Genomic epitopes of Xinjiang hemorrhagic fever virus and the evaluation of its laboratory testing value / 中华检验医学杂志

Objective:To grasp the distribution of fine antigenic epitope profiles of nucleoprotein (NP) and glycoprotein (GP) fragments of Crimean-Congo hemorrhagic fever virus (CCHFV) and to clarify the value of dominant antigenic epitopes in laboratory testing of Crimean-Congo hemorrhagic fever (CCHF).Methods:In a minimal synthetic short peptide consisting of 8 amino acids was segmentally expressed by CCHFV YL04057 strain using a modified bio-peptide synthesis method from 2014 to 2021 in the laboratory of Xinjiang University, College of Life Sciences. Using CCHFV polyclonal antibody or monoclonal antibody 14B7 (IgM) or CCHFV-positive sheep serum as antibodies, the minimal antigenic epitopes (BCEs) with antigenic activity on NP and GP fragments were identified by immunoblotting, and the obtained BCEs with sequence polymorphism were spatially clustered with CCHFV from different regions using the neighbor-joining method to determine the combination mode of BCEs with geographical correlation of regional distribution, to explore its application in establishing serological diagnosis. A prokaryotic expression plasmid (pET-32a), an E. coli expression plasmid (pGEX-KG) and a prokaryotic expression plasmid with an incomplete glutathione (GST188) tag (pXXGST-ST-1) were used to construct and express six dominant antigenic epitopes of different peptide lengths on NP fragments, and an indirect Enzyme-linked immunosorbent assay (ELISA) was established. CCHF sheep serum identified by immunofluorescence assay (IFA) was used as a control, and the specificity, sensitivity and overall compliance of the recombinant proteins with different peptide lengths of antigenic epitopes with IFA assay results were statistically analyzed. Results:CCHFV, NP and GP fragments had a total of 30 antigenically active BCEs, among which the core intermediate fragment NP2 (aa 170 th-305 th), which had a concentration of antigenic epitopes in the NP fragment, has 6 BCEs, and the NP1 (aa 1 st-200 th) and NP3 (aa 286 th-482 nd) at both ends have 9 BCEs; the Gc (aa 1 st-558 th) and Gn (aa 533 th-708 th) fragments of the GP fragment have 14 BCEs and a long antigenic peptide (AP) containing 15 amino acids, and the amino acid sequence homology of the NP fragment BCEs was 97.1% and that of the GP fragment BCEs was 89.1%. There was a significant difference ( P=0.0281, P<0.05). Among the 9 BCEs with sequence polymorphism in the GP fragment, 6 combined BCEs from GnEc1, GnE2, GnE4, GcE3, GcE6 and GcAP-4 (Ap) could cluster 15 CCHFV strains from different regions of the world into 5 geographical taxa, AsiaⅠ, AsiaⅡ, AficaⅠ, AficaⅡ and Europe. The constructs expressing PET-32a-NP (full length), PGEX-KG-NP2 (aa 170 th-305 th), pGEX-KG-NP2-1 (aa 235 th-275 th), PGEX-KG-NP2-1-1 (aa 237 th-256 th), pXXGST-1-NP2-1-2 (aa 250 th-265 th) and PGEX KG-NP2-1-3 (aa 260 th-276 th), six recombinant proteins CCHFV NP rabbit polyclonal antiserum (pAb) Western Blotting reaction positive, 33 sheep sera tested by IFA XHF as a reference, the sensitivity of the assay established by indirect ELISA using the recombinant proteins constructed from two fragments of NP2 and NP2-1 as antigens. The sensitivity, specificity and overall compliance were the best, with 73.4% (11/15) and 66.7% (10/15) for sensitivity, 100% (18/18) and 94.4% (17/18) for specificity, and 87.9% (29/33) and 81.8% (27/33) for overall compliance. Conclusion:CCHFV NP and GP are distributed with a high number of BCEs with antigenic immunoreactivity, among which the dominant antigenic epitopes are of high value in the laboratory serological diagnosis of CCHF.

Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying

Monoclonal antibodies (MAbs) are widely applied in disease diagnoses. Herein, we report a MAb, WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in an immunohistochemistry (IHC) assay. WF-4 was screened by immunofluorescence assay (IFA). The results showed that its reactivity with baculovirus-expressed full-length recombinant NP (rNP) in Western blot (WB), indicating its IHC applicability. Fifteen Influenza A virus (reference subtypes H1 to H15) infected chicken embryonated chorioallantoic membranes (CAM), fixed by formalin, were all detectable in the WF-4-based IHC assay. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreaks of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, a by-product of Influenza A virus preparation, is helpful for Influenza A virus-specific MAb characterization, and that the WF-4 MAb recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigens via IHC and may be suitable for developing various tests for diagnosis of Influenza A virus and, especially, AIV infection.

