The impact of high nicotine concentrations on the viability and cardiac differentiation of mesenchymal stromal cells: a barrier to regenerative therapy for smokers.

Background: Current treatment methods are not successful in restoring the lost cardiomyocytes after injury. Stem cell-based strategies have attracted much attention in this regard. Smoking, as a strong cardiovascular risk factor, not only affects the cardiac cells adversely but also deteriorates the function of stem cells. Since mesenchymal stem cells (MSCs) are one of the popular candidates in cardiovascular disease (CVD) clinical trials, we investigated the impact of nicotine on the regenerative properties (viability and cardiac differentiation) of these cells. Methods: MSCs were isolated from rat bone marrow and characterized based on morphology, differentiation capability, and the expression of specific mesenchymal markers. The MTT assay was used to assess the viability of MSCs after being exposed to different concentrations of nicotine. Based on MTT findings and according to the concentration of nicotine in smokers' blood, the growth curve and population doubling time were investigated for eight consecutive days. Cells were treated with 5-azacytidine (an inducer of cardiac differentiation), and then the expressions of cardiac-specific markers were calculated by qPCR. Results: MSCs were spindle-shaped, capable of differentiating into adipocyte and osteocyte, and expressed CD73 and CD90. The viability of MSCs was reduced upon exposure to nicotine in a concentration- and time-dependent manner. The growth curve showed that nicotine reduced the proliferation of MSCs, and treated cells needed more time to double. In addition, the expressions of GATA4 and troponin were downregulated in nicotine-treated cells on day 3. However, these two cardiac markers were overexpressed on day 7. Conclusion: Nicotine decreased normal growth and reduced the expression of cardiac markers in MSCs. This aspect is of eminent importance to smokers with cardiovascular disease who are candidates for stem cell therapy.

The impact of diabetic glucose concentration on viability and cardiac differentiation of mesenchymal stem cells.

INTRODUCTION: Hyperglycemia may be a stumbling block for delivery of regenerative benefits of mesenchymal stem cells (MSCs) to diabetic patients with cardiovascular diseases. Our study aims to assess the viability and cardiac differentiation potential of MSCs after being exposed to diabetic glucose concentration. METHODS: MSCs were extracted from rat bone marrow. Cells were characterized based on morphology, differentiation potential, and expression of mesenchymal specific markers. MTT assay was done to evaluate the viability of MSCs after treatment with different glucose concentrations. Case group was MSCs treated with diabetic concentration of glucose versus cells treated with PBS as the control group. Growth curve and population doubling time were calculated in both groups. Expression of GATA4 and troponin, as the early and late markers during cardiac differentiation, were measured following 5-azacytidine exposure. RESULTS: Proliferated cells at passage three had fibroblastic-shape, was able to differentiate into adipocytes or osteocytes, and expressed CD73 and CD90. MSCs viability was gradually decreased by increasing glucose concentration. Irrespective of nicotine concentration, three-day exposure imposed more severe detrimental effects on viability compared with one-day treatment. Proliferation rate of the MSCs was lower in the case group, and they need more time for population doubling. Expression of both cardiac markers were downregulated in the case group at day three. However, their expression became higher at day seven. CONCLUSION: Diabetic glucose concentration inhibits normal proliferation and cardiac differentiation of MSCs. This effect should be considered in stem cell therapy of cardiovascular patients who are concurrently affected by hyperglycemia, a common comorbidity in such individuals. Why carry out this study? What was learned from the study?

Protective Effects of Xanthine Derivatives Against Arsenic Trioxide-Induced Oxidative Stress in Mouse Hepatic and Renal Tissues.

In this study, the protective efficacy of pentoxifylline (PTX) as a xanthine derivative against arsenic trioxide (ATO)-induced kidney and liver damage in mice was investigated. Thirty-six mice were divided into six groups, receiving intraperitoneal injections of saline, ATO, PTX, or a combination for four weeks. Blood samples were analyzed for serum biochemistry, while hepatic tissue underwent examination for histopathological changes and assessment of oxidative stress markers and antioxidant gene expression through Real-Time PCR. ATO exposure significantly increased serum markers (creatinine, ALT, BUN, ALP, AST) and induced histopathological changes in the liver. Moreover, it elevated renal and hepatic nitric oxide (NO) and lipid peroxidation (LPO) levels, and reduced antioxidant enzyme expression (CAT, GSR, GPx, MPO, SOD), total thiol groups (TTGs), and total antioxidant capacity (TAC). Conversely, PTX treatment effectively lowered serum hepatic and renal markers, improved antioxidant markers, and induced histopathological alterations. Notably, PTX did not significantly affect renal and hepatic NO levels. These findings suggest that PTX offers therapeutic potential in mitigating liver and acute kidney injuries induced by various insults, including exposure to ATO.

