A molecular and computational study of galbanic acid as a regulator of Sirtuin1 pathway in inhibiting lipid accumulation in HepG2 cells.

INTRODUCTION: Sirtuin1 (SIRT1) plays a crucial role in the pathophysiology of non-alcoholic fatty liver disease. We investigated the mechanistic role of galbanic acid (Gal) as a regulator of SIRT1 in silico and in vitro. METHODS: HepG2 cells were treated with Gal in the presence or absence of EX-527, a SIRT1-specific inhibitor, for 24 h. Sirtuin1 gene and protein expression were measured by RT-PCR and Western blotting, respectively. It has been docked to the allosteric reign of SIRT1 (PDB ID: 4ZZJ) to study the effect of Gal on SIRT1, and then the protein and complex molecular dynamic (MD) simulations had been studied in 100 ns. RESULTS: The semi-quantitative results of Oil red (p < .03) and TG level (p < .009) showed a significant reduction in lipid accumulation by treatment with Gal. Also, a significant increase was observed in the gene and protein expression of SIRT1 (p < .05). MD studies have shown that the average root mean square deviation (RMSD) was about 0.51 Å for protein structure and 0.66 Å for the complex. The average of radius of gyration (Rg) is 2.33 and 2.32 Å for protein and complex, respectively, and the pattern of root mean square fluctuation (RMSF) was almost similar. CONCLUSION: Computational studies show that Gal can be a great candidate to use as a SIRT1 ligand because it does not interfere with the structure of the protein, and other experimental studies showed that Gal treatment with SIRT1 inhibitor increases fat accumulation in HepG2 cells.

Phylogenetic analysis and antimony resistance of Leishmania major isolated from humans and rodents.

Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred to health centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears were subjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products were sequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTT assay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148 rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L. major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L. major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis for planning future control strategies.

Engineering HEK293T cell line by lentivirus to produce miR34a-loaded exosomes.

BACKGROUND: RNA (ribonucleic acid) antisense is developing as a possible treatment option. As an RNA, miR-34a is involved in P53 function and cancer cell apoptosis. Although the therapeutic applications of miRNAs have several limitations, such as structural instability and susceptibility to nucleases. To resolve these issues, this study aims to apply exosomes as a delivery vehicle for miR-34a. AIMS: This study aims to create a cell factory to generate miR34a-enriched exosomes. The produced nanoparticles act as a delivery system and improve the structural stability of miR34a. METHODS: First exosome specific sequences were inserted into miR34a. The resulting miR34a oligonucleotide was transduced HEK293T cells genome with a lentiviral system. In the structure of miR34a oligonucleotide, six nucleotides were substituted to increase its packaging rate into exosomes. To maintain the secondary structure, stability, and expression of the miRNA gene, changes to the miR34a oligonucleotide were made using PCR (polymerase chain reaction) Extension. The forward-34a (5-TGGGGAGAGGCAGGACAGG-3) and Reverse-34a primers (5-TCCGAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA. RESULTS: The results confirmed that the changes in miR34a oligonucleotide do not affect its secondary structure. The energy level of the manipulated miR34a oligonucleotide was kept the same compared to the original one. Moreover, the loading of miR34a to the exosomes was increased. CONCLUSION: Our findings revealed that normal HEK293T did not express miR34a. However, lentiviral transduced miR34a oligonucleotide induced the loading of miR34a into the exosome. Moreover, replacing six nucleic acids in the 3' end of miR34a increased the loading of miR34a to exosome.

Frequency of significant virulence genes in gastric biopsies of Helicobacter pylori-positive patients with gastritis.

Helicobacter pylori is one of the most common bacteria affecting human societies worldwide, and is mainly associated with gastrointestinal complications due to different virulence factors. This study aimed to investigate some virulence genes of H. pylori in gastric biopsies of patients with gastritis in Sari city, North of Iran. Informed consent forms were obtained from the studied patients, and those who needed endoscopy were included in the study. To evaluate the prevalence of cagA, iceA1, iceA2, vacA, dupA, and oipA genes, gastric biopsies with positive or negative rapid urease test were collected from 50 patients (25 in each group) with gastro-duodenal diseases. The bacterial DNAs were extracted by a specific kit, and the presence of the genes was analyzed by PCR using specific primers. Eighteen (72%) biopsies from 25 H. pylori-positive samples were cagA-positive, while 17 (68%) biopsies contained the vacA gene, and 11 (44%) samples had both vacA and cagA genes. However, 16 (64%), 12 (48%), 13 (52%), and 14 (56%) biopsies contained dupA, iceA1, iceA2, and oipA genes, respectively. Due to the significant role of the studied virulence factors in the pathogenicity of H. pylori, the high prevalence of these factors in biopsies of patients with gastritis is a concern needing to the management in this region.

Investigating Facilitatory Effects of Lithium on Methamphetamine-induced Spatial Memory Impairments in Rat.

