OBJECTIVE:To establish the reference intervals of plasma Plasminogen, Factor XII activity, and Factor XIII Antigen in healthy adults in Guangzhou. METHODS:A total of 168 young people (75 males and 93 females, aged 18-65 years) who underwent physical examination in Zhujiang Hospital of Southern Medical University from 2020 to 2022 were recruited. Sysmex CS5100 automatic coagulation analyzer and matching reagents were used to detect Plasminogen. Factor XII activity and Factor XIII Antigen were detected using the ACL TOP 700 (Instrumentation Laboratory, Bedford, MA, USA) automatic coagulation analyzer and matching reagents; reference intervals were established. RESULTS:Plasma Plasminogen and Factor XIII Antigen were normally distributed, and plasma Factor XII activity showed a skewed distribution with no statistical significance in gender. The established reference intervals were as follows: Plasminogen: 71.6-123.0%; Factor XIII Antigen: 55.1-113.1%; Factor XII: 42.3-144.1%. CONCLUSION:The reference intervals for special coagulation items of the laboratory population in a particular area should be established to provide results that align with the population characteristics for assessing the coagulation status of clinical patients.

Stroke is the second leading cause of death worldwide. Acute ischemic stroke (AIS) patients often exhibit hypercoagulable state and gut microbiota dysbiosis. However, the association between coagulation abnormalities and gut microbiota dysbiosis in AIS patients and their predictive value for poor functional outcomes in AIS has not been investigated. Our study enrolled 95 AIS patients and 81 healthy controls, using 16S rRNA sequencing to analyze gut microbiota composition. Baseline fibrinogen level was found to be an independent risk factor for poor functional outcomes at 90-day follow-up (odds ratio = 2.16, 95% confidence interval: 1.02-4.59, P = 0.044). AIS patients showed significant gut microbiota dysbiosis, with significantly increased Parabacteroides and Alistipes, and decreased Prevotella and Roseburia, associated with coagulation indices. Furthermore, compared with AIS patients with normal coagulation function, those in a hypercoagulable state exhibited a significant increase in Alistipes and a decrease in Prevotella. We identified gut microbial biomarkers consisting of 15 bacteria that predicted poor functional outcome in AIS patients at 90-day follow-up. Coagulation indices improved the predictive performance of these biomarkers. In training and validation cohorts, area under the curve (AUC) values were 0.930 and 0.890 for microbial biomarkers alone, 0.691 and 0.751 for coagulation indices alone, and 0.943 and 0.944 for coagulation indices combined with gut microbial biomarkers. Our study showed that AIS patients with hypercoagulable state had gut microbiota dysbiosis, with Alistipes and Prevotella significantly associated with coagulation indices. A classification model based on coagulation indices and gut microbial biomarkers accurately predicted poor functional outcome in AIS patients at 90-day follow-up. IMPORTANCE:Acute ischemic stroke (AIS) patients often exhibit hypercoagulable state and gut microbiota dysbiosis. However, the relationship between hypercoagulable state and gut microbiota dysbiosis in AIS patients and their predictive value for poor functional outcomes has not been fully explored. Our study of 95 AIS patients showed that baseline fibrinogen level was an independent risk factor for poor functional outcome at 90-day follow-up in AIS patients. Hypercoagulable state in AIS patients correlates with gut microbiota dysbiosis. AIS patients with hypercoagulable state had increased Alistipes abundance and decreased Prevotella abundance. A classification model based on coagulation indices and gut microbial biomarkers accurately predicted poor functional outcome in AIS patients at 90-day follow-up.