Detection of in vitro Inhibitory Effects of Boehmeria nivea Leaves Extracts on Influenza A Virus （H1N1）and Its Antioxidant Activity / 中国药房

OBJECTIVE： To study the in vitro inhibitory effects and antioxidant activity of different solvent extracts of Boehmeria nivea leaves against influenza A virus（H1N1）， and to expand the medicinal parts of B. nivea and develop natural antiviral and antioxidant drugs. METHODS： The leaves of B. nivea were extracted with 95％ ethanol. The ethanol extract was dissolved by water heating， and extracted with different solvents to obtain petroleum ether phase， trichloromethane phase， ethyl acetate phase， n-butanol phase and aqueous phase extracts of B. nivea leaves. The toxicity of aqueous extract of B. nivea leaves （50-400 μg/mL） on Madin-Darby canine kidney （MDCK） cell line was investigated. Using ribavirin as positive control， MDCK cells were attacked by influenza A virus（H1N1）. Western blotting assay was used to detect the expression of nucleoproteins （NP） in viral infected cells after treated with same concentrations of petroleum ether phase， trichloromethane phase， ethyl acetate phase， n-butanol phase and aqueous phase extracts of B. nivea leaves （100 μg/mL）， different concentrations of aqueous phase extract solution of B. nivea leaves （50， 100， 200， 400 μg/mL） and different concentrations of ribavirin solution （0.31， 0.63， 1.25 μg/mL）. Using vitamin C as a positive control， hydroxyl radical（·OH） scavenging test， DPPH radical scavenging test and reduction test were used to investigate in vitro antioxidant activity of the extracts. RESULTS： Aqueous phase extract of B. nivea leaves with concentration less than 400 μg/mL was nontoxic to MDCK cells. The petroleum ether phase， trichloromethane phase， ethyl acetate phase and aqueous phase extracts at 100 g/mL could significantly reduce the expression of NP protein in influenza A virus（H1N1） infected cells （P＜0.01）. Different concentrations （50-400 μg/mL） of aqueous extract could significantly reduce the protein expression of NP （P＜0.01） in concentration-dependent manner. The in vitro antioxidant activity of petroleum ether phase and ethyl acetate phase was similar to that of vitamin C. CONCLUSIONS： B. nivea leaves extract have better anti-influenza A virus（H1N1） effects in vitro， and the extracts of petroleum ether phase and ethyl acetate phase show good antioxidant activity in vitro.

Nucleoprotein vaccine induces cross-protective cytotoxic T lymphocytes against both lineages of influenza B virus

PURPOSE: The influenza B virus diverges into two antigenically distinct lineages: B/Yamagata and B/Victoria. Influenza B is the dominant circulating virus during some influenza seasons, and recent data demonstrated that influenza A and B infection similarly cause severe clinical symptoms in hospitalized patients. Nucleoprotein (NP) is a good target for a universal influenza vaccine. This study investigated whether NP epitope variation within two lineages affects the dominant cytotoxic T lymphocyte (CTL) responses induced by vaccination and the resultant protective immunity. MATERIALS AND METHODS: The NP of B/Yamagata/16/1988, the representative strain of the Yamagata lineage, includes a dominant CTL epitope, FSPIRITFL, while B/Shangdong/7/1997 from the Victoria lineage has one amino acid difference in this sequence, FSPIRVTFL. Two recombinant replication-deficient adenovirus (rAd)-vectored vaccines expressing either NP were prepared (rAd/B-NP(I) and rAd/B-NP(V), respectively) and administered to BALB/c mice intranasally. To examine the efficacy of vaccination, antibody responses, CTL responses, and morbidity/mortality after challenge were measured. RESULTS: Both vaccines induce similar antibody and CD8 T-cell responses cross-reacting to both epitopes, and also confer cross-protection against both lineages regardless of amino acid difference. CONCLUSION: The rAd-vectored vaccine expressing the NP could be developed as universal influenza B vaccine which provides broader protection.

Expression of Nupr1 in Gastric Cancer and its Clinical Significance / 胃肠病学

Background:The morbidity and mortality of gastric cancer remain high worldwide,and it is urgent to explore new targets for the diagnosis and treatment of gastric cancer. Nuclear protein 1 (Nupr1)has a variety of biological functions, especially in the tumorigenesis and development of the malignancies. Aims:To investigate the expression and clinical significance of Nupr1 in gastric cancer. Methods:Quantitative RT-PCR was used to determine the mRNA expression of Nupr1 in 72 cases of gastric cancerous tissue and the paired paracancerous tissue. Western blotting and immunohisto-chemistry were employed to detect the protein expression and cellular localization of Nupr1. The correlation of Nupr1 with the clinicopathological characteristics of gastric cancer was analyzed. Results:Expressions of Nupr1 mRNA and protein in gastric cancer were both significantly higher than those in paracancerous tissue (P 0. 05). Conclusions:Nupr1 is overexpressed and correlated with invasion, metastasis and progression of gastric cancer. It can be used as a novel biomarker for early diagnosis and prognosis evaluation,and as a potential target for treatment of gastric cancer.

