Objective:To establish a digital reverse transcription polymerase chain reaction（dRT-PCR）detection method for hepatitis E virus（HEV）using nanoplates，and to provide technical reference for HEV monitoring in shellfish by combining virus enrichment pretreatment methods.Methods:The annealing temperature，primer and probe concentrations of HEV dRT-PCR were optimized，and the specificity of the method was evaluated；the sensitivity of this method for detecting HEV in water samples and oyster extracts was compared. The inhibition rate and recovery rate of HEV detection in artificially contaminated oyster samples were calculated，commercially available oyster samples were tested，and compare them with real-time fluorescence quantitative RT-PCR（qRT-PCR）method.Results:The optimized annealing temperature for HEV dRT-PCR was determined to be 60 ℃，and the final concentrations of primers and probes were 0.4 μmol/L，0.4 μmol/L，and 0.2 μmol/L，respectively，indicating good specificity. The sensitivity of both methods for detecting HEV RNA in water samples was higher than that in oyster extracts. The recovery rates of HEV in oyster specimens contaminated with HEV fecal suspension by dRT-PCR and qRT-PCR were 18.76% and 18.36%，respectively，with no statistically significant difference（ P>0.05）；the inhibition rates were 17.26% and 9.58%，respectively，with statistically significant differences（ P<0.05）；55 commercially available oyster samples were tested，and both methods detected HEV RNA positivity in the same sample. Conclusion:The dRT-PCR method established in this study，combined with “proteinase K digestion，PEG/NaCl precipitation，and chloroform/n-butanol extraction” pretreatment，has a good recovery effect on HEV in shellfish food containing a large amount of PCR inhibitors，and can achieve absolute quantification. It has certain application value in monitoring and risk assessment of HEV in shellfish food.

Objective:To establish a digital reverse transcription polymerase chain reaction（dRT-PCR）detection method for hepatitis E virus（HEV）using nanoplates，and to provide technical reference for HEV monitoring in shellfish by combining virus enrichment pretreatment methods.Methods:The annealing temperature，primer and probe concentrations of HEV dRT-PCR were optimized，and the specificity of the method was evaluated；the sensitivity of this method for detecting HEV in water samples and oyster extracts was compared. The inhibition rate and recovery rate of HEV detection in artificially contaminated oyster samples were calculated，commercially available oyster samples were tested，and compare them with real-time fluorescence quantitative RT-PCR（qRT-PCR）method.Results:The optimized annealing temperature for HEV dRT-PCR was determined to be 60 ℃，and the final concentrations of primers and probes were 0.4 μmol/L，0.4 μmol/L，and 0.2 μmol/L，respectively，indicating good specificity. The sensitivity of both methods for detecting HEV RNA in water samples was higher than that in oyster extracts. The recovery rates of HEV in oyster specimens contaminated with HEV fecal suspension by dRT-PCR and qRT-PCR were 18.76% and 18.36%，respectively，with no statistically significant difference（ P>0.05）；the inhibition rates were 17.26% and 9.58%，respectively，with statistically significant differences（ P<0.05）；55 commercially available oyster samples were tested，and both methods detected HEV RNA positivity in the same sample. Conclusion:The dRT-PCR method established in this study，combined with “proteinase K digestion，PEG/NaCl precipitation，and chloroform/n-butanol extraction” pretreatment，has a good recovery effect on HEV in shellfish food containing a large amount of PCR inhibitors，and can achieve absolute quantification. It has certain application value in monitoring and risk assessment of HEV in shellfish food.

Objective:To compare enrichment and nucleic acid detection method for hepatitis E virus (HEV) in simulated cola samples.Methods:Cola samples experimentally contaminated with HEV were enriched using positively charged filter membrane-direct lysis (Method 1), tangential flow ultrafiltration membrane-direct lysis (Method 2), and Method 3 and 4 (with the addition of a PCR inhibitor removal step on the basis of Method 1 and 2, respectively), and were assayed by real-time fluorescence quantitative RT-PCR(RT-qPCR), and the recoveries and inhibition rates were compared. Digital RT-PCR(RT-dPCR) and RT-qPCR were applied to detect the recovery of HEV in different medium and low concentrations of experimentally contaminated cola samples; and the inhibition rate and sensitivity of HEV RNA detection in different matrices.Methods 3 was selected for virus enrichment of 8 commercially available cola specimens, RT-qPCR and RT-dPCR for HEV RNA detection.Results:The HEV recoveries of method 3 and 4 (10.44% and 10.16%) were higher than those of method 1 and 2 (4.89% and 0.32%), and the differences were statistically significant ( P<0.05). The inhibition rates of method 3 and 4 were smaller than the inhibition rates of method 1 and 2. The recoveries of HEV in medium concentration artificially contaminated cola samples by RT-qPCR and RT-dPCR were 17.04% and 16.28%, respectively, and for low concentration artificially contaminated cola samples were 6.91% and 4.65%, respectively, and the differences in recoveries between the two assays at the same concentration were not statistically significant ( P=0.260, P=0.107 ); Cola matrix inhibits the detection of both RT-qPCR and RT-dPCR assays. Eight commercially available cola specimens were negative for HEV. Conclusions:Detection of HEV in cola beverages can be done by positively charged filter membrane-direct lysis + inhibitor removal (method 3) or tangential flow ultrafiltration membrane-direct lysis + inhibitor removal (method 4) enrichment, followed by RT-dPCR or RT-qPCR, with a high recovery of virus detection.

