ObjectiveTo investigate the characteristic of pathology in Chinese patients with Nonaka myopathy.MethodsThirteen patients (7 males and 6 females) diagnosed with Nonaka myopathy in our laboratory from January 2002 to March 2011 were included in this study.Their mean age was 39.5 years old and the mean duration of illness was 4.15 years.The most common symptoms were weakness of raising feet with sparing of quadriceps femoris muscles in the early stage of disease.One patient presented the initial symptoms of upper limb weakness. Muscles biopsies were obtained from all these 13 patients. Histology study including immunohistochemical (IHC) staining with antibody against amyloid 3,phosphorylated tau protein,ubiquitin,glucose-regulated protein of molecular weight 78 000(GRP78),calnexin,caspase-12and Bax were performed.Skeletal muscle samples from 3 chronic fatigue syndrome patients,2 myofibrillar myopathy patients were used for control in the IHC staining. All coding exons of uridinediphospho-N-acetylglucosamine 2-epimerase gene were directly sequenced in genomic DNA from these patients.Results The main pathological changes of tibialis anterior muscle in 12 cases were muscle dystrophy with rimmed vacuoles.The rimmed vacuoles were positive for anti-β-amyloid,tau protein and ubiquitin in IHC studies.In the atrophy fibers,IHC showed the increased expression of endoplasmic reticulum stress related proteins GRP78 and calnexin,and apoptosis proteins of caspase-12 and Bax.ConclusionsThere is accumulation of abnormal proteins in muscle fibers in Chinese patients with Nonaka myopahty.These proteins may stimulate endoplasmic reticulum stress and apoptosis,which may be a mechanism responsible for muscle damage.

Hepatic eosinophilic granuloma (HEG) is a rare benign liver disease,which belongs to histocytosis.Preoperative diagnosis of HEG was difficult because its clinical manifestation was not characteristic.In this article,the clinical data of 1 patient with HEG who was treated at the Jinhua Municipal Central Hospital of Zhejiang Province in September 2012 were retrospectively analyzed,and the diagnosis,differential diagnosis and treatment for HEG were investigated.

Objective To investigate the epidemiological characteristics of mycoplasma pneumoniae (MP),Epstein-Barr virus (EB virus) and cytomegalovirus (CMV) infection in children with acute upper respiratory tract infections in Hangzhou.Methods Throat swabs and sputum samples were collected from 5942 children with acute upper respiratory tract infections in Hangzhou First People's Hospital during January 2011 and December 2012.MP,EB virus and CMV were detected using quantitative PCR.The distribution and seasonal changes of the above pathogens in children of different ages were analyzed using Chi-square tests.Results MP,EB virus and CMV were positive in 29.91％ (1777/5942),22.92％ (1362/5942) and 8.55％ (508/5942) children,respectively.Mixed infections were found in 556 (9.36％) children.The positive rates of MP varied among different age groups (x2 =113,P =0.000),and the highest one was detected in children ＞ 6-year old (448/1012,44.36％).EB virus infection was rare in age group 0-1 year,and the positive rate was of statistical difference from those in other age groups (x2 =167,181 and 187,P =0.000).The highest positive rate of CMV (23.78％) was found in children aged 0-1 year old.The positive rates of MP varied in different months of the year (x2 =208 and 211,P =0.000),and the highest positive rate was found in July and August.Conclusion The predominant pathogen of acute upper respiratory tract infection in children is MP in Hangzhou,and MP plus EB virus infection is common,particularly in older children;while CMV infection more likely occures in 0-1 year old babies,and usually in summer.

Objective To identify a rapid and efficient fungal genomic DNA extraction method for PCR amplification.Methods Genomic DNA was extracted from Penicillum marneffei,Rhizopus microsporus,Cryptococcus neoformans and Candida albicans by heating pyrolysis,microwave,repeated freezing and thawing,lysozyme digestion,overnight snail enzymatic and Qiagen kit methods.DNA electrophoretogram was observed by gel imaging system.The concentration and purity of extracted DNA were determined with an ultramicro nucleic acid protein tester and the yields were calculated.PCR amplification and sequencing were also performed.ANOVA and SNK-q test were used for data analysis.Results There were statistical differences in concentrations and yields of the fungal DNA extracted from Penicillum marneffei (hyphal phase and yeast phase),Rhizopus microsporus,Coptococcus neoformans and Candida albicans by six methods (F=750.83,220.95,669.35,132.01,510.20 and 1658.35,287.10,963.64,1147.77,4521.22,all P ＜0.01).Of six methods,microwave method gained the highest DNA concentration and yield,followed by heating pyrolysis method,while Qiagen kit method obtained the lowest concentration and yield.All DNA extracted by 6 kinds of methods were positive in PCR amplification.Conclusion All of the six methods can be used for fungal DNA extraction which is sufficient for PCR amplification,but microwave and heating pyrolysis methods are more easy and simple to perform.

