Objective:To establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.Methods:Recombinant plasmid, specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA (sgRNA). Using recombinant plasmid as amplification templates, the optimal reaction conditions and reaction system were screened according to the Ct value, fluorescence intensity, and shape of amplification curve and was evaluated for sensitivity, reproducibility, and specificity. Meanwhile, the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples. Results:A qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established. The detection limit of the assay was 20 copies/mL, and the variation coefficients of in-batch (0.002%~0.767%) and batch to batch repetition (0.016%~0.752%) were less than 1%. Only N_sgRNA recombinant plasmid was detected in the specificity assay. So the method is more highly sensitive, specific and reproducible. The RatiosgRNA/ gRNA of clinical samples HK.3, EG.5.1, JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different ( P<0.05). There is a strong correlation between the median of RatiosgRNA/ gRNA in clinical samples and sewage samples in the same period (correlation coefficient r=1.000, P=0.010). Conclusions:In this study, a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity, stronger specificity and better repeatability, and it can be used to detect SARS-CoV-2 infectivity.

Objective:To establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.Methods:Recombinant plasmid, specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA (sgRNA). Using recombinant plasmid as amplification templates, the optimal reaction conditions and reaction system were screened according to the Ct value, fluorescence intensity, and shape of amplification curve and was evaluated for sensitivity, reproducibility, and specificity. Meanwhile, the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples. Results:A qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established. The detection limit of the assay was 20 copies/mL, and the variation coefficients of in-batch (0.002%~0.767%) and batch to batch repetition (0.016%~0.752%) were less than 1%. Only N_sgRNA recombinant plasmid was detected in the specificity assay. So the method is more highly sensitive, specific and reproducible. The RatiosgRNA/ gRNA of clinical samples HK.3, EG.5.1, JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different ( P<0.05). There is a strong correlation between the median of RatiosgRNA/ gRNA in clinical samples and sewage samples in the same period (correlation coefficient r=1.000, P=0.010). Conclusions:In this study, a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity, stronger specificity and better repeatability, and it can be used to detect SARS-CoV-2 infectivity.

Objective To investigate the effects of endostatin combined with icotinib on chemotherapy sensitivity,tumor markers,and prognosis in patients with advanced non-small cell lung cancer(NSCLC).Methods A total of 88 patients with advanced NSCLC admitted to our hospital from January 2019 to August 2020 were selected and randomly assigned to two groups,with 44 patients in each group.The patients in both groups were treated with pemetrexed+cisplatin(PP)chemotherapy,whereas those in the control group were administered echitinib and those in the observation group was treated with endostatin combined with icotinib.Patients in the two groups were assessed for clinical efficacy,tumor markers[carbohydrate antigen(CA)50,carcinoembryonic antigen(CEA),cytokeratin 19 fragment(CYFRA)21-1],T lymphocyte subsets,microRNA expression levels(miR-34b,miR-204-5p,and miR-158-5p)and tyrosine protein excitation.We also examined janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathways,along with adverse reactions.Results We detected no significant differences between the two groups with respect the total response rate(P>0.05).Following treatment,the levels of serum CA50,CEA,and CYFRA21-1 in the observation group patients were found to be lower than those of patients in the control group(P<0.05).Similarly,the levels of CD3+,CD4+,CD4+/CD8+in observation group patients were higher than those in the control group patients,whereas the levels of CD8+were lower than those in the control patients(P<0.05).Furthermore,the expression levels of miR-34b,miR-204-5p,and miR-158-5p in the observation group patients were higher than those in the control group patients,whereas contrastingly,the levels of J AK2 and STAT3 mRNA expression were lower than those in the control group(P<0.05).In addition,the progression-free survival of the observation group patients was higher than that of control group patients(P<0.05).Conclusion The combination of endostatin and icotinib has clear therapeutic efficacy in patients with advanced NSCLC,which can con-tribute to enhancing their sensitivity to chemotherapy and immune function.We speculate that the underlying mechanisms of action may be associated with a reduction in the activity of the JAK2/STAT3 signaling pathway,which is beneficial for patient prognosis.

Objective:To retrieve, evaluate and integrate the best evidence for cognitive behavioral therapy for improving psychosomatic symptoms in chronic obstructive pulmonary disease (COPD) patients.Methods:According to the "6S" model, evidence related to the cognitive behavioral therapy of COPD patients, including clinical practice guidelines, evidence summaries, expert consensus, systematic reviews and original research, was searched in UpToDate, Joanna Briggs Institute Center for Evidence-Based Health Care database, BMJ Best Practice, Cochrane Library, Guidelines International Network, National Institute for Health and Clinical Excellence, Registered Nurses' Association of Ontario, Medlive, Global Initiative for Chronic Obstructive Lung Disease, PubMed, Embase, Web of Science, China Biology Medicine disc, China National Knowledge Infrastructure, Wanfang database and VIP. The search deadline was from the establishment of the databases to November 21, 2022. Two researchers independently evaluated the quality of the literature and extracted and summarized evidence from the literature that met the quality standards.Results:A total of 13 articles were included, including 2 clinical decision-making articles, 3 guidelines, 1 expert consensus, 3 systematic evaluations, 1 Meta-analysis and 3 randomized controlled trials. Finally, 21 pieces of evidence were formed from three aspects, such as establishing trust relationships, cognitive therapy and behavioral therapy.Conclusions:This study summarizes the best evidence for cognitive behavioral therapy to improve psychosomatic symptoms of COPD patients, in order to provide evidence-based evidence for cognitive behavioral therapy in COPD patients.

