Donors with a serum sodium concentration of >155 mmol/L are extended criteria donors for liver transplantation (LT). Elevated serum sodium of donors leads to an increased incidence of hepatic dysfunction in the early postoperative period of LT; however, the exact mechanism has not been reported. We constructed a Lewis rat model of 70% hepatic parenchymal area subjected to ischemia-reperfusion (I/R) with hypernatremia and a BRL-3A cell model of hypoxia-reoxygenation (H/R) with high-sodium (HS) culture medium precondition. To determine the degree of injury, biochemical analysis, histological analysis, and oxidative stress and apoptosis detection were performed. We applied specific inhibitors of the epithelial sodium channel (ENaC) and Na⁺/Ca²⁺ exchanger (NCX) in vivo and in vitro to verify their roles in injury. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels and the area of hepatic necrosis were significantly elevated in the HS+I/R group. Increased reactive oxygen species (ROS) production, myeloperoxidase (MPO)‍-positive cells, and aggravated cellular apoptosis were detected in the HS+I/R group. The HS+H/R group of BRL-3A cells showed significantly increased cellular apoptosis and ROS production compared to the H/R group. The application of amiloride (Amil), a specific inhibitor of ENaC, reduced ischemia-reperfusion injury (IRI) aggravated by HS both in vivo and in vitro, as evidenced by decreased serum transaminases, inflammatory cytokines, apoptosis, and oxidative stress. SN-6, a specific inhibitor of NCX, had a similar effect to Amil. In summary, hypernatremia aggravates hepatic IRI, which can be attenuated by pharmacological inhibition of ENaC or NCX.
Animals ; Reperfusion Injury/drug therapy* ; Hypernatremia/complications* ; Rats ; Liver/metabolism* ; Rats, Inbred Lew ; Male ; Apoptosis ; Sodium-Calcium Exchanger/antagonists & inhibitors* ; Reactive Oxygen Species/metabolism* ; Oxidative Stress ; Epithelial Sodium Channel Blockers/pharmacology* ; Epithelial Sodium Channels ; Cell Line ; Liver Transplantation

Objective:To compare the detection method of hepatitis E virus (HEV) in simulated water samples, and to provide a reference for the detection of HEV in water.Methods:HEV fecal suspension was added to tap water or distilled water simulated water samples, and pretreatment was carried out by electropositive filter-organic eluent elution method (Method 1) to compare the extraction effect of the three nucleic acid extraction kits, A, B, and C. The simulated water samples were pre-treated by Method 1, 2 (electropositive filter-direct lysis method), 3 (tangential-flow ultrafiltration membrane-organic eluent elution method), and 4 (tangential-flow ultrafiltration membrane-direct lysis method) for pretreatment, A kit for nucleic acid extraction, Real time RT-PCR method for detection and comparison of the recovery rate; comparison of the recovery rate of different concentrations of HEV in simulated water samples; comparing the inhibitory effects of inhibitors in tap water samples on real time RT-PCR; and detection of HEV in different batches of tap water specimens.Results:Kit A nucleic acid extraction was better; the recoveries of method 1, 2, 3 and 4 were 7.31%, 39.88%, 6.85% and 64.88%, respectively, which showed a statistically significant difference in the recoveries ( F=114.069, P<0.001). The recoveries of method 4 with the addition of high, medium and low concentrations of HEV were 65.26%, 42.76% and 32.79%, respectively. The inhibition of all four pre-treatment method was less than 75%, which meets the requirements of ISO (15216-2∶2019). Twenty tap water specimens were tested for HEV and the result were negative. Conclusions:This study showed that the two membranes better recovered in combination with direct lysis, respectively; Methods 4 had a higher recovery in the detection of HEV in small volumes of distilled or tap water, but it was limited by the volume of water samples, turbidity, and so on. Suitable method can be selected for different water quality and laboratory conditions.

Objective:To optimize and compare method for hepatitis E virus (HEV) nucleic acid detection from pig liver, and provide technical references for HEV detection in animal viscera specimens.Methods:Three methods (PBS homogenization treatment, proteinase K treatment, chloroform extraction method) were used to pretreat and extract viral nucleic acid form pig liver, which was artificially contaminated with HEV fecal suspensions, and HEV RT-qPCR was used to compare the HEV recovery rate and inhibition rate. The optimized HEV method was applied to commercially available pig liver specimens, and HEV genotyping was performed on positive specimens.Results:The HEV recovery rate of PBS homogenization treatment, proteinase K treatment and chloroform extraction method was 9.88%, 0.19% and 17.28%, respectively. The recovery rate of proteinase K treatment was less than 1%, and it was discarded; t-test was performed to compare recovery rates of the other two methods, which showed statistically significant differences ( t=26.801, P<0.001), the chloroform extraction method had a higher recovery rate. The inhibition rates of the three methods were all less than 75%, within the range of the ISO/TS 15216-2∶2019 standard. Among 192 commercially available pig liver specimens, 17 specimens were detected positive for HEV RNA, with a nucleic acid positive rate of 8.85%; five specimens were successfully genotyped for HEV, all of which were genotype 4. Conclusions:The virus recovery effect was good when chloroform extraction method was used for pig liver pretreatment; moreover, this method could detect HEV RNA from commercially available pig livers, which indicate that it can be used for virus detection in food.

