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Junqi Liao,1,&ast; Yuan Zhu,1,2,&ast; Aimei Zhang,1 Dan Wu,1 Xiaohui Yan,1 Qiuhua He,1 Fantao Song,1 Jingyi Chen,1 Yunze Li,1 Li Li,1 Zhaoyao Chen,1 Wenlei Li,1 Qin Yang,2 Zhuyuan Fang,3 Minghua Wu1 1Department of Neurology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, 210029, People’s Republic of China; 2Department of Medicine, Physiology and Biophysics, UC Irvine Diabetes Center, University of California Irvine (UCI), Irvine, CA, USA; 3Department of Cardiology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, 210029, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Minghua Wu, Department of Neurology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, 155 Hanzhong Road, Nanjing, 210029, People’s Republic of China, Email yfy0069@njucm.edu.cn Zhuyuan Fang, Department of Cardiology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, 155 Hanzhong Road, Nanjing, 210029, People’s Republic of China, Email fangzhuyuan@njucm.edu.cnBackground: The relationship between insulin resistance-related indices and the outcomes of acute ischemic stroke (AIS) is still unclear. This study aimed to explore the association between the Apo B/Apo A-1 ratio and the Prognostic Nutritional Index (PNI) with the 90-day outcomes of AIS.Methods: A total of 2011 AIS patients with a 3-month follow-up were enrolled in the present study from January 2017 to July 2021. Multivariate logistic regression modeling was performed to analyze the relationship between Apo B/Apo A-1 ratio, PNI, and AIS poor outcomes. The mediating effect between the three was analyzed using the Bootstrap method with PNI as the mediating variable.Results: Among the 2011 included AIS patients, 20.3% had a poor outcome. Patients were categorized according to quartiles of Apo B/Apo A-1 ratio and PNI. Multivariate logistic regression revealed that the fourth Apo B/Apo A-1 ratio quartile had poorer outcomes than the first quartile (OR 1.75,95%CL 1.21– 2.53, P=0.003), and the fourth PNI quartile exhibited a lower risk of poor outcomes than the first quartile (OR 0.40, 95%CL 0.27– 0.61, P< 0.001). PNI displayed a significant partially mediating effect (21.4%) between the Apo B/Apo A-1 ratio and poor AIS outcomes.Conclusion: The Apo B/Apo A-1 ratio is a risk factor for poor AIS outcomes, whereas PNI acts as a protective factor. The association between the ApoB/ApoA-1 ratio and poor AIS outcomes was partially mediated by PNI.Keywords: insulin resistance, Apo B/Apo A-1 ratio, prognostic nutritional index, acute ischemic stroke, outcomes

Background: Phospholipids are significant constituents of bacterial membranes. Bacteria can alter their cytoplasmic membrane fatty acid composition in response to changing environmental stress conditions. Carvacrol, a mono-terpenoid phenol, has excellent antibacterial properties against Streptococcus pyogenes. Purpose: This study examined modifications of the membrane phospholipid composition in the presence of sublethal carvacrol concentrations using erythromycin-resistant S. pyogenes. Methods: The cell membrane isolates (CMIs) were prepared from S. pyogenes strain Spy 1558, erm. The ultrastructural morphology was studied using transmission electron microscopy (TEM). Carvacrol-treated CMIs were analyzed for fluidity changes using fluorescence-anisotropy. Changes in membrane lipid composition were assessed using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS). Results: Fluidity of the bacterial membrane increased time-dependently when exposed to carvacrol. The CMI isolated from cells treated with carvacrol showed a change in cardiolipins (CL), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) percentages compared to the untreated control. Conclusion: Carvacrol induces modulation of specific membrane phospholipid and fatty acid composition of S. pyogenes. Further investigations are necessary to understand how fatty acid and phospholipid biosynthesis can be influenced by carvacrol.

Yi Lou,1,2,&ast; Fanglong Song,3,&ast; Yifan Kang,2 Yaozeng Xu1 1Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou, 215006, People’s Republic of China; 2Department of Orthopaedics, Shanghai Eastern Hepatobiliary Surgery Hospital, Naval Military Medical University, Shanghai, 201805, People’s Republic of China; 3Department of Orthopaedics, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yaozeng Xu, Department of Orthopaedics, The First Affiliated Hospital of Soochow University, No. 899 Pinghai Road, Gusu District, Suzhou, People’s Republic of China, Email xuyaozeng@163.com Yifan Kang, Department of Orthopaedics, Shanghai Eastern Hepatobiliary Surgery Hospital, Naval Military Medical University, Shanghai, 201805, People’s Republic of China, Email kangyf11@163.comPurpose: The development of osteoarthritis (OA) has been linked to mechanical factors. Studies suggest that periodic mechanical stress (PMS) may be a factor contributing to cartilage repair and the onset of OA. Therefore, this study was designed to explore the effects and underlying mechanisms of PMS on OA development.Patients and Methods: Firstly, surgery and interleukin (IL)-1β were used for the establishment of rat/cell models of OA, respectively. Subsequently, activating transcription factor (ATF) 3 expression was knocked down in OA rats, and OA chondrocytes were treated with different heights (0, 1, 2, 4, 8 cm) of PMS or si-ATF. Safranin O staining was used to observe the histological changes in the rat knee joint, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of tumor necrosis factor (TNF)-α, IL-6, and IL-8 in vivo and in vitro. Further, the expression of extracellular matrix (ECM) proteins in the rat knee joint was assessed immunohistochemistry. Flow cytometry was used to evaluate chondrocyte apoptosis. Lastly, Western blot was performed to detect the expression of related proteins of the protein kinase B (Akt) signaling pathway and ECM.Results: The OA rat model was successfully constructed. Further experiments indicated that the knockdown of ATF3 not only alleviated joint swelling, pain, inflammatory response and pathological damage, but also promoted ECM synthesis and the phosphorylation of Akt in OA rats. In vitro experiments showed that PMS (4 cm) effectively inhibited cell apoptosis, decreased the levels of TNF-α, IL-6 and IL-8, promoted ECM synthesis, and activated the Akt signaling pathway in osteoarthritic chondrocytes. However, ATF3 overexpression reversed the positive effects of PMS on osteoarthritic chondrocytes.Conclusion: PMS can effectively inhibit the development of OA, and its protective effects may be attributed to the down-regulation of ATF3 expression and activation of the Akt signaling pathway.Keywords: periodic mechanical stress, osteoarthritis, ATF3-Akt axis, joint damage, pain, apoptosis

