Protective effects of engineered Lactobacillus crispatus strains expressing G-CSF on thin endometrium of mice.

STUDY QUESTION: Does recombinant Lactobacillus expressing granulocyte colony-stimulating factor (G-CSF) have a better protective effect than the current treatment of thin endometrium (TE)? SUMMARY ANSWER: This study suggested that the intrauterine injection of Lactobacillus crispastus (L. crispastus)-pPG612-G-CSF has a positive effect on preventing TE induced by 95% alcohol in mice. WHAT IS KNOWN ALREADY: TE has a negative impact on the success rate of ART in patients, and is usually caused by intrauterine surgery, endometrial infection, or hormone drugs. Exogenous G-CSF can promote endometrial vascular remodelling and increase endometrial receptivity and the embryo implantation rate. Moreover, Lactobacillus plays a crucial role in maintaining and regulating the local microecological balance of the reproductive tract, and it could be a delivery carrier of the endometrial repair drug G-CSF. STUDY DESIGN, SIZE, DURATION: We constructed engineered L. crispastus strains expressing G-CSF. The mice were divided into five groups: (i) Control group (C, n = 28), uteri were treated with preheated saline solution via intrauterine injection on the third and sixth day of oestrus; (ii) Model group (M, n = 35), where uteri were treated with 95% alcohol on the third day of oestrus and preheated saline solution on the sixth day of oestrus via intrauterine injection; (iii) L. crispatus-pPG612-treatment group (L, n = 45), where uteri were treated with 95% alcohol on the third day of oestrus and 0.1 ml × 108 CFU/ml L. crispatus-pPG612 on the sixth day of oestrus via intrauterine injection; (iv) L. crispatus-pPG612-treatment group (LG, n = 45), where uteri were treated with 95% alcohol on the third day of oestrus and 0.1 ml × 108 CFU/ml L. crispatus-pPG612-G-CSF on the sixth day of oestrus via intrauterine injection; (v) G-CSF-treatment group (G, n = 52), where uteri were treated with 95% alcohol on the third day of oestrus and 30 µg/kg G-CSF on the sixth day of oestrus via intrauterine injection. Then, we compared the effects of L. crispastus, L. crispatus-pPG612-G-CSF and G-CSF on endometrial thickness, angiogenesis, fibrosis, and inflammation in the TE mouse. PARTICIPANTS/MATERIALS, SETTING, METHODS: We collected uterine tissues for haematoxylin-eosin staining, immunohistochemical staining, Western blot and RT-PCR, as well as serum for ELISA and uterine flushing solution for high-throughput sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with those in the M group (the mice of the group were intrauterine injected 95% alcohol and treated with saline solution), the L. crispatus-pPG612-G-CSF strain increased the thickness of the endometrium (P < 0.001) and the number of blood vessels and glands (both P < 0.001), enhanced the expression of cytokeratin 19 (CK19) (P < 0.001), vimentin (Vim) (P < 0.001), vascular endothelial growth factor-A (P < 0.001), and CD34 (P < 0.001), and decreased fibrosis levels (P = 0.004). In addition, the high-throughput sequencing results indicated that the L. crispatus-pPG612-G-CSF strain could decrease the abundance of Pseudomonas (P = 0.044) and Actinomyces spp. (P = 0.094) in TE mice and increased the average number of embryos (P = 0.036). Finally, the L. crispatus-pPG612-G-CSF strain was preliminarily confirmed to activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signalling pathway and enhance the mRNA expression of hypoxia-inducible factor-1α (P < 0.001), vascular endometrial growth factor (P = 0.003), and endothelial cell nitric oxide synthase (P = 0.003) in mouse uterine tissue. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Therapy with the L. crispatus-pPG612-G-CSF strain has tremendous potential to accelerate the reparative processes of TE. However, we have reported only the expression of genes and proteins related to the PI3K/AKT pathway, and numerous other mechanisms may also be involved in the restoration of the endometrium by L. crispatus-pPG612-G-CSF. WIDER IMPLICATIONS OF THE FINDINGS: The results from the study provide new ideas and suggest new methods for TE treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by the Project of Science and Technology Development Plan of Jilin Province (grant number 20210101232JC), the Science and Technology Plan Item of Jilin Provincial Education Department (grant number JT53101022010), and the Doctoral Research Start-up Fund of Jilin Medical University (grant numbers JYBS2021014LK and 2022JYBS006KJ). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Clinical outcomes following discontinuation of metformin in patients with type 2 diabetes and advanced chronic kidney disease in Hong Kong: a territory-wide, retrospective cohort and target trial emulation study.

