noorossadat seyyedi

In clinical diagnostics, molecular methods are used to detect Mycobacterium tuberculosis bacilli (MTB) and to distinguish them from non-tuberculous mycobacteria (NTM). They are also used to make the right treatment decision for the patient as soon as possible. The aim of this study was to establish a rapid and novel multiplex PCR (mPCR) assay for the detection and differentiation of MTB and NTM in a single tube.

100 sputum samples positive for acid-fast bacilli (AFB) were included in this study. Mycobacterial culture, biochemical tests, and antibiotic susceptibility testing were performed on samples. After alkaline decontamination, total DNA was extracted from the samples. A primer pair targeting the rpoB gene, encoding the beta-subunit of RNA poly- merase, was used to detect MTB and NTM, amplifying a 235-bp fragment of MTB and a 136-bp sequence of NTM. A pair of primers targeting a 190-bp fragment of the IS6110 region of MTB was also used to confirm the results. The sensitivity and specificity of the mPCR assay were evaluated using DNA extracted from standard strains. The amplified products were then analyzed by conventional agarose gel electrophoresis.

Of 100 AFB smear-positive sputum samples, 92 MTB DNA, 7 NTM DNA, and one mixed-infection sample were identified in a single tube using mPCR assay. There was no correlation between the AFB degree of smear positivity and PCR results. Of seven NTM isolates, 6 (86%) were resistant to rifampin, isoniazid, and ethambutol, the three first-line anti-tuber- culosis drugs.

A single-tube mPCR assay based on the rpoB gene provides a rapid and reliable means of detecting and differ- entiating MTB and NTM in sputum specimens.

Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat.

Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity.

A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control.

MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.

Although the liver is the main site for the Hepatitis C Virus (HCV) replication, there is still an essential debate about extrahepatic HCV reservoirs.

It has been proposed that Peripheral Blood Mononuclear Cells (PBMCs) could be the possible virus replication sites. Therefore, PBMCs may be candidates for recurrent HCV infection after achieving Sustained Virologic Response (SVR). In this study, we designed a lymphocyte culture to explore more about virus replication in PBMCs collected from patients with chronic hepatitis C.

Plasma and PBMC samples were collected from 16 randomly selected seropositive patients for the anti-HCV antibody. Four out of 16 (25%) patients received combination therapy with alpha interferon and ribavirin. PBMCs were isolated from whole blood. Between 106 -107 cells were cultured with optimized concentrations of IL-2 (10 mg/ml) and phytohemagglutinin A (5 mg/ml). Total RNA was extracted from the first collected sera and harvested lymphocytes. Constructed plasmids containing the NCR coding region were used to plot the standard curve for the relative quantification of SYBR green real-time PCR. The sensitivity and specificity of the detection were established by using plasmids containing cDNA.

With this plasmid containing the NCR coding region, the Limit of Detection (LOD) of in-house-developed real-time RT-PCR sensitivity was 2×101 copies. Using primers for the NCR region, 10 out of 16 (62.5 %) PBMCs were positive for negative-strand HCV RNA. Among the four samples collected from patients with SVR, negative-strand HCV RNA was found in two patient samples.

Our results indicated that cultured lymphoid cells from patients with chronic hepatitis, even with SVR, in the presence of IL-2 and PHA, markedly enhanced the detection of HCV RNA replica-tive strands. Therefore, PBMCs may be reservoirs for recurrent hepatitis infection after SVR and antiviral treatment. However, more clinical samples and control groups (lymphocyte culture without mitogen) should be examined to support the data presented in this study

Semaphorins play prominent roles in physiological and pathological processes such as vascular development, tumor growth and immune responses. Semaphorins have different roles in various kinds of cancers, but there is no study concerning their expression in the chronic lymphocytic leukemia (CLL). This study aimed to assess the SEMA3A, SEMA4A and SEMA4D expression in patients with CLL. Peripheral blood specimens were collected from 30 newly-diagnosed untreated patients with CLL and 30 healthy subjects as a control group. The SEMA3A, SEMA4A and SEMA4D expression was determined by real-time PCR method. The fold change expression of SEMA3A and SEMA4D was 7.58 ± 2.66 and 3.20 ± 0.99 in patients with CLL, and was 1.01 ± 0.31 and 1.00 ± 0.27 in healthy subjects, respectively. The CLL patients expressed higher amounts of SEMA3A and SEMA4D in comparison with healthy subjects (P<0.02 and P<0.03, respectively). The fold change expression of SEMA3A in patients with stage II (11.12 ± 5.35) was also higher than patients with stage I (4.49 ± 1.61, P<0.05). No significant difference was also observed in the expression of SEMA4A and SEMA4D between patients with stage I and stage II CLL. In both CLL and control groups, the fold change expression of SEMA3A was higher in men than in women (P<0.03 and P<0.02, respectively). The results of the study indicated elevated expression of the SEMA3A and SEMA4D in patients with CLL. The SEMA3A expression was influenced by tumor stage and gender of participants.

