Real-time Quantitative Transcriptase-polymerase Research Articles

Reduction of some viral protein's gene expression of recombinant influenza A/H1N1-PR8 virus upon treatment with Punica granatum crude extract

BackgroundIt was we known that Flu infections have a place with the class Orthomyxoviridae furthermore, three distinct sorts (A, B and C sort infections) are known. Among these, influenza viral infections display the broadest host range (birds, people, and different well evolved creatures) also, can develop into profoundly pathogenic strains. So, the scientists tried to find a way to restrict this invasion which can cause certain death to human raise. Subjects & methodsDifferent concentrations of Punica granatum extract (2.5, 5, 10 and 20 μg/ml) prepared from the stock solution were used against human influenza A/Puerto Rico/8/34 H1N1 (IAV/PR8) to test its ability to prevent the virus to grow on MDCKII and A549 cells to check the effect of this plant extract on viral replication titter using plaque assay, gene expression of some viral proteins like non-structural protein NS1, nucleoprotein NP and Protein basic PB1 using reverse transcriptase quantitative real time polymerase chain reaction. Also, localization of NP protein was checked using immune-florescent staining technique. ResultsOur finding showed that there was significant decrease in IAV/PR8 ability to grow and replicate in MDCKII cells at multiplicity of infection (MOI) in both of 0.1 and 0.01 MOI. this finding was confirmed upon checking of expression of NS1, PB1 and NP proteins of IAV/PR8 that were reduced significantly at 10 and 20 μg/ml. Interestingly the localization of viral NP protein affected negatively upon the treatment of cells with elected concentration of plant extract by the weak localization outside the nucleus. ConclusionMethanolic extract of Punica granatum produced a strong anti-viral activity against IAV/PR8 replication ability and that was due to the severe effect on viral proteins expression especially on PB1 and NP which responsible for viral protein synthesis and NS1 protein which is a strong IFN antagonistic protein thus; weakening its replication ability in different cell lines.

Circulating miR-146a -5p in Egyptian patients with Rheumatoid Arthritis

We indented to evaluate of the role miRNA-146a-5p expression level as a possible biomarker associated with the incidence of rheumatoid arthritis and the detection of its relevance with different disease parameters. The study included 60 clinically diagnosed RA patients fulfilling 2010 American College of Rheumatology / European League Against Rheumatism (ACR/EULAR) classification criteria for Rheumatoid Arthritis and 45 age and sex matched control subjects. Disease activity was assessed by Disease Activity Score 28 (DAS28). The expression levels of miR-146a in whole blood was measured using reverse transcriptase quantitative real time polymerase chain reaction (RT-PCR). There median expression level of miRNA-146a-5p was significantly higher in RA patients compared with the control group (P = 0.036). MiR-146a expression level was positively correlated with individual activity markers, including erythrocyte sedimentation rate (ESR) (P = 0.04), visual analogue scale (VAS) (P = 0.047) and Modified Health Assessment Questionnaire (MHAQ) (P = 0.050). A lower expression level of miRNA-146a-5p was detected in RA patients on antimalarial drugs as compared to patients whose treatment protocol did not include antimalarial drugs (P = 0.016). In conclusion, data of this study suggested a possible association between miRNA 146a-5p expression level and the incidence of RA in Egyptian patients.

Serum level and gene expression of monocyte chemoattractant protein-1 (MCP-1) as a strong predictor for myocardial infarction in a sample of Iraqi population

BackgroundMyocardial infarction (MI) also referred as a “heart attack” is the sudden death of myocardial tissue caused by ischemia and typically caused by thrombotic occlusion of a coronary artery caused by the breakdown of a fragile plaque. Monocyte Chemoattractant Protein-1 (MCP-1) is a chemokine that belongs to the small inducible cytokine family. It is essential for monocyte and T lymphocyte recruitment into tissues. The aim of this work to see if there was a relationship between the level of MCP-1 in the human peripheral circulation and the development of myocardial infarction. Subject and methodsWe studied 150 patients with MI (24 female and 126 male) were recruited from the Iraqi center for heart disease at (“Ghazi Al-Hariri hospital”) in Baghdad, Iraq between January 2019 and June 2019 with a mean age (58.81 ± 8.21). Another 150 healthy subjects with no history of MI (52 female and 98 male) were enrolled as controls with a mean age (56.67 ± 9.25). Anthropometric measurements, clinical laboratory measurement include serum lipid profile, a quantitative enzyme-linked immune sorbent assay (ELISA) include MCP-1 and oxLDL serum levels and the echocardiographic data were determined. The total RNA was extracted from leukocytes and the relative quantification of MCP-1 gene was assessed by using the reverse transcriptase quantitative real time polymerase chain reaction (RT-qPCR) technique. ResultIn the current study, it was observed insignificant differences in anthropometric parameters except smoking status. The significant data (p < 0.05) was showed in ejection fraction, lipid profile and oxidize low density lipoprotein (oxLDL) between the two studied groups. Serum MCP-1 level increased significantly by 2 folds (310.8 ± 52.91 pg/ml) in MI patients when compared to control (180 ± 36.4 pg/ml). Significant low level of MCP-1 observed in patients with anterior MI (301.1 ± 40.2 pg/ml) compared with inferior MI (3.11 ± 49.6 pg/ml) or other sites (312.7 ± 51.3). Fold expression was statically differences and high unregulated of MCP-1 in MI patients (2-ΔΔCt = 2. 56 ± 0. 99).The diagnostic efficiency of serum MCP-1 and fold expression were demonstrated using a ROC curve study in MI patients versus control group at cutoff value 226.29 pg/ml with (specificity 99.2%; sensitivity 98.1%) and 1.79 with (specificity 85.7%; sensitivity 88.2%) respectively. Pearson's Correlation Coefficients study of MCP-1 serum level and fold expression revealed positive correlation with oxLDL (r = 0.4; 0.35) at p value <0.05 respectively. Also the positive correlation between serum level and fold expression of MCP-1 (r = 0.31) at the corresponding level p < 0.05 was observed. ConclusionFrom a clinical standpoint, MCP-1 concentration provided independent prognostic value and, was found to be one of the best indicators of clinical activity in MI patients. It is a new, independent measure of clinical myocardial infarction status that also accurately reflects the significant correlated with the sensitive cardiovascular risk biomarker.