Objective:To compare enrichment and nucleic acid detection method for hepatitis E virus (HEV) in simulated cola samples.Methods:Cola samples experimentally contaminated with HEV were enriched using positively charged filter membrane-direct lysis (Method 1), tangential flow ultrafiltration membrane-direct lysis (Method 2), and Method 3 and 4 (with the addition of a PCR inhibitor removal step on the basis of Method 1 and 2, respectively), and were assayed by real-time fluorescence quantitative RT-PCR(RT-qPCR), and the recoveries and inhibition rates were compared. Digital RT-PCR(RT-dPCR) and RT-qPCR were applied to detect the recovery of HEV in different medium and low concentrations of experimentally contaminated cola samples; and the inhibition rate and sensitivity of HEV RNA detection in different matrices.Methods 3 was selected for virus enrichment of 8 commercially available cola specimens, RT-qPCR and RT-dPCR for HEV RNA detection.Results:The HEV recoveries of method 3 and 4 (10.44% and 10.16%) were higher than those of method 1 and 2 (4.89% and 0.32%), and the differences were statistically significant ( P<0.05). The inhibition rates of method 3 and 4 were smaller than the inhibition rates of method 1 and 2. The recoveries of HEV in medium concentration artificially contaminated cola samples by RT-qPCR and RT-dPCR were 17.04% and 16.28%, respectively, and for low concentration artificially contaminated cola samples were 6.91% and 4.65%, respectively, and the differences in recoveries between the two assays at the same concentration were not statistically significant ( P=0.260, P=0.107 ); Cola matrix inhibits the detection of both RT-qPCR and RT-dPCR assays. Eight commercially available cola specimens were negative for HEV. Conclusions:Detection of HEV in cola beverages can be done by positively charged filter membrane-direct lysis + inhibitor removal (method 3) or tangential flow ultrafiltration membrane-direct lysis + inhibitor removal (method 4) enrichment, followed by RT-dPCR or RT-qPCR, with a high recovery of virus detection.

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: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.

Zn²⁺ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn²⁺ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn²⁺. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn²⁺ exporter. Loss of SLC-30A9 leads to mitochondrial Zn²⁺ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn²⁺ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn²⁺ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn²⁺ pool from which mitochondrial Zn²⁺ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn²⁺ levels for normal mitochondrial structure and functions.
Animals ; Caenorhabditis elegans/metabolism* ; Cation Transport Proteins/genetics* ; Homeostasis ; Mitochondria/metabolism* ; Zinc/metabolism*

Objective:To evaluate the clinical effects of antagonist protocol and progestin-primed ovarian stimulation (PPOS) protocol in patients with low prognosis.Methods:A total of 1560 controlled ovarian stimulation cycles of 1419 patients consistent with POSEIDON low prognosis with antagonist protocol or PPOS protocol in the treatment of in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) from January 2016 to December 2018 in the Reproductive Medicine Center of Henan Provincial People's Hospital were collected in a retrospective cohort study. The essential characteristic, clinical characteristics, laboratory index and clinical outcomes of patients in the antagonist protocol group and the PPOS protocol group were compared. Multivariate logistic regression analysis was used to compare cumulative live rate per ovulation cycle after adjusting for confounders of the two controlled ovarian stimulation protocols. Results:The comparison of the general conditions of the patients with the two controlled ovarian stimulation protocols showed that anti-Müllerian hormone (AMH) [1.45(0.68, 3.28) μg/L] and antral follicle count (AFC) [7.00(4.00,11.00) μg/L] in the antagonist protocol group were significantly higher than those in the PPOS protocol group [1.10(0.55, 2.71) μg/L, P=0.002; 6.00(3.00, 9.00) μg/L, P=0.010], and basal follicle-stimulating hormone (FSH) [7.65(6.26, 9.99) U/L] was significantly lower than that in the PPOS protocol group [7.88(6.29, 10.58) U/L, P=0.007]. The results of laboratory and clinical outcomes showed that the estrogen level [726.20(415.30,1 095.00) ng/L] on human chorionic hormone (hCG) injection day in the antagonist protocol group was significantly lower than that in the PPOS protocol group [738.00(412.55, 1 187.75) ng/L, P=0.028], and the endometrial thickness [(9.31±2.67) mm] on hCG injection day, the cumulative pregnancy rate [49.35% (379/768)] and the cumulative live birth rate per ovulation cycle [38.04% (291/765)] were significantly higher than those in the PPOS protocol group [(6.81±2.26) mm, P<0.001; 37.62% (298/792), P<0.001; 26.08% (206/790), P<0.001]. After adjusting for confounder factors, the cumulative pregnancy rate ( OR=1.58, 95% CI=1.24-2.01, P<0.001) and the cumulative live birth rate ( OR=1.68, 95% CI=1.30-2.17, P<0.001) per ovulation cycle in the antagonist protocol group were higher than those in the PPOS protocol group in patients with POSEIDON low prognosis. The results of stratified analysis showed that the cumulative pregnancy rate and the cumulative live birth rate per ovulation cycle of antagonist protocol group per ovulation cycle was higher than that of PPOS protocol group. The cumulative pregnancy rate and the cumulative live birth rate per ovulation cycle in different age ( P<0.001, P<0.001), insemination method ( P<0.001, P<0.001), AMH ( P<0.001, P<0.001) and POSEIDON group 1 ( P=0.001, P<0.001) and POSEIDON group 3 ( P=0.008, P=0.024) were statistically different. Conclusion:In patients with low prognosis of POSEIDON, the antagonist protocol improved the cumulative live birth rate per ovulation cycle compared with the PPOS protocol, especially for patients in POSEIDON group 1 and POSEIDON group 3.

