Real-time Quantitative Reverse Transcriptase-polymerase Research Articles

Respiratory virus associated with surgery in children patients

BackgroundViral respiratory infection (VRI) is a common contraindication to elective surgery. Asymptomatic shedding among pediatric surgery patients (PSPs) could potentially lead to progression of symptomatic diseases and cause outbreaks of respiratory diseases. The aim of this study is to investigate the incidence of infection among mild symptomatic PSP group and asymptomatic PSP group after surgical procedure.MethodsWe collected the induced sputum from enrolled 1629 children (under 18 years of age) with no respiratory symptom prior to pediatric surgery between March 2017 and February 2019. We tested 16 different respiratory virus infections in post-surgery mild symptomatic PSP group and asymptomatic PSP group using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay panel. We analyzed symptom data and quantitative viral load to investigate the association between viruses, symptoms and viral quantity in qRT-PCR-positive PSPs.ResultsOut of 1629 children enrolled, a total of 204 respiratory viruses were present in 171 (10.50%) PSPs including 47 patients with mild symptoms and 124 with no symptoms after surgery. Commonly detected viruses were human rhino/enterovirus (HRV/EV, 42.19%), parainfluenza virus 3 (PIV3, 24.48%), coronavirus (CoV NL63, OC43, HKU1, 11.46%), and respiratory syncytial virus (RSV, 9.9%). PIV3 infection with a higher viral load was frequently found in PSPs presenting with mild symptoms, progressing to pneumonia with radiographic evidence after surgery. HRV/EV were the most commonly detected pathogens in both asymptomatic and mild symptomatic PSPs. CoV (OC43, HKU1) infections with a higher viral load were mostly observed in asymptomatic PSPs progressing to alveolar or interstitial infiltration.ConclusionsOur study suggested that PIV3 is a new risk factor for VRI in PSPs. Employing a more comprehensive, sensitive and quantitative method should be considered for preoperative testing of respiratory viruses in order to guide optimal surgical timing.

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions.

Elymus sibiricus, which is a perennial and self-pollinated grass, is the typical species of the genus Elymus, which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in E. sibiricus. The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in E. sibiricus. Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that TBP2 and HIS3 were the most stable reference genes in different genotypes. TUA2 and PP2A had the most stable expression in different developmental stages. TBP2 and PP2A were suitable reference genes in different tissues. Under salt stress, ACT2 and TBP2 were identified as the most stable reference genes. ACT2 and TUA2 showed the most stability under heat stress. For cold stress, PP2A and ACT2 presented the highest degree of expression stability. DNAJ and U2AF were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene CSLE6 in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in E. sibiricus.

PS-04-006 Immunomodulatory effects of Dihydrotestosterone (DHT) in rat vaginal smooth muscle cells

Androgens, in particular Dihydrotestosterone (DHT), show immunomodulatory protective effects in experimental models of chronic inflammation. We aimed to evaluate the possible immunomodulatory effects of DHT in smooth muscle cells (SMCs) isolated from vagina of mature intact Sprague-Dawley rats. SMCs derived from distal vagina were isolated and characterized with α-smooth muscle actin (a-SMA) and myosin heavy chain 11 (MHCII). Then they were left untreated (control) or treated in vitro with different stimuli: Lipopolysaccharides (LPS; 100 ng/ml), the strongest immunogenic ligand for TLR; DHT (30nM); and DHT+LPS (30nM, 100ng/ml) for 24 hours. With Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction, we evaluated mRNA expression of the main pro-inflammatory markers: interleukin-6 (IL-6), IL-1b, chemokine (C-X-C motif) ligand (1CXCL1), cyclooxygenase-2 (COX-2), CD4, RAR-related orphan receptor C (RORC), chemokine for recruitment of macrophages [monocyte chemotactic protein 1 (MCP1) and membrane receptors which mediate the cell inflammatory response, such as Toll-Like Receptors (TLRs). In addition, immunofluorescence studies were performed to analyze the nuclear translocation of NF-kB, an important transcriptional mediator of the inflammatory response.