Fei Song,1,2,&ast; Yan Wang,1,&ast; Xiao-ge Wei,1,2 Nan Yang,1 Wenjing Sun,1 Kaiying Li,1 Huisheng Ma,1,2 Jing Mu1 1College of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 2Key Laboratory of Modernization of Minority Medicine, Ministry of Education, Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jing Mu; Huisheng Ma, Email mujing930@163.com; madoctor@163.comObjective: In this study, we analyzed the differential expression and key signaling pathways of proteins in the skin of guinea pigs with melanin deposition caused by two different modeling methods by utilizing proteomics techniques.Methods: Guinea pig skin melanin deposition models were: (1) induced by ultraviolet (UV) irradiation alone (U group), (2) induced by UV combined with progesterone injection (P group), and guinea pigs treated without any treatment were used as blank group (B group). H&E staining and Masson staining were used to observe the extent of skin damage and melanin deposition in guinea pigs. The differentially expressed proteins (DEPs) in the skin tissues of melanin-deposited guinea pigs were screened by proteomic techniques, the functions of DEPs were analyzed, and a protein-protein interaction network (PPI) was constructed.Results: There was a significant difference in grayscale between the U and P groups of guinea pig skin before and after modeling (P < 0.01). H&E and Masson staining showed that the U and P groups both exhibited incomplete keratinization of the stratum corneum, increased proliferation of epidermal cells with large nuclei and disordered arrangement, neovascularization of the dermis, and increased the number of melanin particles in the epidermis of the U and P groups of guinea pigs compared with the B group. Proteomics analysis showed that there were 171 DEPs between the U and P groups. These DEPs focused on biological processes such as fibrillar collagen trimer, extracellular matrix containing collagen proteins, metalloproteinase activity, and peroxidase activity.Conclusion: The melanin pigmentation model induced solely by UV radiation negatively regulates biological processes such as extracellular matrix and collagen synthesis, while inducing significant skin photoaging. The combination of progesterone injections and UV radiation-induced melanin pigmentation model can cause abnormal protein expression in fatty acid and phospholipid metabolism, possibly being closer to the environment of melasma formation.Keywords: melasma, skin melanin, melanin deposition, ultraviolet, progesterone

Zhan Gao,1,2 Yun Ti,1 Bin Lu,1 Fang-qiang Song,1,3 Lei Zhang,1 Bo-ang Hu,1 Jia-ying Xie,1 Wei Zhang,1 Lu Han,1,4 Ming Zhong1 1The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital, Qilu College of Medicine, Shandong University, Jinan, People’s Republic of China; 2Department of Cardiology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China; 3Department of Critical Care Medicine, Tengzhou Central People’s Hospital, Tengzhou, People’s Republic of China; 4Department of General Practice, Qilu Hospital, Qilu College of Medicine, Shandong University, Jinan, People’s Republic of ChinaCorrespondence: Lu Han; Ming Zhong, Email luhan_sdu@163.com; zhongming2sdu@163.comBackground: Previous studies have reported that six transmembrane protein of prostate 2 (STAMP2) attenuates metabolic inflammation and insulin resistance in diabetes mellitus. However, the role of STAMP2 in the diabetic heart is still unclear.Methods: A diabetic rat cardiomyopathy model was established via intraperitoneal STZ injection. STAMP2 was overexpressed in the treatment group using adeno-associated virus. Rat heart diastolic function was measured using echocardiography and a left ventricular catheter, and cardiac interstitial fibrosis was detected by immunohistochemistry and histological staining. Insulin sensitivity and NF-κB expression were shown by Western blotting. NMRAL1 distribution was illustrated by immunofluorescence.Results: STAMP2 expression in the diabetic rat heart was reduced, and exogenous overexpression of STAMP2 improved glucose tolerance and insulin sensitivity and alleviated diastolic dysfunction and myocardial fibrosis. Furthermore, we found that NF-κB signaling is activated in the diabetic heart and that exogenous overexpression of STAMP2 promotes NMRAL1 translocation from the cytoplasm to the nucleus and inhibits p65 phosphorylation.Conclusion: STAMP2 attenuates cardiac dysfunction and insulin resistance in diabetic cardiomyopathy, likely by promoting NMRAL1 retranslocation and NF-κB signaling inhibition.Keywords: STAMP2, diabetic cardiomyopathy, NADPH, NMRAL1, NF-κB

Xiubin Ma,1– 3 Qing Liu,1– 3 Fangying Song,1– 3 Yusen Huang1– 3 1Department of Ophthalmology, Eye Institute of Shandong First Medical University, Qingdao Eye Hospital of Shandong First Medical University, Qingdao, People’s Republic of China; 2State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Qingdao, People’s Republic of China; 3Department of Ophthalmology, School of Ophthalmology, Shandong First Medical University, Qingdao, People’s Republic of ChinaCorrespondence: Yusen Huang, Department of Ophthalmology, Qingdao Eye Hospital of Shandong First Medical University, Qingdao, 266071, People’s Republic of China, Tel +86-532-85876380, Email huang_yusen@126.comBackground: Pseudomonas aeruginosa (P. aeruginosa) is the second-most common commensal bacterium in healthy conjunctival sacs. When the corneal epithelial barrier is damaged, P. aeruginosa in a healthy conjunctival sac can cause infectious keratitis, which can result in the loss of vision. This study was designed to investigate the differentially expressed genes (DEGs) of P. aeruginosa isolates from eyes with keratitis and from healthy conjunctival sacs to predict their functions and pathways through Illumina high-throughput RNA sequencing (RNA-seq).Methods: P. aeruginosa isolates from keratitis and healthy conjunctival sacs were obtained. The transcriptome profile of P. aeruginosa was characterized by a high throughput RNA-seq strategy using the Illumina HiSeq 2500 platform. The DEGs were analyzed with DESeq and validated through quantitative real-time polymerase chain reaction (PCR) and with experimental mice. GO enrichment and the KEGG pathway were also analyzed.Results: In genome-wide transcriptional analysis, 557 genes (332 upregulated and 225 downregulated) were found to be differentially expressed (fold change ≥ 2, p ≤ 0.05) in the strains from keratitis. GO enrichment analysis suggested that DEGs tended to be associated with cellular and metabolic processes. KEGG pathway analysis revealed the DEGs were typically associated with the pathways of the bacterial secretion system and pyoverdine metabolism. Eleven DEGs were validated using quantitative reverse-transcription PCR and verified with experimental mice. The results were consistent with those obtained in RNA-seq.Conclusion: The DEGs related to pilin, T2SS, T3SS, and pyoverdine metabolisms were significantly altered in the strains from keratitis. The findings may be helpful for further investigations on genes or pathways related to the pathogenesis of and therapeutic targets for P. aeruginosa keratitis.Keywords: P. aeruginosa, genotype, keratitis, conjunctival sac, microflora