Background: Current labelling advises discontinuation of metformin when estimated glomerular filtration rate (eGFR) < 30 ml/min/1.73 m2 due to increased risk of lactic acidosis. However, in real-world practice, the risk-benefit ratios remain uncertain. We examined the risk associations of discontinued-metformin use with cardiorenal and clinical outcomes in patients with type 2 diabetes (T2D) and advanced chronic kidney disease. Methods: In this territory-wide, retrospective cohort and target trial emulation study, we included Chinese patients attending the Hong Kong Hospital Authority (HA) and enrolled in the Risk-Assessment-and-Management-Programme-for-Diabetes-Mellitus (RAMP-DM) from 2002 to 2019. Patients were stratified by discontinuation of metformin within six months after reaching eGFR < 30 ml/min/1.73 m2 from January 1, 2002 to December 31, 2018, and followed up until December 31 2019. We excluded patients who had observational time <6 months from eGFR < 30 ml/min/1.73 m2, and had their eGFR measured during a hospitalisation episode due to acute kidney injury, or missing diagnosis date of diabetes. We compared the risk associations of metformin discontinuation with clinical outcomes. The primary outcomes were major adverse cardiovascular events (MACE), end-stage kidney disease (ESKD), cancer, and all-cause mortality. A Cox-model with time-dependent exposure and covariates was used to estimate the hazard ratio (HR) of outcomes in a propensity-score overlap-weighted cohort. The risk of occurrence of lactic acidosis (serum lactate > 5.0 mmol/L with a concomitant blood pH < 7.35 or ICD-9 codes of 276.2) in discontinued-metformin versus continued-metformin users was assessed in a separate register-based cohort. Findings: A total of 33,586 metformin users with new-onset eGFR < 30 ml/min/1.73 m2 were included in the study, 7500 (22.3%) of whom discontinued metformin within 6 months whereas 26,086 (77.7%) continued use of metformin. During a median follow-up of 3.8 (IQR: 2.2-6.1) years, 16.4% (5505/33,586), 30.1% (10,113/33,586), and 7.1% (2171/30,682) had incident MACE, ESKD, and cancer respectively, and 44.4% (14,917/33,586) died. Compared to continued-metformin use, discontinuation was associated with higher risk of MACE (weighted and adjusted HR = 1.40, 95% CI: 1.29-1.52), ESKD (HR = 1.52, 1.42-1.62), and death (HR = 1.22, 1.18-1.27). No association was observed for cancer (HR = 0.93, 0.85-1.01). Discontinued-metformin users had higher change in HbA1c change at 6-month of follow-up versus continued-metformin users (weighted mean HbA1c level change: 0.5% [0.4-0.6%] versus 0.2% [0.1-0.2]). In the separate register-based cohort (n = 3235), null association was observed between metformin use and risk of lactic acidosis (weighted HR = 0.94 [0.53-1.64]). Interpretation: Our results suggest that discontinuation of metformin in patients with T2D and chronic kidney disease may be associated with increased risk of cardiovascular-renal events. Use of metformin below eGFR of 30 ml/min/1.73 m2 may be associated with cardiovascular, renal, and mortality benefits that need to be weighed against the risk of lactic acidosis, but further research is needed to validate these findings. Funding: CUHK Impact Research Fellowship Scheme.

Trends in all-cause and cause-specific mortality in older adults with and without diabetes: A territory-wide analysis in one million older adults in Hong Kong.

AIMS: Direct comparisons of population-level trends in all-cause and cause-specific mortalities among older adults with and without diabetes are lacking. METHODS: We performed a territory-wide analysis of 1,142,000 unique older adults aged ≥ 65 years (31.7 % with diabetes) with at least one attendance in the Hong Kong Hospital Authority in 2014-2018. We used Joinpoint regression to describe trends of age- and sex-standardised all-cause and cause-specific mortalities (cardiovascular disease [CVD], cancer, and non-CVD and non-cancer) in older adults with and without diabetes. RESULTS: All-cause mortality decreased in older adults with (average annual percent change [AAPC] = -1.6, 95 % confidence interval [-2.7, -0.4]) and without (AAPC = -3.1 [-4.2, -2.1]) diabetes. Largest declines were seen for CVD-cause mortalities for people with and without diabetes (AAPC = -5.5 [-6.8, -4.1] vs AAPC = -5.8 [-8.6, -2.9], respectively). Cancer-cause mortalities were similar in both groups with no change. Men with diabetes showed less favourable improvements. An increasing mortality trend was seen only in the 65-69 age-group regardless of diabetes status. CONCLUSIONS: Mortality continued to decline in older adults with and without diabetes, mainly driven by a decline in CVD deaths, with no narrowing of the mortality gap. Our findings call for continued actions to address excess mortalities especially in older men with diabetes and younger older adults.

Genome assembly and annotation of Periplaneta americana reveal a comprehensive cockroach allergen profile.

BACKGROUND: One of the most common cockroach types in urban areas, the American cockroach (Periplaneta americana), has been reported to impose an increased risk of allergies and asthma. Limited groups of allergens (Per a 1-13) have been identified in this species due to the lack of genome-related information. METHODS: To expand the allergen profile of P. americana, genomic, transcriptomic, and proteomic approaches were applied. With the support of a high-quality genome assembled using nanopore, Illumina, and Hi-C sequencing techniques, potential allergens were identified based on protein homology. Then, using enzyme-linked immunosorbent assay, selected allergens were tested in Thai patients allergic to P. americana. RESULTS: A chromosomal-level genome of P. americana (3.06 Gb) has been assembled with 94.6% BUSCO completeness, and its contiguity has been significantly improved (N50 = 151 Mb). A comprehensive allergen profile has been characterized, with seven novel groups of allergens, including enolase (Per a 14), cytochrome C (Per a 15), cofilin (Per a 16), alpha-tubulin (Per a 17), cyclophilin (Per a 18), porin3 (Per a 19), and peroxiredoxin-6 (Per a 20), showing IgE sensitivity in enzyme-linked immunosorbent assay. A new isoallergen of tropomyosin (Per a 7.02) and multiple potential isoallergens of Per a 5 were revealed using bioinformatics and proteomic approaches. Additionally, comparative analysis of P. americana with the closely related Blattodea species revealed the possibility of cross-reaction. CONCLUSION: The high-quality genome and proteome of P. americana are beneficial in studying cockroach allergens at the molecular level. Seven novel allergen groups and one isoallergen in Per a 7 were identified.

