Prognostic value of m6 A “reader” YTHDF1 in OSCC / 安徽医科大学学报

Objective @# To investigate the correlation between the expression level of YTHDF1 in oral squamous cell carcinoma ( OSCC) and clinicopathologic features and its potential prognostic value．@*Methods @#The expression of YTHDF1 in 132 OSCC tissues and 66 paracancerous tissues was detected by immunohistochemistry (IHC) ，and the expression of YTHDF1 protein in OSCC cell lines was detected by Western blot．The correlation between YTHDF1 and clinicopathological features was analyzed by chi-square test．Kaplan-Meier and Cox factors were used to analyze the factors affecting the survival time of the patients and draw the survival curves of the YTHDF1 gene to evaluate its potential clinical significance. @*Results @#The expression of YTHDF1 in OSCC tissues was higher than that in para- cancerous tissues (P＜0. 001) ，and the expression of YTHDF1 protein increased in OSCC cell lines compared with normal oral epithelial keratinocytes (P ＜0. 001) ．The expression of YTHDF1 was correlated with the TNM stage and T stage of patients with OSCC (P＜0. 05) ，and the patients with high expression of YTHDF1 had a shorter sur- vival time compared with those with low expression (P ＜0. 001) ．@*Conclusion @# High expression of YTHDF1 may be associated with poor patient prognosis and YTHDF1 may be able to serve as a target for OSCC treatment.

Construction and gene identification of myeloid specific Spi1 knockout mice / 安徽医科大学学报

Objective @#To construct myeloid specific Spi1 gene knockout mice and analyze their genotypes , so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets .@*Methods @#According to the principle of CRISPR/Cas9 technology and C re/LoxP system , sgRNA and Donor vectors were de signed and constructed . The transcript of Exon 2 ( Exon 2) was used as the knockout region , and Loxp elements were placed on both sides of Exon 2 . Cas9 protein , sgRNA and Donor vector were mixed and microinj ected into the fertilized eggs of C57BL/6J mice , the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice , and F0 generation was obtained after 19 ~ 20 days . Positive F0 mice were mated with C57BL/6J mice to ob tain stable F1 Spi1 flox/ + mice . Spi1 flox/ + mice of F1 generation were selfed to obtain Spi1 flox/flox mice . Spi1 flox/flox mated with Lyz2-Cre + mice to obtain Spi1 flox/ + /Lyz2-Cre + mice , and then mated with Spi1 flox/flox , the Spi1 flox/flox/Lyz2-Cre + mice were myeloid specific Spi1 gene knockout ( KO) mice . Spi1 flox/flox/Lyz2-cre - mice were used as wild type (WT) mice . DNA of WT and KO mice was extracted , and the genotypes were identified by agarose gel electro phoresis after PCR amplification . Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene , Spi - 1 /purine rich box - 1(PU . 1) in immune cells of WT and KO mice .@*Results@#The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1 flox/flox homozygote , and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre + . Western blot showed that compared with WT group , the protein PU . 1 was not expressed in bone marrow derived macropha ges (BMDMs ) and peritoneal macrophages (PM) in KO group (P < 0.01) . There was no significant difference of statistics in the expression level of PU . 1 in T cells between KO mice and WT mice . The results of PCR and West ern blot showed that myeloid specific Spi1 KO mice were successfully constructed . @*Conclusion @#The myeloid spe cific Spi1 gene KO mice are successfully constructed and identified , which provides animal model basis for further revealing the potential mechanism of PU . 1 inimmune regulation .

