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Objective: To analyze the correlation between the histological classification of hepatocellular carcinoma (HCC), especially macrotrabecular-massive (MTM), tumor invasiveness, and prognosis. Methods: The clinical and follow-up data of 246 consecutive HCC cases who met the inclusion criteria from 2015 to 2018 were retrospectively analyzed. They were divided into trabecular/pseudoglandular, trabecular/patchy, and MTM types according to the histological classification. The relationship between each type and related clinicopathological features was analyzed. The Kaplan-Meier method was used to plot tumor-free survival (DFS) and overall survival (OS) curves. Log rank tests, COX univariate, and multivariate regression analyses were conducted to analyze the relationship between clinical features, including histological classification, DFS, and OS. Results: Trabecular/pseudoglandular, trabecular/nodular, and MTM type proportions were 44.7% (110 cases), 32.9% (81 cases), and 22.4% (55 cases), respectively. The results of the clinicopathological features showed that MTM-type HCC was significantly more invasive than the other two types, with alpha-fetoprotein (AFP) ≥400 ng/ml, tumor diameter≥8 cm, no tumor capsule, poor differentiation, and MVI positivity proportions, and the differences were statistically significant (P < 0.05).The proportion of MTM-type HCC patients with American Joint Committee on Cancer TNM Stage III to IV and Chinese Liver Cancer Staging (CNLC) II to II was significantly higher than that of the first two types, and the differences were statistically significant (P < 0.05). In addition, the proportion of MTM subtypes undergoing transcatheter arterial chemoembolization was also raised (P < 0.05). The DFS and OS were significantly lower for MTM-type HCC compared to trabecular/pseudoductal-type HCC at 1-, 3-, and 5-years, and the differences were statistically significant (P < 0.05). Univariate analysis indicated that strongly invasive clinical pathological features such as alpha fetoprotein (AFP) ≥400 ng/ml, tumor diameter ≥ 8 cm, no tumor capsule, poor differentiation, positive microvascular invasion, tumor stage, and MTM staging were all risk factors affecting DFS and OS (P < 0.05). Multivariate COX analysis showed that MTM histological staging, AFP ≥ 400 ng/ml, tumor non-encapsulation, satellite nodules, CNLC stages II~III, and TNM stages III~IV were independent risk factors for DFS (P < 0.05), while AFP ≥ 400 ng/ml, tumor non-encapsulation, and CNLC stage II~III were independent risk factors for OS ( P < 0.05). Conclusion: Histological classification is highly correlated with tumor invasiveness and HCC prognosis. Trabecular/pseudoglandular types have lower malignancy and a better prognosis, while MTM types exhibit strong invasive features and a poor prognosis.

Background: Radical resection is a curative treatment for patients with hepatocellular carcinoma (HCC), but the incidence of recurrence remains high. We aimed to explore the performance of predicting HCC recurrence by longitudinal surveillance of the protein induced by vitamin K absence (PIVKA-II), alpha- fetoprotein (AFP), and lectin-reactive AFP (AFP-L3) during postoperative follow-up., Methods: Patients who underwent radical resection for HCC at the Ningbo Medical Centre Lihuili Hospital between January 2015 and December 2020 were included. All enrolled patients regularly monitor PIVKA-II, AFP, AFP-L3 every 3 months during postoperative follow-up. The surveillance performance of PIVKA-II, AFP, AFP-L3 during follow-up for the prediction of HCC recurrence was compared in patients. The generalized estimation equation (GEE) was used to analyze the trends of the tumor biomarkers and interactions with time. Area under the receiver operator characteristic (AUROC) curves, the optimal cut-off value, the sensitivity and specificity were calculated to evaluate the performance of the three biomarkers. The recurrence-free survival (RFS) and overall survival (OS) of patients with any of the elevated biomarkers was analyzed by Kaplan-Meier curves and the log-rank test. Multivariate logistic regression models were used to analyze potential risk factors for recurrence., Results: The GEE analysis indicated that PIVKA-II, AFP, AFP-L3 in the recurrence patients were higher than the no recurrence patients during follow-up, PIVKA-II and AFP showed increasing trends from 6 months before recurrence. In predicting recurrence, the AUROCs for PIVKA-II, AFP, AFP-L3 and their combination were 0.885, 0.754, 0.781 and 0.885 respectively, the optimal cut-off value for PIVKA-II, AFP, AFP-L3 was 29.5 mAU/ml, 10.7 ng/L, 1.5% respectively. The sensitivity in predicting recurrence for PIVKA-II, AFP, AFP-L3 and combination were 75.0, 54.7, 57.8 and 79.7% respectively. The RFS and the OS of patients with any of the biomarkers elevated during the follow-up was significantly shorter than that without elevated biomarkers ( P  < 0.001). Multivariate analysis showed that any of the biomarkers elevated was the independent risk factor of recurrence., Conclusion: Longitudinal surveillance of PIVKA-II, AFP and AFP-L3 can effectively predict recurrence of HCC after operation., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

