Objective To investigate the application of orthogonal design to optimize real-time quantitative PCR conditions and perform it for ICAM-1.Methods After model of a skin injury was established in rats,total RNA was isolated from the injured skin tissue and reverse transcriptase reactions were carried out.Using orthogonal design real-time quantitative PCR conditions,the expression level of ICAM-1 gene was studied by use of cDNA as template.The standard of score was set up to amplification and dissociation curves and statistical analysis was done based on the optimized conditions.Results Through 25 tests the best real-time quantitative PCR conditions for ICAM-1 are anneal temperature at 61 ℃,0.4?g of cDNA and concentration of 150?mol/L each primer in the final reaction.There are no interaction among above factors.Conclusion Orthogonal design is a feasible,quick and economical method to optimize real-time quantitative PCR conditions.

BACKGROUND:Previous studies have shown that calcitonin gene-related peptide can be released from cardiac sensory afferent terminals fol owing coronary artery occlusion in rats, indicating the involvement of calcitonin gene-related peptide during pathological process of acute myocardial ischemia.
OBJECTIVE:To investigate the effect of calcitonin gene-related peptide on neonatal rat cardiomyocytes apoptosis induced by oxidized low-density lipoprotein and hypoxia/reoxygenation.
METHODS:Twenty wel s of primary cultured neonatal rat myocardial cel s were randomly divided into five groups:normal control group, oxidized low-density lipoprotein group, oxidized low-density lipoprotein hypoxia/reoxygenation group, calcitonin gene-related peptide group and calcitonin gene-related peptide 8-37 group. The cel s in the last four groups were incubated with oxidized low-density lipoprotein for 24 hours before establishing the myocardial hypoxia/reoxygenation model. At 30 minutes prior to hypoxia/reoxygenation, 10-8 mol/L calcitonin gene-related peptide were added into the culture fluid in calcitonin gene-related peptide group;10-7 mol/L competitive antagonist calcitonin gene-related peptide 8-37 of calcitonin gene-related peptide-1 receptor were added at 30 minutes before calcitonin gene-related peptide administration in calcitonin gene-related peptide 8-37 group. Myocardial apoptotic rate and caspase-3 activity were detected respectively.
RESULTS AND CONCLUSION:Calcitonin gene-related peptide could significantly attenuate apoptosis of neonatal rat myocardial cel s through inhibiting the caspase-3 activation induced by oxidized low-density lipoprotein and hypoxia/reoxygenation. And this effect could be partial y reversed by competitive antagonist calcitonin gene-related peptide 8-37 of calcitonin gene-related peptide 1 receptor, indicating that calcitonin gene-related peptide has anti-apoptotic effect in combination with the calcitonin gene-related peptide 1 receptor to inhibit cardiomyocyte apoptosis in neonatal rats induced by oxidized low-density lipoprotein and hypoxia/reoxygenation.

Objective In order to understand which kind of function genes play an important role for es-timating wound age, the variation of difference genes’ mRNA expression were compared after injury. Methods T he mRNA expression levels of seven candidate genes (ICAM-1, NF-κB, MX2, MT1, MT2, sTnI, and Cox6c) were analyzed in contused rat skeletal muscle at different time points using real-time fluorescent quantitative PC R (R T-qPC R ). T he rawC t values were normalized relative to that of RPL32 mRNA , and converted to standard C t values. A t each time point after injury, the standard deviations (SD ) of the standard C t values were calculated by SPSS. Results T he expression trends of the seven genes were all found to be related to wound age, but there were lower variation coefficients and greater reliability of sTnI and Cox6c when compared with other genes. Conclusion T he genes encoding struc-tural proteins or proteins that performbasic functions can be suitable for wound age estimation.

Objective T o investigate the relationship betw een the expression of secreted frizzled-related protein 5 (SFR P5) m RNA and the tim e interval after skeletal m uscle injury in rats by real-tim e PC R . Methods A total of ninety SD rats w ere random ly divided into the contusion groups at different tim es including 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h after contusion, incision groups at different tim es including 4h and 8h after incision and the control group. T he sam ples w ere taken from the contused zone at different tim e points. T he total RNA w as isolated from the sam ples and re-versely transcribed to analyze the expression levels of SFRP5 m RNA . Results C om pared to the control group, the expression of SFRP5 m RNA in contusion groups w ere dow n-regulated w ithin 48 h after con-tusion and reached the low est level at 20 h, and the expression of SFRP5 m RNA gradually increased from 20 h to 48 h after contusion. T he expression of SFRP5 m RNA in the incised groups w ere signifi-cantly low er than that of the contusion groups at 4 h after injury. A t the tim e of 8 h, the expression levels betw een the contusion and incision groups show ed no statistically significant difference. Conclusion It is suggested that SFRP5 m RNA analysis m ay show regular expression and can be a m arker for estim ation of skeletal m uscle injury age.