Objective To investigate the potential active ingredients and the mechanism of Coreopsis tinctoria Nutt. in the prevention and treatment of hyperlipidemia. Methods Ultra-high performance liquid chromatography-Quadrupole-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap MS) was used to qualitatively analyze the fractions and blood components of flavones in Coreopsis tinctoria Nutt. The intersection targets of flavones in Coreopsis tinctoria Nutt. and hyperlipidemia were screened,and the protein-protein interaction network was constructed and analyzed by the STRING 12.0 database. Finally,the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for enrichment analysis. Results A total of 25 compounds were detected from the flavones in Coreopsis tinctoria Nutt.,and their structures were identified,including ten chalcones,nine flavanones,four flavonols,one aurone,and one biflavone. The analysis of blood components showed that marein,flavanomarein,okanin,isookanin and 5,7,3',5'-tetrahydroxyflavanone-7-O-β-D-glucopyranoside were the main components of the flavones in Coreopsis tinctoria Nutt. in blood. Network pharmacological GO and KEGG enrichment analysis showed that the flavones in Coreopsis tinctoria Nutt. may regulate phosphatidylinositol 3-kinase/protein kinase B,tumor necrosis factor,hypoxia-inducible factor-1 signaling pathway and other signaling pathways in the regulation and prevention of hyperlipidemia. Conclusion Coreopsis tinctoria Nutt. can prevent and treat hyperlipidemia,and the mechanism may be related to the five blood components of the flavones in Coreopsis tinctoria Nutt.,including marein,flavanomarein,okanin,isookanin and 5,7,3',5'-tetrahydroxyflavanone-7-O-β-D-glucopyranoside.

Objective To investigate the bacterial community diversity in human Demodex mites, so as to provide insights into unraveling the role of human Demodex mites in them caused infectious diseases. Methods From June to July 2023, Demodex mites were collected from the faces of college students in a university in Wuhu City using the adhesive tape method, and the V4 region of 16S ribosomal RNA (16S rRNA) gene and the internal transcribed spacer (ITS) gene of nuclear ribosomal DNA were amplified on an Illumina PE250 high-throughput sequencing platform. Sequencing data were spliced according to the overlapping relations and filtered to yield effective sequences, and operational taxonomic units (OTUs) was clustered. The diversity index of obtained OUTs was analyzed, and the structure of the bacterial community was analyzed at various taxonomic levels. Results A total of 57 483 valid sequences were obtained using 16S rRNA gene sequencing, and 159 OUTs were classified according to similarity. Then, OUTs at a 97% similarity were included for taxonomic analyses, and the bacteria in Demodex mites belonged to 14 phyla, 20 classes, 51 orders, 72 families, and 94 genera. Proteobacteria was the dominant phylum, and Vibrio, Bradyrhizobium and Variovorax were dominant genera. A total of 56 362 valid sequences were obtained using ITS gene sequencing, and 147 OTUs were obtained, which belonged to 5 phyla, 17 classes, 34 orders, 68 families, and 93 genera and were annotated to Ascomycota, Basidiomycota and Chytridiomycota, with Ascomycota as the dominant phylum, and Alternaria alternata, Epicoccum, Penicillium, and Sarocladium as dominant genera. Conclusions There is a high diversity in the composition of bacterial communities in human Demodex mites, with multiple types of microorganisms and high species abundance.

Objective To observe the value of prenatal ultrasound measuring fetal cauda equina nerve parameters for diagnosing tethered cord syndrome(TCS).Methods Forty six fetuses with TCS(TCS group)and 591 healthy fetuses(control group)were retrospectively enrolled.The length,area and angle of cauda equina nerve were measured with prenatal ultrasound and compared between groups,and the value for diagnosing TCS was analyzed.Results Significant differences of the length,area and angle of fetal cauda equina nerve were found between groups(all P＜0.05),with the area under the curve for diagnosing TCS of 0.924,0.809 and 0.972,respectively.Conclusion Prenatal ultrasound measuring fetal cauda equina nerve parameters had high value for diagnosing TCS.

