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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

Abstract In this research, palladium (II) and platinum (II), as well as their bimetallic nanoparticles were synthesized using medicinal plants in an eco-friendly manner. Rosemary and Ginseng extracts were chosen due to their promising anticancer potential. The synthesized nanoparticles underwent characterization through FT-IR spectroscopy, DLS, XRD, EDX, SEM, and TEM techniques. Once the expected structures were confirmed, the performance of these nanoparticles, which exhibited an optimal size, was evaluated as potential anticancer agents through in vitro method on colon cancer cell lines (Ls180, SW480). MTT assay studies showed that the synthesized nanoparticles induced cell death. Moreover, real-time PCR was employed to investigate autophagy markers and the effect of nanoparticles on the apoptosis process, demonstrating a significant effect of the synthesized compounds in this regard.

Abstract Introduction Biochemistry is one of the main courses of basic sciences in the medical curriculum, along with other difficult subjects that are difficult to learn. The emergence of new technologies has made it possible to test new methods such as e-Learning. In this study, we compared two methods of Flex-Flipped Classroom (FFC) and face-to-face. Method A quasi-experimental research was done which involved both medical and dental students studying the clinical biochemistry course in the joint semester in 2019. A total of 100 medical students were trained in biochemistry through face-to-face teaching, and 60 dental students were trained in the same course through the FFC model. Three researcher-made tools were used to compare the two groups to assess the student’s satisfaction, scores, and self-evaluation. The content validity of the tools was checked using the opinions of 10 experts through the CVI index. The results were analyzed using one-sample t-tests, independent t-tests, and ANOVA. Results Both groups scored significantly more than the cut-off-point (Mean > 3.5) in their average scores of the total and sub-components of the self-evaluation questionnaire (P 0.05). The students’ knowledge score in the FFC was slightly higher than that in the face-to-face method, but this difference was not significant(P = 0.758). Conclusion Both face-to-face and FFC methods were effective according to the students, but the level of satisfaction with the face-to-face method was higher. It seems that teacher-student interaction is an important factor in students' preferences. However, the students preferred the flexibility of multimedia. It seems necessary to use the advantages of each method in a model appropriate to the students' conditions and available facilities.

Background: Emerging evidence suggests that epigenetic mechanisms contribute to 5-fluorouracil (5-FU) resistance in colorectal cancer (CRC). However, there is limited research on the direct impact of 5-FU on epigenetic alterations in CRC. This study aimed to investigate how 5-FU treatment affects the expression of enzymes involved in epigenome regulation and promoter DNA methylation in human CRC cells. Methods: The viability of CRC cell lines (SW48, HCT116, LS180, and HT29) was evaluated after 48 hours of 5-FU treatment using MTT assay in both monolayer and hanging drop spheroid cultures. The cells were treated with an IC20 concentration of 5-FU and then the relative expressions of histone deacetylases (HDAC) and DNA methyltransferas1 (DNMT1) in 5-FU-treated and untreated cells were measured by quantitative RT-PCR (qRT-PCR). The status of promoter methylation of selected genes was analyzed using the methylation-specific PCR (MSP) method. Results: The 3D cultures of cells were more resistant to 5-FU than their 2D counterparts. The effect of 5-FU on HDAC1 expression was greater in 3D cultures compared to 2D cultures. 5-FU downregulated SIRT1 and DNMT1 in 2D culture of HCT116 and SW48 and upregulated them in 3D cultures of HT29 and LS180 cells. In both monolayer and spheroid cultures, 5-FU downregulated HDAC2 in HCT116, LS180, and HT29 and HDAC4 in HCT116, LS180, and SW48 cells. 5-FU primarily changed promoter methylation in monolayer cultures. Conclusion: The epigenetic response to 5-FU is cell line-specific and depends on the culture method. 5-FU modulates epigenome in CRC cells by regulating DNMT1 and HDAC expressions. 3D cultures were found to be considerably more resistant to 5-FU-induced cytotoxicity and promoter DNA methylation changes than 2D cultures. 5-FU downregulated HDAC and DNMT1, particularly in the drug-sensitive cells, and increased the levels of DNMT1 in the drug-resistant cells.

