The Potential Role of Autophagy in Progression of Liver Fibrosis in Chronic Hepatitis B Patients Receiving Antiviral Treatment: A Brief Report.

Despite antiviral treatment, some patients with chronic hepatitis B (CHB) progress to cirrhosis. Enhancement of autophagy was implicated in the proliferation of hepatitis B in hepatocytes. This study aimed to evaluate the potential role of autophagy in the progression of liver fibrosis in patients receiving antiviral treatments and having completely inhibited viral replication. This descriptive-analytical study was designed and conducted in 2020 at Mottahhari Hepatitis Clinic affiliated with Shiraz University of Medical Science (Shiraz, Iran). Patients who were on anti-hepatitis B nucleotide treatments for at least two years, and those who were not cirrhotic at baseline but later progressed to cirrhosis were identified to be included in the case group. Besides, for the control group, patients on the nucleotide regimens who did not have cirrhosis at baseline or during follow-up were randomly selected. Ultimately, 16 cases and 14 controls were included in the study. Data were analyzed using SPSS software, and P<0.05 was considered statistically significant. Serum Beclin-1 and LC3 levels were compared between the two groups using enzyme-linked immunosorbent assays. The t test was used to assess the statistical differences between the case and control groups. Beclin-1 level was significantly higher in cirrhosis patients than the control group (1283±244 vs. 1063±257, P=0.024). However, there was no statistical difference between the level of LC3 in the cirrhotic group (168±31) and the control group (150±16) (P=0.065). Autophagy may have a role in the progression of cirrhosis in patients with CHB. Future larger prospective studies are required to determine the effect of blocking on the progression of liver disease in this population A preprint of this study was published at https://www.researchsquare.com/article/rs-1435490/v1.pdf.

Outcomes of Different Donor and Recipient Vessel and Ureteral Anastomoses in En Bloc Kidney Transplant From Pediatric Deceased Donors: A Retrospective Cohort.

OBJECTIVES: This study aimed to investigate the possible role of different donor and recipient vessel and ureteral anastomoses on survival and functional outcomes in en bloc kidney transplants. MATERIALS AND METHODS: This retrospective cohort included 99 en bloc kidney transplants performed from December 2005 to March 2022. Recipients were grouped based on donor's vessel (distal [n = 84] or proximal [n = 15] abdominal aorta), recipient's vessel (abdominal aorta [n = 3], external [n = 21], internal [n = 50], or common [n = 25] iliac artery), and ureteral anastomosis (separate [n = 32] or common [n = 67]). Patient and graft survival, complication rates, and estimated glomerular filtration rate trends were compared between groups. RESULTS: Pediatric brain dead donors had a mean age and weight of 37 ± 22 months and 14 ± 4 kg, respectively. Donor and recipient vessel and ureteral anastomoses did not affect overall survival (P = .306, .296, and .225), graft survival (P = .720, .172, and .124), and vascular (P = .347, .689, and .264) and urinary (P = .587, .172, and .385) complication rates. Lymphoceles requiring intervention were significantly more prevalent in the recipient external iliac artery group (P = .008) but were independent of donor vessel and ureteral anastomosis (P = .587 and 1.00). Estimated glomerular filtration rate trend was independentofdonor(P=.921) andrecipient vessel(P=.878 and .536). CONCLUSIONS: We found that different arterial and ureteral anastomoses appear to have comparable outcomes in en bloc kidney transplant with the exception of recipient external iliac artery, which may be slightly inferior because of the relatively higher rate of lymphoceles requiring intervention.

Effect of Mir-4270 Inhibitor and Mimic on Viability and Stemness in Gastric Cancer Stem-Like Cells Derived from MKN-45 Cell Line.

