Therapeutic Effect of Canagliflozin on Nephrotic Syndrome and Its Ultrasonic Evaluation / 中山大学学报(医学科学版)

ObjectiveTo investigate the therapeutic effect of antidiabetic drug canagliflozin （CGLZ） on adriamycin-induced nephrotic syndrome （NS） in rats， and the evaluation of contrast-enhanced ultrasound （CEUS） combined with color Doppler flow imaging （CDFI） during the treatment. MethodsA total of 56 male SD rats were randomly divided into normal group （NG）， model group （MG）， prednisone （PAT） group （PG）， low-dose single CGLZ group （LSCG）， high-dose single CGLZ group （HSCG）， low-dose CGLZ + PAT group （LUCG） and high-dose CGLZ + PAT group （HUCG）， with 8 rats in each group. The NS model in rats was induced by injecting adriamycin twice into the tail vein， and then the NS rats were treated by intragastric administration daily for 6 weeks with reference of PAT. Twenty-four hour urine total protein （24 h-UTP） was assessed one day before the start of oral administration and at the end of 2， 4 and 6 weeks after oral administration， respectively. CDFI and CEUS were performed on the right renal artery at the end of 6 weeks after oral administration， and the blood of abdominal aorta was taken for serological test the next day. ResultsCompared with those detection index of NG rats， the 24-hour UTP of MG rats increased （P＜0.01）， the serum ALB decreased and TG， TC， LDL increased （P＜0.01）， and CDFI shows that RRCT was thinner （P＜0.01） and the renal artery blood flow indicators RA-PI， RA-RI， RA-S/D all increased （P＜0.05）， and CEUS image shows that the TIC curve parameters TTP， AT， AUC all increased and DPI decrease in MG rats （P＜0.01）. After drug treatment， compared with those detection index of MG rats， 24 h-UTP decrease in LSCG after 2 weeks （P＜0.01）， and decrease significantly in all drug groups after 6 weeks （P＜0.01）； the serological test results show that the serum ALB in all CGLZ groups increased （P＜0.05）， TG decrease in LSCG （P＜0.01）， TC and LDL also decrease in LUCG after 6 weeks （P＜0.05）； CDFI shows that the RRCT thinning degree in all CGLZ is reduced （P＜0.01）， and the RA-PI in LSCG， RA-RI in PG， and RA-S/D in PG， LSCG， HSCG and LUCG rats all decreased （P＜0.05）； CEUS shows that the TTP， AT and AUC of renal TIC curve in drug treatment groups all decreased （P＜0.01）， and the DPI in PG， HSCG， LUCG and HUCG rats increased （P＜0.01）. ConclusionsCGLZ has the effect of treating NS， and the small dose is the best. CEUS combined with CDFI can be used to evaluate the renal morphology and hemodynamic changes of NS model rats before and after drug treatment， which is helpful to guide clinical application.

Porcine circovirus 3 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Porcine circovirus 3 (PCV3) has been detected in major pig-producing countries around the world since its first report in the US in 2016. Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs. We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid (Cap) protein expression on autophagic response in human embryonic kidney cell line 293T (HEK293T). We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin (mTOR) in HEK293T cells. The ubiquitin-proteasome pathway is also involved in the autophagy process. These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

Phosphorylation residue T175 in RsbR protein is required for efficient induction of sigma B factor and survival of Listeria monocytogenes under acidic stress / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. RsbR, an upstream regulator of the sigma B (SigB) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion (ΔrsbR), complementation (C-ΔrsbR), and phosphorylation site mutations in the rsbR (RsbR-T175A, RsbR-T209A, and RsbR-T175A-T209A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsbR reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175A disabled RsbR complementation, while RsbR-T209A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of RsbR are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.

Effect of Regulating PPARγ by mTOR Signaling on Adipogenesis of Bone Marrow Mesenchymal Stem Cells from Aplastic Anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the effect and mechanism of mTOR signaling on adipogenesis of bone marrow mesenchymal stem cells(BM-MSCs) from aplastic anemia (AA) patients through regulation of PPARγ.</p><p><b>METHODS</b>BM-MSCs were isolated from 24 newly diagnosed AA patients and 24 healthy controls. The surface antigen expression of BM-MSCs was identified by flow cytometry. The capacity of adipogenic differentiation of BM-MSCs was determined by lipid droplets based on Oil Red O staining and by the expression of FABP4 based on Western blot. Protein levels of mTOR signaling and PPARγ were tested by immunofluorescence and Western blot.</p><p><b>RESULTS</b>AA BM-MSCs displayed an enhanced capacity of differentiating into adipocytes, compared with control BM-MSCs. It was found that mTOR was activated in AA BM-MSCs. Moreover, the expression levels of p-mTOR and PPAR-γ in AA BM-MSCs showed a parallel differentiation-dependent increase during adipogenic differentiation, which were significantly higher than that of control BM-MSCs at the same time point of adipogenic differentiation. mTOR inhibitor rapamycin did not only inhibit the adipogenic differentiation of BM-MSCs from AA pateints at the early-middle stage, but also partly reversed the adipogenic differention of BM-MSCs from AA pateints at the late stage by PPARγ regulation.</p><p><b>CONCLUSION</b>mTOR signaling may play a critical role in the adipogenic differentiation of BM-MSCs from AA patients by positively regulating PPARγ expression.</p>

Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κB nuclear translocation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.