Initiation of sodium-glucose cotransporter-2 inhibitors at lower HbA1c threshold attenuates eGFR decline in type 2 diabetes patients with and without cardiorenal disease: A propensity-matched cohort study.

AIM: To examine the association of initiation of sodium-glucose cotransporter-2 inhibitors (SGLT2i) at lower glycemic threshold with decline in estimated-glomerular filtration rate (eGFR). METHODS: We analyzed a prospective cohort of Chinese patients with type 2 diabetes from Hong Kong. Patients initiating SGLT2i at HbA1c < 7.5 % (lower-HbA1c) versus ≥ 7.5 % (higher-HbA1c) were matched using 1:1 propensity score. We compared annual eGFR changes in the lower-HbA1c and higher-HbA1c groups using linear mixed-effect models. Binary logistic regression was used to explore associations of SGLT2i initiation at lower HbA1c with odds of rapid eGFR decline (>4% per year). RESULTS: Among 3384 patients with a median follow-up of 1.9 years, the mean age was 60.2 ± 11.5 years and 62.1 % were male. The lower-HbA1c and higher-HbA1c groups had baseline HbA1c (%) of 6.9 ± 0.5 and 9.0 ± 1.3 respectively, with similar pre-index annual eGFR decline. The lower-HbA1c group had a slower post-index annual eGFR decline than the higher-HbA1c group (-0.99 versus -1.63 mL/min/1.73 m2, p < 0.001). Overall, the lower-HbA1c group had lower odds of rapid eGFR decline (OR = 0.15, 95 % CI: 0.07-0.29). Greater renoprotection from SGLT2i initiation at lower-HbA1c was observed in those with baseline eGFR < 60 mL/min/1.73 m2, albuminuria and/or treatment with renin-angiotensin-system inhibitors or insulin. CONCLUSIONS: In this real-world study, SGLT2i initiation at HbA1c < 7.5 % was associated with slower eGFR decline especially in high risk patients, supporting the potential renal benefits of SGLT2i initiation at lower glycemic thresholds.

The role of age on the risk relationship between prediabetes and major morbidities and mortality: Analysis of the Hong Kong diabetes surveillance database of 2 million Chinese adults.

Background: Intensive lifestyle modification showed variable success in the prevention of major clinical events and mortality among people with prediabetes. We propose that age may partly explain the heterogeneity and that health hazards related to prediabetes are age-specific. Methods: We conducted a retrospective analysis of a territory-wide diabetes surveillance dataset from the Hong Kong Hospital Authority between 2000 and 2019. Prediabetes was defined according to the American Diabetes Association criteria. Proportional Cox regression was performed, stratified by baseline age categories (20-39, 40-59, 60-79 and ≥80 years). Findings: 1,630,942 individuals were included in the analysis. Compared with normoglycaemia, prediabetes was associated with greater hazards for cardiovascular disease (CVD) and all-cause mortality in most age groups but the effect size attenuated with ascending age (p value for trend <0·05). In the youngest and in the oldest age categories, the respective hazard ratios (95% confidence interval) of prediabetes vs normoglycaemia were 1·79 (1·59, 2·01) and 1·00 (0·95, 1·05) for CVD, and 1·36 (1·20, 1·55) and 0·99 (0·97, 1·02) for all-cause mortality. Similar associations were found for chronic kidney disease, end-stage kidney disease, all-site cancer, all-site infection, subtypes of CVD, and cause-specific mortality. The associations became attenuated but remained after excluding people who later developed diabetes and adjusting for metabolic factors. Similar associations were observed in prediabetes defined by impaired fasting glucose, but not HbA1c. Interpretation: Prediabetes is associated with higher risk of major clinical events, even excluding subsequent development of diabetes and adjusting for metabolic factors. The risk relationships are stronger in young than older people. Funding: This study did not receive any specific funding.

Clinical outcomes following discontinuation of renin-angiotensin-system inhibitors in patients with type 2 diabetes and advanced chronic kidney disease: A prospective cohort study.