Background: MicroRNAs (miRNAs) are significant regulatory factors in stem cell proliferation, and change in miRNA expression influences the cancer stem cell viability and gene expression. Herein, we evaluated the effect of the hsa-miR-4270 inhibitor and its mimic on the expression of stem cell markers in gastric cancer (GC) stem-like cells. Methods: GC stem-like cells were isolated from the MKN-45 cell line by a non-adherent surface system. The cells were confirmed by differentiation assays using dexamethasone and insulin as adipogenesis-inducing agents and also Staurosporine as a neural-inducing agent. Isolated GC stem-like cells were treated with different concentrations (0, 15, 20, 25, 30, 40, 50, and 60 nM) of hsa-miR-4270 inhibitor and its mimic. The quantity of cell viability was determined by trypan blue method. Transcription of the stem cell marker genes, including CD44, OCT3/4, SOX2, Nanog, and KLF4, was evaluated by real-time RT-PCR. Results: The results showed that GC stem-like cells were differentiated into both adipose cells using dexamethasone and insulin and neural cells by Staurosporine. Treatment of GC stem-like cells with hsa-miR-4270 inhibitor decreased cell viability and downregulated OCT3/4, CD44, and Nanog to 86%, 79%, and 91% respectively. Also, SOX2 and KLF4 were overexpressed to 8.1- and 1.94-folds, respectively. However, hsa-miR-4270 mimic had opposite effects on the cell viability and gene expression of the stem cell markers. Conclusion: The effect of hsa-miR-4270 inhibitor and its mimic on the expression of the stem cell markers in GCSCs indicated that hsa-miR-4270 stimulates the stemness property of GCSCs, likely through stimulating the development of gastric stem cells.

Clinical characteristics of 365 hospitalized COVID-19 patients with neurological symptoms: an observational study.

OBJECTIVE: Since the beginning of the COVID-19 pandemic, a number of COVID-related neurological manifestations have been reported. We aimed to categorize the features of hospitalized COVID-19 patients who experienced neurological symptoms. METHODS: In this descriptive, cross-sectional study, we enrolled all patients hospitalized with COVID-19 who experienced neurological symptoms in two hospitals in Tehran. Diagnosis of COVID-19 was established by PCR tests or computed tomography of the chest combined with COVID-19 clinical findings. The clinical characteristics, laboratory data, and imaging findings from 365 patients were analyzed. RESULTS: The average patient age was 59.2 ± 16.7 years and included 213 males and 152 females. The most prevalent neurological symptoms were headache (56.2%), impaired consciousness (55%), and dizziness (20.5%). During hospitalization, most of the patients did not require mechanical ventilation (81.9%). The percentage of patients with end-organ damage was 9% and mortality was 15%. Regression analysis on the neurological symptoms indicated that the mortality rate of patients with headaches was 84% lower than for the other neurological symptoms. Hyperglycemia was significantly related with end-organ damage and mortality (p = 0.029, p = 0.08, respectively). New vascular lesions were evident on brain MRIs of 9 patients and brain CTs of 16 patients. CONCLUSION: Among the neurological symptoms of patients with COVID-19, headache appeared to indicate a protective factor against development of end-organ damage as well as mortality.

Oncological Outcomes of Neoadjuvant Gemcitabine plus Carboplatin versus Gemcitabine plus Cisplatin in Locally Advanced Bladder Cancer: A Retrospective Analysis.