This experiment aimed to investigate the effects of emodin on the total bacterial count and immune response in various tissues of Wuchang bream infected with A. hydrophila . The experimental diets were made by supplementing emodin at 0, 30, 100, and 150 mg kg -1 to basal (control) diet, respectively, and fed to fish with an initial weight of 50.4 ± 2.35 g. All fish were divided into five experimental groups: uninfected fish fed with basal control diet (negative control, NC), infected fish fed with the diet supplemented with 0 (positive control group, PC), 30 (30), 100 (100), and 150 mg/kg (150) of emodin. The fish were reared for 14 days and sampled at different time points. The results showed that the total bacterial count in the kidney, blood, and liver tissues of Wuchang bream infected with A. hydrophila was significantly affected by the supplementation and feeding time of emodin. At the beginning of the experiment, the difference in total bacterial count among the groups was not significant. On day 1, the total bacterial count in all groups was significantly higher ( p < 0.05) than that in the negative control group. On day 4, the total bacterial count in all the emodin groups was significantly reduced, and the best bactericidal effect was observed in the 100 mg kg -1 group. In addition, emodin had a significant effect on the immune response of Wuchang bream after infection with A. hydrophila ( p < 0.05). Compared with the other groups, the respiratory burst activity, tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) content, and white blood cell count (WBC) in the 100 and 150 mg kg -1 groups could be restored to normal levels in the shortest time ( p < 0.05). Furthermore, this study also measured the complement alternative pathway activity (ACH 50 ), plasma superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content of the fish. The results showed that supplying 100 mg kg -1 emodin to the diet could significantly ( p < 0.05) increase the ACH 50 activity of the fish. Compared with the positive control (PC) group, the addition of emodin to the diet can inhibit the decrease in SOD activity and the increase in MDA content in the plasma of infected Wuchang bream. In conclusion, supplying 100 mg kg -1 emodin to the diet can enhance the ability of Wuchang bream to resist A. hydrophila infection by reducing the total bacterial count in tissues, increasing the activity of related immune enzymes, and promoting the secretion of cytokines. This provides a theoretical basis for production practice.

Objective: To understand the incidence of delayed vaccination with the national immunization program vaccines among children aged 0-6 years in Xuhui District, Shanghai, and to evaluate the safety of delayed vaccination. Methods: A stratified random sampling was used to obtain six vaccination clinics in Xuhui District, Shanghai. The vaccination records of children 0-6 years from these six vaccination clinics were collected from the Shanghai Immunization Program Information Management System. Adverse events following immunization (AEFI) data were collected from the China Information System for Disease Control and Prevention. Descriptive epidemiology was used to analyze the data. Children were divided into the timely vaccination group and delayed vaccination group according whether they were delayed in vaccination (received one month or more after the recommended age among children aged ≤1 year; received three months or more after the recommended age among children aged >1 year). The safety of four vaccination methods-individual vaccination, simultaneous vaccination, routine vaccination and combined vaccination-were further compared. Differences between groups were compared using chi-square test. Results: From 2019 to 2021, six vaccination clinics in Xuhui District administered 124 031 doses of the national immunization program vaccines among children aged 0-6 years, and delayed vaccinations accounted for 25.99% (32 234/124 031) of these doses. In 2020, the delayed vaccination rate during the first-level COVID-19 public health emergency response period in Shanghai was significantly higher than that in the same period in 2019 (34.70% vs . 24.19%, χ 2 =136.23, P <0.05). The delayed vaccination rate during the COVID-19 vaccination campaign in 2021 was significantly higher than that in the same period in 2019 (25.27% vs . 22.55%, χ 2 =82.80, P <0.05). From 2019 to 2021, a total of 475 cases of AEFI were reported in six vaccination clinics, with a reported incidence of 382.97 per 100 000 doses, including 421 cases of common adverse reaction (88.63%, 339.43 per 100 000 doses), 51 cases of rare adverse reaction (10.74%, 41.12 per 100 000 doses) and 3 cases of coincidences (0.63%, 2.42 per 100 000 doses). The reported incidence of AEFI among delayed vaccinations was significantly lower than that among timely vaccinations (291.62 per 100 000 doses vs . 415.05 per 100 000 doses). The incidence of AEFI for the four delayed vaccination methods (individual vaccination, simultaneous vaccination, routine vaccination and combined vaccination) was lower than that for timely vaccination. There were significant differences between the groups except for the routine vaccination group ( χ 2 =9.82, P <0.05; χ 2 =5.46, P <0.05; χ 2 =2.97, P >0.05; χ 2 =11.89, P <0.05). Conclusions: In Xuhui District of Shanghai, 25.99% of doses of the national immunization program vaccines administered to children 0-6 years were delayed. Delayed vaccination does not increase the risk of AEFI compared with timely vaccination.

