Single-Cell Microwell Platform Reveals Circulating Neural Cells as a Clinical Indicator for Patients with Blood-Brain Barrier Breakdown.

Central nervous system diseases commonly occur with the destruction of the blood-brain barrier. As a primary cause of morbidity and mortality, stroke remains unpredictable and lacks cellular biomarkers that accurately quantify its occurrence and development. Here, we identify NeuN+/CD45-/DAPI+ phenotype nonblood cells in the peripheral blood of mice subjected to middle cerebral artery occlusion (MCAO) and stroke patients. Since NeuN is a specific marker of neural cells, we term these newly identified cells as circulating neural cells (CNCs). We find that the enumeration of CNCs in the blood is significantly associated with the severity of brain damage in MCAO mice (p < 0.05). Meanwhile, the number of CNCs is significantly higher in stroke patients than in negative subjects (p < 0.0001). These findings suggest that the amount of CNCs in circulation may serve as a clinical indicator for the real-time prognosis and progression monitor of the occurrence and development of ischemic stroke and other nervous system disease.

Interferon characterization associates with asthma and is a potential biomarker of predictive diagnosis.

Interferon (IFN) plays a role in immune and inflammation responses. However, the effect of IFN in asthma is still not fully clear. The present study was conducted to better understand the role of IFN signatures in asthma. Blood samples from case-control studies (study 1: 348 asthmas and 39 normal controls and validation study 2: 411 asthmas and 87 normal controls) were enrolled. The single-sample gene set enrichment analysis (ssGSEA) method was used to quantify the levels of 74 IFN signatures. Gene Ontology analysis and pathway function analysis were performed for functional analysis and a protein-protein interaction (PPI) network was constructed. The area under the curve (AUC) value was used to evaluate the diagnostic ability. In our work, IFN-γ response-DN, negative regulation of IFN-γ secretion, IFNG pathway, negative regulation of response to IFN-γ, and type 1 IFN biosynthetic process showed higher levels in asthma. Functional analysis demonstrated that pathway and biological process involved in IFN signaling pathway, regulation of type 1 IFN production and response to IFN-γ. Hub IFN-related genes were identified, and their combination as biomarker exhibited a good diagnostic capacity for asthma (AUC = 0.832). These findings offered more insight into the underlying mechanism of how IFN signatures affected asthma. The use of the easy-to-apply IFN-related genes might serve as a promising blood-based biomarker for early diagnosis of asthma.

Oxytocin treatment prevents marrow adiposity observed in alloxan-induced diabetic rabbits using proton MR spectroscopy.

INTRODUCTION: Oxytocin might be used therapeutically as an ally to rescue osteopathy resulting from diabetes. However, the in vivo effects of oxytocin on marrow adipogenesis in diabetes remain unknown. In this longitudinal study, we aimed to investigate the protective ef-fects of oxytocin on diabetes-induced marrow adiposity in rabbits using proton MR spectroscopy. MATERIAL AND METHODS: Forty-five female New Zealand rabbits were randomly divided into controls, diabetes, and diabetes treated with oxytocin (ip, 0.78 mg/kg) for six months. Marrow fat fraction (FF) was determined by proton MR spectroscopy at baseline, and at three and six months. Bone mineral density was measured by dual-energy X-ray absorptiometry. Serum biomarkers, glycolipid metabolism, and histological analysis of marrow adipocytes were determined. RESULTS: Oxytocin treatment had positive metabolic effects in diabetic rabbits, which was based on the changes in glucose metabolism, insulin sensitivity, and lipid profiles. The diabetic rabbits demonstrated dramatic marrow adiposity in a time-dependent manner; at three and six months the FF percentage changes from baseline were 10.1% and 25.8%, respectively (all P < 0.001). Moreover, oxytocin treatment significantly reversed FF values and quantitative parameters of marrow adipocyte in diabetic rabbits to levels of naive control rabbits. Oxytocin improved bone formation marker in diabetic rabbits compared to the saline group. Also, treatment of diabetic rabbits with oxytocin significantly mitigated bone deterioration when compared with the saline-treated diabetic group (all P < 0.05). CONCLUSIONS: Oxytocin appears to alleviate harmful effects of hyperglycaemia on marrow adiposity. Proton MR spectroscopy may be a valuable tool, providing complementary information on efficacy assessments.

Progressive genomic convergence of two Helicobacter pylori strains during mixed infection of a patient with chronic gastritis.

