Quantitative Real-time Reverse Transcriptase Research Articles

Functionalizable bacterial cellulose composite membrane for guided tissue regeneration

This research aims to develop guided tissue regeneration (GTR) membranes from bacterial cellulose (BC), a natural polysaccharide-based biopolymer. A double-layered BC composite membrane was prepared by coating the BC membrane with mixed carboxymethyl cellulose/poly(ethylene oxide) (CMC/PEO) fibers via electrospinning. The CMC/PEO-BC membranes were then characterized for their chemical and physical characteristics. The 8 % (wt/v) CMC/PEO (1:1) aqueous solution yielded well-defined electrospun CMC/PEO nanofibers (125 ± 10 nm) without beads. The CMC/PEO-BC membranes exhibited good mechanical and swelling properties as well as good cytocompatibility against human periodontal ligament cells (hPDLs). Its functionalizability via carboxyl entities in CMC was tested using the calcium-binding domain of plant-derived recombinant human osteopontin (p-rhOPN-C122). As evaluated by enzyme-linked immunosorbent assay, a 98–99 % immobilization efficiency was achieved in a concentration-dependent manner over an applied p-rhOPN-C122 concentration range of 7.5–30 ng/mL. The biological function of the membrane was assessed by determining the expression levels of osteogenic-related gene transcripts using quantitative real-time reverse-transcriptase polymerase chain reaction. Mineralization assay indicated that the p-rhOPN-C122 immobilized CMC/PEO-BC membrane promoted hPDLs osteogenic differentiation. These results suggested that the developed membrane could serve as a promising GTR membrane for application in bone tissue regeneration.

RNA interference-mediated knockdown of genes involved in sugar transport and metabolism disrupts psyllid Bactericera cockerelli (Order: Hemiptera) gut physiology and results in high mortality.

The causal agent of zebra chip of potato and vein-greening diseases of tomato is "Candidatus Liberibacter solanacearum" (CLso), a fastidious bacterium transmitted by the potato psyllid. In the absence of disease-resistant cultivars, disease management has relied on minimizing vector population size to reduce CLso transmission, which requires frequent insecticide applications. There is growing interest in the use of RNA interference (RNAi) technology to supplant traditional insecticides with biopesticides. This requires knowledge of genes essential for insect livelihood whose knockdown leads to significant mortality or other phenotypes. Such candidate genes can be evaluated by reverse genetics approaches to further corroborate predicted gene function. Here, five potato psyllid genes involved in sugar homeostasis in the potato psyllid gut, α-glucosidase1 (AGLU1), aquaporin2 (AQP2), facilitated trehalose transporter1 (TRET1), Trehalase1 (TRE1), and Trehalase2 (TRE2), were investigated as candidates for effective gene silencing. Potato psyllid dsRNAs were designed to optimize knockdown of gene targets. Third instar PoP nymphs were given a 48-hr ingestion-access period (IAP) on individual or groups of dsRNA in 20% sucrose. Mortality was recorded 0, 3, 5, 7, and 9 days post-IAP. Gene knockdown was analyzed 9 days post-IAP by quantitative real-time reverse-transcriptase polymerase chain reaction amplification. The individual or stacked dsRNA combinations resulted in 20-60% and 20-40% knockdown, respectively, while subsequent psyllid mortality ranged from 20-40% to >60% for single and stacked dsRNA combinations, respectively. Reverse genetics analysis showed that simultaneous knockdown of the five selected candidate genes with predicted functions in pathways involved in sugar-homeostasis, metabolism, and -transport yielded the highest mortality, when compared with single or combinations of targets. Results confirmed the functions afforded by psyllid gut genes responsible for osmotic homeostasis and sugar metabolism/transport are essential for livelihood, identifying them as potentially lucrative RNAi biopesticide targets and highlighted the translational relevance of targeting multiple nodes in a physiological pathway simultaneously.

