HSP90AB1 is a host factor that promotes porcine deltacoronavirus replication.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus. It causes mortality in neonatal piglets and is of growing concern because of its broad host range, including humans. To date, the mechanism of PDCoV infection remains poorly understood. Here, based on a genome-wide CRISPR screen of PDCoV-infected cells, we found that HSP90AB1 (heat shock protein 90 alpha family class B1) promotes PDCoV infection. Knockdown or KO of HSP90AB1 in LLC-PK cells resulted in a significantly suppressed PDCoV infection. Infected cells treated with HSP90 inhibitors 17-AAG and VER-82576 also showed a significantly suppressed PDCoV infection, although KW-2478, which does not affect the ATPase activity of HSP90AB1, had no effect on PDCoV infection. We found that HSP90AB1 interacts with the N, NS7, and NSP10 proteins of PDCoV. We further evaluated the interaction between N and HSP90AB1 and found that the C-tail domain of the N protein is the HSP90AB1-interacting domain. Further studies showed that HSP90AB1 protects N protein from degradation via the proteasome pathway. In summary, our results reveal a key role for HSP90AB1 in the mechanism of PDCoV infection and contribute to provide new host targets for PDCoV antiviral research.

Porcine deltacoronavirus enters ST cells by clathrin-mediated endocytosis and does not require Rab5, Rab7, or Rab11.

IMPORTANCE: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus threatening pig industries worldwide. Our previous work showed that PDCoV enters porcine kidney (PK-15) cells through a caveolae-dependent pathway, but the entry mechanism for PDCoV into swine testicle (ST) cells remains unclear. Mechanisms of virus entry can be different with different virus isolates and cell types. Here, we determined that PDCoV enters ST cells via clathrin-mediated endocytosis. Additionally, we found that PDCoV entry does not require Rab5, Rab7, or Rab11. These findings provide additional understanding of the entry mechanisms of PDCoV and possible antiviral targets.

Characterization, phylogenetic analysis, and pathogenicity of a novel genotype 2 porcine Enterovirus G.

Enterovirus G belongs to the family Picornaviridae and are associated with a variety of animal diseases. We isolated and characterized a novel EV-G2 strain, CHN-SCMY2021, the first genotype 2 strain isolated in China. CHN-SCMY2021 is about 25 nm diameter with morphology typical of picornaviruses and its genome is 7341 nucleotides. Sequence alignment and phylogenetic analysis based on VP1 indicated that this isolate is a genotype 2 strain. The whole genome similarity between CHN-SCMY2021 and other EV-G genotype 2 strains is 78.3-86.4%, the greatest similarity is to EVG/Porcine/JPN/Iba26-506/2014/G2 (LC316792.1). Recombination analysis indicated that CHN-SCMY2021 resulted from recombination between 714,171/CaoLanh_VN (KT265894.2) and LP 54 (AF363455.1). Except for ST cells, CHN-SCMY2021 has a broad spectrum of cellular adaptations, which are susceptible to BHK-21, PK-15, IPEC-J2, LLC-PK and Vero cells. In piglets, CHN-SCMY2021 causes mild diarrhea and thinning of the intestinal wall. The virus was mainly distributed to intestinal tissue but was also found in heart, liver, spleen, lung, kidney, brain, and spinal cord. CHN-SCMY2021 is the first systematically characterized EV-G genotype 2 strain from China, our results enrich the information on the epidemiology, molecular evolution and pathogenicity associated with EV-G.

A Comparative Transcriptomic Analysis Reveals That HSP90AB1 Is Involved in the Immune and Inflammatory Responses to Porcine Deltacoronavirus Infection.