PURPOSE: Cisplatin-based neoadjuvant chemotherapy (NAC) is the standard of care in non-metastatic muscle-invasive bladder cancer (MIBC). There are limited data regarding the alternative choices for cisplatin-ineligible patients. This study has investigated the oncological outcomes of gemcitabine plus cisplatin (Gem/Cis) and gemcitabine plus carboplatin (Gem/Carbo) in this setting. MATERIALS AND METHODS: One hundred forty consecutive patients with MIBC (cT2-T4a) receiving neoadjuvant Gem/Cis or Gem/Carbo before chemoradiation (CRT) or radical cystectomy (RC) were retrospectively evaluated between April 2009 and April 2019. Patients with ECOG performance status 2, creatinine clearance < 60 mL/min, hydronephrosis, ejection fraction < 50%, or single kidney received Gem/Carbo. The complete clinical response (cCR) and overall survival (OS) of NAC regimens were compared. Prognostic significance was assessed with Cox proportional hazards model. RESULTS: In total, 79 patients (56.4%) received Gem/Cis. The cCR was not significantly different between Gem/Cis and Gem/Carbo regimens (38.7% vs. 36.2%, P = .771). After NAC, 79 patients (56.4%) received CRT, and other cases underwent RC. After a median follow-up of 43 months, patients in the Gem/Cis group had significantly better OS than Gem/Carbo (median OS: 41.0 vs. 26.0 months, P = .008). Multivariable Cox proportional hazards models identified cT4a stage (95% confidence interval [95% CI]: 1.001-4.85, hazard ratio [HR] = 2.08, P = .03) and cCR (95% CI: 0.26-0.99, HR = 0.51, P = .04) as the only independent prognostic factors of OS, and ruled out the type of NAC regimen. CONCLUSION: The choice of NAC (between Gem/Cis and Gem/Carbo) is not the predictor of survival and both regimens had similar cCR.

Translation and validation of the Persian version of Godin Leisure-Time Exercise Questionnaire in patients with multiple sclerosis.

STUDY DESIGN: Psychometric study. OBJECTIVE: The purpose of this study is to translate, culturally adapt and evaluate the validity and reliability of the Persian (Farsi) version of GLTEQ in patients with multiple sclerosis. METHODS: This study had three phases, including translation of the questionnaire into Persian and making cultural adaptation, evaluation of pre-final version of questionnaire's comprehensibility in a pilot study, and investigation of reliability and validity of the final version of the translated questionnaire. Content validity, and convergent validity (correlations among the Persian version of GLTEQ and Global physical activity questionnaire (GPAQ), and international physical activity questionnaire (IPAQ)) and after all test-retest reliability were studied. RESULTS: The subjects were 87 MS patients. The Persian version demonstrated moderate to good convergent validity; the correlation coefficient between the Persian version and GPAQ was r=0.64 (p<0.001), and between the Persian version and IPAQ was r=0.59 (p<0.001). The test-retest reliability was strong (Intra-class Correlation (ICC) value ranged between 0.908 and 0.992). Besides, its face validity and content validity were acceptable. CONCLUSIONS: The Persian version of GLTEQ is a valid and reliable instrument to assess physical activity in patients with MS. This questionnaire can be a step toward standardization of physical activity measurement in patients with MS. Also, in research, it provides the possibilities to carry on a comparative study across cultures using the same outcome measure.

Correlation between the clinical disability and T1 hypointense lesions' volume in cerebral magnetic resonance imaging of multiple sclerosis patients: A systematic review and meta-analysis.

BACKGROUND: To evaluate the correlation between T1 hypointense lesions' mean volume on cerebral MRI with disability level of patients with multiple sclerosis. METHODS: We included studies testing the desired outcome in adult patients diagnosed with RRMS or SPMS. In Feb 2021, we searched PubMed, Embase, CENTRAL, and Web of Science to find relevant studies. All included studies were assessed for the risk of bias using a tailored version of the Quality in Prognosis Studies (QUIPS) tool. Extracted correlation coefficients were converted to the Fisher's z scale, and a meta-analysis using a random-effects model was performed on the results. RESULTS: We included 27 studies (1919 participants). Meta-analysis revealed a correlation coefficient of 0.32 (95% CI 0.26-0.37) between T1 hypointense lesions' mean volume and EDSS score. DISCUSSION: The correlation between T1 hypointense lesions' mean volume and EDSS was interpreted as low to slightly moderate. The certainty of the evidence was judged to be high.

Neuromyelitis optica spectrum disorder (NMOSD) associated with cancer: A systematic review.

