Metagenomic next-generation sequencing for the diagnosis of invasive pulmonary aspergillosis in type 2 diabetes mellitus patients.

Invasive pulmonary aspergillosis (IPA) in patients with diabetes mellitus has high incidence, especially in Type 2 diabetes mellitus (T2DM). The aim of this study was to evaluate the diagnostic efficacy of metagenomic next-generation sequencing (mNGS) for IPA in patients with T2DM. A total of 66 patients with T2DM were included, including 21 IPA and 45 non-IPA patients, from January 2022 to December 2022. The demographic characteristics, comorbidities, laboratory test results, antibiotic treatment response, and 30-day mortality rate of patients were analyzed. The diagnostic accuracy of mNGS and conventional methods was compared, including sensitivity, specificity, positive predictive value and negative predictive value. The sensitivity and specificity of mNGS were 66.7% and 100.0%, respectively, which were significantly higher than those of fluorescence staining (42.1% and 100%), serum 1,3-ß-D-glucan detection (38.1% and 90.9%), serum galactomannan detection (14.3% and 94.9%) and BALF galactomannan detection (47.3% and 70.7%). Although the sensitivity of BALF culture (75.0%) was higher than that of mNGS (66.7%), the turnover time of mNGS was significantly shorter than that of traditional culture (1.6 days vs. 5.0 days). The sensitivity of mNGS combined with BALF culture reached 100.0%. In addition, mNGS has a stronger ability to detect co-pathogens with IPA. 47.6% of T2DM patients with IPA were adjusted the initial antimicrobial therapy according to the mNGS results. This is the first study to focus on the diagnostic performance of mNGS in IPA infection in T2DM patients. MNGS can be used as a supplement to conventional methods for the diagnosis of IPA in patients with T2DM.

Application of peripheral blood routine parameters in the diagnosis of influenza and Mycoplasma pneumoniae.

OBJECTIVES: Influenza and Mycoplasma pneumoniae infections often present concurrent and overlapping symptoms in clinical manifestations, making it crucial to accurately differentiate between the two in clinical practice. Therefore, this study aims to explore the potential of using peripheral blood routine parameters to effectively distinguish between influenza and Mycoplasma pneumoniae infections. METHODS: This study selected 209 influenza patients (IV group) and 214 Mycoplasma pneumoniae patients (MP group) from September 2023 to January 2024 at Nansha Division, the First Affiliated Hospital of Sun Yat-sen University. We conducted a routine blood-related index test on all research subjects to develop a diagnostic model. For normally distributed parameters, we used the T-test, and for non-normally distributed parameters, we used the Wilcoxon test. RESULTS: Based on an area under the curve (AUC) threshold of ≥ 0.7, we selected indices such as Lym# (lymphocyte count), Eos# (eosinophil percentage), Mon% (monocyte percentage), PLT (platelet count), HFC# (high fluorescent cell count), and PLR (platelet to lymphocyte ratio) to construct the model. Based on these indicators, we constructed a diagnostic algorithm named IV@MP using the random forest method. CONCLUSIONS: The diagnostic algorithm demonstrated excellent diagnostic performance and was validated in a new population, with an AUC of 0.845. In addition, we developed a web tool to facilitate the diagnosis of influenza and Mycoplasma pneumoniae infections. The results of this study provide an effective tool for clinical practice, enabling physicians to accurately diagnose and differentiate between influenza and Mycoplasma pneumoniae infection, thereby offering patients more precise treatment plans.

Elevated serum levels of human epididymis protein 4 in adult patients with proliferative lupus nephritis.

