Intrauterine and postnatal overnutrition program hyperphagia, adiposity and glucose intolerance in offspring. Single-nucleotide polymorphisms (SNPs) of the fat mass and obesity associated (FTO) gene have been linked to increased risk of obesity. FTO is highly expressed in hypothalamic regions critical for energy balance and hyperphagic phenotypes were linked with FTO SNPs. As nutrition during fetal development can influence the expression of genes involved in metabolic function, we investigated the impact of maternal obesity on FTO.Female Sprague Dawley rats were exposed to chow or high fat diet (HFD) for 5 weeks before mating, throughout gestation and lactation. On postnatal day 1 (PND1), some litters were adjusted to 3 pups (vs. 12 control) to induce postnatal overnutrition. At PND20, rats were weaned onto chow or HFD for 15 weeks. FTO mRNA expression in the hypothalamus and liver, as well as hepatic markers of lipid metabolism were measured.At weaning, hypothalamic FTO mRNA expression was increased significantly in offspring of obese mothers and FTO was correlated with both visceral and epididymal fat mass (P<0.05); body weight approached significance (P = 0.07). Hepatic FTO and Fatty Acid Synthase mRNA expression were decreased by maternal obesity. At 18 weeks, FTO mRNA expression did not differ between groups; however body weight was significantly correlated with hypothalamic FTO. Postnatal HFD feeding significantly reduced hepatic Carnitine Palmitoyltransferase-1a but did not affect the expression of other hepatic markers investigated. FTO was not affected by chronic HFD feeding.Maternal obesity significantly impacted FTO expression in both hypothalamus and liver at weaning. Early overexpression of hypothalamic FTO correlated with increased adiposity and later food intake of siblings exposed to HFD suggesting upregulation of FTO may contribute to subsequent hyperphagia, in line with some human data. No effect of maternal obesity was observed on FTO in adulthood. Obesity rates are rising dramatically around the globe and its consequences represent a major public health concern The intrauterine environment plays a key role and suboptimal maternal nutrition during gestation is implicated in the development of adult metabolic diseases. It is now accepted that nutritional changes during fetal development can predispose individuals to obesity and metabolic disease in adult life, resetting the expression of genes involved in energy homeostasis and consequently altering metabolic function Recent interest has turned to the gene-environment interactions that underlie obesity and several genes predisposing to obesity have been characterized The hypothalamus is the main brain region regulating food intake, integrating peripheral signals such as leptin and insulin. In rodents, brain FTO levels are influenced by fasting or starvation In rats, we previously showed that intrauterine and early postnatal overnutrition altered the expression of hypothalamic appetite stimulator neuropeptide Y (NPY) and suppressor pro-opiomelanocortin (POMC) in offspring at weaning It is not known whether FTO is regulated by different levels of maternal nutrition, nor whether food intake is influenced by expression levels of FTO from an early age. To this end we examined the impact of maternal obesity and early life overnutrition induced by litter size reduction on FTO expression in the hypothalamus. As FTO has been linked to energy intake, we examined the association between hypothalamic FTO mRNA expression at weaning, and subsequent energy intake in groups of siblings weaned onto either low fat chow or HFD. As FTO expression in the liver has been linked to blood glucose levels All animal work was approved by the Animal Care and Ethics Committee of the University of New South Wales, #06/60B.Virgin female Sprague Dawley rats aged 9 weeks  were housed at 20±2°C and maintained on a 12∶12 h light/dark cycle. Rats were assigned to two groups of equal average starting body weight and were fed either a control or a high fat diet (HFD) during gestation and lactation. Mothers in the control (n = 11) group were fed standard laboratory chow , whereas those in the HFD (n = 9) group were fed a cafeteria diet consisting of a range of western foods  supplemented with chow with added condensed milk and saturated animal fat  as described previously On day 1 after birth, some litters were adjusted to 3 pups , versus normal litters maintained at 12 pups, yielding 4 groups where first letter corresponds to the diet during gestation and the second letter to the litter size adjustment; (CN) chow-fed dam with normal-size litter; (CS) chow-fed dam with small-size litter; (HN) HFD-fed dam with normal-size litter; (HS) HFD-fed mum with small-size litter. At 20 days, half the male pups from each litter were weaned onto chow, while the other half were given HFD .Two time points were examined. At 20 days pups were deeply anesthetized  prior to decapitation. At this age 12, 13, 12 and 12 pups were sampled from CN, CS, HN and HS groups respectively. At 18 weeks group sizes ranged from 10 to 12 rats. Animals were fasted overnight (14 h), then anesthetized , naso-anal (N-A) length measured, blood was collected by cardiac puncture and rats were decapitated. Brains were rapidly removed and whole hypothalamus was dissected at 20 days while at 18 weeks the ventral hypothalamus containing ARC was dissected At 18 weeks a section of liver from each rat (n = 10–12 per group) was placed in 4% formaldehyde overnight, transferred to 70% ethanol, embedded in paraffin wax and stained for Hematoxylin and eosin. Fatty change in the liver was graded by an observer blinded to the treatment. The amount and size of lipid filled vacuoles present was graded with 0 if no evidence of lipid vacuoles; 1 few small lipid vacuoles present within hepatocytes; 2 increased number and larger lipid vacuoles within hepatocytes. The average of the grades within each treatment group was calculated.ΔΔCT method RNA was extracted using Tri-reagent  and stored at −80C (n = 6–8 per group). Spectrophotometric quantification using Biospec-nano spectrophotometer  determined RNA concentration and purity. One µg of RNA was reverse transcribed simultaneously to cDNA using the Omniscript Reverse Transcription kit  following manufacturer's instructions, and stored at −20°C. Preoptimized TaqMan probe/primers labeled with FAM  were used for quantitative real-time PCR  on the following target genes: Fat mass and obesity associated (FTO), Rn01538187_m1; Sterol regulatory element binding protein 1c (SREBP-1C), Rn01446563_m1; Fatty acid synthase (FAS), Rn00569117_m1; Carnitine palmitoyltransferase 1a (CPT1a) Rn00580702_m1. Probes for the gene of reference (Beta Actin) were labeled with VIC. Target gene expression was quantified by single-plexing reaction and normalized by the gene of reference (beta actin). A sample from the control group was used as a calibrator. Analysis was performed using the Statistical analysis were performed using the SPSS 17.0 Windows software  and results were expressed as mean ± SEM. Anthropomorphic, hormone and gene expression data were analyzed by one or two-way ANOVA depending on number of treatment groups, followed by a post hoc analysis using the least significant difference test (LSD) as appropriate. Liver score was analyzed by Wilcoxon matched-pairs signed rank test. Correlations between parameters measured were assessed by Pearson tests. Results were considered statistically significant when P<0.05.As previously reported HFD mothers weighed 23% more than control at the time of mating and their offspring were heavier by seven days of age At 18 weeks, scoring of liver sections revealed that post-weaning HFD increased lipid accumulation within hepatocytes in all groups independently of maternal diet and litter size adjustment P<0.05, .Hypothalamic FTO mRNA expression was increased in offspring of obese mothers , but not in those fed chow. We also observed a significant correlation between FTO mRNA expression at weaning and adiposity in HFD fed siblings at 18 weeks . In adult rats fed HFD, Area Under the Curve (AUC) of glucose during the Glucose Tolerance Test At 18 weeks, no effect of maternal diet, litter size adjustment as well as postweaning diet on FTO mRNA expression was observed . HoweverWe measured key hepatic genes involved in lipid metabolism: SREBP-1c, FAS and CPT-1a . Liver FAt 18 weeks, maternal diet and litter size adjustment as well as postnatal overnutrition did not significantly affect hepatic FTO mRNA expression . FTO mRNIn this study we examined the impact of maternal obesity and early life overnutrition on FTO expression in the hypothalamus and liver_ENREF_18. The novel finding of this study is that maternal obesity impacts FTO expression in young animals, with reduced impact in adulthood. Moreover, the cohort who had increased hypothalamic FTO expression at weaning consumed more energy in adulthood, but notably, this occurred only in offspring consuming palatable HFD. Modest postweaning overfeeding induced by litter size reduction had no impact on FTO, and there was no effect of HFD consumption on FTO expression.In animals, FTO is expressed in feeding related areas and overexpression leads to increased food intake Early in life hypothalamic FTO expression was correlated with fat mass, but in adulthood, the relationship between FTO and adiposity appeared to be less distinct. At 18 weeks our data did not show a significant correlation of FTO with total fat mass, in line with some human studies where the effects of FTO on body composition weakened with age Hypothalamic FTO mRNA expression at weaning was significantly correlated with AUC of glucose of their adult siblings consuming HFD, but it is difficult to draw conclusions regarding causality. Indeed increased body weight in siblings of rats from obese mothers fed HFD may explain the reduction in glucose tolerance compared to those eating standard chow FTO is also expressed in metabolic tissues At 18 weeks, no marked changes in hepatic FTO were observed in response to any of the interventions. Few reports of physiological alterations in hepatic FTO mRNA expression exist, with one study showing fasting increased the expression of hepatic FTO in mice In summary, our data suggest that maternal obesity may have an impact in programming the metabolic state of subsequent generations through modulation of liver and hypothalamic FTO early in life. Our findings link the deleterious impact of a suboptimal intrauterine environment induced by pre-existing maternal obesity with early increased hypothalamic FTO and subsequent development of a hyperphagic phenotype when challenged with a palatable HFD. Thus, FTO may contribute to the programming of obesity through both central and peripheral mechanisms. Additional studies are required to fully elucidate the role of FTO in liver.