Xiaoliang Liang,1,&ast; Fei Song,2,&ast; Wanzhi Fang,1 Yu Zhang,1 Zihan Feng,1 Zeyin Chen,1 Lu Han,3 Zhong Chen1 1Department of Hepatobiliary Surgery, Affiliated Hospital of Nantong University, Nantong, 226001, People’s Republic of China; 2Department of General Surgery, Yancheng TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Yancheng, 224002, People’s Republic of China; 3Department of Medicine, Jiangsu Vocational College of Medicine, Yancheng, 224005, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhong Chen, Department of Hepatobiliary Surgery, Affiliated Hospital of Nantong University, Nantong, 226001, People’s Republic of China, Email chenz9806@163.comPurpose: C-type lectin domain family 1 member B (CLEC1B) is a protein-coding gene involved in various processes, such as platelet activation, tumor cell metastasis and separation of blood/lymphatic vessels. However, how CLEC1B plays its role in hepatocellular carcinoma (HCC) has not been well studied. The purpose of this study was to investigate the clinical significance and biological function of CLEC1B in HCC.Patients and Methods: Based on (The Cancer Genome Atlas) TCGA database, CLEC1B expression matrix and corresponding clinical information were extracted. ROC curves and Kaplan–Meier method were generated to evaluate the value of CLEC1B as a diagnostic and prognostic biomarker. Moreover, single-gene difference analysis constructed by DESeq2 method and then the related genes were used to predict CLEC1B-related signaling pathways. The ssGSEA algorithm was conducted for studies related to immune infiltration. CLEC1B protein expression was evaluated and immunohistochemistry in HCC tissues through tissue microarray. Finally, the relationship between CLEC1B expression and T cell infiltration was assessed according to tissue microarray.Results: The mRNA and protein levels of CLEC1B were significantly down-regulated in HCC compared to paired normal tissues, which were further verified in clinical tissue samples. ROC curves and Kaplan–Meier survival analysis suggested the significant diagnostic and clinical prognostic value of CLEC1B. Meanwhile, downregulation of CLEC1B was significantly associated with clinical parameters such as clinical tumor vascular invasion and distant metastasis. Moreover, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment (GSEA) analysis indicated that CLEC1B has significant association with immune function. Finally, immune infiltration analysis indicated that CLEC1B was significantly associated with immune cell subsets and affected the efficacy of immunotherapy in cancer patient.Conclusion: Collectively, our findings suggested that CLEC1B could be a promising prognostic biomarker in HCC and its expression was related to immune cell infiltration.Keywords: CLEC1B, hepatocellular carcinoma, prognostic value, tumor immune infiltration, bioinformatics analysis

Qi Sun,1,2,&ast; Zhen Liu,1,2,&ast; Xiangya Xu,1,&ast; Yujuan Yang,1,2 Xiao Han,1,2 Cai Wang,1– 3 Fei Song,1,2,4 Yakui Mou,1,2 Yumei Li,1,2 Xicheng Song1,2 1Department of Otorhinolaryngology, Head and Neck Surgery, Yantai Yuhuangding Hospital, Qingdao University, Yantai, People’s Republic of China; 2Shandong Provincial Clinical Research Center for Otorhinolaryngologic Diseases, Yantai Yuhuangding Hospital, Yantai, People’s Republic of China; 3School of Clinical Medicine, Weifang Medical University, Weifang, People’s Republic of China; 4Department of Binzhou Medical University, Clinical Medical College Second, Binzhou Medical University, Yantai, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xicheng Song; Yumei Li, Department of Otolaryngology, Head and Neck Surgery, Yantai Yuhuangding Hospital, Qingdao University, Yantai, 264000, People’s Republic of China, Tel +860535-6691999, Fax +860535-6240341, Email drxchsong@163.com; myheart1263@163.comPurpose: To explore the mechanisms by which circRNA/miRNA/mRNA competitive endogenous RNAs (ceRNA) networks regulate CRSwNP.Methods: The expression profiles of circRNAs, miRNAs, and mRNAs from patients with CRSwNP and control subjects were acquired from the Gene Expression Omnibus database. The circRNA/miRNA/mRNA ceRNA network was constructed based on the predicted circRNA–miRNA interactions and miRNA–mRNA interactions. Hub-mRNAs were screened by protein–protein interaction network analysis and Cytoscape molecular complex detection. The expression of factors in tissue and in hsa_circ_0031594 siRNA transfection cells was verified by RT-qPCR and the association between them was revealed by Spearman correlation analysis. Receiver operating characteristic curve analysis was performed with the pROC R package.Results: The differential expression of 5423 circRNAs, 415 miRNAs, and 3673 mRNAs was identified in CRSwNP subjects compared to control subjects. Among these, 9 circRNAs, 39 miRNAs, and 78 mRNAs were screened to construct a ceRNA network. Ultimately, a subnetwork including circRNA hsa_circ_0031594, hsa-miR-1260b, hsa-miR-6507-5p, NCAPG2, RACGAP1, CHEK1 and PRC1 was screened out. RT-qPCR validated that the expression of hsa_circ_0031594, NCAPG2, PRC1 was significantly increased, and hsa-miR-1260b and hsa-miR-6507-5p were expressed significantly less in patients with CRSwNP than in control subjects. In addition, the AUCs of hsa_circ_0031594, hsa-miR-1260b, hsa-miR-6507-5p, NCAPG2, and PRC1 to discriminate CRSwNP patients were 0.995, 0.842, 0.862, 0.765, and 0.816. Spearman correlation showed that the expression of hsa_circ_0031594 was negatively correlated with hsa-miR-1260b and hsa-miR-6507-5p, and positively correlated with NCAPG2 and PRC1. In human nasal epithelial cell (HNEpC) line, knocking down hsa_circ_0031594 could increase the expression of hsa-miR-1260b and hsa-miR-6507-5p, and reduce the expression of NCAPG2 and PRC1.Conclusion: CeRNA networks including hsa_circ_0031594, hsa-miR-1260b, and NCAPG2, and hsa_circ_0031594, hsa-miR-6507-5p, and PRC1 may be key regulators for CRSwNP occurrence, and may be potential targets for the pathogenesis and treatment development of CRSwNP.Keywords: circRNA, miRNA, mRNA, ceRNA network, CRSwNP