Objective:To investigate the effect of endometrial thickness (EMT) on the clinical outcome of hormone replacement frozen-thawed embryo transfer (HRT-FET) cycle in different body mass index (BMI) groups, and to analyze the threshold and optimal EMT and EMT interval corresponding to the ideal clinical pregnancy rate.Methods:A retrospective cohort study was conducted on 10 239 HRT-FET cycles in the Reproductive Medicine Center of Henan Provincial People's Hospital from January 2013 to December 2017, and they were divided into low weight group (BMI<18.5 kg/m 2), normal weight group (BMI=18.5-24.9 kg/m 2), overweight group (BMI=25.0-29.9 kg/m 2) and obese group (BMI≥30.0 kg/m 2). Four subgroups were divided according to EMT, respectively EMT<8.0 mm, 8.0 mm≤EMT<10.0 mm, 10.0 mm≤EMT<12.0 mm, EMT≥12.0 mm. The clinical characteristics and outcome indicators of different EMT subgroups in different BMI groups were compared. To achieve the ideal clinical pregnancy rate, multiple regression analysis, curve fitting and threshold effect analysis were used to find the best EMT and thickness interval. Results:1) After adjusting for confounding factors, multiple regression analysis showed that, there were no significant differences in clinical pregnancy rate and live birth rate among subgroups with the increase of EMT (all groups P>0.05). The clinical pregnancy rate and the live birth rate increased with the increase of EMT between subgroups of normal body weight group and super-recombinant subgroups (all P<0.001 for normal body weight subgroups, P=0.123, P=0.009, P=0.016 and all P<0.001 for super-recombinant subgroups). In the obesity group, with the increase of EMT, the clinical pregnancy rate did not increase significantly except EMT≥12.0 mm subgroup ( P=0.449, P=0.279, P=0.021), while the live birth rate increased significantly ( P=0.014, P=0.005, P<0.001). 2) Curve fitting showed that in the population of low weight and obese, influence of EMT on clinical pregnancy rate was a straight line, in the population of normal weight and overweight, influence of EMT on clinical pregnancy rate was a curve, as EMT increased the clinical pregnancy rate raised and then decreased, the impact on the live birth rate appeared similar. 3) According to the curve fitting, the threshold effect analysis of the normal weight group showed that the endometrial inflection point of EMT on the clinical pregnancy rate and the live birth rate was 10.0 mm. When EMT was lower than 10.0 mm, the clinical pregnancy rate and the live birth rate increased by 20% and 19% for every 1.0 mm increase in endometrial thickness ( OR=1.20, 95% CI=1.13-1.26; OR=1.13,95% CI=1.13-1.26). In overweight group, the inflection point of EMT on the clinical pregnancy rate and the live birth rate was also 10.0 mm. When EMT was lower than 10.0 mm, the clinical pregnancy rate and the live birth rate increased by 24% and 26% for every 1.0 mm increase in EMT ( OR=1.24, 95% CI=1.13-1.26; OR=1.26, 95% CI=1.14-1.40). When EMT exceeded 10.0 mm, the clinical pregnancy rate and the live birth rate did not increase significantly with the increase of EMT. Conclusion:In HRT-FET cycle, the endometrial thickness has an effect on the clinical pregnancy rate and the live birth rate in the normal weight group and the overweight group. The clinical pregnancy rate and the live birth rate were the best when the EMT was between 10.0-13.5/10.0-12.7 mm and 10.0-14.0/10.0-12.5 mm, respectively. Whether the endometrium was too thin or too thick would affect the clinical pregnancy outcome. The influence of EMT on clinical pregnancy rate and live birth rate was linear between the low weight group and the obese group, but further study is needed.