SIRT7-Induced PHF5A Decrotonylation Regulates Aging Progress Through Alternative Splicing-Mediated Downregulation of CDK2.

Dysregulation of protein posttranslational modification (PTM) can lead to a variety of pathological processes, such as abnormal sperm development, malignant tumorigenesis, depression, and aging process. SIRT7 is a NAD+-dependent protein deacetylase. Besides known deacetylation, SIRT7 may also have the capacity to remove other acylation. However, the roles of SIRT7-induced other deacylation in aging are still largely unknown. Here, we found that the expression of SIRT7 was significantly increased in senescent fibroblasts and aged tissues. Knockdown or overexpression of SIRT7 can inhibit or promote fibroblast senescence. Knockdown of SIRT7 led to increased pan-lysine crotonylation (Kcr) levels in senescent fibroblasts. Using modern mass spectrometry (MS) technology, we identified 5,149 Kcr sites across 1,541 proteins in senescent fibroblasts, and providing the largest crotonylome dataset to date in senescent cells. Specifically, among the identified proteins, we found SIRT7 decrotonylated PHF5A, an alternative splicing (AS) factor, at K25. Decrotonylation of PHF5A K25 contributed to decreased CDK2 expression by retained intron (RI)-induced abnormal AS, thereby accelerating fibroblast senescence, and supporting a key role of PHF5A K25 decrotonylation in aging. Collectively, our data revealed the molecular mechanism of SIRT7-induced k25 decrotonylation of PHF5A regulating aging and provide new ideas and molecular targets for drug intervention in cellular aging and the treatment of aging-related diseases, and indicating that protein crotonylation has important implications in the regulation of aging progress.

Senescent Cell-Secreted Netrin-1 Modulates Aging-Related Disorders by Recruiting Sympathetic Fibers.

Cellular senescence is implicated in several lines of aging-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibers (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SFs densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cells induce recruitment of SFs in vitro. Additionally, subcutaneous transplantation of 2BS fibroblasts in nude mice shows that transplanted senescent 2BS fibroblasts promote SFs infiltration. Intra-articular senolytic molecular injection can reduce SFs density and inhibit SFs infiltration caused by senescent cells in osteoarthritis (OA), suggesting senescent cells promote the infiltration of SFs in vivo in aged tissues. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective ß receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective ß receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE-/- mice) by reducing hepatic SNA. Taken together, this study concludes that senescent cell-secreted netrin-1 mediated SFs outgrowth and infiltration, which contributes to aging-related disorders, suggesting that clearing senescent cells or inhibiting SNA is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced aging-related pathologies.

Circular RNA CircCCNB1 sponges micro RNA-449a to inhibit cellular senescence by targeting CCNE2.

Circular RNAs (CircRNAs) are a novel subset of non-coding RNA widely present in eukaryotes that play a central role in physiological and pathological conditions. Accumulating evidence has indicated that CircRNAs participated in modulating tumorigenesis by acting as a competing endogenous RNA (CeRNA). However, the roles and functions of CircRNAs in cellular senescence and aging of organisms remain largely obscure. We performed whole transcriptome sequencing to compare the expression patterns of circular RNAs in young and prematurely senescent human diploid fibroblast 2BS cells, and identified senescence-associated circRNAs (SAC-RNAs). Among these SAC-RNAs, we observed the significantly downregulated expression of CircRNAs originating from exons 6 and 7 circularization of the cyclin B1 gene (CCNB1), termed CircCCNB1. Reduced CircCCNB1 expression triggered senescence in young 2BS cells, as measured by increased senescence associated-beta-galactosidase (SA-ß-gal) activity, enhanced expression of cyclin-dependent kinase inhibitor 1A (CDKN1A)/P21 and tumor protein 53 (TP53) expression, and reduced cell proliferation. Mechanistically, reduced CircCCNB1 level inhibited cyclin E2 (CCNE2) expression by modulating micro RNA (miR)-449a activity, which repressed cellular proliferation. Our data suggested that CircCCNB1may serve as a sponge against miR-449a to delay cellular senescence by targeting CCNE2. Targeting CircCCNB1 may represent a promising strategy for aging and age-related disease interventions. Furthermore, we also identified and characterized several kinds of the CircCCNB1-binding proteins (CBPs), which may contribute to the degradation of CircCCNB1.