PDCoV is an emerging enteropathogenic coronavirus that mainly causes acute diarrhea in piglets, seriously affecting pig breeding industries worldwide. To date, the molecular mechanisms of PDCoV-induced immune and inflammatory responses or host responses in LLC-PK cells in vitro are not well understood. HSP90 plays important roles in various viral infections. In this study, HSP90AB1 knockout cells (HSP90AB1KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1WT and HSP90AB1KO cells was conducted using RNA sequencing to explore the effect of HSP90AB1 on PDCoV infection. A total of 1295 and 3746 differentially expressed genes (DEGs) were identified in PDCoV-infected HSP90AB1WT and HSP90AB1KO cells, respectively. Moreover, most of the significantly enriched pathways were related to immune and inflammatory response-associated pathways upon PDCoV infection. The DEGs enriched in NF-κB pathways were specifically detected in HSP90AB1WT cells, and NF-κB inhibitors JSH-23, SC75741 and QNZ treatment reduced PDCoV infection. Further research revealed most cytokines associated with immune and inflammatory responses were upregulated during PDCoV infection. Knockout of HSP90AB1 altered the upregulated levels of some cytokines. Taken together, our findings provide new insights into the host response to PDCoV infection from the transcriptome perspective, which will contribute to illustrating the molecular basis of the interaction between PDCoV and HSP90AB1.

Porcine Deltacoronavirus (PDCoV) Entry into PK-15 Cells by Caveolae-Mediated Endocytosis.

(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NH4Cl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-ß-cyclodextrin (MßCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.

Study of the inhibitory effect of STAT1 on PDCoV infection.

Porcine deltacoronavirus (PDCoV) is an enteropathogen found in many pig producing countries. It can cause acute diarrhea, vomiting, dehydration, and death in newborn piglets, seriously affecting the development of pig breeding industries. To date, our knowledge of the pathogenesis of PDCoV and its interactions with host cell factors remains incomplete. Using Co-IP coupled with LC/MS-MS, we identified 67 proteins that potentially interact with PDCoV in LLC-PK1 cells; five of the identified proteins were chosen for further evaluation (IMMT, STAT1, XPO5, PIK3AP1, and TMPRSS11E). Five LLC-PK1 cell lines, each with one of the genes of interest knocked down, were constructed using CRISPR/cas9. In these knockdown cells lines, only STAT1KD resulted in a significantly greater virus yield. Knockdown of the remaining four genes resulted, to varying degrees, in a lower virus yield that wild-type LLC-PK1 cells. The absence of STAT1 did not significantly affect the attachment of PDCoV to cells, but did result in increased viral internalization. Additionally, PDCoV infection stimulated expression of interferon stimulated genes (ISGs) downstream of STAT1 (IFIT1, IFIT2, RADS2, ISG15, MX1, and OAS1) while knockdown of STAT1 resulted in a greater than 80 % decrease in the expression of all six ISGs. Our findings show that STAT1 interacts with PDCoV, and plays a negative regulatory role in PDCoV infection.

HSP90 inhibitors 17-AAG and VER-82576 inhibit porcine deltacoronavirus replication in vitro.

Porcine deltacoronavirus (PDCoV) is highly pathogenic to piglets, and no specific drugs or vaccines are available for the prevention and treatment of PDCoV infection, the need for antiviral therapies is pressing. HSP90 inhibitors have potent inhibitory effects against the replication of numerous viruses, hence we evaluated three HSP90 inhibitors, 17-AAG, VER-82576, and KW-2478, for their effects on PDCoV infection in vitro. We evaluated their effectivenesses at suppressing PDCoV by qRT-PCR, western blot, and TCID50 assay, and found that 17-AAG and VER-82576 inhibited PDCoV at the early stage of replication, while KW-2478 showed no significant antiviral activity at any stage of infection. These results indicated that the PDCoV-inhibitory effects of 17-AAG and VER-82576 might be exerted by targeting host cell factor HSP90AB1 but not HSP90AA1. Further study showed that HSP90AB1 mRNA and protein levels were not significantly different in 17-AAG and VER-82576-treated cells versus control cells. 17-AAG and VER-82576 were also evaluated for their effects on the expressions of TNF-α, IL-6, and IL-12, which are PDCoV-induced proinflammatory cytokines. We found that both 17-AAG and VER-82576 inhibited the expressions of TNF-α, IL-6, and IL-12 to varying degrees, but in a dose dependent manner. From our data we can conclude that the HSP90 inhibitors 17-AAG and VER-82576 are promising candidates for the treatment of PDCoV infection.

The midterm outcomes of 1-stage versus 3-stage laparoscopic-assisted anorectoplasty in anorectal malformations with rectoprostatic fistula and rectobulbar fistula: A retrospective cohort study.