Objective(s): Oncogenic RAS mutations occur in nearly 50% of colorectal cancer cases and are usually dependent on the autophagy mechanism to maintain tumorigenesis. We have recently demonstrated that CK1α controls autophagy machinery possibly through the AKT/p-ß-catenin (S552) signaling in colorectal cancer cells harboring RAS mutation. It has been found that a lipid-protein complex comprising oleic acid binds to human α-lactalbumin, known as HAMLET (human α -lactalbumin made lethal to tumor cells), targets a broad range of kinases including CK1α. Therefore, this study was designed to investigate the effects of BAMLET (bovine α -lactalbumin made lethal to tumor cells, the bovine counterpart of HAMLET) on CK1α expression, AKT/Phospho-ß-catenin (S552) pathway, and autophagy flux in RAS-mutated human colorectal HCT 116 cells.Materials and Methods: For this purpose, HCT116 cells were treated with BAMLET and casein kinase 1 inhibitor (D4476), and quantitative real-time polymerase chain reaction (RT-qPCR) and western blot analysis were used to measure the proteins and genes of the AKT/Phospho-ß-catenin (S552) pathway and autophagy. Apoptosis was measured by flow-cytometry. Results: We found that BAMLET significantly reduced cell viability and decreased the expression of CK1α. Additionally, BAMLET inhibited autophagy flux and enhanced the ability of CK1α inhibitor D4476 to impair autophagy flux, which was accompanied by an increase in the apoptosis percentage. We also observed that BAMLET empowered D4476 to down-regulate the AKT/Phospho-ß-catenin (S552) axis. Conclusion: BAMLET hampers autophagy flux and leads to apoptosis induction, possibly, by reducing the expression of CK1α and attenuation of the AKT/Phospho-ß-catenin (S552) axis.

Background: Autophagy and the unfolded protein response (UPR) are important pathways in colorectal tumorigenesis and drug resistance, rendering them as potential therapeutic targets for treating this cancer. As an ionophoric polyether antibiotic, salinomycin has anti-cancer effects and overcomes drug resistance in cancer cells. Considering the minimal information on the molecular action mechanism of salinomycin in colorectal cancer (CRC), this study was designed to investigate the effect of this compound on autophagy and UPR pathways in CRC cells. Methods: The in vitro cytotoxicity of salinomycin on CRC cell line HCT116 was determined using the MTT assay by treating the cells with different concentrations of salinomycin for 24 and 48 h. The gene expression analysis of three main autophagy biomarkers (Beclin1, LC3, and P62) and two UPR biomarkers (XBP-1s and CHOP) was performed using quantitative real-time polymerase chain reaction (RT-PCR). Data were analyzed with GraphPad Prism 8 software. Results: Salinomycin had cytotoxic effects on HCT116 cells in a time- and dose-dependent manner. The expression analysis of the UPR and autophagy-related genes showed UPR activation at both 24 h and 48 h (increase of XBP-1s and CHOP), autophagy activation at 24 h (increase of Beclin 1, LC3II, and decrease of P62), and autophagy flux inhibition at 48 h (increase of Beclin 1, LC3II and P62). Conclusion: The anti-cancer activity of salinomycin against the HCT116 cell line seems to be through triggering cell death by targeting UPR and autophagy pathways. Further studies are required to confirm our results.

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Context: The uninterrupted dissemination and transmission of COVID-19 brought enormous jeopardy and public-health urgency during past three years. Some patients, who recovered from the infection, develop persistent symptoms and organ dysfunction for weeks or even months; conditions that are called long COVID. Among multiple COVID-related complications, positive individuals suffer from lesser to greater degrees of intrahepatic and extrahepatic complications that principally mediated by ACE2 receptors. Evidence acquisition: We reviewed manuscripts from PubMed, Google Scholar and Web of Science on underlying COVID-19-linked clinical relevance and potential pathogenesis of liver complications during short COVID, as well as long COVID with no time limitation. Results: Liver impairment needs a large-scale and persistent follow-up as it may be multivariate in nature. During COVID-19, physicians must assess whether hepatopathy is associated with hepatic disorders, medications utilized for the COVID-19 therapy, viral antigenic outcomes progression to a complicated course. In the context of COVID-19, physicians report that potential pathophysiological approaches to hepatic failure in critical patients further could be leading to deep vein thrombosis, myocardial infarction, venous thromboembolism, and acute kidney injury. These complications might be either reversible, or irreversible with extended manifestations that mostly occur as a consequence of long COVID in the post-COVID era. Moreover, pre-existing cardiovascular and digestive tract problems tend to be highly correlated with adverse clinical outcomes and the highest fatality rate. Potential drug-disease interactions adversely influencing COVID-19 subjects and persistent comorbidities are also getting a major consideration. Conclusion: The assessment of whether the upshot of hepatic-associated co-morbidities in COVID-19 status can improve an acceptable personalized therapeutic strategy considering these specific tropisms.