FTO Inhibits Insulin Secretion and Promotes NF-κB Activation through Positively Regulating ROS Production in Pancreatic ß cells.

FTO (Fat mass and obesity-associated) is associated with increased risk of obesity and type 2 diabetes incurrence. Pancreas islet ß cells dysfunction and insulin resistance are major causes of type 2 diabetes. However, whether FTO plays an important functional role in pancreatic ß cells as well as the related molecular mechanism is still unclear. In the present study, the tissue expression profile of FTO was firstly determined using quantitative PCR and western blot. FTO is widely expressed in various tissues and presented with relative high expression in pancreas tissue, especially in endocrine pancreas. FTO overexpression in MIN6 cells achieved by lentivirus delivery significantly inhibits insulin secretion in the presence of glucose stimulus as well as KCl. FTO silence has no effect on insulin secretion of MIN6 cells. However, FTO overexpression doesn't affect the transcription of insulin gene. Furthermore, reactive oxygen species (ROS) production and NF-κB activation are significantly promoted by FTO overexpression. Inhibition of intracellular ROS production by N-acetyl-L-cysteine (NAC) can alleviate NF-κB activation and restore the insulin secretion mediated by FTO overexpression. A whole transcript-microarray is employed to analyze the differential gene expression mediated by FTO overexpression. The genes which are modulated by FTO are involved in many important biological pathways such as G-protein coupled receptor signaling and NF-κB signaling. Therefore, our study indicates that FTO may contribute to pancreas islet ß cells dysfunction and the inhibition of FTO activity is a potential target for the treatment of diabetes.

Significance of ST-segment deviation in patients with acute pulmonary embolism and negative T waves.

BACKGROUND: Common electrocardiogram (ECG) manifestations in acute pulmonary em-bolism (APE) include ST-segment deviation (STDV) along with negative T-waves (NTW). STDV could occur in 3 typical ischemic patterns: (i) the left ventricular (LV) subendocardial ischemic pattern; (ii) the right ventricular (RV) transmural ischemic pattern; and (iii) the LV subendocardial plus RV transmural ischemic pattern. The purpose of this study was to evalu-ate the relationship of STDV and adverse clinical outcomes and to identify the relationship of relatively normal ECG and favorable clinical outcomes. METHODS: Retrospective analysis of electronic charts in APE patients was performed in a tertiary hospital. ECGs on admission were obtained and classified as with or without STDV. Adverse clinical outcomes were defined as need to intensify therapy and 30-day mortality. Relatively normal ECG was defined as without any STDV, abnormal QRS morphology in lead V1 and S1Q3T3. RESULTS: From a total of 210 patients with NTW, 131 had STDV ≥ 0.1 mV, while 79 did not. Patients with STDV had worse evolution: higher incidence of dyspnea, hypotension, cardiogen-ic shock, intensification of therapy, and death compared to patients without STDV (p = 0.001 for each variable). The majority (89%) of the patients with STDV presented with 1 of the 3 typical ischemic ECG patterns. LV subendocardial ischemic pattern (OR = 4.963, p = 0.004), RV transmural ischemic pattern (OR = 3.128, p = 0.021) and LV subendocardial plus RV transmural ischemic pattern (OR = 3.036, p = 0.017) independently predicted the need to intensify therapy. RV transmural ischemic pattern (OR = 4.227, p = 0.031) and LV subendocardial plus RV transmural ischemic pattern (OR = 4.022, p = 0.032) independently predicted 30-day mortality. Compared to the patients with abnormal ECG, the patients with relatively normal ECG had a significant lower incidence of death (0% vs. 16%; p = 0.001) and need to intensify therapy during hospitalization (6% vs. 30%; p = 0.002). CONCLUSIONS: Ischemic ECG patterns are common ECG manifestations of APE and predict worse evolution and 30-day mortality. Additionally, relatively normal ECGs may associate with favorable clinical outcomes.

Changes of naturally occurring CD4(+)CD25(+) FOXP3(+) regulatory T cells in patients with acute coronary syndrome and the beneficial effects of atorvastatin treatment.