Silicon dioxide nanoparticles inhibit the effects of cold exposure on metabolism and inflammatory responses in brown adipocytes

Abstract Objective Nanoparticles (NPs) in haze are potentially hazardous to health, which is more severe in the winter. Brown adipose tissue (BAT) plays important roles in obesity, insulin resistance, and diabetes. Though the toxicology of NPs has been intensively studied, few studies have been reported on the antagonistic effects between Silicon dioxide(SiO2) NPs and cold exposure in brown adipocytes. Materials and methods We evaluated changes by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) on metabolism genes, plasticity genes and the inflammatory responses genes in brown adipocytes in vitro. Results The expression of adipogenic genes PRDM16, Dio2, PGC-1α and UCP1 was upregulated upon cold exposure (P < 0.05), but downregulated by SiO2 NPs (P < 0.05). The results demonstrated that there was antagonistic effect between SiO2 NPs and cold exposure on the plasticity genes and metabolism genes in brown adipocytes, where the main effects of SiO2 NPs or cold exposure on the plasticity genes and metabolism genes were significant (P < 0.05). Moreover, the levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were upregulated by SiO2 NPs or cold exposure (P < 0.05). The factorial analysis indicated that there was also antagonistic effect between SiO2 NPs and cold exposure on the toxic effects in brown adipocytes, in which the main effects of cold exposure and/or SiO2 NPs on the toxic effects were significant (P < 0.05). Conclusion SiO2 NPs inhibit the effect of cold exposure on metabolic genes and inflammatory responses genes in brown adipocytes.

P101 Circulating MicroRNA-16 in Inflammatory Bowel Disease Patients - Potential Biomarker to Assess Inflammation

Abstract Background miR-16 is important for the inflammatory processes and the signal pathways that participate in the immune system regulation. As there is an immune mucosal inflammation in IBD, miR-16 could also serve as a potential biomarker to assess inflammation. Methods The study aims to assess the expression of serum miR-16 in IBD patients and to correlate its expression with clinical parameters such as disease extent, activity and severity. 35 patients with UC and 35 patients with CD were included in the study. Serum miR-16 expression was assessed with reverse transcriptase quantitative real time PCR (RT-qPCR), and was then correlated with the miR-16 levels in a group of 30 healthy subjects. Results miR-16 expression is increased in patients with CD, localized in the small intestine (3.54±2.33) and in pancolitis Е3 of UC (2.01±1.54). There is a significant difference in miR-16 expression in inflammatory (B1), stenotic (B2) and penetrating (B3) form of CD (р=0.010) with higher expression levels in the more severe B2 and B3 forms. There is no significant difference in miR-16 expression according to the forms of evolution in patients with UC. In patients with CD, there is a moderate direct correlation between the expression of miR-16 and the Crohn’s disease activity index CDAI (r=0.424; p=0.015). miR-16 expression levels increase progressively in correlation with the activity of CD. Patients with moderate activity of their CD have significantly higher miR-16 expression (4.27±3.00) as compared to patients with mild activity (2.38±1.06) or in remission (2.41±1.23) of the disease (р=0.048). The increased expression levels of miR-16 are a risk factor for CDAI > 150 levels (OR=4.80 (0.506-45.495); p=0.015). In patients with UC, there is no correlation between the miR-16 expression levels and the severity (S) index, measured as per the Montreal classification or the endoscopic activity, measured as per the Partial Mayo score. In patients with severe disease, measured by both S index (1.38±0.89) and Partial Mayo score (1.80±1.12), there is a decrease in miR-16, close to normal controls. MiR-16 expression levels do not correlate with any treatment that was given in patients with either CD or UC. Conclusion The increased miR-16 serum expression corelates with the localization of CD in the small intestine and the stenotic and penetrating form of the disease. Increased miR-16 levels are also found in extensive UC. In patients with CD there is a positive correlation between the expression of miR-16 and the activity of the disease as measured by the CDAI.