The aim of this study was to compare the midterm outcomes of 1-stage and 3-stage surgical procedures to treat anorectal malformations (ARMs) with rectoprostatic and rectobulbar fistula using laparoscopic-assisted anorectoplasty (LAARP).A total of 56 patients with ARMs and rectoprostatic and rectobulbar fistula who underwent LAARP from January 2011 to May 2014 in our institution were included in the study. They were divided into 2 groups according to the stage of procedure. The patients' data and postoperative complications were compared between the 2 groups. The Krickenbeck classification was used for assessing the bowel functions.About 20 ARM newborns (rectoprostatic fistula [12], rectobulbar fistula [8]) successfully underwent a 1-stage LAARP, and about 36 ARM children (rectoprostatic fistula [20], rectobulbar fistula [16]) underwent a 3-stage LAARP (colostomy, LAARP, and closure of colostomy). The average age at the LAARP procedure in 1-stage group was significantly lower than that in 3-stage group (39.8 ±â€Š8.1 hours vs 4.9 ±â€Š1.2 months; P = .00). The average operative time during the definitive procedure was 132.2 ±â€Š15.9 minutes in the 1-stage group and 120.5 ±â€Š12.7 minutes in the 3-stage group (P = .13). There was only 5 to 10 mL of blood loss during the LAARP procedure both the groups (P = .75). There were no significant differences between the 2 groups in postoperative hospital stay during the definitive procedure (10.2 ±â€Š2.3 days vs 8.5 ±â€Š2.2 days; P = .22). The rate of surgical site infection and dehiscence was 5% (1/20) in the 1-stage group and 5.6% (2/36) in 3-stage group (P = 1.00). During the period of follow-up, the rate of voluntary bowel movement was 90% (18/20) in 1-stage group and 94.4% (34/36) in 3-stage group (P = .94). Free from soiling or grade I soiling was 80% (16/20) in 1-stage group and 83.3% (30/36) in 3-stage group (P = 1.00); grade II soiling was found in 3 (10%) patients in 1-stage group and 85.7% in 3-stage group (P = .75); grade III soiling was found in 3 (10%) patients in 1-stage group and 85.7% in 3-stage group (P = 1.00). Three patients (15%) in 1-stage group and 5 patients (13.9%) in 3-stage group suffered from grade I constipation (P = 1.00); while 3 (15%) patients in 1-stage group and 4 patients (11.1%) in 3-stage group had grade II constipation (P = 1.00); no patients in the 2 groups suffered from grade III constipation.The 1-stage LAARP procedure for neonate with rectoprostatic and rectobulbar fistula can achieve comparable midterm outcomes as the conventional 3-stage LAARP procedure. It provides an alternative method to rectify the ARMs with rectoprostatic fistula and rectobulbar fistula without colostomy.

Inhibition of KRAS-dependent lung cancer cell growth by deltarasin: blockage of autophagy increases its cytotoxicity.

Deltarasin is a recently identified small molecule that can inhibit KRAS-PDEδ interactions by binding to a hydrophobic pocket on PDEδ, resulting in the impairment of cell growth, KRAS activity, and RAS/RAF signaling in human pancreatic ductal adenocarcinoma cell lines. Since KRAS mutations are the most common oncogene mutations in lung adenocarcinomas, implicated in over 30% of all lung cancer cases, we examined the ability of deltarasin to inhibit KRAS-dependent lung cancer cell growth. Here, for the first time, we document that deltarasin produces both apoptosis and autophagy in KRAS-dependent lung cancer cells in vitro and inhibits lung tumor growth in vivo. Deltarasin induces apoptosis by inhibiting the interaction of with PDEδ and its downstream signaling pathways, while it induces autophagy through the AMPK-mTOR signaling pathway. Importantly, the autophagy inhibitor, 3-methyl adenine (3-MA) markedly enhances deltarasin-induced apoptosis via elevation of reactive oxygen species (ROS). In contrast, inhibition of ROS by N-acetylcysteine (NAC) significantly attenuated deltarasin-induced cell death. Collectively, these observations suggest that the anti-cancer cell activity of deltarasin can be enhanced by simultaneously blocking "tumor protective" autophagy, but inhibited if combined with an anti-oxidant.

[Vitamin D level in children with bloodstream infection].