Abstract Background Ischemia-reperfusion (I/R) of the liver is a multifactorial condition that happens during transplantation and surgery. The deleterious effects of I/R result from the acute production of reactive oxygen species (ROS), which can trigger immediate tissue damage and induce a series of destructive cellular responses, including apoptosis organ failure and inflammation. The production of ROS in the I/R process can damage the antioxidant system and cause liver damage. Resveratrol has been shown to have antioxidant properties in several investigations. Here, we address the therapeutic effect of resveratrol on I/R-induced liver injury by focusing on unfolded protein response (UPR) signaling pathway. Methods Five minutes before reperfusion, resveratrol was injected into the tail vein of mice. They were ischemic for 1 h and then re-perfused for 3 h before being slaughtered (I/R). The activity of liver enzymes and the expression levels of genes involved in the unfolded protein response pathway were used to measure the hepatic damage. Results Our results revealed that the low dose of resveratrol (0.02 and 0.2 mg/kg) post-ischemic treatment significantly reduced the ALT and AST levels. In addition, compared with the control group, the expression of UPR pathway genes GRP78, PERK, IRE1α, CHOP, and XBP1 was significantly reduced in the resveratrol group. In the mice that received lower doses of resveratrol (0.02 and 0.2 mg/kg), the histopathological changes induced by I/R were significantly improved; however, the highest dose (2 mg/kg) of resveratrol could not significantly protect and solve the I/R damage. Conclusion The findings of this study suggest that hepatic ischemia occurs after liver transplantation and that receiving low-dose resveratrol treatment before reperfusion may promote graft survival through inhibition of UPR arms, especially PERK and IRE1α.

Background: The AKT/PKB (protein kinase B) kinase is the main regulator of autophagy in mammalian cells, which consists of three isoforms, including AKT-1, AKT-2, and AKT-3. Rat sarcoma viral oncogene homolog (RAS), known as the most frequently mutated oncogene in colorectal cancers, is one of the major activators of AKT signaling. However, the relationship between AKT isoforms expression and autophagy level in RAS-driven cancer cells has not been fully investigated. Method: In this experimental in vitro study, RAS mutated colon cancer cell lines (HCT116, SW480, and LS180) and HT29 cells, which are the wild type of RAS, were cultured and real-time polymerase chain reaction (RT-PCR) was utilized to determine the mRNA level of AKT-1, AKT-2, and autophagy markers, including microtubule-associated protein 1 light chain-3B (LC3B) and p62/sequestosome-1 (p62). In addition, Western blotting was performed to assess the protein expression of p62 and LC3B lipidation. Results: We found that RAS mutated colon cancer cells up-regulate basal autophagy. Moreover, highly expressed AKT-1 was observed in RAS mutated colon cancer cells. However, no significant differences were found in AKT-2 expression between RASdriven cells and HT29 cells. Conclusion: Our obtained data suggested that RAS-driven colon cancer cells regulated the autophagy machinery, possibly, through the upregulation of AKT-1 isoform.

Background Colorectal cancer (CRC) is one of the more diffuse reasons for malignancy-associated death worldwide. According to the evidence that CRC is predominantly symptomless as it advances to the highest stages, the administration of screening schemes planned at initial diagnosis is required to lower the prevalence and fatality rate . We aimed to review the literature on different molecular procedures for detection of stool-based biomarkers in CRC. Methods and Evidence Acquisition In the current study we reviewed papers of Google Scholar and PubMed database with no time limitation. Conclusion An integrative framework of all epigenetic and genetic modifications compared to other traditional procedures were studied which represented a more acceptable specificity and sensitivity for the diagnosis, treatment, evaluating drug response and also the clinical outcome of CRC. Differential expression analysis of stool-derived RNAs (sRNAs) and stool DNA (sDNA) testing for ultrasensitive mutations, methylation, and fragmentation patterns can lead to an early-stage diagnosis and a better prognosis for CRC patients in a higher accuracy.

Abstract Background: Cancer stem cells (CSCs) have critical roles in tumor initiation, progression, metastasis, resistance to chemoradiotherapy, and recurrence in various cancers such as colon cancer. Therefore, extensive studies are required to understand the CSCs mechanism of action and design novel cancer therapies. Successful isolation and characterization of CSCs from tumor tissues or cancer cell lines provide the proper opportunity for these kinds of studies. This study aimed to isolate and characterize CSCs from HT29 colon cancer cell lines. Methods: Sphere formation assay was used to isolate CSCs from the HT29 cell line. The expression of four stemness genes, including LGR5, SOX2, c-Myc, and Oct4, was assessed by real-time PCR. The CSC markers, CD 44 and CD 24, were also evaluated and compared between the parental HT29 cell line and HT29-derived spheres by flow cytometry. Statistical analysis was performed using GraphPad Prism version 6.0 and the Mann-Whitney U test. Results:HT29-derived sphere cells were successfully formed in serum-free media. The proportion of CD 24 and CD 44-expressing cells and the expression of LGR5, SOX2, c-Myc, and Oct4 stemness genes were significantly higher in CSCs isolated from HT29 spheres compared with parental HT29 cells. Conclusion:Sphere formation assay is a proper method for the isolation of CSCs from the HT29 cell line. The stemness markers LGR5, SOX2, c-Myc, Oct4,CD 44,andCD 24are suitable for the characterization of these cells.