Fuxi Song,1 Yanwu Jin,1 Peng Li,1 Chao Zheng,2 Xin Zhao1 1Department of Anesthesiology, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, People’s Republic of China; 2Department of Breast Surgery, The Second Hospital, School of Medicine, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, People’s Republic of ChinaCorrespondence: Xin ZhaoDepartment of Anesthesiology, The Second Hospital, Cheeloo College of Medicine, Shandong University, No. 247 Beiyuan Street, Jinan, Shandong, 250033, People’s Republic of ChinaFax +86-531-88962544Email lujnzx@sohu.comPurpose: The aim of this study was to investigate the efficacy of esmolol on intraoperative hemodynamic and perioperative analgesic management.Methods: Totally, 125 patients undergoing colectomy were randomly divided into three groups. Group S (saline group) was administered 0.75 mL/kg/h of normal saline for 5 min before anesthesia induction and maintenance of 0.25 mL/kg/h; Group E1 and Group E2 were administered 0.5 mg/kg and 1.0 mg/kg esmolol for 5 min before anesthesia induction, and maintained of 0.5 mg/kg/h and 2.0 mg/kg/h, respectively. Several parameters including indexes of hemodynamics variation (primary outcome), intra- and postoperative analgesic usage, and pain score were measured.Results: Group E1 and Group E2 had significantly lower intubation response than Group S (P = 0.007, P = 0.001), and extubation response of Group E2 was significantly lower than Group S (P = 0.007). The opioid consumption in Group E1 and Group E2 was significantly lower than in Group S intraoperatively (P = 0.020 and 0.007). The incidence of postoperative adverse reactions among the three groups was not statistically significant (P = 0.368 and 0.772).Conclusion: Esmolol 0.5 mg/kg and 1.0 mg/kg infusion before intubation both can effectively inhibit the intubation response, while only maintenance with 2.0 mg/kg/h of esmolol can reduce the incidence of extubation response. At the same time, esmolol can decrease intraoperative opioid requirement without increasing the risk of adverse reactions.Trial Registration: ChiCTR1900024538 and the date of registration was July 15, 2019 at http://www.chictr.org.cn.Keywords: esmolol, hemodynamics, analgesia, intubation response, extubation response

Fei Song,1,2 Hanny Al-Samkari2,3 1Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; 2Harvard Medical School, Boston, MA, USA; 3Division of Hematology, Massachusetts General Hospital, Boston, MA, USACorrespondence: Hanny Al-SamkariDivision of Hematology, Massachusetts General Hospital, Suite 118, Room 112, Zero Emerson Place, Boston, MA, 02114, USATel +617-643-6214Fax +617-643-8840Email hal-samkari@mgh.harvard.eduAbstract: Immune thrombocytopenia (ITP) is an autoimmune process resulting in increased destruction and inadequate production of platelets that can result in bleeding, fatigue, and reduced health-related quality of life. While treatment is not required for many patients with ITP, the occurrence of bleeding manifestations, severe thrombocytopenia, and requirement for invasive procedures are among the reasons necessitating initiation of therapy. Corticosteroids, intravenous immunoglobulin, and anti-RhD immune globulin are typical first-line and rescue treatments, but these agents typically do not result in a durable remission in adult patients. Most patients requiring treatment therefore require subsequent line therapies, such as thrombopoietin receptor agonists (TPO-RAs), rituximab, fostamatinib, splenectomy, or a number of other immunosuppressive agents. In this focused review, we discuss management of adult ITP in the acute and chronic settings.Keywords: platelets, immune thrombocytopenia, ITP, treatment, corticosteroids, IVIG, splenectomy, thrombopoietin receptor agonist, rituximab, fostamatinib

Jianliang Chen,1,2,* Shengqiang Cai,1,2,* Tianchun Gu,1,2 Fei Song,1,2 Yingchun Xue,1,2 Di Sun1,2 1Department of General Surgery, People’s Hospital of Jingjiang, Taizhou, 214500, Jiangsu, People’s Republic of China; 2Seventh Clinical Medical College of Yangzhou University, Taizhou, 214500, Jiangsu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Di SunDepartment of General Surgery, People’s Hospital of Jingjiang, 28 Zhongzhou Road, Jingjiang, Taizhou, 214500, Jiangsu, People’s Republic of ChinaTel +86-18052633030Email 1093730063@qq.comIntroduction: MiRNAs have been proven to modulate the progression of gastric cancer (GC). In this field, we evaluated the role and mechanism of miR-140-3p in GC.Methods: Western blotting and qRT-PCR were used to detect the levels of miR-140-3p and BCL2. The interaction of miR-140-3p and BCL2 was confirmed by dual-luciferase reporter and miRNA pull-down assays. CCK-8, EdU, wound healing, and Transwell invasion assays were performed to evaluate cell proliferation, migration and invasion. Autophagy was analyzed using Western blot analysis of the LC3-II/I ratio and immunofluorescence staining. A xenograft model was established to reveal the role of miR-140-3p in tumorigenesis.Results: In GC cell lines and tissues, miR-140-3p was highly expressed, and BCL2 was expressed at low levels. MiR-140-3p directly inhibited BCL2 expression and indirectly promoted BECN1 expression, and BCL2 inhibited BECN1 expression. MiR-140-3p overexpression or silencing restrained or facilitated migration, invasion and EMT in GC cells. Moreover, we noticed that overexpression or downregulation of miR-140-3p promoted or suppressed BECN1-dependent autophagy in GC cells. BCL2 introduction or BECN1 silencing in GC cells partially blocked the effects of miR-140-3p. In conclusion, miR-140-3p directly downregulated the expression of BCL2, BCL2 downregulation further activated BECN1-dependent autophagy, and autophagy activation further inhibited EMT.Conclusion: miR-140-3p may act as a tumor suppressor by targeting BCL2 and regulating downstream BECN1-induced autophagy and metastasis in GC progression.Keywords: miR-140-3p, gastric cancer, BCL2, BECN1, autophagy, EMT