Development and evaluation of a recombinant Bartonella henselae outer membrane protein (BHp26)-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of cat scratch disease

Aims@#The diagnosis of cat scratch disease (CSD), a disease caused by Bartonella henselae, is challenging and often hampered by the lack of appropriate laboratory assays in developing countries due to limited resources. Currently, the indirect immunofluorescence assay (IFA) is the mainstay for CSD diagnosis. However, IFA kits are costly as limited samples can be tested on one slide and reading of the immunofluorescence results is subjective. In this study, the sensitivity and specificity of a recombinant B. henselae outer membrane protein (BHp26)-based enzyme-linked immunosorbent assay (ELISA) was assessed for serodiagnosis purposes. @*Methodology and results@#Bartonella henselae outer membrane protein (BHp26) gene was cloned into a pBAD-TOPO expression plasmid and transformed into a TOP10 Escherichia coli host. The recombinant protein BHp26 was purified using an affinity chromatography approach in an AKTA purifier 10 system. The immunogenicity of the purified recombinant protein was evaluated using Western blot (WB). A recombinant outer membrane protein-based enzyme-linked immunosorbent assay (ELISA) was developed for detection against B. henselae antibodies in human sera. The recombinant protein-based ELISA demonstrated 57.7% agreement and 25% sensitivity as compared to IFA. A high specificity (94%) was exhibited when the ELISA was tested against 50 patients’ sera with positive findings to other infectious causes, including dengue, rickettsiae, leptospira, legionella and mycoplasma. Using the ELISA developed in this study, 14% (7/50) of urban blood donors and 9.1% (5/55) of healthy individuals from rural areas had IgG antibodies detected against B. henselae, suggesting previous exposure to the pathogen.@*Conclusion, significance and impact of study@#In view of the rising incidence of CSD, the recombinant outer membrane protein-based ELISA will be helpful for screening a large sample size of human sera for serosurveillance study.

Expression of TRIB3 and Prognostic Analysis in Siewert Type Ⅱ Adenocarcinoma of Esophagogastric Junction / 医学研究杂志

Objective To investigate the expression of pseudokinase Tribbles homology 3(TRIB3)and its clinical prognostic value in Siewert type Ⅱ adenocarcinoma of esophagogastric junction(AEG).Methods Western blot and immunohistochemical method were used to detect the expression of TRIB3 in R0 resected Siewert type Ⅱ AEG and its corresponding adjacent tissues,and analyze its rela-tionship with clinical parameters,survival and prognosis.Results Western blot analysis showed that the expression level of TRIB3 in Siewert type Ⅱ AEG tissues was significantly lower than that in the adjacent tissues(P<0.05).The immunohistochemical Results showed that the positive expression rate of TRIB3 in cancer tissues was significantly lower than that in adjacent tissues(P<0.01).The expression of TRIB3 was significantly correlated with the degree of differentiation,clinical TNM stage and lymph node metastasis(P<0.05),but not with age,gender and pathological morphology(P>0.05).Kaplan-Meier survival analysis showed that the long-term survival of patients with positive TRIB3 expression was significantly better than that of patients with negative TRIB3 expression(P<0.01).Univariate(HR =0.290,95%CI:0.110-0.761,P =0.012)and multivariate(HR =0.179,95%CI:0.051-0.630,P = 0.007)COX regression analysis showed that TRIB3 could be used as an independent prognostic factor for patients with Siewert type ⅡAEG(P<0.05).Conclusion TRIB3 may be involved in the occurrence and development of Siewert typeⅡ AEG.It is expected to be-come a new target for early diagnosis and treatment of AEG,and can be used as an important indicator for judging the prognosis of patients.

False positive reaction of COVID-19 antibody ELISA screening: internal logic of various methods / 中国输血杂志

【Objective】 To analyze the results of different methods for reactive samples screened by the enzyme linked immunosorbent assay (ELISA) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in blood donors. 【Methods】 From March to April 2020, a total of 8 632 blood samples in Shenzhen were screened for SARS-CoV-2 total antibodies (TAb, including IgG, IgM, IgA) in plasma using ELISA(PC group), the antibody reactivity samples and their follow up plasma samples (FC group), and samples of disease control group(DC group) from January to April 2020 were detected using the following methods: 1) ELISA method for detecting IgG, IgM, and (or without detection) TAb; 2) pseudovirus neutralizing antibody test(pVNT); 3) western blot (WB) of SARS-CoV-2 antibody. The negative control group(NC group) from February to April 2020 performed ELISA and WB testing. 【Results】 Among the 34 total antibody positive samples, 2 were positive for pVNT test, and the total antibody, IgG and WB in the initial screening and tracking testing were positive. Thereafter, it was determined to be confirmed positive. The other 2 cases were positive for pVNT test, while the samples with positive WB results were in the follow-up stage. The TAb, IgG, and pVNT results did not conform to the dynamic evolution of antibodies, and cannot be determined as confirmed positive. 【Conclusion】 The infection status of antibody reactivity samples screened by SARS-CoV-2 ELISA can be judged by the logic of pVNT, WB and the dynamic change of antibody.