OBJECTIVE:Recent studies indicated that transmembrane protein 40 (TMEM40) is associated with several types of cancers but is not clear in cervical cancer (CC). The study aimed to examine the role of TMEM40 in CC and related mechanisms.METHODS:The expression of TMEM40 in CC tissues and cell lines was studied with western blot and real-time quantitative RT-PCR. The effect of TMEM40 on proliferation was evaluated by CCK-8, EdU and colony formation assay. The migration, invasion, cell cycle and apoptosis of CC cells were studied with wound healing, transwell assays and flow cytometry. Tumor growth was evaluated in vivo using a xenogenous subcutaneously implant model.RESULTS:The results revealed that the TMEM40 elevation in CC tissues and cell lines was closely correlated with tumor size and lymph node metastasis in clinical patients. Upregulation of TMEM40 with OE-TMEM40 vector promoted the invasion, migration and proliferation, inhibited the apoptosis and led to distinct S cell cycle arrest in CC cell lines. Silencing TMEM40 with shRNA inhibited the invasion, migration and proliferation, promoted apoptosis and led to a G0/G1 cell cycle arrest in CC cell lines. Silence of TMEM40 downregulated the expression of c-MYC, Cyclin D1, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-9 (MMP-9), but in contrast, activated p53 and several apoptosis related proteins such as p53, Caspase-3, Caspase-9 and PARP1. In addition, TMEM40 silencing dramatically decreased tumor growth in mice models.CONCLUSION:The present study demonstrates that TMEM40 upregulation can be a potential prognostic biomarker and contribute to CC development.

Background: Enterovirus D68 (EV-D68) is an emerging pathogen in humans. EV-D68 causes a wide range of respiratory symptoms in children and has the propensity to cause severe complications. EV-D68 outbreaks are rarely investigated in mainland China. Therefore, in this study, we aimed to investigate the prevalence of EV-D68 in children and to describe the clinical manifestations as well as the phylogeny of EV-D68 in Guangdong Province from 2014 to 2018. Methods: Nasopharyngeal swabs were collected from hospitalized children with respiratory symptoms and screened for respiratory pathogens by fluorescence quantitative PCR and culture. The EV-positive samples were subsequently typed by sequencing the 5 '-untranslated region and EV-D68-specific VP1 capsid gene. A phylogenetic tree was constructed by the maximum-likelihood method based on the VP1 gene using ClustalW. Results: A total of 1,498 (59.8%) out of 2,503 children were screened positive for >= 1 virus species. Among the 158 (6.31%) EV-positive samples, 17 (0.68%) were identified as EV-D68. Most EV-D68 cases (n = 14) were diagnosed with pneumonia and bronchial pneumonia. No deaths were found in EV-D68 cases. Wheezing occurred in EV-D68 cases more frequently (70.59% vs. 43.26%, P = 0.040) than that of other EVs. All the EV-D68 were of clade B3, which were highly similar to the strains circulating in China. Conclusion: EV-D68 was the predominant enterovirus type in hospitalized children with respiratory symptoms in Guangdong Province. All the EV-D68 strains belong to clade B3. The development of diagnostic tools is warranted in order to monitor EV-D68 infections in China.

Nonstructural protein 1 (NS1) of the yellow fever virus (YFV) is an immunogenic protein. It is present predominantly as a dimer in the membranes of infected cells, and secreted from them as a soluble, detergent-labile hexamer. Little is known about the dynamics of NS1 secretion in animal and YFV-infected cells. After infection by YFV-17 D, the NS1 protein and RNA of the YFV in susceptible cells (C6/36 and Vero) and suckling mice models were detected by NS1 capture enzyme-linked immunosorbent assay and real-time reverse-transcription polymerase chain reaction, respectively. For suckling mice infected by the YFV, NS1 could be found in sera 2 days and RNA could be found 3 days after infection. For infected C6/36 and Vero cells, YFV NS1 could be detected 36 hafter infection whereas YFV RNA could be detected at48 hin culture supernatants. YFV NS1 protein can be used as a biomarker for the early diagnosis of YFV infection.