Background: This study aimed to access whether serum human epididymis protein 4 (HE4) level could identify lupus nephritis (LN) pathological classes in adults and children. Methods: The serum HE4 levels of 190 healthy subjects and 182 patients with systemic lupus erythematosus (SLE) (61 adult-onset LN [aLN], 39 childhood-onset LN [cLN], and 82 SLE without LN) were determined using Architect HE4 kits and an Abbott ARCHITECT i2000SR Immunoassay Analyzer. Results: Serum HE4 level was significantly higher in the aLN patients (median, 85.5 pmol/L) than in the patients with cLN (44 pmol/L, P < 0.001) or SLE without LN (37 pmol/L, P < 0.001), or the healthy controls (30 pmol/L, P < 0.001). Multivariate analysis showed that serum HE4 level was independently associated with aLN. Stratified by LN class, serum HE4 level was significantly higher in the patients with proliferative LN (PLN) than in those with non-PLN, and this difference was found only in aLN (median, 98.3 versus 49.3 pmol/L, P = 0.021) but not in cLN. Stratified by activity (A) and chronicity (C) indices, the aLN patients with class IV (A/C) possessed significantly higher serum HE4 levels than those with class IV (A) (median, 195.5 versus 60.8 pmol/L, P = 0.006), and this difference was not seen in the class III aLN or cLN patients. Conclusion: Serum HE4 level is elevated in patients with class IV (A/C) aLN. The role of HE4 in the pathogenesis of chronic lesions of class IV aLN needs further investigation.

Metagenomic next-generation sequencing for the diagnosis of Pneumocystis jirovecii Pneumonia in critically pediatric patients.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of metagenomic next-generation sequencing (mNGS) for the diagnosis of Pneumocystis jirovecii Pneumonia (PCP) in critically pediatric patients. METHODS: Seventeen critically pediatric patients with PCP and sixty patients diagnosed with non-PCP pneumonia who were admitted in pediatric intensive care unit between June 2018 and July 2021 were enrolled. Conventional methods and mNGS for detecting Pneumocystis jirovecii (P. jirovecii) were compared. The patients' demographics, comorbidities, laboratory test results, antibiotic treatment response and 30 day mortality were analyzed. RESULT: The mNGS showed a satisfying diagnostic performance with a sensitivity of 100% in detecting P. jirovecii compared with Gomori methenamine silver staining (5.9%), serum (1,3)-ß-D-glucan (86.7%) and and LDH (55.6%). The diagnostic specificity of mNGS for PCP was higher than that of serum BDG (56.7%) and LDH (71.4%). In PCP group, over one thirds' cases had mixed infections. Compared with survivors, non-survivors had higher stringently mapped read numbers (SMRNs) in bronchoalveolar lavage fluid (BALF) sample (P < 0.05), suggesting SMRNs were closely associated with the severity of response. The detection for P. jirovecii by mNGS both in BALF and blood samples reached a concordance rate of 100%, and the SMRNs in the BALF were remarkably higher than that in blood samples. Initial antimicrobial treatment was modified in 88.2% of PCP patients based on the mNGS results. CONCLUSION: The mNGS is a potential and efficient technology in diagnosing PCP and shows a satisfying performance in the detection of co-pathogens. Both blood and BALF samples for mNGS are suggested for the presumptive diagnosis of PCP.

An online alpha-thalassemia carrier discrimination model based on random forest and red blood cell parameters for low HbA2 cases.

BACKGROUND: Since screening of α-thalassemia carriers by low HbA2 has a low positive predictive value (PPV), the PPV was as low as 40.97% in our laboratory, other more effective screening methods need to be devised. This study aimed at developing a machine learning model by using red blood cell parameters to identify α-thalassemia carriers from low HbA2 patients. METHODS: Laboratory data of 1213 patients with low HbA2 used for modeling was randomly divided into the training set (849 of 1213, 70%) and the internal validation set (364 of 1213, 30%). In addition, an external data set (n = 399) was used for model validation. Fourteen machine learning methods were applied to construct a discriminant model. Performance was evaluated with accuracy, sensitivity, specificity, etc. and compared with 7 previously published discriminant function formulae. RESULTS: The optimal model was based on random forest with 5 clinical features. The PPV of the model was more than twice the PPV of HbA2, and the model had a high negative predictive value (NPV) at the same time. Compared with seven formulae in screening of α-thalassemia carriers, the model had a better accuracy (0.915), specificity (0.967), NPV (0.901), PPV (0.942) and area under the receiver operating characteristic curve (AUC, 0.948) in the independent test set. CONCLUSION: Use of a random forest-based model enables rapid discrimination of α-thalassemia carriers from low HbA2 cases.

Age-related changes in the TRB and IGH repertoires in healthy adult males and females.