Expression of miR-let7b and miR-19b in progressive familial intrahepatic cholestasis (PFIC) children.

BACKGROUND: MicroRNAs (miRNAs) are a group of small non-coding RNAs that bind to the target mRNA and regulate gene expression. Recently circulating microRNAs were investigated as markers of diseases and therapeutic targets. Although various studies analyze the miRNA expression in liver disease, these studies on PFIC are few. Progressive familial intrahepatic cholestasis (PFIC) is a rare liver disease with autosomal recessive inheritance. Most children with PFIC progress to cirrhosis and liver failure and consequently need to have a liver transplant. The aim of this study is the investigation of the miR-19b and miR-let7b expression levels in Iranian PFIC children. METHODS: 25 PFIC patients, 25 healthy children and 25 Biliary Atresia patients were considered as case and two control groups respectively. Blood samples were obtained and Liver function tests (LFTs) were measured. After RNA extraction and cDNA synthesis, quantitative PCR was performed using specific primers for miR-19b and miR-let7b. The U6 gene is used as an internal control. RESULTS: qPCR on PFIC patients' samples demonstrated that the miR-19b and the miR-let7b expression were significantly decreased in patients compared to the control groups, with a p-value<0.0001 and p-value=0.0006 receptively. CONCLUSION: In conclusion, circulating micro-RNA like miR-19b and miR-let7b have a potential opportunity to be a non-invasive diagnostic marker or therapeutic target for PFIC in the future.

Phenyl-quinoline derivatives as lead structure of cholinesterase inhibitors with potency to reduce the GSK-3ß level targeting Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to cognitive decline and memory loss. Unfortunately, there is no effective treatment for this condition, so there is a growing interest in developing new anti-AD agents. In this research project, a series of phenyl-quinoline derivatives were designed as potential anti-AD agents. These derivatives were substituted at two different positions on benzyl and phenyl rings. The structures of the derivatives were characterized using techniques such as IR spectroscopy, 1H NMR, 13C NMR, and elemental analysis. During the in vitro screening, the derivatives were tested against both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). It was observed that most of the derivatives showed higher selectivity against BChE compared to AChE. Among the derivatives, analog 7n (with a methoxy group at R1 and a 4-bromine substituent at R2 exhibited the highest potency, with a 75-fold improvement in the activity compared to the positive control. Importantly, this potent analog demonstrated no toxicity at the tested concentration on SH-SY5Y cells, indicating its potential as a safe anti-AD agent. The level of GSK-3ß was also reduced after treatments with 7n at 50 µM. Overall, this study highlights the design and evaluation of phenyl-quinoline derivatives as promising candidates for developing novel anti-AD agents.

Pyridoxamine protects human granulosa cells against advanced glycation end-products-induced steroidogenesis disturbances.

BACKGROUND: Ovarian advanced glycation end-products (AGEs) accumulation is associated with ovarian granulosa cells (GCs) dysfunction. Vitamin B6 derivatives positively affected reproduction. The current study was conducted to elucidate the AGEs effects on human luteinized mural GCs steroidogenesis in the presence or absence of pyridoxamine (PM). METHODS AND RESULTS: Isolated GCs of 50 healthy women were divided into four parts and treated with media alone (Control), PM alone, or human glycated albumin (HGA) with/without PM. Main steroidogenic enzymes and hormones were assessed by qRT-PCR and ELISA. The AGE receptor (RAGE) protein was also determined using Western blotting. The non-toxic concentration of HGA increased the expression of RAGE, StAR, 3ß-HSD, and 17ß-HSD (P < 0.0001 for all) but decreased the expression of CYP19A1 at mRNA levels. The increased RAGE protein expression was also confirmed by western blot analysis. These effects resulted in declined estradiol (E2), slightly, and a sharp rise in progesterone (P4) and testosterone (T) levels, respectively. PM, on its own, ameliorated the HGA-altered enzyme expression and, thereby, corrected the aberrant levels of E2, P4, and T. These effects are likely mediated by regulating the RAGE gene and protein expression. CONCLUSION: This study indicates that hormonal dysfunctions induced by the AGEs-RAGE axis in luteinized GCs are likely rectified by PM treatment. This effect is likely acquired by reduced expression of RAGE. A better understanding of how AGEs and PM interact in ovarian physiology and pathology may lead to more targeted therapy for treating ovarian dysfunction.