Plasma oxidized low-density lipoprotein level and miRNA-146a gene expression, as a strong predictor for atherosclerotic coronary artery disease and its associated response to atorvastatin in a sample of the Iraqi population

BackgroundAtherosclerosis is considered to be a chronic inflammatory disease of the artery wall. Complications of atherosclerosis, such as myocardial infarction (MI) or ischemic stroke, are the major causes of disability and death in the modern world. Atorvastatin is broadly used as statin therapy to lower the serum lipid level with better safety and tolerance and is a class of statin medication that used in Iraqi healthcare services. The aim of the present study is to explore the expression level of miRNA146a and OX-LDL as a potential diagnostic biomarker of atherosclerotic coronary artery disease which depends upon vessel affected and its associated with the lipid lowering medication (atorvastatin).Subjects and methodsA total of 200 blood specimens were collected from adults with atherosclerosis comprise 100 patients subdivided to patients with 20 mg atorvastatin (n = 52) and patients with 40 mg atorvastatin (n = 48), and healthy control subjects which comprise 100. The age range between 30‐ and 60 years old. The disease activity of patients᾿ groups to determine by vessel disease affected. OxLDL determined by competitive ELISA, relative quantification (RQ) of miRNA146a expression, in whole blood was estimated using reverse transcriptase quantitative real time polymerase chain reaction. ResultThe statistical analysis showed significant statistical differences between patients and healthy controls in multiple studied parameters. Regarding lipid profile and oxLDL levels in patients taking 20 mg atorvastatin were significantly higher than controls (P < .05). A significant difference in total cholesterol (TC) and low density lipoprotein (LDL-C) between two classes of patients with double disease affected and a significant decrease in the rate of oxidizing LDL (oxLDL) in patients with 40 mg compared to 20 mg. Highly significant upregulated gene expression of miRNA-146a in patients with 20 mg versus 40 mg and the highest folds in a single vessel was observed. OxLDL and miRNA146a demonstrated better performance in atherosclerotic coronary artery disease (ACAD) diagnosis, showing sensitivity (95.3; 75%) (AUC: 0.81; 0.83 at cutoff value 57.94; 1.17) P 0.05. The level of miRNA-146a is positively correlated with oxLDL (p < .05). ConclusionThe applicability of miRNA146a and ox-LDL as potential diagnostic biomarkers of ACAD, by using different protocols. Its diagnostic performance was better than lipid variable and other traditional risk factor. Therefore, our data strongly suggest measuring both parameters for more specific prediction of ACAD.

Monitoring PRRSV-1 in suckling piglets in an endemic herd using reverse transcriptase quantitative real time polymerase chain reaction: comparison of the rate of detection in serum and oral fluid samples and evaluation of pooling

