Objective:To explore the short-term outcomes of 3D-printing stand-alone artificial vertebral body(AVB)in the surgical procedure of anterior cervical corpectomy and fusion(ACCF).Methods:Following the proposal of IDEAL(idea,development,exploration,assessment,and long-term follow-up)framework,we designed and conducted this single-armed,retrospective cohort study.The patients with cervical spondylotic myelopathy were recruited,and these patients exclusively received the surgical procedure of single-level ACCF in our single center.After the process of corpectomy,the size was tailored using different trials and the most suitable stand-alone AVB was then implanted.This AVB was manufactured by the fashion of 3D-printing.Two pairs of screws were inserted in an inclined way into the adjacent vertebral bodies,to stabilize the AVB.The participants were regularly followed-up after the operation.Their clinical data were thoroughly reviewed.We assessed the neurological status according to Japanese Orthopedic Association(JOA)scale.We determined the fusion based on imaging examination six months after the operation.The recorded clinical data were analyzed using specific software and they presented in suitable styles.Paired t test was employed in comparison analysis.Results:In total,there were eleven patients being recruited eventually.The patients were all followed up over six months after the operation.The mean age of the cohort was(57.2±10.2)years.The mean operation time was(76.1±23.1)min and the median bleeding volume was 150(100,200)mL.The postoperative course was uneventful for all the cases.Dysphagia,emergent hematoma,and deterioration of neurological func-tion did not occur.Mean JOA scores were 13.2±2.2 before the operation and 16.3±0.8 at the final follow-up,which were significantly different(P＜0.001).The mean recovery rate of neurological func-tion was 85.9％.By comparing the imaging examinations postoperatively and six months after the opera-tion,we found that the average subsidence length was(1.2±1.1)mm,and that there was only one ca-ses(9.1％)of the severe subsidence(＞3 mm).We observed significant improvement of cervical lor-dosis after the operation(P=0.013).All the cases obtained solid fusion.Conclusion:3D-printing stand-alone AVB presented favorable short-term outcome in one-level ACCF in this study.The fusion rate of this zero-profile prosthesis was satisfactory and the complication rate was relatively low.

OBJECTIVE To set up normal ranges for indexes in embryo-fetal development toxicity studies in Sprague-Dawley(SD)rats and to establish a background database to provide reference for the embryo-fetal development toxicity evaluation of drugs.METHODS The data on embryonic develop-ment and fetal growth from embryo-fetal development toxicity studies(11 items)conducted by our center between 2013 and 2022 was statistically analyzed,involving 205 pregnant rats and 3037 fetuses in total,with the mean and standard deviation,coefficient of variation and 95%confidence interval calculated.The indexes included body mass,body mass gain and food consumption during pregnancy,pregnancy outcomes(pregnancy rate,average corpora lutea,average Implant sites,average live conceptuses,live conceptuse rate,resorption rate and dead conceptuse rate),fetal growth and development(fetal mass,placental mass and sex ratio),appearance abnormality rate,visceral abnormality rate,and skeletal abnormality rate.RESULTS The mass of pregnant rats trended up during gestation,with significant increases in the late period.Food consumption increased along with gestation.Caesarean section was conducted on gestation day 20,and the pregnancy rate was 93.2%.The average corpora lutea,Implant sites and live conceptuses were 18.0±3.2,15.9±2.8 and 14.8±3.0,respectively.The live conceptuse rate was 93.4%while the total dead embryo rate was 6.6%.The average mass of fetuses and placenta were respectively 3.6±0.3 and(0.6±0.3)g,and the fetal sex ratio(male/female)was 0.94.The incidence of fetal appearance abnormalities was about 0.2%,and that of soft tissue abnormalities was approximately 0.8%.The rate of skeletal abnormalities was about 1.2%,with higher incidence of non-ossification and incomplete ossification mostly identified on sternum and hyoid bone.The numbers of ossifications of metacarpal bones,metatarsal bones and sacrococcygeal vertebrae were 7.0±0.7,8.0±0.1 and 7.4±0.5,respectively.The rate of ossification of sternumⅠtoⅣwas higher,with an average of about 98.6%-99.9%.The ossification rates of sternum Ⅴ and Ⅵ were(68.0±28.4)%and(82.8±23.9)%.CONCLUSION The background database of indexes in the embryo-fetal development toxicity study on SD rats is established for our GLP laboratory,which provides reference for reproductive toxicity studies.