Post-Weaning Exposure to Sunset Yellow FCF Induces Changes in Testicular Tight and Gap Junctions in Rats: Protective Effects of Coenzyme Q10.

Studies on adverse health consequences of azo dyes are limited and conflicting. Coenzyme Q10 (CoQ10) supplementation has been shown to have benefits associated with antioxidant and anti-inflammatory characteristics on several body systems. This work investigates the possible toxic effects of the widely used food additive sunset yellow and the probable protective effects of CoQ10 on testicular tight and gap junctions in rats by assessing molecular, immunohistochemical, and histopathological changes. Sixty Sprague-Dawley male weanling rats were randomly divided into six groups (n = 10). The rats received their treatments via daily oral gavages for 6 weeks. The treatments included as follows: low dose of sunset yellow (SY-LD) (2.5 mg/kg/day), high dose of sunset yellow (SY-HD) (70 mg/kg/day), CoQ10 (10 mg/kg/day), CoQ10 with low dose of sunset yellow (CoQ10 + LD), CoQ10 with high dose of sunset yellow (CoQ10 + HD), and distilled water as the control treatment. At the end of the experiment, the rats were anesthetized, and the testes were removed for molecular (real-time quantitative PCR), immunohistochemical, and histopathological (H & E staining) assessments. Claudin 11 and occludin gene expression significantly decreased in HD and CoQ10 + HD groups compared with the controls. Connexin 43 (Cx43) expression in the control and CoQ10 groups was significantly higher than in the HD group. The immunohistochemical and histopathological data were largely in line with these findings. The results showed that exposure to a high dose of sunset yellow led to disturbances in cell-to-cell interactions and testicular function. Simultaneous treatment with CoQ10 had some beneficial effects but did not completely improve these undesirable effects.

NRXN3 mutations cause developmental delay, movement disorder, and behavioral problems: CRISPR edited cells based WES results.

NRXN3geneencodesneurexin-III which is a Neural Cell Adhesion Molecule (NCAM) with important synaptic functions in the brain. Neurexin-III deficiency could affect synapse development, synaptic signaling and neurotransmitter release. Hitherto, there is no related disorder in the OMIM due to NRXN3 mutation. In this study, two unrelated Iranian families with homozygous (NM_001330195.2:c.3995G>A, p.Arg1332His) and compound heterozygous (NM_001330195.2:c.4442G>A, p.Arg1481Gln; c.3142+3A>G) variants in theNRXN3gene were detected for the first time. The proband of the first family manifested learning disability, developmental delay, inability to walk, and behavioral problems such as difficulty in social communication. Also, global development delay, intellectual disability, abnormal gait, severe speech problems, muscle weakness, and behavioral problems were observed in the affected individual in the second family. In addition, deciphering the pathogenicity of NRXN3 variants was done by functional studies such as CRISPR edited cells, in-silico analysis, and NGS results. All of these data together with phenotype similarity between observed phenotypes in our patients and manifested symptoms in the homozygousNrxn3α/ß knockout mice, demonstrate the homozygous and compound heterozygous mutations of NRXN3 could cause a novel syndromic mendelian genetic disorder with autosomal recessive inheritance. The main phenotype of patients with neurexin-III deficiency includes developmental delay, learning disability, movement disorder, and behavioral problems.

Premature Death, Underlying Reasons, and Preventive Experiences in Iran: A Narrative Review.

Premature mortality (PM) has emerged as a global health challenge. This is of eminent importance in low- and middle-income countries, where nearly three fourths of the deaths occur. The concerning issue is the early occurrence of fatal events in productive age. Fatal events before the age of 70 are called PM, which mainly result from cardiovascular diseases (CVDs). Iran as a middle- income country greatly suffers from the cardiovascular burden, which accounts for almost 50% of all PM. Despite substantial success in reducing mortality due to communicable diseases across different age ranges, urbanization and pervasiveness of cardiovascular risk factors have increased the death rate in adults in recent years. Undoubtedly, such lifestyles have imposed heavy costs on the healthcare system; it is possible that PM reduction, as one of the fundamental elements of sustainable development goals defined by the World Health Organization (WHO), would not be reached by the due date. Recently, researchers have introduced a cost-effective fixed-dose drug combination, the so-called polypill, in order to attenuate the detrimental effects of hypertension and hyperlipidemia, as two strong cardiovascular risk factors. PolyIran and PolyIran-Liver studies are two pivotal clinical trials that revealed the feasibility of primary and secondary prevention of premature cardiovascular mortality, both in an urban and a rural population. In the present narrative review, we tried to present a comprehensive appraisal on PM status, its underlying reasons, and the impact of polypill strategy on PM prevention in Iran.