Dandan Chen,1 Feng Song,2 Nianqi Cui,3 Leiwen Tang,1 Hui Zhang,4 Jing Shao,1 Ruolin Qiu,1 Dan Wang,1 Xiyi Wang,1 Zhihong Ye1 1Zhejiang University School of Medicine, Affiliated Sir Run Run Shaw Hospital, Hangzhou, Zhejiang, People’s Republic of China; 2Yunnan College of Business Management, Medical Faculty, Kunming, Yunnan, People’s Republic of China; 3The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China; 4Guizhou Provincial People’s Hospital, Department of Cardiology, Guiyang, Guizhou, People’s Republic of ChinaCorrespondence: Zhihong YeAffiliated Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou, Zhejiang, 310016, People’s Republic of ChinaTel +86 136 0661 2119Email yezh@zju.edu.cnIntroduction: With the effective treatments of novel coronavirus disease 2019 (COVID-19), thousands of patients have recovered from COVID-19 globally. The public perceptions and views are vital to facilitate recovered COVID-19 patients reintegrate into society. In China, the rural population accounts for nearly 70% of the total population. Therefore, we chose to evaluate perceptions and views of rural residents towards COVID-19 recovered patients in China.Methods: Fifteen participants were sampled from a village with the severe COVID-19 epidemic in Zibo city, Shandong Province. The fifteen participants who lived in the village with COVID-19 recovered patients were included. They were over 18 years of age and were voluntary to participant in the study. A descriptive qualitative design using semi-structured telephone interviews was undertaken. Thematic analysis was undertaken.Results: Five main themes emerged from the data: (1) Perceived personal characteristics of COVID-19 recovered patients; (2) Perceived difficulties faced by COVID-19 recovered patients; (3) Perceptions on the social relationship with COVID-19 recovered patients; (4) Views on COVID-19 recovered patients going to public venues; (5) Views on helping COVID-19 recovered patients. Each theme was supported by several subthemes.Conclusion: Our study showed that discrimination and reduced social intimacy exist among rural residents. To improve their views or the situation, relevant departments could lead health educational programs and encourage supportive social connections. Through these strategic messaging, rural residents are expected to recognize that COVID-19 recovered patients need more social support, rather than discrimination and resistance, which helps recovered patients better return to society.Keywords: rural residents, perceptions, views, COVID-19 recovered patients, China

Abstract Streptococcus pyogenes is an important human pathogen worldwide. The identification of natural antibacterial phytochemicals has renewed interest due to the current scarcity of antibiotic development. Carvacrol is a monoterpenoid found in herbs. We evaluated carvacrol alone and combined with selected antibiotics against four strains of S. pyogenes in vitro. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of carvacrol against S. pyogenes were 125 µg/mL (0.53 mM) and 250 µg/mL (1.05 mM), respectively. Kill curve results showed that carvacrol exhibits instantaneous bactericidal activity against S. pyogenes. We also demonstrated the potential mechanism of action of carvacrol through compromising the cell membrane integrity. Carvacrol induced membrane integrity changes leading to leakage of cytoplasmic content such as lactate dehydrogenase enzymes and nucleic acids. We further confirmed dose-dependent rupturing of cells and cell deaths using transmission electron microscopy. The chequerboard assay results showed that carvacrol possesses an additive-synergistic effect with clindamycin or penicillin. Carvacrol alone, combined with clindamycin or penicillin, can be used as a safe and efficacious natural health product for managing streptococcal pharyngitis.

Fangbin Song,1,2 Hui Du,1,2 Aimin Xiao,1,2 Chun Wang,1,2 Xia Huang,1,2 Peisha Yan,1,2 Zhihong Liu,1,2 Xinfeng Qu,3 Jerome L Belinson,4,5 Ruifang Wu1,2 1Department of Obstetrics and Gynecology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, People’s Republic of China; 2Shenzhen Key Laboratory on Technology for Early Diagnosis of Major Gynecological Diseases, Shenzhen, People’s Republic of China; 3Sanming Project of Medicine in Shenzhen, Peking University Shenzhen Hospital, Shenzen, People’s Republic of China; 4Preventive Oncology International, Inc., Cleveland Heights, OH, USA; 5Gynecologic Oncology Division, Women’s Health Institute, Cleveland Clinic, Cleveland, OH,USACorrespondence: Ruifang Wu; Hui DuDepartment of Obstetrics and Gynecology, Peking University Shenzhen Hospital, No. 1120 Lianhua Road, Shenzhen, Guangdong 518036, People’s Republic of ChinaEmail wurfpush@126.com; duhui_107108@163.comPurpose: When used for cervical cancer primary screening, liquid-based cytology (LBC) has a high specificity but a low sensitivity. For histological diagnosis of high-grade lesions, p16INK4a immunostaining has proven to be useful. Therefore, our objective was to evaluate the use of p16INK4a immuno-cytology as a primary screen and a secondary screen after primary high-risk human papillomavirus (hrHPV) screening or LBC screening.Methods: A total of 1197 cytology slides were immuno-stained using automatic p16INK4a staining system (PathCIN®p16INK4a) in two studies from cervical screening programs. In the primary screening study, 875 slides were randomly selected and analyzed for p16INK4a. In the secondary screening study, 322 of the remaining slides were chosen by virtue of being HPV 16/18+, other hrHPV+/LBC≥ASC-US, or HPV-negative/LBC ≥LSIL. The sensitivity and specificity for detection of cervical intraepithelial neoplasia 2/3 or worse (CIN2+/CIN3+) were compared based on p16INK4a, LBC and HPV test results.Results: In combining two studies, there were 431 cases with biopsy pathology. They included 83 cases with CIN2+ and 41 cases with CIN3+. The p16 positivity rate increased with pathologic and cytologic severity (P< 0.0001). For primary screening: p16 immuno-cytology was more specific than HPV testing and was similar in sensitivity. Also, p16 immuno-cytology compared favorably with routine LBC (≥ASC-US or ≥LSIL) in sensitivity and specificity. For secondary screening: after LBC screening, “Triaging ASC-US with p16” gave a higher specificity and a similar sensitivity as compared to the “Triaging ASC-US with hrHPV” algorithm. After HPV primary screening, p16 immuno-cytology was more specific than LBC (≥ASC-US); the calculated colposcopy referral rate was also decreased by using p16 immuno-cytology as triage. Triage of “HPV16/18 and p16” had higher specificity and similar sensitivity as compared to triage of “HPV16/18 and LBC ≥ASC-US”.Conclusion: For primary screening, p16INK4a immuno-cytology compares favorably to routine LBC and HPV testing. p16INK4a immunostaining could be an efficient triage to reduce the colposcopy referral rate after primary hrHPV screening or LBC screening. Therefore, p16INK4a immuno-cytology may be applicable as a favorable technology for cervical cancer screening.Keywords: cervical intraepithelial neoplasia, human papillomavirus, p16, immunochemical staining, cervical cancer screening