Relation between sperm protamine transcripts with global sperm DNA methylation and sperm DNA methyltransferases mRNA in men with severe sperm abnormalities

This study aimed to evaluate the relationship between mRNA expression of DNA methyltransferases (DNMTs) such as DNMT1, DNMT3A and DNMT3B mRNA and sperm global DNA methylation with protamine transcripts in the sperm from men with severe sperm abnormalities. Sperm from each semen sample were isolated using a standard gradient isolation procedure by layering 1 mL of 40% (v/v) density gradient medium over 1 mL of 80% (v/v). A total of 30 oligoasthenoteratozoospermic ejaculates (OAT) and 30 normozoospermic ejaculates as controls were compared using real-time quantitative reverse transcriptase polymerase chain reaction for mRNA expression of DNMT1, 3A, 3B, protamine1 (P1) and protamine2 (P2). The enzyme-linked immunosorbent assay was used to detect global DNA methylation in sperm. A p-value of <0.05 was considered statistically significant. In OAT ejaculates, the increased level of DNMT3A, 3B mRNA, sperm global methylation, P1 plus P2 mRNA and decrease of P1–P2 ratio were significantly different. Also the content of protamine transcript was not correlated with sperm parameters. The increased total protamine transcript levels were associated with increased mRNA methyltransferases. The increase of DNMT1 may lead to an increased level of global methylation.

Seroepidemiology of dengue virus infection among adults during the ending phase of a severe dengue epidemic in southern Taiwan, 2015

BackgroundA severe dengue epidemic occurred in 2015 which resulted in over 22,000 laboratory-confirmed cases. A cross-sectional seroprevalence study was conducted during the ending phase of this epidemic to evaluate the true incidence of dengue virus (DENV) infection and the level of herd immunity.MethodsAdult residents in three administrative districts with high dengue incidence were recruited; workers in two districts with intermediate dengue incidence were also recruited for comparison. DENV-specific IgM and IgG were tested using commercial enzyme-linked immunosorbent assays. DENV RNA was detected using commercial quantitative real-time reverse transcriptase polymerase chain reaction assay. Univariate and multivariate logistic regressions were performed to identify risk factors for recent and past DENV infection.ResultsThe overall seroprevalence of anti-DENV IgM and IgG in 1391 participants was 6.8 and 17.4%, respectively. The risk of recent DENV infection increased with age, with the elderly having the highest risk of infection. Living in areas with high incidence of reported dengue cases and having family members being diagnosed with dengue in 2015 were also independent risk factors for recent DENV infection. One sample was found to have asymptomatic viremia with viral load as high as 105 PFU/ml.ConclusionsComparing the seroprevalence of anti-DENV IgM with the incidence of reported dengue cases in 2015, we estimated that 1 out of 3.7 dengue infections were reported to the surveillance system; widespread use of rapid diagnostic tests might contribute to this high reporting rate. The results also indicate that the overall herd immunity remains low and the current approved Dengvaxia® is not quite suitable for vaccination in Taiwan.

Obestatin/Ghrelin Ratio as a New Activity Index in Ulcerative Colitis

Background and Aim: Ulcerative colitis (UC) is an immune-mediated systemic inflammatory process that destroys the intestinal mucosa. Ghrelin, an appetite-regulatory hormone, has anti-inflammatory effects including a decrease in circulating cytokines. Some reports demonstrated a strong relationship between the serum ghrelin level and the severity of mucosal inflammation in the gastrointestinal tract. The aim is to investigate serum levels and colonic mucosal mRNA expression of ghrelin, obestatin, and obestatin/ghrelin ratio in patients with UC and to determine their potential as markers for UC disease activity. Patients and Methods: seventy-five outpatients with UC and 45 age- and sex-matched healthy volunteers were enrolled in this study after written conscious consent and approval by the Institutional Review Board of Mansoura University. UC was diagnosed by conventional clinical, radiological, endoscopic, and histopathological criteria. Serum ghrelin, obestatin levels, and their mucosal mRNA expression were measured by ELISA kits and a real-time quantitative reverse transcriptase polymerase chain reaction according to the manufacturer’s protocols. Results: Serum levels and mucosal mRNA expression of ghrelin were significantly higher in patients with active UC than patients in remission p˂0.0001). Obestatin/ghrelin ratio was significantly lower in patients with active UC (0.26±0.08) than those in remission (0.523±0.16; p˂0.0001). Obestatin/ghrelin ratio was negatively and significantly correlated with inflammation and endoscopic scores, colitis activity index, serum ghrelin level, and its mucosal mRNA expression (p˂0.05). Conclusion: obestatin/ghrelin ratio might be a reliable surrogate non-invasive marker of disease activity in UC with significantly high sensitivity, specificity, predictive values, and diagnostic accuracy.

