Laboratory validation of an RNA/DNA hybrid tagmentation based mNGS workflow on SARS-CoV-2 and other respiratory RNA viruses detection

BackgroundAcute respiratory infection (ARI) caused by RNA viruses is still one of the main diseases all over the world such as SARS-CoV-2 and Influenza A virus. mNGS was a powerful tool for ethological diagnosis. But there were some challenges during mNGS implementation in clinical settings such as time-consuming manipulation and lack of comprehensive analytical validation. MethodsWe set up CATCH that was a mNGS method based on RNA and DNA hybrid tagmentation via Tn5 transposon. Seven respiratory RNA viruses and three subtypes of Influenza A virus had been used to test CATCHs capabilities of detection and semiquantification. Analytical performance of SARS-CoV-2 and Influenza A virus had been determined with reference standards. We compared accuracy of CATCH with quantitative real time PCR by using clinical 98 samples from 64 COVID-19 patients. ResultsWe minimized the library preparation process to 3 hours and handling time to 35 minutes. Duplicate filtered RPM of 7 respiratory viruses and 3 Influenza A virus subtypes were highly correlated with viral concentration (r=0.60, p<0.001, Spearman correlation test). LOD of SARS-CoV-2 was 39.2 copies/test and of Influenza A virus was 278.1 copies/mL Comparing with qR-TPCR, the overall accuracy of CATCH was 91.4%. Sensitivity was 84.5% and specificity was 100%. Meanwhile, there were significant difference of microbial profile in oropharyngeal swabs among critical, moderate patients and healthy controls (p<0.001, PERMANOVA test). ConclusionAlthough further optimization is needed before CATCH can be rolled out as a routine diagnostic test, we highlight the potential impact of it advancing molecular diagnostics for respiratory pathogens.

Neutralizing Antibodies Responses to SARS-CoV-2 in COVID-19 Inpatients and Convalescent Patients

BackgroundCOVID-19 is a pandemic with no specific antiviral treatments or vaccines. The urgent needs for exploring the neutralizing antibodies from patients with different clinical characteristics are emerging. MethodsA total of 117 blood samples were collected from 70 COVID-19 inpatients and convalescent patients. The presence of neutralizing antibody was determined with a modified cytopathogenic assay based on live SARS-CoV-2. The dynamics of neutralizing antibody levels at different with different clinical characteristics were analyzed. ResultsThe seropositivity rate reached up to 100.0% within 20 days since onset, and remained 100.0% till day 41-53. The total GMT was 1:163.7 (95% CI, 128.5 to 208.6), and the antibody level was highest during day 31-40 since onset, and then decreased slightly. Individual differences in changes of antibody levels were observed among 8 representative convalescent patients. In multivariate GEE analysis, patients at age of 31-60 and 61-84 had a higher antibody level than those at age of 16-30 ({beta}=1.0518, P=0.0152; {beta}=1.3718, P=0.0020). Patients with a worse clinical classification had a higher antibody titer ({beta}=0.4639, P=0.0227). ConclusionsThe neutralizing antibodies were detected even at the early stage of disease, and a significant response showed in convalescent patients. Moreover, changes on antibody levels ware individual specific.

Serological diagnostic kit of SARS-CoV-2 antibodies using CHO-expressed full-length SARS-CoV-2 S1 proteins

WHO has declared COVID-19 a pandemic with more than 300,000 confirmed cases and more than 14,000 deaths. There is urgent need for accurate and rapid diagnostic kits. Here we report the development and validation of a COVID-19/SARS-CoV-2 S1 serology ELISA kit for the detection of total anti-virus antibody (IgG+IgM) titers in sera from either the general population or patients suspected to be infected. For indirect ELISA, CHO-expressed recombinant full length SARS-CoV-2-S1 protein with 6* His tag was used as the coating antigen to capture the SARS-CoV-2-S1 antibodies specifically. The specificity of the ELISA kit was determined to be 97.5%, as examined against total 412 normal human sera including 257 samples collected prior to the outbreak and 155 collected during the outbreak. The sensitivity of the ELISA kit was determined to be 97.5% by testing against 69 samples from hospitalized and/or recovered COVID-19 patients. The overall accuracy rate reached 97.3%. Most importantly, in one case study, the ELISA test kit was able to identify an infected person who had previously been quarantined for 14 days after coming into contact with a confirmed COVID-19 patient, and discharged after testing negative twice by nucleic acid test. With the assays developed here, we can screen millions of medical staffs in the hospitals and people in residential complex, schools, public transportations, and business parks in the epidemic centers of the outbreaks to fish out the "innocent viral spreaders", and help to stop the further spreading of the virus.