Background: Renin-angiotensin-system inhibitors (RASi), that include angiotensin converting enzyme inhibitors (ACEis) and angiotensin receptor blockers (ARBs) reduce proteinuria, delay chronic kidney disease (CKD) progression, protect against cardiovascular events and heart failure hospitalizations. We examined the associations of discontinuation of ACEi/ARBs with risk of clinical outcomes in Chinese patients with type 2 diabetes (T2D) and advanced-CKD (estimated-glomerular filtration rate [eGFR] <30 ml/min/1.73 m2). Methods: We conducted a prospective, population-based cohort study including 10,400 patients with T2D in Hong Kong stratified by continuation of ACEi/ARBs within 6 months after reaching eGFR <30 ml/min/1.73 m2 from January 01, 2002 to December 31, 2018 and observed until December 31, 2019. The primary outcomes were death, major-adverse cardiovascular events (MACE), heart failure, end-stage kidney disease (ESKD), and all-cause mortality. Cox-model with time-dependent exposure and covariates was used to estimate the hazard ratio (HR) of outcomes in a propensity-score overlap-weighted cohort. The risk of occurrence of hyperkalemia (plasma potassium >5.5 mmol/L) in discontinued-ACEi/ARBs versus continued-ACEi/ARBs users was assessed in a register-based cohort. Findings: In the population-based cohort of 10,400 ACEi/ARBs users with new-onset eGFR<30 ml/min/1.73 m2, 1766 (17.0%) discontinued ACEi/ARBs and 8634 (83.0%) persisted with treatment. During a median follow-up of 3.6 (interquartile range, IQR: 2.11-5.8) years (41,623 person-years), 13.5%, 12.9%, and 27.6% had incident MACE, heart failure and ESKD respectively, and 35.8% died. Discontinued-ACEi/ARBs use was associated with higher risk of MACE (HR = 1.27, 95% CI: 1.08-1.49), heart failure (HR = 1.85, 95% CI: 1.53-2.25) and ESKD (HR = 1.30, 95% CI: 1.17-1.43), and neutral risk of all-cause mortality (HR = 0.93, 95% CI: 0.86-1.01) compared to counterparts with continued use. In the register-based cohort (583 discontinued-ACEi/ARBs users and 3817 continued-ACEi/ARBs users), discontinued-ACEi/ARBs had neutral risk of hyperkalemia (HR = 0.95, 95% CI: 0.84-1.08). Interpretation: Discontinuation of ACEi/ARBs was associated with increased risk of cardiovascular-renal events supporting their continued use in patients with T2D and advanced-CKD. Funding: CUHK Impact Research Fellowship Scheme.

Effects of RAS inhibitors on all-site cancers and mortality in the Hong Kong diabetes surveillance database (2002-2019).

BACKGROUND: Cancer is replacing cardiovascular-disease as a leading cause of death in type 2 diabetes (T2D). The association of RAS-inhibitors (RASi) and cancer, including differences between angiotensin-converting-enzyme-inhibitor (ACEi) and angiotensin-receptor-blocker (ARBs) as well as their associations independent of blood pressure lowering, remains inconclusive in T2D. METHODS: We conducted a cohort study with new-user design in 253,491 patients in the Hong-Kong-Diabetes-Surveillance-Database (HKDSD) in 2002-2019. We evaluated the associations of time-varying RASi use (ACEi and ARBs) with all-site cancer, diabetes-related cancers, and cancer-specific mortality including comparison with new-users of calcium-channel-blockers (CCBs) as an active-comparator group. FINDINGS: Of 253,491, 133,730 (52.8%) were new-RASi and 119,761 (47.2%) were non-RASi users with a median follow-up period of 6.3 (interquartile ragne: 3.4-9.2) years (1,678,719 patient-years). After propensity-score weighting and adjustment for time-varying covariables, RASi use was associated with lower risk of all-site cancer (HR=0.76, 95%CI: 0.74-0.79), diabetes-related cancer (HR=0.79, 95%CI: 0.75-0.84), cancer-specific mortality (HR=0.50, 95%CI: 0.47-0.53), and diabetes-related cancer mortality (HR=0.49, 95%CI: 0.45-0.54) versus non-RASi. Amongst RASi users, ARBs use was associated with lower risk of cancer-specific mortality versus ACEi (HR=0.77, 95%CI: 0.66-0.91). Use of RASi was associated with an estimated-prevention of 2.6 (95%CI: 2.3-3.0) all-site cancer per-1000-person-years and 2.2 (95%CI: 2.0-2.5) cancer-related mortality per-1000-person-years. Lower risk of cancer-specific mortality was similarly observed in new-RASi compared with new-CCBs users. INTERPRETATION: RASi use was independently associated with lower cancer risk in T2D with stronger associations in users of ARBs than ACEi. The benefits of RASi in patients with diabetes might go beyond cardiovascular-renal protection if confirmed by other real-world studies and trials. FUNDING: Dr. Aimin Yang was supported by a CUHK Impact-Research-Fellowship Scheme.

Risk Associations of Glycemic Burden and Obesity With Liver Cancer-A 10-Year Analysis of 15,280 Patients With Type 2 Diabetes.

Liver is a major site for glucose metabolism. Patients with type 2 diabetes (T2D) and obesity have increased risk of liver cancer. We explored the association of glycemic burden (GB) and obesity with liver cancer in T2D in the prospective Hong Kong Diabetes Register (1995-2019). We calculated GB using the area under the curve above hemoglobin A1c (HbA1c) of 5.7% and defined obesity as body mass index (BMI) ≥ 25 kg/m2 . We used Cox proportional hazards models to evaluate the association between GB and liver cancer. We included 15,280 patients with at least 10 years of disease duration before liver cancer occurred or censor date, ≥3 years of observation, and ≥5 HbA1c measurements (64% male, age: 58.23 ± 12.47 years, HbA1c: 7.60 ± 1.65%, BMI: 25.58 ± 4.10 kg/m2 ). We excluded 3 years of HbA1c values before liver cancer to avoid reverse causality. Every 1-SD increase in GB was associated with an adjusted hazard ratio (aHR) of liver cancer of 1.22 (95% confidence interval [CI]: 1.01-1.47). The top GB quartile group (range: >2.41) had aHR of 1.78 (1.01-3.13) versus the lowest quartile group (0-1.19). The aHRs for each SD increase in GB were 1.34 (1.05, 1.70) in the obese group and 1.12 (0.81-1.53) in the nonobese group, but no interaction (Pinteraction  = 0.120). When stratified by GB median (1.69 [1.13, 2.43]) and obesity, obese patients with high GB had the highest aHR of 2.51 (1.44-4.37) for liver cancer versus the nonobese group with low GB, but no interaction (Pinteraction  = 0.071). Subgroup analysis of patients with available hepatitis B surface antigen status (n = 9,248) yielded similar results. Conclusion: Our results emphasized the importance of glycemic and weight control for reducing the risk of liver cancer in T2D.