To determine the number and function of naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells (nTregs) in patients with acute coronary syndrome (ACS) and to determine the effects of a low dose of atorvastatin treatment (20 mg/day) on nTregs.Patients with ACS were randomly divided into a group receiving conventional therapy (n = 60) or conventional therapy supplemented with atorvastatin (20 mg/day) (n = 60). A group of healthy volunteers that did not suffer from ACS was used as controls (n = 60). T lymphocytes were isolated from ACS patients, both before and 4 weeks after treatment, or control patients and the percentage of nTregs was determined by flow cytometry. Serum levels of cytokines were determined by enzyme-linked immunosorbent assays. A mixed lymphocyte reaction was used to determine the ability of nTregs to inhibit proliferation of effector T cells. Quantitative PCR and Western blot were used to analyze (forkhead box P3) FOXP3 mRNA transcript levels and the expression of FOXP3 protein.In ACS patients, the percentage and inhibitory properties of nTregs were reduced, IFN-γ and hsCRP levels were increased, and IL-10 and TGF-ß1 levels were lowered. Atorvastatin treatment increased the percentage and inhibitory ability of nTregs, decreased serum IFN-γ and hsCRP levels, and decreased IL-10 and TGF-ß1 levels, as compared with the non-atorvastatin group.Our findings suggest that nTregs play an atheroprotective role in atherosclerosis. The inhibitory effects of atorvastatin on inflammation in ACS may be due to its beneficial effects on nTregs and restoration of immune homeostasis.

Mesenchymal stem cells alleviate atherosclerosis by elevating number and function of CD4(+)CD25 (+)FOXP3 (+) regulatory T-cells and inhibiting macrophage foam cell formation.

Atherosclerosis is a chronic inflammatory disease characterized by the formation of plaques inside arteries, leading to narrowing and blockage. Potential therapeutic strategies include expanding the population of regulatory T-cells (Tregs) to enhance atheroprotective immunity, and inhibiting the formation of macrophage foam cells. Here, we studied the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) on atherosclerotic plaque formation in Apolipoprotein E(-/-) (ApoE-KO) mice, and elucidated the underlying mechanism. BM-MSCs isolated from 4 week-old ApoE-KO mice were evaluated by flow cytometry for expression of MSC-specific markers. Thirty eight week-old ApoE-KO mice were randomly divided into three experimental groups (n = 10 per group): 1. MSC group-received BM-MSCs intravenously; 2. Vehicle group-received DMEM; 3. Control group-did not receive any treatment. Administration of MSCs resulted in a marked decrease in the size of atherosclerotic plaques 3 months after treatment. In addition, the number and function of CD4(+)CD25(+)FOXP3(+) regulatory T-cells (Tregs) in cultured splenocytes, and the expression of FOXP3 at both mRNA and protein levels, was significantly increased in the MSC group. In vitro experiments further indicated that the formation of macrophage foam cells was inhibited by treatment with MSCs, accompanied by a significant downregulation in CD36 and scavenger receptor A (SRA). Our findings suggest that MSCs play an atheroprotective role by enhancing the number and function of Tregs and inhibiting the formation of macrophage foam cells. Hence, administration of MSCs to atherosclerotic patients might have significant clinical benefits.

Annual financial impact of thyroidectomies for nodular thyroid disease in China.

A large proportion of patients with thyroid nodules in China undergo thyroidectomy in order to get confirmatory histology diagnosis. The financial impact of this modality remains to be investigated. To evaluate rationality of performing thyroidectomy without a routine FNA preoperatively from the economic perspective, we conducted a retrospective, observational study of all archival thyroidectomies with records of cost per stay (CPS), cost per day (CPD) and length of stay (LOS) from 2008 to 2013 in the First Affiliated Hospital of Nanjing Medical University. We compared all the parameters between cancer and non-cancer thyroidectomies. We recruited 6, 140 thyroidectomies with valid records of CPS, CPD and LOS in this period. The CPS of cancer thyroidectomy was significantly higher than non-cancer thyroidectomy. The percentage of cancer thyroidectomy increased from 26.5% to 41.6%. The percentage of annual cost of cancer thyroidectomies rose from 30.2% to 45.2%. The LOS for cancer and non-cancer thyroidectomy decreased while the CPD increased in the past six years. The estimated national cost in 2012 for all thyroidectomies would be USD 1.86 billion with USD 1.09 billion for non-cancer thyroidectomies. We have witnessed great improvement in the healthcare for patients with thyroid nodules in China. However, given limited healthcare resources, currently thyroid FNA for more precise preoperative diagnosis may help to curb the rapidly increasing demand in healthcare costs in the future for nodular thyroid disease in China.