A diverse immune repertoire is capable of recognizing the enormous universe of foreign antigens encountered over life. Aging has a profound impact on the immune repertoires. However, whether continuous age-related changes in the immune repertoires differ between sexes is unclear. In this study, the characteristics of immune repertoires stratified by sex during aging are deciphered by analyzing T-cell receptor ß-chain (TRB) and immunoglobulin heavy chain (IGH) sequences in 361 healthy adults. A similar change was observed between males and females across their lifespan, whereas age-subgroup analysis revealed sex-specific signatures in TRB and IGH repertoires. In regard to TRB, in males, repertoire richness and evenness increases slightly before the age of 32 years and 45 years respectively, and decreases sharply thereafter. Intriguingly, in females, they decrease significantly until around the age 57 years old, and subsequently undergo a stable stage up to the age of 83 years. Although IGH repertoire evenness increases significantly with age in both sexes, richness decreases significantly with age in males but remains stable in females. Moreover, average length of IGH CDR3 increases with age. In conclusion, these findings provide fundamental insights into the mechanisms underlying sex differences in adaptive immunity.

Prenatal Genetic Counseling in a Chinese Pregnant Woman With Rare Thalassemia: A Case Report.

Background: Prenatal genetic counseling can be difficult, especially when it is related to fetuses with a rare thalassemia. An intronic variant located far from obvious regulatory sequences in the HBB gene could be very difficult to evaluate as it may affect the mRNA processing or cause ß-thalassemia (ß-thal). In the present study, a Chinese pregnant woman with HbJ-Bangkok and a very rare change in the second intron of the HBB gene [IVS-II-806(G>C), NM_000518.4, HBB: c.316-45G>C] in combination with α+-thalassemia was reported, which can assist in prenatal genetic counseling. Case Report: A 26-year-old pregnant woman presented at the obstetric clinic for a routine pregnancy check at 12 weeks of gestation. Red blood counts and high-performance liquid chromatography (HPLC) were consistent with clinical manifestations of anemia. Multiplex gap-polymerase chain (gap-PCR) displayed rightward deletion (-α3.7/αα). Direct DNA sequencing of the δ-globin gene showed no mutation. Sanger sequencing of the ß-globin gene showed a previously undescribed condition of double heterozygosity for HbJ-Bangkok and a very rare change in the second intron of the HBB gene [IVS-II-806(G>C), NM_000518.4, HBB: c.316-45G>C] that has not been previously reported in the HbVar database. Thus, a rare combination of α+-thal and a compound heterozygosity of HbJ-Bangkok and [IVS-II-806(G>C)] with α+-thal (-α3.7/αα) was finally diagnosed. Prenatal genetic counseling was made based on the genotype and phenotype analyses. Conclusion: This study enlarges the mutation spectrum of ß-globin gene and emphasizes DNA analysis in resolving unusual patterns in Hb analysis and the importance of sharing the observed rare undefined mutations and the possible interactions with known molecular defects, which can assist in prenatal genetic counseling.

High Efficiency and Problems of Chemiluminescence Assay-Detected Aldosterone-To-Renin Ratio in Practical Primary Aldosteronism Screening.

Primary aldosteronism is a main cause of secondary hypertension which can be effectively treated. The screening test for primary aldosteronism is benefit for minimizing damage to the patient. In the previous retrospective study, we obtained the optimal cutoff value of aldosterone-to-renin ratio detected by chemiluminescence assay, a newly developing method, and prompted its high efficiency in primary aldosteronism screening in upright position. In this study, we want to evaluate its efficiency in practical work. We used this ratio to continuously screen 238 patients, and 58 patients were finally diagnosed with primary aldosteronism. We found it had 86.13% accuracy rate in the upright position compared with the final clinical diagnosis. False negative and positive rates were 13.79% and 13.89%. Diagnostic sensitivity and specificity were 86.21% and 86.11%, which are slightly different from results in our previous study. False negative rate can be improved by combining the aldosterone-to-renin ratio with aldosterone concentration. We also found impaired glucose tolerance may be a reason for high false positive rate. Besides, chemiluminescence assay may be interfered in aldosterone detection. Although it has some shortcomings, chemiluminescence assay-detected aldosterone-to-renin ratio is a highly effective index for screening primary aldosteronism in practice.

Clinical value of interferon-γ release assay in the diagnosis of active tuberculosis.