Risk associations of long-term HbA1c variability and obesity on cancer events and cancer-specific death in 15,286 patients with diabetes - A prospective cohort study.

BACKGROUND: Obesity, cancer and diabetes frequently coexist. The association of glycaemic variability (GV) and obesity with cancer events had not been explored in diabetes. METHODS: In the prospective Hong Kong Diabetes Register cohort (1995-2019), we used cox proportional hazards models to examine the risk associations of GV with all-site cancer (primary outcome) and cause-specific death (secondary outcome). We also explored the joint association of obesity and GV with these outcomes and site-specific cancer. We expressed GV using HbA1c variability score (HVS) defined as percentage of HbA1c values varying by 0.5% compared with values in preceding visit. FINDINGS: We included 15,286 patients (type 2 diabetes: n=15,054, type 1 diabetes: n=232) with ≥10 years of diabetes and ≥3 years of observation (51.7% men, age (mean±SD): 61.04±10.73 years, HbA1c: 7.54±1.63%, body mass index [BMI]: 25.65±3.92 kg/m2, all-site cancer events: n=928, cancer death events: n=404). There were non-linear relationships between HVS and outcomes but there was linearity within the high and low HVS groups stratified by the median (IQR) value of HVS (42.31 [27.27, 56.28]). In the high HVS group, the adjusted hazard ratios (aHR) of each SD of HVS was 1.15 (95% CI: 1.04, 1.26) for all-site cancer (n=874). The respective aHRs for breast (n=77), liver (n=117) and colorectal (n=184) cancer were 1.44 (1.07, 1.94), 1.37 (1.08, 1.74), and 1.09 (0.90, 1.32). In the high GV group, the respective aHRs were 1.21 (1.06, 1.39), 1.27 (1.15, 1.40), and 1.15 (1.09, 1.22) for cancer, vascular, and noncancer nonvascular death. When stratified by obesity (BMI ≥25 kg/m2), the high HVS & obese group had the highest aHRs of 1.42 (1.16, 1.73), 2.44 (1.24, 4.82), and 2.63 (1.45, 4.74) respectively for all-site, breast, and liver cancer versus the low GV & non-obese group. The respective aHRs were 1.45 (1.07, 1.96), 1.47 (1.12, 1.93), and 1.35 (1.16, 1.57) for cancer, vascular, and noncancer nonvascular death. INTERPRETATION: Obesity and high GV were associated with increased risk of all-site, breast, liver cancer, and cancer-specific death in T2D. FUNDING: The Chinese University of Hong Kong Diabetes Research Fund.

Increased co-expression of PSMA2 and GLP-1 receptor in cervical cancer models in type 2 diabetes attenuated by Exendin-4: A translational case-control study.

BACKGROUND: Type 2 diabetes (T2D) increases the risk of many types of cancer. Dysregulation of proteasome-related protein degradation leads to tumorigenesis, while Exendin-4, a glucagon-like peptide 1 receptor (GLP-1R) agonist, possesses anti-cancer effects. METHODS: We explored the co-expression of proteasome alpha 2 subunit (PSMA2) and GLP-1R in the Cancer Genome Atlas (TCGA) database and human cervical cancer specimens, supplemented by in vivo and in vitro studies using multiple cervical cancer cell lines. FINDINGS: PSMA2 expression was increased in 12 cancer types in TCGA database and cervical cancer specimens from patients with T2D (T2D vs non-T2D: 3.22 (95% confidence interval CI: 1.38, 5.05) vs 1.00 (0.66, 1.34) fold change, P = 0.01). psma2-shRNA decreased cell proliferation in vitro, and tumour volume and Ki67 expression in vivo. Exendin-4 decreased psma2 expression, tumour volume and Ki67 expression in vivo. There was no change in GLP-1R expression in 12 cancer types in TCGA database. However, GLP-1R expression (T2D vs non-T2D: 5.49 (3.0, 8.1) vs 1.00 (0.5, 1.5) fold change, P < 0.001) was increased and positively correlated with PSMA2 expression in T2D-related (r = 0.68)  but not in non-T2D-related cervical cancer specimens. This correlation was corroborated by in vitro experiments where silencing glp-1r decreased psma2 expression. Exendin-4 attenuated phospho-p65 and -IκB expression in the NF-κB pathway. INTERPRETATION: PSMA2 and GLP-1R expression in T2D-related cervical cancer specimens was increased and positively correlated, suggesting hyperglycaemia might promote cancer growth by increasing PSMA2 expression which could be attenuated by Exendin-4. FUNDING: This project was supported by Postdoctoral Fellowship Scheme, Direct Grant, Diabetes Research and Education Fund from the Chinese University of Hong Kong (CUHK).

Mutational Signature Analysis Reveals Widespread Contribution of Pyrrolizidine Alkaloid Exposure to Human Liver Cancer.

