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1.  Molecular mechanisms underlying genotype-dependent responses to dietary restriction 
Aging cell  2013;12(6):10.1111/acel.12130.
Summary
Dietary restriction (DR) increases lifespan and attenuates age-related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here we describe a large-scale effort to define molecular mechanisms that underlie genotype-specific responses to DR. The effect of DR on lifespan was determined for 166 single-gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR) which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype-dependent effects of DR that may be important modulators of DR in higher organisms.
doi:10.1111/acel.12130
PMCID: PMC3838465  PMID: 23837470
aging; replicative lifespan; longevity; yeast; dietary restriction; mitochondria; mitochondrial unfolded protein response
2.  Dietary restriction and mitochondrial function link replicative and chronological aging in Saccharomyces cerevisiae 
Experimental gerontology  2012;48(10):1006-1013.
Chronological aging of budding yeast cells results in a reduction in subsequent replicative life span through unknown mechanisms. Here we show that dietary restriction during chronological aging delays the reduction in subsequent replicative life span up to at least 23 days of chronological age. We further show that among the viable portion of the control population aged 26 days, individual cells with the lowest mitochondrial membrane potential have the longest subsequent replicative lifespan. These observations demonstrate that dietary restriction modulates a common molecular mechanism linking chronological and replicative aging in yeast and indicate a critical role for mitochondrial function in this process.
doi:10.1016/j.exger.2012.12.001
PMCID: PMC3604125  PMID: 23235143
chronological lifespan; replicative lifespan; caloric restriction; calorie restriction; dietary restriction; glucose; mitochondria
3.  Contribution of TIP30 to chemoresistance in laryngeal carcinoma 
Zhu, M | Yin, F | Yang, L | Chen, S | Chen, R | Zhou, X | Jing, W | Fan, X | Jia, R | Wang, H | Zheng, H | Zhao, J | Guo, Y
Cell Death & Disease  2014;5(10):e1468-.
Laryngeal squamous cell carcinoma (LSCC) is one of the most common carcinomas of the head and neck. Despite advances in diagnosis and treatment, the survival of patients with LSCC has not improved in the past two decades. TIP30, a newly identified tumour suppressor, appears to be involved in multiple processes during tumour development. Here, we investigated the involvement of TIP30 in chemoresistance of LSCC in vitro and in vivo. We showed that TIP30 expression decreased significantly in drug-selected cells (DSCs) of laryngeal carcinoma. Suppressing TIP30 enhanced resistance capability to multiple chemotherapy drugs, cell proliferation and self-renewal in Hep2 cells. Additionally, decreased self-renewal capacity and chemotherapeutic resistance were observed in DSCs overexpressing TIP30. Furthermore, TIP30 negatively regulated tumourigenesis and chemoresistance in LSCC cells subcutaneously transplanted into nude mice. Moreover, decreased TIP30 expression contributed to chemoresistance, self-renewal and proliferation of LSCC cells via nuclearlisation of β-catenin, a cell–cell adhesion and stem cell renewal regulator. Consistently, Kaplan–Meier and Cox proportional hazards regression modelling analyses showed that decreased TIP30 expression independently predicted poor survival in patients with LSCC. Taken together, our results reveal that TIP30 has a crucial role in chemoresistance of LSCC through the AKT/glycogen synthase kinase-3β/β-catenin signalling pathway and may be a promising candidate for improving LSCC chemotherapy.
doi:10.1038/cddis.2014.424
PMCID: PMC4237250  PMID: 25321475
4.  Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro  
Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.