NMOSD: is a disease shown to be highly associated with other diseases such as autoimmune diseases. There are a few reports of this association with cancer. So, this systematic review aimed to obtain a broad understanding on the cancers associated in NMOSD, including the source of common perceptions and assumptions in this regard. METHODS: in this study, we systematically searched the PubMed, EMBASE, SCOPUS, Web of Sciences, Proquest, Ovid, conference proceedings, and reference lists of the retrieved articles. All NMOSD cases who met the last version of criteria for its diagnosis, which reported the patients with a history of cancer before or after the onset of neurological symptoms without time limitations, and those who were referred as paraneoplastic neuromyelitis optica in articles published in English language (both the abstract & full text), were assessed. Finally, each study was critically appraised. RESULTS: Only 47 studies met the inclusion criteria, so they were assessed for qualitative synthesis. Considering the Euro network criteria, only 62 cases met this issue. The mean age of 52.21 ± 17.14 and 52.16 ± 17.21 was reported for cancer and NMOSD cases with female predominance (79%), respectively. The most reported organ in the cancer population were genitourinary (n = 14, 22.3%), breast (n = 12, 19.4%), lung (n = 12, 19.3%), gastrointestinal (n = 7, 11.3%), and hematology (n = 6, 9.7%), respectively. CONCLUSION: In older NMOSD patients without suspicious symptoms, we recommend paying more attention to lung, breast and genitourinary (especially ovary) cancer screening. Also, cancer resection had positive effect on the attack numbers after receiving treatment and NMOSD recovery.

Biofluid Biomarkers in Traumatic Brain Injury: A Systematic Scoping Review.

Emerging evidence suggests that biofluid-based biomarkers have diagnostic and prognostic potential in traumatic brain injuries (TBI). However, owing to the lack of a conceptual framework or comprehensive review, it is difficult to visualize the breadth of materials that might be available. We conducted a systematic scoping review to map and categorize the evidence regarding biofluid-based biochemical markers of TBI. A comprehensive search was undertaken in January 2019. Of 25,354 records identified through the literature search, 1036 original human studies were included. Five hundred forty biofluid biomarkers were extracted from included studies and classified into 19 distinct categories. Three categories of biomarkers including cytokines, coagulation tests, and nerve tissue proteins were investigated more than others and assessed in almost half of the studies (560, 515, and 502 from 1036 studies, respectively). S100 beta as the most common biomarker for TBI was tested in 21.2% of studies (220 articles). Cortisol was the only biomarker measured in blood, cerebrospinal fluid, urine, and saliva. The most common sampling time was at admission and within 24 h of injury. The included studies focused mainly on biomarkers from blood and central nervous system sources, the adult population, and severe and blunt injuries. The most common outcome measures used in studies were changes in biomarker concentration level, Glasgow coma scale, Glasgow outcome scale, brain computed tomography scan, and mortality rate. Biofluid biomarkers could be clinically helpful in the diagnosis and prognosis of TBI. However, there was no single definitive biomarker with accurate characteristics. The present categorization would be a road map to investigate the biomarkers of the brain injury cascade separately and detect the most representative biomarker of each category. Also, this comprehensive categorization could provide a guiding framework to design combined panels of multiple biomarkers.

Variations in IL-22, IL-27 and IL-35 serum levels in untreated and treated hepatitis C patients.

BACKGROUND: Hepatitis C virus (HCV) is the leading cause of chronic liver diseases including hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. We aimed to assess serum levels of interleukin (IL)-22, IL-27 and IL-35 in patients with hepatitis C and healthy controls to investigate their possible relationship with viral genotypes and liver enzyme levels. METHOD: A total of 30 newly diagnosed hepatitis C patients with no history of antiviral therapy and 30 healthy individuals participated in this study. Serum levels of IL-22, IL-27 and IL-35 were determined by ELISA in peripheral blood samples from patients prior to and following treament with pan-genotypic direct-acting anti-viral therapy. Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were measured to determine any possible association between hepatic enzymes and cytokine serum levels concentrations. RESULT: The results show elevated serum levels of of IL-35 in HCV-infected patients compared to treated cases and healthy controls, whereas there was no significant difference in IL-22 and IL-27 serum levels among the three groups. Additionally, the cytokine levels were not significantly correlated with certain genotypes and levels of liver enzymes. CONCLUSION: Our findings indicate a potential role for IL-35 in chronic HCV infection and therapeutic management of patients with hepatitis C infection.