IGF2BP1 promotes LPS-induced NFκB activation and pro-inflammatory cytokines production in human macrophages and monocytes

BackgroundLipopolysaccharide (LPS)-induced macrophage/monocyte activation and pro-inflammatory cytokines production are important mediators for periodontitis progression. The current study tested the potential role of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in the process. MethodsTHP-1 human macrophages and primary human peripheral blood mononuclear cells (PBMCs) were treated with LPS. mRNA and protein expression of IGF2BP1 were tested by qPCR and Western blotting assay. IGF2BP1 expression was altered by shRNAs or CRISPR/Cas-9 gene editing methods. LPS-induced cytokine production was tested by ELISA assay. Cytokine mRNA expression was tested by the quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay. ResultsIn THP-1 human macrophages and PBMCs, treatment with LPS induced mRNA and protein expression of IGF2BP1. IGF2BP1 silencing (by targeted shRNAs) or CRISPR/Cas-9 knockout largely inhibited LPS-induced production of multiple pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Conversely, forced over-expression of IGF2BP1 facilitated LPS-induced pro-inflammatory cytokines production in THP-1 cells. For the mechanism study, we show that IGF2BP1 co-immunoprecipitated with p65-p52 nuclear factor kappa B (NFκB) complex in nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced p65-p52 nuclear translocation and NFκB activation were inhibited by IGF2BP1 silencing or CRISPR/Cas-9 knockout. ConclusionIGF2BP1 promotes LPS-induced NFκB signalling and transcriptional activation in human macrophages and monocytes.

The effect of autophagy on the survival and invasive activity of Eimeria tenella sporozoites

Autophagy is a cellular process that is vital for the maintenance of homeostasis in eukaryotic cells. Currently, autophagy-related genes (atgs) in the Eimeria tenella genome database have been reported, but very little is known about the effects of autophagy on the survival and invasive activity of this protozoan. In this study, we investigated the autophagy in E. tenella sporozoites under starvation and autophagy-modulators treatments and evaluated the autophagy influence on cellular adenosine triphosphate (ATP) levels, the survival rate and the invasive activity of the sporozoites. The results showed that the autophagy could be induced in the sporozoites by starvation or inducer rapamycin (RP), but it could be inhibited by 3-methyladenine (3-MA) treatment. The sporozoites after starvation and RP-treatment displayed punctate signals of EtATG8 and formed autophagosomes. The survival rate of the sporozoites under starvation was significantly lower than that in the control group, whereas the ATP levels in sporozoite were far greater than those in the control. The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) showed that the invasive activity of the sporozoites was up- and down-regulated by RP and 3-MA induction, respectively. Our results indicate that autophagy has effects on the survival and invasive activity of E. tenella sporozoites, which may provide new insights into anti-coccidial drugs.

Enhanced Th17 Responses in Patients with Autoimmune Hepatitis.

BACKGROUND T cells are major players in chronic inflammatory diseases such as autoimmune hepatitis (AIH). However, it is not clear which subset of T cells participates in the pathophysiology of the disease. The aim of this study was to assess the expression profile of signature transcription factor and cytokines of T helper 17 (Th17) cells in patients with AIH. METHODS A total of 24 patients with AIH and 24 normal subjects were recruited in the study. Comparison of gene expression patterns between the patients and normal subjects was done by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS The results showed that retinoic acid receptor-related orphan receptors gamma (RORɣt), interleukin-17A (IL-17A), and interleukin-22 (IL-22) mRNA expression were increased greatly in the patients group compared with the normal controls group (p < 0.05). CONCLUSION Deregulated production of Th17-related molecules may be associated with the pathogenesis of AIH.

Validation of reference genes for gene expression studies in tartary buckwheat (Fagopyrum tataricum Gaertn.) using quantitative real-time PCR.

Quantitative real-time reverse transcriptase polymerase chain reaction is a sensitive technique for quantifying gene expression levels. By implementing three distinct algorithms (geNorm, normFinder and BestKeeper), we have validated the stability of the expression of seven candidate reference genes in tartary buckwheat, including FtSAND, FtCACS, FtExpressed1, FtGAPDH, FtActin, FtEF-1a and FtH3. In this study, the results indicated that FtCACS and FtSAND were the best reference genes for ‘abiotic cotyledons’, FtExpressed1 and FtEF-1α were the best reference genes for aluminium treatment, FtCACS and FtExpressed1 performed the best for the immature seed stage, FtCACS was best for the abiotic treatment, and FtH3 appeared to be the most suitable reference gene for the abiotic treatment in hypocotyls and all samples in this study. In contrast, FtActin and FtGAPDH are unsuitable genes. Our findings offer additional stable reference genes for gene expression research on tartary buckwheat at the immature seed stage and under abiotic treatment.