Tick saliva is a reservoir of bioactive proteins. Saliva protein compositions change dynamically during blood-feeding. Decipherment of protein profiles in different blood-feeding stages may bring deeper insight into tick feeding physiology and provide targets for immunologic control alternatives. However, having the infancy of tick genome sequencing, assembly, annotation, and limited knowledge of tick salivary proteins restrain the data interpretation. Here, we aimed to depict the saliva protein profile in partially- (PE) and fully-engorged (FE) Haemaphysalis flava ticks, with a special focus on the analysis of those uncharacterized proteins. Saliva was collected from PE and FE adult female H. flava ticks. Saliva proteins were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS-MS). MS data were searched against an in-house salivary gland transcriptome library for identification of tick-derived proteins. Abundances of proteins were compared between PE and FE ticks. The uncharacterized proteins detected in saliva were further bioinformatically analyzed. In total, 614 proteins were identified including 94 host proteins and 520 tick-derived proteins. The 226 tick-derived high-confidence proteins were classified into 10 categories: transporters, enzymes, protease inhibitors, immunity-related proteins, lipocalins, glycine-rich proteins, muscle proteins, secreted proteins, uncharacterized proteins and others. A total of 98 proteins were shared in both PE and FE with 74 only in PE and 54 only in FE. Abundances of 24 shared proteins were significantly higher in PE. The profile of top 15 most abundant proteins was also different between PE and FE ticks. The 65 uncharacterized proteins detected in tick saliva were branched into subclusters 1 A, 1B, 2, 3 A, 3B and 3 C based on particular motifs like RGD, LRR, indicating their diverse predicted functions like anti-coagulation, regulation of innate immune, or other functions. This study provides and compares saliva proteomes of H. flava ticks in two feeding stages with special cluster analysis on the uncharacterized proteins. Further investigations are needed to confirm the roles of these uncharacterized proteins in ticks., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: To evaluate the efficacy of in-situ full size split liver transplantation(fSLT) for adult recipients using the living donor liver transplantation(LDLT) technique and to compare the characteristics of the left hemiliver graft (LHG) and the right hemiliver graft(RHG)transplantation. Methods: Deceased donor and recipient data of 25 consecutive cases of fSLT at Department of Hepatopancreatobiliary Surgery, Ningbo Medical Center Lihuili Hospital from March to December 2021 was retrieved and the patients divided into two groups:LHG group and RHG group. Among the 13 donors,11 were male and 2 were female,aged ( M (IQR))38(19) years(range: 25 to 56 years),with height of 168(5) cm(range:160 to 175 cm) and weight of 65(9) kg(range: 50 to 75 kg). The median age of the 25 recipients was 52(14) years(range:35 to 71 years),17 were male and 8 were female,15 had primary liver cancer and 10 had benign end-stage liver disease,model for end-stage liver disease score was 10(9) points(range:7 to 23 points). Of the 25 recipients,10 recipients had previously undergone hepatobiliary surgery. The follow-up period was to January 2022. Demographic,clinicopathological,surgical outcomes and postoperative complications were evaluated and compared between the two groups. Continuous quantitative data were compared using Mann-Whitney U test. Classification data were expressed as frequencies,and were compared between groups using χ 2 test or Fisher exact probability method. Results: Using LDLT technique,in-situ full-left/full-right liver splitting was performed and 13 viable pairs of hemiliver grafts were harvested with acquisition time of 230(53) minutes(range:125 to 352 minutes) and blood loss of 250(100) ml(range:150 to 1 000 ml). A total of 25 hemiliver grafts(13 LHG and 12 RHG) were allocated to patients listed for liver transplantation in our center by China Organ Transplant Response System. In the LHG group(13 cases),there were more females and more patients with benign end-stage liver disease than in the RHG group(12 cases)( P <0.05). The body weight and graft weight of recipients in the LHG group were lower than that in RHG group(both P <0.05). There were no significant differences in other baseline data between the two groups(all P> 0.05). The graft to recipient weight ratio(GRWR) was 1.2(0.4)%(range:0.7% to 1.9%) for 25 recipients,1.1(0.5)%(range:0.7% to 1.6%)for the LHG group and 1.3(0.5)%(range:0.9% to 1.9%)for the RHG group. There was no significant difference between the two groups ( P >0.05). Sharing patterns of hepatic vessels and the common bile duct are as follows:all the trunk of middle hepatic vein were allocated to the LHG group. The proportion of celiac trunk,main portal vein and common bile duct assigned to LHG and RHG was 10∶3 ( P =0.009), 9∶4 ( P >0.05) and 4∶9 ( P =0.027),respectively. The vena cava of 12 donors in early stage retained in LHG and that of last one was shared between LHG and RHG ( P< 0.01). The median cold ischemia time of 25 hemiliver grafts was 240(90) minutes(range:138 to 420 minutes). For the total of 25 fSLT,the median anhepatic phase was 50(16) minutes(range:31 to 98 minutes) and the operation time was 474(138)minutes(range:294 to 680 minutes) with blood loss of 800(640) ml(range:200 to 5 000 ml). There were no significant differences in all of operation data between two groups. In the LHG group,3 patients with GRWR≤0.8% had postoperative small-for-size syndrome which improved after treatment. Postoperative Clavien-Dindo grade≥Ⅲ complications were observed in 6 cases(24.0%),4 cases(4/13) in the LHG group and 2 cases(2/12) in the RHG group,respectively. The difference was not statistically significant. Among them,5 cases improved after re-operation and intervention,1 case in LHG group died of secondary infection 2 weeks after operation,and the mortality was 4.0%. Analysis of serious postoperative complications and death has suggested that conventional caval interposition should not be used for LHG transplantation. Conclusion: Relying on accurate donor-recipient evaluation and the apply of LDLT technique,the morbidity and mortality of in-situ fSLT in adults is acceptable.

