Branched-chain amino acids attenuate early kidney injury in diabetic rats.

Diabetic nephropathy (DN) is the most severe diabetic microvascular complication. The pathogenesis of diabetic nephropathy is complex, and oxidative stress plays an important role in the development of diabetic nephropathy. Elevated reactive oxygen species (ROS) levels activate various signaling pathways and influence the activities of transforming growth factor-ß (TGF-ß) and matrix metalloproteinase-9 (MMP-9), which contributes to glomerular hypertrophy. Branched-chain amino acids (BCAAs) are widely used in clinical treatment, and BCAAs can reduce the oxidative stress associated with the diabetic pancreas and some liver diseases. Thus, the aim of the present study was to determine whether BCAAs could attenuate oxidative stress in the kidneys of streptozotocin (STZ)-induced diabetic rats to prevent early diabetic kidney injury. Male Wistar rats were fed for two weeks with a normal chow diet or a high-fat diet in which 40% of calories were derived from fat. After this two-week period, the mice fed normal chow were injected with vehicle, while the high-fat diet group was injected intraperitoneally (i.p.) with 40 mg/kg STZ. The STZ-treated group was randomly divided into four subgroups that were treated with different doses of BCAAs or vehicle for two months by oral gavage. Plasma glucose, plasma creatinine, urinary protein and JNK, TGF-ß, and MMP-9 mRNA and protein expression levels were measured in the rats. The ROS levels and proteinuria in the STZ-induced diabetic rats were significantly higher than those in the control groups. Moreover, early kidney injury occurred in the STZ-induced diabetic rats. However, BCAAs treatment decreased ROS levels, proteinuria and kidney injury. Moreover, JNK, TGF-ß and MMP-9 mRNA and protein levels were significantly increased in the diabetic rats when compared with the control rats, and BCAAs treatment reversed these changes. Our results suggest that BCAAs counter oxidative stress in the kidneys of diabetic rats and alleviate diabetic kidney injury via the JNK/TGF-ß/MMP-9 pathway.

Surveillance of pathogens causing gastroenteritis and characterization of norovirus and sapovirus strains in Shenzhen, China, during 2011.

Viral gastroenteritis is one of the most common diseases in humans, and it is primarily caused by rotaviruses (RVs), astroviruses (AstVs), adenoviruses (AdVs), noroviruses (NoVs), and sapoviruses (SaVs). In this study, we determined the distribution of viral gastroenteritis and human calicivirus (HuCVs) in acute gastroenteritis patients in Shenzhen, China, during 2011. Real-time RT-PCR was used to detect norovirus (NoV), group A rotavirus (RV), adenovirus (AdV), and astrovirus (AstV). From a total of 983 fecal samples, NoV was detected in 210 (21.4 %); RoV in 173 (17.6 %); AstV in 10 (1.0 %); and AdV in 15 (1.5 %). Mixed infections involving two NoVs were found in 21 of the 387 pathogen-positive stool specimens. NoV and SaV genotypes were further tested using RT-PCRs and molecular typing and phylogenetic analysis were then performed based on the ORF1-ORF2 region for NoV and a conserved nucleotide sequence in the capsid gene for SaV. Of the 68 typed strains that were sequenced and genotyped, five were NoV G1 (7.5 %) and 63 were NoV GII (96.6 %). GII strains were clustered into five genotypes, including GII.4 (65.1 %; 36 GII.4 2006b and five GII.4 New Orleans), GII.3 (28.6 %), GII.2 (3.2 %), GII.6 (1.6 %), and GII.1 (1.6 %). While all fecal specimens were tested for SaVs, 15 (1.5 %) were positive, and of these, 12 isolates belonged to G1.2, and the remaining three SaV strains belonged to the SaV GII genogroup. Although various HuCVs were detected in acute gastroenteritis patients, NoV GII.4 2006b was more prevalent than the other HuCVs.

Emergence, circulation, and spatiotemporal phylogenetic analysis of coxsackievirus a6- and coxsackievirus a10-associated hand, foot, and mouth disease infections from 2008 to 2012 in Shenzhen, China.