Efficacy of Rose Oil Soft Capsules on Clinical Outcomes in Ulcerative Colitis: A Pilot Randomized, Double-Blinded, Placebo-Controlled Clinical Trial.

BACKGROUND: Ulcerative colitis is the most common form of inflammatory bowel disease worldwide, which presents with superficial ulcers in the rectum and colon. The aim of this study was to assess the effectiveness of rose oil soft capsules over placebo on the clinical outcomes in moderate to severe ulcerative colitis. MATERIALS AND METHODS: This study was a pilot randomized, double-blind clinical trial, and the 40 patients were assigned into rose oil and placebo groups (n=20 per group). All patients were instructed to use their prescribed two soft capsules three times daily for two months. The clinical symptoms, quality of life the patients, and calprotectin level were evaluated via partial Mayo clinic score, irritable bowel disease questionnaire (IBDQ-9), and calprotectin kit as primary outcome measures. RESULTS: The mean age of the participants was 41±10 years. Most of them (53.6%) were male, and the remaining (46.4%) were female. The demographic and baseline data showed no differences between the two groups. Partial Mayo clinic scores decreased in both groups after the treatment, but the difference between the rose oil and placebo groups was not statistically significant (P=0.99). IBDQ-9 score also increased in both interventions before and after the treatment (P=0.012), though the differences between these two groups were not statistically significant (P=0.61). There were no significant differences between the two study groups either in terms of calprotectin level (P=0.219). CONCLUSION: This study showed that rose oil might improve ulcerative colitis clinical outcomes, but for a better evaluation, it is imperative to conduct experiments with a large sample size and longer follow-up observation.

Effect of ursodeoxycholic acid and vitamin E in the prevention of liver injury from methotrexate in pediatric leukemia.

BACKGROUND/AIMS: Ursodeoxycholic acid (UDCA) and antioxidants such as vitamin E are considered to have a protective role in preventing chemotherapy-induced liver damage. The aim of this study was to assess the efficacy of these agents for hepatoprotection in pediatric patients with B-cell acute lymphoblastic leukemia (ALL), who were treated with methotrexate in their maintenance phase of treatment. MATERIALS AND METHODS: Eighty children with B-cell ALL were randomly divided into four groups. Group 1 was administered oral vitamin E (400 mg/day); group 2 was administered oral UDCA (15 mg/kg/day); group 3 was administered a combination of the two drugs; and group 4 served as a control group and was administered no drug except their chemotherapy drugs. Complete blood count, liver function test, liver ultrasonography, and liver fibroscan were requested, and the results were compared. RESULTS: Group 1 showed a slight increase in total bilirubin levels compared to baseline levels during the study (P=0.036). Group 2 showed a decline in aspartate aminotransferase and alanine aminotransferase levels during the study and at 6 months after discontinuing the drug; however, these differences were not statistically significant (P=0.051 and 0.083, respectively). None of the patients showed the evidence of significant fibrosis on liver fibroscan. Eight patients showed some evidence of mild-to-moderate fibrosis (F1, F2), but the results were not different between the groups as well as between pre- and post-study periods in each group. CONCLUSION: Low-dose methotrexate does not cause significant liver fibrosis in pediatric leukemia. UDCA and vitamin E have minimal roles in hepatoprotection among pediatric patients with ALL.

Effect of RGD coupled MDA-7/IL-24 on apoptosis induction in a hepatocellular carcinoma cell line.