Sensitivity and specificity of the interferon-γ release test for active tuberculosis screening were evaluated. Due to the high-test cost of imported IGRAs, QFT-GIT and T-SPOT.TB, we applied a cheaper domestic TB-IGRA which was approved in China recently. We recruited 740 patients and performed tuberculosis interferon release test (IGRAs), detection of Mycobacterium tuberculosis IgG antibody (TB-IgG) and tuberculin skin test (TST). The sensitivity of the three methods are 90.8, 40.0 and 75.45%, with specificity of 76.62, 74.47 and 72.27%. The area under the ROC curve according to the value of T-N detected by IGRAs was 0.878 (95% CI, 0.839-0.917), with the area under the curve for the diagnosis of active pulmonary tuberculosis and extrapulmonary tuberculosis being 0.839 and 0.841 respectively. The interferon-γ release test seems to be superior to TST and TB-IgG as a screening tool for the detection of active tuberculosis in China.

miR-300 regulates the epithelial-mesenchymal transition and invasion of hepatocellular carcinoma by targeting the FAK/PI3K/AKT signaling pathway.

Several microRNAs (miRNAs) have been closely correlated with the development of hepatocellular carcinoma (HCC). However, the involvement of miR-300 in the development of HCC remains unknown. This study elucidated the potential molecular mechanisms of miR-300 in the modulation of the epithelial-mesenchymal transition (EMT) and invasion of HCC. The expression levels of miR-300 in HCC cells and clinical samples were detected by quantitative real-time PCR and in situ hybridization. The in vitro function of miR-300 in HCC was evaluated using a migration/invasion assay. Quantitative real-time PCR, western blotting, immunofluorescence and immunohistochemistry were used to validate the roles of miR-300 and FAK/PI3K/AKT in EMT progression. A dual-luciferase reporter assay was performed to confirm the target gene. miR-300 was down-regulated in HCC and significantly correlated with a poor prognosis in HCC patients. The down-regulation of miR-300 increased the invasiveness of the HCC cells, and promoted the EMT in both HCC tissues and HCC cells. In contrast, up-regulation of miR-300 led to the opposite results. Ectopic overexpression of miR-300 reversed TGF-ß1-induced EMT in SMMC-7721 cells, and according to a dual-luciferase reporter assay and rescue assay, miR-300 inhibits the EMT-mediated migration and invasion of HCC cells via the targeted modulation of FAK and the downstream PI3K/AKT signaling pathway. miR-300 targeting modulates FAK, and the PI3K/AKT signaling pathway inhibits the EMT and suppresses the migration and invasion of HCC cells. Thus, miR-300 represents a promising therapeutic target for HCC.

Prognostic significance of c-Met, ß-catenin and FAK in patients with hepatocellular carcinoma following surgery.

The present study aimed to investigate the prognostic value of specific molecular markers in patients with hepatocellular carcinoma (HCC) who had received surgery. Immunohistochemical analysis was used to measure the expression of hepatocyte growth factor receptor (c-Met), ß-catenin and focal adhesion kinase (FAK) in patients with HCC. c-Met expression was identified to be high in patients with larger tumors, higher α-fetoprotein (AFP) levels, higher Edmondson grades, portal vein invasion and higher tumor-node-metastasis (TNM) stages. FAK expression was high in patients with portal vein invasion, higher Edmondson grades and higher TNM stages. ß-catenin expression was high in patients with larger tumors, hepatitis B virus (HBV) infection, portal vein invasion, higher Edmondson grades and higher TNM stages. Following multivariate analysis, FAK (P=0.002) and ß-catenin (P=0.006) expression levels were demonstrated to be significantly associated with Edmondson grade. Additionally, the tumor size (P=0.009) and HBV infection status (P=0.002) were revealed to be associated with ß-catenin expression. Kaplan-Meier survival curve analysis demonstrated that patients with HCC with higher FAK expression, higher ß-catenin expression, portal vein invasion, higher Edmondson grades, higher TNM stages, younger ages and higher AFP levels had significantly poorer prognoses. Cox's regression analysis revealed that the survival period was correlated with the Edmondson grade, age, AFP level, and FAK and ß-catenin expression. Univariate analysis of c-Met, ß-catenin and FAK identified a significant correlation between FAK and ß-catenin (P=0.015). Correlation analysis revealed no significant correlation between the three molecular markers, but ß-catenin and c-Met were markedly correlated (P=0.052). No significant correlation between FAK, c-Met or ß-catenin expression was identified. FAK and ß-catenin expression demonstrated a correlation with a range of clinicopathological factors, and high FAK and ß-catenin expression levels were identified to be correlated with a poor survival rate of patients with HCC. Thus, patients with higher FAK and ß-catenin expression may require more aggressive therapy. The results of the present study suggest that FAK and ß-catenin expression possess more prognostic value than c-Met expression in patients with HCC.

miR-338-3p inhibits epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma cells.