BackgroundDefining shedding and exposure status for PRRSV is essential in herd stabilisation protocols and weaning-age pigs is a key subpopulation. Oral fluid (OF) sampling is a welfare-friendly and cost saving promising alternative to blood sampling. The first objective of our study was to compare the rate of detection of PRRSV-1 in individual serum sample, individual OF sample, litter-based OF sample, collected the day before weaning. The second objective was to evaluate the interest of pooling samples.ResultsThe study was performed on a 210-sows, PRRSV-1 exposed, with confirmed shedding, non-vaccinated against PRRSV, herd. 80 litters were sampled and 26 were viropositive and therefore included. The rate of detection of PRRSV-1 with RT-qrtPCR in blood samples, iOF and cOF was 67, 23 and 77%, respectively. The Ct values from RT-qrtPCR on collective OF were statistically lower if the serum of the piglet of the litter was positive. The lower the Cycle threshold (Ct) value of RT-qrtPCR on collective OF, the higher the probability that the serum sampled in the same litter was positive. Ability to detect PRRSV RNA after pooling was 67% for sera and 58% for cOF.ConclusionsThe rate of detection of PRRSV-1 was about the same in cOF and blood samples. Virus sequencing, if required, should be performed on individual serum samples. The smaller the Ct of a cOF sample from a litter, the greater the likelihood that the serum sample from a piglet of that litter is positive.A cost-effective and representative sampling protocol to monitor sow herds stabilisation of a sow batch could be: to collect both cOF and one serum sample per litter; to perform firstly RT-qrtPCR on pooled cOF; in case of negative results to consider the batch negative; in case of positive results in a unvaccinated herd or a killed vaccine vaccinated one to consider the batch positive; in case of positive result in a herd vaccinated with a modified live vaccine serum samples of litters with positive cOF should be tested for sequencing (selecting the litters with the lowest Ct for cOF).

MIR-146A AND TLR4 GENE EXPRESSION IN PREDICTING RHEUMATOID ARTHRITIS DISEASE

Disease Activity Score 28 (DAS28) isthe most widely used method for rheumatoid arthritis(RA) disease activity evaluation. However, it may not reflect disease activity accurately being a subjective tool dependent on patient's self-health assessment and evaluation of swollen and tender joint counts. Therefore, finding a suitable blood genetic marker for accurate monitoring of RA disease activity has become essential. Therefore, the aim of this study was to assess miR- 146a and TLR4 expression in PBMCs of RA patients and to study their value as potential molecular biomarkers for RA disease activity in comparison to DAS28 score. This study was conducted on 51 RA patients and 15 age and sex matched healthy subjects as control group. The relative gene expression levels of miR-146a and TLR4 were determined by reverse transcriptase quantitative real time polymerase chain reaction. There were statistically significant differences between patients and healthy controls as regards miR-146a and TLR4 expression. In addition, statistically significant differences were observed between different patients’ subgroups as regards miR-146a and TLR4 expression. Moreover, miR-146a and TLR4 expression levels showed significant positive correlationswith those of morning stiffness durations, tender joints counts, swollen joints counts, visual analogue scale values, erythrocyte sedimentation rates, CRP, Anti- CCP antibody and DAS28.MiR-146aillustrated the best performance characteristics particularly in differentiating between high and moderate disease activity grades, showing highest sensitivity and specificity.Furthermore, there was a statistically significant increase in the expression levels of miR-146a and TLR4 in PBMCs of RA patients with ankles and/or feet joints involvement, as compared with those without involvement.Conclusion: MiR-146a and TLR4 were overexpressed in PBMCs of RA patients and were correlated with disease activity and radiographic progression especially miR-146a.Keywords: DAS28, Larsen score, RA, reverse transcriptase quantitative real time PCR.

MicroRNA-146a expression as a potential biomarker for rheumatoid arthritis in Egypt

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs, whose role in regulating diverse immune functions, suggests they might play a role as biomarkers for immune mediated disorders. Studies showed that miRNA-146a (miR-146a) expression is increased by proinflammatory cytokines and is an important modulator of differentiation and function of cells of innate and adaptive immunity. Aim of the workThe current study aimed to evaluate the expression of miR-146a as a potential biomarker for diagnosis of rheumatoid arthritis (RA) and to explore its association with disease activity. Subjects and methodsThe study enrolled 50 Egyptian subjects divided into a patient group, which comprised 25 RA patients, and a control group which comprised 25 healthy individuals. The disease activity for the patients’ group was determined by simplified disease activity index. Relative quantification of miR-146a expression in whole blood was determined using reverse transcriptase quantitative real time polymerase chain reaction. ResultsThere were highly significant statistical differences between patients and healthy controls as regards miR-146a relative expression, erythrocyte sedimentation rate (ESR) and anti-cyclic citrullinated peptide (anti-CCP) (p<0.001). Highly significant statistical differences (p<0.001) were also found between different patients’ subgroups as regards miR-146a relative expression and ESR. miR-146a levels correlated positively with those of ESR, C-reactive protein and anti-CCP (p<0.001).miR-146a illustrated best performance in diagnosing RA, showing the highest sensitivity and specificity (96% and 100%, respectively) (AUC: 0.992 at a cut off value of ⩾2.16) compared to anti-CCP (sensitivity: 68%, specificity: 100% and AUC: 0.87 at a cut off value of ⩾22U/ml) and RF (sensitivity: 56%, specificity: 80% and AUC: 0.992 at a cut off value of ⩾13U/ml). ConclusionThis study demonstrated that miR-146a expression was highly significantly elevated in whole blood of patients with RA. Its diagnostic performance was better than anti-CCP and RF and its level of expression correlates with disease activity.