Evaluation of Autophagy Process in Differentiation of Human Induced Pluripotent Stem Cells toward Insulin Producing Cells.

Background: Autophagy is an intracellular self-degradative homeostasis process which eliminates undesirable and harmful macromolecules and organelles. Autophagy is also involved in self-renewal and differentiation of induced pluripotent stem cell (iPSCs). Objective: In this study, we investigated the expression profile of autophagy marker genes in human iPSCs during their differentiation induction toward insulin producing ß-like cells. Methods: Human iPSC line, R1-hiPSC1, was used for differentiation induction toward ß-like cells. The mRNA expression of Nanog, OCT4 (pluripotency markers), SOX17, FOXA2 (endodermic markers), PTF1A, NKX6.1 (exocrine/endocrine determinants), and PDX1 were measured during differentiation stages. Autophagy was monitored by genes expression study of four autophagy markers, MAP1LC3B, BECN1, SQSTM1/P62 and ATG5, along with protein expression profile of LC3b-II during differentiation stages. Results: The mRNA expression measurement of pluripotency, endoderm and exocrine/endocrine marker genes confirmed that hiPSCs skipped pluripotency, differentiated into endoderm, passed through the pancreatic lineage commitment stage and successfully generated insulin producing ß-like cells. Expression profile of autophagy genes during differentiation stages indicated the decreased expression levels at the early stages (EB and MEI) and then increased at the definitive endoderm stages (DEI 1, DEI 2 and DE) followed by a subtractive pattern toward the end of differentiation. The results of protein expression of LC3b-II were consistent with gene expression data. Conclusion: This study demonstrated the high contribution of key autophagy genes/proteins during the differentiation of hiPSC toward ß-like cells. The enhanced autophagy levels were a prominent feature of early stages of differentiation and DE rather than the later stages.

Ultrasound elastography using a regularized modified error in constitutive equations (MECE) approach: a comprehensive phantom study.

Many of the current techniques in transient elastography, such as shear wave elastography (SWE) assume a dominant planar shear wave propagating in an infinite medium. This underlying assumption, however, can be easily violated in real scenarios in vivo, leading to image artifacts and reconstruction errors. Other approaches that are not bound to planar shear wave assumption, such solutions based on the partial differential equation, can potentially overcome the shortcomings of the conventional SWE. The main objective of this paper is to demonstrate the advantages of the modified error in constitutive equations (MECE) formulation with total variation regularization (MECE + TV) over SWE in reconstructing the elastic moduli of different tissue-mimicking phantoms. Experiments were conducted on phantoms with inclusions of well-defined shapes to study the reconstruction of specific features relevant to practical applications. We compared the performances of MECE + TV and SWE in terms of quantitative metrics to estimate reconstruction accuracy, inclusion shape recovery, edge preservation and edge sharpness, inclusion size representation, and shear elasticity and contrast accuracies. The results indicate that the MECE + TV approach outperforms SWE based on several of these metrics. It is concluded that, with further development, the proposed method may offer elastography reconstructions that are superior to SWE in clinical applications.

IBD Patients Could Be Silent Carriers for Novel Coronavirus and Less Prone to its Severe Adverse Events: True or False?

Inflammatory bowel diseases (IBDs) are chronic disorders of the gastrointestinal tract. The goal of IBD treatment is to reduce the inflammation period and induce long-term remission. Use of anti-inflammatory drugs including corticosteroids, immunosuppressants and biologicals, is often the first step in the treatment of IBD. Therefore, IBD patients in pandemic of infectious diseases are considered a high-risk group. The public believes that IBD patients are at a higher risk in the current coronavirus 2 pandemic. Nevertheless, these patients may experience mild or moderate complications compared to healthy people. This might be because of particular anti-TNF-α treatment or any immunosuppressant that IBD patients receive. Moreover, these patients might be silent carrier for the virus.

Autophagy activation is required for influenza A virus-induced apoptosis and replication.

