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 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 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 explore the clinical strategies to improve the clinical outcomes of patients with thin endometrium.Methods:A retrospective cohort study was conducted to analyze 1924 frozen-thawed embryo transfer (FET) cycles of 2452 patients with thin endometrium at the Reproductive Medicine Center of Henan Provincial People's Hospital from January 2013 to December 2019. According to the type of embryos transferred, they were divided into the embryo group at cleavage stage (cleavage embryo group) and embryo group at blastocyst stage (blastocyst group). Univariate analysis, multivariate regression analysis, curve fitting and threshold effect analysis were used to compare the clinical outcomes of the cycles transferring cleavage stage embryo and blastocyst.Results:The percentage of cycles with two embryos transferred [87.17% (1298/1489)], the rate of ectopic pregnancy [3.35% (16/477)] and the rate of multiple births [23.32% (73/313)] in cleavage embryo transfer cycle were higher than those of blastocyst transfer cycle [40.71% (392/963), P<0.001; 0.76% (4/524), P=0.003; 16.27% (55/338), P=0.024]. The clinical pregnancy rate [54.41% (524/963)] and the live birth rate [35.10% (338/963)] in blastocyst transfer cycle were significantly higher than those of cleavage embryo transfer cycle [32.03% (477/1489), P<0.001; 21.02% (313/1489), P<0.001]. After adjusting for confounders, the clinical pregnancy rate ( OR=3.42, 95% CI=2.71-4.31, P<0.001) and the live birth rate ( OR=2.35, 95% CI=1.84-3.00, P<0.001) of blastocyst transfer cycle were higher than those of cleavage stage embryo transfer cycle. The results of stratified analysis showed that the clinical pregnancy rate (<6 mm: OR=2.94, 95% CI=1.32-6.51, P=0.008; 6-7 mm: OR=3.41; 95% CI=2.13-5.45, P<0.001; >7 mm: OR=3.56, 95% CI=2.67-4.74, P<0.001) and the live birth rate (<6 mm: OR=2.50, 95% CI=1.01-6.22, P=0.049; 6-7 mm: OR=2.56, 95% CI=1.56-4.21, P<0.001; >7 mm: OR=2.30, 95% CI=1.71-3.10, P<0.001) of blastocyst transfer cycle in different endometrial thickness stratifications were higher than those of cleavage stage embryo transfer cycle. The results of stratified curve fitting analysis showed that with increasing endometrial thickness among patients with thin endometrium, the clinical pregnancy rate and the live birth rate of both cleavage stage embryo cycle and blastocyst transfer cycle increased, and in all of the different endometrial thickness stratifications, the clinical pregnancy rate and the live birth rate in blastocyst transfer cycle were higher than those of cleavage embryo transfer cycle. Compared with the blastocyst transfer cycle, the slope of curve fitting between endometrial thickness and clinical pregnancy rate was larger in cleavage embryo transfer cycle. Conclusion:The clinical pregnancy rate and the live birth rate of the blastocyst transfer cycle in FET were higher than those of cleavage stage embryo transfer cycle. The increasement of endometrial thickness on the first day of progesterone administration can improve the clinical pregnancy rate and the live birth rate in patients with thin endometrium.

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.