Sangjie Yu

Extensive nuclear localization of α-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody

Synuclein was initially named for its localization in both presynaptic nerve terminals and portions of nuclear envelope. However, subsequent studies only confirmed the presynaptic localization of this protein in the brain; its nuclear localization in the neurons remained elusive. Here, two new monoclonal antibodies against alpha-synuclein (alpha-SYN) were produced. Epitope mapping using phage peptide display showed that the epitopes of the two antibodies were localized in two distinct specific sequences of the C-terminal domain of alpha-SYN. One antibody named 3D5 recognized amino acids 115-121 of alpha-SYN and the other antibody named 2E3 identified the amino acids 134-138 of the protein. Western blot analysis demonstrated that both 2E3 and 3D5 detected a 19 kD protein from rat and human brain homogenates, which was identical to the molecular size of recombinant alpha-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of alpha-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic alpha-SYN but only 3D5 detected the nuclear alpha-SYN. The nuclear localization of alpha-SYN was further confirmed by Western blot analysis in isolated nuclear fraction where the same size of alpha-SYN was detected, and by immunoelectron microscopy using colloidal gold probes where gold particles were specifically localized in portions of peri- and intra-nucleus. The nuclear positive neurons were distributed extensively in almost all the brain regions. This is the first report well characterizing the extensive localization of alpha-SYN in the neuronal nuclei throughout the brain in normal conditions. This finding indicates an important physiological function of this molecule in the nuclei of brain neurons, which deserves further investigations.

Epidemiology and molecular characteristics of community-associated methicillin-resistant and methicillin-susceptible Staphylococcus aureus from skin/soft tissue infections in a children's hospital in Beijing, China

To evaluate the epidemiology and molecular features of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and methicillin-sensitive S. aureus (MSSA) from children with skin and soft tissue infections (SSTIs) in Beijing, China, prospective community-acquired S. aureus SSTIs surveillance was conducted at the Beijing Childrens Hospital, Beijing, China, for a 12-month period from August 1, 2008, to July 30, 2009. Susceptibility to 12 antimicrobials was determined by the agar dilution method. Genotypic characteristics of CA-MRSA isolates were tested by SCCmec typing, spa typing, and multilocus sequence typing. Panton-Valentine leukocidin gene was detected. Of 1104 cases, 31.8% (351) were community-acquired S. aureus. CA-MRSA accounted for 4% (14) of S. aureus. Among 14 CA-MRSA and 120 MSSA isolates tested, 100% and 91.7% were multidrug resistant, respectively. ST59-MRSA-IVa-t437 (42.9%) was the most common form of CA-MRSA. Spa typing analysis of 120 MSSA isolates was performed, followed by pulsed-field gel electrophoresis and multilocus sequence typing of a selected number of isolates. The most common spa types among MSSA were t084 (8.3%), t091 (5.8%), t034 (5%), t127 (4.2%), t002 (4.2%), and t796 (4.2%). No predominant spa type was seen. Of the MSSA isolates that could be classified into spa-CCs, 15.0% had a genetic background observed in CA-MRSA clones (spa-CC437, spa-CC342, and spa-CC377). Panton-Valentine Leukocidin (PVL)-positive community-acquired S. aureus strains were more commonly associated with skin abscesses than other SSTIs (29.4% versus 5.9%, P < 0.01).In conclusion, CA-MRSA infections are not common among Chinese children with SSTIs. Our findings show that MSSA strains in China have diverse genetic backgrounds.

Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Isolates That Cause Invasive Disease among Chinese Children

A total of 171 Streptococcus pneumoniae isolates causing invasive disease were isolated from Chinese children. The serotype distribution and antimicrobial resistance were tested. The results suggested that the 7-valent pneumococcal conjugate vaccine has a preventive effect among children and that there should be long-term surveillance for serotype 19A.

Superantigen gene profiles and presence of exfoliative toxin genes in community-acquired meticillin-resistant Staphylococcus aureus isolated from Chinese children.

This study aimed to evaluate the distribution of superantigen gene profiles and the presence of exfoliative toxin genes in community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) isolated from Chinese children, and simultaneously to assess virulence gene profiles and genetic background. Of the CA-MRSA isolates, 88.9 % (88/99) harboured toxin genes, with sek as the most frequent toxin gene (62.6  %), followed by seq (61.6  %), seb (60.6  %) and sea (35.4  %). The eta gene was detected only in one ST398-IVa-spa t034 strain. The sed and etd genes were not found in any of the isolates tested. A total of 38 virulence genotypes were observed, of which the genotype seb-sek-seq (27.3  %, 24/88) comprised the majority, followed by sea-seb-sek-seq (18.2  %, 16/88). The enterotoxin gene cluster including seg-sei-sem-sen-seo-seu predominated at a rate of 15.1  %. The relationship among toxin genotypes, toxin genes encoding profiles of mobile genetic elements and genetic background was analysed. Among 66 clonal complex (CC) 59 isolates, 87.9 % (58/66) were positive for toxin genes, and 75.8  % (50/66) harboured the toxin gene combination seb-sek-seq. Among seb-sek-seq-positive CC59 strains, 42.0  % (21/50) also carried the sea gene. CC59 corresponded exclusively to accessory gene regulator 1 (agr-1). The data presented here enhance our current knowledge on the virulence determinants of CA-MRSA.