OBJECTIVE: To study the detailed nature of genomic microevolution during mixed infection with multiple Helicobacter pylori strains in an individual. DESIGN: We sampled 18 isolates from a single biopsy from a patient with chronic gastritis and nephritis. Whole-genome sequencing was applied to these isolates, and statistical genetic tools were used to investigate their evolutionary history. RESULTS: The genomes fall into two clades, reflecting colonisation of the stomach by two distinct strains, and these lineages have accumulated diversity during an estimated 2.8 and 4.2 years of evolution. We detected about 150 clear recombination events between the two clades. Recombination between the lineages is a continuous ongoing process and was detected on both clades, but the effect of recombination in one clade was nearly an order of magnitude higher than in the other. Imputed ancestral sequences also showed evidence of recombination between the two strains prior to their diversification, and we estimate that they have both been infecting the same host for at least 12 years. Recombination tracts between the lineages were, on average, 895 bp in length, and showed evidence for the interspersion of recipient sequences that has been observed in in vitro experiments. The complex evolutionary history of a phage-related protein provided evidence for frequent reinfection of both clades by a single phage lineage during the past 4 years. CONCLUSIONS: Whole genome sequencing can be used to make detailed conclusions about the mechanisms of genetic change of H. pylori based on sampling bacteria from a single gastric biopsy.

[Detection of WU polyomavirus in children with low respiratory tract infections using real-time fluorescent quantitative PCR].

OBJECTIVE: Development and application of a real time fluorescent quantitative PCR (FQ-PCR) assay for detecting WU polyomavirus in children with low respiratory tract infections. METHODS: The VP2 gene of WU polyomavirus was selected as the detection target, from which the real time primers and probes were designed. The standard curve was established by using recombinant plasmid as template. And the FQ-PCR assay for specific detection of WU polyomavirus was established. The specificity, sensitivity and reproducibility of the method were evaluated. Furthermore, the clinical specimens from children with respiratory tract infections collected in Wenling First People's Hospital were quantitatively detected using this method. RESULTS: In this study, the FQ-PCR method was established to detect a specific fragment in VP2gene of WU polyomavirus. The standard curve coefficient R2 was 0.998. And this method can detect as low as 50 copies recombinant plasmid. The clinical specimens of sputum and throat swab from children with respiratory tract infections were quantitatively detected using this method. 7 sputum specimens were detected as WU polyomavirus positive in 700 sputum specimens, the positive ratio was 1.00%. No positive specimens were detected in 146 specimens of throat swabs and 846 blood samples from same patient population. CONCLUSION: The results indicated that the FQ-PCR assay method established in this study was specific, rapid and sensitive for detecting WU polyomavirus in children with lower respiratory tract infections. The sputum specimen is more suitable to be used for gene detection of WU polyomavirus than throat swab or blood.

[Association of human papillomavirus infection with other microbial pathogens in gynecology].

OBJECTIVE: To Investigate correlation between screening assay of human papillomavirus (HPV) and microbial pathogens in gynecology. METHODS: Cervical samples were collected to search for HPV, bacteria and yeast infections in gynecologic outpatients. HPV typing was carried out by PCR and sequencing on cervical brush specimens. Chlamydia trachomatis was identified by strand displacement amplification (SDA) and the other microorganisms were detected by conventional methods. All data were analyzed to investigate the correlation among them. RESULTS: In this cross-sectional study, among 857 enrolled outpatients, there were 266 cases with positive HPV DNA, and the rate of infection was 31.0% (266/857). HPV genotype showed that thirty-five different HPV types were identified, of which HPV16 was the most prevalent (14.5%, 38/262), followed by HPV58 (9.2%, 24/262), HPV53 (8.0%, 21/262) and HPV42 (6.1%, 16/262); while other genotypes were present in less than 5% of HPV positive women. According to the reclassification, the aggregated percentage (high-risk and probably high-risk) of detected HPV was 58.8% (154/262), 27.9% (73/262) for low-risk and 13.4% (35/262) for unknown-risk HPV types. Among HPV positive women, cervical brush specimens results showed that more than 60% cases with normal cytology, 3.8% (10/266) with high-grade squamous intraepithelial lesions (HSIL), 29.7% (79/266) with low-grade squamous intraepithelial lesions (LSIL) and 3.0% (8/266) with atypical squamous cells of undetermined significance (ASCUS), respectively. Statistical analyses revealed there was a significant association between the infected HPV and Chlamydia trachomatis or Ureaplasma urealyticum (> 10,000 CCU/ml; all P < 0.01), while no correlation was found between HPV infection and bacterial vaginosis, streptococcus agalactiae, candida, Trichomonas vaginalis or Ureaplasma urealyticum (≤ 10 000 CCU/ml; all P > 0.05). Among the cases with bacterial vaginosis, the positive rate of HPV infected was 42.6%. Chlamydia trachomatis was one of the high-risk factors for the infection of HPV (OR = 2.82, 95% CI: 1.74 - 4.57). Mycoplasma hominis was isolated only in 2 cases, no patient was infected with Neisseria gonorrhoeae. CONCLUSIONS: Although bacterial vaginosis was not significantly associated with HPV, it was more common among the HPV positive women. There is the significant association between HPV and Chlamydia trachomatis or Ureaplasma urealyticum which may be increase the infection of HPV. These data suggest that it may be important to screen for the simultaneous presence of different microorganisms which may have synergistic pathological effects.