OBJECTIVE:To optimize the formulation of ferulic acid ligustrazine (FATM) solid lipid nanoparticles (FATM-SLN),and conduct the quality evaluation. METHODS:Emulsification ultrasonic method was used to prepare FATM-SLN. Using particle size and entrapment efficiency as indexes,amount of glyceryl monostearate,egg yolk lecithin (PC),poloxamer 188 (P188),and sodium stearate as factors,single factor test and orthogonal test were used to optimize the formulation;and verifica-tion test was conducted. The appearance morphology,distribution of particle size,Zeta potential,stability and in vitro release de-gree of prepared FATM-SLN were investigated. RESULTS:The optimal formulation was as follows as FATM of 10 mg,glyceryl monostearate of 300 mg,PC of 200 mg,P188 of 200 mg,sodium stearate of 10 mg,and purified water of 20 mL. The prepared FATM-SLN showed spherical solid particles,appearance morphology was round,distribution of particle size was 40-800 nm,parti-cle size was 106.23 nm,polydispersity index was 0.254,Zeta potential was -34.8 mV,entrapment efficiency was 73.32%,drug loading was 1.20%;the appearance had no obvious changes within 10 d in 4 ℃(RSD<2%). The drug-release in 0.5-1 h was the fastest,the cumulative release degree reached to 60.47%;it tended to be stable after 8 h,the cumulative release degree reached to 93.46%,and drugs were basically released completely. CONCLUSIONS:FATM-SLN formulation is successfully optimized,and the prepared FATM-SLN has small particle size,high entrapment efficiency and good stability.

ObjectiveFrom the changes of expression of cold-inducible RNA-binding protein (CIRP) in rats with traumatic brain injury under mild hypothermia treatment with Shenfu decoction as a subsidiary, to speculate the mechanism of protective effect of the decoction on the injury.Methods Ninety Sprague-Dawley (SD) rats were divided into three groups by random number table: non-transfection control group, adenovirus mediated immune flourescent reverse transcription virus group (blank AD5-GFP transfection group) and adenovirus mediated immune flourescent reverse transcription virus carrying CIRP silent expression gene group (AD5-GFP-CIRP-SiRNA transfection group), 30 rats in each groups. Then, each group was subdivided into three subgroups: model group, traditional Chinese medicine (TCM) low and high dose groups, 10 rats in each subgroup. After the mild hypothermia treatment for 48 hours, in the TCM low dose group and high dose group, a dose of TCM 1 mL/kg and 5 mL/kg was injected via a tail vein into the rat respectively, while in the model group, 1 mL/kg normal saline was injected into the same vein, once a day for consecutive 2 days in all the groups. Before modeling in the blank AD5-GFP transfection group and AD5-GFP-CIRP-SiRNA transfection group, virus transfection models were reproduced at first by one-time intrathecal injection of 0.1 mL AD5-GFP and 0.1 mL (1×1010 pfu/mL) AD5-GFP-CIRP-SiRNA virus vector respectively, and in model group, 0.1 mL normal saline was given. The rat cortex, hippocampus and hypothalamus part were collected, the brain cell apoptosis was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), the CIRP mRNA expression in the cortex, hippocampus and hypothalamus part was measured by reverse transcription-polymerase chain reaction (RT-PCR), the protein expressions of rat sarcoma protein Raf, Ras, extracellular signal-regulated kinase (ERK), phosphorylation ERK (p-ERK), mitogen activated protein kinase (MEK), p-MEK were determined by Western Blot.Results The brain tissue cell apoptosis indexes (AI) in the cortex, hippocampus and hypothalamus part in TCM low, high dose group of non-transfection control and blank AD5-GFP transfection group were lower than those in model group, and the expressions of CIRP mRNA were higher than those in model group, there were no significant differences in AI and CIRP mRNA in the cortex, hippocampus and hypothalamus between model, TCM low and high dose groups of AD5-GFP-CIRP-SiRNA transfection group, but AI was significantly higher and CIRP mRNA was significantly lower than that in corresponding subgroups of AD5-GFP transfection control group and blank AD5-GFP transfection group. Western Blot detection showed that: Raf/Ras, p-MEK/MEK protein expressions revealed no statistical significant differences in different parts of each group (allP > 0.05), the p-ERK/ERK protein expression in the cortex, hippocampus, and hypothalamus part was significantly lower in TCM low and high dose group than that in the model group of non-transfection control group and blank AD5-GFP transfection group, the degree of descent in the TCM high dose group being more significant (the cortex: non-transfection control group was 7.2±1.0 vs. 15.3±1.8, AD5-GFP transfection group was 8.1±0.7 vs. 16.2±1.5; hippocampus part: non-transfection control group was 6.6±0.8 vs. 14.7±2.0, AD5-GFP transfection group was 6.8±1.0 vs. 14.9±1.3; hypothalamus part: non-transfection control group was 9.4±1.1 vs. 12.7±1.7, AD5-GFP transfection group was 10.6±1.3 vs. 9.4±1.1, allP < 0.05). There were no significant statistical differences in p-ERK/ERK protein expression in above brain parts between AD5-GFP-CIRP-SiRNA transfection subgroups (allP > 0.05).Conclusions The Shenfu decoction used in rats with brain trauma under treatment of mild hypothermia is possibly by promoting CIRP over-expression, lowering ERK expression and inhibiting the initiation of signal transduction of the secondary transcription factor phosphorylation, thereby the neural cell apoptosis is decreased and play a subsidiary role of anti-apoptosis of mild hypothermia.

The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5-80 μmol/L) for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π-π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.