Community-acquired, methicillin-resistant Staphylococcus aureus isolated from children with community-onset pneumonia in China.

Community‐acquired, methicillin‐resistant Staphylococcus aureus (CA‐MRSA) has been associated with morbidity and mortality in various countries. In this study, we characterized the molecular and clinical features of pediatric CA‐MRSA pneumonia in China. Between June 2006 and February 2008, 55 previously healthy children confined in eight hospitals countrywide were found to be afflicted with CA‐MRSA pneumonia. A total of 55 strains collected from these children were analyzed by multilocus sequence typing (MLST), Staphylococcus cassette chromosome mec (SCCmec) typing, and spa typing. The Panton–Valentine leukocidin (PVL) gene was also detected. Overall, nine STs were obtained, with ST59 (40.4%) established to be the most prevalent type. We first registered the new ST1409 from a child with necrotizing pneumonia. SCCmecIVa was the most predominant type, followed by SCCmec type V. Twelve spa types were identified, of which one new spa type, t5348, was first detected and registered. One typical livestock‐associated spa type, t034, was found in a 4‐month‐old girl living in the countryside. We also found that 40% of those isolates were PVL‐positive. In addition, the median age of the children in this study was 10 months. A total of 69% (38/55) of the children with community‐acquired pneumonia (CAP) had preceding influenza or influenza‐like illness, and three ST910‐MRSA‐IV strains (PVL gene‐positive) were associated with severe necrosis. The results indicated that the recent CA‐MRSA found in Chinese children with CAP was largely associated with the spread of the ST59‐MRSA‐IV clone, and most of the PVL‐positive strains in this study did not cause necrotic cases. Pediatr Pulmonol. 2010; 45:387–394.

Serotype distribution and antibiotic resistance of 140 pneumococcal isolates from pediatric patients with upper respiratory infections in Beijing, 2010

In the present study, the serotype distribution and antibiotic resistance of S. pneumoniae from pediatric patients with upper respiratory infections in Beijing, 2010 were described. 140 pneumococcal isolates were obtained, and the prevailing five serotypes were 19F (18.6%), 23F (9.3%), 14 (9.3%), 15 (9.3%), and 6A (7.1%). The vaccine coverage of PCV7, PCV10, and PCV13 were 43.6%, 43.6%, and 60.0%, respectively. According to the CLSI 2010 criteria, 99.3% of the S. pneumoniae isolates were susceptible to penicillin. The resistance rates to erythromycin and azithromycin were 96.4% and 97.1%, respectively. Meanwhile, 64.3% (90/140) of all pneumococcal isolates were multidrug-resistant S. pneumoniae (MDRSP). PCV13 covered 68.9% (62/90) of MDRSP strains, whereas it was 47.8% (43/90) for PCV7. ErmB was the dominant macrolide-resistance gene, whereas 30.4% pneumococcal isolates expressed both ermB and mefA. No isolate expressed ermTR. The potential coverage of PCV13 is higher than PCV7 and PCV10 because high rates of serotypes 6A and 19A, and the conjugate vaccines could prevent the spread of MDRSP. S. pneumoniae is still sensitive to penicillin. The resistance rate of S. pneumoniae to macrolides is high and ermB is the dominant macrolide-resistance gene in China, so continued surveillance of the antimicrobial susceptibility of S. pneumoniae may be necessary.

Epidemiological and molecular characteristics of clinical isolates of Streptococcus pyogenes collected between 2005 and 2008 from Chinese children

The aim of this study was to explore the epidemiological and molecular characteristics of Streptococcus pyogenes in children from different cities in mainland China who were diagnosed with scarlet fever, impetigo and pharyngitis, as well as to detect asymptomatic carriers, between 2005 and 2008, and to compare the results with isolates from rural Chinese children with acute glomerulonephritis in 2005 and in the 1990s. Susceptibility tests to determine MICs and analysis of the presence of erythromycin-resistant genes (mefA, ermB and ermA) and emm gene typing were performed on 466 S. pyogenes isolates from Beijing, Shanghai, Chongqing and Shenzhen. Superantigen genes (speA and speC) were examined by performing PCR on isolates with the most prevalent emm genotype. All isolates were sensitive to penicillin, cefradine and ofloxacin. The highest rate of resistance was against clarithromycin (98.1 %), followed by erythromycin (97.6 %), azithromycin and clindamycin (both 97.2 %), and tetracycline (94.0 %). Among the 466 isolates, 421 (90.3 %) harboured the ermB gene, 145 (31.1 %) were speA-positive and 273 (58.6 %) were speC-positive. The speA gene was common in emm1.0 (88.8 %) and emm6.5 (83.3 %) genotypes. The speC gene was frequently observed in emm4.0 (90.0 %), emm12.0 (69.6 %), emm18.0 (66.7 %), emm22.0 (75.9 %) and emm80.0 (80.0 %) genotypes. The most prevalent emm genotypes in mainland China in recent years were emm1.0 and emm12.0. All isolates remained sensitive to β-lactams and quinolone.