Molecular mechanism of inbreeding effects based on RNA-Seq analysis of the adductor muscle of bay scallop (Argopecten irradians)

Understanding the effects of inbreeding is critical for the long-term viability of shellfish breeding and for understanding inbreeding depression (ID) of the offspring of selfed individuals and full-sib crosses. The bay scallop (Argopecten irradians) is a simultaneous hermaphroditic marine bivalve with distinct male and female gonads and is a species with external fertilization. It is a canonical model invertebrate for analysis of the genetics of ID and evolution of outcrossing mating system. ID in bivalves is well documented with regard to the phenotype and molecular markers aspects, but little is known on how inbreeding affects genome-wide gene expression. Here, we compare the gene expression profiles of adductor muscle of A. irradians lines inbred to F=0.5 with those of its counterpart outbred lines which are originated from the same paternal individual. Inbred and outbred individuals have some differences in growth traits. The shell length and the total weight of inbred scallops are lower than the counterpart of outbred lines. We found that 1 175 genes were up-regulated while 1 390 were downregulated in inbred lines. Regarding components, differentially expressed genes (DEGs) were concentrated on sarcomeres, contractile filaments, and cell membranes. Among others, whereas regarding their function, the Gene Ontology (GO) was concentrated on transferase, phosphotransferase, peptidase, and others. The DEGs-enriched metabolic pathways mainly belonged to the oxidative phosphorylation pathway and apoptosis pathway. We used another two families to verify our bioinformatic results. The selfed and the outbred family originated from different ancestors, but the selfed family showed obvious ID. By using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we verified that DEGs with inbreeding are involved in the oxidative phosphorylation and cell apoptosis pathways, suggesting that inbreeding may affect energy metabolism and homeostasis.

Nigella sativa essential oil and its bioactive compounds as resistance modifiers against Staphylococcus aureus.

Nigella sativa essential oil (EO) and its compounds (thymoquinone, carvacrol, and p-cymene) have a broad antimicrobial spectrum. The aim of this study was to investigate the antimicrobial and resistance modifying activity of N.sativa EO, thymoquinone, carvacrol, and p-cymene against one methicillin susceptible and one methicillin resistant Staphylococcus aureus strain. N.sativa EO, thymoquinone, carvacrol, and p-cymene were assessed for antimicrobial activity and modulation of antimicrobial resistance (by broth microdilution), inhibition of antimicrobial efflux (by ethidium bromide [EtBr] accumulation assay), relative expression of mepA gene (by real-time reverse transcriptase quantitative polymerase chain reaction), membrane disrupting effect (by LIVE/DEAD BacLight™ Kit), and finally antibiofilm activity (by the crystal violet assay). Both strains of S.aureus were susceptible to N.sativa EO, thymoquinone, and carvacrol. N.sativa EO and carvacrol induced the increase of EtBr accumulated by both S.aureus strains. Membrane integrity of ATCC strain was disrupted by carvacrol and p-cymene, whereas for the methicillin resistant S.aureus (MRSA) strain the membrane integrity was disrupted by each compound. N.sativa EO and its bioactive compounds such as carvacrol and p-cymene could be applied as resistance modifiers in MRSA strains.

Custodiol-N™ cardioplegia lowers cerebral inflammation and activation of hypoxia-inducible factor-1α.