Aims: We explored the relationship between the mutation at the p.G245S site in TP53 and the short-term recurrence of hepatocellular carcinoma (HCC). Materials and Methods: 101 HCC patients were included in this study. The TP53 p.G245S mutation frequency spectrum was examined by direct sequencing of genomic DNA from tissue specimens of HCC patients. Univariate and multivariate Cox regression analyses were performed to evaluate the independent prognostic factors of tumor recurrence. ROC curve analysis was applied to determine the cut-off value for the p.G245S mutation frequency and to verify the predictive ability of the Cox model compared with single risk factor indices. Results: A multivariate Cox regression analysis showed that TP53 p.G245S mutation frequency ( HR  = 1.231, 95% CI: 1.006-1.505, p  = 0.043), AFP ( HR  = 2.432, 95% CI: 1.297-4.561, p  = 0.006), MTM ( HR  = 2.656, 95% CI: 0.930-7.583, p  = 0.068), and PVTT ( HR  = 14.297, 95% CI: 3.085-66.243, p  = 0.001) were independent prognostic factors for short-term recurrence. The cut-off value for the TP53 p.G245S mutation frequency (18.5%) was determined by ROC analysis. A predictive model integrating the TP53 p.G245S mutation frequency with PVTT, MTM, and AFP values appears to an excellent predictive indicator of short-term recurrence in HCC patients (AUC = 0.849, 95% CI = 0.748-0.950, p  = 0.000001). Survival analysis indicated that the probability of short-term recurrence-free survival was significantly different among different TP53 p.G245S mutation frequency, MTM, PVTT, and AFP risk groups ( p  < 0.05). Conclusion: The mutation frequency of the p.G245S site is a novel prognostic risk factor for the short-term recurrence of HCC.