doi:10.1590/1414-431X20143935
PMCID: PMC4165290  PMID: 25003544
Sulfamonomethoxine sodium; Gallic acid; Pro-chondrogenic agent; Chondrocyte; Rabbit articular cartilage; Dedifferentiation
5.  Effects of a Chelated Copper as Growth Promoter on Performance and Carcass Traits in Pigs 
Three studies were conducted to investigate whether a chelated Cu can replace CuSO4 as a growth promoter in pigs. In Exp. 1, a total of 240 piglets (Large White×Landrace, 7.36±0.10 kg) were randomly allocated to 1 of 3 treatments with 8 replicates and 10 piglets per pen. Treatments included a NRC control (CuSO4, 6 mg/kg), two Cu supplementations from either CuSO4 or Cu(HMTBa)2 at 170 mg/kg. Pigs fed Cu(HMTBa)2 were 6.0% heavier than pigs fed either the NRC control or 170 mg/kg CuSO4 (p = 0.03) at the end of the experiment. During the 42 days of experimental period, pigs fed Cu(HMTBa)2 gained 9.0% more (p = 0.01), tended to eat more feed (p = 0.09), and had better feed efficiency (p = 0.06) than those fed CuSO4. Compared with the 6 mg/kg CuSO4 NRC control, liver Cu was increased 2.7 times with 170 mg/kg CuSO4 supplementation, and was further increased with Cu(HMTBa)2 (4.5 times, p<0.05). In Exp. 2, a total of 616 crossbred piglets (PIC, 5.01±0.25 kg) were randomly allocated to 1 of 4 treatments with 7 replicates and 22 piglets per pen. Treatments included a NRC control (from CuSO4), and three pharmaceutical levels of Cu (150 mg/kg) supplemented either from CuSO4, tri-basic copper chloride (Cu2[OH]3Cl), or Cu(HMTBa)2. Pigs fed CuSO4 or Cu(HMTBa)2 had better feed efficiency (p = 0.01) and tended to gain more (p = 0.08) compared with those fed the NRC control. Pigs fed Cu2(OH)3Cl were intermediate. Pigs fed Cu(HMTBa)2 had the highest liver Cu, which was significantly higher than those fed (Cu2[OH]3Cl) or the negative control (p = 0.01). In Exp. 3, a total of 1,048 pigs (PIC, 32.36±0.29 kg) were allotted to 6 treatments with 8 replicates per treatment and 20 to 22 pigs per pen. The treatments included a NRC control with 4 mg/kg Cu from CuSO4, a positive control with 160 mg/kg Cu from CuSO4, and incremental levels of Cu(HMTBa)2 at 20, 40, 80, and 160 mg/kg. During the overall experimental period of 100 days, no benefit from 160 mg/kg CuSO4 was observed. Pigs fed Cu(HMTBa)2 had increased ADG (linear and quadratic, p≤0.05) and feed efficiency (linear and quadratic, p≤0.05) up to 80 mg/kg and no further improvement was observed at 160 mg/kg for the whole experimental period. Pigs fed 80 mg/kg Cu(HMTBa)2 weighed 1.8 kg more (p = 0.07) and were 2.3 kg heavier in carcass (p<0.01) compared with pigs fed 160 mg/kg CuSO4. In addition, loin depth was increased with increased Cu(HMTBa)2 supplementation with pigs fed 80 mg/kg Cu(HMTBa)2 had the greatest loin depth (p<0.05). In summary, Cu(HMTBa)2 can be used to replace high CuSO4 as a growth promoter in nursery and grower-finisher pigs.
doi:10.5713/ajas.2013.13416
PMCID: PMC4093573  PMID: 25050038
Chelated Trace Mineral; Cu(HMTBa)2; Copper; Growth Promoter; Swine
6.  Somatostatin Receptor 1, a novel EBV-associated CpG hypermethylated gene, contributes to the pathogenesis of EBV-associated gastric cancer 
British Journal of Cancer  2013;108(12):2557-2564.
Background:
Somatostatin receptor 1 (SSTR1) was preferentially methylated in Epstein-Barr virus (EBV)-positive gastric cancer using promoter methylation array. We aimed to analyse the epigenetic alteration and biological function of SSTR1 in EBV-associated gastric cancer (EBVaGC).
Methods:
Promoter methylation was examined by combined bisulphite restriction analysis (COBRA) and pyrosequencing. The biological functions of SSTR1 were evaluated by loss- and gain-of-function assays.
Results:
Promoter hypermethylation of SSTR1 was detected in EBV-positive gastric cancer cell lines (AGS-EBV) with SSTR1 transcriptional silence, but not in EBV-negative gastric cancer cell lines with SSTR1 expression. Expression level of SSTR1 was restored in AGS-EBV by exposure to demethylating agent. Moreover, methylation level of SSTR1 was significantly higher in EBV-positive primary gastric cancers compared with EBV-negative gastric cancers (P=0.004). Knock-down of SSTR1 in gastric cancer cell lines (AGS and BGC823) increased cell proliferation and colony formation ability, and promoted G1 to S-phase transition, enhanced cell migration and invasive ability. In contrast, ectopic expression of SSTR1 in gastric cancer cell lines (MKN28 and MGC803) significantly suppressed cell growth in culture conditions and reduced tumour size in nude mice. The tumour suppressive effect of SSTR1 was associated with upregulation of cyclin-dependent kinase inhibitors (p16, p15, p27 and p21); downregulation of oncogenes (MYC and MDM2), key cell proliferation and pro-survival regulators (PI3KR1, AKT, BCL-XL and MET); and inhibition of the migration/invasion-related genes (integrins, MMP1 (matrix metallopeptidase 1), PLAUR (plasminogen activator urokinase receptor) and IL8 (interleukin 8)).