Cardioplegic solutions induce cardiac arrest and protect cardiac tissue from ischaemia-reperfusion injury. However, the effects on the brain, which is vulnerable to cardiopulmonary bypass (CPB) surgery and ischaemia-reperfusion injury, mostly remain unknown. We investigated if cardioplegic solutions differ in their effects in altered oxygen conditions and in their ability to induce cerebral inflammation. Thirty pigs were subjected to a midline sternotomy and CPB at 34°C with 90 min cardiac arrest followed by 120 min reperfusion. Following randomization on a 1:1:1 basis, they received either a single shot of histidine-tryptophan-α-ketoglutarate (HTK)-Bretschneider solution (n = 10), histidine-tryptophan-α-ketoglutarate-N (HTK-N; n = 10) or HTK plus 1.2 mg/l cyclosporine A (HTK/CsA; n = 10). Brain regions of interest (frontal cortex, cerebellum, brain stem, diencephalon, colliculus superior) were analysed by real time quantitative reverse transcriptase polymerase chain reaction for hypoxia-inducible factor-1α (HIF-1α), tumour necrosis factor-α, interleukin (IL)-10, IL-1β and IL-1β receptor as well as by immunohistochemical analysis for HIF-1α. Blood gas and electrolyte analyses were performed. Comparisons between baseline and reperfusion period levels revealed that HTK-N cardioplegia induced a smaller reduction of the haemoglobin content and blood calcium concentrations (hbbaseline: 5.97 ± 0.63 mmol/l; hbreperfusion: 6.16 ± 0.66 mmol/l; P = 0.428; Cabaseline2+: 1.36 ± 0.05 mmol/l; Careperfusion2+: 1.28 ± 0.05 mmol/l; P < 0.001) compared to HTK (hbbaseline: 5.93 ± 0.45 mmol/l; hbreperfusion: 4.72 ± 0.79 mmol/l; P = 0.001; Cabaseline2+: 1.34 ± 0.07 mmol/l; Careperfusion2+: 1.24 ± 0.06 mmol/l; P = 0.004) and HTK/CsA cardioplegia (hbbaseline: 5.88 ± 0.44 mmol/l; hbreperfusion: 5.14 ± 0.87 mmol/l; P = 0.040; Cabaseline2+: 1.38 ± 0.04 mmol/l; Careperfusion2+: 1.20 ± 0.14 mmol/l; P = 0.001). Brain region-specific regulation of the HIF-1α expression, no general HIF-1α activation and a lower tumour necrosis factor-α expression (pto HTK = 0.050, pto HTK/CsA = 0.013) were documented for HTK-N cardioplegia. HTK-N (Custodiol-N) induced fewer cerebral effects and less inflammation during CPB surgery than HTK and HTK/CsA cardioplegia. These data suggest that HTK-N exerts brain protective effects during and after CPB surgery.

Neuroinflammation in the dorsolateral prefrontal cortex in elderly chronic schizophrenia

Cognitive deterioration and symptom progression occur in schizophrenia over the course of the disorder. A dysfunction of the immune system/neuroinflammatory pathways has been linked to schizophrenia (SZ). These altered processes in the dorsolateral prefrontal cortex (DLPFC) could contribute to the worsening of the deficits. However, limited studies are available in this brain region in elderly population with long-term treatments. In this study, we explore the possible deregulation of 21 key genes involved in immune homeostasis, including pro- and anti-inflammatory cytokines, cytokine modulators (toll-like receptors, colony-stimulating factors, and members of the complement system) and microglial and astroglial markers in the DLPFC in elderly chronic schizophrenia. We used quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) on extracts from postmortem DLPFC of elderly subjects with chronic SZ (n = 14) compared to healthy control individuals (n = 14). We report that CSF1R, TLR4, IL6, TNFα, TNFRSF1A, IL10, IL10RA, IL10RB, and CD68 were down-regulated in elderly SZ subjects. Moreover, we found that the expression levels of all the altered inflammatory genes in SZ correlated with the microglial marker CD68. However, no associations were found with the astroglial marker GFAP. This study reveals a decrease in the gene expression of cytokines and immune response/inflammation mediators in the DLPFC of elderly subjects with chronic schizophrenia, supporting the idea of a dysfunction of these processes in aged patients and its possible relationship with active microglia abundance. These findings include elements that might contribute to the cognitive decline and symptom progression linked to DLPFC functioning at advanced stages of the disease.

Quantification of bovine viral diarrhoea virus ribonucleic acid in serum of infected animals by one-step reverse transcriptase quantitative real-time polymerase chain reaction

Bovine viral diarrhoea virus (BVDV) can cause either acute transient or persistent infection. Identification and removal of persistently infected animals from infected herds is a crucial component to control BVDV infection. Only limited data on serum virus concentration in infected animals are available to date. Using one-step reverse transcriptase quantitative real-time polymerase chain reaction, we quantified the serum viral load in 40 BVDV infected animals. To control nucleic acid extraction, complementary DNA synthesis and polymerase chain reaction amplification, each serum sample was spiked with a known small amount of reference canine coronavirus. Detected ribonucleic acid copy number ranged from 2.2 × 106to 7.4 × 108per 1 ml of serum of persistently infected animals and from 6.6 × 104to 3.3 × 107of transiently infected animals. These findings support the idea that it is impossible to accurately distinguish between transiently and persistently infected animals just from a single blood sample. To use this testing as a means of declining costs of BVDV control programmes cannot be recommended and paired serum samples have to be investigated to confirm persistent infection.