Objective T o investigate the relation betw een injury tim e and the expression of cytochrom e c oxidase subunitⅥc (COX6C) m R N A in skeletal m uscle of rat after contusion. Methods A total of fifty-four SD rats w ere divided into the control group and the contusion groups (0.5,1,6,12,18, 24, 30, and 36 h after contusion), random ly. T he contusion m odel w as established by free fall drop of gravity ham-m er. A t corresponding tim e point after contusion, the regular histology w as exam ined and expression level of COX6C m R N A w as tested by real-tim e PC R after extraction of total R N A from the tissues. Results T he m ain pathological features of 6 h after injury included edem a and hem orrhage in m yocytes w ith no inflam m atory cells found. A fter 6 hours, the findings included m yocyte degeneration and necro-sis, inflam m atory cells infiltration, and fibrous connective tissue proliferation in the contused zone. T he expression level of COX6C m R N A w as higher than that of the control group w ithin 6 h after contusion. T he expression level w as low er than that of the control group from 6-36 h after contusion. Conclusion T he level of COX6C m R N A expresses in a regular w ay after contusion. It m ay be useful for estim ating w ound age in com bination w ith the results of pathological features.

Objective Take frizzled class receptor 4(FZD4) as an example for exploring whether the transmembrane receptor protein is a suitable marker for wound age estimation. Methods A total of 78 Sprague-Dawley rats were divided randomly into the control group and the contusion groups of 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, and 48h after injury (n=6). Using a drop-ball technique, a contusion was produced in the right lamb of rats. The samples were dissected from injury zones. Then the expression of FZD4 was detected using immunofluorescence, real-time PCR and western blot, respectively. Results FZD4 was located on the membrane of skeletal muscle cells. Compared to the control group, FZD4 mRNA showed a signiifcant increase (over 2-fold) in 8h, 12h, 36h, and 40h after injury by Real-time PCR (P<0.05), and FZD4 protein showed a statistical up-regulation (less than 2-fold) in 8h, 36h, 40h, 44h, and 48h post contusion by western blotting (P<0.05). Conclusion The expression of FZD4 mRNA and protein are both time-dependent during contused skeletal muscle repair. To some degree, FZD4 mRNA was more suitable than corresponding protein for determining wound age.

The modified early warning score (MEWS), as a rapid assessment and early warning scoring tool, has been widely used in patients in China, which can help nurses to identify potentially critical patients early, but not in all clinical fields. Through the retrieval of the relevant literature of the clinical application of the MEWS in Wanfang Medical Database from 2011 to 2018, the SWOT analysis was used in this paper [strength (S), weakness (W), opportunities (O) and threats (T)] methods, to systematically analyze the advantages, problems existence, implementation opportunities and challenges in the clinical application of the MEWS in our country. The purpose of this study was to obtain the best cut-off value of MEWS in different diseases and to formulate standard early-warning intervention measures for MEWS, which may provide reference for clinical workers to carry out relevant research.

Objective To address the challenges of poor performance and lack of interpretability in existing models,the SHAP algorithm is used to develop an interpretable machine learning model that offers a novel approach to wound age estimation,Methods Based on the previous discovery of the expression of 35 wound age healing-related genes in contused skeletal muscle,the woun age estimaton model was constructed using four algorithms,namly,Multilayer Perceptron(MLP),Random Forest(RF),LightGBM(LGBM),and Support Vector Machine(SVM).The SHAP(Shapley Additive Explanation)algorithm was used to rank the importance of genetic features,eliminate redundant attributes,and optimize the model for accurate wound age estimation.the genetic features of the optimal model were analyzed using SHAP's local interpretation capabilities.Results The best results were obtained using model of MLP(area under the curve(AUC)=0.99)The wound ages were classified into four categories:4～12 h,16～24 h,28～36 h,and 40～48 h,using only 15 gene features.According to SHAP analysis,Fam210a was identified as the most relevant gene.Local analysis revealed that high expression of Fam210a contributed to an increase in the predicted probability of 4 h～12 h,while high expression of Rae1 contributed to an increase in the predicted probability of 16 h～24 h.Additionally,low expression of Tbx18 contributed to an increase in the predicted probability of 28 h～36 h,whereas high expression of Tbx18 contributed to an increase in the predicted probability of 40 h～48 h.Conclusions The combined MLP and SHAP model can be used to predict wound age.Using the SHAP interpreter can better understand the degree of contribution of feature genes to the model prediction,and lay the foundation for further in-depth study of wound age estimation.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Humans ; Antiviral Agents/chemistry* ; COVID-19 ; COVID-19 Drug Treatment ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Protease Inhibitors/chemistry* ; SARS-CoV-2/enzymology* ; Viral Nonstructural Proteins