MiR-448 regulates potassium voltage-gated channel subfamily A member 4 (KCNA4) in ischemia and heart failure

MicroRNA miR-448 mediates some of the effects of ischemia on arrhythmic risk. Potassium voltage-gated channel subfamily A member 4 (KCNA4) encodes a Kv1.4 current that opens in response to membrane depolarization and is essential for regulating the action potential duration in heart. KCNA4 has a miR-448 binding site. We investigated whether miR-448 was involved in the regulation of KCNA4 messenger RNA expression in ischemia. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to investigate the expression of KCNA4 and miR-448. Pull-down assays were used to examine the interaction between miR-448 and KCNA4. miR-448 decoy and binding site mutation were used to examine the specificity of the effect for KCNA4. The expression of KCNA4 is diminished in ischemia and human heart failure tissues with ventricular tachycardia. Previously, we have shown that miR-448 is upregulated in ischemia and inhibition can prevent arrhythmic risk after myocardial infarction. The 3'-untranslated region of KCNA4 has a conserved miR-448 binding site. miR-448 bound to this site directly and reduced KCNA4 expression and the transient outward potassium current. Inhibition of miR-448 restored KCNA4. These findings showed a link between Kv1.4 downregulation and miR-448-mediated upregulation in ischemia, suggesting a new mechanism for the antiarrhythmic effect of miR-448 inhibition.

Association of epidemiological and clinical features with PCR cycle threshold values of SARS-CoV-2 infection: a cross-sectional study

ABSTRACT The cycle threshold (Ct) in quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) is inversely correlated to the amount of viral nucleic acid or viral load and can be regarded as an indicator of infectivity. We examined the association of socio-demographic and clinical characteristics of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) polymerase chain reaction (PCR) positive cases with PCR cycle threshold (Ct) values at the time of diagnosis. SARS-CoV-2 cases reported between 12 October 2020 and 24 January 2021 in Regensburg were analyzed employing bivariate and multivariable methods. We included 3,029 SARS-CoV-2 cases (31% asymptomatic at diagnosis) and analyzed the association of case characteristics with Ct values in 2,606 cases. Among symptomatic patients, cough (38.0%), rhinitis (32.4%), headache (32.0), and fever/chills (29.9%) were the most frequent complaints. Ct values ≤20 were more frequent in symptomatic cases (20.9% vs. 11.3%), whereas Ct values >30 were more common in asymptomatic cases (32.6% vs. 18.0%). Ct values >20 and ≤30 were most common in symptomatic and asymptomatic cases (48.0% vs 40.7%). We observed lower median Ct values of E and N gene in symptomatic cases. In a random forest model, the total number of symptoms, respiratory symptoms, and age were most strongly associated with low Ct values. In conclusion, certain symptoms and age were associated with lower Ct values. Ct values can be used as a pragmatic approach in estimating infectivity at the first notification of a case and, thus, in guiding containment measures.

Full-Length Genome of the Equine Influenza A Virus Subtype H3N8 from 2019 Outbreak in Saudi Arabia.

Simple SummaryEquine influenza is a highly contagious respiratory viral disease. The current study is the first to provide a description of the full-length genome sequence and surveillance of recent exposure to the equine influenza virus (EIV) during the 2019 epidemic in Saudi Arabia. This epidemic was benign, since it resulted in low case fatality (0.45%, 1/224). The viruses detected in the current study were found to be related to subtype H73N8 clade 1 of the Florida sublineage. Full-length genome sequencing revealed no evidence of major genetic changes or of reassortment among the eight segments of the viral genome. However, the Saudi strains showed a considerable number of amino acid substitutions in the signal peptide (2 amino acid substitutions), HA1 (10 amino acid substitutions) and HA2 (4 amino acid substitutions) in the haemagglutinin glycoprotein in comparison to clade 1 Florida sublineage vaccinal strains. These findings should be considered during selection of the equine influenza vaccine strains approved for use in Saudi Arabia.Equine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. This study aimed to screen the incidence of equine influenza virus (EIV) and molecularly characterize the haemagglutinin and neuraminidase from positive EIV field samples collected from Saudi Arabia. Six-hundred twenty-one horses from 57 horse barns were screened for the presence of the clinical signs, suggestive for equine influenza, from different parts of Saudi Arabia. Nasopharyngeal swabs were collected from each horse showing respiratory distress. Samples from the same horse barn were pooled together and screened for the presence of the influenza A virus using quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR). Selective positive samples were subjected to full-length genome sequencing using MiSeq Illumina. Out of the total 57 pools, 39 were found positive to EIV using qRT-PCR. Full-length gene sequences were compared with representative EIV strains selected from the GenBank database. Phylogenetic analysis of the HA and NA genes revealed that the identified virus strains belong to H3N8 clade 1 of the Florida sublineage and were very similar to viruses identified in USA in 2019, with no current evidence for reassortment. This is one of the first reports providing detailed description and characterization of EIVs in Saudi Arabia. Detailed surveillance and genetic information sharing could allow genetic evolution of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.