A study of natural IgG antibodies against ATP-binding cassette subfamily C member 3 in oral squamous cell carcinoma

ATP-binding cassette subfamily C member 3 (ABCC3) is involved in multidrug resistance and is overexpressed in some solid tumors. Recent work revealed an increase in circulating anti-ABCC3 antibodies in lung and esophageal cancers. This in vitro study was undertaken to investigate the effects of the natural IgG antibody against the ABCC3-derived peptide antigen on proliferation of oral squamous cell carcinoma (OSCC) cells and augment the development of efficient and effective treatments in patients with OSCC. An in-house enzyme-linked immunosorbent assay was applied to detect anti-ABCC3 IgG antibody in human plasma. Two OSCC cell lines, CAL27 and SCC15, were cultured with 20% plasma either positive or negative for anti-ABCC3 IgG. Cell proliferation was quantified by the CCK-8 method, and cell apoptosis and cell cycle distribution were analyzed by flow cytometry. The expression of the ABCC3 gene in the cell lines was analyzed by reverse transcriptase quantitative real-time polymerase chain reaction. The results showed that plasma anti-ABCC3 IgG significantly inhibited the proliferation of CAL27 cells but not SCC15 cells, although ABCC3 was expressed in both cell lines. The proportion of apoptotic cells was significantly higher in CAL27 cells treated with anti-ABCC3 IgG-positive plasma than in those treated with IgG-negative plasma. Cell cycle progression was arrested in CAL27 cells treated with anti-ABCC3 IgG-positive plasma. Our data suggest that human plasma anti-ABCC3 IgG may be a promising agent in anti-OSCC therapy, although further studies are needed to arrive at a definitive conclusion.

Lactobacillus rhamnosus biosurfactant inhibits biofilm formation and gene expression of caries-inducing Streptococcus mutans

It is cleared that some probiotic strains inhibit biofilm formation of oral bacteria, but its mechanisms are not clearly understood yet. It is proposed that one of the mechanisms can be biosurfactant production, a structurally diverse group of surface-active compounds synthesized by microorganisms. Hence, this study focused on the evaluation of the anti-biofilm and antiadhesive activities of the L. rhamnosus derived-biosurfactant against Streptococcus mutans and its effect on gtfB/C and ftf genes expression level. In this in vitro study Lactobacillus rhamnosus ATCC7469 overnight culture was used for biosurfactant production. The biosurfactant effect on the surface tension reduction was confirmed by drop collapse method. Chemical bonds in the biosurfactant were identified by Fourier transform infrared (FTIR). Anti-biofilm and antiadhesive activities of the biosurfactant were determined on glass slides and in 96-well culture plates, respectively. The effect of the biosurfactant on gtfB/C and ftf genes expression level was also investigated after biofilm formation, total RNA extraction, and reverse transcription by quantitative real-time reverse transcriptase polymerase chain reaction (PCR) assay (quantitative PCR). The data were assessed by one-way analysis of variance in the Tukey-Kramer postdeviation test for all pairs. P < 0.05 was considered statistically significant. The FTIR results of biosurfactant showed that it was protein rich. It also showed anti-biofilm formation activity on the glass slide and antiadhesive activity till 40% on microtiter plate wells. It also showed a significant reduction (P < 0.05) in gtfB/C and ftf genes expression level. L. rhamnosus-derived biosurfactant exhibits a significant inhibitory effect on biofilm formation ability of S. mutans due to downregulation of biofilm formation associated genes, gtfB/C and ftf. L. rhamnosus-derived biosurfactant with substantial antiadhesive activity is suitable candidates for use in new generations of microbial antiadhesive agents.

