qScript One-Step RT-qPCR Kit

Background The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. Methods We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. Findings We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3−/− mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. Interpretation Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease.

Targeted wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proposed by the United States Centers for Disease Control and Prevention's National Wastewater Surveillance System as a complementary approach to clinical surveillance to detect the presence of Coronavirus Disease 2019 (COVID-19) at high-density facilities and institutions such as university campuses, nursing homes, and correctional facilities. In this study we evaluated the efficacy of targeted wastewater surveillance of SARS-CoV-2 RNA together with individual-level testing for outbreak mitigation on a university campus during Fall 2020 semester. Wastewater samples (n = 117) were collected weekly from manholes or sewer cleanouts that receive wastewater inputs from dormitories, community-use buildings, and a COVID-19 isolation dormitory. Quantitative RT-PCR N1 and N2 assays were used to measure SARS-CoV-2 nucleocapsid genes in wastewater. Due to varying human waste input in different buildings, pepper mild mottle virus (PMMV) RNA was also measured in all samples and used to normalize SARS-CoV-2 N1 and N2 RNA wastewater concentrations. In this study, temporal trends of SARS-CoV-2 in wastewater samples mirrored trends in COVID-19 cases detected on campus. Normalizing SARS-CoV-2 RNA concentrations using human fecal indicator, PMMV enhanced the correlation between N1 and N2 gene abundances in wastewater with COVID-19 cases. N1 and N2 genes were significant predictors of COVID-19 cases in dormitories, and the N2 gene was significantly correlated with the number of detected COVID-19 cases in dormitories. By implementing several public health surveillance programs include targeted wastewater surveillance, individual-level testing, contact tracing, and quarantine/isolation facilities, university health administrators could act decisively during an outbreak on campus, resulting in rapid decline of newly detected COVID-19 cases. Wastewater surveillance of SARS-CoV-2 is a proactive outbreak monitoring tool for university campuses seeking to continue higher education practices in person during the COVID-19 pandemic.

Background The emergence of Zika virus (ZIKV) as an important cause of congenital and childhood developmental disorders presents another challenge to global health. Efforts to develop a Zika vaccine have begun although vaccine development against flaviviruses, of which ZIKV belongs to, has proven to be time-consuming and challenging. Defining the vaccine attributes that elicit adaptive immune response necessary for preventing ZIKV infection could provide an evidence-based guide to Zika vaccine development. Methods We used a previously described attenuated ZIKV DN-2 strain in a type-I interferon receptor deficient mouse model and tested the hypothesis that duration of vaccine burden rather than peak level of infection, is a determinant of immunogenicity. We quantified both humoral and cellular responses against ZIKV using plaque reduction neutralisation test and flow cytometry with ELISPOT assays, respectively. Vaccinated mice were challenged with wild-type ZIKV (H/PF/2013 strain) to determine the level of protection against infection. Findings We found that the overall vaccine burden is directly correlated with neutralising antibody titres. Reduced duration of vaccine burden lowered neutralising antibody titres that resulted in subclinical infection, despite unchanged peak vaccine viraemia levels. We also found that sterilising immunity is dependant on both neutralising antibody and CD8+ T cell responses; depletion of CD8+ T cells in vaccinated animals led to wild-type ZIKV infection, especially in the male reproductive tract. Interpretation Our findings indicate that duration of attenuated virus vaccine burden is a determinant of humoral and cellular immunity and also suggest that vaccines that elicit both arms of the adaptive immune response are needed to fully prevent ZIKV transmission. Funding This study was supported by the National Medical Research Council through the Clinician-Scientist Award (Senior Investigator) to E.E.O. Salary support for S.W. was from a Competitive Research Programme grant awarded by the National Research Foundation of Singapore.