Objectives To investigate the properties and distributions of ampC gene among different drug-resistant strains of Serratia marcescens,and the relationship of control gene ampR with AmpC enzymes′ expressions.Methods According to the results of inducting experiment with 1/2 MIC of beta-lactam antibiotics (CTX),three-dimensional testing and isoelectric focusing electrophoresis testing,143 strains of S.marcescens were classified into three groups:including induction group, continuous low-production group and hyperproduction group. In each group, the sequences of ampC and ampR genes were amplified using the method of PCR. The products of PCR were analyzed. The plasmid-mediated beta-lactamases were detected using the method of conjugation experiment.Results Among 143 strains of S.marcescens, the continuous low -production strains, induction strains and hyperproduction strains were 14,103,and 18, respectively.125 and 99 strains were ampC and ampR gene positive, respectively.The detection rate of ampR in hyperproduction group was lower than other groups.5 sites of ampC genes and 4 sites in the Open Reading Frame (ORF) of ampR gene were easily mutated in 5 induction strains and 2 hyperproduction strains.Conclusions The production of inducing drug-resistance of some S.marcescens might be related to mutation of ampC gene encoding AmpC beta-lactamases and the ORF mutation in ampR. The continuous hyperproduction drug-resistance had something to do with deletion mutation in ampR in segmental hyperproduction strains.The plasmid-mediated AmpC enzymes hadn′t been found in S.marcescens.

Objective To explore phenotypes and genotypes in 6 Chinese Han families with oculopharyngeal muscular dystrophy (OPMD).Methods There were 28 OPMD patients in 6 Chinese Han families, aged between 32 and 70 years old.The initial symptom was dysphagia in 13 patients, ptosis in 4 patients,lower limb weakness in 1 patient.Most OPMD patients displayed dysphagia and ptosis in 3-20 years after the initial symptom appeared.Biceps brachii biopsies were made in the 6 OPMD probands.The specimens were examined with histological, histochemical stainings and ultrastructural examination.The exon 1 of PABPN1 gene was sequenced in all probands of OPMD and some of their family members.Haplotype analysis was executed in the 6OPMD probands.Results Mild hypertrophy and hypotrophy of fibers and rimmed vacuoles were found in each probands of OPMD muscle biopsies.Intranuclear palisading filamentous inclusions only appeared in 4OPMD probands.Gene analysis identified (GCG)9 in exon 1 of PA BPN 1 gene was detected in 3OPMD families, while (GCG) 6 (GCA) 1 (GCG) 3, (GCG) 10 and (GCG) 8were in one family respectively.Two families with (GCG) 9 expansion shared a common rs2239579 (C) -(GCG) 9-SNP2622 (C) haplotype.Conclusions Dysphagia and ptosis may be the common initial symptoms in Chinese Han OPMD patients.Rimmed vacuoles and intranuclear inclusions may be the common pathological feature in the muscle fibers.Both (GCG) expansions and (GCA) insertion of PABPN1 gene could be detected in Chinese patients.The genetic heterogeneity and the haplotype of three (GCG)9 families in our patients indicate that OPMD arises from different origin within the Han population.Some families with the same (GCG)9 expansions probably come from a common ancestor.

Objective To establish a rapid,accurate and specific method to detect the common mycobacteria based on multiplex real-time PCR.Methods The dual priming oligonucleotide ( DPO)primers and TaqMan probes labeled with FAM,ROX,HEX or JOE fluoresceins at 5' end and eclipse at 3' end respectively were designed to detect the 16S rRNA of mycobacteria.Both specificity and sensitivity were estimated on multiplex real-time PCR detecting genome DNA from 4 mycobacterial model species.Sixty eight early morning sputum specimens collected from hospitalized patients in the Red Cross Hospital of Hangzhou were detected by multiplex real-time PCR,bacterial culture and smear microscopy simultaneously.The positive rates were analyzed by chi-square.Results Mycobacteria including Mycobacterium tuberculosis and three common non-tuberculosis mycobacteria spp.were identified by multiplex real-time PCR accurately and specifically,with the limited load at 101 cfu/ml.In 68 sputum specimens,31 were positive (positive rate 45.6％ ) by this method,which was significant higher than that by smear microscopy ( positive rate 14.7％,x2 =15.4,P ＜0.05 ).The positive cases were identified as 28 Mycobacterium tuberculosis,1 Mycobacterium avium and 2 Mycobacterium intracellulare in agreement with the culture results.One case,which is detected by culture,but not by PCR,was identified as Mycobacterium chelonae by sequencing.Conclusion The multiplex real-time PCR characterizing as sensitive,specific and time-saving for Mycobacterium tuberculosis and common non-tuberculosis mycobacteria could be chosen as the rapid laboratory test of mycobacterial infection.