Objective To investigate the distribution of enterovirus (EV) serotypes of 759 cases in Guangzhou during 2014-2015.Methods Rectal swabs were collected from children with suspected infection of enterovirus and detected for pan-EV-,EV71-and Coxsackie virus A group 16 (CA16)-specific nucleic acid using real-time reverse transcription-polymerase chain reaction (RT-PCR).The serotype of EV-positive samples was identified using semi-nested RT-PCR and sequencing for VP1 gene product.Results A total of 759 (71.6 ％) were detected EV positive among the 1 060 specimens during 2014-2015.Eleven serotypes were identified circulating in both of years.The top five predominant serotypes were CA16,EV71,CA4,CA10,CB5 in2014 and CA6,EV71,CA10,CA16 and CA2 in 2015 respectively.CA16 and EV71 accounted for 77.1％ in 2014,contrasting that CA6 accounted for 51.7％ in 2015.The sex ratio ranged from 1.8 to 5.3.Children under 3 years old were more likely suffering from infection (60.1％,456/759).EV could be detected throughout the year with CA16,EV71,CA10 and CA2 peaked in May while CA6 in July.Clinical manifestation was mild in most infection cases.However,severe cases were observed in 12.7％ of EV71 infection and occasionally in CA16,CA10,CA4 and CA6 infections.Conclusion Eleven serotypes of EV,with different spectrum,circulated in Guangzhou during 2014-2015.Clinical manifestation in most EV infection were mild.

Objective:To ascertain whether the immune complexes (ICs) formed by Dengue virus 1 non-structure protein 1 (DENV1 NS1)and its IgG antibodies could mediate passive systemic anaphylaxis (PSA) and to explain the pathogenesis of Dengue hemorrhagic fever or Dengue shock syndrome (DHF/DSS).Methods:The monoclonal antibodies (mAbs) or mAb cocktails from 20 IgG mAbs of DENV1 NS1 prepared in this lab were screened to initiate PSA and passive cutaneous anaphylaxis (PCA) in mice.Meanwhile, the effects of GdCl3 and platelet activating factor ( PAF) antagonist CV-3988 on PSA induced by the NS1-IgG ICs were observed.Results:Two groups of monoclonal antibody cocktails with purified NS 1 were proved to be capable of provoking PCA and PSA in mice,whereas the other mAbs or mAb cocktails could be not .The murine PSA initiated by NS1-IgG(5D25+3B1) ICs could be sig-nificantly inhibited by in vivo treatment with GdCl3 or PAF antagonist CV-3988.Conclusion: The NS1-IgG ICs formed with DENV1 NS1 and IgG mAb cocktails can mediate PSA and PCA ,but not all of ICs formed by DENV 1 NS1 mAbs or mAb cocktails with DENV 1 NS1 can induce PSA ,indicating that it may be related to the special epitopes of DENV 1 NS1.The monocyte/macrophages and PAF may be as major effector cells and the major mediator for PSA induced by NS 1-IgG ICs,respectively.

Objective To study the viral etiologic characteristics of acute respiratory viruses associated with adults influenza like illness (ILI) case.Methods ILI cases and non-ILI cases of nose swabs specimen were collected from Zhujiang Hospital of Southern Medical University from Nov 2009 to Oct 2014.qRT-PCR was used to test for 18 respiratory viruses.Results 201 virus positive responses were detected in samples from 420 (47.9％) patients with influenza like illness,while only 3 cases (2.6％) were found to be infected with virus in 115 non-ILI cases.The most common virus identified was influenza virus (IFV) in frequency of 51.2％ (103/201),followed by human rhinovirus (RHV) and coronaviruses (Coy).There was no significant difference among each age group in virus detection rate,however,there were obvious differences in viruses detected of several age groups,which showed that the main inflect crowd of influenza was 18 to 30 age group,the main inflect crowd of human coronavirus was 51-77 age group,and the main inflect crowd of human rhinovirus was 51-90 age group.Conclusion ILI cases in adult mosdy were caused by infection with several kinds of respiratory virus,and the dominant virus for ILI was proved to be the seasonal influenza viruses.