Chapare Hemorrhagic Fever and Virus Detection in Rodents in Bolivia in 2019

BackgroundIn June 2019, the Bolivian Ministry of Health reported a cluster of cases of hemorrhagic fever that started in the municipality of Caranavi and expanded to La Paz. The cause of these cases was unknown.MethodsWe obtained samples for next-generation sequencing and virus isolation. Human and rodent specimens were tested by means of virus-specific real-time quantitative reverse-transcriptase–polymerase-chain-reaction assays, next-generation sequencing, and virus isolation.ResultsNine cases of hemorrhagic fever were identified; four of the patients with this illness died. The etiologic agent was identified as Mammarenavirus Chapare mammarenavirus, or Chapare virus (CHAPV), which causes Chapare hemorrhagic fever (CHHF). Probable nosocomial transmission among health care workers was identified. Some patients with CHHF had neurologic manifestations, and those who survived had a prolonged recovery period. CHAPV RNA was detected in a variety of human body fluids (including blood; urine; nasopharyngeal, oropharyngeal, and bronchoalveolar-lavage fluid; conjunctiva; and semen) and in specimens obtained from captured small-eared pygmy rice rats (Oligoryzomys microtis). In survivors of CHHF, viral RNA was detected up to 170 days after symptom onset; CHAPV was isolated from a semen sample obtained 86 days after symptom onset.ConclusionsM. Chapare mammarenavirus was identified as the etiologic agent of CHHF. Both spillover from a zoonotic reservoir and possible person-to-person transmission were identified. This virus was detected in a rodent species, O. microtis. (Funded by the Bolivian Ministry of Health and others.)

Adenovirus 5 Vectors Expressing SARS-CoV-2 Proteins.

ABSTRACTSARS-CoV-2 coronavirus is a recently identified novel coronavirus that is the causative agent of the COVID-19 pandemic that began in 2020. An intense research effort has been undertaken by the research community in order to better understand the molecular etiology of this virus and its mechanisms of host cell subjugation and immune system evasion. To facilitate further research into the SARS-CoV-2 coronavirus we have generated adenovirus 5-based viral vectors that express SARS-CoV-2 proteins—S, N, E, NSP7, NSP8, and NSP12 as hemagglutinin (HA)-tagged and untagged variants. We have also engineered two additional viruses that express the S protein receptor binding domain and a fusion of the receptor binding domain to the N protein. We show that these vectors are expressed in several different cell lines by Western blotting and real-time quantitative reverse transcriptase (qRT-PCR), we evaluate the subcellular localization of these viral proteins, and we show that these coronavirus proteins bind to a variety of cellular targets. The flexibility of adenovirus vectors allows them to be used in a variety of cell models and, importantly, in animal models as well.IMPORTANCE The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has brought untold personal and economic suffering to the world. Intense research has made tremendous progress in understanding how this virus works, yet much research remains to be done as new variants and continued evolution of the virus keep shifting the rules of engagement on the pandemic battlefield. Therefore, wide availability of resources and reagents to study SARS-CoV-2 is essential in overcoming the pandemic and for the prevention of future outbreaks. Our viral vectors provide additional tools for researchers to use in order to better understand the molecular biology of virus-host interactions and other aspects of SARS-CoV-2.

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.

Circulating microRNA 9-3p and serum endocan as potential biomarkers for hepatitis C virus-related hepatocellular carcinoma.