Expression ratio of circular to linear ANRIL in hypertensive patients with coronary artery disease.

Atherosclerotic lesions of the coronary arteries are still in charge of significant annual morbidity and mortality despite intense therapeutic advancements. Genome-born elements contribute substantially to the atherosclerosis process. ANRIL is one of the long non-coding RNAs with outstanding functions particularly regulation of genes involved in atherosclerosis development. In this study, we measured ANRIL expression (circular-, linear-, and circular/linear ratio) in hypertensive patients with coronary artery disease (CAD) compared with peers without CAD. Among hypertensive patients who were candidates of angiography, 25 subjects with CAD and the equal number without CAD were considered as the case and control groups, respectively. Different categories of data were recorded through a predefined questionnaire. Before angiography, blood samples were obtained. After RNA extraction and cDNA synthesis, quantitative PCR was performed using specific primers for circular and linear ANRIL. Age and gender were not different between the groups. Most of the parameters of the lipid profile besides creatinine and blood urea nitrogen were remarkably worse in the case group. Circular ANRIL was significantly lower in the case group while linear counterparts were significantly higher in this group. Circular/linear ratio was also significantly lower in the case group. To overcome growing devastating trend of CAD, scrutinizing different factors involved in the initiation and development of atherosclerosis is a must. Atheroprotective role of circular ANRIL and atheroprogressive role of linear ANRIL were shown in our patients with hypertension.

Novel RNA extraction method from human tears.

Human tears can be used as a noninvasive source of genetic materials and biomarkers in the prognosis and diagnosis of ocular and non-ocular diseases. The present protocol is a novel direct RNA extraction method from tears. This study aims to provide a suitable method for direct extraction of RNA from tears with high quality and quantity. In this study, we develop a TRIzol base protocol for direct RNA extraction from human tears. quality and quantity of extracted RNA measured by calculation of 260/280 UV absorption ratio using Nanodrop and real-time PCR. RNA was extracted with this modified method and a purified (260/280 UV absorption ratio between 1.8 to 2 and a high yield of total RNA, on average 95 µg, from tears was extracted. In conclusion, we developed an easy and suitable method for direct extraction of total RNA from tears with high quality and quantity.

Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC).

OBJECTIVES: Duchene muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the DMD gene, resulting in the absence of dystrophin expression leading to membrane fragility and myofibril necrosis in the muscle cells. Because of progressive weakness in the skeletal and cardiac muscles, premature death is inevitable. There is no curative treatment available for DMD. In recent years, advances in genetic engineering tools have made it possible to manipulate gene sequences and accurately modify disease-causing mutations. CRISPR/Cas9 technology is a promising tool for gene editing because of its ability to induce double-strand breaks in the DNA. MATERIALS AND METHODS: In this study for the exon-skipping approach, we designed a new pair of guide RNAs (gRNA) to induce large deletion of exons 48 to 53 in the DMD gene in the human skeletal muscle cell line (HSkMC), in order to correct the frame of the gene. RESULTS: Data showed successful editing of DMD gene by deletion of exons 48 to 53 and correction of the reading frame in edited cells. Despite a large deletion in the edited DMD gene, the data of real-time PCR, immune florescent staining demonstrated successful expression of truncated dystrophin in edited cells. CONCLUSION: This study demonstrated that the removal of exons 48-53 by the CRISPR / Cas9 system did not alter the expression of the DMD gene due to the preservation of the reading frame of the gene.

Using CRISPR/Cas9 System to Knock out Exon 48 in DMD Gene.