BACKGROUND AND AIMS: Mutational signature analyses are an effective tool in identifying cancer etiology. Humans are frequently exposed to pyrrolizidine alkaloids (PAs), the most common carcinogenic phytotoxins widely distributed in herbal remedies and foods. However, due to the lack of human epidemiological data, PAs are classified as group II hepatocarcinogens by the World Health Organization. This study identified a PA mutational signature as the biomarker to investigate the association of PA exposure with human liver cancer. APPROACH AND RESULTS: Pyrrole-protein adducts (PPAs), the PA exposure biomarker, were measured and found in 32% of surgically resected specimens from 34 patients with liver cancer in Hong Kong. Next, we delineated the mode of mutagenic and tumorigenic actions of retrorsine, a representative PA, in mice and human hepatocytes (HepaRG). Retrorsine induced DNA adduction, DNA damage, and activation of tumorigenic hepatic progenitor cells, which initiated hepatocarcinogenesis. PA mutational signature, as the unique molecular fingerprint of PA-induced mutation, was derived from exome mutations in retrorsine-exposed mice and HepaRG cells. Notably, PA mutational signature was validated in genomes of patients with PPA-positive liver cancer but not patients with PPA-negative liver cancer, confirming the specificity of this biomarker in revealing PA-associated liver cancers. Furthermore, we examined the established PA mutational signature in 1,513 liver cancer genomes and found that PA-associated liver cancers were potentially prevalent in Asia (Mainland China [48%], Hong Kong [44%], Japan [22%], South Korea [6%], Southeast Asia [25%]) but minor in Western countries (North America [3%] and Europe [5%]). CONCLUSIONS: This study provides a clinical indication of PA-associated liver cancer. We discovered an unexpectedly extensive implication of PA exposure in patients with liver cancer, laying the scientific basis for precautionary approaches and prevention of PA-associated human liver cancers.

IL-6/STAT3-mediated autophagy participates in the development of age-related glomerulosclerosis.

The standard of age-related glomerulosclerosis is unclear. Both signal transducer and activator of transcription 3 (STAT3) and autophagy are involved in age-related kidney disease. Therefore, we aimed to explore the standard, as well as the potential mechanism(s). A total of 44 patients who underwent radical nephrectomy were enrolled. Pearson analysis was performed to investigate the parameters with ages. The patients were divided into the young- and aged-kidney groups. Kidney morphological changes were evaluated by histology staining, senescence was evaluated by senescence-associated-ß-galactosidase (SA-ß-gal) staining, and autophagosome was measured by transmission electron microscopy. Moreover, Western blot and/or immunohistochemistry were accomplished to assess the expression of p16, STAT3, and glycoprotein130 (GP130) and autophagy-related proteins. Furthermore, human glomerular mesangial cells were administrated with tocilizumab (TCZ) and/or IL-6, and then the above indexes were tested again. Sclerotic glomerular density and glomerular sclerosis rate were significantly higher in individuals more than 40 years old, and they were strongly correlated with ages. Moreover, the expression of p16, STAT3, GP130, and p62 was significantly increased, while LC3II and autophagosome were statistically decreased in the aged-kidney. Glomeruli were hardly to stain with SA-ß-gal. For the in vitro experiments, we observed that IL-6 significantly increased p16, STAT3, GP130, and p62, induced higher SA-ß-gal staining, while downregulated LC3II and autophagosome. Furthermore, TCZ statistically reversed the effects of IL-6 on the above expression of proteins. Glomerular sclerosis rate might be one standard for natural renal aging, and IL-6/STAT3-mediated autophagy may participate in the development of age-related glomerulosclerosis.

The effect of glucocorticoids on serum cystatin C in identifying acute kidney injury: a propensity-matched cohort study.

BACKGROUND: Glucocorticoids may impact the accuracy of serum cystatin C (sCysC) in reflecting renal function. We aimed to assess the effect of glucocorticoids on the performance of sCysC in detecting acute kidney injury (AKI) in critically ill patients. METHODS: A prospective observational cohort study was performed in a general intensive care unit (ICU). Using propensity score matching, we successfully matched 240 glucocorticoid users with 960 non-users among 2716 patients. Serum creatinine (SCr) and sCysC were measured for all patients at ICU admission. Patients were divided into four groups based on cumulative doses of glucocorticoids within 5 days before ICU admission (Group I: non-users; Group II: 0 mg < prednisone ≤50 mg; Group III: 50 mg < prednisone ≤150 mg; Group IV: prednisone > 150 mg). We compared the performance of sCysC for diagnosing and predicting AKI in different groups using the area under the receiver operator characteristic curve (AUC). RESULTS: A total of 240 patients received glucocorticoid medication within 5 days before ICU admission. Before and after matching, the differences of sCysC levels between glucocorticoid users and non-users were both significant (P <  0.001). The multiple linear regression analysis revealed that glucocorticoids were independently associated with sCysC (P <  0.001). After matching, the group I had significantly lower sCysC levels than the group III and group IV (P <  0.05), but there were no significant differences in sCysC levels within different glucocorticoids recipient groups (P > 0.05). Simultaneously, we did not find significant differences in the AUC between any two groups in the matched cohort (P > 0.05). CONCLUSIONS: Glucocorticoids did not impact the performance of sCysC in identifying AKI in critically ill patients.

Pan-cancer analysis of differential DNA methylation patterns.