Effect of overexpression of apoptosis-stimulating protein 2 of p53 on activation and apoptosis of hepatic stellate cells induced by transforming growth factor-β1 and its mechanism / 中华肝脏病杂志

Objective@#To study the effect of apoptosis-stimulating protein 2 of p53 (ASPP2) on the activation and apoptosis of hepatic stellate cells induced by transforming growth factor-β1 (TGF - β1), and to explore the role of autophagy in this process.@*Methods@#Mouse hepatic stellate cells were primarily isolated and cultured with green fluorescent protein (GFP) expressing empty vector adenovirus (Ad-GFP) and ASPP2 expressing adenovirus (Ad-ASPP2) for 12 h by transfection kit, and then treated with TGF-β1 (10ng/ml) for 24 h. The experiments were grouped as follows: control group: green fluorescent protein (GFP) expressing empty vector adeno (Ad-GFP); experimental group 1: transfected with Ad-GFP and added with TGF-β1; experimental group 2: transfected with Ad-ASPP2 and induced by TGF-β1. Western blot and quantitative fluorescence PCR were used to detect the expression of ASPP2, α-smooth muscle actin (SMA). At the same time, autophagy was determined by microtubule-associated protein 1 light chain 3-β (LC3). Autophagy and apoptosis of MHSc were observed by immunocytochemistry and RNA interference (RNAi). Multiple pairwise-comparisons between the mean of groups was performed by one-way ANOVA.@*Results@#The relative expression of α-SMA mRNA in mHSC of TGF-β1 + Ad-GFP group (16.83 ± 2.41) was significantly higher than Ad-GFP group (3.62 ± 0.56) (P < 0.05), while the relative expression of α-SMA mRNA (4.22 ± 0.48) in TGF-β1 + Ad-GFP group was significantly lower than TGF-β1 + Ad-GFP group (P < 0.05). The expression of α-SMA protein in each group was consistent with mRNA expression. The proportion of mHSC autophagy in TGF-β1 + Ad-GFP group (80%) was significantly higher than Ad-GFP group (35%); however, there was no statistically significant difference between the two groups. The proportion of mHSC autophagy in TGF-β1 + Ad-ASPP2 group was 42%, which was significantly lower than TGF-β1 + Ad-GFP group, but the apoptotic rate was significantly increased. Cells were simultaneously treated with autophagy inhibitors 3-MA and TGF-β1. The level of autophagy was not statistically significantly different from that of TGF-β1 + Ad-ASPP2 group, but the apoptotic rate was increased. In addition, the RNAi group added with ASPP2 had increased autophagy (LC3-II/LC3-I) than control RNAi group, and the rate of apoptosis was significantly decreased.@*Conclusion@#Overexpression of ASPP2 can alleviate the activation of mHSC and promote the apoptosis of HSC by inhibiting autophagy, so as to alleviate liver fibrosis.

Research and practice on strengthening the integration of pathophysiology teaching and national medical licensing examination / 中华医学教育探索杂志

The National Medical Licensing Examination has become one of the most important indicator s to measure the teaching quality of medical colleges and universities. In this paper, by analyzing the status of pathophysiology in National Medical Licensing Examination and the current problems existing in pathophysiology teaching, the author proposed a scheme of reform in the pathophysiology teaching based on Medical Licensing Examination, including changing teaching idea, optimizing teaching content, reforming teaching means and adjusting the assessment methods . This reform aims to make the pathophysiology teaching really serve the needs of clinical application.