Oxidative stress due to excessive production of reactive oxygen species (ROS) and subsequent lipid peroxidation plays a critical role in renal ischemia/reperfusion (IR) injury. The purpose of current study is to demonstrate the effect of antecedent ethanol exposure on IR-induced renal injury by modulation of oxidative stress.Bilateral renal warm IR was induced in male C57BL/6 mice after ethanol or saline administration. Blood ethanol concentration, kidney function, histological damage, inflammatory infiltration, cytokine production, oxidative stress, antioxidant capacity and Aldehyde dehydrogenase (ALDH) enzymatic activity were assessed to evaluate the impact of antecedent ethanol exposure on IR-induced renal injury.After bilateral kidney ischemia, mice preconditioned with physiological levels of ethanol displayed significantly preserved renal function along with less histological tubular damage as manifested by the reduced inflammatory infiltration and cytokine production. Mechanistic studies revealed that precondition of mice with physiological levels of ethanol 3 h before IR induction enhanced antioxidant capacity characterized by significantly higher superoxidase dismutase (SOD) activities. Our studies further demonstrated that ethanol pretreatment specifically increased ALDH2 activity, which then suppressed lipid peroxidation by promoting the detoxification of Malondialdehyde (MDA) and 4-hydroxynonenal (HNE).Our results provide first line of evidence indicating that antecedent ethanol exposure can provide protection for kidneys against IR-induced injury by enhancing antioxidant capacity and preventing lipid peroxidation. Therefore, ethanol precondition and ectopic ALDH2 activation could be potential therapeutic approaches to prevent renal IR injury relevant to various clinical conditions. Ischemia/reperfusion (IR), a common pathological process, occurs under various clinical conditions such as trauma, hypovolemic shock, sepsis, and most importantly renal transplantation. Surgical procedures requiring cross clamping of the aorta and renal vessels are associated with a renal failure rate up to 30% The pathophysiology of renal IR injury includes both direct cellular damage caused by ischemic insult, and delayed dysfunction/damage due to the activation of inflammatory pathways The deleterious effects of lipid peroxidation might be prevented by enhancing the scavenging systems. Indeed, studies in cardiac ischemia suggested that pretreatment with physiological levels of ethanol can provide cardioprotection, which is consistently correlated with the phosphorylation status of Aldehyde Dehydrogenase 2 (ALDH2), a key metabolic enzyme in the oxidation and detoxification of reactive aldehydes in a range of organs and cell types Male C57BL/6 mice  were obtained from Joint Ventures Sipper BK Experimental Animal . All animal experiments were performed in accordance with the guidelines of National Institute of Health for the Care and Use of Laboratory Animals, and approved by the Scientific Investigation Board of Second Military Medical University, Shanghai, China.i.p., 50 mg/kg). After a midline laparotomy, renal pedicles on both sides were closed for 30 min with nontraumatic microaneurysm clamps . The incision was temporarily closed during ischemia. The abdomen was closed after visual verification of reperfusion upon the removal of microaneurysm clamps. Mice with sham surgery  were used as controls. Body temperature was maintained with an adjustable heating pad at 37°C. All mice were i.p. injected with 500 µl 0.9% NaCl upon completion of the surgery, and were sacrificed 24 h or day 7 after reperfusion. At the time of sacrifice, blood was collected through inferior vena cava into a heparinized syringe for plasma biochemistry within 24 h. Kidneys were fixed in 10% neutral-buffered formalin for paraffin embedding or snap frozen for later use. Some mice were housed individually in metabolism cages in the first 24 h after reperfusion for collection of urine samples. For ethanol pretreatment, mice were injected different dose of ethanol  before the induction of kidney IR. The control group received the largest volume of saline as that in the 2 g/kg body weight dose group.Mice were anesthetized with sodium pentobarbital  at 37°C for 30 min and washed through a sieve (mesh diameter 75 µm) in DMEM/F12 medium (Invitrogen) to obtain single cell suspensions. After centrifugation at 300 g for 5 min, the pellet was resuspended in DMEM/F12 medium supplemented with 1% penicillin/streptomycin and 10% FCS (Invitrogen), and incubated at 37°C for 3 h in a Petri dish to allow contaminated glomerular cells to adhere. The non-adherent tubular cells were next collected and cultured in 6 - well plates in DMEM/F12 medium supplemented with 10% FCS until epithelial colonies were established. TECs were identified by cobblestone-shaped morphology and expression of cytokeratin 18 (CK18) using an anti-CK18 antibody (Santa Cruz) as described by Wu et al. Primary mouse renal tubular epithelial cells (TEC) were cultured following a method described by Wuthrich et al. IR was simulated by immersing cell monolayer in mineral oil as previously described Cr), urea, sodium (PNa), urine creatinine (UCr) and sodium (UNa) were assessed using a standard autoanalyzer at the Central Laboratory, Changzheng Hospital, Shanghai. Urinary osmolality (UOsm) was measured using an automatic freezing point osmometer . Fractional excretion of sodium  was calculated from the standard formula FENa = PCr×UNa×100/(UCr×PNa).Plasma creatinine (PThiobarbituric acid-reactive substances (Sigma) were employed to assess lipid peroxidation. Samples were evaluated for MDA production via a spectrophotometric assay for thiobarbituric acid-reactive substances Kidneys embedded in paraffin were sectioned at 2 µm and stained with H&E by standard methods. Tubular damage was estimated using a grading scale of 0–4, as outlined by Jablonski et al. GAPDH. Specificity was verified by melting curve analysis and agarose gel electrophoresis. Primers used RT-PCR analysis are: TNF-α ; IL-6 ; IL-8 ; IL-10 ; CD3 ; CD11b ; iNOS ; eNOS ; and GAPDH . Data were analyzed using the comparative Ct (2−ΔΔCt) method.Total kidney RNA was extracted using TRIzol (Invitrogen) reagent according to the manufacturer's instructions. cDNA was synthesized using oligo d(T) (Applied Biosystems) and a SuperScript III Reverse Transcriptase Kit (Invitrogen). A StepOne™ Real-Time PCR System (Applied Biosystems) and a SYBR RT-PCR kit (Takara) were used for quantitative real-time RT-PCR analysis. All reactions were conducted in a 20 µl reaction volume in triplicate. The relative expression levels for a target gene were normalized by For measurement of myeloperoxidase (MPO) activity in renal homogenates, kidneys were homogenized in a buffer containing 50 mM Tris–HCl, 1 mM EDTA and 5% Sucrose (pH 7.4). The detailed steps were in accordance with manufacturer's protocol of the kit . MPO activity was expressed as ΔOD per minute per milligram total protein in renal lysates.Snap-frozen renal tissues or cultured cells were lysed with the M-PER Protein Extraction Reagent (Pierce) supplemented with a protease inhibitor ‘cocktail’. The samples were incubated for 30 min at 4°C and centrifuged at 16,000 g at 4°C for 15 min. Lysates were collected and stored at −70°C until use. Protein concentration in the lysates was measured with a bicinchoninic acid assay (Pierce). Equal amount of lysates were separated by SDS-PAGE, and then transferred onto nitrocellulose membranes for immunoblot analysis.Ready-to-use Q.E.D. A150 Alcohol Test kit  was used to quantify alcohol levels in the plasma samples.+ to NADH at 340 nm as previously described Enzymatic activity for ALDH was determined spectrophotometrically by monitoring the reductive reaction of NADMouse primary cultured TECs were transfected with a negative control siRNA (NC) or an ALDH2-siRNA, using the INTERFERin™ transfection reagent (Polyplus). TECs treated with the INTERFERin™ transfection reagent only were served as another negative control. The assays were conducted 48 h after transfection.Lactate dehydrogenase (LDH) in the culture medium and primary renal TECs was measured using the modified colorimetric routine laboratory method t test or a Mann - Whitney test (two tailed). All data were expressed as mean ± SEM. In all cases, p<0.05 was considered with statistical significance.Data from multiple groups were analyzed using one way ANOVA with post-hoc Bonferroni's correction . Data derived from two groups were analyzed using an unpaired Student's i. p.) injection. To this end, the mice were i.p. injected with 5% ethanol at a dose of 1 g/kg body weight. The mice were next sacrificed at indicated time point and ethanol concentration in the plasma was determined as described. As shown in i.p. injection, and the highest level (approximately 51 mg/dl or 11 mM) was observed 30 min after injection, which is equivalent to the physiologically attainable blood ethanol levels in humans after one to three drinks We first monitored the temporal blood ethanol concentration after intra-peritoneal  and urine osmolality (UOsm). Ethanol pretreatment significantly decreased FENa but increased urine osmolality as compared with that of controls  of TECs 3 h before simulated IR reduced LDH release  have been widely documented in cardiovascular diseases The present studies for the first time demonstrated that moderate ethanol administration antecedent to renal IR induction provides protection in a dose and time dependent manner. After bilateral renal ischemia, ethanol pretreated mice displayed significantly preserved renal function as characterized by less histological tubular damage, reduced leukocyte infiltration and proinflammatory cytokine production. We further demonstrated that the protective effect was associated with ameliorated oxidative stress as manifested by the increase of antioxidant capacity (higher SOD activities) and decrease of lipid peroxidation (less generation of MAD and HNE). Further studies revealed that ethanol administration antecedent to IR insult significantly enhanced the enzymatic activity of ALDH2, which then promoted the detoxification of aldehydic metabolites to prevent lipid peroxidation. By employing an in vitro IR simulation model, we confirmed that the protective effect of antecedent ethanol exposure on IR-induced renal injury dependent on the induction of ALDH2. Transfection of a siRNA specific for ALDH2 into the primary cultured TECs completely abrogated the protective effect. Taken together, our data demonstrate that moderate antecedent ethanol exposure provides protection for kidneys against subsequent IR-induced injury by increasing antioxidant capacity and by preventing lipid peroxidation through increasing the activity of ALDH2.Our data presented in the current report are in agreement with previous studies in the heart, intestine and brain after IR induction Interestingly, we observed a similar biphasic protection against renal IR injury in ethanol antecedent exposed mice, but more powerful in the early phase at 3 h after ethanol pretreatment. We ruled out the possibility that differences in approaches for ethanol administration might affect the results, as we observed almost the same peak levels of ethanol in the blood and same elimination kinetics as compare with that in precious studies in vitro studies demonstrated that the early phase protection was ALDH2 dependent since knockdown of ALDH2 by siRNA abrogated the protective effect.The early phase (60 min) effect of ethanol pretreatment was extensively studied in the cardiac IR model To rule out that direct chemical stimulation due to high concentration of ethanol might affect the results, we kept constant ethanol concentration at 5%. Therefore, the total saline volumes among each ethanol dose groups were different, and the control group received the largest volume of saline as that in the 2 g/kg body weight dose group. Nevertheless, the volumes of saline before kidney IR induction did not show a perceptible effect on the response to IR injury, as we failed to observe any difference on the renal function after IR insult between the mice injected with or without saline at the largest volume (data not shown). Despite that better renal function was observed based on the histological results in the kidneys of ethanol-pretreated mice at day 7 post reperfusion, we cannot exclude the possibility that the well reserved renal function was due to that ethanol treatment accelerated the recovery of renal function after IR insult. As such, further studies would be necessary with focus on to verify whether ethanol pretreatment accelerates renal functional recovery. It is noteworthy that pretreatment with high dose of ethanol (2 g/Kg) failed to protect the animals against IR injury. Given our observations that the predominant protective effect occurs in the short-term period , the protective effect for the high dose of ethanol could be overwhelmed by its damaging impact on the kidneys that concomitantly occurs within the same window of time period. As a consequence, additional mechanistic studies would be important to clarify the narrow therapeutic window for ethanol pretreatment in the prevention of IR-induced renal injury.i.p. injection on biphasic protection of kidneys against IR-induced injury, with much stronger protective effect observed 3 h after ethanol administration (early phase). Moreover, our work suggests that the protective effect is ALDH2 dependent, which serves as a key enzyme in eliminating the toxic aldehydes produced by IR to prevent lipid peroxidation. Altogether, our results demonstrate that moderate ethanol ingestion and ectopic ALDH2 activation could be viable therapeutic approaches for various clinical conditions involving renal IR injury.In summary, our data resulted from a renal IR model provide the first line of experimental evidence for the effect of antecedent ethanol exposure by single bolus