Lymphocytic choriomeningitis virus/physiology* ; Allosteric Regulation ; Nucleotidyltransferases

Heme/metabolism* ; Cryoelectron Microscopy ; Membrane Transport Proteins ; Escherichia coli Proteins/metabolism*

ObjectiveTo systematically evaluate the safety of Chinese medicines combined with Tripterygium wilfordii polyglycoside tablets/Tripterygium wilfordii tablets （TWPT/TWT） in the treatment of rheumatoid arthritis （RA）， and to explore the network regulatory mechanisms of enhancing efficacy and reducing toxicity of commonly used combination regimes. MethodThe literature involving the adverse reactions of TWPT/TWT in treating RA was searched and collected from three Chinese databases （CNKI， Wanfang Data， VIP） and three English databases （PubMed， Cochrane Library， Embase） from the inception of the databases to July 2021. All studies were assessed by the Cochrane risk of bias tool， and the data were extracted and analyzed by Stata 15.0. Furthermore， Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine 2.0 （TCMIP v2.0，http：//www.tcmip.cn/） was used to construct a "drug target-symptom gene of efficacy and toxicity" interaction network， to explore the underlying network regulatory mechanisms of enhancing efficacy and reducing toxicity of common T. wilfordii preparation combinations. ResultA total of 2 132 articles on Chinese medicines combined with TWPT/TWT in the treatment of RA were retrieved， and 18 of them were finally included. The systematic review showed that the adverse reactions of TWPT/TWT against RA mainly occurred in the digestive system， blood system， and reproductive system， of which digestive system had the highest incidence of damages. However， the combination with Chinese medicines effectively alleviated the adverse reactions caused by TWPT/TWT ［RR （95% CI）=0.45 （0.30， 0.66）， P<0.01］. In addition， the subgroup analysis indicated that the age of RA patients， course of disease， combination regimen， medication dosage and duration of treatment all affected the occurrence of adverse reactions of TWPT/TWT. It was found in clinical studies that total glucosides of paeony （TGP） and TWPT/TWT was most widely combined， and the effect of TGP in reducing TWPT/TWT-induced hepatotoxicity was also more significant than that of other Chinese medicines. Moreover， taking this combination regime as an example， this paper explored the "efficacy-toxicity" association mechanisms of TGP-TWPT/TWT against RA. The "drug target-symptom gene of efficacy and toxicity" interaction network revealed that the core network targets of TGP-TWPT/TWT enhanced efficacy and reduced toxicity mainly through regulating immunity-inflammation-related pathways， metabolic pathways and cell signal transduction. Especially， interleukin-4 （IL-4） and interleukin-13 （IL-13）， which were involved in the "immunity-inflammation" module， were the common targets of TGP-TWPT/TWT to enhance efficacy and reduce toxicity. The endogenous sterols， bile acids and bile salts， insulin secretion and other metabolic pathways in the "body metabolism" module were closely associated with the mechanisms of TWPT/TWT inducing hepatotoxicity and TGP reducing hepatotoxicity. While cell function regulation pathways， such as stem cell factor （SCF）/tyrosine kinase receptor （KIT） signaling pathway and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）signaling pathway were involved in both anti-RA effects and hepatotoxicity of TWPT/TWT. ConclusionClinical application of suitable Chinese medicines combined with TWPT/TWT in the treatment of RA can effectively improve the rheumatism and reduce the adverse reactions of TWPT/TWT， and TGP-TWPT/TWT has the most significant toxicity-reducing effect. Further biological network-based investigation indicates that the toxicity-reducing mechanism of TGP-TWPT/TWT may be related to the regulation of interleukin signaling pathway and bile acid metabolism pathway， and the synergistic efficacy-enhancing effect of the combination may be achieved by acting on interleukin signaling pathway and cell function regulation pathway.

Halomonas can grow on diverse carbon sources. As it can be used for unsterile fermentation under high-salt conditions, it has been applied as a chassis for next-generation industrial biotechnology. Short-chain volatile fatty acids, including acetate, propionate, and butyrate, can be prepared from biomass and are expected to be novel carbon sources for microbial fermentation. Halomonas sp. TD01 and TD08 were subjected to shaking culture with 10-50 g/L butyrate, and they were found to effectively synthesize poly-3-hydroxybutyrate with butyrate as the carbon source. The highest yield of poly-3-hydroxybutyrate was achieved at butyrate concentration of 20 g/L (9.12 g/L and 7.37 g/L, respectively). Butyrate at the concentration > 20 g/L inhibited cell growth, and the yield of poly-3-hydroxybutyrate decreased to < 4 g/L when butyrate concentration was 50 g/L. Moreover, Halomonas sp. TD08 can accumulate the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate by using propionate and butyrate as carbon sources. However, propionate was toxic to cells. To be specific, when 2 g/L propionate and 20 g/L butyrate were simultaneously provided, cell dry weight and polymer titer were 0.83 g/L and 0.15 g/L, respectively. The addition of glycerol significantly improved cell growth and boosted the copolymer titer to 3.95 g/L, with 3-hydroxyvalerate monomer content of 8.76 mol%. Short-chain volatile fatty acids would be promising carbon sources for the production of polyhydroxyalkanoates by Halomonas.
Butyrates ; Carbon ; Fatty Acids, Volatile ; Halomonas ; Polyhydroxyalkanoates ; Propionates