Down-regulation of the miRNA miR-338-3p correlates with the invasive ability of hepatocellular carcinoma (HCC) cells. However, it is currently unclear whether down-regulation of miR-338-3p induces epithelial-mesenchymal transition (EMT), which may be the underlying mechanism governing HCC invasion. Here, we demonstrate that restoration of miR-338-3p expression via transfection of a miR-338-3p mimic reversed EMT and inhibited the motility and invasiveness of HCC cells. Conversely, silencing of endogenous miR-338-3p expression with a miR-338-3p-specific inhibitor induced EMT and enhanced HCC cell motility. Additionally, Snail1 (an upstream regulatory protein of EMT) and Gli1 (a key transcription factor in the sonic hedgehog (SHH) signaling pathway) expression was up-regulated in cells treated with the miR-338-3p inhibitor and down-regulated by the miR-338-3p mimic. Further analyses demonstrated that miR-338-3p inhibitor-induced EMT in HCC cells was blocked by treatment with a small interfering RNA (siRNA) targeting Snail1, that the SHH signaling pathway was required for both miR-338-3p inhibitor-induced EMT and up-regulation of Snail1, and that miR-338-3p targeted a sequence within the 3'-untranslated region of N-cadherin mRNA. Notably, miR-338-3p expression was significantly down-regulated in HCC samples from patients with metastases and was associated with poor metastasis-free survival rates. Lastly, correlations between the expression levels of miR-338-3p and E-cadherin, Smoothened (SMO), Gli1, Snail1, N-cadherin, and vimentin were confirmed in HCC xenograft tumors and HCC patient specimens. Our findings suggest that miR-338-3p suppresses EMT and metastasis via both inhibition of the SHH/Gli1 pathway and direct binding of N-cadherin. miR-338-3p is a potential therapeutic target for metastatic HCC.

MicroRNA-24 increases hepatocellular carcinoma cell metastasis and invasion by targeting p53: miR-24 targeted p53.

MicroRNA-24 (miR-24), a member of the miRNA family, functions as an oncogene in various types of human cancer. However, the underlying mechanisms of miR-24 involvement in the development and progression of hepatocellular carcinoma (HCC) remain poorly understood. The present study revealed that miRNA-24 down-regulates p53 through binding to the 3'-UTR of p53 mRNA based on a luciferase reporter assay, and that the expression level of miR-24 could affect the invasion of HCC lines via p53. Down-regulation of p53 significantly attenuated the inhibitory effects of miR-24 knockdown on the invasion of HCC cells, suggesting that miR-24 could be a potential target for HCC treatment. Moreover, our results revealed that miR-24 expression was significantly increased in HCC metastatic tumor tissues compared with matched non-metastatic tumor tissues, and that the up-regulation of miR-24 was significantly associated with down-regulation of p53 in the HCC tissues. In conclusion, this study demonstrates that miR-24 functions as an oncogene in HCC, at least partly by promoting cell invasion through down-regulation of p53. Therefore, miR-24 may be a potential therapeutic target for treatment of HCC.

Schistosoma japonicum calcium-binding tegumental protein SjTP22.4 immunization confers praziquantel schistosomulumicide and antifecundity effect in mice.