Gene Expression Profiling of Human Hepatocytes Grown on Differing Substrate Stiffness

Objective: To study the effects of different substrate stiffness on human hepatocytes using RNA sequencing (RNA-Seq) technology. The stiffness was corresponding to physiology and pathology stiffness of liver tissues. <br/> Results: With the aid of RNA-Seq technology, our study characterizes the transcriptome of hepatocytes cultured on soft, moderate, stiff and plastic substrates. Compared to soft substrate, our RNA-Seq results revealed 1131 genes that were up-regulated and 2534 that were down-regulated on moderate substrate, 1370 genes that were up-regulated and 2677 down-regulated genes on stiff substrate. Functional enrichment analysis indicated that differentially expressed genes were associated with the regulation of actin cytoskeleton, focal adhesion, tight junction, adherens junction as well as antigen processing and presentation. RNA-Seq results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction. <br/> Conclusion: Our study provides a comprehensive picture of the gene expression landscape in hepatocytes grown on different substrate stiffness, offering insights into the role of substrate stiffness in hepatic pathology.

Cytotoxic Effects of Nonionic Iodinated Contrast Agent on Human Adipose-Derived Mesenchymal Stem Cells.

Transplantation of mesenchymal stem cells (MSCs) is a promising therapy for degenerative spine conditions. However, cell therapy for painful spine degeneration presently requires use of contrast agents during fluoroscopy-guided injections, and the effects of these agents on MSCs represents a gap in knowledge. To investigate the biological effects of contrast media (CM) that are coinjected with MSCs. Prospective observational study. Academic medical center. Patient-derived clinical-grade culture expanded MSCs. Iohexol (Omnipaque300) was reduced to 12.5%, 25%, 50%, and 100% of the stock solution and incubated with MSCs for 30 minutes, 4 hours, and 48 hours. We also used complete media and 12.5%, 25%, 50%, 100% of phosphate-buffered saline as a control group. We examined cytotoxicity of iohexol at different concentrations and exposure duration, as well as the potential for recovery over time. Cell counts, mitochondrial activity, and quantitative real time reverse-transcriptase polymerase chain reaction of related genes were analyzed immediately after exposure (day 0) and after 2 days of exposure (day 2). Human MSCs exhibit a time- and concentration-dependent cytotoxic response to iodinated CM. A brief, 30-minute exposure did not affect MSCs function and viability. However, extended treatment with iohexol for 4 hours at 50% or higher concentration had a significant impact on both viability and gene expression in MSCs. CM (Omnipaque300) is cytotoxic to MSCs in a time-and concentration-dependent manner. Hence, the concentration of CM that accompanies MSC injections should be carefully considered during MSC therapy for disk-degenerative diseases. To be determined.

Quercetin inhibits swarming motility and activates biofilm production of Proteus mirabilis possibly by interacting with central regulators, metabolic status or active pump proteins

BackgroundVia its virulence factors such as swarm differentiation, biofilm and hemolysin production, urease enzyme, Proteus mirabilis causes urinary tract infections (UTIs), especially in complicated cases. Anti-pathogenic compounds attenuate the virulence of bacteria without showing ‘cidal’ activity and carry the potential to be used in the prevention and treatment of infectious diseases. PurposeSearch for anti-pathogenic effects of quercetin, which is a widely known and biologically active phytochemical, on Proteus mirabilis was the purpose of this study. In this context, the potential inhibitory activity of quercetin on swarming motility and biofilm production of a wild-type strain, P. mirabilis HI4320, was investigated in both phenotypically and genotypically. MethodsQuercetin's effect on swarming motility was examined on LB agar plates, containing quercetin at various concentrations, by measuring the swarming diameter. The effect on biofilm formation, on the other hand, was analyzed by staining the formed biofilm of the bacterium, exposed to quercetin at various concentrations, with crystal violet and reading spectrophotometrically. Differences in expression levels of selected genes involved in swarming regulation were determined by real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to evaluate the mechanism of inhibitory action on swarming. Further investigations were carried out repeating swarming assays with the clones that derived from the wild-type strain by a TA system kit for direct one-step cloning and overexpressing the relevant genes. ResultsOur study revealed that quercetin inhibited swarming motility while activating biofilm production of P. mirabilis in direct proportion to the dose. Although all selected genes are inhibited in the same manner in liquid medium, and no significant differences could be detected in solid medium as demonstrated by RT-qPCR, experiments repeated with the clones overexpressing flhC (a component of flagellar transcriptional activator), speB (an agmatinase enzyme) and ompF (an outer membrane porin) genes showed that the respective clones could restore swarming, compensating for the inhibitory effect of quercetin. ConclusionQuercetin's inhibitory effect on P. mirabilis swarming was possibly due to interactions with components of swarming regulators, the genes expressing polyamine coding enzymes that trigger swarm differentiation, or active pump proteins.