BACKGROUNDThe high mortality rate of hepatocellular carcinoma (HCC) in Egypt is due mainly to the increasing prevalence of hepatitis C virus infection (HCV) and late diagnosis of the carcinoma. MicroRNAs (miRNA), which regulate tumor proliferation and metastasis in HCC, may serve as a useful diagnostic approach for the early detection of HCC, thus decreasing its mortality. Meanwhile, endocan is a protein with angiogenic and inflammatory properties that are associated with tumor progression and poor outcomes. AIMTo analyze the levels of miRNA 9-3p and endocan in HCV-infected HCC patients and correlate them with clinicopathological parameters.METHODSWe compared levels of endocan and circulating miRNA 9-3p from 35 HCV-related HCC patients to 33 patients with HCV-induced chronic liver disease and 32 age and gender matched healthy controls recruited from inpatient and outpatient clinics of the National Liver Institute, Menoufia University, Egypt in the period from January to March 2021 in a case-control study. Serum samples from all groups were analyzed for HCV. Endocan was measured by enzyme-linked immunosorbent assays, and the expression levels of circulating miRNA 9-3p were measured by real-time quantitative reverse transcriptase PCR.RESULTSThe levels of circulating miRNA 9-3p were significantly lower in the HCC group compared to the chronic liver disease (P < 0.001) and control (P < 0.001) groups, while levels in the chronic liver disease were significantly lower than those in the control group (P < 0.001). The levels of serum endocan were significantly higher in the HCC group compared to the chronic liver disease (P < 0.001) and control (P < 0.001) groups. Moreover miRNA 9-3p and endocan performed better than α-fetoprotein in discriminating HCC patients from cirrhosis and healthy patients. The levels of miRNA 9-3p were significantly inversely correlated to vascular invasion (P = 0.002), stage of advancement of Barcelona Clinical Liver Cancer (P < 0.001) and the metastatic site (P < 0.001) of the HCC group.CONCLUSIONCirculating miRNA 9-3p and endocan can be used as novel biomarkers for the early diagnosis of HCV-related HCC.

Anticancer effects of mifepristone on human uveal melanoma cells

BackgroundUveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds.MethodsThe effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR.ResultsMF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor.ConclusionThis study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

Exosomal circ_0048856 derived from non-small cell lung cancer contributes to aggressive cancer progression through downregulation of miR-1287–5p

AimPresent study was aimed to explore the diagnostic value and mechanism of exosome derived circular RNA (circ)_0048856 in non-small cell lung cancer (NSCLC) development. MethodsExosomes protein markers, CD63 and CD9, were examined by Western blot. Real time quantitative reverse transcriptase - polymerase chain reaction (qRT-PCR) was used to detect the relative expression of circ_0048856 and miR-1287–5p. Proliferation of NSCLC cell was detected by MTT assay. Transwell assay was utilized to evaluated the migration and invasion of NSCLC cells. Apoptosis rate was examined by Flow cytometry. Double luciferase report assay was used to assess the targeting association between circ_0048856 and miR-1287–5p. Diagnostic value of circ_0048856 for NSCLC was estimated by receiver operating characteristic (ROC) curve. Animal models were constructed to explore the function of exosomal circ_0048856 in NSCLC. ResultsDiameter of exosomes in NSCLC serum was ranged between 75 and 150 nm. Exosomes circ_0048856 was enhanced in NSCLC serum and cell lines (P < 0.001). Exosome derived circ_0048856 had high diagnostic value for NSCLC. Area under the curve (AUC) was 0.943 (95%CI=0.904–0.982, P < 0.001). at the optimal cutoff value of 1.800, the sensitivity was 0.880, and specificity was 0.800. Exosome circ_0048856 facilitated proliferation, migration and invasion, inhibited apoptosis of NSCLC cells. MiR-1287–5p could be effaced by circ_0048856. MiR-1287–5p reversed the biological behavior changes of NSCLC cells which induced by circ_0048856. In vivo, exosomal circ_0048856 could significantly promote tumor growth (P < 0.001). ConclusionExosomes derived circ_0048856 was elevated in NSCLC serum and cell lines. Exosome circ_0048856 promoted the NSCLC development by targeting miR-1287–5p. Exosome circ_0048856 had high diagnostic value for NSCLC.

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.