A family of platyhelminth tegument-specific proteins comprising of one or two calcium ion binding EF-hand and a dynein light chain-like domain, termed tegumental proteins, are considered as candidates of vaccine. In this study, we cloned and characterized SjTP22.4, a novel membrane-anchored tegumental protein in Schistosoma japonicum with theoretic MW of 22.4. The recombinant SjTP22.4 could be recognized by S. japonicum infected sera. Immunofluorescence revealed that this protein is not only located on the surface of tegument of adult and schistosomulum and cercaria, but also in the parenchymatous tissues and intestinal epithelium. Circular dichroism (CD) measurement demonstrated rSjTP22.4 had Ca(2+)-binding ability. The rSjTP22.4 vaccination without adjuvants produced comparable high level of antibody with that of immunization with adjuvants together indicated it was an antigen of strong antigenicity. The level of IgG1 is much higher than that of IgG2a and IgE is nearly negative in S. japonicum-infected and rSjTP22.4 immunized mice. In cercaria challenge experiment, mice vaccinated with SjTP22.4 showed no reduction in adult burden and egg production, comparing with the control mice, but 41% decrease in egg mature rate and 32% reduction in liver egg granuloma area. However, the SjTP22.4 immunized mice that received praziquantel treatment at 10d post infection caused 26% reduction in adult burden and 53% decrease in egg mature rate, comparing with the control mice only received praziquantel treatment. In conclusion, SjTP22.4 is a valuable vaccine candidate for S. japonicum of anti-pathogenesis and anti-transmission effect and plays a synergetic role in praziquantel to kill schistosomulum.

The topological structure and function of Echinococcus granulosus lactate dehydrogenase, a tegumental transmembrane protein.

Lactate dehydrogenase (LDH), a terminal glycolytic enzyme, is generally considered as a cytosolic protein. We cloned lactate dehydrogenase from Echinococcus granulosus (EgLDH) and predicted it may be a membrane protein with two transmembrane regions through bioinformatics analysis. Intact worm immunofluorescence with antibodies prepared against linear B cell epitopes predicted in the region inside or outside of the membrane demonstrated that EgLDH spans the tegumental membrane twice, with the N terminal and C terminal all outside, just consistent with the putative topological structure. Then, the enzymatic characteristics and kinetic parameters of recombinant EgLDH were surveyed and the results suggested that EgLDH is responsible for catalyzing the reduction of pyruvic acid into lactic acid under physiological conditions. The enzymatic activity of the recombinant protein was inhibited by antibodies directed against the intact protein or against epitopes that contain key residues in the catalytic center or substrate binding sites. EgLDH is a potential target for drugs and vaccines against E. granulosus.

Suppression of RNA interference pathway in vitro by Grass carp reovirus.

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

Gene cloning, expression, and localization of antigen 5 in the life cycle of Echinococcus granulosus.

Antigen 5 (Ag5) has been identified as a dominant component of cyst fluid of Echinococcus granulosus and is considered as a member of serine proteases family, which in other helminth, plays an important role in the egg hatch and larva invasion. However, whether Ag5 is expressed and secreted in all life stages is unknown. In this study, according to the sequence in GenBank, we cloned and sequenced the open reading frame (ORF) of Ag5 gene from the protoscolices of E. granulosus isolated from the sheep in Qinhai Province of China, and found several substitutions and a base insert and deletion in a short region near the stop code, leading to a frameshift mutation which is conserved with the homologue of other cestode. The ORF is 1,455 bp in length, encoding 484 amino acids with a secretory signal peptide. Bioinformatics analysis predicted several phosphorylation and myristoylation sites and a N-glycosylation site and a species-specific linear B epitope in the protein. The ORF was cloned into the plasmid pET28a(+) vector and expressed in Escherichia coli . The recombinant protein was purified by affinity chromatography. Anti-rEgAg5 antiserum was prepared in rats and used to analyze the localization of Ag5 in protoscolex and adult worm by immunofluorescence technique. Results demonstrated that the Ag5 is strongly expressed in the tegument of protoscolex and the embryonic membrane of egg and surface of oncosphere; meanwhile, it is also weakly expressed in tegument of the adult. This study showed that Ag5 is expressed in all stages of life cycle, secreted from the surface of the worm and may be anchored in membrane by its myristoylation sites; these characteristics make it a candidate antigen for diagnosis and vaccine for both intermediate and definitive hosts.

The use of an in vitro microneutralization assay to evaluate the potential of recombinant VP5 protein as an antigen for vaccinating against Grass carp reovirus.