BACKGROUND: Out of frame mutations in DMD gene cause Duchenne Muscular Dystrophy (DMD) which is a neuromuscular progressive genetic disorder. In DMD patients, lack of dystrophin causes progressive muscle degeneration, which results in heart and respiratory failure leading to premature death. At present, there is no certain treatment for DMD. DMD gene is the largest gene in human genome by 2.2 mega base pairs and contains 79 exons. In the past few years, gene therapy has been considered a promising DMD treatment, and among various gene-editing technologies, CRISPR/Cas9 system is shown to be more precise and reliable. The aim of this study was to assess the possibility of knocking out exon 48 by using a pair of sgRNAs. METHODS: A pair of guide RNAs (gRNAs) was designed to cleave DMD gene and induce deletion of exon 48. gRNAs were transfected to the HEK-293 cell line and then the deletion in genomic DNA was analyzed by PCR and subsequent Sanger sequencing. RESULTS: Exon 48 was successfully deleted and therefore exon 47 was joined to exon 49. CONCLUSION: This result indicated that CRISPR/Cas9 system could be used to edit DMD gene precisely.

Low Rate of babA2 Genotype among Iranian Helicobacter pylori Clinical Isolates.

INTRODUCTION: The Blood Group Antigen-Binding Adhesion (babA), Outer Inflammatory Protein (oipA) and Sialic Acid-Binding Adhesin (sabA) as outer membrane proteins involved in Helicobacter pylori adherence to gastric mucosa have been suggested to have a role in the pathogenesis. AIM: To investigate the frequency of H. pylori isolates babA2, oipA and sabA genes in Iranian dyspeptic patients. MATERIALS AND METHODS: DNAs were extracted from H. pylori -positive cultures taken from 100 different dyspeptic patients. Genotyping was performed by Polymerase Chain Reaction (PCR), using the specific primers for babA2, oipA and sabA genes. Chi square test was used to investigate association between variables, p<0.05 was considered statistically significant. RESULTS: All (100%) isolates possessed oipA and sabA genotypes, whereas babA2 was detected in 22% of isolates. There was no significant relationship between presence of genes with clinical outcome. The combined genotype oipA +/sabA +/ babA2- was correlated with gastritis. The rate of babA2 genotype in our isolates was lower than other Iranian reports. CONCLUSION: Frequency of babA2 genotype among H. pylori isolates from Southwest of Iran is considerably less than other regions of Iran. Due to heterogeneity of H. pylori strains in different geographic regions, further work will be needed to understand the role of these virulence genes in H. pylori pathogenesis and their possible association with disease outcome.

High frequency of hopQ genotypes among Iranian Helicobacter pylori clinical isolates.

Different virulence factors are involved in Helicobacter pylori pathogenesis. H. pylori outer membrane proteins are a family of virulence factors that have diverse members. HopQ (H. pylori outer membrane protein) is the largest of them that contains types I and II alleles. The role of hopQ is not exactly known, but it has been considered in H. pylori adhesion and colonization. The aim of this study was to determine the frequency of hopQ genotypes among H. pylori isolates obtained from patients with gastroduodenal disorders and their association with the clinical outcome. The DNA of 100 H. pylori clinical isolates was investigated by polymerase chain reaction (PCR) method using specific primers for determining the hopQI and hopQII genotypes. hopQI was present in 35%, while hopQII was positive in 55% of the isolates. Amongst the gastritis subjects, the rate of hopQII compared to hopQI was higher, and a statistically significant difference was found between hopQII genotype and the clinical outcome. With respect to the significant difference between the hopQ genotype and clinical outcome in our clinical isolates, it seems that this genotype is a useful marker for evaluating its association with H. pylori-related diseases.

High rate of A2142G point mutation associated with clarithromycin resistance among Iranian Helicobacter pylori clinical isolates.

This study aimed to investigate the clarithromycin resistance and its associated molecular mechanisms among Helicobacter pylori isolates from dyspeptic patients in Shiraz, Iran. From January to May 2014, 100 H. pylori strains were isolated from patients with gastroduodenal disorders. The resistance to clarithromycin was quantitatively evaluated, using Epsilometer (E-test) method. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on all the isolates to detect A2143G and A2142G mutations in 23S rRNA gene. The H. pylori isolation rate was found to be 31.4%. E-test showed that 20% of isolates were resistant to clarithromycin (MIC ≥ 1 mg/L). MIC of clarithromycin ranged between 0.016 and 24 mg/L. Findings of PCR-RFLP showed that the A2142G was the most (90%) frequently point mutation, followed by the A2143G (10%). No statistically significant difference was found between H. pylori clarithromycin resistance point mutations and patients' gender or age. To the best of our knowledge, this is the first report of high frequency of A2142G point mutation in Iran and probably in other regions of the world. Considering the increasing trend of H. pylori resistance to clarithromycin due to these mutations, it is crucial to investigate the new therapeutic approaches against H. pylori infection.