Association of serum uric acid with 2-hour postload glucose in Chinese with impaired fasting plasma glucose and/or HbA1c.

OBJECTIVE: To examine whether serum uric acid (SUA) is associated with 2-hour postload glucose (2-h PG) in Chinese with impaired fasting plasma glucose (IFG) and/or HbA1c (IA1C). RESEARCH DESIGN AND METHODS: Anthropometric and biochemical examinations, such as SUA concentration, were performed in 3763 individuals from all the villages in Baqiao County, China. A 75-g oral glucose tolerance test (OGTT) was conducted in 1197 Chinese with prediabetes as having IFG (110 ≤ fasting plasma glucose [FPG] <126 mg/dl and HbA1c <6.5%), IA1C (5.7% ≤ HbA1c <6.5% and FPG <126 mg/dl), or both. RESULTS: The present study included 1197 participants with IFG and/or IA1C (mean age 56.5 ± 10.3 years; 50.6% men). In multivariate linear regression, after adjustment for gender, age, smoking and drinking, body mass index (BMI), systolic and diastolic blood pressure (SBP, DBP), lipid profiles, logarithmic transformed C-reactive protein (log-CRP), estimated glomerular filtration rate (e-GFR), FPG and HbA1c, with a 1-mg/dl increment of SUA, 2-h PG increased by 5.04 ± 0.72 (P<0.001), 3.06 ± 1.08 (P = 0.001), 5.40 ± 1.26 (P<0.001), and 2.34 ± 2.16 mg/dl (P = 0.056) in all participants, in participants with normal glucose tolerance (NGT), with impaired glucose tolerance (IGT), and with 2-h newly diagnosed diabetes (2-h NDM, with 2-h PG ≥ 200 mg/dl), respectively. In both men and women, 2-h PG increased progressively and significantly from the lower to the upper SUA tertiles (P<0.001). Moreover, in multivariate logistic regression, 1-standard deviation (SD; 1.53 mg/dl) increment of SUA was significantly associated with a 36% higher risk for 2-h NDM (Odds ratio [CI 95%]: 1.36 [1.09-1.99]; P = 0.03). CONCLUSIONS: SUA is significantly associated with 2-h PG in Chinese with IFG and/or IA1C.

Associations of polymorphisms in the apolipoprotein APOA1-C3-A5 gene cluster with acute coronary syndrome.

BACKGROUND: Acute coronary syndromes (ACSs) are clinically cardiovascular events associated with dyslipidemia in common. Single nucleotide polymorphisms (SNPs) and haplotypes in the APOA1/C3/A5 gene cluster are associated with diabetes and familial combined hyperlipidaemia (FCH). Little is known about whether the polymorphisms in these genes affect lipid homeostasis in patients with ACSs. The present paper aimed to examine these associations with 4 SNPs in the APOA1 -75G > A, the APOC3 -455T > C, and APOA5 -1131T > C, c.553G > T variant to ACSs in Chinese Han. METHODS: Chinese Han of 229 patients with ACSs and 254 unrelated controls were analyzed. Four SNPs in APOA1/C3/A5 cluster were genotyped and lipid was determined. RESULTS: Our data show that minor allelic frequencies of APOC3 -455T > C, APOA5 -1131T > C, and c.553G > T polymorphisms in patients with ACSs were significantly higher than control group (P < 0.05). Furthermore, the 3 polymorphic sites were strongly of linkage disequilibrium, and minor alleles of 3 SNP sites had higher TG level than wild alleles (P < 0.05), APOC3 -455C and APOA5 c.553T allele carriers also had lower level of HDL-C. CONCLUSIONS: The minor alleles of APOC3 -455T > C, APOA5 -1131T > C, and c.553G > T polymorphisms are closely associated with ACSs.