The coronavirus disease 2019 (COVID-19) pandemic has become the most serious global public health issue in the past two years, requiring effective therapeutic strategies. This viral infection is a contagious disease caused by new coronaviruses (nCoVs), also called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Autophagy, as a highly conserved catabolic recycling process, plays a significant role in the growth and replication of coronaviruses (CoVs). Therefore, there is great interest in understanding the mechanisms that underlie autophagy modulation. The modulation of autophagy is a very complex and multifactorial process, which includes different epigenetic alterations, such as histone modifications and DNA methylation. These mechanisms are also known to be involved in SARS-CoV-2 replication. Thus, molecular understanding of the epigenetic pathways linked with autophagy and COVID-19, could provide novel therapeutic targets for COVID-19 eradication. In this context, the current review highlights the role of epigenetic regulation of autophagy in controlling COVID-19, focusing on the potential therapeutic implications.

Background: Autophagy and unfolded protein response (UPR) are mechanisms with dual roles in both maintaining the cellular homeostasis and progression of various diseases such as cancer. Therefore, identification of different molecules and proteins involved in the regulation of these pathways may contribute to find new therapeutic targets. A member of the M28 family of the metallopeptidases, Endoplasmic Reticulum Metallo Protease 1 (ERMP1), is overexpressed in cancers such as colorectal cancer. The role of this protein in the UPR activation was previously reported in breast cancer. We aimed to evaluate the role of ERMP1 in the activation of autophagy and apoptosis in colorectal cancer. Methods: ERMP1 Gene silencing was performed using specific small hairpin RNA (shRNA) in HCT-116 colorectal cancer cell line. Then, autophagy associated protein markers including Beclin 1, p62 and LC3II were evaluated using western blot. The effect of ERMP1 knockdown on cellular apoptosis was also assessed by propidium iodide staining flow cytometry analysis. Statistical analysis was performed using SPSS software version 20. Results: All three autophagy markers were increased significantly in the ERMP1-silenced HCT116 cell lines compared with negative control cells (P < 0.05). It seems that ERMP1 silencing inhibits autophagy at the flux stage. However, ERMP1 knockdown had no significant effect on HCT-116 apoptotic cell death (P > 0.05). Conclusion: The oncogenic protein, ERMP1, activates autophagy in colorectal cancer cell line. Targeting of ERMP1 may be considered as a proper approach in colorectal cancer therapy. Further investigations are required to confirm these results.

Abstract Background The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has profoundly affected the lives of millions of people. To date, there is no approved vaccine or specific drug to prevent or treat COVID-19, while the infection is globally spreading at an alarming rate. Because the development of effective vaccines or novel drugs could take several months (if not years), repurposing existing drugs is considered a more efficient strategy that could save lives now. Statins constitute a class of lipid-lowering drugs with proven safety profiles and various known beneficial pleiotropic effects. Our previous investigations showed that statins have antiviral effects and are involved in the process of wound healing in the lung. This triggered us to evaluate if statin use reduces mortality in COVID-19 patients. Results After initial recruitment of 459 patients with COVID-19 (Shiraz province, Iran) and careful consideration of the exclusion criteria, a total of 150 patients, of which 75 received statins, were included in our retrospective study. Cox proportional-hazards regression models were used to estimate the association between statin use and rate of death. After propensity score matching, we found that statin use appeared to be associated with a lower risk of morbidity [HR = 0.85, 95% CI = (0.02, 3.93), P = 0.762] and lower risk of death [(HR = 0.76; 95% CI = (0.16, 3.72), P = 0.735)]; however, these associations did not reach statistical significance. Furthermore, statin use reduced the chance of being subjected to mechanical ventilation [OR = 0.96, 95% CI = (0.61–2.99), P = 0.942] and patients on statins showed a more normal computed tomography (CT) scan result [OR = 0.41, 95% CI = (0.07–2.33), P = 0.312]. Conclusions Although we could not demonstrate a significant association between statin use and a reduction in mortality in patients with COVID19, we do feel that our results are promising and of clinical relevance and warrant the need for prospective randomized controlled trials and extensive retrospective studies to further evaluate and validate the potential beneficial effects of statin treatment on clinical symptoms and mortality rates associated with COVID-19.