OBJECTIVE To investigate macrolide resistance and main molecular mechanisms in Mycoplasma pneumoniae. METHODS Thirty two throat swabs from children infected with M. pneumoniae were cultured by modified Hayflick medium. Antibiotic susceptibility test was used to screen the macrolide-resistant M. pneumoniae. The 23S rRNA gene sequences of the strains were determined with polymerase chain reaction and sequencing. RESULTS Nineteen strains were isolated from 32 throat swabs successfully.Fifteen strains were resistant to macrolide antibiotics according to the results of antibiotic susceptibility test. Once the strain was resistant to one of macrolide antibiotics,it would be resistant to the others. Sequencing results of the sensitive strains and the standard strain FH were completely same. Fifteen resistant strains presented A2063G point mutation in 23 SrRNA region Ⅴ, in which 2 examples showed the coexistence of the sensitive strain and the resistant strain. CONCLUSIONS Macrolide-resistant M. pneumoniae is common and serious at present. The antibiotic resistant isolate carries point mutations of the 23S rRNA region Ⅴ.

Objective To report clinical and pathological features in a Chinese family with desminopathy caused by a novel mutation in desmin gene. Methods There were 8 patients (7 male and 1 female) in a family manifesting desminopathy in an autosomal dominant inheritance pattern. The onset age ranged from 20--50 years old. Heart disease appeared in 7 cases, diarrhea in 1 case and limb weakness in 3 patients. Muscle biopsies were carried out in proband and his elder brother. The histological, enzymhistochemicai staining, ultrastructural examination and immunohistochemical staining with desmin antibody were performed. The sequence of desmin gene was analyzed in DNA from 4 patients, 5 asymptomatic members and 50 healthy controls. Results Muscle pathology in the proband revealed wide variation in size of fibers. Some fibers had eosinophilic changes with rimmed vacuoles. Muscle pathology in the brother revealed multiple small and round eosinophilic inclusions in the fibers. Desmin staining was positive in the inclusions and in the sarcolemma. Electron microscopy revealed deposits of abnormal granulofilamentous materials. A novel S12F mutation in desmin gene has been identified in 4 affected family individuals, but not in unaffected family members and controls. Conclusions The desminopathy caused by desmiu SI2F mutation dominantly affected male individuals. Most of victims had cardiac abnormalities. The myopatholgical features can vary in the different patients in one family.

Objective To observe the effect of Huangxiong Kangshuan capsule on serum high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6) levels in patients with acute cerebral infarction. Methods Ninety-two patients with acute cerebral infarction admitted to the First Affiliated Hospital of Anhui University of Traditional Chinese Medicinefrom July 2013 to December 2016 were enrolled, and they were divided into an observation group (47 cases) and a control group (45 cases) by random number table. The control group was given conventional treatment of ischemic cerebrovascular disease, while the observation group was additionally treated by Huangxiong Kangshuan capsule orally taken, once 3 tablets, 3 times a day, on the basis of routine treatment; the duration of treatment was 2 weeks in both groups. After 2 weeks of treatment, the clinical effects of the two groups and the changes of serum hs-CRP and IL-6 levels were observed.Results After treatment, the levels of serum hs-CRP and IL-6 were decreased significantly compared with those before treatment in the two groups [observation group: hs-CRP (mg/L) was 6.18±2.17 vs. 14.11±3.01, IL-6 (ng/L): 28.10±11.47 vs. 120.83±24.51; control group: hs-CRP (mg/L) was 8.89±2.46 vs. 13.97±2.69, IL-6 (ng/L) was 49.48±16.43 vs. 115.25±24.05], and the degree of decline in the observation group was more significant than that in the control group [hs-CRP (mg/L): 6.18±2.17 vs. 8.89±2.46, IL-6 (ng/L): 28.10±11.47 vs. 49.48±16.43, bothP < 0.01]; the total effective rate of the observation group was significantly higher than that of the control group [87.2% (41/47) vs. 71.1% (32/45),P < 0.05]. Conclusion Huangxiong Kangshuan capsule can decrease the serum hs-CRP and IL-6 levels in patients with acute cerebral infarction, and has a role in brain protection and nerve function defect improvement.