Conclusion:
Somatostatin receptor 1 is a novel methylated gene driven by EBV infection in gastric cancer cells and acts as a potential tumour suppressor.
doi:10.1038/bjc.2013.263
PMCID: PMC3694244  PMID: 23722468
Epstein-Barr virus; gastric cancer; somatostatin receptor 1; tumour suppressor gene; epigenetic alteration
7.  High-Precision Distribution of Highly Stable Optical Pulse Trains with 8.8 × 10−19 instability 
Scientific Reports  2014;4:5109.
The high-precision distribution of optical pulse trains via fibre links has had a considerable impact in many fields. In most published work, the accuracy is still fundamentally limited by unavoidable noise sources, such as thermal and shot noise from conventional photodiodes and thermal noise from mixers. Here, we demonstrate a new high-precision timing distribution system that uses a highly precise phase detector to obviously reduce the effect of these limitations. Instead of using photodiodes and microwave mixers, we use several fibre Sagnac-loop-based optical-microwave phase detectors (OM-PDs) to achieve optical-electrical conversion and phase measurements, thereby suppressing the sources of noise and achieving ultra-high accuracy. The results of a distribution experiment using a 10-km fibre link indicate that our system exhibits a residual instability of 2.0 × 10−15 at1 s and8.8 × 10−19 at 40,000 s and an integrated timing jitter as low as 3.8 fs in a bandwidth of 1 Hz to 100 kHz. This low instability and timing jitter make it possible for our system to be used in the distribution of optical-clock signals or in applications that require extremely accurate frequency/time synchronisation.
doi:10.1038/srep05109
PMCID: PMC4037707  PMID: 24870442
8.  End-of-life cell cycle arrest contributes to stochasticity of yeast replicative aging 
FEMS yeast research  2013;13(3):267-276.
There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in lifespan that can be modeled by the Gompertz-Makeham law of mortality. Here we report that within genetically identical haploid and diploid wild type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean lifespan (25.6 vs. 35.6) and larger coefficient of variance with respect to individual lifespan (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten lifespan and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.
doi:10.1111/1567-1364.12030
PMCID: PMC3960949  PMID: 23336757
replicative lifespan; longevity; yeast; senescence; DNA damage; sir2; cell cycle arrest
9.  Upregulation and nuclear translocation of testicular ghrelin protects differentiating spermatogonia from ionizing radiation injury 
Cell Death & Disease  2014;5(5):e1248-.
Proper control of apoptotic signaling is important for maintenance of testicular homeostasis after ionizing radiation (IR). Herein, we challenged the hypothesis that ghrelin, a pleiotropic modulator, is potentially involved in IR-induced germ cell injury. Lower body exposure to 2 Gy of IR induced a notable increase of ghrelin expression in the nuclear of differentiating spermatogonia at defined stages, with an impairment in the Leydig cells (LCs)-expressing ghrelin. Unexpectedly, inhibition of the ghrelin pathway by intraperitoneal injection of a specific GHS-R1α antagonist enhanced spermatogonia elimination by apoptosis during the early recovery following IR, and thereafter resulted in impaired male fertility, suggesting that the anti-apoptotic effects of evoked ghrelin, although transient along testicular IR injury, have a profound influence on the post-injury recovery. In addition, inhibition of ghrelin signaling resulted in a significant increase in the intratesticular testosterone (T) level at the end of 21 days after IR, which should stimulate the spermatogenic recovery from surviving spermatogonia to a certain extent during the late stage. We further demonstrated that the upregulation and nuclear trafficking of ghrelin, elaborately regulated by IR-elicited antioxidant system in spermatogonia, may act through a p53-dependent mechanism. The elicitation of ghrelin expression by IR stress, the regulation of ghrelin expression by IR-induced oxidative stress and the interaction between p53 and ghrelin signaling during IR injury were confirmed in cultured spermatogonia. Hence, our results represent the first evidence in support of a radioprotective role of ghrelin in the differentiating spermatogonia. The acutely, delicate regulation of local-produced ghrelin appears to be a fine-tune mechanism modulating the balance between testicular homeostasis and early IR injury.
doi:10.1038/cddis.2014.223
PMCID: PMC4047875  PMID: 24853426
ionizing radiation; ghrelin; spermatogonia; oxidative stress; p53
10.  Anticancer compound Oplopantriol A kills cancer cells through inducing ER stress and BH3 proteins Bim and Noxa 
Cell Death & Disease  2014;5(4):e1190-.