Abstract Background ARID1A has been described as a tumor suppressor gene, participating in chromatin re-modeling, epithelial-mesenchymal-transition and many other cellular and molecular processes. It has been cited as a contribute in tumorigenesis. The role of ARID1A in CRC is not yet defined. Aim To investigate the role of ARID1A methylation and CNV in its expression in CRC cell lines and to examine the relationship between ARID1A status with survival and clinicopathologic characteristics in patients with CRC. Methods We used RT-PCR to determine both CNV and expression of ARID1A from six CRC cell lines. We used MSP to evaluate methylation of ARID1A. IHC was used to assess ARID1A protein expression. We also evaluated MSI and EMAST status in 18 paired CRC and adjacent normal tissues. 5AzadC was used to assess effect of DNA demethylation on ARID1A expression. Statistical analysis was performed to establish correlations between ARID1A expression and other parameters. Results Among the 18 CRC tumors studied, 7 (38.8%) and 5 tumors (27.7%) showed no or low ARID1A expression, respectively. We observed no significant difference in ARID1A expression for overall patient survival, and no difference between clinicopathological parameters including MSI and EMAST. However, lymphatic invasion was more pronounced in the low/no ARID1A expression group when compared to moderate and high expression group (33% VS. 16.6% respectively. ARID1A promoter methylation was observed in 4/6 (66%) cell lines and correlated with ARID1A mRNA expression level ranging from very low in SW48, to more pronounced in HCT116 and HT-29/219. Treatment with the methyltransferase inhibitor 5-Azacytidine (5-aza) resulted in a 25.4-fold and 6.1-fold increase in ARID1A mRNA expression in SW48 and SW742 cells, respectively, while there was no change in SW480 and LS180 cells. No ARID1A CNV was observed in the CRC cell lines. Conclusion ARID1A expression is downregulated in CRC tissues which correlates with it being a tumor suppressor protein. This finding confirms ARID1A loss of expression in CRC development. Our in-vitro results suggest high methylation status associates with reduced ARID1A expression and contributes to CRC tumorigenesis. However, there was no significant association between ARID1A loss of expression and clinicopathological characteristics. Future in-vivo analysis is warranted to further establish ARID1A role in colorectal neoplastic transformation.

ARID1A, a bona fide tumor suppressor, is the third most mutated gene in human colorectal cancer. In this paper, we reported the identification of a novel mutation in one of 20 primary human colorectal cancer tumors. Sanger sequencing of the exon 3 of the ARID1A gene revealed a novel heterozygous non-sense mutation of the c.1653C>G. This resulted in the premature termination of the 2285 amino acid protein at the 551st codon. Mutation Taster predicted this novel mutation to be pathogenic. Immunohistochemical analysis revealed the loss of ARID1A protein expression in cancerous tissues harboring ARID1A mutation. As far as we know, this is the first report on the mutation of ARID1A gene in an Iranian patient. This mutation can expand the spectrum of ARID1A gene pathogenic mutations among colorectal cancer patients.

Abstract: 1. Context Endoplasmic reticulum stress (ER stess) is associated with endoplasmic reticulum perturbation homeostasis. Prolonged ER stress conditions may induce cell death. Unfolded protein response (UPR) attempts to restore normal cell conditions. 2. Evidence Acquisition There now exists an emergent body of evidence identifying the WNT signaling network as a regulator of cancer cell metabolism. Given that existing findings show that the WNT pathway and ER stress regulates changes in metabolic activities of cancer cells suggesting these signaling pathways represent critical nodes in the regulation of central metabolism in tumors. 3. Results Findings suggest that the molecular cross-talks between hypoxic ER stress, Wnt/βcatenin signaling, may represent an important mechanism that enables some tumor subtypes to survival and grow in hypoxic conditions. 4. Conclusions The present article disuses differential effects of the activation of the three arms of UPR, namely endoplasmic reticulum kinase (PERK), activation transcription factor -6 (ATF-6), and inositol –requiring enzyme (IRE-1) on cancer. This review also highlights regulators and downstream effectors of Wnt cascade and addresses the increasingly apparent crosstalk of Wnt with other tumorigenic signaling pathways.

Milk protein, alpha-lactalbumin, is one of the proteins that many researchers have done a lot of research on its structure and function. In the late nineties, a completely different activity of α-LA was discovered. When α-LA is present in a conformation that has been partially unfolded, it can form a complex with oleic acid (OA), which specifically has an anti-cancer activity. The complex between human α-LA and OA has been called HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells). The cell death mechanism by HAMLET has not yet been determined. However, it has been shown that HAMLET interacts with various cellular organelles including nuclei, lysosomes, mitochondria, proteases, and ribosomes. Apoptosis, autophagy, and disorders in the structure of chromatin are three different cytotoxic pathways activated in response to HAMLET. In this study, a summary of the structure, antitumor mechanisms, and clinical applications of this complex will be presented.

