[Epidemiological characteristics of Yersinia enterocolitica in patients with diarrhea from 2013 to 2019 in Pudong New Area, Shanghai].

Objective: To investigate the epidemiological characteristics of Yersinia enterocolitica in patients with diarrhea in Pudong New Area, Shanghai. Methods: Active surveillance of diarrhea was conducted in 14 sentinel hospitals (three tertiary-level hospitals, nine secondary-level hospitals, and two primary-level hospitals) from January 2013 to December 2019 in Pudong New Area of Shanghai, China base on their location, catchment area, and patient volume. Cold enrichment method was used to isolate Y. enterocolitica and further detection of bioserotype, virulence genes and antimicrobial susceptibility of the isolates were conducted. The difference of rates was determined using chi-square test or Fisher's exact test. Results: A total of 12 941 diarrhea cases were included, and 0.7% (88/12 941) cases were confirmed with Yersinia enterocolitica infection. 67.0% (59/88) cases were single infection, 33.0% (29/88) cases were mixed infections. Detection rates of Y. enterocolitica increased annually (0.3%-1.2%) and were highest in children<5 years of age (1.1%, 37/3 218) and in spring (1.1%, 32/2 998) (χ2 were 18.64 and 9.76, respectively, P<0.05). 58.0% (51/88) cases had watery diarrhea, 15.9% (14/88) had fever and 14.8% (13/88) had vomiting. The predominant bioserotypes were 3/O:3 (53.4%, 47/88), followed by 1A/O:8 (15.9%, 14/88) and 1A/O:5(6.8%, 6/88), respectively. Bioserotype 3/O:3 counted for the highest proportions (89.2%, 33/37) in children <5 years of age. All the strains of bioserotype 3/O:3 harbored ail, ystA, yadA and virF genes, which encoded pathogenic Y. enterocolitica. 11/14 strain of 1A/O:8 and 4/6 strains of 1A/O:5 harbored ystB gene. Most strains were resistant to ampicillin (80.7%,71/88) and amoxicillin/clavulanic acid (71.6%,63/88), and 63.8% (56/88) strains were multidrug resistance (MDR). The difference of antimicrobial resistance rates between 3/O:3 and non 3/O:3 was statistically significant in ampicillin, cefoxitin, nalidixic acid, tetracycline and ampicillin/sulbactam (χ2 was 14.68, 43.80, 41.86, 30.54 and 5.07, respectively, P<0.05). Conclusion: The detection rate of Yersinia enterocolitica was higher in children than in adults in Pudong New Area, Shanghai. The predominant bioserotype was pathogenic 3/O:3 with multidrug resistance.

[Association of hyperuricemia-induced renal damage with sirtuin 1 and endothelial nitric oxide synthase in rats].

Objective: To investigate the association of hyperuricemia-induced renal damage with sirtuin 1 (SIRT1) and endothelial nitric oxide synthase (eNOS) in rats. Methods: Using the random number table method, 32 Sprague-Dawley rats were randomly divided into 4 groups: control group, model A group (the model was generated using oxonic acid potassium salt alone), model B group (hyperuricemia model was generated using oxonic acid potassium salt combined with uric acid) and resveratrol group, with 8 rats in each group. The experiment lasted 12 weeks. Serum uric acid and cystatin C levels were monitored regularly. In week 12, serum creatinine and urea nitrogen levels were measured, and the kidneys were extracted. The expression of SIRT1 and eNOS in renal tissues was measured and determined by immunohistochemistry, quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blotting. Immunohistochemistry of alpha-smooth muscle actin combined with Masson staining was employed to evaluate the degree of renal fibrosis, and pathological changes were observed based on hematoxylin and eosin staining. Results: In week 12, the uric acid levels in both the model A and model B groups were higher than those in the control group [(316±43) µmol/L, (297±40) µmol/L vs (118±44) µmol/L, both P<0.05]. The levels of cystatin C in the model A, model B, and resveratrol groups were all higher than those in the control group [(156±20) ng/ml, (143±29) ng/ml, (128±26) ng/ml vs (62±18) ng/ml, all P<0.05]. Creatinine levels were higher in the model A and model B groups than those in the control group [(68.5±10.3) µmol/L, (64.5±13.9) µmol/L vs (43.2±10.6) µmol/L, both P<0.05]. The levels of uric acid, cystatin C and creatinine in the resveratrol group were lower than those in the model A group (all P<0.05). Immunohistochemistry, RT-qPCR, and Western blotting for renal SIRT1 and eNOS showed that the expression in the model A and model B groups was inhibited, while the expression in the resveratrol group was not significantly inhibited, compared with that in the control group. Microscopically, obvious abnormalities were not found in the renal tissue of the control group. Renal inflammatory cell aggregation and edema occurred, and interstitial fibrosis was obvious in both the model A and model B groups, while these lesions in the resveratrol group were significantly improved. Conclusions: Hyperuricemia may cause renal injury by inhibiting the expression of SIRT1 and eNOS.