Statistical tests for biomarker identification and classification methods for patient grouping are two important topics in adaptive designs of clinical trials related to genomic studies. In this article, we evaluate four test methods for biomarker identification in the first stage of an adaptive design: a model-based identification method, the popular two-sided t -test, the nonparametric Wilcoxon Rank-Sum test (two-sided), and the Regularized Generalized Linear Models. For patients grouping in the second stage, we examine classification methods such as Random Forest, Elastic-net Regularized Generalized Linear Models, Support Vector Machine (SVM), Gradient Boosting Machine (GBM), and Extreme Gradient Boosting (XGBoost). Simulation studies are carried out to assess the performance of the different methods. The best identification methods are chosen based on the well-known F 1 score, while the best classification techniques are selected based on the area under a receiver operating characteristic curve (AUC). The chosen methods are then applied to the Adaptive Signature Design (ASD) with a real data set from breast cancer patients for the purpose of evaluating the performance of ASD in different situations., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 Informa UK Limited, trading as Taylor & Francis Group.)

Estrogen receptor-α (ER) drives tumor development in ER-positive (ER+) breast cancer. The transcription factor GATA3 has been closely linked to ER function, but its precise role in this setting remains unclear. Quantitative proteomics was used to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, few proteins were lost from the ER complex in the absence of GATA3, with the only major change being depletion of the dioxygenase TET2. TET2 binding constituted a near-total subset of ER binding in multiple breast cancer models, with loss of TET2 associated with reduced activation of proliferative pathways. TET2 knockdown did not appear to change global methylated cytosine (5mC) levels; however, oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) was significantly reduced, and these events occurred at ER enhancers. These findings implicate TET2 in the maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes., Competing Interests: Declaration of interests S.B. is a founder and shareholder of Cambridge Epigenetix, Ltd., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

Cytosine methylation is an important epigenetic mark, but how the distinctive patterns of DNA methylation arise remains elusive. For the first time, we systematically investigated how these patterns can be imparted by the inherent enzymatic preferences of mammalian de novo DNA methyltransferases in vitro and the extent to which this applies in cells. In a biochemical experiment, we subjected a wide variety of DNA sequences to methylation by DNMT3A or DNMT3B and then applied deep bisulfite sequencing to quantitatively determine the sequence preferences for methylation. The data show that DNMT3A prefers CpG and non-CpG sites followed by a 3'-pyrimidine, whereas DNMT3B favors a 3'-purine. Overall, we show that DNMT3A has a sequence preference for a TNC[G/A]CC context, while DNMT3B prefers TAC[G/A]GC. We extended our finding using publicly available data from mouse Dnmt1/3a/3b triple-knockout cells in which reintroduction of either DNMT3A or DNMT3B expression results in the acquisition of the same enzyme specific signature sequences observed in vitro . Furthermore, loss of DNMT3A or DNMT3B in human embryonic stem cells leads to a loss of methylation at the corresponding enzyme specific signatures. Therefore, the global DNA methylation landscape of the mammalian genome can be fundamentally determined by the inherent sequence preference of de novo methyltransferases.

Transmission of varicella occurs frequently in schools and households. We investigated the characteristics of varicella cases derived from within-household transmission and the modes of varicella transmission between school and household settings in Shanghai, China, from 2009 to 2018. Within-household transmission occurred in 278 households, of which 134 transmission events were between children. Sixty-one household varicella transmission events may be attributed to isolation procedures for infected students during school outbreaks, and 7.6% of school outbreaks were caused by schoolchildren cases derived from within-household transmission. The frequency of 'school-household-school' transmission adds an additional layer of complexity to the control of school varicella outbreaks. Administration of varicella vaccine as post-exposure prophylaxis after exposure is considered to be an effective measure to control varicella spread within households and schools.