Purpose: GnRH-DFF40 (gonadotropin releasing hormone - DNA fragmentation factor 40) is a humanized recombinant immunotoxin and serves as a prospective candidate for targeted therapy of gonadotropin releasing hormone receptor (GnRHR) overexpressing malignancies. However, its production in Escherichia coli in a soluble and functional form still remains a challenge. Here we introduce two successful and reproducible conditions for production and purification of "difficult-to-express" GnRH-DFF40 protein. Methods: A synthetic codon optimized GnRH-DFF40 fusion gene was cloned in pET28a plasmid. Two methods including high cell density IPTG induction (HCDI) and autoinduction method (AIM) with a focus on obtaining high cell density have been investigated to enhance the protein production in (E. coli). Moreover, to obtain higher protein production several factors in the AIM method including carbon sources, incubation time and temperature, plasmid stability and double colony selection, were optimized. Results: Remarkable amounts of soluble GnRH-DFF40 protein were achieved by both methods. Cell density and protein yields in AIM was about 1.5 fold higher than that what obtained using HCDI. Initial screening showed that 25ºC is better to achieve higher protein production in both methods. pH alterations in AIM were maintained in a more constant level at 25ºC and 37ºC temperatures without any detrimental effects on cell growth during protein production phase up to 21 hours after incubation. Plasmid stability during growth and expression induction phase was maintained at a high level of 98% and 96% for AIM and HCDI methods, respectively. After parameter optimization and double colony selection in AIM, a very high yield of recombinant protein was achieved (528.3 mg/L). Conclusion: With the optimization of these high cell density expression methods, reproducible manifold enhancement of soluble protein yields can be achieved for "difficult-to-express" GnRH-DFF40 compared to conventional expression methods.

Background: Differentiating oligodendrocyte precursor cells (OPCs) into oligodendrocytes could be improved by inhibiting signaling pathways such as Wnt and Notch. 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) can ameliorate oligodendrogenesis. We investigated whether they could increase oligodendrogenesis by inhibiting the Wnt and Notch signaling pathways. Methods: Cortical neural stem cells were isolated from 14-day-old rat embryos and cultured using the neurosphere assay. The cells were treated in 4 different conditions for 1 week: the negative control group received only the basic fibroblast growth factor, the positive control group received only T3 without growth factors, the RA group was treated with 9-cis-RA, and the Vit D3 group was treated with 1,25(OH)2D3. The effects of 9-cis-RA and 1,25(OH)2D3 on the level of the myelin basic protein (MBP) and the gene expression of the SOX10, MBP gene, HES5, and LRP6 were studied using flow cytometry and real-time PCR. The data were analyzed using one-way ANOVA with GraphPad Prism. A P value less than 0.05 was considered significant. Results: The mRNA expressions of the SOX10, MBP, and MBP gene were significantly increased in the treated groups compared with the negative control group; the increase was similar in the 9-cis-RA group and the positive control group. Furthermore, 9-cis-RA significantly decreased the expression of the HES5 gene, a Notch signaling pathway transcription factor, and 1,25(OH)2D3 significantly reduced the expression of the LRP6 gene, a Wnt signaling pathway co-receptor. Conclusion: It seems that 9-cis-RA and 1,25(OH)2D3 are good candidates to improve the differentiation of OPCs into oligodendrocytes.

Over the recent years, isolation of high quantities of pure, intact, double-stranded, highly concentrated and not contaminated genomic DNA is prerequisite for successful and reliable large scale genetic and epigenetic analysis and also high quantities of pure DNA are required for these types of studies. The DNA extraction methods developed for human somatic cells are not effective for sperm because of the high rating of nuclear compaction and DNA integrity in sperm cells. In the present study, an economic, reliable and simple method of DNA extraction procedures, were examined to ascertain their relative effectiveness for extracting DNA from human sperm. The quality and quantity of the extracted DNA were assessed by spectrophotometric measurements, methylation-specific PCR (MSP) amplification, and gel electrophoresis. This method was applied successfully from sperm samples (N = 50, age = 33.9 ± 7.8 years) with DNAs absorbency ratios of A260/A280 (1.82 ± 0.03) and A260/230 (1.95 ± 0.16). Gel electrophoresis revealed non-degraded and suitable quality genomic DNA for epigenetic and genetic analysis. The purity and quality of this protocol was closer to the optimum value sand no contamination during manual extraction was observed. It concluded that this described method might have a sufficient quality for subsequent molecular analysis and general genetic and epigenetic methods. Keywords: Absorbency ratios, DNA concentration, Genetic and epigenetic studies, Protamine, Proteinase K, Sperm DNA extraction, TRIzol