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris.

The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

Expression analysis of VfDGAT2 in various tissues of the tung tree and in transgenic yeast.

The tung tree (Vernicia fordii Hemsl.; Vf) has great potential as an industrial crop owning to its seed oil that has multiple uses. Diacylglycerol acyltransferases (DGATs) catalyze the last and most committed step of triacylglycerol (TAG) biosynthesis. In order to examine the physiological role of the VfDGAT2 gene in the tung tree, we characterized its expression profiles in different tung tissues/organs and seeds at different developmental stages. Oil content and α-eleostearic acid production during seed development were also examined. Expression studies showed that VfDGAT2 was expressed in all tissues tested, with the highest expression in developing seeds where the expression was about 19-fold more than that in leaves. VfDGAT2 showed temporal-specific expression during seed development and maturation. Notably, the expression of VfDGAT2 in developing seeds was found to be consistent with tung oil accumulation and α-eleostearic acid production. The expression level of VfDGAT2 was lower in the early stages of oil accumulation and α-eleostearic acid biosynthesis, rapidly increased during the peak periods of fatty acid synthesis in August, and then decreased during completion of the accumulation period at the end of September. When the VfDGAT2 gene was transferred to the oleaginous yeast Rhodotorula glutinis, its expression was detected along with fatty acid products. The results showed that VfDGAT2 was highly expressed in transgenic yeast clones, and the total fatty acid content in one of these clones, VfDGAT2-3, was 7.8-fold more than that in the control, indicating that VfDGAT2 contributed to fatty acid accumulation into TAG and might be a target gene for improving tung oil composition through genetic engineering.

[Antimicrobial resistance and molecular epidemiology of foodborne Yersinia enterocolitica in Pudong New District, Shanghai].

Objective: To investigate the antimicrobial resistance and molecular epidemiology of foodborne Yersinia (Y.) enterocolitica in Pudong New District of Shanghai. Methods: Four kinds of raw food samples were collected in retail circulation sites in Pudong from 2012 to 2016. Cold enrichment method was used to isolate Y. enterocolitica and further detection of biotype, serotype, virulent genes, antimicrobial susceptibility of the isolates and pulsed field gel electrophoresis (PFGE) were conducted. Results: A total of 3 900 raw food samples were collected during this period, including poultry product (n=590), livestock product (n=1 074), aquatic product (n=1 488), vegetable (n=748), in which 111 (2.8%) were contaminated by Y. enterocolitica. The detection rates of Y. enterocolitica in poultry product samples (5.3%, 31/590) and livestock product samples (4.5%, 48/1 074) were higher than those in aquatic product samples (1.6%, 24/1 488) and vegetable samples (1.1%, 8/748). The predominant biotype was 1A (95.5%) and predominant serotype was O∶8 (42.3%). All the strains lacked ail, ystA, yadA and virF genes, which encoded pathogenic Y. enterocolitica. Seventy six (68.5%) strains harbored ystB gene, in which 35 (31.5%) belonged to 1A/O∶8/ystB pattern. Most strains were resistant to ampicillin (74.8%) and amoxicillin/clavulanic acid (70.3%), and non-sensitive rate to Cefoxitin was over 50.0%. No third generation cephalosporin or fluoroquinolone resistant strains were detected, but 38.7% (43/111) strains were multidrug resistant (MDR). Serotype O∶8 and O∶5 strains had 44 and 18 PFGE patterns, respectively. Conclusions: The main foodborne exposure sources of Y. enterocolitica in raw food were poultry and livestock products in Pudong New District. 1A/O∶8/ystB was the predominant pattern with potential pathogenicity despite lacks of typical pathogenic virulent genes. The antimicrobial resistant rates of Y. enterocolitica were at a low level, but MDR strains still existed. Molecular types of the isolates showed highly genetic diversity.