Neuronal activity regulates a broad range of processes in the hippocampus, including the precise regulation of translation. Disruptions in proper translational control in the nervous system are associated with a variety of disorders that fall in the autistic spectrum. MicroRNA (miRNA) represent a relatively recently discovered player in the regulation of translation in the nervous system. We have conducted an in depth analysis of how neuronal activity regulates miRNA expression in the hippocampus. Using deep sequencing we exhaustively identify all miRNAs, including 15 novel miRNAs, expressed in hippocampus of the adult mouse. We identified 119 miRNAs documented in miRBase but less than half of these miRNA were expressed at a level greater than 0.1% of total miRNA. Expression profiling following induction of neuronal activity by electroconvulsive shock demonstrates that most miRNA show a biphasic pattern of expression: rapid induction of specific mature miRNA expression followed by a decline in expression. These results have important implications into how miRNAs influence activity-dependent translational control. Fragile X Mental Retardation Protein (FMRP), an RNA binding protein involved in both the transport and translation of specific sets of mRNAs Tuberous sclerosis complex 1 or 2 genes, which in turn regulates mechanistic target of rapamycin- (mTOR) dependent translation. These syndromes directly affect translational control in neurons and lead to cognitive disorders that lie within the autistic spectrum Translational control is essential for the normal function of neurons. Numerous mechanisms exist in neurons to finely tune the translational output of mRNAs Lim kinase 1 mRNA Drosophila homolog of FMRP (dFmr1) physically interacts with components of RISC dFmr and the core RISC component dAgo1 show genetic interactions MicroRNA (miRNA) play a significant role in the translational control of mRNAs in neurons. These small RNAs are loaded into the RNA-induced silencing complex (RISC) where they direct target mRNAs in a sequence-specific fashion for either translational repression or degradation Neuronal activity regulates protein synthesis in a variety of ways including the mobilization of translationally repressed mRNAs, including those regulated by mTOR and FMRP. Though a number of activity-regulated miRNAs in the hippocampus have been identified 6 reads per library resulting in a total of 118 million reads  gene  the sequence is represented in multiple libraries. (2) The sequence maps to a genomic region containing a hairpin that is consistent with Dicer and Drosha processing. (3) The hairpin that the sequence lies within must be considered thermodynamically stable by Randfold. All novel miRNAs were required to have a sequenced star strand. The sequence is rejected as a novel miRNA if the mature sequence has any of the following negative features: the sequences is inconsistent with being a Dicer product, has a high degree of 5′ end variability, a hairpin sequence that is too short, if the sequence maps to greater than 10 locations in the genome, has high G-C content, if the sequence is not in 18–24 nt range, or if the read overlaps the predicted hairpin sequence. With these stringent requirements we identified 15 confident novel miRNAs . The majsequence . Though 2c) gene .in vivo. Electroconvulsive shock (ECS) was chosen as a model for in vivo neuronal activity for several reasons. ECS generates a massive, synchronous depolarization of neurons in the CNS, thus permitting a time-course analysis of miRNA expression following activity. This technique faithfully recapitulates activity-dependent induction of the classic immediate early genes (IEGs) activity-regulated cytoskeletal protein (Arc) and FBJ osteosarcoma oncogene , an approach that generally yields a more stable reference. The results of the TLDA showed no signs of the ‘batch effect’ observed in the deep sequencing data. Rather, individual arrays grouped more closely by time-point than replicate, suggesting that biological instead of technical variation is observed in these data  platform (Life Technologies) followed by extensive low-throughput quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Importantly, the rodent TLDA ‘A’ array includes assays for 46/53 miRNAs that are expressed at levels greater at 0.1% of total miRNA . A cut-o of 0.99 . The TLDese data .TLDA analysis demonstrated that the majority of miRNAs' levels respond to activity . UnsuperTo validate these results and to increase the resolution of the time course of miRNA expression, we conducted a low-throughput analysis using qRT-PCR of select high and low abundance miRNAs . This isHtr2c, bringing the total of intronic miRNAs encoded the Htr2c locus to 7. Additionally, we identified three new members of the mir-410 family. Finally, using a variety of platforms we made the striking observation that the majority of miRNAs expressed in the hippocampus are down-regulated in response to robust neuronal activity at a relatively late time point. This finding in particular is important to how we think about the effects of activity on the programming of RISC and the regulation of target mRNAs.This study defines the breadth of the hippocampal miRNA transcriptome, at the basal state and following robust synchronous neuronal activity. Using deep sequencing, we provide the most in-depth investigation of a single tissue's miRNA expression published to-date. In total, we identified 15novel miRNAs using a stringent set of criteria. Among the novel miRNAs identified, three lay within the introns of bona fide miRNA. A recent study called into question nearly one third of the total miRNAs registered in miRBase 14.0 due to a lack of significant sequence support or absence of characteristic miRNA features We identified a large number of candidate novel miRNAs in our sequencing analysis , cytoplasmic polyadenylation element binding protein 4 (Cpeb4), and phosphodiesterase 4B (Pde4b) among others. Second, though transcribed from a variety of genomic locations  3/15 novel miRNAs are embedded within introns of the serotonin receptor 2c (Htr2c) gene  gene . The intOur investigation of activity-dependent regulation of miRNA expression began with analysis of deep sequencing libraries constructed from mice treated with ECS. Unsupervised hierarchical clustering revealed that the contents of libraries were highly dependent on the replicate, rather than on treatment with ECS . This tyAplysia suggests that these are evolutionarily conserved phenomena Dicer in the forebrain of young adult mice showed that during a period shortly after deletion, the Dicer mutants showed improved performance in memory tasks Dicer deletion, the levels of residual miRNA expression are similar to those found in Class 3 of ECS-treated mice  guidelines as specifically approved as a part of animal protocol # MO08M522. All experiments described in this study were specifically approved as a part of the afore mentioned animal protocol. Mice were treated with electroconvulsive shock (ECS) using an ECT Unit (Ugo Basile) with following settings: 1 sec shock,100 pulses/sec, 0.4 ms pulse width, 22 mA current. To test the requirement of NMDA receptor activation, mice were injected with either PBS alone or CPP (10 mg/ml i.p.) 1 hour prior to treatment with ECS. Following ECS, total hippocampal RNA was isolated from 6–8 week old C57BL/6 males using Trizol (Life Technologies) following the manufacturer's protocol. RNA quality was assessed by electrophoresis and spectrophotometric analysis using a Nano drop spectrophotometer. For histological analysis, animals were first anesthetized using pentobarbital and perfused with 4% paraformaldehyde followed by cryoprotection in sucrose and cryosectioning. Sections were Nissl stained using standard methods and light micrographs capture using an Axioplan 2 (Zeiss).Libraries were constructed using the Illumina Small RNA Digital Gene Expression kit following the manufacturer's protocol. RNA quality was assessed by gel electrophoresis and analysis using a Nanodrop spectrophotometer. In brief, 10 µg of total RNA was separated by denaturing gel electrophoresis, stained with ethidium bromide, and the fragment containing ∼18–25 nt small RNAs was recovered. Following elution, the 5′ adapter was ligated to the small RNA using T4 RNA ligase. Following ligation, the products of this reaction were separated by denaturing gel electrophoresis and the band in the 40–60 nt range was excised and again eluted from the gel fragment. The 3′ adapter was then ligated using T4 RNA ligase to the eluted fragments followed by separation by denaturing gel electrophoresis. The 70–90 nt range was excised from the gel, eluted, and precipitated. The 5′ and 3′ adapted small RNA was then reverse transcribed and subjected to PCR amplification using the Phusion HF (Finnzymes) polymerase. Sequencing was conducted using an Illumina Genome Analyzer IIx at the UCLA Genome Sequencing Center. The miR-Intess pipeline used to process the sequencing output is described in detail elsewhere Rnu6, Snord65, Snord68, Snord87, and Rny1. Unsupervised hierarchical clustering was performed using Cluster 3.0 for the Macintosh and visualized using TreeView 1.1.3 for Macintosh OS X.RNA isolated from the hippocampus was treated with RNase-free DNase I (Ambion) to remove contaminating DNA. For TLDA analysis, 750 ng of total RNA was used for reverse transcription using the Megaplex Pool A primer set (Life Technologies) and the MultiScribe RT system (Life Technologies). Following loading, the TLDA mouse miRNA ‘A’ array was run on an ABI 7900HT Real-time PCR System (Life Technologies). Changes in miRNA expression were quantified using the ΔΔCt method normalizing the to geometric mean of the Ct values of 5 non-coding RNAs Rnu6 or Rps2. All assays were performed on RNA from 3–4 mice. All PCRs were performed in quadruplicate.For low-throughput qRT-PCR, 500 ng of total RNA was reverse transcribed with the miScript RT System (Qiagen), allowing the assessment of both miRNA and mRNA expression. Expression of miRNAs was measured using pre-designed miScript primer sets (Qiagen). Real-time PCR analysis was conducted on an ABI 7900HT using Power SYBR-Green reagents (Qiagen). Changes in expression levels of transcripts were determined using the ΔΔCt method normalizing to All statistical analyses were performed using Prism (Graphpad) or Excel (Microsoft).Figure S1Percent composition of individual libraries in this study. Each class of RNA is represented by a different color .(TIF)Click here for additional data file.Figure S2Heat map displaying the relative expression levels of all miRNAs analyzed by TLDA. Expression profiles of miRNAs were grouped by unsupervised hierarchical clustering into three expression profile classes.(TIF)Click here for additional data file.Table S1Summary of the mapping of reads. “Non-matching” reads were unable to be mapped to genome following removal of the adapter sequences. “Trimmed 3′ end” are reads that matched to the genome after trimming all but the 20 nt of the 5′ end of the read. “Perfect” refers to reads that match perfectly in the genome following removal of adapter sequence.(XLS)Click here for additional data file.Table S2Ssummary of miRNA reads from all deep sequencing libraries.(XLS)Click here for additional data file.Table S3Predicted target sites for novel miRNAs described in this study.(XLS)Click here for additional data file.Table S4Fold changes in miRNA expression of all TLDA experiments for miRNAs with that amplified with a Ct value greater than 32.(XLS)Click here for additional data file.