BACKGROUND: DNA methylation is a key epigenetic regulator contributing to cancer development. To understand the role of DNA methylation in tumorigenesis, it is important to investigate and compare differential methylation (DM) patterns between normal and case samples across different cancer types. However, current pan-cancer analyses call DM separately for each cancer, which suffers from lower statistical power and fails to provide a comprehensive view for patterns across cancers. METHODS: In this work, we propose a rigorous statistical model, PanDM, to jointly characterize DM patterns across diverse cancer types. PanDM uses the hidden correlations in the combined dataset to improve statistical power through joint modeling. PanDM takes summary statistics from separate analyses as input and performs methylation site clustering, differential methylation detection, and pan-cancer pattern discovery. We demonstrate the favorable performance of PanDM using simulation data. We apply our model to 12 cancer methylome data collected from The Cancer Genome Atlas (TCGA) project. We further conduct ontology- and pathway-enrichment analyses to gain new biological insights into the pan-cancer DM patterns learned by PanDM. RESULTS: PanDM outperforms two types of separate analyses in the power of DM calling in the simulation study. Application of PanDM to TCGA data reveals 37 pan-cancer DM patterns in the 12 cancer methylomes, including both common and cancer-type-specific patterns. These 37 patterns are in turn used to group cancer types. Functional ontology and biological pathways enriched in the non-common patterns not only underpin the cancer-type-specific etiology and pathogenesis but also unveil the common environmental risk factors shared by multiple cancer types. Moreover, we also identify PanDM-specific DM CpG sites that the common strategy fails to detect. CONCLUSIONS: PanDM is a powerful tool that provides a systematic way to investigate aberrant methylation patterns across multiple cancer types. Results from real data analyses suggest a novel angle for us to understand the common and specific DM patterns in different cancers. Moreover, as PanDM works on the summary statistics for each cancer type, the same framework can in principle be applied to pan-cancer analyses of other functional genomic profiles. We implement PanDM as an R package, which is freely available at http://www.sta.cuhk.edu.hk/YWei/PanDM.html .

Metformin Reduces the Senescence of Renal Tubular Epithelial Cells in Diabetic Nephropathy via the MBNL1/miR-130a-3p/STAT3 Pathway.

Senescence of renal tubular epithelial cells plays an important role in diabetic nephropathy, but the mechanism is unknown. Metformin may alleviate diabetic nephropathy by reducing this senescence. This study is aimed at clarifying the effects and mechanism of metformin on the senescence of renal tubular epithelial cells in diabetic nephropathy. We found that metformin reduced the expression of senescence-associated gene P21 in high-glucose-induced (30 mmol/L) renal tubular epithelial cells and decreased the ß-galactosidase positive staining rate (decreased 16%, p < 0.01). Metformin was able to reduce senescence by upregulating the expression of RNA-binding protein MBNL1 and miR-130a-3p and reducing STAT3 expression. MBNL1 prolonged the half-life of miR-130a-3p, and miR-130a-3p could negatively regulate STAT3 by binding to its mRNA 3'UTR. In db/db diabetic mice, we found an enhanced senescence level combined with low expression of MBNL1 and miR-130a-3p and high expression of STAT3 compared with db/m control mice during nephropathy development. Meanwhile, metformin (200 mg/kg/day) could increase the expression of MBNL1 and miR-130a-3p and decreased STAT3 expression, thus reducing this senescence in db/db mice. Our results suggest that metformin reduces the senescence of renal tubular epithelial cells in diabetic nephropathy via the MBNL1/miR-130a-3p/STAT3 pathway, which provided new ideas for the therapy of this disease.

The RAGE/STAT5/autophagy axis regulates senescence in mesangial cells.

Renal aging and associated functional decline are associated with an increase in cellular senescence. Previous studies show a direct correlation between advanced glycation end products (AGEs) accumulation and renal aging, chronic kidney disease (CKD) and other nephropathies, although the underlying molecular mechanisms remain largely unclear. We found elevated levels of the receptor of advanced glycation end product (RAGE) as well as STAT5 in aged human kidneys, as well as in human mesangial cells aged artificially through AGEs. Furthermore, genetic and pharmacological ablation of STAT5 significantly downregulated p16 levels and the percentage of ß-Gal-positive senescent cells in mesangial cells and kidneys of SD rats, indicating that AGEs-induced senescence depends on STAT5 signaling. The aged kidney tissues (both in patients and SD rats) and mesangial cells show low levels of LC3 (both LC3-II and LC3-II/I), and cultured mesangial cells also show fewer autolysosomes, autophagosomes, and autophagic vacuoles, which can be partially restored upon STAT5 inhibition. This indicates that AGEs accumulation also obliterates the protective effects of autophagy against aging via the RAGE/STAT5 axis. Direct inhibition of autophagy via 3-methyladenine (3-MA) increases the phenotype of renal aging without activating RAGE, it is inhibition of autophagy caused by RAGE/STAT5 that leads to mesangial aging. In conclusion, we found AGEs induced inhibition of autophagy and cellular senescence in mesangial cells via the RAGE/STAT5 pathway. Moreover, we found that RAGE/STAT5 acts as a key link between autophagy and senescence in the process of mesangial aging in vivo and in vitro.

Translation of the circular RNA circß-catenin promotes liver cancer cell growth through activation of the Wnt pathway.