[Detection of BK virus infection in renal transplant recipients and clinical application].

OBJECTIVE: To study the detection methods of BK virus infection in kidney transplant recipients, and to explore the clinical application. METHODS: 132 cases of renal transplant recipients were undertaken BK virus detection including presence of decoy cells in urinary sediment, urine and serum BKV-DNA to demonstrate the BK virus replication. RESULT: Among 132 cases of renal transplant recipients, urinary decoy cell was found in 37 (28.0%) patients and the median time was 12 months after surgery. 32 (24.2%) patients were diagnosed as BK viruria at a median of 11 months after surgery, and 16 (12.1%) recipients were diagnosed as BK viremia at a median of 15 months after surgery, 5 patients with BK viruria were diagnosed as BK virus associated nephropathy according to allograft biopsy. CONCLUSION: To make early diagnosis of BK virus infection, detection of urine decoy cells and BKV-DNA in urine and plasma sample is important,which provides an important basis for the prevention of BK virus associated nephropathy.

[Expression of recombinant VP2 gene in insect sf9 cells and screening of clinical specimens].

OBJECTIVE: To clone and express VP, gene from HBoV, and the expressed VP, protein was as the antigen in order to detect serum from children in Wenling area with lower respiratory tract infections. METHODS: The VP, gene was recombined with the genome of Baculovirus, which infected the insect cell. The fusion protein with HA tag was applied to confirm the specificity of expressed protein. Furthermore, the recombinant protein was observed using electron microscopy. The 176 serum from children in Wenling area with lower respiratory tract infections was screened using Western blot. RESULTS: The expressed VP2 protein was more than 60% in total proteins from insect cell, and MWt about 60 x 10(3). The virus-like particle (VLP) was observed using electron microscopy, and size about 20 nm. The 176 serum from children in wenling area with lower respiratory tract infections was screened using Western blot. The HBoV positive rate was 2.28% (4/176). CONCLUSION: The VP2 protein from human bocavirus was expressed in insect cell successfully. Through HA tag the VP2 protein was specific, and then the assay using SDS-PAGE with Western blot could detect and screen the antibody in serum from children with lower respiratory tract infections rapidly and accurately.

[Identification of KI polyomavirus in children with lower respiratory tract infections from Zhejiang region of China].

KI polyomavirus, which was firstly discovered in 2007, is a new human polyomavirus belonging to Polyomaviridae and containing circular double-strand genomic DNA. This study was based on identification assay of KI polyomavirus reported. Total 2293 clinical sputum specimens from children under 3-years-old were collected and screened from Wenzhou Medical College affiliated Wenling Hospital, Zhejiang Province. A KI polyomavirus was detected and identified, the positive rate was 0.04%. The sequences of PCR products was identical to that of the viral capsid protein (VP1) gene derived from KI polyomavirus. The results strongly suggested that the KI polyomavirus was found firstly in Chinese children with acute lower respiratory tract infections from Zhejiang region. This study provided new information for further investigation of etiopathogenisis and diagnosis in children with lower respiratory tract infections.

[Characterization of the cytopathic effect in human bronchial epithelial cell after Human Bocavirus Infection (HBoV)].

OBJECTIVE: In this study, human bronchial epithelial cells were inoculated with positive sputum specimens of HBoV. After four days' infection, cytopathic effects (CPE) were observed by inverted microscopy. These viruses all cause typical cell damages such as rounded and shrivelled, fusion and fallout. These damages got quick following increased future degenerations. The other assay result of CPE within the infected cells were observed by inverted microscopy, have typical "owl's eye" plaque and above 90 percent hemadsorption within the infected cells by erythrocytes for hemadsorption technique. The typical fluorescence lump of nucleus within the infected cells was found by indirect immunofluorescence technique. CONCLUSION: Isolation and identification of HBoV could be done in the human bronchial epithelial cell, and we found some characterizing CPE in the human bronchial epithelial cell after HBoV infection. The above studies pave a way for studying pathogenicity of human bocavirus.

[Discovery and identification of WU polyomavirus in children from Zhejing region].