Enterovirus (EV)-related hand, foot, and mouth disease/herpangina (HFMD/HA) has been prevalent in Guangdong Province, China, since 2010.

目的 以西尼罗病毒非结构蛋白(nonstructural protein1,NS1)为免疫原制备西尼罗病毒NS1蛋白特异性单克隆抗体,并鉴定其特异性.方法 在表达具有良好抗原性的重组西尼罗病毒NS1蛋白基础上免疫Balb/c小鼠,取免疫小鼠的脾细胞与小鼠骨髓瘤细胞进行融合,用间接ELISA法筛选阳性的杂交瘤细胞,并结合免疫荧光(IFA)和免疫印迹对所获得单克隆抗体的特异性进行鉴定,通过竞争抑制试验对mAb识别的抗原位点进行分析.结果 获得39株特异性针对西尼罗病毒NS1蛋白的单克隆抗体,Ig亚类测定结果IgG3和IgG2a单抗各2株,IgG2b单抗5株,IgG单抗1株,另外29株均为IgG1.结论 成功获得了特异性针对西尼罗病毒NS1蛋白的单克隆抗体,将为进一步建立西尼罗病毒NS1抗原检测方法及探讨NS1蛋白及抗体在西尼罗病毒发病机制中提供依据.

目的 以Ⅰ型登革病毒(DENV)非结构蛋白1(NS1)为免疫原制备NS1特异性单克隆抗体,建立Ⅰ型登革病毒NS1抗原特异性检测方法,并应用于广东以Ⅰ型登革感染为主的临床血清标本检测.方法 用原核表达的重组登革病毒NS1蛋白和灭活的Ⅰ型登革病毒免疫Balb/c小鼠,通过细胞融合,间接ELISA、免疫荧光和免疫印迹鉴定,获得DENV1NS1特异性单克隆抗体并建立检测方法.结果 共获得26株Ⅰ型登革病毒NS1蛋白特异性结合的单抗.通过对26株1型登革病毒NS1蛋白特异性单抗进行多株抗体组合配对,最终筛选出最佳抗体组合.该组合检测DENV1病毒培养上清的检测限为1∶16 384(12.5 pfu/ml),且与其他3个血清型登革病毒、乙型脑炎病毒和黄热病毒均无交叉反应,对500份健康人血清标本检测中,阴性标本497例,临界3例,对发病0～13d的836份临床确诊的登革热患者血清检测的敏感性为85.6％,明显高于qRT-PCR检测体系(66.3％)(P＜0.001),亦明显高于病毒分离诊断率(P＜0.001).结论 成功获得了1组DENV1-NSl蛋白的特异性单克隆抗体,利用该组抗体建立了1种敏感性和特异性更好的只针对DENV1-NS1蛋白的双抗体夹心检测方法,且比其他方法能够覆盖更宽泛的时间范围,该方法可作为登革热早期诊断的工具.

Human adenoviruses (HAdVs) are highly contagious pathogens causing acute respiratory disease (ARD), such as community-acquired pneumonia. HAdV-7d, a re-emergent genomic variant, has been recently reported in Asia and the United States after a several-decade absence. However, whether HAdV-7d is associated with higher severity than other types is currently unclear. In this study, the clinical and epidemiological investigation showed that fever, cough, and sore throat were the three most common respiratory symptoms of HAdV infections. HAdV-7 caused longer duration of fever, higher morbidity of tachypnea/dyspnea, pleural effusion, diarrhea, hepatosplenomegaly, consciousness alteration, as well as higher rates of pneumonia, mechanical ventilation and higher fatality rate (28.6%) than other types, particularly HAdV-3 and HAdV-2. The genomes of seven HAdV-7d isolates from mild, severe, and fatal cases were sequenced and highly similar with each other. Surprisingly, two isolates (2011, 2012) had 100% identical genomes with an earlier strain from a fatal ARD outbreak in China (2009), which elucidates the virus origin and confirms the unexpected HAdV genomic conservation and stability. Phylogenetic analysis indicated that L1 52/55-kDa DNA packaging protein may be associated with the higher severity of illness and fatality rate of HAdV-7. Clinicians need to be aware of HAdVs in children with ARD.