Oplopantriol-A (OPT) is a natural polyyne from Oplopanax horridus. We show here that OPT preferentially kills cancer cells and inhibits tumor growth. We demonstrate that OPT-induced cancer cell death is mediated by excessive endoplasmic reticulum (ER) stress. Decreasing the level of ER stress either by inactivating components of the unfolded protein response (UPR) pathway or by expression of ER chaperone protein glucose-regulated protein 78 (GRP78) decreases OPT-induced cell death. We show that OPT induces the accumulation of ubiquitinated proteins and the stabilization of unstable proteins, suggesting that OPT functions, at least in part, through interfering with the ubiquitin/proteasome pathway. In support of this, inhibition of protein synthesis significantly decreased the accumulation of ubiquitinated proteins, which is correlated with significantly decreased OPT-induced ER stress and cell death. Finally, we show that OPT treatment significantly induced the expression of BH3-only proteins, Noxa and Bim. Knockdown of both Noxa and Bim significantly blocked OPT-induced cell death. Taken together, our results suggest that OPT is a potential new anticancer agent that induces cancer cell death through inducing ER stress and BH3 proteins Noxa and Bim.
doi:10.1038/cddis.2014.169
PMCID: PMC4001317  PMID: 24763047
ER stress; Oplopantriol-A; unfolded protein response; noxa; bim
11.  Enhanced myeloid differentiation factor 88 promotes tumor metastasis via induction of epithelial–mesenchymal transition in human hepatocellular carcinoma 
Cell Death & Disease  2014;5(3):e1103-.
Metastasis is the leading cause of death in patients with hepatocellular carcinoma (HCC) after curative resection. Therefore, it is critical to understand the mechanisms underlying tumor metastasis in HCC. We have previously shown that elevated expression of myeloid differentiation factor 88 (MyD88) may promote tumor growth and metastasis in HCC. In this study, we reported that enhanced expression of MyD88 promoted epithelial–mesenchymal transition (EMT) properties and tumor-initiating capabilities in HCC cells. MyD88 was found to be able to interact with p85, a regulatory subunit of phosphoinositide 3-kinase (PI3-K), independent of TLR/IL-1R-mediated response and caused PI3-K/v-akt murine thymoma viral oncogene homolog (Akt) activation, which resulted in subsequent phosphorylation of glycogen synthase kinase-3β and stabilization of Snail, a critical EMT mediator. Consistently, we observed a significant correlation between MyD88 expression and p-Akt levels in a cohort of HCC patients, and found that the combination of these two parameters have better prognostic value for HCC patients. Taken together, these results suggest that elevated MyD88 may facilitate HCC metastasis by promoting EMT properties and tumor-initiating capabilities via PI3–K/Akt pathway.
doi:10.1038/cddis.2014.71
PMCID: PMC3973199  PMID: 24603331
tumor metastasis; myeloid differentiation factor 88; epithelial–mesenchymal transition
12.  Stress profiling of longevity mutants identifies Afg3 as a mitochondrial determinant of cytoplasmic mRNA translation and aging 
Aging cell  2012;12(1):156-166.
Summary
While environmental stress likely plays a significant role in promoting aging, the relationship remains poorly understood. In order to characterize this interaction in a more comprehensive manner, we examined the stress response profiles for 46 long-lived yeast mutant strains across four different stress conditions (oxidative, ER, DNA damage, and thermal), grouping genes based on their associated stress response profiles. Unexpectedly, cells lacking the mitochondrial AAA protease gene AFG3 clustered strongly with long-lived strains lacking cytosolic ribosomal proteins of the large subunit. Similar to these ribosomal protein mutants, afg3Δ cells show reduced cytoplasmic mRNA translation, enhanced resistance to tunicamycin that is independent of the ER unfolded protein response, and Sir2-independent but Gcn4-dependent life span extension. These data demonstrate an unexpected link between a mitochondrial protease, cytoplasmic mRNA translation, and aging.