Study of brain perfusion with IVIM-DWI in early hypertension / 医疗卫生装备

Objective To explore the value of intra-voxel incoherent motion diffusion weighted imaging(IVIM-DWI)in brain perfusion of early hypertensive patients. Methods Totally 36 hypertensive patients and 14 volunteers were recruited and scanned using routine MRI sequences including axial T2WI, T1WI, T2FLAIR, TOF-MRA and IVIM-DWI sequence. Perfusion-related diffusion coefficient (D*) values and perfusion fraction (f) values in various regions were measured separately.The independent sample t test was used to analyze the data.Results Compared with the volunteers,both D*values and f values in lenticular nucleus,thalamus,superior frontal gyrus,occipital lobe,genu of corpus callosum(CC)and posterior horns of periventricular WM, were found to be lower (P<0.05) in hypertensive patients. For other regions, there were no significant difference(P>0.05).Conclusion IVIM-DWI has the ability to detect subtle brain perfusion abnormalities at early stages of hypertension.It has an important value to the prevention and treatment of hypertensive encephalopathy.

Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

A one-step dual flow immunochromatographic assay (DICGA), based on a competitive format, was developed for simultaneous quantification of ochratoxin A (OTA) and zearalenone (ZEN) in corn, wheat, and feed samples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53‒12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06‒39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.

Evaluation of four kits for screening HIV antibody / 中华预防医学杂志

<p><b>OBJECTIVE</b>To Evaluate four kits for screening HIV antibody by comparing and analyzing the HIV antibody screening positive results and Western Blot (WB) test results.</p><p><b>METHODS</b>From January 2004 to June 2009, three ELISA kits (Zhongshan, Biomérieux and Livzon) were used for initial screening HIV antibody. The reactive positive samples were reexamed by initial ELISA kit and a rapid kit (Abbot Determine HIV-1/2). All repeatedly reactive positive screening results were followed by WB test.</p><p><b>RESULTS</b>A total of 193 (0.094%) WB confirmed positive results were obtained from 206 151 specimens. The sensitivities and predictive values of negative test result (PVN) of three ELISA kits were all 100% and those of Abbot Determine HIV-1/2 were 93.93%, and 91.67% respectively. All false negative results from Abbot were WB indeterminate. The specificities of Zhongshan, Biomérieux, Livzon and Abbot were 99.88%, 99.89%, 99.96% and 89.38%; the study predictive values of a positive test result (PVP) were 35.58%, 46.46%, 76.61% and 92.20%; the efficiencies were 99.88%, 99.89%, 99.96% and 91.98%; the areas under ROC curve of the three ELISA kits were 0.93, 0.99, and 0.95 respectively. PVP of Livzon was obviously higher than those of Zhongshan (chi(2) = 45.804, P = 0.000), Biomérieux (chi(2) = 25.231, P = 0.000) and Biomérieux was higher than Zhongshan (chi(2) = 2.488, P = 0.115). PVP of Abbot was highest (chi(2) = 18.633, P = 0.000, vs Livzon). There were some specimens with S/CO (optical density of sample/cut off) ratio < 6 or > or = 6 in all three groups with positive, indeterminate and negative WB results. The S/CO ratio from Zhongshan in confirmed positive group (14.29 + or - 2.63) was higher than in positive-negative group (2.80 + or - 3.25) (t = 17.652, P = 0.000). The S/CO ratio from Biomérieux in confirmed positive group(16.09 + or - 2.35) was higher than in positive-negative group (2.14 + or - 1.91) (t = 31.622, P = 0.000). The S/CO ratio from Livzon in confirmed positive group (11.54 + or - 1.95) was higher than in positive-indeterminate group (5.54 + or - 3.57) (t = 6.386, P = 0.000), positive-negative group (3.25 + or - 2.41) (t = 21.772, P = 0.000) and positive-indeterminate group was higher than positive-negative group (t = 2.301, P = 0.033).</p><p><b>CONCLUSION</b>The performances of four HIV antibody screening kits are good but estimating WB confirming result in line with S/CO ratio is not available. All repeated screening positive results should be followed by confirmatory tests.</p>

White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Chinese soft-shelled turtles (Trionyx sinens) in culture farms using an artificial warming system in Zhejiang, China, often show typical signs of white-spot disease such as white spots on their bodies, skin lesions, anorexia and eventually death. The sick turtles were mostly 5~80 g in weight. A suspected fungal pathogen was isolated from the sick turtles and verified as Paecilomyces lilacinus by sequence analysis of the internal transcribed spacer (ITS) of its ribosomal DNA (rDNA). Detailed morphological examinations were also conducted to confirm the white-spot disease.