OBJECTIVE: To investigate the difference in serum 25(OH)D level between children with bloodstream infection and healthy children. METHODS: A case-control study was conducted among 60 children with bloodstream infection who were hospitalized between January 2010 and December 2013 and had positive results of two blood cultures. Meanwhile, 60 aged-matched healthy children who underwent physical examination during the same period of time were enrolled as the healthy control group. Chemiluminescence was applied to measure the serum 25(OH)D level, and the constituent ratios of children with different serum 25(OH)D levels were compared between the two groups. RESULTS: The bloodstream infection group had a significantly lower serum 25(OH)D level than the healthy control group (P<0.01). Compared with the healthy control group, the bloodstream group had significantly lower constituent ratios of children with normal Vitamin D level (8% vs 35%) or vitamin D insufficiency (22% vs 43%) (P<0.05). Compared with the healthy control group, the bloodstream group had significantly higher constituent ratios of children with vitamin D deficiency (42% vs 13%) or severely vitamin D deficiency (28% vs 8%) (P<0.01). CONCLUSIONS: Vitamin D insufficiency prevails among children, and children with bloodstream infection have a significantly lower serum 25(OH)D level than healthy children.

Plasma MASP-1 concentration and its relationship to recovery from coronary artery lesion in children with Kawasaki disease.

BACKGROUND: This study investigated prognostic factors for early recovery of coronary artery lesion (CAL) in children with Kawasaki disease (KD). METHODS: Patients hospitalized for KD were enrolled less than 2 wk from the onset of illness and divided into two groups: KD with CAL and KD without CAL. The CAL group was further divided into two subgroups according to the degree of CAL: mild (n = 31) and moderate/severe (n = 6) and further divided into two subgroups according to the age: younger than 1 y (n = 9) and older than 1 y (n = 28). Lectin pathway-related factors MASP-1, CD59, and C5b-9 were measured, along with C-reactive protein, white blood cell counts, erythrocyte sedimentation rate, and platelet count. Patients were followed up for 3 mo. Correlation between the measured factors and the length of time of recovery from CAL was analyzed. RESULTS: Plasma concentrations of MASP-1 in the CAL group were significantly lower than those without CAL. MASP-1 and gender positively correlated with the recovery time of CAL. There was no difference in MASP-1 between mild and moderate/severe CAL. At 3-mo follow-up, there was a positive correlation between plasma MASP-1 concentration and recovery time of the patients with CAL older than 1 y. CONCLUSION: Plasma MASP-1 concentration at the early stage of KD is predictive of length of time of recovery from CAL.

Early decreased plasma levels of factor B and C5a are important biomarkers in children with Kawasaki disease.

BACKGROUND: The mechanisms underpinning Kawasaki disease (KD) are incompletely understood. There is an unmet need for specific biomarkers for the early diagnosis of KD. METHODS: Eighty-five KD patients suffering from acute-phase and subacute-phase KD, 40 healthy children, and 40 febrile children comprised the study cohort. An enzyme-linked immunosorbent assay was used to measure plasma levels of C1q, C1q-circulating immune complex (C1q-CIC), mannan-binding lectin-associated serine protease (MASP)-1, factor B, C4d, C3d, C5a, C5b-9 and CD59. RESULTS: Plasma concentrations of factor B and C5a in the acute phase were lower than those in healthy and febrile control groups (all P < 0.05). Compared with acute-phase KD patients, plasma concentrations of C1q, factor B, and C3d in KD patients were increased significantly (P < 0.05), but those of C4d, MASP-1 and CD59 decreased significantly (P < 0.05), in patients with sub-acute KD. CONCLUSION: These data suggest that more than one pathway in the complement system is activated in KD. Importantly, decreased plasma concentrations of factor B and C5a in the acute phase (6-10 d) could be employed as biomarkers for the early diagnosis of KD.

[Clinical research of Bordetella pertussis infection in infants with prolonged cough].