Autophagy and apoptosis are two major interconnected host cell responses to viral infection, including influenza A virus (IAV). Thus, delineating these events could facilitate the development of better treatment options and provide an effective anti-viral strategy for controlling IAV infection. We used A549 cells and mouse embryonic fibroblasts (MEF) to study the role of virus-induced autophagy and apoptosis, the cross-talk between both pathways, and their relation to IAV infection [ATCC strain A/Puerto Rico/8/34(H1N1) (hereafter; PR8)]. PR8-infected and mock-infected cells were analyzed by immunoblotting, immunofluorescence confocal microscopy, electron microscopy and flow cytometry (FACS). We found that PR8 infection simultaneously induced autophagy and apoptosis in A549 cells. Autophagy was associated with Bax and Bak activation, intrinsic caspase cleavage and subsequent PARP-1 and BID cleavage. Both Bax knockout (KO) and Bax/Bak double knockout MEFs displayed inhibition of virus-induced cytopathology and cell death and diminished virus-mediated caspase activation, suggesting that virus-induced apoptosis is Bax/Bak-dependent. Biochemical inhibition of autophagy induction with 3-methyladenine blocked both virus replication and apoptosis pathways. These effects were replicated using autophagy-refractory Atg3 KO and Atg5 KO cells. Taken together, our data indicate that PR8 infection simultaneously induces autophagy and Bax/caspase-dependent apoptosis, with autophagy playing a role to support PR8 replication, in part, by modulating virus-induced apoptosis.

ERBB4 gene polymorphisms and the risk of prostate cancer in a sample of Iranian Population.

Genetic polymorphisms in ERBB4 are thought to be associated with cancer susceptibility. In the present study, we aimed to assess the impact of ERBB4 rs12052398 T>C, rs13393577 A>G, rs13424871 A>T, rs16847082 A>G and rs6147150 (12-bp I/D) polymorphisms on risk of prostate cancer (PCa) in a sample of Iranian population. In a case-control study, we enrolled 169 patients with pathologically confirmed PCa and 182 subjects with benign prostatic hyperplasia (BPH). No significant association was found among ERBB4 polymorphisms and risk of PCa. Subjects carrying TT/AA/AA/AG/ID, TC/AA/AA/AA/II, TT/AA/AT/AA/II and TT/AA/AT/AG/ID genotypes are associated with a decreased risk of PCa. Our findings suggest that haplotypes CAAAI and TAAAD (rs12052398, rs13393577, rs13424871, rs16847082 and rs6147150I) of the ERBB4 polymorphisms are associated with a significantly lower risk of PCa. Further studies with a larger sample sizes and diverse ethnicities are necessary to verify our findings.

miR-608 rs4919510 C>G polymorphism decreased the risk of breast cancer in an Iranian subpopulation.

AIM: MicroRNAs (miRNAs) are small noncoding RNAs that function as oncogene or tumor suppressors. The single nucleotide polymorphisms in miRNAs potentially can alter miRNA-binding sites on target genes as well as affecting miRNAs expression. The present study aimed to evaluate the impact of miR-608 rs4919510 C>G variant on breast cancer (BC) risk. MATERIALS AND METHODS: This case-control study conducted on 160 women with BC and 192 age-matched healthy women. Genotyping of miR-608 rs4919510 was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Our findings showed that GC genotype significantly decreased the risk of BC (odds ratio (OR) = 0.49, 95% confidence interval (CI) 0.28-0.88, p = 0.018) compared to CC genotype. Furthermore the G allele decreased the risk of BC (OR = 0.53, 95%CI 0.30-0.92, p = 0.024). No significant association was found between miR-609 genotypes and clinicopathological characteristics of BC patients (p >0.05). CONCLUSION: Our findings indicate that miR-608 polymorphism might be associated with decreased risk of BC in an Iranian subpopulation. Further large-scale studies with different ethnicities are needed to verify our findings.

Autophagy in airway diseases: a new frontier in human asthma?