The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa.Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC).Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels  in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues ; however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues.proColon®), providing further support for the clinical relevance of this biomarker.Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to acquire hypermethylation subsequent to epithelial cells, possibly through field effects. The region in SEPT9 with disease-related hypermethylation also contains the CpGs targeted by a novel blood-based screening test (Epi  Alterations in the DNA methylation profile of cells are among the earliest molecular changes in cancer . Both loa involvement in cancer was first discovered as a fusion product with the MLL gene in leukemia [Septin 9 (SEPT9)leukemia . Subsequleukemia  or amplileukemia ; it was leukemia . MoreoveSEPT9 belongs to a highly conserved family of septin genes coding for GTP-binding proteins. These multidomain proteins assemble into complexes and form filamentous structures which comprise part of the cytoskeleton ,9. The sSeveral analyses have been conducted on the expression of the SEPT9 transcripts. The transcript SEPT9_v1 was overexpressed in breast, ovarian, and prostate cancer while no SEPT9_v1 expression was observed in the normal tissue of these organs ,13. Two proColon®) for colorectal cancer [Recently, SEPT9 was shown to be epigenetically modified in colorectal cancer (CRC) . Subsequl cancer , which il cancer . The tesl cancer . HoweverOur results provide new insights into the cellular origin of aberrant DNA methylation in the SEPT9 gene for CRC and the clinical relevance of DNA methylation for early CRC detection.Biopsies of normal mucosa were obtained from three patients, aged 32 to 72 during routine endoscopy examinations  was performed on normal, adenomatous, and colorectal tumor mucosa specimens after they were embedded in optimal cutting temperature (OCT) media  and prepared for cryosectioning: 6 μm sections were cut on PALM Membrane Slide 1.0 PEN  slides at −20°C and stored at −80°C until used. Consecutive sections were used for H&E and IHC staining for LCM and photo documentation. A PALM laser microdissector  was used to collect a total of 96 specimens with a target of approximately 1000 cells each, as shown in Additional file proColon® 2.0 kit, Epigenomics, Germany) were used with a modified protocol: 45 μl of bisulfite solution plus 10 μl of protection buffer were added to each lysate and incubated at 80°C for 45 minutes in a thermocycler . Next, binding buffer and poly-dA  were added to the lysate solutions, which were then placed on to silica membranes . On-column desulfonation using 0.2 M NaOH for two minutes was followed by two washing steps using commercial buffer solutions AW1 and AW2 , and finally, DNA was eluted using 40 μl of pre-warmed (50°C) PCR-grade water. Eluted DNA was stored at −20°C until used for analysis. The total amount of bisulfite-converted DNA samples was quantified using an in-house duplex qPCR assay which determines genomic and bisulfite DNA simultaneously [Microdissected samples were lysed at 56°C for three hours followed by the addition of fresh proteinase K (ProtK) solution, and then overnight incubation at 37°C to ensure complete digestion of the samples. All lysates were subjected to bisulfite treatment without prior DNA extraction. To further minimize DNA loss while maintaining high DNA conversion rates a non-standard protocol was established. Bisulfite reagents   and sperm DNA, previously reported to be free of methylation at most loci ,26, wereImmunohistochemistry (IHC) staining was done as previously described  with modThe mPCR assay established for this study included primer pairs for 13 different loci , and the region covered by the neighboring amplicon 7 (located outside CGI3), displayed elevated methylation in normal tissue samples with epithelial cells showing higher methylation levels than stromal cells . The observed aberrant methylation in adenoma and tumor samples clearly originated in epithelial cells while in stromal cells, hypermethylation at this locus occurred only after hypermethylation in epithelial cells ; IHC: Immunohistochemistry; LCM: Laser capture microdissection; MLL: Myeloid/lymphoid or mixed-lineage leukemia gene; mPCR: Multiplex PCR; NAT: Normal adjacent tissue; OCT: Optimal cutting temperature media; PBL: Peripheral blood lymphocytes; PBS: Phosphate buffered saline; PMR: Percent of methylated reference; qPCR: Quantitative PCR; SEPT9: Septin 9 gene; sPCR: Singleplex PCR; TSS: Transcription start site; tv: Transcription variant.RW and MK were employees at Epigenomics AG at the time the study was designed and the experiments were performed. AZS was an employee at and currently is consultant to Epigenomics Inc. RW and AZS are shareholders of Epigenomics. All other authors declare that they have no competing interests.BM, MK, and RW conceived the study; MK designed primers and performed a pilot study to establish the approach; SS collected the samples and performed cryosectioning; AK and KT performed LCM and IHC experiments; GV measured and analyzed IHC data; RW supervised and analyzed all methylation related experiments and wrote the manuscript; AZS and ZT contributed to the design and critical review of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/398/prepubPatient characteristics and clinical data.Click here for fileCollected LCM specimens.Click here for filePrimers used for mPCR and sPCR assays. Reverse primers used for locus specific resequencing after mPCR were as shown in the table but had an added ‘cgtcgtcg’-tag at their 5′ end.Click here for fileAmplicon information. Distribution of CpGs within each of the eight amplicons for SEPT9. All amplicons are drawn to the same scale; the real length of each amplicon, given in base pairs, is shown in the second column, and the number of CpGs covered by the respective amplicon in the third column.Click here for file


Skin is an excellent site for vaccine administration due to the abundance of antigen presenting cells and easy access. When DNA or protein is delivered into the skin, uptake and expression of immunogens can lead to a rapid and robust induction of immune responses.Skin tattooing has been suggested as a useful tool delivering vaccines intradermally. We have chosen to use the skin tattooing technique to deliver both DNA and protein immunogens that are focusing the immune response on the highly immunogenic V3 region of HIV-1 gp120.A GFP-containing plasmid was used first to optimize the protocol for skin tattooing on BALB/c mice. Subsequently, BALB/c mice were immunized using a DNA prime/Protein boost regimen. DNA skin tattooing was used first as the prime to deliver a gp120-encoding DNA plasmid intradermally at weeks 0, 2, and 4. The animals were then given a fusion protein, V3-bearing cholera toxin subunit B (V3-CTB), via skin tattooing  or intramuscular injection  at weeks 10 and 14. Two weeks after the final immunization, the sera were collected and analyzed.GFP expressed in skin cells was visualized by confocal microscopy showing that skin tattooing can effectively deliver DNA into these cells. ELISA assays using sera drawn after the final immunization confirmed that tattoo-delivered DNA followed by V3-CTBelicited V3 binding antibodies, and these antibodies have potent neutralizing activities in an HIV pseudovirus neutralization assay.Immunization by skin tattooing is an easy, effective, and economical way to administer both DNA and protein vaccines. Interestingly, the needle perforations may serve as a potent and natural adjuvant for the immunization, making skin tattooing a promising vaccine delivery technique.


L) encoded by ELAC2, and a short form . We studied their subcellular localization by expression of proteins fused to green fluorescent protein. RNase ZS was found in the cytosol, whereas RNase ZL localized to the nucleus and mitochondria. We show that alternative translation initiation is responsible for the dual targeting of RNase ZL. Due to the unfavorable context of the first AUG of ELAC2, translation apparently also starts from the second AUG, whereby the mitochondrial targeting sequence is lost and the protein is instead routed to the nucleus. Our data suggest that RNase ZL is the enzyme involved in both, nuclear and mitochondrial tRNA 3′ end maturation.RNase Z is an endonuclease responsible for the removal of 3′ extensions from tRNA precursors, an essential step in tRNA biogenesis. Human cells contain a long form (RNase Z The primary structure of RNase ZL suggests that it has evolved from RNase ZS by duplication. While all eukarya appear to have at least one RNase ZL gene, some also have RNase ZS, or more than one gene encoding a long and/or short form of RNase Z.RNase Z enzymes belong to the metallo-β-lactamase superfamily. In addition to the diagnostic α–β/β–α β-lactamase-fold, a protruding flexible arm involved in binding tRNA precursors is characteristic for RNase Z structures. There are two forms of RNase Z, one of 280 to 360 amino acids (RNase ZS is encoded by ELAC1 and RNase ZL by ELAC2 (the gene names refer to their homology to the Escherichia coli RNase Z gene elaC). Recombinant proteins derived from both genes have RNase Z activity, i.e., cleave tRNA precursors at their 3′ end in vitroELAC2 suggests that RNase ZL localizes to mitochondria, whereas RNase ZS might accordingly be assumed to be responsible for the processing of nuclear encoded tRNAs. However, due to a lack of systematic approaches, the available experimental evidence is largely inconclusive. RNase ZS was found mainly in the cytosol upon biochemical fractionation L (ELAC2) was either reported to be mostly cytosolic In the human genome RNase ZL (ELAC2) we moreover revealed the basic mechanism underlying the observed dual nuclear/mitochondrial targeting of RNase ZL.To clarify the subcellular distribution of the two human RNase Z isoforms we studied the localization of enhanced green fluorescent protein (EGFP)-tagged variants in human cell lines. By bioinformatics and experimental manipulation of the translation initiation site of RNase ZL to be targeted to mitochondria by its N-terminal amino acid sequence. Moreover, amino acids 28 to 35 are predicted to comprise a nuclear localization signal. In contrast, RNase ZS appears to neither contain a mitochondrial targeting sequence nor a nuclear localization signal.Different bioinformatic tools invariably predicted human RNase ZL localized to the nucleus and the mitochondria of transfected cells, but showed no cytosolic fluorescence .To address the localization of both proteins experimentally we expressed their complete coding sequences tagged by EGFP in human 293 cells. To avoid interfering with putative N-terminal sorting signals we fused the EGFP-tag to the C-termini of the proteins. RNase Zrescence . RNase Z cytosol . Still, L escape the mitochondrial import pathway? The assigned start codon of human RNase ZL (ELAC2) appears to be in a suboptimal configuration for efficient initiation of translation , the slightly shortened RNase ZL (811 amino acids) is no longer predicted to localize to mitochondria by any of the employed bioinformatic tools, whereas the predicted nuclear localization signal is not affected. The peculiar configuration of the two putative start codons is conserved among mammalian ELAC2 homologs (−4 and C−2) conform more closely to the consensus sequence in the case of the second putative initiation site. The second AUG thus invariably appears to be in a better initiation context than the first.How does the nuclear fraction of RNase Znslation . Initiathomologs . While pL is indeed due to alternative translation initiation we prepared two variants of the ELAC2 cDNA and analyzed their subcellular localization. On the one hand, we changed the sequence preceding the first AUG to the more favorable initiation context GTCACC, on the other hand, we deleted the first 15 amino acids to allow translation from the second AUG only. Modification