Antibiotic use in five children's hospitals during 2002–2006: the impact of antibiotic guidelines issued by the Chinese Ministry of Health

To investigate the pattern of antibiotic use in five Chinese childrens hospitals from 2002 to 2006. To see if the Guidelines to encourage rational use of antibiotics issued by the Ministry of Health in October 2004 have any impact on the use.

Clinical and molecular characteristics of invasive community-acquired Staphylococcus aureus infections in Chinese children

BackgroundThis study aims to investigate the clinical features of invasive community-acquired Staphylococcus aureus (CA-SA) infection in Chinese children and analyze its molecular features.MethodsClinical data and invasive CA-SA isolates were prospectively collected. Pediatric risk of mortality (PRISM) score was used for disease severity measurement. Molecular typing was then performed, followed by expression analysis for virulence genes.ResultsAmong 163 invasive CA-SA infection cases, 71 (43.6%) were methicillin-resistant SA (MRSA) infections and 92 (56.4%) were methicillin-susceptible SA (MSSA). A total of 105 (64.4%) children were younger than 1 year old, and 79.7% (129/163) were under 3 years age. Thirteen kinds of diseases were observed, in which bacteremia and pneumonia accounted for 65.6% (107/163) and 52.8% (86/163), respectively. A total of 112 (68.1%) patients had two or more infective sites simultaneously, and four cases (2.5%) died. CA-MSSA more frequently caused multi-sites infections, bacteremia, and musculoskeletal infection than MRSA. A total of 25 sequence types (STs) were detected. MRSA mainly comprised ST59 (49/71, 69%), whereas the most frequent clonotypes were ST88 (15/92, 16.3%), ST25 (13/92, 14.1%), ST7 (13/92, 14.1%), ST2155 (12/92, 13%), and ST188 (9/92, 9.8%) for MSSA. Seven STs were common to both MSSA and MRSA groups. No differences in clinical presentation or PRISM score were found between the two groups or among different ST. The expression levels of the four known virulence genes varied among the six main ST clones.ConclusionsInvasive CA-SA infections were characterized by high incidence and multi-site infections in young children in China. The clinical manifestations of CA-MSSA were more frequently associated with multi-site infections, bacteremia and musculoskeletal infection than those of CA-MRSA. Isolated genotypes may be relevant to the expressions of virulence genes, but not to clinical manifestations.

Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Isolates Causing Invasive Diseases from Shenzhen Children’s Hospital

Objective To provide guidance for clinical disease prevention and treatment, this study examined the epidemiology, antibiotic susceptibility, and serotype distribution of Streptococcus pneumoniae (S. pneumoniae) associated with invasive pneumococcal diseases (IPDs) among children less than 14 years of age in Shenzhen, China. Materials and Methods All the clinical strains were isolated from children less than 14 years old from January 2009 to August 2012. The serotypes and antibiotic resistance of strains of S. pneumoniae were determined using the capsular swelling method and the E-test. Results A total of 89 strains were isolated and 87 isolates were included. The five prevailing serotypes were 19F (28.7%), 14 (16.1%), 23F (11.5%), 19A (9.2%) and 6B (6.9%). The most common sequence types (ST) were ST271 (21.8%), ST876 (18.4%), ST320 (8.0%) and ST81 (6.9%) which were mainly related to 19F, 14, 19A and 23F, respectively. The potential coverage by 7-, 10-, and 13-valent pneumococcal conjugate vaccine were 77.0%, 77.0%, and 89.7%, respectively. Among the 87 isolates investigated, 11.5% were resistant to penicillin, and for meningitis isolates, the resistance rate was 100%. Multi-drug resistance (MDR) was exhibited by 49 (56.3%) isolates. Eighty-four isolates were resistance to erythromycin, among which, 56 (66.7%) carried the ermB gene alone and 28 (33.3%) expressed both the ermB and mefA/E genes. Conclusions The potential coverage of PCV13 is higher than PCV7 and PCV10 because high rates of serotypes 19A and 6A in Shenzhen. The clinical treatment of IPD needs a higher drug concentration of antibiotics. Continued surveillance of the antimicrobial susceptibility and serotypes distribution of IPD isolates may be necessary.