Objective:The two detection method for the detection of hepatitis A virus (HAV) in strawberries were optimized and compared to select the best detection method for the detection of hepatitis A virus in strawberries.Methods:Different concentrations of HAV were inoculated on the surface of known negative frozen strawberry specimens, the concentration of beef extract powder in alkaline elution-PEG concentration method was optimized, the optimal nucleic acid extraction kit was selected, the optimal lysis buffer volume in direct lysis method was optimized, and the recovered viral load was detected by Real-time fluorescence quantitative RT-PCR. SPSS26.0 was used to statistically analyze the data, and the optimized two method were used for the detection of actual specimens.Results:The concentration of beef extract powder by the optimized alkaline elution-PEG concentration method was selected at 3%, and the viral nucleic acid extraction kit was selected as kit B. Six ml of lysis buffer was selected for the optimized direct lysis method. The recovery rates of HAV virus by alkaline elution-PEG concentration and direct lysis were compared with the HAV addition levels, and the HAV virus recovery rates of the two method were 21.50±1.06% and 5.82±0.01%, respectively, and the result showed that the differences were statistically significant. A total of 60 strawberry specimens from four regions were tested at the same time, and the result were all negative.Conclusions:The optimized alkaline elution-PEG concentration method has higher sensitivity and is more suitable for the detection of HAV in strawberry specimens.

Objective:To establish a digital droplet RT-PCR(dRT-PCR) method for Hepatitis A virus (HAV), and compare it with Real time RT-PCR(RT-qPCR) method, and select the best method for detecting hepatitis A virus in strawberry.Methods:Extract HAV vaccine RNA, optimize the reaction conditions of dRT-PCR and evaluate its specificity; Alkaline elution -PEG concentration method was used to extract nucleic acid from strawberry samples. At the same time, dRT-PCR and RT-qPCR method were used to detect the sensitivity and inhibition rate of HAV vaccine RNA in pure water and strawberry matrix, and the recovery rate of HAV in artificially contaminated strawberry was compared, which was applied to the detection of commercially available samples.Results:The optimal annealing temperature for dRT-PCR reaction was 60 ℃, and the optimal concentrations of primers and probes were 0.4 μmol/L、0.4 μmol/L and 0.2 μmol/L, with good specificity. There is no significant difference in sensitivity between the two method in detecting HAV vaccine RNA in pure water and strawberry matrix. The inhibition rate of dRT-PCR is low. The recovery rates of dRT-PCR and RT-qRCR in the detection of strawberry samples contaminated with HAV at higher concentrations were 12.90±0.006% and 30.12±0.02%, respectively. The recovery rates of lower concentrations of HAV contaminated strawberry samples were 18.27±0.07% and 10.85±0.03%, respectively, and the difference was statistically significant ( P<0.05). When strawberry samples on the market were tested, the result of both method were negative. Conclusions:The sensitivity of dRT-PCR method established in this study is not significantly different from that of RT-qPCR in detecting HAV RNA in different substrates, but dRT-PCR has good tolerance to PCR reaction inhibitors and high recovery rate when detecting low concentration HAV. Both detection method can be used for quantitative detection of hepatitis A virus in strawberry, and can be selected according to the actual situation.

Objective:A comparison of method for the detection of hepatitis E virus (HEV) in oysters was performed to provide a technical reference for the detection of HEV in oysters.Methods:After pre-treatment of oyster digestive gland specimens artificially contaminated with HEV fecal suspensions by the proteinase K digestion with reference to the European Union ISO/TS 15216-2∶2019, HEV RNA was extracted by four nucleic acid extraction method and assayed by Real time RT-PCR to compare the HEV recoveries; artificially contaminated oyster digestive gland specimens were pretreated by proteinase K digestion, proteinase K digestion + PEG precipitation, and proteinase K digestion + PEG precipitation + chloroform extraction, respectively, and HEV RNA was extracted by the optimal nucleic acid extraction method, which was assayed by real time RT-PCR to compare the HEV recoveries and inhibition rates of the three pretreatment method. The optimal HEV assay was applied to commercially available oyster specimens.Results:The HEV recoveries of the four nucleic acid extraction methods were 1.37%, 2.50%, 4.24% and 7.56%, respectively, with statistically significant differences ( F=847.220, P<0.001); The HEV recoveries for each of the three pre-treatment method were 6.02%, 13.65% and 21.17%, respectively, with statistically significant differences ( F=16.800, P<0.001), and the proteinase K digestion + PEG precipitation + chloroform extraction method had the highest recovery; the inhibition rates of the three method were 13.38%, 20.98% and 8.66%, respectively, and the differences were statistically significant ( F=20.205, P<0.001), with the lowest inhibition rate for the proteinase K digestion + PEG precipitation + chloroform extraction method. One HEV RNA positive specimen was detected in 120 commercially available oyster specimens. Conclusions:In the HEV detection of oyster specimens, pre-treatment with proteinase K digestion + PEG precipitation + chloroform extraction can improve the recovery of HEV from oysters and is more suitable for pre-treatment of oyster specimens; different manufacturers′ viral nucleic acid extraction method have different HEV recoveries and should be compared and screened for superiority before carrying out the assay.