[Effects of MSCs on the progression of atherosclerosis plaque in ApoE-knock out mice].

AIM: To explore the effects of mesenchymal stem cells in the formation of atherosclerosis plaque in hypercholesterolemic apoliprotein (apo) E -/ - mice. METH-ODS: ApoE -- mice mesenchymal stem cells (MSCs)were isolated and identified. Thirty ApoE -/ - mice were divided into negative control group (Neg, n = 10), positive control group (Pos, n = 10) and MSCs group ( n = 10).MSCs were injected through caudal vein into the body ofPos and MSCs groups. The plaque area of all subjects were compared, the percentage of CD4 CD25' regulatory T cells in different tissues were analyzed by FACS, proliferation response of splenocytes to mesenchymal stem cells and cyto-kines in the supernatant were determined by ELISA. RE-SULTS: Compared with controls, MSCs resulted in a significant decrease of the atherosclerotic plaques size (P <0.05), and a significant increase of CD4 CD25 regulatory T cells in spleen (P<0.05). Specific proliferation response of CD4' CD25' regulatory T cells in splenocytes to MSCswas significantly suppressed. The supernatant levels ofTGF-f3 and IL-10 in MSCs group were increased while IFN-y decreased significantly. CONCLUSION: MSCs play an important role in regulating the inflammatory response and may significantly inhibit the formation of the atherosclerosis plaque in ApoE-'- mice.

[Effects of FOXP3 on the progression of atherosclerosis plaque in ApoE-knock out mice].

AIM: To explore effects of FOXP3 on the progression of atherosclerosis plaque in hypercholesterolemic apoliprotein(apo)E-/- mice. METHODS: At 8 weeks of age, 32 male ApoE-/- mice were randomly divided into four groups of eight. Labeled: negative control group, positive control group, small interfering RNA (siRNA) group, and regulatory T cells transfer (Tregs) group. Lentivirus-mediated (siRNA) identified its function by Western blot was used to knock down FOXP3 and Foxp3(high+);CD4(+); CD25(+); Tregs acquired through magnetic activated cell sorting adoptive transfer assays in high fat diet ApoE-/- mice were done. The resulting atherosclerotic lesions were assessed by determining the number and function of CD4(+);CD25(+); Tregs, FOXP3 transcript levels and investigating the expression of Foxp3 protein in different tissues. Inflammatory cytokines were determined by ELISA. RESULTS: Animals treated with siRNA of FOXP3 showed a significant increase in atherosclerotic lesion formation and a reduction in the number and function of Foxp3(+);CD4(+);CD25(+); Tregs compared with other groups. Transfer of Foxp3(high+);CD4(+);CD25(+); Tregs significantly decreased atherosclerotic plaque formation and increased the number and function of Foxp3(+); CD4(+); CD25(+); Tregs. Foxp3 protein levels and FOXP3 transcript levels were lowest in the siRNA group, and were highest in tissues from the Tregs transfer group. CONCLUSION: FOXP3 plays an important role in regulating the inflammatory response within the atherosclerotic lesion. It can inhibit significant the progression of the atherosclerosis plaque in ApoE-/- mice.

[Analysis of apolipoprotein A5 gene polymorphisms in Hubei Han people].

Polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP) was used to explore the distribution of apolipoprotein A5 gene -1131T>C and 56C>G polymorphisms in 257 healthy Hubei Han people. The following results were calculated: the frequency of -1131TT genotype was 50.9%, far more than that of -1131TC and -1131CC genotypes (32.9% and 16.2%, respectively). The number of T allele carriers was higher than that of C carriers, and their respective frequencies were 0.675 and 0.325. There were 56GG and 56GC genotypes, but only 2 individuals in all subjects carried the G allele, the frequency of which was low than 5%. Furthermore, the frequency of genotypes and alleles in apoa5 -1131T>C and 56C>G polymorphisms was clearly different from other races and areas. We conclude that the apoa5 -1131T>C variation should be considered a single nucleotide polymorphism, but the 56C>G variation should be considered as a mutation instead.