of the nucleotides preceding the first AUG to conform to the consensus gave rise to an RNase ZL exclusively localized to mitochondria  initiation context-optimized cDNA too L variant exclusively found in the nucleus . All eukarya also have one RNase ZS indicates that it is not involved in the processing of tRNA primary transcripts. This conclusion is consistent with its low tRNA processing efficiency in vitroELAC1) is part of a homozygous deletion in a lung cancer cell line, and thus apparently dispensable for cell survival and growth S.The cytosolic localization of human RNase ZL suggests a role in the 3′ end maturation of nuclear and mitochondrial encoded tRNAs. Such a dual role has in fact been demonstrated for D. melanogaster RNase ZLL is generally the only nuclease responsible for tRNA 3′ end formation. While there is evidence for alternative, exonucleolytic pathways for nuclear tRNA maturation in different eukarya A. thalianaL are targeted to mitochondria, only the double mutant is lethal in this case In contrast, the subcellular localization of human RNase ZL appears to be the alternative use of two translation initiation sites. The first AUG of mammalian RNase ZL is in a poor context for initiation and thus apparently passed by most scanning ribosomes, while the second AUG more closely conforms to a consensus initiation site and appears to be used preferentially. This mechanism of alternative translation initiation has been described as context-dependent leaky scanning L downstream initiation results in the loss of most of the mitochondrial targeting signal, whereby the protein translated from the second AUG is no longer imported into the mitochondrial matrix, but routed to the nucleus via its nuclear localization signal. Accordingly, experimental optimization of the context of the first AUG resulted in an RNase ZL exclusively targeted to mitochondria, whereas its elimination produced nuclear RNase ZL only. This moreover implies that the mitochondrial targeting signal is either dominant over the nuclear localization signal, or the latter masked by the presence of the former The basic mechanism underlying the dual nuclear/mitochondrial localization of human RNase ZL. We previously reported a reduction in mitochondrial RNase Z activity in human cells lacking mitochondrial DNA (ρ0 cells) L coding ELAC2 mRNA is in fact down-regulated in ρ0 cells L to the nucleus or affects the cellular RNase ZL pool as a whole and thereby possibly contributes to the slower growth of ρ0 cells.Context-dependent leaky scanning does not exclude that there are other, currently unknown mechanisms involved in regulating the balance between nuclear and mitochondrial RNase ZL in the cytosol. Previously, others had reported a predominantly cytosolic localization of ELAC2 upon biochemical fractionation L by immunofluorescence L, prevented. The N-terminal tag might have additionally masked the nuclear localization signal L in our study prevented its cytosolic localization, as this was neither the case with EGFP alone nor with EGFP tagged RNase ZS.We did not observe any RNase ZL is localized to both compartments of tRNA biogenesis, and considering that only a single gene (TRNT1) for ATP(CTP):tRNA nucleotidyltransferase, the CCA-adding enzyme has been identified in the human genome, it appears reasonable to assume it is shared as well L .Animal cells use two radically different enzymes for the processing of tRNA 5′ ends in the nucleus and mitochondria For the prediction of subcellular localization we used the following tools: LOCtree L gene sequences were retrieved from Ensembl Mammalian RNase ZELAC1 (NM_018696) and ELAC2 (NM_018127) including 15 nucleotides of 5′ untranslated region preceding the start codon were PCR amplified from HeLa cell (ATCC# CCL-2) cDNA using primers containing BamHI and in-frame XbaI restriction sites. PCR products were cloned into a pcDNA4/TO (Invitrogen) that was modified by insertion of the EGFP coding sequence from pEGFP-N3 (Invitrogen) into its ApaI site. Proteins expressed from these plasmids are composed of their complete native amino acid sequence fused at their C-terminus via a short linker peptide (SerArgGlyProAlaThr) to EGFP.The coding sequences of ELAC2 expression plasmid was modified by site-directed mutagenesis using the QuikChange (Stratagene) protocol: GTCACC replaced (i) the natural 5′ untranslated region, or (ii) the 5′ untranslated region plus the first 45 nucleotides of coding sequence.The native Stable T-REx-293 (Invitrogen) cell lines were generated as described recently c oxidase (Molecular Probes A6403) at 5 ng/µl. For detection biotinylated anti mouse IgG and Texas Red conjugated streptavidin were used. A conventional epifluorescent and a dual-channel confocal laser scanning microscope (Olympus FluoView) were used. Pictures were processed with the FluoView application software (Olympus).Cells were grown on cover slips and expression induced with tetracycline at 0.2 µg/ml. Paraformaldehyde fixed cells were stained with bisbenzimid H33342 and embedded in Kaiser's glycerol gelatin. For mitochondrial staining cells were fixed, permeabilized with 0.1% Triton X-100, and incubated with a monoclonal antibody specific for subunit I of cytochrome c oxidase subunit VIII was selected. This cell line was transfected with the ELAC expression plasmids by a magnet assisted transfection procedure according to the instructions of the supplier (IBA BioTAGnology).A 143B (ATCC# CRL-8303) derived cell line stably expressing DsRed2 (Clontech) targeted to mitochondria by the signal peptide of cytochrome Figure S1S, RNase ZL, and RNase ZL variants with modified translation initiation sites in 143B cells.Subcellular localization of EGFP-tagged RNase Z (A) RNase ZS-EGFP; (B) RNase ZL-EGFP with native translation initiation context; (C) RNase ZL-EGFP variant with an optimized translation initiation context of the first AUG; (D) RNase ZL-EGFP variant without the first 15 amino acids; (E–H) DsRed2 labeled mitochondria of cells shown in the same row; (I–L) nuclear staining of cells shown in the same row.(TIF)Click here for additional data file.


Neuropathological and molecular characterisation of Bv109ICWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv109ICWD was characterised by a mild and discrete distribution of spongiosis and relatively low levels of protease-resistant PrPSc (PrPres) in the same brain regions. Despite the low PrPres levels and the short time lapse available for its accumulation, end-point titration revealed that brains from terminally-ill voles contained up to 108,4 i.c. ID50 infectious units per gram. Bv109ICWD was efficiently replicated by protein misfolding cyclic amplification (PMCA) and the infectivity faithfully generated in vitro, as demonstrated by the preservation of the peculiar Bv109ICWD strain features on re-isolation in Bv109I. Overall, we provide evidence that the same CWD strain was isolated in Bv109I from the three-cervid species. Bv109ICWD showed unique characteristics of “virulence”, low PrPres accumulation and high infectivity, thus providing exceptional opportunities to improve basic knowledge of the relationship between PrPSc, neurodegeneration and infectivity.In order to assess the susceptibility of bank voles to chronic wasting disease (CWD), we inoculated voles carrying isoleucine or methionine at codon 109  with CWD isolates from elk, mule deer and white-tailed deer. Efficient transmission rate (100%) was observed with mean survival times ranging from 156 to 281 days post inoculation. Subsequent passages in Bv109I allowed us to isolate from all CWD sources the same vole-adapted CWD strain (Bv Myodes glareolus), a wild rodent species that we demonstrated to be susceptible to many animal and human prion diseases, is also very susceptible to CWD from elk, mule deer and white-tailed deer. Adaptation of CWD to bank vole led to the isolation of a prion strain with peculiar characteristics: unprecedented short incubation and survival times, respectively of 25–28 and ∼35 days, low PrPSc levels compared with other vole-adapted prion strains and high infectious titre. These features were all faithfully maintained upon the generation of this strain in vitro by protein misfolding cyclic amplification. The development of a model for prion diseases that led to disease in less than one month accumulating high infectious titres but low PrPSc levels, represents a significant tool for investigating the still unclear relationship between PrPSc, neurodegeneration and infectivity in prion diseases.Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids and is expanding increasingly in the USA and Canada. Animal models are of key importance in the study of prion diseases but their development for CWD has long been hampered by its very inefficient transmission to wild-type mice. Significant progress was made following the generation of transgenic mice over-expressing cervid PrP. Here we show that the bank vole ( Sc) of the host-encoded cellular prion protein (PrPc). Prion diseases also include Creutzfeldt-Jakob disease (CJD) of humans, scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle.Chronic wasting disease (CWD) of cervids belongs to the family of transmissible spongiform encephalopathies (TSE) or prion diseases, a group of fatal neurodegenerative pathologies affecting animals and humans. They are characterised by spongiform changes, gliosis and the deposition in the brain of a post-translational misfolded isoform (PrPCervus elaphus nelsoni), mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus) and moose  CWD is the only prion disease known to affect free-ranging wild animals. It was first described in the United States in the late 1960s Epidemiological evidence indicates that CWD spreads naturally with relative efficiency and recent trend analyses suggest that its prevalence is increasing The development of laboratory models for CWD has long been hampered by the very inefficient transmission of CWD to wild-type mice Myodes glareolus) to a wide range of human and animal prion diseases. Two lines of voles, one homozygous for methionine and the other for isoleucine at codon 109 of PrP – here designated Bv109M and Bv109I, respectively – were investigated. Bv109M was shown to be susceptible to sporadic and genetic CJD In recent years, with the aim of developing new animal models for prion diseases, we have studied the susceptibility of the bank vole .In the present study, we investigated the susceptibility of bank voles to CWD. To this purpose, we inoculated Bv109M and Bv109I with various CWD sources and found that CWD replicated faster in Bv109I compared to Bv109M. We then focused on the Bv109I line, which showed unprecedented short survival times upon CWD adaptation. We describe a thorough characterization of Bv109I-adapted CWD, showing that in spite of their short survival time, high infectious titres accumulate in the brains of affected animals. Furthermore, we provide evidence that Bv109I-adapted CWD can be easily and faithfully replicated All CWD isolates transmitted with 100% attack rate and short survival times in both Bv109I and Bv109M . In someCWD isolates transmitted in Bv109I with mean survival times ranging from 156 to 281 d.p.i. . All isoAll infected voles showed moderate spongiform changes at brain histopathology, accompanied by gliosis and neuronal loss . All groSc was readily detected in all infected voles by IHC, PET-blot and WB. IHC showed punctate PrPSc deposition  or found dead for intercurrent disease , were found PrPIn order to study the adaptation of CWD prions in Bv109I and to compare more closely the biological properties of the various CWD isolates, vole-passaged CWD isolates were further propagated in Bv109I. On the second passage we observed a dramatic decrease in the incubation times, with behavioural alterations already evident at 25–28 d.p.i.. Following a short clinical phase all CWD isolates produced strikingly short mean survival times of between 34 and 44 d.p.i. . SurvivaSc distribution by PET-blot was the same in all groups, characterised by PrPSc deposition mainly in the thalamus, substantia