The clinical significance of anti-nuclear protein B23 in systemic sclerosis / 中华风湿病学杂志

Objective To explore the clinical significance of anti-nuclear protein B23 antibody in systemic sclerosis. Methods Enzyme-linked immunosorbent assay was employed to detect the serum antinuclear B23 autoantibody. Mann-Whitney U test was used to compare the clinical and autoantibody profiles between SSc patients with B23 antibody and those without B23 antibody. Logistic regression analysis was employed to analyze the correlation between B23 antibody and clinical manifestations and autoantibody profiles. Results Mann-Whitney U test showed that, forced vital capacity (FVC) diffusion capacity of CO (DLco) in B23 positive SSc was significantly lower than that in B23 negative counterparts, pulmonary artery hypertension was more prevalent in B23 positive SSc patients. While anti-fibrillarin, anti-U1RNP, and antic entromere antibodies were more prevalent in B23 positive SSc. Multivariate logistic regression showed that anti-B23 antibody positivity was an independent risk factor for pulmonary artery hypertension in SSc (OR=123.92, 95％CI 26.67～575.66, P＜0.01), and a protective factor for severe gastrointestinal involvement (OR=0.08, 95％CI 0.01 ～0.70, P＜O.05). Logistic analysis showed that anti-B23 antibody was correlated with antifibrillarin (OR=11.50, 95％CI3.85～34.37, P＜0.01) and anti-U1RNP antibodies (OR=3.43, 95％CI 1.01～11.63, P＜0.05), and correlated with different degree of pulmonary artery hypertension. Conclusion The pulmonary artery pressure should be monitored closely in those SSc patients with a positive B23 antibody.

Dinámica del cmpplejo del poro nuclarr / Dinamicsof nucleear pore compexx

El complejo del poro nuclear (CPN) es un conjunto supra-molecular compuesto de múltiples copias de 30 familias de proteínas diferentes, siendo 456 nucleoporinas (Nups) en total que atraviesan la envoltura nuclear de todos los organismos pertenecientes al dominio Eukaria. El CPN es la compuerta del núcleo por lo tanto, todas las macromoléculas deben atravesarla para transitar del núcleo al citoplasma y viceversa. Durante los últimos años, se han propuesto varios modelos para explicar la regulación y el transporte de macromoléculas a través del CPN. En este escrito se describe la estructura, los mecanismos y procesos involucrados durante el transporte a través del CPN, y cómo estos procesos son regulados por interacciones macromoleculares altamente dinámicas.

The nuclear pore complex (NPC) is a large supramolecular assemble made up of multiple copies of about 30 different families proteins, so in total 456 nucleoporines (Nups) span the nuclear envelope of all Eukariotic organisms. The NPC is considered the gate through which all macromolecules must pass in order to achieve nucleo-cytoplasmic transport. The aim of this review is to describe the structure, mechanisms and processes involved during the transportation of molecules and how of these processes are regulated by highly dynamic macromolecular interactions with molecules of the NPC which have been described during the past years.

The effect of STAT3 decoy oligodeoxynucleotides on the expression of IFI16 in peripheral blood mononuclear cell of patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate the effect of signal transducer and activator of transcription 3 (STAT3) decoy oligodeoxynucleotides (ODNs) on the expression of IFI16 in the peripheral blood mononuclear cell of patients with systemic lupus erythematosus (SLE).Methods STAT3 decoy ODNs was transfected to PSMC in vitro,and RT-PCR was used to semiquantitatively analyze the IFI16 mRNA in the peripheral blood mononuclear cell (PBMC) before and after the transfection.Results The mean level of IFI16 mRNA in ac- tive SLE group and inactive SLE group were much higher than that of normal control.The difference was sig- nificant (P＜0.05).However,there was no statistical difference between active and inactive SLE group (P＞0.05).After being transfected by STAT3 decoy ODNs,the level of IFI16 mRNA in active and inactive SLE group were markedly decreased comparing with negative controls.The difference was significant (P＜0.05). However,the difference was not significant in normal control (P＞0.05).Conclusions The abnormal overex- pression of IFI16 mRNA in PBMC of patients with SLE suggests that IFI16 may contribute to the pathogenesis of SLE and the overexpression of IFI16 may be mediated through JAK-STAT3 pathway.

A clinical evaluation of nuclear matrix protein 22 in urine as an adjuvant diagnostic marker for transitional cell carcinoma of the upper urinary tract / 中华泌尿外科杂志

Objective To evaluate urinary nuclear matrix protein(NMP 22) as an adjuvant diagnostic marker for transitional cell carcinoma of the upper urinary tract. Methods 24 patients with transitional cell carcinoma of the upper urinary tract and 20 patients with benign urinary diseass were evaluated with urinary NMP 22(cutoff level 10U/ml) and voided urinary cytology.NMP 22 was determined using a commercial test kit. Results The sensitivity and specificity of NMP 22 were 87.5% and 85.0% respectively whereas these of cytology were 58.3% and 95.0%. Conclusions Urinary NMP 22 might be an useful adjuvant diagnostic marker for transitional carcinoma of the upper urinary tract.