Background: Discriminating latent tuberculosis infection (LTBI) from active TBI may be challenging. The objective of this study was to produce the recombinant L-alanine dehydrogenase (AlaDH) antigen and evaluate individuals with LTBI, those with active TBI, and uninfected individuals by enzyme-linked immunospot assay (ELISPOT) in order to distinguish LTBI from active TBI. Methods: This exploratory study was performed in the Iranian city of Shiraz from 2014 to 2015. The study population (N=99) was divided into 3 groups: individuals with newly diagnosed active TBI (n=33), their household contacts (n=33), and controls (n=33). AlaDH was produced through PCR and cloning methods. The diagnostic characteristics of AlaDH vs. ESAT-6/CFP-10 were evaluated in responses to interferon-γ (IFN-γ) and interleukin-2 (IL-2) with ELISPOT. Differences between the groups were assessed with the Kruskal–Wallis and Mann–Whitney tests for nonparametric data analysis. The statistical analyses were performed with SPSS, version 16. Results: IFN-γ responses to both ESAT-6/CFP-10 (P=0.81) and AlaDH (P=0.18) revealed that there were no significant differences between the individuals with LTBI and those with active TBI. The same results were determined for IL-2 responses to ESAT-6/CFP-10 between the 2 groups, while significantly higher IL-2 responses to AlaDH were observed in LTBI than in active TBI. According to the ROC curve analysis, a cutoff value of 275 SFC showed sensitivity of 75.8% and specificity of 78.8% for distinguishing LTBI from active TBI by IL-2 responses to AlaDH. Conclusion: The current study suggests that it may be possible to discriminate LTBI from active TBI by IL-2 responses to AlaDH.

Cellular protein homeostasis in the lungs is constantly disrupted by recurrent exposure to various external and internal stressors, which may cause considerable protein secretion pressure on the endoplasmic reticulum (ER), resulting in the survival and differentiation of these cell types to meet the increased functional demands. Cells are able to induce a highly conserved adaptive mechanism, known as the unfolded protein response (UPR), to manage such stresses. UPR dysregulation and ER stress are involved in numerous human illnesses, such as metabolic syndrome, fibrotic diseases, and neurodegeneration, and cancer. Therefore, effective and specific compounds targeting the UPR pathway are being considered as potential therapies. This review focuses on the impact of both external and internal stressors on the ER in idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) and discusses the role of the UPR signaling pathway activation in the control of cellular damage and specifically highlights the potential involvement of non-coding RNAs in COPD. Summaries of pathogenic mechanisms associated with the ER stress/UPR axis contributing to IPF and COPD, and promising pharmacological intervention strategies, are also presented.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor with a very poor survival rate. Temozolomide (TMZ) is the common chemotherapeutic agent used for GBM treatment. We recently demonstrated that simvastatin (Simva) increases TMZ-induced apoptosis via the inhibition of autophagic flux in GBM cells. Considering the role of the unfolded protein response (UPR) pathway in the regulation of autophagy, we investigated the involvement of UPR in Simva–TMZ-induced cell death by utilizing highly selective IRE1 RNase activity inhibitor MKC8866, PERK inhibitor GSK-2606414 (PERKi), and eIF2α inhibitor salubrinal. Simva–TMZ treatment decreased the viability of GBM cells and significantly increased apoptotic cell death when compared to TMZ or Simva alone. Simva–TMZ induced both UPR, as determined by an increase in GRP78, XBP splicing, eukaryote initiation factor 2α (eIF2α) phosphorylation, and inhibited autophagic flux (accumulation of LC3β-II and inhibition of p62 degradation). IRE1 RNase inhibition did not affect Simva–TMZ-induced cell death, but it significantly induced p62 degradation and increased the microtubule-associated proteins light chain 3 (LC3)β-II/LC3β-I ratio in U87 cells, while salubrinal did not affect the Simva–TMZ induced cytotoxicity of GBM cells. In contrast, protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibition significantly increased Simva–TMZ-induced cell death in U87 cells. Interestingly, whereas PERK inhibition induced p62 accumulation in both GBM cell lines, it differentially affected the LC3β-II/LC3β-I ratio in U87 (decrease) and U251 (increase) cells. Simvastatin sensitizes GBM cells to TMZ-induced cell death via a mechanism that involves autophagy and UPR pathways. More specifically, our results imply that the IRE1 and PERK signaling arms of the UPR regulate Simva–TMZ-mediated autophagy flux inhibition in U251 and U87 GBM cells.

Background: Optimized RNA extraction from tissues and cell lines consists of four main stages regardless of the method of extraction: 1) homogenizing, 2) effective denaturation of proteins from RNA, 3) inactivation of ribonuclease, and 4) removal of any DNA, protein, and carbohydrate contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of RNase. Various technical difficulties occur during extraction of RNA from pancreatic tissue due to spontaneous autolysis. Since standard routine protocols yield unacceptable results in pancrease, we have designed a simple method for RNA extraction by comparing different protocols. Methods: We obtained 20-30 mg pancreatic tissues in less than 2 min from 30 rats. Several methods were performed to extract RNA from pancreatic tissue and evaluate its integrity. All methods were performed three times to obtain reproducible results. Results: Immersing pancreatic tissue in RNA-later for 24 h at -80ºC yielded high quality RNA by using the TriPure reagent which was comparable to the commercial RNeasy Micro Kit. The quality of RNA was evaluated by spectrophotometer, electrophoresis and RT-PCR. We separated intact 28S and 18S ribosomal RNA (rRNA) when our procedure was compared with the RNeasy Micro Kit. Finally, full length of the actin gene was amplified by RT-PCR. Conclusion: We designed a simple, fast, cost-effective method for complete RNA extraction from the least amount of quantitatively intact pancreatic tissue