BACKGROUND: Lung cancer is a malignant with high incidence and mortality and adenocarcinoma is among the most popular subtypes. Epidermal growth factor receptor (EGFR) mutation is one of the most important driver mutations for lung adenocarcinoma and EGFR-tyrosine kinase inhibitor (TKI) will benefit those patients with sensitive EGFR mutations. Recently, immune checkpoint inhibitor (ICI) therapy, provide a new breakthrough treatment for lung cancer patients. Whereas immunotherapy as an emerging treatment does not benefit patients with EGFR mutations, for which mechanistic studies are poorly defined and focused on the link of EGFR mutations and programmed cell death-ligand 1 (PD-L1) expression, we speculate that the different immune microenvironment associated with the two classes of patients. METHODS: Lung adenocarcinoma datasets were collected from the Cancer Genome Atlas (TCGA) database, and clinical information and gene expression profiles were downloaded. The immune related lymphocyte infiltration in TCGA database were generated through timer 2.0 GSEA was used to analyze the difference of pathway expression between EGFR mutant patients and wild type patients. RESULTS: EGFR mutation was more frequently among women and never smokers. Immunoinfiltration analysis showed that patients with EGFR mutation tends to have more tumor associated fibroblasts, common myeloid progenitor cells, hematopoietic stem cells, effector CD4⁺ T cells and natural killer T cells infiltration, and less memory B cells, naïve B cells, plasma B cells, plasmacytoid dendritic cells, memory CD4⁺ T cells, CD4⁺ helper T cells 2, naive CD8⁺ T cells, CD8⁺ T cells and central memory CD8⁺ T cells infiltration. Moreover, patients with more infiltration of CD8⁺ T cells, natural killer T cells, memory B cells and hematopoietic stem cells, tends have better prognosis (Log-rank test, P=0.017, 0.0093, 0.018, 0.016). However, the patients with more CD4⁺ T th2 infiltration in the tumor tends to have worse prognosis (Log-rank test, P=0.016). Furthermore, the results of gene set enrichment analysis showed that compared with the lung adenocarcinoma patients with EGFR wild type, the three pathways positive regulation of natural killer (NK) cell-mediated immune response to tumor cells, NK cell activation involved in immune response, and NK cell-mediated immune response to tumor cells related to natural killer cells in patients with EGFR mutation were down regulated, while the pathway the positive regulation of cytokine secretion involved in immune response was up-regulated in EGFR mutation patients. CONCLUSIONS The tumour microenvironment of patients with EGFR mutations lacks potent tumour killing effector cells and appears dysfunctional with effector cells. This may be a potential reason for the poor efficacy of immunotherapy in patients with EGFR mutations.

OBJECTIVE：To provide reference f or the qualit y sta ndard establishment of Amaranthus retroflexus. METHODS ： Taking 7 batches of A. retroflexus medicinal materials as the research object ，the appearance properties of the medicinal materials were investigated ，and the microscopic characteristics of the medicinal powders were observed. TLC method was adopted to qualitatively identify rutin ，valine and leucine in A. retroflexus medicinal materials. According to the relevant methods of the 2015 edition of Chinese Pharmacopoeia （part Ⅳ），water content ，total ash content ，acid-insoluble ash content and water-soluble extract content were determined. HPLC method was used to determine the content of rutin in the medicinal material of A. retroflexus . The determination was performed on Agilent 5 TC-C18（2）column with mobile phase consisted of methanol- 0.3％ phosphoric acid solution（40∶60，V/V），at the flow rate of 1.0 mL/min. The detection wavelength was set at 358 nm，and the column temperature was 30 ℃. The sample size was 10 μL. RESULTS：The appearance and microstructure characteristics of the medicinal materials were consistent with the existing description. The identification results of TLC meth od showed that 7 batches of medicinal materials and each reference substance （rutin，valine，leucine）showed spots of the same color at the same position. The moisture content of 7 batches of A. retroflexus medicinal materials was 7.43％-8.72％，the total ash content was 11.82％-13.78％，the acid-insoluble ash content was 0.15％-0.55％，and the water-soluble extract content was 17.27％-24.74％. The linear range of rutin was 10-200 μg/mL（R 2＝1.000 0）. RSDs of precision test ，stability test （24 h）and repeatability test were all less than 2.0％ （n＝6）. The average recovery rates of rutin were 99.14％，97.98％ and 98.80％ in low ，medium and high concentration of samples，and RSDs were 0.97％，0.95％，0.96％（n＝3）. The contents of rutin in 7 batches of A. retrophylla were 0.314-1.102 mg/g. CONCLUSIONS：In this study ，character observation ，microscopic identification ，moisture content ，total ash content ，acid- insoluble ash content and water-soluble extract content of A. retroflexus are investigated ；TLC method was established for qualitative identification of leucine ，valine and rutin in A. retroflexus ，and the HPLC method was established for content determination of rutin. It provides reference for the quality standard establishment of A. retroflexus .

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M
Antiviral Agents/therapeutic use* ; Binding Sites ; COVID-19/virology* ; Coronavirus Papain-Like Proteases/metabolism* ; Crystallography, X-Ray ; Drug Evaluation, Preclinical ; Drug Repositioning ; High-Throughput Screening Assays/methods* ; Humans ; Imidazoles/therapeutic use* ; Inhibitory Concentration 50 ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Naphthoquinones/therapeutic use* ; Protease Inhibitors/therapeutic use* ; Protein Structure, Tertiary ; Recombinant Proteins/isolation & purification* ; SARS-CoV-2/isolation & purification*