The melanoma differentiation-associated gene­7 (MDA-7) gene, also termed interleukin­24 (IL­24), is a tumor suppressor gene that induces apoptosis in a broad scope of malignant neoplastic cells. The apoptosis induction capacity of the MDA­7/IL­24 gene is partially associated with adhering to cognate receptors. The current study aimed to enhance the antitumor effect of IL­24. The intrinsic signal sequence of IL-24 replaced with a fused artificial signal (secrecon)-RGD4C sequence and its impact was evaluated in HepG2 cells. The modified SP.RGD.IL­24 and native IL­24 cDNA sequences were cloned into the pcDNA3.1 expression vector. Subsequently, the expression level, secretion efficacy and targeting propensity of the modified SP.RGD.IL­24 product compared with normal IL­24 by were determined by enzyme­linked immunosorbent assay. The constructs were then transfected into HepG2 and LX­2 cells as tumor and normal hepatic cell lines, respectively. The expression level of the pro­apoptotic DNA damage inducible transcript 3 (Gadd153) and BCL2 associated X apoptosis regulator (Bax) genes in the different groups were compared by reverse transcription-quantitative polymerase chain reaction. Additionally, the rate of apoptosis induction of modified and intact IL­24 sequences was compared by flow cytometry analysis of cells following their propidium iodide/annexin V staining. SP.RGD-IL-24 protein was expressed and secreted in a similar manner to native IL­24, however, the modified SP.RGD.IL-24 adhered to tumor cells more efficiently than IL­24 (P<0.05). SP.RGD.IL­24 significantly induced upregulation of Gadd153 and Bax in HepG2 cells compared with native IL­24 (P<0.05). However, neither had a significant impact on the expression level of pro-apoptotic genes in LX­2 cells. Flow cytometry analysis also indicated that modified SP.RGD.IL-24 induced apoptosis more than native IL­24 in HepG2 cells (P<0.05). In conclusion, the novel generated RGD­coupled IL-24 construct exhibited sufficient anticancer activity compared with the native IL­24. The results of the current study provide novel insights for the future of cytokine targeting and indicates its potential capacity as a valuable candidate for gene therapy methods.

Analysis of TLR7, SOCS1 and ISG15 immune genes expression in the peripheral blood of responder and non-responder patients with chronic Hepatitis C.

AIM: To evaluate the baseline expression of the immune genes in PBMCs of responder and non-responder patients with chronic Hepatitis C. BACKGROUND: Although the contribution of peripheral blood mononuclear cell (PBMC) gene expression in treatment outcome of hepatitis C virus (HCV) infection is supposed, it has remained to be distinctly delineated. The baseline expression of the immune genes inside PBMCs may reflect the responsiveness status following IFN treatment. METHODS: Totally, 22 chronic HCV encompasses 10 responders and 12 non-responsive cases enrolled randomly regarding medical records. The PBMCs from the peripheral blood samples were isolated and then incubated for 6 hours in the culture media. The baseline expression of TLR7, SOCS1 and ISG15 was measured by Real time PCR. RESULTS: The gene expression pattern in PBMCs of both groups showed a similar trend. The expression of SOCS1 and TLR7 genes showed higher levels in non-responder group (P>0.05). The result of ISG15 showed a higher but non-significant expression in the responder group (P>0.05). CONCLUSION: The similar pattern of TLR7, SOCS1 and ISG15 expression in the responder and non-responder patients indicated their poor discriminating and predictive value in PBMCs sample.

Metabolic and endocrine effects of bisphenol A exposure in market seller women with polycystic ovary syndrome.