Circular RNA circZFPM2 promotes epithelial-mesenchymal transition in endometriosis by regulating miR-205-5p/ZEB1 signalling pathway

Endometriosis is a debilitating gynecological disease affecting millions of women worldwide, but its exact pathogenesis remains unclear. Circular RNAs (circRNAs) have been demonstrated to be important regulators in multiple diseases. Nonetheless, the potential regulatory mechanism of aberrant circRNA expression in endometriosis has been elusive. The up-regulated circZFPM2 in ectopic endometrial tissues was previously screened by circRNA high-throughput sequencing and was furtherly validated by quantitative real time reverse transcriptase polymerase chain reaction (RT-qPCR). Overexpression of circZFPM2 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in Ishikawa and End1/E6E7 cells, whereas silencing circZFPM2 produced the opposite effect. Luciferase reporter assays validated that circZFPM2 could directly target miR-205-5p and miR-205-5p target ZEB1. RT-qPCR results showed that miR-205-5p was underexpressed while ZEB1 was overexpressed in ectopic endometrial tissues compared with their expression in eutopic endometria and non-endometriosis control endometria. The expression level of miR-205-5p was inversely proportional and that of ZEB1 was directly proportional with the proliferative, migrative, and invasive ability of endometrial cells. Further in vitro investigation indicated that miR-205-5p could inhibit EMT by targeting ZEB1. Subsequent rescue experiments confirmed that circZFPM2 could induce EMT and promote cell proliferation, migration, and invasion cascades through the miR-205-5p /ZEB1 signaling pathway. Conclusively, circZFPM2 may present a promising biomarker in the diagnosis and treatment of endometriosis.

Clinical Significance of Chemokine Receptor CXCR4 and CCR7 mRNA Expression in Patients With Colorectal Cancer.

The chemokine receptors C-X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 7 (CCR7) play an important role in the invasion and metastasis of cancer. This study investigated the relationship between relative expression of CXCR4 and CCR7 mRNA, clinicopathological factors, and outcomes in patients with colorectal cancer (CRC). We studied 202 patients who underwent surgery for CRC. The expression levels of CXCR4 and CCR7 mRNA in cancerous tissue were measured using quantitative real-time reverse-transcriptase polymerase chain reaction. High CCR7 mRNA expression levels in CRC tissues were positively associated with tumour size and were more frequently associated with cancer of the rectum than of the colon. Moreover, outcomes were significantly poorer in patients with high CCR7 mRNA expression than in those with low expression. On multivariate Cox regression analysis, a higher CCR7 mRNA expression level was a significant independent predictor of poorer overall survival in patients with CRC. Overexpression of CCR7 mRNA may be a useful independent prognostic factor in patients with CRC.

Frequent expression of a novel cancer testis antigen, protein kinase human monopolar spindle 1 (hMps1/TTK) in human urinary bladder transitional cell carcinoma.

Urothelial bladder cancer (UBC) is a frequently occurring malignancy of the urinary tract. The present study was undertaken to evaluate the mRNA and immunohistochemical (IHC) expression of protein kinase human monopolar spindle 1 (hMps1/TTK) gene in transitional cell carcinoma (TCC) of the bladder and correlate its expression with the clinicopathological characteristics of patients. In the present study, quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate TTK mRNA expression in TCC. IHC analysis of TTK was also evaluated. Independent Student's t, ANOVA and chi-square (χ2) tests were used to analyze the data statistically. The frequency of TTK mRNA over expression was detected in 50% of UBC (38/76) by qRT-PCR. Relative mean fold expression of TTK mRNA was found significantly (p < 0.05) higher in muscle-invasive bladder cancer (MIBC) as compared to non-muscle-invasive bladder cancer (NMIBC) patients (8.96 ± 4.51 vs. 5.64 ± 3.53, p = 0.03). Moreover, IHC reveals heterogenous immunostaining pattern of TTK in TCC tissues. The frequency of TTK protein over expression was detected in 56.9% (37 of 65) UBC patients. No significant IHC expression of TTK was detected among adjacent noncancerous tissues (ANCTs) and benign prostatic hyperplasia (BPH) used as control. Collectively our study observations conclude that TTK is a novel cancer/testis antigen (CTA) as a diagnostic marker for early diagnosis of UBC.