OBJECTIVE: To explore the prevalence of Bordetella pertussis (B. pertussis) infection in unvaccinated or incomplete vaccinated infants with cough for a prolonged duration. METHODS: The serum samples and nasopharyngeal secretions were collected from 176 patients with cough for a prolonged duration ( ≥ 2 weeks) from 2011 to 2012 at Children's Hospital Affiliated to Capital Institute of Pediatrics. Multiplex PCR of nasopharyngeal secretion was employed to identify B.pertussis. And enzyme-linked immunosorbent assay(ELISA) was used to detect antibody to pertussis toxin(PT-IgG). Total bacterial DNA was enacted from nasopharyngeal secretion and two-target IS481/PT of B.pertussis was detected by PCR. The sera and nasopharyngeal secretions were also collected from household contacts with cough for a prolonged duration. Their clinical characteristic and epidemiological profiles were collected and analyzed. RESULTS: B.Pertussis infection was demonstrated in 51 cases (29.0%). The patients ages were from 23 days to 4 years. Among them, 46 cases (90.2%) were aged under 12 months and 5 cases (9.8%) over 12 months. And 40 cases were unvaccinated (31 cases <3 months old, 4 cases 3-12 months old, 5 cases >5 years old) and 11 cases incompletely vaccinated. There were 31 males and 20 females. More patients were found in spring and summer than those in autumn and winter. Nine infant cases had 12 household contacts. Among 12 household contacts, 3 were PCR positive and 12 PT-IgG positive. Pertussis was remarkably critical in infants. Serious complications included failure to thrive, pneumonia, respiratory failure and seizures. CONCLUSIONS: B.pertussis infection is an important cause in unvaccinated or incomplete vaccinated infants with prolonged cough. Peak seasons of pertussis are spring and summer. Undiagnosed adolescents and adults with pertussis may be a significant source for transmission of B.pertussis to other susceptible children. Infants aged under 1 year are at risk for severe pertussis and life-threatening complications. As a rapid and sensitive method of detecting B.pertussis, PCR may be used in early phase.

Hydrogen sulfide suppresses the expression of MMP-8, MMP-13, and TIMP-1 in left ventricles of rats with cardiac volume overload.

AIM: To study the effects of hydrogen sulfide (H2S) on the left ventricular expression of MMP-8, MMP-13, and TIMP-1 in a rat model of congenital heart disease. METHODS: Male SD rats underwent abdominal aorta-inferior vena cava shunt operation. H2S donor NaHS (56 µmol·kg(-1)·d(-1), ip) was injected from the next day for 8 weeks. At 8 weeks, the hemodynamic parameters, including the left ventricular systolic pressure (LVSP), the left ventricular peak rate of contraction and relaxation (LV ± dp/dtmax) and the left ventricular end diastolic pressure (LVEDP) were measured. The left ventricular tissues were dissected out, and hydroxyproline and collagen I contents were detected with ELISA. The expression of MMP-8, MMP-13, and a tissue inhibitor of metalloproteinase-1 (TIMP-1) in the tissues was measured using real-time PCR, Western blotting, and immunohistochemistry, respectively. RESULTS: The shunt operation markedly reduced LVSP and LV ± dp/dtmax, increased LVEDP, hydroxyproline and collagen I contents, as well as the mRNA and protein levels of MMP-8, MMP-13, and TIMP-1 in the left ventricles. Chronic treatment of the shunt operation rats with NaHS effectively prevented the abnormalities in the hemodynamic parameters, hydroxyproline and collagen I contents, and the mRNA and protein levels of MMP-13 and TIMP-1 in the left ventricles. NaHS also prevented the increase of MMP-8 protein expression, but did not affect the increase of mRNA level of MMP-8 in the shunt operation rats. CONCLUSION: H2S suppresses protein and mRNA expression of MMP-8, MMP-13, and TIMP-1 in rats with cardiac volume overload, which may be contributed to the amelioration of ventricular structural remodeling and cardiac function.

Study on the resistant genes to carbapenems and epidemiological characterization of multidrug-resistant Acinetobacter baumannii isolates.