Sporadic hand, foot, and mouth disease (HFMD) outbreaks and other infectious diseases in recent years have frequently been associated with certain human enterovirus (HEV) serotypes. This study explored the prevalences and genetic characteristics of non-HEV71 and non-coxsackievirus A16 (CV-A16) human enterovirus-associated HFMD infections in Shenzhen, China. A total of 2,411 clinical stool specimens were collected from hospital-based surveillance for HFMD from 2008 to 2012. The detection of HEV was performed by real-time reverse transcription-PCR (RT-PCR) and RT-seminested PCR, and spatiotemporal phylogenetic analysis was performed based on the VP1 genes. A total of 1,803 (74.8%) strains comprising 28 different serotypes were detected. In the past 5 years, the predominant serotypes were HEV71 (60.0%), followed by CV-A16 (21.2%) and two uncommon serotypes, CV-A6 (13.0%) and CV-A10 (3.3%). However, CV-A6 replaced CV-A16 as the second most common serotype between 2010 and 2012. As an emerging pathogen, CV-A6 became as common a causative agent of HFMD as HEV71 in Shenzhen in 2012. Phylogenetic analysis revealed that little variation occurred in the Chinese HEV71 and CV-A16 strains. The genetic characteristics of the Chinese CV-A6 and CV-A10 strains displayed geographic differences. The CV-A6 and CV-A10 strains circulating in Shenzhen likely originated in Europe. It was found that human enteroviruses have a high mutation rate due to evolutionary pressure and frequent recombination (3.2 × 10(-3) to 6.4 ×10(-3) substitutions per site per year for HEV71, CV-A6, CV-A16, and CV-A10). Since certain serotypes are potential threats to the public health, this study provides further insights into the significance of the epidemiological surveillance of HFMD.

[2009 evolution for VP4 of enterovirus 71 strains in Shenzhen].

OBJECTIVE: To analyze the genetic evolution for the common causative agent of hand, foot and mouth disease(HFMD) that VP4 of human enterovirus 71 in Shenzhen district. METHOD: 491 sttol specimen were collected from, children with hand, foot and mouth diease in Shenzhen Children's Hospital 2009. After cell culture, VP4 gene of eight EV71 strains were amplified by reverse-transcriptase PCR( RT-PCR), phylogenetic analysis of the VP4 gene was constructed by using MEGA 4. 0. RESULT: The VP4 gene of eight EV71 strains encoding 69 amino acids with full length 207 bp. The nucleotide homology of VP4 gene among eight EV71 strains was 94. 2% -98. 1%, compared with VP4 gene of EV71 strains retrieved from Shenzhen 2001 to 2004 and GenBank was 89. 1%-98. 1% and 79.2%-100% respectively. Asian epidemic strain Fuyang had the highest nucleotide homology, representative strain C4 and Shenzhen strain (AY895144) with 94. 2% -98. 1% secondly. Except for the 54th amino acid of VP4 gene of India reported strain and one of the eight EV71 strains, the homology of the rest amino acids between the eight EV71 strains and those in GenBank was 100%. Compared with representative strain C4,there were seventeen differences in nucleotide sequences of VP4 of the eight EV71 strains. All of the different nucleotides were located at the degenerate password sites except one. There was no significant difference in VP4 gene between the severe and the mild cases of strainS. The eight Shenzhen EV71 strains were classified as sub-genotype C4 in the phylogenetic tree. CONCLUSIONS: The epidemic of EV71 in Shenzhen 2009 was sub-genotype C4. VP4 gene of EV71 was very conservative which dose not belong to the variation section. The variation of most of nucleotide was invalid variation. The amino acids encoded by VP4 gene which variation was almost zero.

[Genetic evolution of VP1 of enterovirus type 71 in Shenzhen].