ST3GAL5 inhibited malignant biological behavior of bladder cancer by activation of the PPAR pathway and inhibition of the PI3K/AKT pathway / 安徽医科大学学报

Objective @# To the effects and potential mechanisms of ST3GAL5 on biological behaviors of Bladder Urothelial Carcinoma(BLCA) . @*Methods @# Differentially expressed genes related to bladder cancer were identified using microarray analysis . Suitable bladder cancer cell lines were then screened . In vitro experimental measurements , including CCK8 , EdU , colony formation assays , transwell migration , flow cytometry apoptosis experiments , scratch assay , were used to evaluate the effects of ST3GAL5 on biological behaviors of BLCA . ST3GAL5 gene Kyoto Encyclopedia of Genes and Genomes ( KEGG) , gene set enrichment analysis ( GSEA) were analyzed using The Cancer Genome Atlas (TCGA) database . Finally , Western blot technology was used to verify the classical proliferation and metastasis related pathway factors . @*Results @# The combination of bioinformatics analyses and experimental measurements demonstrate that ST3GAL5 expression is aberrantly down⁃regulated in human cell lines of BLCA . Through Cancer Cell Line Encyclopedia (CCLE) database , HT⁃1376 cell lines were successfully screened for vitro test . Upregulation of ST3GAL5 was found to suppress the malignant biological behaviour of bladder cancer. GSEA enrichment analyses exhibited that ST3GAL5 and its co⁃expressed genes inhibited cell proliferation , invasion and metastasis of bladder urothelial carcinoma by activation of the PPAR pathway and inhibition of the PI3K/AKT pathway . The results of Western blot experiments verified that the key proteins of the PPAR signaling pathway showed a significant increase and the key proteins of the PI3K/AKT signaling pathway showed a significant decrease ( P <0. 05) after ST3GAL5 overexpression in bladder cancer. @*Conclusion @#ST3GAL5 gene might act as an oncogenic suppressor gene in bladder cancer , possibly inhibit the proliferation , invasion and metastasis of bladder cancer cells by activating the PPAR signaling pathway and inhibiting related molecules in the PI3K/AKT signaling pathway .

HIV prevalence and Western blot analysis of voluntary blood donors in Wuhu area / 中国输血杂志

【Objective】 To investigate the confirmatory status of HIV-1 antibody detection and Western blot (WB) test among voluntary blood donors in Wuhu, and to explore the strategies and methods to further ensure blood quality and safety. 【Methods】 Blood samples were preliminarily screened by ELISA and NAT, and the reactive samples were sent to Wuhu CDC for further WB test of HIV-1 antibody. The confirmation results of HIV-1 antibodies of voluntary blood donors in Wuhu in the past 10 years were retrospectively collected. The characteristics of WB bands of positive samples were analyzed, and the demographic characteristics of HIV-infected voluntary blood donors were sorted out. 【Results】 A total of 354 864 blood samples from voluntary blood donors in Wuhu during January 2011 to May 2021 were investigated, among which 42 were confirmed HIV positive (HIV-1 antibody positive in 41, and solo HIV-RNA reactive in 1), with a total HIV positive rate of 11.8/100 000(42/354 864). Statistical differences were found in gender [males 97.6% (41/42) vs females 2.4% (1/42)], marital status [unmarried 17.3/100 000 vs married 8.0/100 000] and occupation [staff/workers 37.5/100 000 vs students11.4/100 000 vs others 7.7/100 000]. Among the positive samples, the yield rate of WB bands gp160 was 100% (41/41), both gp41 and p24 were 97.6% (40/41),, and p55 was the lowest 46.3% (19/41). P51 and P66 presented the highest yield consistency (Kappa=1.000, P5 000 cps/mL by viral load (VL) testing, indicating HIV window period infection. 【Conclusion】 HIV infection statistically affected male donors more than females in Wuhu area, and most were early infection that revealed by WB band analysis. NAT plays an important role in the detection and confirmation of HIV infection during the window period, and is essential for blood safety.