Background: A series of complications caused by enteroviruses, including meningitis, encephalitis, acute flaccid paralysis, acute cardiopulmonary failure, respiratory infection, and myocardial injury have been reported in hand, foot and mouth disease/herpangina (HFMD/HA). However, the complication of diarrhoea caused by enteroviruses has been neglected, and a summary of its clinical features and impact on HFMD/HA is unavailable.Methods: We included inpatients with HFMD/HA admitted to the Paediatric Department of Zhujiang Hospital during 2009-2012. We summarised and compared clinical data for cases with and without diarrhoea, and determined enterovirus serotypes by reverse transcriptase polymerase chain reaction and genotyping based on a partial-length fragment of viral protein 1 or the 5'-untranslated region.Results: There were 804 inpatients with HFMD/HA and 28 (3.5 %) presented with diarrhoea. Gastrointestinal symptoms were mild in most cases of diarrhoea (82.1 %), with high prevalence of no dehydration (82.1 %), short duration of diarrhoea (78.6 %) and watery stools (75.0 %). The prevalence of multi-organ dysfunction syndrome (10.7 vs 0.40 %) (p = 0.001), hepatic injury (14.3 vs 3.4 %) (p = 0.019), myocardial injury (21.4 vs 6.1 %) (p = 0.002) and convulsion (21.4 vs 7.2 %) (p = 0.016) was significantly higher in the diarrhoea than no diarrhoea group. There was no significant difference between the two groups regarding prevalence of death, altered consciousness, paralysis, central nervous system involvement, or acute respiratory infection.Conclusions: Most patients with diarrhoea caused by enteroviruses circulating in Guangdong Province in 2009-2012 had mild or moderate gastrointestinal symptoms. Although enterovirus-related diarrhoea caused additional multi-organ dysfunction syndrome, hepatic injury and myocardial injury in children with HFMD/HA, timely intervention efficiently reduced disease severity and improved outcome.

目的 初步了解广州地区儿童和成人病毒性腹泻的病原学构成和分布情况及相关病毒的分子特征.方法 收集2012年11月-2013年5月于珠江医院就诊的腹泻患者的粪便标本,采用免疫层析法检测轮状病毒和腺病毒抗原,实时荧光PCR检测诺如病毒GⅠ/GⅡ,上述检测阳性标本进行病毒基因测序分型.结果 290例患者中,3种常见病毒的检出率分别为21.4％、15.5％和1.7％;男性患者病毒阳性率显著高于女性患者(x2=0.017,P＜0.05);≤5岁患者轮状病毒阳性率最高,为28.49％(x2=0.017,P＜0.05),＞5岁患者诺如病毒阳性率最高,为21.62％;轮状病毒性腹泻秋冬季节高发;轮状病毒G/P分型以G1P8、G9P8、G2P4和G3P8为主,占92.16％,诺如病毒共检测出8种基因型,以GⅡ.4型为主,占53.33％;病毒合并其他常见腹泻病原体感染占病毒阳性标本的15.89％.结论 轮状病毒和诺如病毒分别是广州地区婴幼儿和成人病毒性腹泻的主要病原体,本地区流行的轮状病毒和诺如病毒优势株分别是G1P8和GⅡ.4,诺如病毒基因型多样性更丰富;混合感染常见,需引起重视.