doi:10.1111/acel.12032
PMCID: PMC3687586  PMID: 23167605
aging; stress response; translation; mitochondria; ER stress; replicative lifespan; longevity; yeast; epistasis; phenotype mapping
13.  Predicting cooperative drug effects through the quantitative cellular profiling of response to individual drugs 
Quantitative prediction of cellular responses to drugs and drug combinations is a challenging and valuable topic in pharmaceutical research. In the past decade, microarray technology has become a routine tool for monitoring genome-wide expression changes and has been widely adopted for exploring drug response in the pharmaceutical field. However, how to predict the synergistic effect of drug combinations using microarray data is a challenging task. In this article, we report a simple prediction framework based on the genome-wide and quantitative profiling of cellular responses to individual drugs. By exploring the differential expression profiles, our correlation-based strategy can reveal the synergistic effects of drug combinations. The comparison with gold-standard experimental results demonstrates the strengths and weaknesses in relation to prediction based only on cellular response to individual drugs. Specifically, the prediction strategy may work for a drug combination whose individual drugs show related transcriptomic mechanisms but not for others.
doi:10.1038/psp.2013.79
PMCID: PMC3944117  PMID: 24573337
14.  Highly Stable Wideband Microwave Extraction by Synchronizing Widely Tunable Optoelectronic Oscillator with Optical Frequency Comb 
Scientific Reports  2013;3:3509.
Optical frequency combs (OFCs), based on mode-locked lasers (MLLs), have attracted considerable attention in many fields over recent years. Among the applications of OFCs, one of the most challenging works is the extraction of a highly stable microwave with low phase noise. Many synchronisation schemes have been exploited to synchronise an electronic oscillator with the pulse train from a MLL, helping to extract an ultra-stable microwave. Here, we demonstrate novel wideband microwave extraction from a stable OFC by synchronising a single widely tunable optoelectronic oscillator (OEO) with an OFC at different harmonic frequencies, using an optical phase detection technique. The tunable range of the proposed microwave extraction extends from 2 GHz to 4 GHz, and in a long-term synchronisation experiment over 12 hours, the proposed synchronisation scheme provided a rms timing drift of 18 fs and frequency instabilities at 1.2 × 10−15/1 s and 2.2 × 10−18/10000 s.
doi:10.1038/srep03509
PMCID: PMC3863813  PMID: 24336459
15.  All-trans retinoic acid-induced hypothalamus–pituitary–adrenal hyperactivity involves glucocorticoid receptor dysregulation 
Translational Psychiatry  2013;3(12):e336-.
Clinical reports have highlighted a role for retinoids in the etiology of mood disorders. Although we had shown that recruitment of the nuclear receptor retinoic acid receptor-α (RAR-α) to corticotropin-releasing hormone (CRH) promoter is implicated in activation of the hypothalamus–pituitary–adrenal (HPA) axis, further insight into how retinoids modulate HPA axis activity is lacking. Here we show that all-trans retinoic acid (RA)-induced HPA activation involves impairments in glucocorticoid receptor (GR) negative feedback. RA was applied to rats chronically through intracerebroventricular injection. A 19-day RA exposure induced potent HPA axis activation and typical depression-like behavior. Dexamethasone failed to suppress basal corticosterone (CORT) secretion, which is indicative of a disturbed GR negative feedback. In the hypothalamic paraventricular nucleus, increased CRH+ and c-fos+ cells were found while a negative R−2+/ER+ correlation was present between the number of RAR-α+ and GR+ cells. This was paralleled by increased RAR-α and decreased GR protein expression in the hypothalamus. Additional in vitro studies confirmed that RA abolished GR-mediated glucocorticoid-induced suppression of CRH expression, indicating a negative cross-talk between RAR-α and GR signaling pathways. Finally, the above changes could be rapidly normalized by treatment with GR antagonist mifepristone. We conclude that in addition to the ‘classic' RAR-α-mediated transcriptional control of CRH expression, disturbances in GR negative feedback constitute a novel pathway that underlies RA-induced HPA axis hyperactivity. The rapid normalization by mifepristone may be of potential clinical interest in this respect.
doi:10.1038/tp.2013.98
PMCID: PMC4030330  PMID: 24346134
all-trans retinoic acid; corticotropin-releasing hormone; depression; glucocorticoid receptor; hypothalamus–pituitary–adrenal axis; mifepristone (RU38486)
16.  Targeting annexin A7 by a small molecule suppressed the activity of phosphatidylcholine-specific phospholipase C in vascular endothelial cells and inhibited atherosclerosis in apolipoprotein E−/− mice 
Li, H | Huang, S | Wang, S | Zhao, J | Su, L | Zhao, B | Zhang, Y | Zhang, S | Miao, J
Cell Death & Disease  2013;4(9):e806-.
Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key factor in apoptosis and autophagy of vascular endothelial cells (VECs), and involved in atherosclerosis in apolipoprotein E−/− (apoE−/−) mice. But the endogenous regulators of PC-PLC are not known. We recently found a small chemical molecule (6-amino-2, 3-dihydro-3-hydroxymethyl-1, 4-benzoxazine, ABO) that could inhibit oxidized low-density lipoprotein (oxLDL)-induced apoptosis and promote autophagy in VECs, and further identified ABO as an inhibitor of annexin A7 (ANXA7) GTPase. Based on these findings, we hypothesize that ANXA7 is an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO may inhibit atherosclerosis in apoE−/− mice. In this study, we tested our hypothesis. The results showed that ABO suppressed oxLDL-induced increase of PC-PLC level and activity and promoted the co-localization of ANXA7 and PC-PLC in VECs. The experiments of ANXA7 knockdown and overexpression demonstrated that the action of ABO was ANXA7-dependent in cultured VECs. To investigate the relation of ANXA7 with PC-PLC in atherosclerosis, apoE−/− mice fed with a western diet were treated with 50 or 100 mg/kg/day ABO. The results showed that ABO decreased PC-PLC levels in the mouse aortic endothelium and PC-PLC activity in serum, and enhanced the protein levels of ANXA7 in the mouse aortic endothelium. Furthermore, both dosages of ABO significantly enhanced autophagy and reduced apoptosis in the mouse aortic endothelium. As a result, ABO significantly reduced atherosclerotic plaque area and effectively preserved a stable plaques phenotype, including reduced lipid deposition and pro-inflammatory macrophages, increased anti-inflammatory macrophages, collagen content and smooth muscle cells, and less cell death in the plaques. In conclusion, ANXA7 was an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO inhibited atherosclerosis in apoE−/− mice.
doi:10.1038/cddis.2013.317
PMCID: PMC3789175  PMID: 24052074
apoptosis; autophagy; PC-PLC; ANXA7; ABO; atherosclerosis
17.  pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast 
Cell Cycle  2012;11(16):3087-3096.
Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.
doi:10.4161/cc.21465
PMCID: PMC3442919  PMID: 22871733
chronological lifespan; longevity; replication stress; replicative lifespan; yeast
18.  Application of Micro-Computed Tomography with Iodine Staining to Cardiac Imaging, Segmentation and Computational Model Development 
Micro-computed tomography (micro-CT) has been widely used to generate high-resolution 3D tissue images from small animals non-destructively, especially for mineralized skeletal tissues. However, its application to the analysis of soft cardiovascular tissues has been limited by poor inter-tissue contrast. Recent ex vivo studies have shown that contrast between muscular and connective tissue in micro-CT images can be enhanced by staining with iodine. In the present study, we apply this novel technique for imaging of cardiovascular structures in canine hearts. We optimize the method to obtain high resolution X-ray micro-CT images of the canine atria and its distinctive regions - including the Bachmann’s bundle, atrioventricular node, pulmonary arteries and veins - with clear inter-tissue contrast. The imaging results are used to reconstruct and segment the detailed 3D geometry of the atria. Structure tensor analysis shows that the arrangement of atrial fibres can also be characterised using the enhanced micro-CT images, as iodine preferentially accumulates within the muscular fibres rather than in connective tissues. This novel technique can be particularly useful in non-destructive imaging of 3D cardiac architectures from large animals and humans, due to the combination of relatively high speed (~1 hour/scan of a large canine heart) and high voxel resolution (36 μm) provided. In summary, contrast micro-CT facilitates fast and non-destructive imaging and segmenting of detailed 3D cardiovascular geometries, as well as measuring fibre orientation, which are crucial in constructing biophysically detailed computational cardiac models.
doi:10.1109/TMI.2012.2209183
PMCID: PMC3493467  PMID: 22829390
19.  Bright betatron X-ray radiation from a laser-driven-clustering gas target 
Scientific Reports  2013;3:1912.