Xin Cheng,1,2,* Yu Zhang,1,2,* Fei Song,1,2 Fengliang Song,1,2 Cheng Gao,1,2 Xiaoliang Liang,1,2 Feiran Wang,1,2 Zhong Chen1 1Department of Hepatobiliary Surgery, Affiliated Hospital of Nantong University, Nantong 226001, People’s Republic of China; 2Medical College of Nantong University, Nantong 226001, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhong Chen Department of Hepatobiliary SurgeryAffiliated Hospital of Nantong University, No. 20 Xisi Road, Nantong 226001, People’s Republic of ChinaEmail chenz9806@163.comObjective: Ubiquitin-related modifier 1 (URM1) is a member of the ubiquitin-like regulator family, which acts as a post-translational protein modifier in the oxidative emergency response mechanism. Previous studies have shown that URM1 may be involved in the process of apoptosis and may play a role in JNK signaling pathway. In this study, we aimed to investigate the role and possible mechanism of URM1 in HCC progression.Patients and Methods: Expression of URM1 was determined in 90 pairs of matched liver cancer and adjacent non-cancerous tissues by immunohistochemistry. The impacts of URM1 on HCC cell proliferation, apoptosis, migration and invasion capacities were verified by CCK-8, colony formation, TUNEL staining, wound healing assay and transwell, respectively. Then, the effect of URM1 on subcutaneous tumor formation in vitro was explored by nude mouse xenograft model of liver cancer. Finally, the expression of apoptosis-related proteins was analyzed in URM1 knockdown samples by Western blotting.Results: In this study, compared with paired adjacent non-cancerous tissues, the expression of URM1 was higher in liver cancer tissues (P < 0.01). Kaplan–Meier survival analysis showed that high URM1 expression was significantly associated with poor prognosis (P < 0.05). Moreover, URM1 knockdown inhibited liver cancer cell proliferation and migration. Furthermore, URM1 knockdown promoted apoptosis of liver cancer cells. At the same time, URM1 knockdown inhibited tumor growth in nude mouse xenograft model of liver cancer. In addition, URM1 knockdown downregulated the expression of the apoptosis-related factors JNK1/2 and TP53 and upregulated the phosphorylation of JNK1/2 and P53.Conclusion: In summary, our results suggested that URM1 expression is increased in liver cancer tissues, and URM1 knockdown inhibits the proliferation and migration of liver cancer cells and accelerates apoptosis. High URM1 expression is associated with poor prognosis in patients with HCC.Keywords: hepatocellular carcinoma, URM1, proliferation, migration, apoptosis, JNK signaling pathway

Shanbao Li,1,2,* Lei Li,1,* Junyi Wu,1,* Fangbin Song,1 Zhiwei Qin,1 Lei Hou,2 Chao Xiao,1 Junyong Weng,1 Xuebin Qin,3 Junming Xu1 1Department of General Surgery, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200080, People’s Republic of China; 2Department of General Surgery, Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences, Shanghai, People’s Republic of China; 3Division of Pathology, Tulane National Primate Research Center, Health Sciences Campus, Covington, LA 70433, USA*These authors contributed equally to this workCorrespondence: Junming XuDepartment of General Surgery, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200080, People’s Republic of ChinaTel +86 18616364978Email junmingxushdy@163.comPurpose: Tryptophan 2,3-dioxygenase (TDO), encoded by the gene TDO2, is an enzyme that catalyses the first and rate-limiting step of tryptophan (Try) degradation in the kynurenine (Kyn) pathway in the liver. Recently, TDO has been demonstrated to be expressed in various human tumours, especially hepatocellular carcinoma (HCC). However, the role of TDO in HCC is still not very clear. Here, we studied the role of TDO in HCC.Methods: We demonstrated that TDO is overexpressed in human HCC tissues and is significantly correlated with malignant phenotype characteristics, including tumour size, tumour differentiation, vascular invasion, etc. Kaplan–Meier analysis showed a poor overall survival rate in patients with TDO-overexpressing tumours. In addition, the effects of TDO on HCC tumour growth and metastasis were detected both in vivo and in vitro. TDO overexpression facilitated HCC cell growth, invasion and migration.Conclusion: Our results suggest that TDO positively regulates HCC proliferation and invasion and acts as a new prognostic biomarker of HCC.Keywords: TDO, tryptophan 2, 3-dioxygenase, hepatocellular carcinoma, tryptophan

Xiaoyong Xu,1,* Feihong Song,2,* Xinwei Jiang,1,* Han Hong,1 Qiang Fei,1 Zhengkang Jin,1 Xiang Zhu,1 Binghua Dai,2 Jiamei Yang,2 Chengjun Sui,2 Minhui Xu1 1Department of Hepato-Pancreato-Biliary Surgery, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou 215001, People’s Republic of China; 2Department of Special Treatment and Liver Transplantation, Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai 200438, People’s Republic of China*These authors contributed equally to this workCorrespondence: Minhui XuDepartment of Hepato-Pancreato-Biliary Surgery, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou 215001, People’s Republic of ChinaEmail xuminhui11@aliyun.comChengjun SuiDepartment of Special Treatment and Liver Transplantation, Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai 200438, People’s Republic of ChinaEmail suichengjun1978@163.comBackground: Thousands of long non-coding RNAs (lncRNAs) have been functionally verified as crucial regulators of physiological processes and disease progressions, yet their roles in hepatocellular carcinoma (HCC) have not been clearly illuminated.Methods: We analyzed the expression of lncRNA-SNHG14 in TCGA data via bioinformatic analysis and detected its expression in HCC specimens by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Loss-of-function experiments were used to study the biological function of SNHG14 in HCC cells. RT-qPCR, Western blotting and dual-luciferase reporter assay were carried out to investigate the molecular mechanism of SNHG14 in HCC.Results: The upregulation of lncRNA-SNHG14 was observed in HCC tissues compared with normal tissues via RT-qPCR and bioinformatic analysis of TCGA data. Silencing of SNHG14 inhibited cell proliferation and induced cell apoptosis in HCC cells. microRNA-217 (miR-217), the tumor-suppressive miRNA in HCC, was predicted and confirmed as a miRNA sponged by SNHG14 in HCC cells. Via downregulation of miR-217, SNHG14 increased the expression of several miR-217-related oncogenes and subsequently activated oncogene-related signaling pathways in HCC cells. In addition, inhibition of miR-217 reversed SNHG14 silencing induced decrease of cell proliferation and increase of cell apoptosis. Their association was verified in the published microarray dataset and the collected HCC samples.Conclusion: In summary, SNHG14 is involved in the development of HCC via sponging miR-217 and it may be a biomarker for patients with HCC.Keywords: SNHG14, hepatocellular carcinoma, miR-217