Comparative Biological Properties and Mineralization Potential of 3 Endodontic Materials for Vital Pulp Therapy: Theracal PT, Theracal LC, and Biodentine on Human Dental Pulp Stem Cells

IntroductionThe aim of this study was to assess the biological properties and mineralization potential of the new Theracal PT (Bisco Inc, Schaumburg, IL) compared with its predecessor Theracal LC (Bisco Inc) and the hydraulic silicate–based cement Biodentine (Septodont, Saint-Maur-des-Fossés, France) on human dental pulp stem cells (hDPSCs) in vitro. MethodsStandardized sample discs were obtained for each material (n = 30) together with 1:1, 1:2, and 1:4 material eluates. Previously characterized hDPSCs were cultured with the different materials in standardized conditions, and the following assays were performed: a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a wound healing assay, Annexin-V-FITC and 7-AAD staining (BD Biosciences, San Jose, CA), reactive oxygen species production analysis, cell adhesion and morphology evaluation via scanning electron microscopy and immunofluorescence, quantification of the expression of osteo/odontogenic markers via real-time quantitative reverse-transcriptase polymerase chain reaction, and alizarin red S staining. Statistical significance was established at P < .05. ResultsAll of the tested dilutions of Theracal LC exhibited a significantly higher cytotoxicity and reactive oxygen species production (P < .001) and a lower cell migration rate than the control group (hDPSCs cultured in growth medium without material extracts) at all of the measured time points (P < .001). Both 1:4 Theracal PT and Biodentine-treated hDPSCs exhibited similar levels of cytocompatibility to that of the control group, a significant up-regulation of at least 1 odontogenic marker (Biodentine: dentin sialophosphoprotein (P < .05); Theracal PT: osteonectin and runt-related transcription factor 2 [P < .001]), and a significantly higher mineralized nodule formation (P < .001). ConclusionsThe newly introduced TheraCal PT offers an improved in vitro cytocompatibility and mineralization potential on hDPSCs compared with its predecessor, TheraCal LC, and comparable biological properties to Biodentine.

Biomineralization potential and biological properties of a new tantalum oxide (Ta2O5)\u2013containing calcium silicate cement

ObjectiveThe present study evaluated the biological effects and biomineralization potential of a new tantalum oxide (Ta2O5)–containing material designed for vital pulp therapy or perforation repair (NeoMTA 2), compared to NeoMTA Plus and Bio-C Repair.Material and methodsHuman dental pulp stem cells (hDPSCs) were exposed to different eluates from NeoMTA Plus, NeoMTA 2, and Bio-C Repair. Ion release from each material was determined using inductively coupled plasma-optical emission spectrometry (ICP-MS). The biological experiments performed were MTT assays, apoptosis/necrosis assays, adhesion assays, migration assays, morphology evaluation, and reactive oxygen species (ROS) production analysis. Biomineralization was assessed by Alizarin red S staining. Finally, osteo/odontogenic gene expression was determined by real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test.ResultsNeoMTA 2 displayed a significantly higher calcium release compared to the other materials (p < 0.05). When hDPSCs were cultured in presence of the different material eluates, all groups exhibited similar hDPSC viability and migration rates when compared to untreated cells. Substantial cell attachment and spreading were observed in all materials’ surfaces, without significant differences. hDPSCs treated with NeoMTA 2 displayed an upregulation of ALP, Col1A1, RUNX2 (p < 0.001), ON, and DSPP genes (p < 0.05), and showed the highest mineralization potential compared to other groups (p < 0.001). Finally, the more concentrated eluates from these materials, specially NeoMTA Plus and NeoMTA 2, promoted higher ROS production in hDPSCs compared to Bio-C Repair and control cells (p < 0.001), although these ROS levels did not result in increased cell death.ConclusionsThe new tantalum oxide (Ta2O5)–containing material shows an adequate cytocompatibility and the ability to promote biomineralization without using chemical osteogenic inducers, showing great potential as a new material for vital pulp therapy.Clinical relevanceNeoMTA 2 seems to be a promising material for vital pulp therapy. Further studies considering its biocompatibility and biomineralization potential are necessary.