BACKGROUND: Grass carp reovirus (GCRV) is the causative pathogen of grass carp hemorrhagic disease, one of the major diseases damaging grass carp Ctenopharyngon idellus breeding industry in China. Prevention and control of the disease is impeded largely due to the lack of research in economic subunit vaccine development. This study aimed to evaluate the potential of viral outer shell protein VP5 as subunit vaccine. METHODS: The vp5 gene was isolated from the viral genome through RT-PCR and genetically engineered to express the recombinant VP5 protein in E coli. The viral origin of the recombinant protein was confirmed by Western blot analysis with a monoclonal antibody against viral VP5 protein. Polyclonal antibody against the recombinant VP5 protein was prepared from mice. A microneutralization assay was developed to test its neutralizing ability against GCRV infection in cell culture. RESULTS: The GST-VP5 fusion protein (rVP5) was produced from E. Coli with expected molecular weight of 90 kDa. The protein was purified and employed to prepare anti-VP5 polyclonal antibody from mice. The anti-VP5 antibody was found to neutralize GCRV through in vitro microneutralization assay and viral progeny quantification analysis. CONCLUSIONS: The present study showed that the viral VP5 protein was involved in viral infection and bacterially-expressed VP5 could be suitable for developing subunit vaccine for the control of GCRV infection.

Cloning and expression of 21.1-kDa tegumental protein of Clonorchis sinensis and human antibody response to it as a trematode-nematode pan-specific serodiagnosis antigen.

A complete cDNA encoding a 21.1-kDa tegumental protein (CsTP21.1) was recognized from Clonorchis sinensis adult full-length cDNA plasmid library by bioinformatics analysis. Recombinant CsTP21.1 was highly expressed in Escherichia coli, purified by affinity chromatography, and identified by Western blotting. Immunohistochemistry demonstrated that CsTP21.1 is localized in the tegument of the adult worm. The rCsTP21.1-specific IgG1, IgG2, and IgG4 subclasses could be detected in the sera of clonorchiasis patients by ELISA, but their sensitivity was much lower than that of total IgG. The sensitivity and specificity of IgG in 66 serum samples of clonorchiasis patients were 100% and 95.5%, and the sensitivity was independent of worm loads; the cross-reaction rates in 86, 24, and 31 serum samples from patients infected with Fasciola hepatica, Schistosoma japonicum, and nematode were 98.8%, 83.3%, 93.3%, respectively, whereas no cross-reactions with Toxoplasma gondii and sparganum. This study demonstrated that CsTP21.1 is a trematode-nematode pan-specific antigen that is valuable in the development of a universal immunodiagnostic kit for human trematode and nematode infections.

Reverse vaccinology approach identify an Echinococcus granulosus tegumental membrane protein enolase as vaccine candidate.

Applying reverse vaccinology strategy, we employed a sequence encoding an enolase from Taenia asiatica to search its homolog in the expression sequence tag (EST) database of Echinococcus granulosus and found two EST sequences (Access number: CN653186 and CN649593) of a clone Eg_PSGRS_13B09 from E. granulosus protoscolex full-length cDNA library, which are responding for the 5' and 3' partial cds of E. granulosus enolase, respectively. Primers are designed according to the 5' end and 3'end of the putative encoding sequence to amplify the genomic DNA of E. granulosus strain isolated from sheep in Qinghai province of China by polymerase chain reaction (PCR). A sole product of 1,449 bp in length was obtained, which contains two little introns of 78 bp and 69 bp, respectively. The introns were excised by unsymmetrical PCR with combined flank sequences of introns as primers. The structural, functional, and immunological characteristics of putative amino acid sequence were predicted by bioinformatics analysis. The complete coding sequence was predicted to encode 433 residues and contain a transmembrane region aa(104-124), with the N terminus outside and C terminus inside. The inside part is quite the functional domain. SWISS-MODEL modulated its 3D structure as a barrel which constitutes of alternatively arranged alpha helix-beta sheet, with the key sites such as substrate binding region, active sites, Mg(2+)-binding sites closely located at the center. The protein contains a potential nuclear localization sequence aa(190-199) and several linear B cell epitopes and CTL T cell epitopes, of which the outside epitope aa(49-57) and inside epitope aa(228-236) are facultative T cell and B cell epitope, and the linear B cell epitope aa(206-213) contains the active center site Glu(210), suggesting the putative protein is a potential membrane with strong immunogenicity. The complete cds was expressed in Escherichia coli, and the recombinant protein can be recognized by the serum from patient infected with E. granulosus. Reverse vaccinology process identified E. granulosus tegumental membrane protein enolase as vaccine candidate.