BACKGROUND: Circular RNAs are a class of regulatory RNA transcripts, which are ubiquitously expressed in eukaryotes. In the current study, we evaluate the function of a novel circRNA derived from the ß-catenin gene locus, circß-catenin. RESULTS: Circß-catenin is predominantly localized in the cytoplasm and displays resistance to RNase-R treatment. We find that circß-catenin is highly expressed in liver cancer tissues when compared to adjacent normal tissues. Silencing of circß-catenin significantly suppresses malignant phenotypes in vitro and in vivo, and knockdown of this circRNA reduces the protein level of ß-catenin without affecting its mRNA level. We show that circß-catenin affects a wide spectrum of Wnt pathway-related genes, and furthermore, circß-catenin produces a novel 370-amino acid ß-catenin isoform that uses the start codon as the linear ß-catenin mRNA transcript and translation is terminated at a new stop codon created by circularization. We find that this novel isoform can stabilize full-length ß-catenin by antagonizing GSK3ß-induced ß-catenin phosphorylation and degradation, leading to activation of the Wnt pathway. CONCLUSIONS: Our findings illustrate a non-canonical function of circRNA in modulating liver cancer cell growth through the Wnt pathway, which can provide novel mechanistic insights into the underlying mechanisms of hepatocellular carcinoma.

Distribution of human papillomavirus genotypes in cervical lesions.

The aims of the present study were to investigate the distribution of human papillomavirus (HPV) genotypes in cervical lesions, and the association between different HPV genotypes and cervical lesions. Between January 2013 and June 2014, the HPV type determinations of nucleic acid by use of fluorescence polymerase chain reaction (PCR) method of 15,192 outpatients in China-Japan Friendship Hospital were performed and the infection status was analyzed. The results showed that: i) 2,366 Cases were HPV positive and 12,826 cases were HPV negative, the overall infection rate was 15.57% (2,366/15,192), in which a single genotype of HPV infection rate was 11.63% (1,767/15,192), and multiple genotypes of HPV infection rate was 3.94% (599/15,192); ii) HPV16, HPV52 and HPV58 infections were the most common HPV genotypes, the infection rates were 3.95% (600/15,192), 2.86% (435/15,192) and 2.67% (406/15,192), respectively; and iii) According to the gold standard of histopathological analysis via hematoxylin-eosin staining, HPV16, HPV52 and HPV58 accounted for 58.80% (154/267) of all CIN2 or above squamous epithelial lesions. Furthermore, three cases with pathological changes of the cervical severe glandular epithelium were all HPV18 infection. The difference was statistically significant (χ2=60.74, P<0.001). Single HPV subtype infection was primarily associated with HPV16, HPV52 and HPV58. In conclusion, HPV type detection had a may be important in screening of cervical lesions as a difference in pathogenic ability was noted among different HPV genotypes. As cervical cancer is an infectious disease, HPV testing may help detect more precancerous lesions, thus reducing the morbidity and mortality of cervical cancer. HPV16, HPV52 and HPV58 were associated with severe cervical squamous epithelial lesions; HPV18 was associated with cervical severe glandular cell pathological changes, although it was not the most common HPV genotype in China. When positive, a clinical cervical examination should be conducted, including colposcopy and biopsy.

[Relationship between Life Habits and Constitution Types of Chinese Medicine in the Senile Living at Home in Beijing Downtown].

OBJECTIVE: To investigate the distribution of constitution types of Chinese medicine (CM) in the elderly living at home in Beijing downtown, and to explore its relationship with life habits. METHODS: A total of 3894 senile more than 60 years old were enrolled in this study. Their constitution types of CM were typed using CM constitution questionnaire. Meanwhile, their demographic features, disease condition, diet habits, exercise habits, sleep habits, and so on were investigated. Multivariate Logistic regression analysis was used to evaluate the relationship between life habits and constitution types of CM. RESULTS: The number of mild type constitution senile was 1111 (28.53%) and the number of biased constitutions 2783 (71.47%). Biased constitutions of the top three were qi deficiency constitution (662, 17.00%), yang deficiency constitution (445, 11.43%), and blood stasis constitution (363, 9.32%). Univariate analysis showed that different habits of diet, exercise, and sleep exist among the senile of different constitutions (P < 0.05). By taking mild type constitution, multivariate Logistic regression analysis (except demographic indices and chronic history) showed that significantly positive correlation existed between qi deficiency constitution and favor for hot food (OR = 1.349, P = 0.015), yang deficiency constitution and favor for hot food (OR = 2.448, P < 0.01), phlegm-wetness constitution and favor for barbecue food (OR = 2.144, P = 0.003), wet-heat constitution and favor for sweet food (OR = 1.355, P = 0.032), wet-heat constitution and favor for tea (OR = 1.359, P = 0.047), blood stasis constitution and favor for hot food (OR = 1.422, P = 0.017), and qi depression constitution and favor for hot food (OR = 1.446, P = 0.031). Regular exercise had negative correlation with qi deficiency constitution (OR = 0.397, P < 0.01), yang deficiency constitution (OR = 0.522, P < 0.01) , phlegm-wetness constitution (OR = 0.475, P < 0.01), wet-heat constitution (OR = 0.647, P = 0.015), blood stasis constitution (OR = 0.608, P = 0.001), qi depression constitution (OR = 0.541, P = 0.001), and special diathesis constitution (OR = 0.466, P < 0.01). Early sleep and rise habit had negative with phlegm-wetness constitution (OR = 0.414, P < 0.01), wet-heat constitution (OR = 0.536, P = 0.015), blood stasis constitution (OR = 0.515, P = 0.004), and special diathesis constitution (OR = 0.526, P = 0.039). CONCLUSIONS: Different constitution types of CM might be highly related to specific life habits. Cultivating better life habits can improve biased constitutions of CM.