Hard X-ray sources from femtosecond (fs) laser-produced plasmas, including the betatron X-rays from laser wakefield-accelerated electrons, have compact sizes, fs pulse duration and fs pump-probe capability, making it promising for wide use in material and biological sciences. Currently the main problem with such betatron X-ray sources is the limited average flux even with ultra-intense laser pulses. Here, we report ultra-bright betatron X-rays can be generated using a clustering gas jet target irradiated with a small size laser, where a ten-fold enhancement of the X-ray yield is achieved compared to the results obtained using a gas target. We suggest the increased X-ray photon is due to the existence of clusters in the gas, which results in increased total electron charge trapped for acceleration and larger wiggling amplitudes during the acceleration. This observation opens a route to produce high betatron average flux using small but high repetition rate laser facilities for applications.
doi:10.1038/srep01912
PMCID: PMC3665959  PMID: 23715033
20.  Sir2 deletion prevents lifespan extension in 32 long-lived mutants 
Aging cell  2011;10(6):1089-1091.
SUMMARY
Activation of Sir2-orthologs is proposed to increase lifespan downstream of dietary restriction (DR). Here we describe an examination of the effect of 32 different lifespan-extending mutations and four methods of dietary restriction on replicative lifespan (RLS) in the short-lived sir2Δ yeast strain. In every case, deletion of SIR2 prevented RLS extension; however, RLS extension was restored when both SIR2 and FOB1 were deleted in several cases, demonstrating that SIR2 is not directly required for RLS extension. These findings indicate that suppression of the sir2Δ lifespan defect is a rare phenotype among longevity interventions and suggest that sir2Δ cells senesce rapidly by a mechanism distinct from that of wild-type cells. They also demonstrate that failure to observe life span extension in a short-lived background, such as cells or animals lacking sirtuins, should be interpreted with caution.
doi:10.1111/j.1474-9726.2011.00742.x
PMCID: PMC3215821  PMID: 21902802
aging; replicative lifespan; longevity; yeast; epistasis
21.  Ghrelin Contributes to Derangements of Glucose Metabolism Induced by Rapamycin in Mice 
Xu, G | Wang, Z | Li, Y | Li, Z | Tang, H | Zhao, J | Xiang, X | Ding, L | Ma, L | Yuan, F | Fei, J | Wang, W | Wang, N | Guan, Y | Tang, C | Mulholland, M | Zhang, W
Diabetologia  2012;55(6):1813-1823.
Aims/hypothesis
Rapamycin impaired glucose tolerance and insulin sensitivity. Our previous study demonstrated that rapamycin significantly increases the expression of gastric ghrelin, which is critical in the regulation of glucose metabolism. Here, we investigated whether ghrelin contributes to derangements of glucose metabolism induced by rapamycin.
Methods
The effects of rapamycin on glucose metabolism were examined in mice receiving ghrelin receptor antagonist or with ghrelin receptor gene deletion. Changes in Glut4, JNK, and pS6 were investigated by immnuofluorescent staining or Western. Related hormones were detected by radioimmuno-assay kits.
Results
Rapamycin impaired glucose metabolism and insulin sensitivity not only in normal C57BL/6J mice but also in both obese mice induced by high fat diet and db/db mice. This was accompanied by elevation of plasma acylated ghrelin. Rapamycin significantly increased the levels of plasma acylated ghrelin in normal C57BL/6J mice, high fat diet induced obese mice, and db/db mice. Elevation in plasma acylated ghrelin and derangements of glucose metabolism upon administration of rapamycin was significantly correlated. The deterioration in glucose homeostasis induced by rapamycin was blocked by D-Lys3-GHRP-6, a ghrelin receptor antagonist, or by deletion of ghrelin receptor gene. Ghrelin receptor antagonism and ghrelin receptor gene deletion blocked the up-regulation of JNK activity, and GLUT4 expression and translocation in the gastrocnemius muscle induced by rapamycin.
Conclusions
The current study demonstrates that ghrelin contributes to derangements of glucose metabolism induced by rapamycin via altering the expression and translocation of GLUT4 in muscles.
doi:10.1007/s00125-012-2509-1
PMCID: PMC3496261  PMID: 22391948
Ghrelin; glucose metabolism; rapamycin
22.  Enhanced Osseointegration of Titanium Implant Through the Local Delivery of Transcription Factor SATB2 
Biomaterials  2011;32(33):8676-8683.