Analysis of Lamivudine-resistant variants mutation patterns in patients with chronic hepatitis disease / 中华实验和临床病毒学杂志

Objective@#To investigate the genetic characteristics of Lamivudine-resistant mutation patterns and HBV S gene mutants in patients with chronic hepatitis disease of different disease progression.@*Methods@#Blood samples of LAM-resistant patients with chronic hepatitis disease were collected. HBV RT gene nucleotide sequences were obtained, and then differences in drug-resistant mutation patterns, drug susceptibility and HBV S gene mutants characteristics between the two groups were analyzed.@*Results@#Forty-seven chronic hepatitis B (CHB) patients and 16 HBV-related liver cirrhosis (LC)/HBV-related hepatocellular carcinoma (HCC) patients were included in this study. M204I single point mutation and L180M+ M204I/V were the most common pattern during patients with chronic hepatitis disease (35/63, 55.56%). The numbers of resistant to three nucleos(t)ide analogues in LC/HCC group was higher than CHB group’s (62.50% vs 34.04%, P=0.046). In HBV S gene, more immune associated HBsAg-escape mutations were detected in LC/HCC group than that in CHB group (62.50% vs 31.91%, P=0.031). I126T/V and G145A (for LCC/HCC group, 60%), I126S/T and S117T (for CHB group, 46.67%) were showed as the most common form for HBsAg escape mutations in the two groups. The two groups both detected RT mutations concomitantly with stop codon mutations in S gene (rtA181T/sW172* and rtM204I/sW196*).@*Conclusions@#Different characteristics in Lamivudine-resistant mutations and associated HBV S gene mutants were found in patients with chronic hepatitis disease of different disease progression, and LC/HCC patients exhibit more multi-drug resistant variants and immune associated HBsAg-escape mutants than CHB patients.

Inhibitory effect of siRNA-mediated MSK1-silencing on the proliferation of CNE2 cells and its mechanisms / 医学研究生学报

Objective Abnormal activation of mitogen-and stress-activated kinase (MSK1) plays an important role in the development of various cancers.This study was to explore the effect of small interfering RNA (siRNA)-mediated MSK1-silencing on the proliferation of human nasopharyngeal carcinoma (NPC) cells and its underlying mechanism.Methods The siRNA vector targeting MSK1 was constructed and transfected into CNE2 cells, and the NPC cell line stably expressing MSK1 was established.Then the cells were divided into a blank control (without transfection of the plasmid), a negative control (with stable transfection of the negative control plasmid), and an experimental group (with stable transfection of the positive recombinant plasmid).The expressions of MSK1 mRNA and protein were detected by real-time quantitative PCR and Western blot, respectively, the proliferation of the cells determined by CCK-8 and colony formation assays, the cell cycles analyzed by flow cytometry, the level of histone H3 phosphorylation at Ser10 examined by Western blot, and The transcriptional activity and expression of the c-jun protein measured by dual-luciferase reporter gene assay and Western blot.Results Compared with the blank control, the inhibition rates of cell proliferation at 48, 72 and 96 hours were significantly reduced in the experimental group (P<0.05), and so were the colony formation ability of the cells (P<0.01) and the expression and transcriptional activity of the c-jun protein (P<0.05).In comparison with the negative control, the experimental group showed significant decreases in the rate of cell growth after 24 hours, the inhibition rates of cell proliferation at 48, 72 and 96 hours (P<0.05), the number of formed colonies ([221.00±20.08] vs [99.67±15.57] / 300 cells, P<0.01), the proportion of S-phase cells (P<0.01), and the expression of the c-jun protein in the CNE2 cells ([100.00±0.00] vs [48.77±10.71] %, P<0.05), but a remarkable increase in the percentage of G0/G1-phase cells (P<0.01).Furthermore, histone H3 phosphorylation at Ser10 was markedly reduced (P<0.01) but no significant change was observed in the expression of the total c-jun protein in the experimental group.Conclusion Knockdown of MSK1 using siRNA can significantly inhibit the growth and proliferation of CNE2 cells, which may be closely related to the decreased phosphorylation of histone H3 and subsequently down-regulated transcriptional activity of c-jun.