The study of autophagy ('self-eating'), a fundamental cell fate pathway involved in physiological and pathological subcellular processes, opens a new frontier in the continuous search for novel therapies for human asthma. Asthma is a complex syndrome with different disease phenotypes. Autophagy plays a central role in cell physiology, energy and metabolism, and cell survival. Autophagy's hallmark is the formation of double-membrane autophagic autophagosomes, and this process is operational in airway epithelial and mesenchymal cells in asthma. Genetic associations between autophagy genes and asthma have been observed including single nucleotide polymorphisms in Atg5 which correlate with reduced lung function. Immune mechanisms important in asthma such as Th2 cells and eosinophils also manifest autophagy. Lastly, we address the role of autophagy in extracellular matrix deposition and fibrosis in asthmatic airways remodeling, a pathologic process still without effective therapy, and discuss potential pharmacologic inhibitors. We end by offering two opposing but plausible hypotheses as to how autophagy may be directly involved in airway fibrosis.

Autophagy is a regulator of TGF-ß1-induced fibrogenesis in primary human atrial myofibroblasts.

Transforming growth factor-ß(1) (TGF-ß(1)) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-ß(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-ß(1)-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-ß(1) to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-ß(1) promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-ß(1) in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3ß indicated the localization of punctate LC3ß with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-ß(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

Association study of hsa-mir-603 rs11014002 polymorphism and risk of breast cancer in a sample of Iranian population.

Accumulated evidence have proposed that single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) are connected to breast cancer (BC) risk. We have done a case-control study with 258 BC patients and 209 control women to examine the potential association of Hsa-mir-603 rs11014002 C>T polymorphisms with BC susceptibility. The polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Our findings showed that the rs11014002 C>T variant was not associated with an increased risk of BC in codominant (OR=0.67, 95%CI=0.42-1.08, P=0.121, CT vs CC; and OR=0.18, 95%CI=0.02-1.67, P=0.170, TT vs CC), dominant (OR=0.64, 95%CI=0.41-1.01, P=0.062, CT+TT vs CC), and recessive (OR=0.20, 95%CI=0.02-1.81, P=0.178, TT vs CC+CT) inheritance models tested. While, the T allele significantly decreased the risk of BC (OR= 0.63; 95% CI =0.41-0.95; P=0.032) compared to C allele. In conclusion, the findings indicated that Mir603 rs11014002 T allele might contribute to decrease the risk of BC in a sample of Iranian population. Further studies with larger sample sizes and different ethnicities are warranted to confirm our findings.

Autophagy and heart disease: implications for cardiac ischemia-reperfusion damage.

Survival of myocytes and mesenchymal cells in the heart is tightly regulated by a number of adaptive processes that are invoked with the changes that occur within the parenchyma and stroma. Autophagy is implicated in cellular housekeeping duties and maintenance of the integrity of the intracellular milieu by removal of protein aggregates and damaged organelles, whereas under pathophysiological conditions, the chronic up-regulation of autophagy may lead to significant disturbance of homeostatic conditions. Nonetheless, the role of autophagy in heart disease in the context of cardiac ischemia-reperfusion injury is currently unclear. This review will focus upon the role of autophagy as it pertains to ischemia reperfusion damage in the heart.

Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells.

Malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew; these cells are known as cancer stem-like cells (CSCs) or tumor-initiating cells. Primitive mammary CSCs/progenitor cells can be propagated in culture as floating spherical colonies termed 'mammospheres'. We show here that the expression of the autophagy protein Beclin 1 is higher in mammospheres established from human breast cancers or breast cancer cell lines (MCF-7 and BT474) than in the parental adherent cells. As a result, autophagic flux is more robust in mammospheres. We observed that basal and starvation-induced autophagy flux is also higher in aldehyde dehydrogenase 1-positive (ALDH1(+)) population derived from mammospheres than in the bulk population. Beclin 1 is critical for CSC maintenance and tumor development in nude mice, whereas its expression limits the development of tumors not enriched with breast CSCs/progenitor cells. We found that decreased survival in autophagy-deficient cells (MCF-7 Atg7 knockdown cells) during detachment does not contribute to an ultimate deficiency in mammosphere formation. This study demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance, and that Beclin 1 plays a dual role in tumor development.

Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts.

3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins.

R620W functional polymorphism of protein tyrosine phosphatase non-receptor type 22 is not associated with pulmonary tuberculosis in Zahedan, southeast Iran.