Designing a cavity with a high quality factor for omnidirectionally emitting laser (OEL) can extend its potential applications in optical communication and biomedical detection. We demonstrate a method including five steps to design a high-Q cavity for OEL using a one-dimensional topological photonic crystal heterostructure. A Si/SiO 2 fiber cavity for OEL with solid gain medium Er-doped SiO 2 is designed following our design steps. The designed fiber can axially transmit the pump energy at low confine loss and act as a cavity for the radial emission of the exited beam, simultaneously. The quality factor of this fiber cavity is on the order of magnitude of 10 8 . Moreover, a method of further improving the Q-factor is proposed. The results in this paper are not restricted to the solid gain medium, and they also can be applied to designing a cavity for optofluidic OEL or quantum dot OEL. Our study may provide not only the reference for OEL manufacture, but also a route for improving the performance of OEL.

Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated, or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.

Essential hypertension (EH) is a multifactorial disease. Interferon-γ (IFN-γ) plays an important role in the onset of EH through cytokine-mediated systemic inflammatory responses. We aimed to determine whether the methylation status of the IFN-γ gene (IFNG) promoter is involved in the pathogenesis of EH. Six copies of CpG dinucleotides are distributed between 3,203 bp and 3,121 bp upstream from the transcription initiation site of IFNG, termed CpG1 to CpG6 in the 5'-to-3' direction. We recruited 96 patients with EH and 96 sex- and age-matched healthy subjects as controls. Using bisulfate pyrosequencing datasets, we analyzed the methylation status of the six CpG sites and thus found that CpG5 was consistently methylated in all of the 96 EH patients and 96 control subjects. Among the remaining five CpG sites, there was no significant difference in the methylation levels of CpG4 and CpG6 between the two groups. By contrast, CpG1 (P = 0.003) and CpG3 (P = 5.87 × 10 -7 ) were highly methylated among the EH subjects compared with the controls, whereas CpG2 (P = 1.24 × 10 -12 ) was significantly less methylated in among EH subjects. The methylation levels of CpG2 were still lower after adjustment with logistic regression (adjusted P = 0.032). The CpG2 methylation level was an effective marker of EH (area under curve = 0.384; P = 1.40 × 10 -15 ). The present study shows that hypomethylation of the IFNG promoter is significantly related to the risk of EH, providing new insights into the pathogenesis of EH.

Essential hypertension (EH) is a chronic disease with clear epigenetic component. Inflammation and endothelial dysfunction have a great role in the development of persistent blood pressure elevation. The aim of this study was to explore an association between EH and DNA methylation in pro-inflammation cytokine gene interleukin-6 (IL-6) during the inflammatory process. We performed methylation analysis of peripheral blood DNA using bisulphite pyrosequencing technology between 96 EH patients and 96 age- and gender-matched healthy controls. The present results showed that three cytosine-phosphate-guanine (CpG) sites of IL-6 promoter CpG island had different lower methylation in EH group compared with controls, but only CpG2 (58.43±7.53 versus 62.34±9.65, P=0.004) and CpG3 (51.52±6.18 versus 57.45±8.29, P<0.001) had statistical difference. Logistic regression analysis found CpG3 hypomethylation was a risk factor of EH (odds ratio=1.111, adjusted P=0.004). In addition, we found hypermethylation of CpG1 (64.84±7.06 versus 61.84±8.61) and CpG2 (62.04±7.40 versus 59.30±9.57) in male compared with female. In male, we found hypomethylation of CpG2 (60.41±7.74 versus 64.28±6.36) and CpG3 (53.70±8.62 versus 57.78±7.87) of smoker versus non-smoker and hypomethylation of CpG2 (60.89±7.32 versus 64.70±7.03) and CpG3 (53.23±7.99 versus 60.48±7.58) of drinker versus non-drinker. Furthermore, the CpG2 and CpG3 had a negative correlation with systolic blood pressure and diastolic blood pressure (P<0.05). Receiver operating characteristic curve analysis showed that CpG2 (area under curve: 0.638, P=0.001) and CpG3 (area under curve: 0.704, P<0.001) had a diagnostic value to predict the risk of EH. In summary, our study revealed hypomethylation of IL-6 was correlated with the risk of EH in the population assessed and we found the differences of IL-6 promoter methylation in gender, smoking and drinking.