nigra, geniculate nuclei, vestibular nuclei and the deep layers of the cerebral cortex  or the original Bv109ICWD used as seed. Bv109ICWDPMCA induced terminal disease in 42±2 d.p.i., a survival time similar to that of the control Bv109ICWD inoculum (38±2 d.p.i.). The second passage of Bv109ICWDPMCA produced disease in 34±2 d.p.i.. The clinical phenotype and the lesion profile of animals infected with Bv109ICWDPMCA, after primary transmission and second passage, were indistinguishable from the control group  and PET-blot . Transmission studies of CWD to Bv109M have not yet completed but we have observed that also CWD adapts to Bv109M with survival times longer than Bv109ICWD (60–100 d.p.i.) (unpublished data). The short survival time of Bv109ICWD is also unprecedented when compared with those found in transgenic mouse models, in which PrP overexpression greatly fosters prion diseases 109ICWD. Interestingly, it was recently shown that TgS3581 mice overexpressing vole PrP encoding for Isoleucine at position 109, undergo spontaneous prion disease and that it adapts to the same model with mean survival time of 35 days 109ICWDA striking feature of Bv109I-adapted CWD was the short incubation time of less than one month. In an earlier work, we showed that bank voles and related rodent species have peculiarly short survival times after infection with adapted prions, and presumably support equally fast prion replication kinetics, possibly due to the previously mentioned 150N–170N PrP residues Sc deposition showed a discrete brain distribution, involving specific neuronal populations such as those in the medulla and thalamus, might suggest that Bv109ICWD replication primarily involves the so called clinical target areas (CTAs) which, once colonised by prions, trigger the clinical signs and death of the animals res and infectivity in the brain of ip-inoculated mice. In Bv109ICWD we also observed unusually low levels of PrPres, compared with those observed in most of the vole-adapted prion strains  to the rate of its maturation into PrPSc. Thus fast prion replication in Bv109ICWD might have triggered the production of high levels of PrPL in short time periods, leading to rapid disease onset and animal death. The low levels of PrPSc and the fast replication kinetics observed in Bv109ICWD are consistent with this interpretation.Several observations suggest that prion infectivity and toxicity might be uncoupled C did not affect the overall level of prion infectious titres at terminal disease, while it was directly related to the incubation time, suggesting that the production of PrPL is directly proportional to PrPC concentrations 109ICWD, i.e. the unusually short survival time and the high prion infectious titre in a model that does not overexpress PrPC, suggest that the kinetics of prion propagation and toxicity are governed by mechanisms that cannot be interpreted solely on the basis of the amount of available substrate (PrPC).A recent work showed that in mice inoculated with the RML scrapie strain the concentration of PrP109ICWD prompted us to pursue its in vitro propagation by saPMCA. Bv109ICWD PrPSc was indeed easily propagated in vitro, which allowed us to produce Bv109ICWDPMCA PrPSc, theoretically devoid of any PrPSc formed in vivo. Bv109ICWDPMCA was highly infectious and faithfully reproduced the peculiar phenotype of Bv109ICWD. These findings confirm that CWD prions can be generated in vitro, as already demonstrated by others using transgenic mice expressing cervid PrP The unique and easily distinguishable features of Bv109ICWD was isolated not only from natural cases of disease in elk  and mule deer (CWD3), but also from white-tailed deer experimentally inoculated with CWD-affected white-tailed deer, mule deer and elk . Along with previous findings in transgenic mice expressing cervid PrP res similar to the other voles of their groups. However this hypothesis is excluded when these results are compared with the survival times observed in the endpoint titration experiment, which showed that even 10−5 dilution of Bv109ICWD had a mean survival time <80 d.p.i.. In addition, the slightly deviant lesion profile observed in outlier voles was preserved for 2 sub-passages, before both the survival time and the neuropathological profile converged with the fast Bv109ICWD strain. Overall, our findings strongly suggest that a second CWD strain was propagated in vole outliers, which was progressively outcompeted by the extremely rapid Bv109ICWD strain. The “evanescent” presence of a second strain might be also interpreted, according to the quasi species nature proposed for prions Elk, mule deer and white-tailed deer are the species most affected by CWD. The homogeneous and peculiar phenotypes observed in Bv109I inoculated with CWD isolates from these three cervid species indicate that the same CWD strain was isolated from all species. Interestingly, Bv109ICWD provide exceptional opportunities to improve basic knowledge of the relationship between PrPSc, neurodegeneration and infectivity. The short survival time of Bv109ICWD, coupled with its high infectious titre, offers useful advantages for titration studies, while its unique clinico-pathological phenotype makes Bv109ICWD one of the best options for studies aimed at investigating strain fidelity in experimental conditions.Here we demonstrate the high susceptibility of Bv109I to CWD, which adds Bv109I to the portfolio of animal models useful for the study of CWD strains. The unique properties of BvBv109I and Bv109M were obtained from the breeding colony at the Istituto Superiore di Sanità (ISS). The research protocol, approved by the Service for Biotechnology and Animal Welfare of the ISS and authorised by the Italian Ministry of Health, adhered to the guidelines contained in the Italian Legislative Decree 116/92, which transposed the European Directive 86/609/EEC on Laboratory Animal Protection.Brain tissues from CWD affected elk (n = 3), mule deer (n = 1) and white-tailed deer (n = 3) were used for primary transmissions. Two isolates from elk, CWD1 and CWD2, were provided by the United States Department of Agriculture (Dr. A.L. Jenny) and carried the wild type PRNP genotype. The third elk isolate, CWD4, was heterozygous methionine/leucine at codon 132. It was provided by the University of Wyoming (Dr. J.E. Jewel) as the mule deer isolate, CWD3, which was homozygous for serine at codon 225. The three isolates from white-tailed deer derived from an intracerebral experimental challenge An Italian scrapie isolate from Sarda sheep (SS7B) carrying the ARQ/ARQ genotype previously characterised into Bv109M res amount estimated by Western blot was comparable in all the homogenates (data not shown).Tissues were homogenised at 10% (wt/vol) in phosphate buffered saline (PBS) and stored at −80°C. The PrPGroups of eight-week-old Bv109I and Bv109M were inoculated intracerebrally with 20 µ of homogenate into the left cerebral hemisphere, under ketamine anaesthesia (ketamine 0.1 µg/g). The animals were examined twice a week until neurological signs appeared, after which they were examined daily. Diseased animals were culled with carbon dioxide at the terminal stage of the disease, but before neurological impairment was such as to compromise their welfare, in particular, their ability to drink and feed adequately. Survival time was calculated as the interval between inoculation and culling or death. After that, the brain was cut parasagittally into two parts. The smaller portion was stored at −80°C and the larger one was fixed in formalin.The inocula for the second and third passages were prepared, as 10% wt/vol homogenates in PBS, using the brain of one of the first animals of each group that developed the disease. Supplementary second and subsequent passages from two voles that showed much longer survival times compared with the rest of their groups (outliers) were also performed.109ICWD was assessed in Bv109I by end-point titration using tenfold dilutions of brain homogenates from the third passage of CWD1 and calculated as ID50 U g−1 according to the Spearman and Kärber method The specific infectivity of BvHistology, immunohistochemistry (IHC) and PET-blot analysis were performed on formalin-fixed tissues as previously described CWD inocula (10% w/v homogenates in PBS) were added to PBS/sarcosyl up to a final 2% sarcosyl (Sigma) concentration and incubated for 20 min at room temperature before digestion with proteinase K (200 µg/ml) for 1 hour at 37°C with gentle shaking. Protease treatment was stopped with 3 mM PMSF.Brain homogenates (10% w/v) from individual voles were prepared in 100 mM Tris-HCl, pH 7.4, containing 2% sarcosyl, incubated for 20 min at room temperature and then digested with proteinase K (50 µg/ml) for 1 hour at 37°C with gentle shaking. Protease treatment was stopped with 3 mM PMSF.g for 5 min each sample was loaded onto 12% bis-Tris polyacrylamide gels (Invitrogen). After electrophoresis and Western blotting on PVDF membranes , the blots were processed by SNAP i.d. Protein Detection System (Millipore) in accordance with the manufacturer's instructions. Vole PrPres was detected using monoclonal antibodies SAF84  and 12B2 . Horseradish peroxidase-conjugated anti-mouse immunoglobulin  was used as secondary antibody . The membranes were developed using an enhanced chemiluminescence method . The chemiluminescence signal was detected using the VersaDoc imaging system (Bio-Rad) and was quantified by QuantityOne software (Bio-Rad).Electrophoresis and Western blotting were performed as previously described −15 final dilution of the initial CWD infected brain homogenate. The detailed protocol for saPMCA has been published elsewhere res was evaluated by Western blot  and saPMCA was performed as previously described ern blot . Given tFigure S1Survival times of Bv109I and Bv109M following primary transmission of CWD and sheep scrapie. Outliers are visible among Bv109I inoculated with CWD2, 3 and 4; one outlier is visible among Bv109M with CWD1. Symbols represent individual survival times. Bars indicate the median for each group.(TIF)Click here for additional data file.Figure S2Lesion profiles of Bv109I inoculated with outliers from CWD3 and CWD4. A) Lesion profiles of Bv109I inoculated with the third passage of CWD3outlier (red line and open circles) and CWD4outlier (green line and closed triangles). B) Lesion profiles of Bv109I inoculated with the fourth passage of CWD3outlier (red line and open circles) and CWD4outlier (as shown in 109ICWD (black line and closed circles). Brain areas are as in (TIF)Click here for additional data file.Figure S3res amount in Bv109I inoculated with scrapie and Bv109ICWD.Comparison of PrP The amount of PrPres in Bv109I inoculated with Bv109I-adapted sheep scrapie (SS7B) and with Bv109ICWD was estimated by direct comparison of brain homogenate dilutions. The homogenates were treated with 50 µg/ml PK. Each sample was diluted in loading buffer after the denaturation step. The original samples (1∶1) were loaded as 0.5 mg of equivalent brain tissue. Membrane was probed with SAF84. Molecular weight markers are shown on the right.(TIF)Click here for additional data file.Figure S4In vitro109ICWD. amplification of Bv Western blot of Bv109ICWD following saPMCA. Bv109ICWD was diluted 10-fold into healthy Bv109I brain homogenate and submitted to a round (48 cycles) of PMCA. The amplified material was diluted 10-fold into healthy brain homogenate repeating this procedure to reach a 10−13 dilution of Bv109ICWD. Amplified samples from rounds 4 to13 were digested with 80 µg/ml of proteinase K and analysed by Western blot using monoclonal antibody D18. Control: Normal Bv109I brain homogenate.(TIF)Click here for additional data file.Figure S5C levels in the brain of voles, mouse and hamster.Comparison of PrP The amount of PrPC in the brain of Bv109M, Bv109I, RIII mice and hamster was assessed by direct comparison of brain homogenate dilutions . Brain homogenates were loaded as 0.1 mg (1∶1), 0.05 mg (1∶2) and 0.025 mg (1∶4) tissue equivalents. Membrane was probed with 12B2.(TIF)Click here for additional data file.