Introduction: It has long been known that Methamphetamine (MA), as a psychostimulant, leads to long-lasting cognitive deficits. Previous studies have shown that lithium, a mood stabilizer, could facilitate cognitive ability in most of brain diseases. In current study the effects of lithium on spatial memory, hippocampal apoptosis and brain edema in METH-exposed rats are investigated. Methods: The present study 32 Wistar rats were used to examine the effects of lithium on spatial memory by the Morris water maze, hippocampal apoptosis using the TUNEL assay, and brain edema following MA administrations. Results: The findings indicated that treatment with lithium significantly ameliorated spatial learning and memory impairment in MA-treated rats. In addition, the findings showed that treatment with lithium significantly reduced brain edema and apoptosis in the CA1 neurons in MA -exposed rats. Conclusion: The results show that treatment with lithium can partially ameliorate the MA-induced neurocognitive deficits in rats, which may be related to its protective effect in the hippocampus.

Patient-related post-ERCP pancreatitis (PEP) risk factors between two groups of patients: Prophylactic pancreatic stent and rectal indomethacin.

Background: Pancreatitis is one of the most crucial complications following endoscopic retrograde cholangiopancreatography (ERCP). The purpose of the current study was to investigate patient-related post-ERCP pancreatitis (PEP) risk factors in two groups of patients: prophylactic pancreatic stent and rectal indomethacin. Methods: Two different prophylactic modalities were planned and complications were assessed based on the defined inclusion criteria. In this study, the patients were evaluated for the procedure and patient-related risk factors in post-ERCP pancreatitis in the recipient groups of the prophylactic pancreatic stent and rectal indomethacin. Results: Pancreatitis was confirmed in 27 of all 170 selected patients after ERCP. By univariate analysis, two variables were significant with the development of PEP. Regarding the patient-related risk factors, unique subjects with common bile duct (CBD) dilated 10mm were more exposed to an increased chance of PEP (P=0. 015); meanwhile, other factors did not correlate with the increased possibility of PEP in both groups. The only procedure-related risk factor for PEP was the deep cannulation of the pancreatic duct in both groups during the procedure with an incremental significant incidence of pancreatitis (P=0.005). Comparison of prophylactic pancreatic stent and rectal indomethacin showed no effects in term of post ERCP pancreatitis reduction. Additionally, there was no significant difference between these two strategies in the rate of PEP. Conclusion: Prophylactic pancreatic duct stents and administration of rectal indomethacin cannot have particular approaches for reducing the possible occurrence of PEP. The increase in time of deep cannulation and the presence of CBD dilation <10mm could be considered as important risk factors.

Effects of clozapine and risperidone antipsychotic drugs on the expression of CACNA1C and behavioral changes in rat 'Ketamine model of schizophrenia.

Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C) is one of the most important genes associated with schizophrenia. In this study, 45 male Wistar rats were divided into 5 groups of saline, control, ketamine, clozapine, and risperidone. Animals in ketamine, risperidone, and clozapine groups received ketamine (30 mg/kg-i.p.) for 10 days. After the last injection of ketamine, we started injecting clozapine (7.5 mg/kg-i.p.), risperidone (1 mg/kg-i.p.), up to 28 days. Twenty-four hours after the last injection, open field, social interaction, and elevated plus-maze tests and gene expression in hippocampus were performed. The results of the social interaction test revealed a significant decrease in cumulative time with ketamine, compared with the saline group, and an increase with clozapine and risperidone compared with the ketamine group. Moreover, results from the elevated plus-maze test demonstrated a critical decrease in open arm time and increase in close arm time with ketamine compared with saline, as well as increased in open arm time with risperidone compared with ketamine. Further results revealed a significant increase in rearing and grooming with ketamine compared to saline, as well as a decrease with risperidone and clozapine compared to ketamine. There were no significant differences in CACNA1C gene expression between groups in the rat hippocampus. In brief, the results of this study indicated that clozapine and risperidone could partially improve cognitive impairments in the rat. However, our findings demonstrated that this treatment is not related to CACNA1C gene expression.

Circulatory microRNAs: promising non-invasive prognostic and diagnostic biomarkers for parasitic infections.

MicroRNAs (miRNAs) are a non-coding subclass of endogenous small regulatory RNAs, with about 18-25 nucleotides length which play a critical role in the regulation of gene expression at the post-transcriptional level in eukaryotes. Aberrant expression of miRNAs has the potential to become powerful non-invasive biomarkers in pathological diagnosis and prognosis of different disorders including infectious diseases. Parasite's life cycle may require the ability to respond to environmental and developmental signals through miRNA-mediated gene expressions. Over the last years, thousands of miRNAs have been identified in the helminthic and protozoan parasites and many pieces of evidence have demonstrated the functional role of miRNAs in the parasites' life cycle. Detection of these miRNAs in biofluids of infected hosts as prognostic and diagnostic biomarkers in infectious diseases is growing rapidly. In this review, we have highlighted altered expressions of host miRNAs, detected parasitic miRNAs in the infected hosts, and suggested some perspectives for future studies.

Naloxone Ameliorates Spatial Memory Deficits and Hyperthermia Induced by a Neurotoxic Methamphetamine Regimen in Male Rats.

