001). CONCLUSIONS: This is the first systematic study of the incidence and epidemiology of HPeV-1 among children with acute gastroenteritis in Iran. The results revealed that RT-PCR is a more practical, sensitive, and rapid technique for the detection of HPeV-1 directly from clinical samples than cell culture, which makes this a valuable method for epidemiology. Also, the rapid detection of virus can decrease both the unnecessary use of antibiotics and costs in clinical practice.

BMSC and CoQ10 improve behavioural recovery and histological outcome in rat model of Parkinson's disease.

OBJECTIVE: This study assessed the ability of a combination treatment of bone marrow stromal cell (BMSC) graft and oral coenzyme (CoQ10) in a rat model of Parkinson's disease (PD) as an appropriate substitute for current Parkinson treatments. The combination treatment was compared to sole treatments of BMSC and CoQ10. MATERIALS AND METHODS: In this experimental study, there were six groups of male Wistar rats: control, sham, lesion, CoQ10, graft BMSC and graft BMSC plus CoQ10. Oral administration of CoQ10 began 1 week before the PD and continued during the entire treatment period. To simulate PD, we injected 6 hydroxydopamine (6OHDA) in rats. BMSC were labelled by 5-bromo-2'-deoxyuridine (Brdu) before transplantation. We assessed behaviour before PD, 2 weeks after PD and 8 weeks after cell transplantation. At the end of the second month of treatment, immunohistochemistry, histology and molecular studies were performed. RESULTS: Behavioural assessment of the CoQ10 group and BMSC group indicated equal recovery in comparison with the lesion group (P<0.01), while the combined treatment of BMSC and CoQ10 showed considerably better recovery compared with the lesion group (P<0.001). There were no signs of gliosis and graft rejection. Immunohistochemistry analysis of Brdu indicated that cells were alive after 2 months of application in host tissue. Cell counts showed significantly greater numbers of neural cells in the combination treatment of BMSC and CoQ10 compared to the other groups. Tyrosine hydroxylase (TH) gene expression levels in the combined therapy group was significantly more than the other experimental groups (P<0.001). CONCLUSION: The combined use of two neuroprotective treatments and cell replacement therapy can be effective in the treatment of PD, at least in experimental settings.

Comparing invasive and non-invasive of isolated Shigella flexneri by electron microscopy of cell culture, SDS-PAGE and Congo red method.

BACKGROUND: The aim of this study was to compare invasive and non-invasive strains of Shigella flexneri isolated from Tehran by a 120 kDa protein band by SDS-PAGE, electron microscopy of cell culture and Congo red dye methods. METHODS: S. flexneri strains were isolated by standard bacterial methods from fecal specimens of children attending to the 3 children's hospitals. Phenotype analysis for screening virulent of strains of S. flexneri was done on a plate of tryptic soy agar contained 0.003% Congo red dye. Whole membrane protein preparations were used to examine the protein profiles of the inner and outer membrane of these Gram-negative bacteria. The protein mixture was electrophoresed through a polyacrylamide gel. The gel was stained with Coomassie brilliant blue R250 and destained with ethanol and acetic acid. HeLa cell culture was done by two-step preparations: one for light microscopy and the other for electron microscopy. RESULTS: Some of S. flexneri (46%) were Congo red positive colonies. S. flexneri with negative Congo red phenotype could not enter the HeLa cell culture. A 120 kDa protein band was found in 46% of these bacteria which could enter into HeLa cell culture. Pseudopod structures which facilitate bacterial cell-to-cell spread were readily identified by electron microscopy. DISCUSSION: Since the existence of 120-kDa protein band was corresponded to enter of S. flexneri into the HeLa cell culture and correlated with Congo red dye positive, for identification of invasive and non-invasive S. flexneri strains, the use of a 120-kDa protein band by SDS-PAGE or a simple, rapid and very cheap Congo red dye method is recommended. Because, there are some deaths due to Shigella sp. in our country, notification on the isolation of these bacteria in both children hospitals laboratories and private clinical laboratories is important.

Comparison between molecular epidemiology, geographical regions and drug resistance in Mycobacterium tuberculosis strains isolated from Iranian and Afghan patients.

The aim of this study was to determine the prevalence of drug-resistant tuberculosis (TB) and its associated risk factors. The susceptibilities of Mycobacterium tuberculosis isolates were tested against four first-line antituberculous drugs and were typed by spoligotyping. Spoligotyping of M. tuberculosis strains resulted in 95 different patterns that were divided into three evolutionary groups (1-3). Eighty-six (90%) of the isolates had unique patterns that were reported for the first time. Interestingly, 9.4% of the strains belonged to the Beijing family. Multidrug resistance (MDR) was seen in group 1 of the evolutionary scenario. All M. tuberculosis isolates belonging to the Beijing family were associated with a resistance pattern. MDR was much higher in bacteria isolated from Afghan TB patients residing in Iran.