Background: Colorectal Cancer (CRC) is the third common cancer in the world. One of the pathways in colorectal tumor genesis is Microsatellite Instability (MSI+). MSI is detected in about 15% of all colorectal cancers. Colorectal tumors with MSI have dis-tinctive features compared with Microsatellite Stable (MSS) tumors. Due to the high percentage of MSI+ in patients with CRC in Iran, screening of this type of CRC is im-perative. In current study, two markers (BAT-26 and BAT-25) were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose MSI status in patients with CRC. Methods: Allelic variation in two markers (BAT-26 and BAT-25) was analyzed in tis-sues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic colorectal cancer by Real Time PCR (Hy-bridization probe) and High-Performance Liquid Chromatography (HPLC) techniques. The sensitivity and specificity of Real Time PCR and HPLC compared with sequencing as gold standard. The data were statistically analyzed using Student’s t-test and 2 or fisher exact test, where applicable with (P

Context: The incidence of colorectal cancer has significantly increased in Iran during the last decade. Accumulating evidence suggeststhat there is a significant correlation between genetic variations such as polymorphisms and colorectal cancer. Therefore,identification of critical polymorphisms related to colorectal cancer can contribute to find individuals at high risk of CRC.Evidence Acquisition: The focus of this review was on published articles in English about the association between different singlenucleotide polymorphisms and colorectal cancer in the Iranian population. Evidences were gathered by searching online medicaldatabases including Google scholar, Pubmed, Scopus and Science Direct.Conclusions: Various single nucleotide polymorphisms of critical genes indicated significant association with colorectal cancer inthe Iranian population. New polymorphism markers for high risk individuals have been recognized through further investigationsto reduce the incidence and mortality of colorectal cancer.

Different DNA aberrations processes can cause colorectal cancer (CRC). Herein, we conducted a comprehensive molecular characterization of 27 CRCs from Iranian patients.Array CGH was performed. The MSI phenotype and the methylation status of 15 genes was established using MSP. The CGH data was compared to two established lists of 41 and 68 cancer genes, respectively, and to CGH data from African Americans. A maximum parsimony cladogram based on global aberrations was established.The number of aberrations seem to depend on the MSI status. MSI-H tumors displayed the lowest number of aberrations. MSP revealed that most markers were methylated, except RNF182 gene. P16 and MLH1 genes were primarily methylated in MSI-H tumors. Seven markers with moderate to high frequency of methylation (SYNE1, MMP2, CD109, EVL, RET, LGR and PTPRD) had very low levels of chromosomal aberrations. All chromosomes were targeted by aberrations with deletions more frequent than amplifications. The most amplified markers were CD248, ERCC6, ERGIC3, GNAS, MMP2, NF1, P2RX7, SFRS6, SLC29A1 and TBX22. Most deletions were noted for ADAM29, CHL1, CSMD3, FBXW7, GALNS, MMP2, NF1, PRKD1, SMAD4 and TP53. Aberrations targeting chromosome X were primarily amplifications in male patients and deletions in female patients. A finding similar to what we reported for African American CRC patients.This first comprehensive analysis of CRC Iranian tumors reveals a high MSI rate. The MSI tumors displayed the lowest level of chromosomal aberrations but high frequency of methylation. The MSI-L were predominantly targeted with chromosomal instability in a way similar to the MSS tumors. The global chromosomal aberration profiles showed many similarities with other populations but also differences that might allow a better understanding of CRC's clinico-pathological specifics in this population.

Mutations and promoters' methylation of a set of candidate cancer genes (CAN genes) are associated with progression of colorectal cancer (CRC). We hypothesized that these genes' promoters are inactivated through epigenetic silencing and may show a different profile in high-risk populations. We investigated the status of CAN gene methylation and CHD5 protein expression in African American CRC tissue microarrays (TMA) using immunohistochemical staining.The promoter methylation status of the CAN genes was studied by methylation-specific PCR (MSP) in 51 Iranians (a white population) and 51 African Americans (AA). Microsatellite instability (MSI) was analyzed as well. The differential frequency of methylation for each gene was tested by chi-square analysis between the two groups based on matched age and sex. CHD5 protein expression was evaluated in moderate to well differentiated and poorly differentiated carcinomas compared to matched normal tissue using TMA. In addition, the correlation between these epigenetic biomarkers and various clinicopathological factors, including, age, location, and stage of the disease were analyzed. Seventy-seven and 34% of tumors were distal in Iranian and African American patients, respectively. In both populations, the percentage of methylation was >65% for SYNE1, MMP2, APC2, GPNMB, EVL, PTPRD, and STARD8, whereas methylation was