Short telomere length (TL) is an independent predictor of mortality in patients with coronary heart disease (CHD). However, the relationship between physical fitness and TL has not been explored in these patients.In a cross sectional study of 944 outpatients with stable CHD, we performed exercise treadmill testing, assessed self-reported physical activity, and measured leukocyte TL using a quantitative PCR assay. We used generalized linear models to calculate mean TL (T/S ratio), and logistic regression models to compare the proportion of patients with short TL (defined as the lowest quartile), among participants with low, medium and high physical fitness, based on metabolic equivalent tasks achieved (METs).229 participants had low physical fitness (<5 METS), 334 had moderate physical fitness (5–7 METS), and 381 had high physical fitness (>7 METS). Mean ± T/S ratio ranged from 0.86±0.21 (5349±3781 base pairs) in those with low physical fitness to 0.95±0.23 (5566±3829 base pairs) in those with high physical fitness (p<.001). This association remained strong after adjustment for numerous patient characteristics, including measures of cardiac disease severity and physical inactivity (p = 0.005). Compared with participants with high physical fitness, those with low physical fitness had 2-fold greater odds of having TL in the lowest quartile . This association was similar after multivariable adjustment . Self-reported physical inactivity was associated with shorter TL in unadjusted analyses, but not after multivariable adjustment.We found that worse objectively-assessed physical fitness is associated with shorter leukocyte telomere length in patients with CHD. The clinical implications of this association deserve further study. Telomeres are tandem repeat DNA sequences (TTAGGG) and associated proteins which form the protective caps at the ends of eukaryotic chromosomes 2max) than 27 endurance-trained adults Some studies suggest that self-reported physical activity is associated with longer leukocyte telomere length among healthy individuals The Heart and Soul Study is a prospective cohort study investigating the influence of psychosocial factors on cardiovascular events in patients with stable coronary artery disease. The enrollment process has been previously described The study protocol was approved by: the University of California San Francisco Committee on Human Research, the Research and Development Committee at the San Francisco VA Medical Center, the Medical Human Subjects Committee at Stanford University, the Human Subjects Committee at the VA Palo Alto Health Care System, and the Data governance Board of the Community Health Network of San Francisco. All participants provided written informed consent. Between September 2000 and December 2002, a total of 1024 participants enrolled in the study. Of these, 944 provided DNA samples and completed exercise treadmill testing.Genomic DNA was isolated according to standard procedures from peripheral blood leukocytes and stored at −70°C. Purified DNA samples were diluted in 96-well microtiter source plates to a fixed concentration of 3 ng/µL. Relative mean telomere length was measured from DNA by a quantitative polymerase chain reaction (qPCR) assay that compares mean telomere repeat sequence copy number (T) to a reference single-copy gene copy number (S) in each sample, as previously described and validated by comparison with Southern blot terminal restriction fragment analysis 5′-CGGTTT[GTTTGG] 5GTT-3′) and tel2b (5′-GGCTTG [CCTTAC]5CCT-3′), used at final concentrations of 100 nM and 900 nM respectively. Human beta-globin gene qPCR primers were hbg1 (5′-GCTTCTGACACAACTGT-GTTCACTAGC-3′), used at a final concentration of 300 nM, and hbg2 (5′-CACCAACTTCATCCACGTTCACC-3′), used at a final concentration of 700 nM. All PCRs were carried out on a Roche Lightcycler 480 real-time PCR machine .The primers for the telomere qPCR were tel1b . The intra-assay coefficient of variability was 2.5% .To determine the conversion factor for the calculation of approximate telomere length in base-pairs  from the T/S ratio, the above method was used to determine the T/S ratios, relative to the same reference DNA, for a set of genomic DNA samples from the human fibroblast primary cell line IMR90 at different population doublings, as well as with the telomerase protein subunit gene (hTERT) transfected via a lentiviral vector construct to elongate the bulk telomere population. For each of these DNA samples the mean TRF length was determined using Southern blot analysis, and the slope of the plot of mean TRF length versus T/S for these samples served as the conversion factor for calculation of telomere length in base pairs from the T/S ratio. The equation for conversion from T/S ratio to base pairs for this study was base pairs = 3274+2413*(T/S). Measurement of leukocyte telomere length was performed in a blinded fashion by Dr. Jue Lin without knowledge of the clinical data.Participants were instructed to fast for at least 4 hours before exercise, except for taking their usual medications. All subjects completed a graded exercise treadmill test according to a standard Bruce protocol, under the direct supervision of a cardiology technician and fellow. To achieve maximum heart rate, subjects unable to continue the standard Bruce protocol (for orthopedic or other reasons) were switched to slower settings on the treadmill and encouraged to exercise for as long as possible. Continuous electrocardiographic monitoring was performed throughout exercise. The only indications for stopping were those required for safety  and duration of exercise, as total number of METs achieved at peak exercise (1 MET = 3.5 mL/kg/min of oxygen consumption). We evaluated exercise capacity both as a continuous variable and as a categorical variable, defined as low (<5 METS), moderate (5–7 METS) or high (>7 METS) exercise capacity To assess self-reported physical activity, we asked, “Which of the following statements best describes how physically active you have been during the last month; that is, done activities such as 15–20 minutes of brisk walking, swimming, general conditioning, or recreational sports?” Participants chose from one of the following 6 categories: not at all active, a little active (1 to 2 times per month), fairly active (3 to 4 times per month), quite active (1 to 2 times per week), very active (3 to 4 times per week), or extremely active (5 or more times per week). We evaluated physical activity both as a 6-point continuous variable (corresponding to the above categories) and as a binary variable. For the binary variable, participants who reported that they were not at all or a little active were considered physically inactive; all other participants were considered physically active.We conducted annual telephone follow-up interviews with participants (or their proxy). Death was determined by review of death certificates and coroner's reports.Age, sex, race, education, smoking, alcohol use, and medical history were determined by self-report questionnaire. We measured height and weight and calculated body mass index. Participants brought all of their medication bottles to the study appointment, and study personnel recorded all current medications. Depressive symptoms were assessed using the 9-item Patient Health Questionnaire, and depression was defined as a score of ≥10 on this questionnaire Baseline characteristics of participants were compared across categories of exercise capacity using ANCOVA for continuous and chi-squared tests for dichotomous variables. We used generalized linear models to calculate mean telomere length (T/S ratio) in patients with low, moderate and high exercise capacity. Models were adjusted for differences in patient characteristics associated with exercise capacity. Logistic regression models were used to evaluate the association between exercise capacity as a continuous variable and short telomere length as a binary variable (defined as having telomere length in the lowest quartile). Finally, we used Cox proportional hazards models to evaluate the association between exercise capacity and mortality, with and without adjustment for telomere length. We verified the proportional hazards assumption of these models. Analyses were performed using SAS Version 9.2 .Of the 944 participants, 229 had low (<5 METS), 334 had moderate (5–7 METS) and 381 had high (>7 METS) exercise capacity . ParticiLower exercise capacity was significantly associated with shorter mean telomere length , Table 2Participants who reported being physically inactive had a mean telomere length (T/S ratio) of 0.88 (5397 base pairs), while those who reported being physically active had a mean telomere length of 0.91  . After aSelf-reported physical inactivity was not independently associated with short telomere length . HoweverDuring an average of 6.27+/−2.11 years follow-up, each SD decrease in METS was associated with a two-fold increased risk of mortality . This association was unchanged after adjustment for telomere length, sex, race, education, comorbid conditions, LV ejection fraction, use of cardioprotective medications, BMI, physical inactivity, and depression . Likewise, each SD increase in baseline telomere length was associated with an 18% decreased risk of mortality , but this association was unchanged after adjustment for exercise capacity .We found a strong association between physical fitness and telomere length in 944 patients with CHD. After adjustment for other patient characteristics, including markers of cardiac disease severity and physical inactivity, participants with low exercise capacity (<5 METS) had a 94% greater odds of having short telomere length than those with high exercise capacity (>7 METS). In addition, participants with low exercise capacity had shorter mean telomere length than those with high exercise capacity . This is equivalent to a difference of 169 base pairs (5325 vs. 5494 base pairs). Given that telomeres in this population decrease at an average rate of approximately 42 base pairs/year Our study demonstrates for the first time a strong relationship between physical fitness and telomere length in a large sample of patients with existing CHD, after controlling for many other patient characteristics, such as severity of CHD. We extend the findings of previous studies on cardiovascular disease and telomere length The causal direction of the association between fitness and telomere length cannot be determined by our analyses. Short telomeres could reduce exercise capacity by decreasing the function of the cardiovascular system, an idea supported by the fact that functional telomeres are required for viability of cardiovascular cells in-vitro and that deficient telomeres have been shown to cause cardiovascular disease in mice Several studies have suggested mechanisms by which physical inactivity may lead to shorter telomere length. The strongest evidence comes from research by Werner et al. showing that inactivity in mice alters the protein complexes that regulate leukocyte telomere length and structure. In this study, mice were randomized to running or no running wheel conditions for 3 weeks. The sedentary mice then showed lower telomerase activity, lower expression of telomerase reverse transcriptase (TERT), and lower telomere-stabilizing telomere repeat binding factor TRF2 compared with the aerobically conditioned mice Previous studies have shown that both exercise capacity and telomere length are powerful independent predictors of mortality among men with cardiovascular disease Among the strengths of the present study is the characterization of numerous clinical, biological, and psychosocial covariates that enable us to exclude possible confounders. However, our study has several limitations that should be considered in the interpretation of our results. First, the association reported in this study is cross-sectional, so no conclusions can be reached regarding causality. Next, no genetic polymorphisms were analyzed in our study, but genetics are known to impact telomere length in people with coronary artery disease In summary, our study shows a strong relationship between exercise capacity and telomere length in a population of patients with stable coronary heart disease. The association between self-reported physical activity and telomere length became non-significant after multivariable adjustment. Whether poor physical fitness leads to shorter telomeres, or vice versa, and whether common genetic or other factors may reduce both telomere length and exercise capacity, deserve further study.