A survey on porcine circovirus type 2 infection and phylogenetic analysis of its ORF2 gene in Hangzhou, Zhejiang Province, China / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands's isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.

Detection of PCV2 DNA by SYBR Green I-based quantitative PCR / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 10(3) to 10(11) copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.

Protection of Carassius auratus Gibelio against infection by Aeromonas hydrophila using specific immunoglobulins from hen egg yolk / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.

The study of the pro-nucleating activity of bacteria identified in cholesterol gallstones in model bile systems / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the relationship of bacteria identified in cholesterol gallstones and gallstone formation.</p><p><b>METHODS</b>Observe the bacteria activity in model bile and the influence of bacteria on the cholesterol nucleation time (NT).</p><p><b>RESULTS</b>(1) Model bile were suitable for the growth of E. coli, Pseudomonas aeruginosa, staphylococcus aureus, enterococcus faecalis, clostridium difficile and Clostridium. Propionibacterium acne grew weakly and the growth of Bacteroides fragilis was restrained in model bile. (2) Only pseudomonas aeruginosa and enTerococcus faecalis could ly shorten the cholesterol nucleation time. (3) With pseudomonas aeruginosa or enTerococcus faecalis added in model bile, the formation of cholesterol crystals presented a progressive course of evolution.</p><p><b>CONCLUSIONS</b>Pseudomonas aeruginosa and enterococcus faecalis, not propionibacterium acne, have pro-nucleating ability in model bile.</p>

Expression of the infectious bursal disease virus polyprotein in Vero cells using attenuated Salmonella typhimurium as transgenic carrier / 生物工程学报

To examine if polyprotein gene (VP2/VP4/VP3) of Infectious Bursal Disease Virus (IBDV) could be delivered into mammalian cells and expressed using attenuated Salmonella typhimurium as vector. The IBDV polyprotein gene was amplified by RT-PCR and inserted in to pCI, an eukaryotic expression plasmid. The resulting recombinant pCI-VP2/VP4/VP3 was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam- and phoP-), which was then use to transfect the Vero cells. Gene specific RT-PCR revealed that VP2/VP4/VP3 was transcribed into mRNA in the Vero cells. Indirect immunofluorscence assay, SDS-PAGE and Western-blot analysis showed that VP2/VP4/VP3 was expressed and the product was immuno-reactive with anti-IBDV serum. This work provides essential precondition for developing a new oral DNA vaccine against IBDV.

Safety, stability and immunogenicity of an oral DNA vaccine against Newcastle disease / 生物工程学报

Mice and 3-day-old chickens were orally inoculated with the recombinant attenuated Salmonella typhimurium strain ZJ111 carrying pcDNA3-F expression plasmid encoding the fusion protein of Newcastle disease virus (NDV). The results showed that ZJ111/pcDNA3-F was relatively safe. The recombinant plasmid pcDNA3-F was stable within the host stain ZJ111 in vitro and in vivo as shown by restriction enzyme analysis and PCR identification of the F gene. In an experimental vaccination study, 3-day-old chickens were orally immunized with ZJ111/pcDNA3-F with a dose of 108 cfu per chicken and boosted two weeks later. At week 4 post boosting, all chickens were challenged with a lethal dose of a virulent NDV strain F48 E9. The results showed that oral vaccination with ZJ111/pcDNA3-F induced stronger humoral and cellular immune responses than intramuscular immunization with naked pcDNA3-F plasmid. It also exhibited higher protection rate than the latter (66.7% vs 50%). This study indicates that the DNA vaccine using attenuated Salmonella typhimurium as delivery carrier had good safety, stability and immunogenicity and exhibited good potential of low cost and convenience for poultry disease control.

Breeding of High Streptolydigin-Producing Strain by Complex Mutagenesis and Resistance Selection / 微生物学通报

A high Streptolydigin-producing strain Streptomyces lydicus AS 4.2501-L8 with genetic stability was obtained from the original strain S. lydicus AS 4.2501-P28 by mutagenesis with UV and acridine orange, followed by resistance selection. Its yield of Streptolydigin was 177.0 ?g/mL, 96.2% higher than the original strain.