BACKGROUND: Methamphetamine (METH) as a synthetic psychostimulant is being increasingly recognized as a worldwide problem, which may induce memory impairment. On the other hand, it is well established that naloxone, an opiate antagonist, has some beneficial effects on learning and memory. The present research aimed at evaluating naloxone effects on spatial learning and memory impairment triggered by a neurotoxic regimen of METH in male rats. MATERIALS AND METHODS: The animals received the subcutaneous (sc) regimen of METH (4×6 mg/kg at 2-h intervals), intraperitoneal (ip) naloxone (4×1 mg/kg at 2-h intervals), or normal saline at four events. The Nal-METH group of rats received four naloxone injections (1 mg/ kg, ip) 30 min before each METH injection (6 mg/kg, sc) at 2-h intervals. Seven days later, they were evaluated for spatial learning and memory in the Morris Water Maze (MWM) task. RESULTS: METH regimen induced hyperthermia, as well as a poor performance, in the acquisition and retention phases of the task, indicating spatial learning and memory impairment compared to the controls. Naloxone administration (1 mg/kg, ip) before each METH injection led to significant attenuations of both hyperthermia and METH adverse effects on the rat performance in the MWM task. CONCLUSION: The results revealed that pretreatment with the opiate antagonist naloxone could prevent METH adverse effects on body temperature and memory performance. It seems that the opioidergic system and hyperthermia may, at least partially, be involved in METH effects on spatial memory.

Relationship between ureB Sequence Diversity, Urease Activity and Genotypic Variations of Different Helicobacter pylori Strains in Patients with Gastric Disorders.

Association of the severity of Helicobacter pylori induced diseases with virulence entity of the colonized strains was proven in some studies. Urease has been demonstrated as a potent virulence factor for H. pylori. The main aim of this study was investigation of the relationships of ureB sequence diversity, urease activity and virulence genotypes of different H. pylori strains with histopathological changes of gastric tissue in infected patients suffering from different gastric disorders. Analysis of the virulence genotypes in the isolated strains indicated significant associations between the presence of severe active gastritis and cagA+ (P = 0.039) or cagA/iceA1 genotypes (P = 0.026), and intestinal metaplasia and vacA m1 (P = 0.008) or vacA s1/m2 (P = 0.001) genotypes. Our results showed a 2.4-fold increased risk of peptic ulcer (95% CI: 0.483-11.93), compared with gastritis, in the infected patients who had dupA positive strains; however this association was not statistically significant. The results of urease activity showed a significant mean difference between the isolated strains from patients with PUD and NUD (P = 0.034). This activity was relatively higher among patients with intestinal metaplasia. Also a significant associa-tion was found between the lack of cagA and increased urease activity among the isolated strains (P = 0.036). While the greatest sequencevariation of ureB was detected in a strain from a patient with intestinal metaplasia, the sole determined amino acid change in UreB sequence (Ala201Thr, 30%), showed no influence on urease activity. In conclusion, the supposed role of H. pylori urease to form peptic ulcer and advancing of intestinal metaplasia was postulated in this study. Higher urease activity in the colonizing H. pylori strains that present specific virulence factors was indicated as a risk factor for promotion of histopathological changes of gastric tissue that advance gastric malignancy.

Relationship between ureB Sequence Diversity, Urease Activity and Genotypic Variations of Different Helicobacter pylori Strains in Patients with Gastric Disorders.

Association of the severity of Helicobacter pylori induced diseases with virulence entity of the colonized strains was proven in some studies. Urease has been demonstrated as a potent virulence factor for H. pylori. The main aim of this study was investigation of the relationships of ureB sequence diversity, urease activity and virulence genotypes of different H. pylori strains with histopathological changes of gastric tissue in infected patients suffering from different gastric disorders. Analysis of the virulence genotypes in the isolated strains indicated significant associations between the presence of severe active gastritis and cagA+ (P = 0.039) or cagA/iceA1 genotypes (P = 0.026), and intestinal metaplasia and vacA m1 (P = 0.008) or vacA s1/m2 (P = 0.001) genotypes. Our results showed a 2.4-fold increased risk of peptic ulcer (95% CI: 0.483-11.93), compared with gastritis, in the infected patients who had dupA positive strains; however this association was not statistically significant. The results of urease activity showed a significant mean difference between the isolated strains from patients with PUD and NUD (P = 0.034). This activity was relatively higher among patients with intestinal metaplasia. Also a significant association was found between the lack of cagA and increased urease activity among the isolated strains (P = 0.036). While the greatest sequence variation of ureB was detected in a strain from a patient with intestinal metaplasia, the sole determined amino acid change in UreB sequence (Ala201Thr, 30%), showed no influence on urease activity. In conclusion, the supposed role of H. pylori urease to form peptic ulcer and advancing of intestinal metaplasia was postulated in this study. Higher urease activity in the colonizing H. pylori strains that present specific virulence factors was indicated as a risk factor for promotion of histopathological changes of gastric tissue that advance gastric malignancy.