Bisphenol A (BPA) is one of the synthetic monomer which can be found in the environment. Limited animal and human studies have demonstrated that BPA alters endocrine and or metabolic functions. The aims of the present study were to evaluate serum BPA level in marketing seller women with polycystic ovary syndrome (PCOS) and hormonal and metabolic effects of this exposure compared to a control paired group. In a case-control study, 62 PCOS women who work as marketing sellers and 62 healthy women with similar jobs were included. The two groups were body mass index (BMI)- and age-matched. Serum samples were analyzed for BPA content, fasting blood sugar (FBS), triglyceride, cholesterol, HDL and LDL levels, thyroid stimulating hormone (TSH) concentration, and LH:FSH ratio. Significant higher serum BPA content (0.48 ± 0.08 vs. 0.16 ± 0.04 ng/ml), triglyceride (103.05 ± 13.10 vs. 91.65 ± 12.52 mg/dl), cholesterol (165.05 ± 10.79 vs. 161.21 ± 10.31 mg/dl) levels and LH:FSH ratio (3.64 ± 0.86 vs. 0.62 ± 0.14) and significant lower TSH concentration (1.56 ± 0.68 vs. 2.15 ± 1.09 IU/ml) were detected in case against control group, respectively (P < 0.05). No significant differences were detected in FBS, LDL, and HDL levels between the two groups. Also, there were no significant associations between serum TSH concentration and BPA level neither in case (P = 0.269) nor in control (P = 0.532) groups. In BPA-exposed PCOS women, BPA level was higher than healthy women and this difference maybe the cause of significant differences in levels of triglyceride, cholesterol, TSH, and LH:FSH ratio. These observations confirm the potential role of BPA in PCOS pathophysiology.

Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting.

OBJECTIVES: Up to now, many researches have been performed to improve the antitumoral effect of melanoma differentiation-associated gene-7 (mda-7) protein. The purpose of our research was to construct 3 expression vectors producing mda-7 in fusion with RGD (Arginine-Glycine-Aspartic acid) peptide and evaluate their expression. MATERIALS AND METHODS: mda-7 gene with two different RGD sequences was amplified by PCR then was cloned by TA-cloning system. The colonies including these genes were selected by blue-white screening, colony PCR, and sequencing, respectively. Afterward, the genes were sub-cloned into the expression vector following confirmation by colony PCR and sequencing. In addition, these constructs were transfected into 293 and Huh-7 cells for further expression analysis. The mda-7 gene expression was evaluated by RT-PCR and IF (immunofluorescence assay). DNA laddering test and trypan blue exclusion assays were performed to screen cytotoxicity of prepared plasmids. RESULTS: Three different mda-7 genes with terminal RGD peptide were cloned correctly into the expression vectors and their expression was confirmed to be suitable by RT-PCR and IF assay. It was shown that expressions were limited to those transfected, GFP shining cells. No significant cytotoxicity was observed by simple assays in all plasmid treated cells. In expressing cells, all forms of mda-7 protein were localized mainly around ER prenuclear compartment while GFP protein was distributed evenly among them. CONCLUSION: Theoretically RGD tagged mda-7 would be able to induce apoptosis with more specificity and stronger than the standard one, therefore, these new constructs may have the potential for further researches.

Analysis of Naturally Occurring Resistant Mutations to Hepatitis C Virus NS3 Protease Inhibitors: A Preliminary Study in South of Iran.

BACKGROUND: Exploring the rate of naturally occurring NS3 protease mutants in HCV infected population is influential in the future therapeutic approaches. OBJECTIVES: This study explored naturally occurring resistant mutations to protease inhibitors in a pilot study. PATIENTS AND METHODS: We analyzed NS3 gene sequences in 7 HCV infected patients, referred to the central liver center, south of Iran. The protease domain was amplified by PCR followed by product extraction. Amplified NS3 genes were cloned by TA/cloning system. For each patient, clonal-sequencing was performed to improve mutation detection sensitivity. Then, the obtained sequences were compared with the reference sequences and final phylogenic tree was constructed. Afterwards, the sequences were studied to investigate point mutations. RESULTS: Phylogenetic analysis between reference and amplified sequences demonstrated high similarity of all sequences with genotype 1. Interestingly, crucial protease resistant mutations were detected in V36 and R155 positions in one patient's sequence. Checking different clones of this patient confirmed V36L, as the dominant mutation while R155K was detected only in a few cases. CONCLUSIONS: As revealed, naturally occurring resistant mutations, especially R155K in protease sequence were identified in 1 out of the 7 patients, so the rate of such mutations is estimated to be high. It seems that checking HCV patients before protease inhibitor treatment are necessary in the region.