Mechanism of antiplatelet aggregation of active fraction from sorghum roots / 中国药理学通报

Aim To study the mechanism of anti-plate- let aggregation of sorghum root active parts. Methods The effects of active fraction (WEAE-M 30%) from sorghum roots on platelet aggregation induced by collagen, thrombin and adenosine diphosphate were investigated in vitro. Western blot, enzyme-linked immunoas-say, flow cytometry and fluorescence techniques were used to explore the mechanism of the antiplatelet aggregation effect of WEAE-M 30% . Results WEAE-M 30% had a significant inhibitory effect on platelet aggregation induced by the three agonists mentioned above. The inhibitory effect on platelet aggregation induced by collagen was the most significant, with an inhibitory rate of (72. 91 ±2. 42)%. It was found that WEAE-M 30% had a significant inhibitory effect on the collagen- mediated platelet (IPVI signaling pathway protein Src, MAPK signaling pathway protein p38 and ERK phosphorylation. It also significantly inhibited the levels of ATP, P-selection and Ca2+ in platelets. Conclusions It is suggested that the mechanism of WE-AE-M 30% antiplatelet aggregation may be related to the inhibition of platelet activation pathway GPV1, MAPK and the release of typical platelet representative particles.

Prevalencia de cisticercosis en porcinos de la provincia de Tambopata, Perú / Cysticercosis Prevalence in Pigs in the Tambopata Province, Perú

Resumen La cisticercosis es una de las principales enfermedades zoonóticas parasitarias que es causada por el establecimiento de la forma larvaria de Taenia solium. Esta enfermedad se desarrolla principalmente en cerdos que son criados en granjas sin tecnificación, donde el uso de la tecnología y las condiciones sanitarias son mínimas. Este tipo de crianza es muy usual, por lo que representa un riesgo de la salud pública. En ese sentido, se determinó la prevalencia de cisticercosis en porcinos de la provincia de Tambopata, donde fue evaluado un total de 98 porcinos. Se tomaron aproximadamente 5 ml de sangre de la vena cava en animales mayores de 6 meses y hembras que no estuviesen preñadas; posteriormente, se obtuvo el plasma para ser procesado mediante la prueba de enzyme-linked inmunoelectrotransfer bloot assay (EITB) o Western Blot. Se determinó que el 17 % de los cerdos evaluados dio positivo para cisticercosis; con respecto al sexo, se obtuvo una seroprevalencia de 5,21 % ± 0,82 % para machos y 11,45 % ± 1,93 % para hembras. Finalmente, se determinó una seroprevalencia de 10,41 % ± 1,75 % para animales jóvenes de 6 a 11 meses y 6,25 % ± 1,01 % para animales adultos de 12 meses a más. Estos resultados reflejan la importancia de la vigilancia y control de las enfermedades parasitarias en los animales de producción ya que pudo corroborarse que la cisticercosis porcina constituye un serio problema de salud pública.