The effect of radiofrequency ablation with sublethal temperature on the stem cells of hepatocellular carcinoma / 介入放射学杂志

Objective To study the effect of radiofrequency ablation (RFA) with sublethal temperature on the production of liver cancer stem cells (LCSCs) and on the expression of LCSCs-related transcriptional factors.Methods Mouse hepl-6 hepatoma cell line and clinical samples of patients with hepatocellular carcinoma (HCC) were used to test the expressions of LCSCs-related markers and transcriptional factors.Results Different temperatures were used to stimulate Hep1-6 cells,and it was proved that the temperature of 45℃ was a sublethal temperature that could not induce cell death.Flow cytometry testing showed that treatment with 45℃ could obviously increase CD13+,CD44+,CD90 and CD133+ Hep1-6 cells,suggesting that treatment with 45℃ could increase the production of above mentioned types of LCSCs in hep1-6 cells.Real-time quantitative polymerase chain reaction (RT-qPCR) assay indicated that the temperature of 45℃could cause significant increase in CD13,CD90 and CD133 mRNA.In all 5 HCC patients,CD13 mRNA in the recurrent HCC lesions was remarkably increased,CD133 mRNA was increased in 4 patients with recurrent HCC,and CD90 mRNA was increased in only one patient with recurrent HCC.Flow cytometry testing revealed that CD13+ LCSCs were strikingly increased in 4 recurrent HCC patients,while CD133+LCSC was increased in only one patient,suggesting that more close correlation existed between the increase of CD13+ LCSCs and the temperature of 45℃.RT-qPCR assay showed that in 4 recurrent HCC patients with increased CD13+ LCSC,the Sox2 and Stat2 among 13 LCSCs-related transcriptional factors were obviously increased.Flow cytometry testing showed that 45℃ treatment also increased the expression of Sox2 and Stat1 mRNA in Hep1-6 cells.Finally,Sox2 and Stat1 could be knockdown by siRNAs,indicating that both Sox2 and Stat1 transcriptional factors were involved in 45℃-induced production of CD13+ LCSCs in Hep1-6 cells.Conclusion In RFA therapy,the use of sublethal temperature of 45℃ can increase CD13+LCSCs,which is related to the promotion of Sox2 and Stat1 expression.The results of this study can be used for reference in the research of liver cancer recurrence.

Endoplasmic reticulum stress collaborates with lipopolysaccharide to promote the inflammatory response in macrophages / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the protective mechanism of endoplasmic reticulum stress (ERS) inhibition against inflammation-induced acute liver injury using a mouse model.</p><p><b>METHODS</b>Marrow-derived stem cells were isolated from mouse femur and used to derive macrophages for analysis in experimental inflammation conditions, established by exposure to LPS and consequent activation of TLR4. Tunicamycin, an ERS chemical inducer, was applied to interfere the inflammation model condition.Affect on the inflammation-related factor MAPK was detected by western blot, and affects on gene expression of inflammatory factors were measured by real-time quantitative PCR. Affect on TNFa was also measured by ELISA.</p><p><b>RESULTS</b>Expression of TNFa, IL-6 and IL-1b was induced upon exposure to LPS, with the peak levels being reached at 4 hours of exposure (TNFa, 0.82+/-0.24; IL-1 b, 2.20+/-0.69; IL-6, 0.330+/-0.150). Tunicamycin significantly enhanced the LPS-induced up-regulation of TNFa, IL-6 and IL-1b expression (TNFa, 1.44+/-0.38, t=2.8, P<0.05; IL-1b, 16.063.40, t =7.93, P<0.05; IL-6, 31.1610.60, t=5.08, P<0.05). The tuniamycin treatment also enhanced the LPS-induced up-regulation of the protein expression of phospo-p38, phospho-JNK and phoshpo-ERK.</p><p><b>CONCLUSION</b>ERS collaborates with LPS to promote the TLR4-mediated inflammatory response of macrophages, and this collaboration may be a pathogenic mechanism underlying progressive development of acute liver injury.</p>

Relationship between systemic lupus erythematosus and autoimmune thyroid disease / 中华全科医师杂志