Xue Leng,1 Geli Liu,1 Sen Wang,2 Jing Song,3 Wanfeng Zhang,3 Xianqin Zhang,1 Li Rong,1,4 Yongping Ma,1 Fangzhou Song1 1Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, People’s Republic of China; 2Laboratory Department, Affiliated Hospital of Jining Medical College, Shandong, 272029, People’s Republic of China; 3Department of Bioinformatics, The Basic Medical School of Chongqing Medical University, Chongqing 400016, People’s Republic of China; 4Department of Gastroenterology, Chongqing Public Health Medical Center, Chongqing, 400036, People’s Republic of ChinaCorrespondence: Fangzhou SongMolecular Medicine and Cancer Research Center, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, People’s Republic of ChinaTel +86 23 68485958Email fzsongcq@163.comPurpose: Gastric cancer (GC) is the fifth most common tumor in the world, and most patients with GC have a poor prognosis. This study aimed to explore the biological influence and mechanism of LINC01272 in GC.Materials and Methods: Using bioinformatic analyses, we investigated the expression of LINC01272 in TCGA database and predicted the biological functions and mechanism of LINC01272 in GC. Then, we detected the expression of LINC01272 in GC cell lines, GC tissues, and corresponding normal tissues using real-time polymerase chain reaction (RT-PCR). Finally, we explored the migration and invasion ability of LINC01272 by wound-healing and Transwell assays and examined the expression of epithelial-mesenchymal transition (EMT)-related proteins through Western blotting.Results: We found that LINC01272 was upregulated in GC and was associated with GC staging and lymph node metastasis. The results of wound-healing and Transwell assays revealed that the LINC01272 was closely related to GC cell migration and invasion. LINC01272 knockdown inhibited the migration and invasion ability of GC cells by reducing the expression of EMT-related proteins. Overexpression of LINC01272 had the opposite effect.Conclusion: Together, our results showed that LINC01272 promoted GC metastasis ability by regulating the expression of EMT-related proteins and could serve as a potential diagnostic biomarker for GC.Keywords: gastric cancer, long non-coding RNA, migration, invasion, LINC01272

Wei-hua Wang,1,* Min Lin,2,* Hai-liang Li,3 Jun-yun Huang,1 Jiang-tao Chen,4,5 Xian-song Fang,6 Dong-mei Huang,1 Xu-xiang Xi,1 Qing-fei Zhao,1 Fang-li Song,7 Shao Huang,7 Tian-yu Zhong1 1Department of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi Province, People’s Republic of China; 2School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, Guangdong Province, People’s Republic of China; 3Department of Hematology, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi Province, People’s Republic of China; 4The Chinese Medical Aid Team to the Republic of Equatorial Guinea, Guangzhou, Guangdong Province, People’s Republic of China; 5Department of Medical Laboratory, Huizhou Central Hospital, Huizhou, Guangdong Province, People’s Republic of China; 6Department of Blood Transfusion, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi Province, People’s Republic of China; 7Jiangxi Shiningmed Medical Technology Ltd, Ganzhou, Jiangxi Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shao HuangJiangxi Shiningmed Medical Technology Ltd, Ganzhou, Jiangxi Province, People’s Republic of ChinaTel +86-18602004914Email huangshao@shiningmed.comTian-yu ZhongDepartment of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou 341000, Jiangxi, People’s Republic of ChinaTel +86-797-8680632Email zhongtianyu@126.comPurpose: Currently, thalassemia is commonly detected using gap-polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) reverse dot blot, which have high requirements of space, instruments, and personnel. Therefore, it is necessary to develop a new method for thalassemia detection with high sensitivity, low cost, and simple and fast operation. In this study, we aimed to design and evaluate a new method for detecting three α-thalassemia genes including –Southeast Asian (SEA), -α 3.7, and -α 4.2 and five β-thalassemia genes including 654M, 41/42M, − 28M, 17M, and 27/28M based on loop-mediated isothermal amplification (LAMP).Methods: Primer sequences were designed using Primer Explorer V4 software. Blood samples (5 mL) were collected from all participants in EDTA. DNA was extracted using Chelex 100 and was subjected to LAMP. LAMP products were detected by fluorescence development in ultraviolet light.Results: We found that LAMP assays for positive samples of thalassemia reached a plateau before 60 minutes, whereas the negative control samples entered the plateau after 70 minutes or showed no amplification. The concentration range of positive reactions was between 20– 60 pg/μL and 20– 60 ng/μL. Additionally, there were no cross-reactivities among 8 thalassemia subtypes. For clinical samples, the positive sample tube showed strong green fluorescence, whereas the negative tube showed light green fluorescence. According to these results, the LAMP method has high sensitivity for detecting thalassemia (252/254). However, 43 false-positive results were obtained in the LAMP test. The LAMP assay was also of low cost and with simple and fast operation.Conclusion: The novel LAMP assay can be completed within 60 min using a heating block or a water bath, and the result can be read visually based on color change to detect thalassemia. The LAMP assay fulfills the requirements of field application and resource-limited areas, especially those with primary hospitals and rural areas.Keywords: loop-mediated isothermal amplification, diagnosis, thalassemia genes, fast, low cost