In spite of recent progress in mRNA technologies and their potential applications for treatment of human diseases, problems such as the transient nature of mRNA limit the stability of gene up-regulation and, thus, potentially reduce mRNA efficiency for gene therapy. Using human β-globin 5′ and 3′ untranslated regions (UTRs), this study aimed to develop the different chimeric constructs of mRNAs to increase the stability of destabilized green fluorescent protein (EGFPd2) in HEK 293 cells. Purified human β-globin (HBG) 5′-3′UTRs, and the coding sequence of destabilized green fluorescent protein (EGFPd2) were amplified separately and ligated to each other using SOEing PCR method in a different format. As controls, the original construct of EGFPd2 under the control of T7 promoter was used. Following in vitro transcription, HEK 293 cells were then transfected with several constructs and incubated at 37oC in a CO2 incubator. They were monitored under a fluorescence microscope every four hours for the first 24 hr, then every 12 hr afterwards. The resulting fluorescence was measured as a surrogate for translation efficiency and duration. By monitoring the HEK cells over 48 hr, cells transfected with mRNA with various HBG UTRs showed significantly different fluorescence intensity and stability in comparison with the pEGFPd2 prototype (control transcript) overtime. Overall, the images show that replacement of the 3′ UTR end of the prototype vector pGFPd2 with the 3′ end of β- globin mRNA increases the half-life of the chimeric mRNA for more than 32 hr. This result indicates that β-globin 3′ UTR would definitely increase the half-life of mRNA and may help to decrease the mRNA therapeutic dosage in the treatment of diseases associated with mRNA therapy.

There are few studies indicating the post-neonatal HBV vaccination level of anti-HBs antibody in first-year enrolled university students in Iran. In addition, anti- HBc antibody detection, which is a good indicator of virus exposure, has not been reported in vaccines. Hence, this study was conducted to determine the level of anti-HBs and anti-HBc antibodies in the serum sample of medical laboratory students who had received primary infantile HBV vaccination.
This study was conducted on first-year students enrolled in the department of laboratory sciences at Shiraz University of Medical Sciences, Iran. For determining anti-HBs and anti-HBc titers, 5 mL of venous blood was aseptically collected. Anti-HBs and anti-HBc antibody levels were determined by enzyme-linked immunosorbent assay. HBV DNA was also performed on DNA extracted from individuals positive for an anti-HBc antibody test.
Of the 257 vaccinated individuals (188 females and 69 males) who participated in this study, 36.2% showed a non-protective anti-HBs response (anti-HBs Our results indicate that a substantial number of our study population vaccinated against HBV during childhood showed non-protective anti-HBs antibody level. Therefore, a booster dose of vaccine needs to be scheduled for students with anti-HBs level

Pseudomonas aeruginosa is a Gram negative ubiquitous opportunistic organism and one of the more problematic drug-resistant pathogens encountered today.
The aims of the current multicenter research were to assess antibiotic resistance profiles, carbapenemase production, and detection of antibiotic resistance IMP gene as well as virulence factors genes including exoA, algD, lasB, and plcH among the clinical isolates of P. aeruginosa.
A total of 80 nonduplicate isolates of P. aeruginosa were recovered from inpatients. Bacterial identification was done by standard diagnostic tests. Species was confirmed by detection of the exoA gene using the PCR technique. Antimicrobial susceptibility test was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Carbapenemase production among the isolates was determined by the modified Hodge test. Virulence genes were detected by PCR.
A total of 42 (52.5%) isolates recovered from wound specimens. Colistin was the most effective antibiotic against isolates (97.5% isolates were susceptible) and levofloxacin was the least effective drug (67.5% isolates were resistant). The most common antibiotic resistance pattern was CIPR-CPMR-GEMR (47 isolates). In total, 47 (58.75%) isolates were identified as multidrug resistance (MDR), while 30% of isolates were carbapenemase producer (MHT). Among studied isolates, plcH and lasB genotypes (100% isolates) were the most common virulence gene patterns. Of 80 P. aeruginosa isolates, 39 (48.75%) showed algD, plcH, lasB, and IMP genotype. The blaIMP resistance gene was detected in all MHT positive and MDR isolates.
In our study, the emergence of potentially highly pathogenic and carbapenem-resistant strains in joining with a MDR phenotype is alarming, as a feasible outcome would be a severe clinical result concomitant with critical restrictions in antibiotic therapy.