The aim of the present study was to investigate whether methylation of the angiotensin I converting enzyme 2 (ACE2) promoter increases the risk of essential hypertension (EH). A total of 96 patients with EH were recruited and 96 sex‑ and age‑matched healthy controls. Methylation of 5 CpG dinucleotides in the ACE2 promoter was quantified using bisulfite pyrosequencing. Logistic regression and multiple linear regression were used to adjust for confounding factors and the generalized multifactor dimensionality reduction (GMDR) method was applied to investigate high‑order interactions. Methylation of CpG4 (adjusted P=0.020) and CpG5 (adjusted P=0.036) was significantly higher in patients with EH, with frequency 97.56±5.65% and 12.75±4.15% in EH individuals and 95.73±9.11% and 11.47±3.67% in healthy controls. GMDR detected significant interaction among the 5 CpG sites (odds ratio=7.33, adjusted P=0.01). Furthermore, receiver operating characteristic curves identified that CpG5 methylation was a significant predictor of EH. Notably, CpG2 methylation was significantly higher in males than in females (adjusted P=0.018). Conversely, CpG5 methylation was significantly lower in males (adjusted P=0.032). These results indicated that aberrant methylation of the ACE2 promoter may be associated with EH risk. In addition, sex may significantly influence ACE2 methylation.

Utilizing the plankton 1 (505 Μm), 2 (160 Μm), 3 (77 Μm) nets to seasonally collect zooplankton samples at 10 stations and the corresponding abundance data was obtained. Based on individual zooplankton biovolume, size groups were classified to test the changes in spatiotemporal characteristics of both Sheldon and normalized biovolume size spectra in thermal discharge seawaters near the Guohua Power Plant, so as to explore the effects of temperature increase on zooplankton size spectra in the seawaters. The results showed that the individual biovolume of zooplankton ranged from 0.00012 to 127.0 mm 3 ·ind -1 , which could be divided into 21 size groups, and corresponding logarithmic ranges were from -13.06 to 6.99. According to Sheldon size spectra, the predominant species to form main peaks of the size spectrum in different months were Copepodite larvae, Centropages mcmurrichi, Calanus sinicus, fish larvae, Sagitta bedoti, Sagitta nagae and Pleurobrachia globosa, and minor peaks mostly consisted of individuals with smaller larvae, Cyclops and Paracalanus aculeatus. In different warming sections, Copepodite larvae, fish eggs and Cyclops were mostly unaffected by the temperature increase, while the macrozooplankton such as S. bedoti, S. nagae, P. globosa, C. sinicus and Beroe cucumis had an obvious tendency to avoid the outfall of the power plant. Based on the results of normalized size spectra, the intercepts from low to high occurred in November, February, May and August, respectively. At the same time, the minimum slope was found in February, and similarly bigger slopes were observed in May and August. These results indicated that the proportion of small zooplankton was highest in February, while the proportions of the meso- and macro-zooplankton were relatively high in May and August. Among different sections, the slope in the 0.2 km section was minimum, which increased with the increase of section distance to the outfall. The result obviously demonstrated that the closer the distance was from outfall of the power plant, the smaller the zooplankton became. On the whole, the average intercept of normalized size spectrum in Xiangshan Bay was 4.68, and the slope was -0.655.