The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, which encodes an intracellular lymphoid-specific phosphatase, is considered an important regulator of T-cell activation. We investigated a possible association between the PTPN22 C1858T (R620W) polymorphism and pulmonary tuberculosis in an Iranian population. Single nucleotide polymorphisms of PTPN22 C1858T (rs2476601) were genotyped in 172 pulmonary tuberculosis cases and 204 normal subjects from Zaheden, Iran. Frequencies of genotypes CC, CT and TT of the PTPN22 C1858T polymorphism were 98.3, 1.7 and 0% in the pulmonary tuberculosis patients, and 96.1, 3.9 and 0% in the control group, respectively (P = 0.239). The frequency of the minor (T) allele was 0.8% in pulmonary tuberculosis patients and 2.0% in controls. Significant differences were not observed in genotype or allele frequencies of PTPN22 C1858T in the comparison between pulmonary tuberculosis patients and healthy subjects in our Iranian population sample.

Lack of association between rs1024611 (-2581 A/G) polymorphism in CC-chemokine Ligand 2 and susceptibility to pulmonary Tuberculosis in Zahedan, Southeast Iran.

Approximately 5-10% of subjects infected with Mycobacterium tuberculosis develop active tuberculosis. It has been proposed that genetic factors determine the host's vulnerability to tuberculosis. Chemokine (C-C motif) ligand 2 (CCL2), commonly known as monocyte chemoattractant protein-1 (MCP-1), plays a key role in protective immunity against M. tuberculosis. The present study was aimed to determine if there was an association between -2581 A/G single nucleotide polymorphism of CCL2 and pulmonary tuberculosis (PTB) in a sample of Iranian subjects. This case-control study was performed on 142 PTB and 166 healthy subjects. The polymorphism of CCL2 (rs1024611) was determined using tetra amplification refractory mutational system-polymerase chain reaction (tetra ARMS-PCR). There were no significant differences between PTB patients and control subjects regarding -2581 A/G single nucleotide polymorphism of CCL2. In conclusion, our results do not support an association of -2581 A/G polymorphism of CCL2 with PTB susceptibility.

Virus-triggered autophagy in viral hepatitis - possible novel strategies for drug development.

Autophagy is a very tightly regulated process that is important in many cellular processes including development, differentiation, survival and homoeostasis. The importance of this process has already been proven in numerous common diseases such as cancer and neurodegenerative disorders. Emerging data indicate that autophagy plays an important role in some liver diseases including liver injury induced by ischaemia reperfusion and alpha-1 antitrypsin Z allele-dependent liver disease. Autophagy may also occur in viral infection, and it may play a crucial role in antimicrobial host defence against pathogens, while supporting cellular homoeostasis processes. Here, the latest findings on the role of autophagy in viral hepatitis B and C infection, which are both serious health threats, will be reviewed.

Functional polymorphism of interferon-γ (IFN-γ) gene +874T/A polymorphism is associated with pulmonary tuberculosis in Zahedan, Southeast Iran.

Concerning the key role of interferon-γ (IFN-γ) in the protective immunity against Mycobacterium tuberculosis, we aimed to find the possible association between single nucleotide polymorphism of IFN-γ +874T/A (rs61923114) and pulmonary tuberculosis (PTB). This case-control study was performed on 142 PTB patients and 166 healthy subjects. Genotype analysis was done using amplification refractory mutation system-PCR (ARMS-PCR). We found that the AA genotype of +874A/T IFN-γ is a risk factor for PTB (OR = 3.333, 95% CI = 1.537-7.236, p=0.002). The results showed that the +874A allele frequency was higher in PTB than in normal subjects (OR = 1.561, 95% CI = 1.134-2.480, p=0.007). In conclusion, significant association was found between the IFN-γ +874T/A polymorphism (rs61923114) and susceptibility to PTB in a sample of Iranian population.

The L55M polymorphism of paraoxonase-1 is a risk factor for rheumatoid arthritis.

Paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme that exhibits antioxidant and antiatherogenic activities. We examined a possible association between T172A (L55M) and T(-107)C polymorphisms and rheumatoid arthritis. These polymorphisms were determined in 88 rheumatoid arthritis patients and 78 healthy subjects, using the tetra-amplification refractory mutation system-PCR method. The prevalence of the PON1 55MM genotype was significantly greater among rheumatoid arthritis patients (17%) when compared to control subjects (5.2%) (odds ratio (OR) = 3.75; 95% confidence interval (CI) = 1.87-11.8, P = 0.025). In addition, the M allele was more frequent in rheumatoid arthritis patients (40%) than in healthy subjects (24.7%) (OR = 1.997; 95%CI = 1.243-3.210, P = 0.005). There were no significant differences in the -107C/T polymorphism in the promoter sequence of PON1 between rheumatoid arthritis and normal subjects (chi(2) = 0.861, P = 0.650). In conclusion, the PON1 55MM genotype is a risk factor for rheumatoid arthritis.

Lack of association between paraoxonase-1 Q192R polymorphism and rheumatoid arthritis in southeast Iran.

Decreased paraoxonase-1 (PON1) activity has been associated with rheumatoid arthritis. There are two polymorphisms in serum PON1; one differs in the amino acid at position 192 (Q192R) and the other one differs at position 55 (L55M). We looked for a possible association between Q192R polymorphism and rheumatoid arthritis. The Q192R polymorphism in 88 rheumatoid arthritis patients and 78 healthy subjects was determined using tetra amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and PCR-restriction fragment length polymorphism (RFLP) methods. We found no significant differences between rheumatoid arthritis patients and control subjects regarding PON1 Q192R polymorphism. PON1 Q192R polymorphism was not found to be correlated with increased risk for rheumatoid arthritis in this Iranian population.

Adenosine deaminase activity, trypsin inhibitory capacity and total antioxidant capacity in psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by pathological skin lesions because of various exogenous and endogenous factors and associated with a number of biochemical and immunological disturbances. OBJECTIVE: The aim of the present study was to determine the level of adenosine deaminase activity, serum trypsin inhibitory capacity and total antioxidant capacity of plasma in psoriatic patients. SUBJECTS AND METHODS: The study was performed in controls (n = 46) and in psoriatic patients (n = 40). The patients were scored with PASI (psoriasis area and severity index). The serum ADA activity was determined using Aguisti and Galanti method and serum trypsin inhibitory capacity (sTIC) were measured by enzymatic assay. Besides, serum total antioxidant capacity was measured using ferric reducing ability of plasma. RESULTS: The serum ADA activity of the psoriatic patients was found to be significantly higher (P < 0.001) than that of the healthy control. We also found that the trypsin inhibitory capacity was significantly higher in patients than in control group (P < 0.001). Total antioxidant capacity of plasma was significantly lower in psoriatic patients than in healthy controls (P = 0.025). There were no significant correlations among ADA, TAC and TIC. CONCLUSION: Serum ADA activity and sTIC were increased in psoriatic patients. In parallel, serum total anti-oxidant activity was decreased in these patients.

Apoptosis and cancer: mutations within caspase genes.

The inactivation of programmed cell death has profound effects not only on the development but also on the overall integrity of multicellular organisms. Beside developmental abnormalities, it may lead to tumorigenesis, autoimmunity, and other serious health problems. Deregulated apoptosis may also be the leading cause of cancer therapy chemoresistance. Caspase family of cysteinyl-proteases plays the key role in the initiation and execution of programmed cell death. This review gives an overview of the role of caspases, their natural modulators like IAPs, FLIPs, and Smac/Diablo in apoptosis and upon inactivation, and also in cancer development. Besides describing the basic mechanisms governing programmed cell death, a large part of this review is dedicated to previous studies that were focused on screening tumours for mutations within caspase genes as well as their regulators. The last part of this review discusses several emerging treatments that involve modulation of caspases and their regulators. Thus, we also highlight caspase cascade modulating experimental anticancer drugs like cFLIP-antagonist CDDO-Me; cIAP1 antagonists OSU-03012 and ME-BS; and XIAP small molecule antagonists 1396-11, 1396-12, 1396-28, triptolide, AEG35156, survivin/Hsp90 antagonist shephedrin, and some of the direct activators of procaspase-3.