The purpose of this study was to examine the carbapenemase-encoding resistance genes and analyze homologous of multidrug-resistant Acinetobacter baumannii (MRAB) isolates from nosocomial infections. Seventy-six A. baumannii strains were collected from inpatients and object surface of devices in intensive care units from May 2008 to February 2011. Antibiotic susceptibility testing of 18 antimicrobial agents was performed. The presence of carbapenemase-encoding resistance genes was investigated by polymerase chain reaction. Genotyping and dendrogram analysis of A. baumannii strains from nosocomial infections were performed using the DiversiLab System. All of the 76 clinical A. baumannii isolates were shown multidrug resistance. The bla(OXA-23) gene was identified in the 76 MRAB strains, while bla(OXA-24), bla(OXA-58), VIM, IMP-1, IMP-4, SIM, and blaNDM-1 were absent in all. Twenty-four A. baumannii strains from the samples with nosocomial infections were classified into four unrelated groups and nine patterns. In conclusion, production of bla(OXA-23) in MRAB is one of the molecular mechanisms responsible for carbapenem resistance. The MRAB strains from unrelated groups show different drug resistance, but the homologous strains also have different drug resistance. Homologous analysis can provide scientific evidence for evaluation of epidemic status of nosocomial infection caused by MRAB.

Hydrogen sulfide upregulates heme oxygenase-1 expression in rats with volume overload-induced heart failure.

The present study investigated the role of hydrogen sulfide (H2S), a novel gaseous transmitter, in chronic heart failure (CHF) induced by left-to-right shunt, leading to volume overload. Thirty male Sprague-Dawley rats were randomly divided into four groups: the shunt group, the sham group, the shunt + sodium hydrosulfide (NaHS) group and the sham + NaHS group. CHF was induced in the rats by abdominal aorta-inferior vena cava shunt operation. Rats in the shunt + NaHS and sham + NaHS groups were injected intraperitoneally with NaHS (H2S donor). Haemodynamic parameters were measured 8 weeks after surgery. In addition, left ventricular heme oxygenase (HO)-1 mRNA expression was measured by real-time PCR. Protein expression of HO-1 was evaluated by western blot analysis. Eight weeks after surgery, compared to the sham group, the left ventricular systolic pressure (LVSP) and left ventricular peak rate of contraction and relaxation (LV±dp/dtmax) were significantly reduced; the left ventricular end-diastolic pressure (LVEDP) was significantly increased in the shunt group (all P<0.05). However, NaHS increased LVSP and LV±dp/dtmax (all P<0.05) and decreased LVEDP (P<0.05). Protein expression of HO-1 was significantly decreased in the shunt group compared to that in the sham group (P<0.05). NaHS increased protein expression of HO-1 compared to that in the shunt group (P<0.05). HO-1 mRNA expression was significantly increased in the shunt + NaHS group compared to that in the shunt group (P<0.01). The present study demonstrated that H2S may play a protective role in volume overload-induced CHF by upregulating protein and mRNA expression of HO-1.

[Survey of pertussis infection in infants aged under 3 months with persistent cough].

OBJECTIVE: To explore the prevalence of pertussis in hospitalized infants aged under 3 months with persistent cough. METHODS: The nasopharyngeal secretions and serum samples were collected from hospitalized infants aged under 3 months with cough for over 2 weeks from January 2011 to January 2012. The samples of nasopharyngeal secretion were suctioned and collected. Multiplex PCR assay was employed to identify Bordetella pertussis (B. pertussis) and enzyme-linked immunosorbent assay used to detect antibody to pertussis toxin (PT-IgG). Total bacterial DNA was exacted from nasopharyngeal secretion and two-target IS481/PT of B. pertussis was detected by PCR. RESULTS: Fifty-nine infants (32 boys and 27 girls) were enrolled. None of them was pre-immunized with diphtheria-pertussis-tetanus vaccine. Seventeen infants (28.8%) were B. Pertussis positive. Among 17 cases, 3 infants under 1 month, 4 infants 1 -2 months, and 10 infants 2 - 3 months. Three infants had household contacts with persistent cough and their serum antibodies to pertussis toxin were positive. Sixteen infants with pertussis had the paroxysms of frequent and rapid coughs while another 5 with pertussis had long inspiratory effort accompanied by a high-pitched "whoop" at the end of paroxysms. Seven infants with pertussis had conjunctiva bleeding, a special sign of pertussis. Ten infants had lymphocytosis with a predominant elevation of lymphocytes. CONCLUSIONS: B. pertussis is an important pathogen for the infants under 3 months with persistent cough. Multiplex PCR may be used to identify B. pertussis with a high sensitivity. The unrecognized close family members of the infants with pertussis are probably an important source of infection.