Breast cancer is the second most common cause of death for women behind lung cancer and the most common cause of cancer deaths for women aged 45–55 years old (CDC.gov 2012). Although there continue to be enormously large numbers of disease incidence, deaths have been declining due to the disease with two hallmark time frames. The first occurred during the mid to late 1980’s when hormonal therapy was introduced as a treatment for ER/PR positive breast cancer. The second occurred in the late 1990’s when trastuzumab was introduced in treating HER2 positive breast cancer. These remarkable accomplishments in developing novel targeted therapies for breast cancer, along with a better understanding of the disease biology have improved disease outcome over the past 20 years.This article reviews the data presented at 2012 American Society of Clinical Oncology and 2012 San Antonio Breast Cancer Symposium regarding progress made in the field of HER2 positive breast cancer and examines the future of HER2 targeted therapy. The HER2 gene (ErbB2) is a member of a group of epithelial tyrosine kinase receptors ,2. TheseHER2 has no identified ligand, which allows it to always be in open confirmation to dimerize with HER1, HER3, or HER4 . The devBriefly, trastuzumab is a humanized monoclonal antibody directed against the extracellular domain of the HER2 receptor which prevents ligand-independent HER2 signaling. It was initially approved by the FDA for metastatic breast cancer in 1998  is an immunoconjugate agent combining trastuzumab with an antimicrotubule cytotoxic chemotherapeutic agent linked together by a covalent bond . In PhasP = 0.035)  . The druTo assist in addressing this issue, there is an ongoing phase III study of patients, previously treated with trastuzumab, which is evaluating vinorelbine plus afatinib versus vinorelbine plus trastuzumab . The PhaBesides this phase III trial, there are three other phase II studies that were presented.Firstly, Hickish et al. presented a phase II study (LUX-breast 2) evaluating the objective response of afatinib at dose of 40 mg/day in metastatic breast cancer patients who progressed on prior trastuzumab or lapatinib as single-agent followed by afatinib ‘beyond progression’ with chemotherapy (weekly Paclitaxel 80 mg/m2 or Vinorelbine 25 mg/m2) . 120 patSecondly, LUX-breast 3 trial presented by Joensuu et al. is an ongoing phase II study, which randomizes HER2 positive breast cancer patients with progressive brain metastases (after trastuzumab or lapatinib based therapy) to single-agent afatinib versus afatinib + vinorelbine versus the investigator’s choice of approved therapy . The stuLastly, Swanton et al. presented a phase II trial evaluating the efficacy and safety of afatinib in inflammatory breast cancer . This st50 values (concentration which inhibits 50% of growth). This study showed significant benefit with neratinib as a single agent and in combination with trastuzumab in both trastuzumab sensitive and lapatinib resistant cell lines  . This drll lines . It was A phase I NSABP study (FB-8) by Jankowitz et al. evaluated weekly paclitaxel with neratinib and trastuzumab in women with metastatic HER2 positive breast cancer. It showed good tolerability and activity in patients pre-treated with anti-HER2 therapy and chemotherapy . This stThe results of an interesting trial [TBCRC 022] presented at the San Antonio Breast Conference looked at Neratinib in CNS disease. Pre-clinical data suggests it may cross the blood brain barrier . This phIn this article, we reviewed studies presented at 2012 American Society of Clinical Oncology and 2012 San Antonio Breast Cancer Symposium on the development of HER2 positive breast cancer treatments. Although great strides have been made in the treatment of breast cancer and in particular, HER2 positive breast cancer, the disease has not been conquered. There are diverse potential mechanisms that make the currently FDA approved HER2 targeted agents, trastuzumab, lapatinib, pertuzumab, and T-DM1 to be refractory. One of the many challenges we struggle with are the multiple pathways of resistance involved. We seek to discover ways in which the combination therapy may assist in blocking these pathways. Many new agents, some discussed in this paper, present new and exciting strategies to combat HER2 positive breast cancer, which provide additional safe and effective treatment options, either as single agents or in combination regimens. Equally as important, further investigation is ongoing in evaluating biological markers to assist in tailoring treatments to the individual and not just to the disease. Identification of potential biomarkers would provide the clinician valuable information with regard to response and clinical benefit -56. ManyIn order to better understand HER2 positive breast cancer, we need further dynamic and innovative clinical trial designs in the form of basic, translational, and clinical research to improve patient outcomes in drug targeted HER2 positive breast cancer. For example, T-DM1 was recently approved by the FDA as second line metastatic therapy. Can T-DM1 be given as a first line agent given its efficacy and low toxicity profile? What about evaluating dual anti-HER2 therapy, such as T-DM1 plus pertuzumab as opposed to T-DM1 alone in the neoadjuvant and adjuvant settings? How effectively does neratinib overcome resistance with combination therapy with, for instance, trastuzumab, pertuzumab or T-DM1? Such pertinent questions are being evaluated by ongoing and future trials and need to be addressed, along with many others by researchers.We foresee that in the future, HER2 breast cancer will no longer be lumped as a single entity in breast cancer, but a multiple different subsets within a single entity of HER2 breast cancer. The ultimate purpose is to be able to customize drug therapy based on a variety of clinical and translational research, and to optimize drug treatment for HER2 positive breast cancer.The authors declare that they have no competing interests.JI, SS, YM and JL drafted the manuscript. All authors read and approved the final manuscript.


Reactive oxygen species (ROS) are essential for survival but also pose serious risks to that survival. A particularly striking example was the demonstration in 2003 by the Campisi group that primary mouse fibroblasts have an indefinite proliferative lifespan in 3% oxygen, the amount found in the capillaries feeding the tissues, but greatly shortened ones under normal in vitro culturing conditions, i. e., 20% oxygen . Now, thROS may be produced in a regulated manner during cellular metabolism but they can also arise in an unregulated manner by metabolic dysfunctions and by exogenous stresses. ROS production is typically localized in cellular compartments by NAD(P)H oxidases, lipoxygenases, cyclooxygenases among others as well as by the mitochondrial electron-transport chain . At physHowever, compromised homeostatic pathways lead to elevated ROS levels that may result in the damage of cellular components . A growing body of evidence implicates oxidative stress in both aging and a wide spectrum of human diseases including diabetes, atherosclerosis, hypertension, cancer, cardiovascular diseases and neurodegenerative diseases among others .2−) to hydrogen peroxide (H2O2), which is in turn, converted to molecular oxygen and water by catalase and peroxiredoxins. In this Aging paper, the Campisi group showed that normal aged mice exhibit impaired mitochondrial complex II activity and increased frequencies of senescent cells. They observed similar phenotypes in SOD2 knockout (KO) mice at very young ages and also reported that these mice exhibit significant epidermal thinning which is an age-associated phenotype in humans as well as mice. The epidermal thinning appeared to be due to decreased cellular proliferation in the senescent skin tissue rather than increased apoptosis. In addition, senescence in SOD2 KO mouse skin correlated with increased levels of two senescence markers, SA-βgal activity and the p16INK4a tumor suppressor, as well as amplified keratinocyte terminal differentiation ). SOD2 is a mitochondrial matrix protein that converts the superoxide anion (OSimilar to the observations made in mice, other studies in yeast and Drosophila also show a predominant role for SOD2 in increasing the lifespan -9. HowevOther studies failed to find a direct link between SOD2 and aging. A study with mice showed that SOD2 overexpression, while resulting in decreased lipid peroxidation, did not alter either lifespan or age-related pathology . In the 2− and H2O2 generation were inversely correlated to maximum life span potential with shorter-lived species producing relatively higher amounts of ROS [The shorter lifespan observed in SOD2 KO mice compared to wild-type and heterozygous mice may be reminiscent of previous studies showing that rates of mitochondrial Os of ROS .Because SOD2 plays a role in aging in mice, it would be interesting to look if a correlation exists between age-related pathologies and SOD2 alterations in human. For example, mitochondrial dysfunction was shown to contribute to the pathogenesis of aging-related neurodegenerative diseases . Moreove


Metastasis is a multistep process characterized by the ability of tumor cells to “communicate” and to interact with their microenvironment to establish tumors in distant organs. A significant proportion of the metastatic microenvironment consists of leukocytes, mostly of the innate immune system, contributing to tumor invasion and outgrowth. Chemokines are instrumental for recruiting immune cells thereby enabling efficient metastasis . Still, in vivo and in vitro. Moreover, blocking of signaling pathways downstream of CCR2 significantly reduced metastasis, supporting the notion that endothelial CCR2 “licenses” colon cancer extravasation. Consequently, our findings define an additional role for chemokines in enabling metastasis that goes beyond the attraction of inflammatory leukocytes.Still, the different roles of CCL2 and CCL5 during metastasis are just at the beginning to be defined. In the tumor microenvironment, stromal cells, infiltrating leukocytes and tumor cells themselves were identified as sources of chemokines. During the initial phase of metastasis, local activation of endothelia by tumor cells induced endothelial CCL5 expression, resulting in monocyte recruitment and promotion of metastasis . Recentl(1)Do other cancer cells also use chemokine-chemokine receptor interactions to accomplish tumor cell extravasation?(2)Can we use chemokine expression analysis in primary tumors of patients to predict increase risk for metastasis?(3)What other chemokine-chemokine receptor pairs exist, having the same or other biological function as CCL2-CCR2?(4)What do we know about the cellular and spatiotemporal denominators of CCL2-induced tumor cell extravasation?We confirmed that metastasis of another tumor cell line, Lewis lung carcinoma (3LL), is also dependent on CCL2 expression. In contrast, the melanoma cell line B16-BL6 could extravasate and metastasize independently of endothelial CCR2 expression. Interestingly, this cell line expresses only little CCL2. Our findings raise several questions:Consequently, further experiments will show how general our recently reported findings are and whether other chemokine receptors on endothelial cells exert similar functions. We hypothesize that other functions of tumor cell-derived chemokines will be identified in the future, surpassing their known role as immune cell attractors, as the currently identified contribution to induced vascular permeability.