TET proteins have been found to play an important role in active demethylation at CpG sites in mammals. There are some reports implicating their functions in removal of DNA methylation imprint at the imprinted regions in the germline. However, it is not well established whether TET proteins can also be involved in demethylation of DNA methylation imprint in embryonic stem (ES) cells. Here we report that loss of TET proteins caused a significant increase in DNA methylation at the Igf2-H19 imprinted region in ES cells. We also observed a variable increase in DNA methylation at the Peg1 imprinted region in the ES clones devoid of TET proteins, in particular in the differentiated ES cells. By contrast, we did not observe a significant increase of DNA methylation imprint at the Peg3, Snrpn and Dlk1-Dio3 imprinted regions in ES cells lacking TET proteins. Interestingly, loss of TET proteins did not result in a significant increase of DNA methylation imprint at the Igf2-H19 and Peg1 imprinted regions in the embryoid bodies (EB). Therefore, TET proteins seem to be differentially involved in maintaining DNA methylation imprint at a subset of imprinted regions in ES cells and EBs., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Zooplankton samples were seasonally collected at 10 stations in thermal discharge seawaters near Guohua Power Plant in Xiangshan Bay. The abundance data from these samples were pooled and further combined with field environmental factors, then generalised dissimilarity modelling (GDM) was used to explore the effects of environmental factors on β diversity of zooplankton community. The results showed that altogether 95 species of zooplankton belonging to 14 taxa were found. In these taxa, small zooplankton with 62.6% of abundance was the main taxa, while copepods dominated in adult groups, which abundance accounted for 35.3%. According to Whittaker's definition and additive partition, a diversity accounted for 36.3% and β diversity 63.7%. Environmental factors explained 43.8% of β diversity, and geographical distance between sampling sites had no effect on β diversity. However, there were still 19.9% of β diversity remained to be explained. After GDM fitting, there were nine environmental variables affecting zooplankton β diversity and explaining 68.8% of β diversity. The variables contributing to β diversity from high to low were seasonal water temperature, dissolved oxygen, seawater temperature increment, conductivity, suspended particulate matter, salinity, transparency, water depth and redox potential, respectively. Seasonal water temperature, dissolved oxygen and seawater temperature increment were the most important factors for driving β diversity changes, and accounted for 23.9%, 13.7% and 9.7% of absolute contribution to the interpretable portion of the β diversity, respectively. When seasonal water temperature, dissolved oxygen and seawater temperature increment were below 25 °C, greater than 5 mg · L(-1) and over 1 °C, respectively, β diversity rapidly increased with the increasing variable gradients. Furthermore, other predictors had little effect on β diversity.

In mammals, cytosine methylation (5mC) is widely distributed throughout the genome but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in the regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.

Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Vitamin C, a micronutrient known for its anti-scurvy activity in humans, promotes the generation of induced pluripotent stem cells (iPSCs) through the activity of histone demethylating dioxygenases. TET hydroxylases are also dioxygenases implicated in active DNA demethylation. Here we report that TET1 either positively or negatively regulates somatic cell reprogramming depending on the absence or presence of vitamin C. TET1 deficiency enhances reprogramming, and its overexpression impairs reprogramming in the context of vitamin C by modulating the obligatory mesenchymal-to-epithelial transition (MET). In the absence of vitamin C, TET1 promotes somatic cell reprogramming independent of MET. Consistently, TET1 regulates 5-hydroxymethylcytosine (5hmC) formation at loci critical for MET in a vitamin C-dependent fashion. Our findings suggest that vitamin C has a vital role in determining the biological outcome of TET1 function at the cellular level. Given its benefit to human health, vitamin C should be investigated further for its role in epigenetic regulation.

DNA hydroxymethylation and its mediated DNA demethylation are critical for multiple cellular processes, for example, nuclear reprogramming, embryonic development, and many diseases. Here, we demonstrate that a vital nutrient ascorbic acid (AA), or vitamin C (Vc), can directly enhance the catalytic activity of Tet dioxygenases for the oxidation of 5-methylcytosine (5mC). As evidenced by changes in intrinsic fluorescence and catalytic activity of Tet2 protein caused by AA and its oxidation-resistant derivatives, we further show that AA can uniquely interact with the C-terminal catalytic domain of Tet enzymes, which probably promotes their folding and/or recycling of the cofactor Fe(2+). Other strong reducing chemicals do not have a similar effect. These results suggest that AA also acts as a cofactor of Tet enzymes. In mouse embryonic stem cells, AA significantly increases the levels of all 5mC oxidation products, particularly 5-formylcytosine and 5-carboxylcytosine (by more than an order of magnitude), leading to a global loss of 5mC (∼40%). In cells deleted of the Tet1 and Tet2 genes, AA alters neither 5mC oxidation nor the overall level of 5mC. The AA effects are however restored when Tet2 is re-expressed in the Tet-deficient cells. The enhancing effects of AA on 5mC oxidation and DNA demethylation are also observed in a mouse model deficient in AA synthesis. Our data establish a direct link among AA, Tet, and DNA methylation, thus revealing a role of AA in the regulation of DNA modifications.

TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here, we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements., (Copyright © 2013 Elsevier Inc. All rights reserved.)