目的 了解我国手足口病常见病毒感染患者血清中抗体交叉反应及保护性,为揭示手足口病二次感染保护机制提供依据.方法 收集2010-2014年珠江医院经荧光定量PCR确诊为手足口病患儿血清,用间接免疫荧光鉴定非EV71肠道病毒感染血清与EV71病毒感染细胞间结合,及EV71感染恢复期血清和抗EV71单克隆抗体与非EV71肠道病毒感染细胞的结合,对存在交叉结合反应的血清检测其交叉中和保护性.结果 共收集149例非EV71肠道病毒感染血清,经鉴定其中54份标本无EV71混合感染及既往感染,血清型分别为CA6、CA10、CA9、CA5、CA16、CB3、CB5及未分型肠道病毒.46份(85.2％)非EV71感染血清IgG抗体与EV71病毒感染细胞结合,具统计学意义(Z=-2.39,P=0.017),并对EV71病毒具弱中和活性.EV71感染血清及5株抗EV71单克隆抗体仅与CA16感染细胞结合.结论 初步确定多种非EV71肠道病毒感染血清与EV71病毒存在交叉反应,且具有弱中和活性;而EV71感染血清对其他肠道病毒无交叉保护作用.首次证明抗EV71 VP2、VP4单抗可结合CA16病毒.

Dengue virus (DENV) envelope [E] protein is the major surface protein of the virions that indued neutralizing antibodies. The domain III of envelope protein (EDIII) is an immunogenic region that holds potential for the development of vaccines; however, the epitopes of DENV EDIII, especially neutralizing B-cell linear epitopes, have not been comprehensively mapped. We mapped neutralizing B-cell linear epitopes on DENV-1 EDIII using 27 monoclonal antibodies against DENV-1 EDIII proteins from mice immunized with the DENV-1 EDIII. Epitope recognition analysis was performed using two set of sequential overlapping peptides (16m and 12m) that spanned the entire EDIII protein from DENV-1, respectively. This strategy identified a DENV-1 type- specific and a group-specific neutralizing epitope, which were highly conserved among isolates of DENV-1 and the four DENV serotypes and located at two regions from DENV-1 E, namely amino acid residues 309-320 and 381-392(aa 309-320 and 381-392), respectively. aa310 -319(310KEVAETQHGT319)was similar among the four DENV serotypes and contact residues on aa 309 -320 from E protein were defined and found that substitution of residues E309 , V312, A313 and V320 in DENV-2, -3, -4 isolates were antigenically silent. We also identified a DENV-1 type-specific strain-restricted neutralizing epitope, which was located at the region from DENV-1 E, namely amino acid residues 329-348 . These novel type- and group-specific B-cell epitopes of DENV EDIII may aid help us elucidate the dengue pathogenesis and accelerate vaccine design.

Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.

Objective To compare the detection efficiency between multiplex RT-PCR method and liquichip technology for screening the viral etiological agents of diarrhea.Methods The development of the multiplex RT-PCR method.A total of 107 feces samples from patients who suffered from diarrhea and attended to Zhujiang Hospital of Southern University from September 2013 to February 2014 were collected and tested in parallel by both multiplex RT-PCR and xTAG Gastrointestinal Pathogen Panel ( xTAG GPP) for Adenovirus, Norovirus genogroupⅠandⅡ, as well as by both multiplex RT-PCR and monoplex RT-PCR for Astrovirus and Sapovirus.To evaluate the sensitivity and specificity of multiplex RT-PCR, xTAG GPP and monoplex RT-PCR were used as reference.Kappa coefficient test was used to evaluate the consistency among the methods.The detection limit and accuracy of multiplex RT-PCR were evaluated by detection of serial dilution of positive plasmids and products sequencing for the five viral agents.Results The multiplex RT-PCR showed high consistency with xTAG GPP and monoplex RT-PCR, in which Kappa value was 0.885 and 1.000 respectively( P=0.000 ).Compared to xTAG GPP, the sensitivity and specificity of the multiplex RT-PCR were at average of 80.8%( 21/26 ) and 100%( 295/295 ) respectively.The detection limit and accuracy of multiplex RT-PCR were 104 copies /μl-106 copies/μl.Conclusion The high consistency indicated that both the multiplex RT-PCR and xTAG GPP are useful as a special,sensitive, high throughput and rapid diagnostic tools for the detection of the major viral pathogens related to diarrhea in clinical laboratory.