Objective To evaluate the incidence of autoimmune thyroid disease (AITD) in systemic lupus erythematosus (SLE) and examine the correlation between AITD and SLE activity.Methods The study group included 220 SLE patients with the screening of thyroid function (FT3,FT4,TSH) and antithyroid autoantibodies (TgAb,TPOAb) were hospitalized into Affiliated Hospital,Jining Medical College between July 2009 and October 2013.The control group included 160 healthy subjects.We compared the prevalence of AITD between SLE patients and normal controls and also the positive rate of anti-thyroid autoantibodies was observed.We also compared the positive rate of anti-thyroid autoantibodies between AITD in SLE and simple SLE group and also analyzed the correlation between two groups of patients and SLE activity (evaluated by the titer of anti-dsDNA,C3,C4,CH50,SLEDAI score).Results Among them,45 patients suffered from AITD (20.5％).There were hyperthyroidism (n =6,13.3％) and hypothyroidism (including subclinical hypothyroidism) (n =26,57.8％),Hashimoto' s thyroiditis (n =13,28.9％).And 74 SLE cases were positive for anti-thyroid autoantibodies.The prevalence of AITD and the positive rate of anti-thyroid autoantibodies in SLE patients (20.5％,33.6％) were higher than that in normal controls (3.13％,7.50％)(P ＜ 0.05).The positive rate of anti-thyroid autoantibodies of SLE with AITD patients (62.2％) was higher than that in simple SLE (21.5％).No significant differences existed in anti-dsDNA titre,C3,C4,CH50 and SLEDAI score between two groups (P ＞ 0.05).Conclusions The SLE patients have a great prevalence of AITD and a positive rate of anti-thyroid autoantibodies.Those with anti-thyroid autoantibodies have a higher incidence of AITD and it has nothing to do with SLE activity.It is essential to monitor thyroid function and thyroid autoantibodies during the follow-ups.

Effects of Sophora flavescens on rat model of chronic Pseudomonas aeruginosa biofilm pneumonia / 中国中药杂志

<p><b>OBJECTIVE</b>To study the therapeutical effects of the Chinese medicinal herb, Sophora flavescens (SFA) on a rat model of chronic Pseudomonas aeruginosa (PA) biofilm pneumonia.</p><p><b>METHOD</b>Rats were challenged intratracheally with alginate embedded PA strain PAO579 at the concentration of 1 x 10(9) colony-forming units per milliliter (CFU x mL(-1)). After challeng on the second day, three different doses SFA or sterile normal saline (NS) were administered by gastric intubation once a day for two weeks. Two weeks post intratracheal challenge with P. aeruginosa, parameters were evaluated.</p><p><b>RESULT</b>Two weeks after challenge, a remarkable serum antibody response and significant infiltration of numerous polymorphonuclear leukocytes (PMN) with lower IFN-gamma production in the lungs were found in the model group. However, milder macroscopic and lower incidence of lung abscesses were found in all the three groups received different doses of SFA treatment compared to the model group (P < 0.001). Meanwhile, the microscopic lung pathology in all SFA-treated groups were characterized by chronic inflammation dominated by mononuclear leukocytes (MN). The rat number with acute inflammation in group II, III was significantly lower than that in the model group (P < 0.05). Furthermore, the serum level of anti-PA IgG was down-regulated in group II and III (P < 0.05 or P < 0.001), and serum IgG level was negatively correlated with the SFA doses (r = -0.95, P < 0.01). In all the SFA-treated groups higher IFN-gamma production in the lung was found compared to the model group (P < 0.001), and the lung IFN-gamma level was positively correlated with the SFA doses (r = 0.9, P < 0.02). These findings indicate that SFA has an effect on inducing Thl type of immune response. The anti-PA activity test of SFA was weakly positive whereas NS was negative.</p><p><b>CONCLUSION</b>SFA treatment significantly reduced pathology, which might be associated with a shift of local immune responding type from a Th2 like to Thl like that might provide a better protection to the rats with chronic P. aeruginosa lung infection. And these results also showed that the SFA dose of 12 g x kg(-1) was the best dosage in this present study.</p>

The reflection of the problems on teaching exploratory experiment of experimental physiology sciences / 中华医学教育探索杂志

According to teaching practice,the authors discuss the problems on teaching exploratory experiment of experimental physiology sciences and propose some reflection and suggestions.