WU polyomavirus, which was firstly discovered in 2007, is a new human polyomavirus belonging to Polyomaviridae and containing circular double-stranded genomic DNA. In this study, the 278 clinical sputum specimens from children under 5 years old were collected from Wenzhou Medical College affiliated Wenling First Hospital, Zhejiang Province. Based on identification assay of WU polyomavirus previously reported, a WU polyomavirus was identified from clinical samples successfully, the positive rate was 0.4%. The sequences of PCR products were identical to that of VP2 gene and large T antigen gene derived from WU polyomavirus reported. The above results strongly suggested that the WU polyomavirus isolated was firstly found in Chinese children with acute lower respiratory tract infections. This study provides a firm basis for further research of WU polyomavirus.

WU polyomavirus in children with acute lower respiratory tract infections, China.

BACKGROUND: WU polyomavirus (WUPyV), a new member of the genus of Polyomavirus in the family Polyomaviridae, has been found and associated with respiratory tract infections recently. However, its clinical role and pathogenicity has not been known. OBJECTIVES: To confirm that WU polyomavirus has been found in Chinese children. STUDY DESIGN: WU polyomavirus was detected and identified using PCR methods. A total of 278 specimens of nasopharyngeal aspirate were collected, and then PCR products were sequenced directly. RESULTS: One of 278 nasopharyngeal aspirates was positive for WUPyV in one child, and the positive rate was 0.4%. The results showed that the sequences of genome, LTAg and VP2 gene was identical to the reference sequences of WU polyomavirus prototype strains. CONCLUSIONS: We confirmed that WU polyomavirus had been found and identified in the respiratory secretions in China.

[Study on relationship between hepatitis B virus DNA load and genotype with large envelope protein].

OBJECTIVE: To explore the relation between hepatitis B virus DNA load and genotype with the level of large envelope protein. METHODS: Serum HBV DNA was quantitively detected by using real time polymerase chain reaction (RT-PCR). The LHBs were detected by using enzyme linked immuno sorbent assay (ELISA) and HBV markers were detected by time differentiate immunofluorescence assay in 140 serum samples collected from chronic hepatitis B patients.The genotypes of HBV were identified by DNA sequencing; and analyze their relationship. RESULTS: There was no significant difference between positive rate of LHBs and that of HBV DNA in HBeAg negative and positive group (P > 0.05); The HBV LHBs absorbency was markedly correlated with the HBV DNA load ( R2 = 0.9267). The difference of HBV LHBs absorbency between HBV genotype B and C was not significant. CONCLUSIONS: The close correlation between HBV LHBs absorbence and HBV DNA load illustrated that he level of serum LHBs can be used to estimate the state of HBV replication; and there is no relationship between HBV LHBs absorbency and genotypes. So HBV LHBs may be used as a new serological marker to detect HBV replication.

[Clinical prospective study on maternal-fetal transmission of human bocavirus].

OBJECTIVE: To investigate maternal-fetal transmission at human bocavirus (HBoV). METHODS: IgG antibody to HBoV in serum samples of 316 mothers were determined with ELISA and HBoV DNA was determined with real time PCR in the sera of the mothers and their infants. RESULTS: HBoV-IgG was positive in 40.20 percent (127/316) of the mothers, while it was positive in 29.43 percent (93/316) of the cord blood specimens of the infants. The difference between the two groups was significant (X2=8.12, P less than 0.005); 93 samples of both the mothers and the infants were positive for HBoV-IgG. CONCLUSION: HBoV-IgG can cross the placenta to the fetuses through placenta. Further study is needed to answer the question whether vertical maternal-fetal transmission occurs.

[Study on PBPs genes of penicillin-resistant Streptococcus pneumonia in Zhejiang province].

To elucidate alternations in gene/amino acid sequence of penicillin-binding proteins (PBPs) 1A, 2B, 2X from clinical isolates of penicillin-resistant Streptococcus pneumonia (PRSP) in Zhejiang Province, 26 strains of Streptococcus pneumonia were collected from November 2001 to January 2004. The antibiotics susceptibility of these strains was detected. PCR amplification and direct sequencing of PBP1A, 2B, 2X genes were performed. The sequence variations of PBP genes of the PRSPs in this region were studied by sequence BLAST analysis. It was shown that the main alternations of PBP1A were the four consecutive amino acid substitutions (Thr574Ala, Ser575Thr, Gln576Gly, Phe577Tyr) following the conservative motif KTG and the amino acid substitution Thr371Ser in the conservative motif STMK. The main alternation of PBP2B was Thr451Ala following the conservative motif SSN, and the main alternation of PBP2X was Thr338Ala in conservative motif STMK. The above mutation sites and drug resistant level were consistent to the data reported previously. Neither new gene mutation specific to these strains nor certain amino acid substitutions related to penicillin resistance reported was identified in the genes.