OBJECTIVE: Genetic evolution of VP1 of enterovirus type 71 in Shenzhen were analyzed. METHODS: All samples were tested by RT-PCR using EV71 specific primer. The VP1 of EV71 were amplified and sequenced. A phylogenetic tree was constructed by comparison of the sequences with subgenotype A, B and C using DNAStar, BioEdit and Mega 3.1 software. RESULTS: Among 35 strains, the homogeneity of the VP1 nucleotide sequence was between 92.1%-100%. The homogeneity of the VP1 nucleotide sequence with subgenotype A and B was between 81.4% -91.1%. The VP1 nucleotide sequence of 35 strains of Shenzhen shared between 93% -97.4% identity with cluster C4. The prevalence strains of EV71 were cluster C4b from 1998 to 2004, and gradually moved to C4a since 2003. All of EV71 were C4b from 2006 to 2008. Also, the homogeneity of the VP1 nucleotide sequence with Anhui FY23 EV71 strain were 94.5% -94.7%, 95.7% -95.8%, 96.2%, 95.4% -97.5%, 96.3% -99.2% from 2003 to 2008. It shows that the homogeneity was increased year by year. There was a mutation (A --> C) at No. 66 nucleotide of VP1 of EV71 that two strains were isolated in 2003 and 8 strains in 2008, that caused amino acid mutation (Q --> H) at No. 22 of VP1. CONCLUSION: EV71 C4b was gradually moved to C4a from 1998 to 2008. There was a missense mutation at No. 66 nucleotide of VP1.

[Identification and sequence analysis of E gene of Dengue virus type 2 strain isolated from patient serum in Shenzhen].

OBJECTIVE: To isolate and identify the pathogen of Dengue fever from Shenzhen city in 2005 - 2006, and to analyze the molecular characteristics of the isolated Dengue virus strain as well as to explore its possible origin. METHODS: IgM and IgG of serum samples taken from 60 suspected Dengue fever patients were detected by ELISA and immunochromatography, and 9 specimens were positive. Nine samples from patients with early stage Dengue fever were used to isolate virus with C6/36 cell line and the positive cell cultures were identified by MGB fluorescent PCR. The type of isolated virus strain was determined by RT-semi-nested-PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of Shenzhen Dengue virus with the strains isolated from other areas were constructed. RESULTS: Of nine antibody-positive serum samples, one strain of Dengue virus was successfully isolated. The isolated virus strain was confirmed as Dengue virus type 2 and designated as DEN2-SZ0521. The homology of nucleotide sequence and the deduced amino acid sequence of E gene of SZ0521 with standard type 2 Dengue virus NGC strain was 94.2% and 98.2%, but the homology with standard Dengue virus 1, 3, 4 in the same fragment were 59.1%, 57.2%, 58.5% and 68.1%, 66.7%, 63.2%, respectively. The phylogenetic tree indicated that SZ0521 had the greatest similarity with the Malay0412a/Tw strain and they lied in the same branch of the phylogenetic tree. The corresponding homology of nucleotide sequence and amino acid sequence was 99.8% and 100%, respectively. The isolated Dengue virus type 2 belonged to genotype IV with Indonesia-76, Somalia-84 and Sri Lanka-90. CONCLUSION: Dengue virus was isolated from Shenzhen for the first time, and it was classified as type 2. It was confirmed that the type 2 Dengue virus may come from the epidemic area in Malaysia.

[Genetype characteristics of enterovirus type 71 in Shenzhen from 2005 to 2008].

Genetic characteristics of enterovirus type 71 in Shenzhen from 2005 to 2008 were analyzed. All samples were detected by RT-PCR using EV71-specific primers. The VP1s of EV71 strains were amplified and sequenced. A phylogenetic tree was constructed by comparison of the sequences with those of subgenotype A, B and C using DNASTAR, BioEdit and Mega 3.1 softwares. The VP1 nucleotide sequences of 17 strains of Shenzhen shared 95.3%-99.4% identities with cluster C4a, and one strain shared 93.0%-95.6% identities with cluster C4b. The homogeneity of the VP1 nucleotide sequence with subgenotype A and B was 81.8%-86.0%. Among 18 strains, the homogeneity of the VP1 nucleotide sequence was between 92.5%-100%. All EV71 strains circulating in Shenzhen were of subgenotype C4. The predominant strain of EV71 belonged to cluster C4a, also there was cluster C4b.