Background:The emergence of Zika virus (ZIKV) as an important cause of congenital and childhood develop-mental disorders presents another challenge to global health. Efforts to develop a Zika vaccine have begunalthough vaccine development againstflaviviruses, of which ZIKV belongs to, has proven to be time-consum-ing and challenging. Defining the vaccine attributes that elicit adaptive immune response necessary for pre-venting ZIKV infection could provide an evidence-based guide to Zika vaccine development.Methods:We used a previously described attenuated ZIKV DN-2 strain in a type-I interferon receptor defi-cient mouse model and tested the hypothesis that duration of vaccine burden rather than peak level of infec-tion, is a determinant of immunogenicity. We quantified both humoral and cellular responses against ZIKVusing plaque reduction neutralisation test andflow cytometry with ELISPOT assays, respectively. Vaccinatedmice were challenged with wild-type ZIKV (H/PF/2013 strain) to determine the level of protection againstinfection.Findings:We found that the overall vaccine burden is directly correlated with neutralising antibody titres.Reduced duration of vaccine burden lowered neutralising antibody titres that resulted in subclinical infec-tion, despite unchanged peak vaccine viraemia levels. We also found that sterilising immunity is dependanton both neutralising antibody and CD8+Tcell responses; depletion of CD8+Tcells in vaccinated animals ledto wild-type ZIKV infection, especially in the male reproductive tract.Interpretation:Ourfindings indicate that duration of attenuated virus vaccine burden is a determinant ofhumoral and cellular immunity and also suggest that vaccines that elicit both arms of the adaptive immuneresponse are needed to fully prevent ZIKV transmission.Funding:This study was supported by the National Medical Research Council through the Clinician-ScientistAward (Senior Investigator) to E.E.O. Salary support for S.W. was from a Competitive Research Programmegrant awarded by the National Research Foundation of Singapore.

The molecular basis of dengue virus (DENV) attenuation remains ambiguous and hampers a targetedapproach to derive safe but nonetheless immunogenic live vaccine candidates. Here, we take advantageof DENV serotype 2 PDK53 vaccine strain, which recently and successfully completed a phase-3 clinical trial,to identify how this virus is attenuated compared to its wild-type parent, DENV2 16681. Site-directed muta-genesis on a 16681 infectious clone identifies a single G53D substitution in the non-structural 1 (NS1) proteinthat reduces 16681 infection and dissemination in bothAedes aegypti, as well as in mammalian cells to pro-duce the characteristic phenotypes of PDK53. Mechanistically, NS1 G53D impairs the function of a knownhost factor, the endoplasmic reticulum (ER)-resident ribophorin 1 protein, to properly glycosylate NS1 andthus induce a host antiviral gene through ER stress responses. Our findings provide molecular insights onDENV attenuation on a clinically tested strain.

During skin healing, periostin facilitates myofibroblast differentiation through a β1 integrin/FAK dependent mechanism and continued expression is associated with scarring. In contrast to skin, gingival tissue does not typically scar upon injury, but the role of periostin in gingival healing has never been investigated. Using a rat gingivectomy model, we show that the gingival architecture is re-established within 14 days of wounding. Periostin mRNA levels peak at day 7 post-wounding, with persistence of periostin protein in the connective tissue through day 14. Collagen type I and lysyl oxidase mRNA levels peak at day 7 post wounding, which corresponded with the peak of fibroblast proliferation. Although α-smooth muscle actin mRNA levels increased 200-fold in the tissue, no myofibroblasts were detected in the regenerating tissue. In vitro, human gingival fibroblast adhesion on periostin, but not collagen, was inhibited by blocking β1 integrins. Fibroblasts cultured on periostin exhibited similar rates of proliferation and myofibroblast differentiation to cells cultured on collagen only. However, human gingival fibroblasts cultured in the presence of periostin exhibited significantly increased fibronectin and collagen mRNA levels. Increases in fibronectin production were attenuated by pharmacological inhibition of FAK and JNK signaling in human gingival fibroblasts. In vivo, mRNA levels for fibronectin peaked at day 3 and 7 post wounding, with protein immunoreactivity highest at day 7, suggesting periostin is a modulator of fibronectin production during gingival healing.

Several Zika virus (ZIKV) vaccines designed to elicit protective antibody (Ab) responses are currently under rapid development, but the underlying mechanisms that control the magnitude and quality of the Ab response remain unclear. Here, we investigated the CD4+ T cell response to primary intravenous and intravaginal infection with ZIKV. Using the LysMCre+Ifnar1fl/fl (myeloid type I IFN receptor-deficient) C57BL/6 mouse models, we identified six I-Ab-restricted ZIKV epitopes that stimulated CD4+ T cells with a predominantly cytotoxic Th1 phenotype in mice primed with ZIKV. Intravenous and intravaginal infection with ZIKV effectively induced follicular helper and regulatory CD4+ T cells. Treatment of mice with a CD4+ T cell-depleting Ab reduced the plasma cell, germinal center B cell, and IgG responses to ZIKV without affecting the CD8+ T cell response. CD4+ T cells were required to protect mice from a lethal dose of ZIKV after infection intravaginally, but not intravenously. However, adoptive transfer and peptide immunization experiments showed a role for memory CD4+ T cells in ZIKV clearance in mice challenged intravenously. These results demonstrate that CD4+ T cells are required mainly for the generation of a ZIKV-specific humoral response but not for an efficient CD8+ T cell response. Thus, CD4+ T cells could be important mediators of protection against ZIKV, depending on the infection or vaccination context.