Titanium implants are widely used in dentistry and orthopaedic surgery. Nevertheless, bone regeneration around the implant is a relatively slow process, after placement. This study assessed whether SATB2 can enhance osseointegration of a titanium implant. To determine the effect of SATB2 in implant integration, two different viruses encoding SATB2 (PBABE-Satb2 virus or RCAS-Satb2 virus) were locally administered to the bone defect prior to titanium implant placement in our established transgenic TVA mice. Seven and 21 days post implantation, the femurs were isolated for quantitative real-time RT-PCR, H&E staining, immunohistochemical (IHC) staining, and microcomputed tomography (microCT) analysis. Quantitative real-time RT-PCR results demonstrated that the in vivo overexpression of SATB2 enhanced expression levels of potent osteogenic transcription factors and bone matrix proteins. We also found that 21 days after implantation, there were no significant differences in the expression levels of SATB2, Osx, Runx2, COLI, OC, and BSP between the RCAS-Satb2 group and the RCAS group. Histological analysis showed that SATB2 overexpression significantly enhanced new bone formation and bone-to-implant contact after implantation. IHC staining analysis revealed that forced expression of SATB2 increased the number of BSP-positive cells surrounding the implant. MicroCT analysis demonstrated that in vivo overexpression of SATB2 significantly increased the density of the newly formed bone surrounding the implant. These results conclude that in vivo overexpression of SATB2 significantly accelerates osseointegration of titanium implants and SATB2 can serve as a potent molecule in promoting tissue regeneration.
doi:10.1016/j.biomaterials.2011.07.072
PMCID: PMC3218567  PMID: 21862122
implant; SATB2; osseointegration; TVA mice
24.  Cross-bridge Kinetics in Myofibrils Containing Familial Hypertrophic Cardiomyopathy R58Q Mutation in the Regulatory Light Chain of Myosin 
Journal of theoretical biology  2011;284(1):71-81.
Familial hypertrophic cardiomyopathy (FHC) is a heritable form of cardiac hypertrophy caused by single-point mutations in genes encoding sarcomeric proteins including ventricular myosin regulatory light chain (RLC). FHC often leads to malignant outcomes and sudden cardiac death. The FHC mutations are believed to alter the kinetics of the interaction between actin and myosin resulting in inefficient energy utilization and compromised function of the heart. We studied the effect of the FHC-linked R58Q-RLC mutation on the kinetics of transgenic (Tg)-R58Q cardiac myofibrils. Kinetics was determined from the rate of change of orientation of actin monomers during muscle contraction. Actin monomers change orientation because myosin cross-bridges deliver to it periodic force impulses. An individual impulse (but not time average of impulses) carries the information about the kinetics of actomyosin interaction. To observe individual impulses it was necessary to scale down the experiments to the level of a few molecules. A small population (~4 molecules) was selected by using (deliberately) inefficient fluorescence labeling and observing fluorescent molecules by a confocal microscope. We show that the kinetic rates are significantly smaller in the contracting cardiac myofibrils from Tg-R58Q mice then in control Tg-wild type (WT). We also demonstrate a lower force per cross-section of muscle fiber in Tg-R58Q versus Tg-WT mice. We conclude that the R58Q mutation-induced decrease in cross-bridge kinetics underlines the mechanism by which Tg-R58Q fibers develop low force and thus compromise the ability of the mutated heart to efficiently pump blood.
doi:10.1016/j.jtbi.2011.06.014
PMCID: PMC3152379  PMID: 21723297
25.  The antitumor natural compound falcarindiol promotes cancer cell death by inducing endoplasmic reticulum stress 
Cell Death & Disease  2012;3(8):e376-.
Falcarindiol (FAD) is a natural polyyne with various beneficial biological activities. We show here that FAD preferentially kills colon cancer cells but not normal colon epithelial cells. Furthermore, FAD inhibits tumor growth in a xenograft tumor model and exhibits strong synergistic killing of cancer cells with 5-fluorouracil, an approved cancer chemotherapeutic drug. We demonstrate that FAD-induced cell death is mediated by induction of endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Decreasing the level of ER stress, either by overexpressing the ER chaperone protein glucose-regulated protein 78 (GRP78) or by knockout of components of the UPR pathway, reduces FAD-induced apoptosis. In contrast, increasing the level of ER stress by knocking down GRP78 potentiates FAD-induced apoptosis. Finally, FAD-induced ER stress and apoptosis is correlated with the accumulation of ubiquitinated proteins, suggesting that FAD functions at least in part by interfering with proteasome function, leading to the accumulation of unfolded protein and induction of ER stress. Consistent with this, inhibition of protein synthesis by cycloheximide significantly decreases the accumulation of ubiquitinated proteins and blocks FAD-induced ER stress and cell death. Taken together, our study shows that FAD is a potential new anticancer agent that exerts its activity through inducing ER stress and apoptosis.
doi:10.1038/cddis.2012.122
PMCID: PMC3434669  PMID: 22914324
ER stress; falcarindiol; apoptosis; unfolded protein response; proteasome

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