Abstract Cysticercosis is one of the main zoonotic parasitic diseases caused by the larval settlement of Taenia solium. This disease develops mainly in pigs that are reared in non-technified farms where the use of technology and the sanitary conditions are poor. It is quite usual to rear pigs this way and, therefore, there is a public health risk. In this sense, the cysticercosis prevalence was determined among pigs in the Tambopata Province, including 98 animals in the evaluation. Approximately 5 ml of blood were taken from the vena cava in more than 6-month-old female pigs that were not pregnant. Next, the plasma was taken in order to be processed under an enzyme-linked inmunoelectrotransfer bloot assay (EITB) or western blot. It was found that 17% of pigs were positive to cysticercosis. Regarding the sex, the seroprevalence was 5.21% ± 0.82% in males and 11.45% ± 1.93% in females. Finally, the seroprevalence was determined at 10.41% ± 1.75% in young animals (6 to 11 months old) and 6.25% ± 1.01% in adult animals (12 months old and above). These results show how important it is to monitor and control the parasitic diseases in production animals as this study confirmed that porcine cysticercosis is a serious problem in public health.

Reactividad de sueros de ratones inmunizados con vesículas de membrana externa derivadas de Salmonella entérica / Reactivity of sera from mice immunized with outer membrane vesicles derived from Salmonella enterica

La fiebre tifoidea causada por Salmonella Paratyphi A (fiebre paratifoidea) es indistinguible de la producida por Salmonella Typhi y el grado de incidencia ha aumentado en los últimos años, especialmente en el sudeste asiático. Por otro lado, la diarrea y otras complicaciones entéricas causadas por Salmonella Enteritidis y Salmonella Typhimurium continúan siendo un problema de salud grave, especialmente en países subdesarrollados. Las vacunas continúan siendo la forma más efectiva de prevenir estas enfermedades. Existen vacunas basadas en el polisacárido capsular de Salmonella Typhi que protegen contra la fiebre tifoidea; sin embargo, no hay vacunas efectivas licenciadas para uso en humanos que prevengan las enfermedades producidas por los serotipos de Salmonella no tifoideas. El desarrollo de una formulación con capacidad para proteger contra estas enfermedades sigue siendo un desafío para la comunidad científica. En este trabajo se evaluó, mediante Western blot, la reactividad de los sueros de ratones inmunizados por vía subcutánea con formulaciones basadas en vesículas de membrana externa derivadas de Salmonella Paratyphi A, Salmonella Enteritidis y Salmonella Typhimurium, contra los respectivos lisados celulares, para identificar la formulación que induce la mejor respuesta inmunológica cruzada. Los resultados obtenidos indicaron una alta reactividad de todos los sueros a los lisados, sin una diferencia aparente entre ellos. Sin lugar a dudas, se deberán realizar pruebas de inmunogenicidad seguidas de pruebas de retos cruzados para identificar un candidato vacunal. Estos resultados sugieren que las vesículas de membrana externa empleadas en este estudio están compuestas por antígenos posiblemente conservados en los tres serotipos de Salmonella y que pueden inducir una respuesta inmune de amplio espectro y protección cruzada(AU)

Typhoid fever caused by Salmonella Paratyphi A (paratyphoid fever) is indistinguishable from that caused by Salmonella Typhi and the degree of incidence has increased in recent years, especially in Southeast Asia. On the other hand, diarrhea and other enteric complications caused by Salmonella Enteritidis and Salmonella Typhimurium continue to be a serious health problem, especially in underdeveloped countries. Vaccines continue to be the most effective way to prevent these diseases. There are vaccines based on Salmonella Typhi capsular polysaccharide, which protects against typhoid fever; however, there are no effective vaccines licensed for use in humans to prevent disease caused by nontyphoidal Salmonella serotypes. Developing a formulation capable of protecting against these diseases remains a challenge for the scientific community. In this work, the reactivity of the sera of mice immunized subcutaneously with formulations based on Outer Membrane Vesicles (OMV) derived from Salmonella Paratyphi A, Salmonella Enteritidis and Salmonella Typhimurium, was evaluated by Western blot, against the respective cell lysates to identify the formulation that induces the best cross immune response. The results obtained indicated a high reactivity of all the sera to the lysates; without an apparent difference between them. Undoubtedly, immunogenicity tests followed by cross-challenge tests should be performed to identify a vaccine candidate. These results suggest that the OMV used in this study are composed of possibly conserved antigens in the three Salmonella serotypes and that they can induce a broad-spectrum immune response and cross protection(AU)