Cervical carcinoma high‑expressed long non‑coding RNA 1 (CCHE1) has been demonstrated to promote several different types of cancer; however, the involvement of CCHE1 in other types of cancer remains unknown. In the present study, the expression levels of CCHE1 and inter‑ leukin (IL)‑17A were increased in the plasma of patients with metastatic and non‑metastatic colon adenocarcinoma (MC and NMC, respectively) compared with the healthy controls. There was no significant difference in the plasma expression levels of CCHE1 and IL‑17A in patients with MC compared with patients with NMC. The plasma expression levels of CCHE1 and IL‑17A were positively associated with the primary tumor diameter. A significant correlation as demonstrated between the serum levels of CCHE1 and IL‑17A in patients with colon adenocarcinoma, but not in the healthy controls. CCHE1 and IL‑17A overexpression promoted colon adenocarcinoma cell proliferation. Transfection of small interfering RNA against IL‑17A partially reversed the effects of CCHE1 overexpres‑ sion on cancer cell proliferation. Upregulation of IL‑17A was observed after CCHE1 overexpression, while IL‑17A overex‑ pression did not significantly change the expression level of CCHE1. Therefore, CCHE1 may promote growth of colon adenocarcinoma through interactions with IL‑17A.

Objective To determine whether vertical sleeve gastrectomy (VSG) attenuates fibrosis in mice on a high-fat high-cholesterol (HFHC) diet. Background Bariatric surgery mitigates non-alcoholic steatohepatitis in 85–90% of obese patients. While animal models demonstrate similar results on a high-fat diet, none have observed the effects of bariatric surgery on a combined HFHC diet. Methods Mice on a HFHC diet were used to confirm the development of hepatic fibrosis at 8 (n = 15) and 24 (n = 15) weeks. A separate cohort of mice on a HFHC diet for 12 weeks was subjected to either VSG (n = 18) or sham (n = 12) operations and remained on a HFHC diet for an additional 20 weeks. Changes in weight, dyslipidemia, and the development of steatosis and fibrosis were documented. Serum was obtained for bile acid analysis by liquid chromatography and mass spectrometry, while hepatic gene expression by RT-PCR was performed to evaluate intrahepatic lipid metabolism. Results Hepatic steatosis and fibrosis developed after 8 weeks on the HFHC diet. After VSG, mice demonstrated a sustained decrease in weight with a significant decrease in fibrosis compared to sham mice. Serum total cholesterol, HDL, and LDL were significantly reduced following surgery, while serum bile acids were significantly elevated. Intra-hepatic cholesterol excretion was not upregulated based on hepatic gene expression of CYP7A1 and ABCG5/8. Conclusions VSG attenuates the development of hepatic fibrosis in diet-induced obese mice, presumably through enhancement of cholesterol elimination at the intestinal level.

NRON mediates the degradation of tat protein to participate in HIV-1 infection. Interestingly, our study observed the down-regulation of NRON in triple-negative breast cancer (TNBC) tissues compared with paired adjacent healthy tissues. In contrast, lncRNA snaR was up-regulated in TNBC tissues and was inversely correlated with NRON. Expression levels of snaR increased, while expression levels of NRON decreased along with the increase of clinical stages. The snaR overexpression resulted in promoted cancer cell proliferation but did not significantly affect NRON expression. NRON overexpression inhibited cancer cell proliferation and down-regulated snaR. The snaR overexpression reduced the effects of NRON overexpression. We therefore conclude that NRON may down-regulate lncRNA snaR to inhibit cancer cell proliferation in TNBC.

Citrus Huanglongbing (HLB) or greening is a devastating disease of citrus worldwide and no effective control measure is currently available. Plant activators represent environment friendly compounds capable of inducing resistance against many plant pathogens. Earlier studies showed that foliar spray of plant defense inducers could slow down HLB disease progress. In this study, eight plant activators and three antibiotics were evaluated in 3 field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that 4 trunk injections of several activators including salicylic acid, oxalic acid, acibenzolar-S-methyl and potassium phosphate provided significant control of HLB by suppressing Las titer and disease progress. Trunk injection of penicillin, streptomycin and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of Las titer and HLB symptom expressions than plant activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of PR-1 and PR-2 genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction of PR-2 or PR-15 gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance (SAR) under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision-making regarding developing an effective HLB management program.