Ganhua You, 1, 2 Xiangshu Long, 3, 4 Fang Song, 3, 4 Jing Huang, 3, 4 Maobo Tian, 3, 4 Yan Xiao, 3, 4 Shiyan Deng, 3, 4 Qiang Wu 3, 4 1Guizhou University School of Medicine, Guiyang 550025, People’s Republic of China; 2Guizhou Institute for Food and Drug Control, Guiyang 550004, People’s Republic of China; 3Department of Cardiology, Guizhou Provincial People’s Hospital, Guiyang 550002, People’s Republic of China; 4Department of Cardiology, People’s Hospital of Guizhou University, Guiyang 550002, People’s Republic of ChinaCorrespondence: Qiang WuDepartment of Cardiology, Guizhou Provincial People’s Hospital, 83 Zhongshan East Road, Guiyang, Guizhou, People’s Republic of ChinaTel +86-0851-85937194Fax +86-0851-85924943Email wqgz0851@126.comBackground: Metformin has been shown to inhibit the proliferation and migration of vascular wall cells. However, the mechanism through which metformin acts on atherosclerosis (AS) via the long non-coding RNA taurine up-regulated gene 1 (lncRNA TUG1) is still unknown. Thus, this research investigated the effect of metformin and lncRNA TUG1 on AS.Methods: First, qRT-PCR was used to detect the expression of lncRNA TUG1 in patients with coronary heart disease (CHD). Then, the correlation between metformin and TUG1 expression in vitro and their effects on proliferation, migration, and autophagy in vascular wall cells were examined. Furthermore, in vivo experiments were performed to verify the anti-AS effect of metformin and TUG1 to provide a new strategy for the prevention and treatment of AS.Results: qRT-PCR results suggested that lncRNA TUG1 expression was robustly upregulated in patients with CHD. In vitro experiments indicated that after metformin administration, the expression of lncRNA TUG1 decreased in a time-dependent manner. Metformin and TUG1 knockdown via small interfering RNA both inhibited proliferation and migration while promoted autophagy via the AMPK/mTOR pathway in vascular wall cells. In vivo experiments with a rat AS model further demonstrated that metformin and sh-TUG1 could inhibit the progression of AS.Conclusion: Taken together, our data demonstrate that metformin might function to prevent AS by activating the AMPK/mTOR pathway via lncRNA TUG1.Keywords: metformin, taurine up-regulated gene 1, AMPK/mTOR, autophagy, atherosclerosis

Xin Wang,1,* Ziwei Feng,1,* Yubei Huang,1 Haixin Li,1,2 Ping Cui,1 Dezheng Wang,3 Hongji Dai,1 Fangfang Song,1 Hong Zheng,1 Peishan Wang,1 Xuchen Cao,4 Lin Gu,5 Jin Zhang,6 Fengju Song,1 Kexin Chen1 1Department of Epidemiology and Biostatistics, National Clinical Research Center for Cancer，Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, People’s Republic of China; 2Department of Cancer Biobank, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, People’s Republic of China; 3Center for Non-Communicable Disease Control and Prevention, Tianjin Centers for Disease Control and Prevention, Tianjin 300011, People’s Republic of China; 4The First Department of Breast Cancer, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, People’s Republic of China; 5The Second Department of Breast Cancer, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, People’s Republic of China; 6The Third Department of Breast Cancer, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fengju Song; Kexin ChenTianjin Medical University Cancer Institute and Hospital, Huanhu Xi Road, Tiyuan Bei, Hexi District, Tianjin 300060, People’s Republic of ChinaTel/Fax +86-22-23372231Email songfengju@163.com; chenkexin@tjmuch.comPurpose: We aim to construct a nomogram to predict breast cancer survival and guide postoperative adjuvant chemotherapy in China.Patients and methods: A total of 5,504 breast cancer patients from the Tianjin Breast Cancer Cases Cohort were included. Multivariable Cox regression was used to investigate the factors associated with overall survival (OS) and a nomogram was constructed based on these prognostic factors. The nomogram was internal and external validated and the performance was evaluated by area under the curve (AUC) and calibration curve. The partial score was also constructed and stratified them into low, moderate and high-risk subgroups for death according to the tripartite grouping method. Multivariate Cox regression analysis and the propensity score matching method were respectively used to test the association between adjuvant chemotherapy and OS in different risk subgroups.Results: Age, diameter, histological differentiation, lymph node metastasis, estrogen, and progesterone receptor were incorporated into the nomogram and validation results showed this nomogram was well-calibrated to predict the 3-year [AUC =74.1%; 95% confidence interval (CI): 70.1–78.0%] and 5-year overall survival [AUC =72.3%; 95% CI: 69.6–75.1%]. Adjuvant chemotherapy was negatively associated with death in high risk subgroup [Hazard Ratio (HR) = 0.54; 95% CI: 0.37–0.77; P

Background: Carvacrol, a mono-terpenoid phenol found in herbs, such as oregano and thyme, has excellent antibacterial properties against Streptococcus pyogenes. However, its mechanism of bactericidal activity on S. pyogenes has not been elucidated. Objectives: This study investigated the bactericidal mechanism of carvacrol using three strains of S. pyogenes. Methods: Flow cytometry (FCM) experiments were conducted to determine carvacrol’s membrane permeabilization and cytoplasmic membrane depolarization activities. Protoplasts of S. pyogenes were used to investigate carvacrol’s effects on the membrane, followed by gel electrophoresis. The carvacrol-treated protoplasts were examined by transmission electron microscopy (TEM) to observe ultrastructural morphological changes. The fluidity of the cell membrane was measured by steady-state fluorescence anisotropy. Thin-layer chromatographic (TLC) profiling was conducted to study the affinity of carvacrol for membrane phospholipids. Results: Increased membrane permeability and decreased membrane potential from FCM and electron microscopy observations revealed that carvacrol killed the bacteria primarily by disrupting membrane integrity, leading to whole-cell lysis. Ultra-structural morphological changes in the membrane induced by carvacrol over a short period were confirmed using the S. pyogenes protoplast and membrane isolate models in vitro. In addition, changes in the other biophysical properties of the bacterial membrane, including concentration- and time-dependent increased fluidity, were observed. TLC experiments showed that carvacrol preferentially interacts with membrane phosphatidylglycerol (P.G.), phosphatidylethanolamine (P.E.), and cardiolipins (CL). Conclusions: Carvacrol exhibited rapid bactericidal action against S. pyogenes by disrupting the bacterial membrane and increasing permeability, possibly due to affinity with specific membrane phospholipids, such as P.E., P.G., and CL. Therefore, the bactericidal concentration of carvacrol (250 µg/mL) could be used to develop safe and efficacious natural health products for managing streptococcal pharyngitis or therapeutic applications.