[Establishment of protein fingerprint database of Salmonella paratyphi A using SELDI-TOF-MS].

AIM: To establish a protein fingerprint database of Salmonella paratyphi A by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). METHODS: Thirty-six clinical bacterial isolates and 96 control bacteria isolates were collected and identified using 16S rDNA sequencing. Bacterial proteins were detected by SELDI-TOF-MS, and all protein fingerprints were analyzed by ProteinChip and Biomarker Wizard software. The analysis results were used to set up a classification tree model by means of BioMarker Patterns software. At the same time, the data were tested by a blinded validation. RESULTS: In the range of M(r); 3 000-20 000, we obtained 104 protein peaks, of which 90 were of statistical significance (P<0.01). A protein peak with mass-to-charge ratio(M/Z) 10 061.7 was chosen to establish the classification tree model of Salmonella paratyphi A, and the sensitivity and specificity of Salmonella paratyphi A diagnosis was 100% as shown by the blinded validation. CONCLUSION: The classification tree model of Salmonella paratyphi A can be not only established using SELDI-TOF-MS technology, but also used for the rapid identification of Salmonella paratyphi A.

Polysaccharides from the Medicinal Mushroom Cordyceps taii Show Antioxidant and Immunoenhancing Activities in a D-Galactose-Induced Aging Mouse Model.

Cordyceps taii, an edible medicinal mushroom native to south China, is recognized as an unparalleled resource of healthy foods and drug discovery. In the present study, the antioxidant pharmacological properties of C. taii were systematically investigated. In vitro assays revealed the scavenging activities of the aqueous extract and polysaccharides of C. taii against various free radicals, that is, 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, and superoxide anion radical. The EC(50) values for superoxide anion-free radical ranged from 2.04 mg/mL to 2.49 mg/mL, which was at least 2.6-fold stronger than that of antioxidant thiourea. The polysaccharides also significantly enhanced the antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase) and markedly decreased the malondialdehyde production of lipid peroxidation in a D-galactose-induced aging mouse model. Interestingly, the immune function of the administration group was significantly boosted compared with the D-galactose-induced aging model group. Therefore, the C. taii polysaccharides possessed potent antioxidant activity closely associated with immune function enhancement and free radical scavenging. These findings suggest that the polysaccharides are a promising source of natural antioxidants and antiaging drugs. Consequently, a preliminary chemical investigation was performed using gas chromatography-mass spectroscopy and revealed that the polysaccharides studied were mainly composed of glucose, mannose, and galactose. Fourier-transform infrared spectra also showed characteristic polysaccharide absorption bands.

[A pathogen analysis within 174 cases of hand-foot-mouth disease in the summer of 2009].

OBJECTIVE: To analyze the pathogen and characteristics of the serum types of enterovirus of hand-foot-and-mouth disease (HFMD) in the summer, 2009. METHODS: Both throat swab and herpes fluids were taken respectively from 174 children with HFMD in the outpatient infection during April to September, 2009. Anti-Cox A16 and anti-EV71 IgMs in the serum were detected with ELISA. And RNA were extracted from each sample followed with real-time fluorescence quantitative RT-PCR kits with three reagents: universal enterovirus primer, Coxsackievirus A16 (CA16) primer and enterovirus 71 (EV71) primer. Parts of positive samples were sequenced and analyzed. RESULTS: (1) EV genes were detected from 167 cases, of which ,112 cases were positive for CA16 and 46 were positive for EV71. CA16: EV71 was 2.43: 1. (2) There were 51 cases with CA16 IgM positive and 25 cases with EV71 IgM positive in the early collected sera, and in the later samples, 98 cases with CA16 IgM positive and 32 cases with EV71 IgM positive. (3)The nucleotide homologies were 88.7%-98.5% of VP1 gene among CA16. The nucleotide homologies were 94.9% - 99.7% of VP1 gene among EV71, and were 92.1% - 95.3% with C4 subtype. CONCLUSION: The mainly pathogen causing HFMD in children in the summer, 2009 were CA16 and EV71. EV71 infection, mainly C4 subtype, was highly elevated according to the earlier reported. Real-time RT-PCR is more appropriate than the serological test.