This study aimed to evaluate the impact of genetic notification of smoking-related disease risk on smoking cessation in the general population. Secondary objectives were to assess the impact of genetic notification on intention-to-quit smoking and on emotional outcomes as well as the understanding and the recall of this notification.A systematic review of articles from inception to August 2011 without language restriction was realized using PubMed, Embase, Scopus, Web of Science, PsycINFO and Toxnet. Other publications were identified using hand search. The pooled-analysis included only randomized trials. Comparison groups were (i) high and low genetic risk versus control, and (ii) high versus low genetic risk. For the pooled-analysis random effect models were applied and sensitivity analyses were conducted.Eight papers from seven different studies met the inclusion criteria of the review. High genetic risk notification was associated with short-term increased depression and anxiety. Four randomized studies were included in the pooled-analysis, which revealed a significant impact of genetic notification on smoking cessation in comparison to controls  in short term follow-up less than 6 months .In short term follow-up, genetic notification increased smoking cessation in comparison to control interventions. However, there is no evidence of long term effect (up to 12 month) on smoking cessation. Further research is needed to assess more in depth how genetic notification of smoking-related disease could contribute to smoking cessation. Smoking is a major public health problem worldwide and the most preventable cause of mortality and morbidity. It increases the risk of many diseases such as lung cancer, chronic obstructive pulmonary diseases and cardiovascular diseases Common diseases are highly dependent on multiple environmental and genomic factors. Both the cigarette consumption and the allele frequencies vary substantially between populations. Multiple single nucleotide polymorphisms (SNPs) are needed for the assessment of each specific smoking-related disease risk. Generally speaking, testing multiple SNPs for diverse smoking-related disease risks will identify smokers to be at higher risk of at least one disease. Different genes seem to be of interest in cancer risk prediction, among them: GSTM1, GSTT1, CYP2D6, L-myc, NQO1, and CYP1A1 Risk communication and health literacy are complex issues dealing with the use, the understanding and the recalling of a notification by the patient Studies reporting the impact of genetic notification on smoking cessation have been conflicting, which could, among other reasons, suggest that it is not a strong motivator of behavioral change.The impact of genetic notification can be either explored in a real or a hypothetical situation. In hypothetical genetic testing, the anticipated reactions of smokers are assessed in view of a hypothetical genetic risk of smoking-related disease. The outcome of interest is intention-to-quit smoking, which is an important precursor of quit attempts that lead to smoking cessation The primary objective of this systematic review and pooled-analysis was to determine the impact of genetic notification on smoking cessation in the general population. We addressed this by the following questions:Is genetic notification of smoking-related disease risk influencing smoking cessation success rate in comparison to clinical notification of smoking-related disease risk (e.g. blood pressure and cardiovascular diseases) or no notification?Is high genetic risk notification of smoking-related disease risk influencing smoking cessation success rate in comparison to low genetic risk notification?The secondary objectives were to review the impact of genetic notification on intention-to-quit smoking and emotional outcome as well as to determine to which extend smokers really understand and recall their genetic notification.First, we conducted a systematic review. Then we carried out further quantitative assessment only on the primary outcome (smoking cessation) by a pooled analysis. For this systematic review and pooled-analysis, we followed the Quality of Reporting of Systematic reviews and meta-analyses (PRISMA) guidelines (PRISMA Flow Diagram S1 and PRISMA Checklist S1) Regarding the systematic review, we included studies incorporating smokers of any age receiving genetic notification of smoking-related disease risk in prospective studies . The only exclusion criterion was studies involving hypothetical genetic notification as intervention.The intervention of interest was the genetic notification of smoking-related disease risk. Notification based on one gene was stratified in high and low genetic risk based on dominant or recessive genetic model.The primary outcome was smoking cessation. To assess the impact of genetic notification, we collected smoking cessation rates at each follow-up that was presented in the studies. Smoking cessation could be biochemically confirmed  or self-reported. The outcomes in the selected studies included prolonged (continuous abstinence during a follow-up period) and point prevalence  cessation rates. Only one study presented a sustained smoking cessation Secondary outcomes were: (a) intention-to-quit smoking; (b) emotional outcome ; (c) recall and understanding of the genetic notification.We searched PubMed, Embase, Scopus, Web of Science, PsycINFO, and Toxnet for studies published until August 2011 without language restriction using the following terms: smoking cessation, genetic testing, and genetic predisposition to disease. The search strategy is available in the supporting information documents (PubMed research S1). In addition, we manually reviewed the reference list of relevant articles and reviews.Two authors (SDV and JVDH) independently screened for title and electronic abstracts identified by the search for relevance to the inclusion criteria. Articles retrieved from this examination were full text screened by the same authors. Reasons for excluding studies were noted . Data weFrom each eligible paper of the systematic review, we extracted information, where available, on first author, year of publication, country, study setting, study design, year of the recruitment, inclusion and exclusion criteria, sample size , description of interventions, characteristics of participants , outcomes , and length of follow-up.Methodological quality and potential risk of bias were assessed using the following criteria: selection criteria clearly described, sample size calculation, adequate allocation concealment for randomized trials, comparability of groups at baseline, presentation of the Hardy-Weinberg Equilibrium ; intention-to-treat analysis, ascertainment of outcome, and control for confounding.In the systematic review, we realized comparisons of sample sizes between studies, genetic risk and gene to understand the difference in the relative proportion of smoking cessation between studies. This was effectuated using Pearson Chi’s square test. Studies included in these analyses were first randomized and non randomized trial Individual study risk ratios (RR) and binomial 95% confidence intervals were computed from event numbers extracted from each study.2 statistic, with a value of 50% or more indicating a substantial level of heterogeneity For the pooled-analysis, we limited included studies to randomized trials for their ability to minimize likelihood of systematic error In studies that had more than two arms, we collapsed arms to obtain one intervention and one control group . Tests were two-sided with a significance rate α of 0.05. All statistical analyses were performed using STATA, version 10.1 .The selection of studies included in our review is summarized in Participants were aged from 18 to 88 years old. The percentage of females was around 50% except in two studies where there were only 6.2% and 14.0% females Regarding the study design, four studies were randomized trials The disease of interest was always cancer. In six studies risk notification was based on a single-gene test  Egger’s two tailed p-value showed no significant publication biases for short follow-up as well as the last follow-up of main analyses (genetic notification versus control) .The impact of genetic notification on smoking cessation was conflicting among randomized and non randomized studies 2 range from 0.0 to 42.9%, p range from 0.17 to 0.54) except a substantial heterogeneity in the low genetic risk notification versus control group .The pooled analysis indicated that, when considering the last follow-up, there were no significant differences in smoking cessation between the following subgroups: genetic notification versus control, low genetic risk notification versus control, and high genetic risk notification versus low-genetic risk  1.03 (0.64–1.65); 0.97(0.33–2.88); 1.48(0.74–2.95)) . However2 = 0.0% for the two analyses). Sensitivity analyses did not identify influential studies.When focusing only on short-term smoking cessation outcome (2 or 6 month), the borderline effect of high genetic risk versus control was still visible (RR = 1.55 (0.94–2.58)). Moreover, genetic notification increased 1.55 times smoking cessation in comparison to control (RR = 1.55 (1.09–2.21) . These tSecondary outcomes were available for most of the studies but not all of them.(a) Intention-to-quit. Six studies observed intention-to-quit smoking after a genetic notification of smoking-related disease risk (b) Emotional outcome. Seven studies reported emotional outcome  (c) Recall and understanding. Smokers seemed to reasonably understand the meaning of their genetic test results Our systematic review indicated that few studies have assessed the impact of genetic notification on smoking cessation. Only 8 papers based on 7 different studies were available and just 4 of them were included in the pooled-analysis.The pooled-analysis suggested a short-term increase of smoking cessation for participants receiving genetic notification in comparison to control group and a borderline increase of smoking cessation for high genetic risk smokers in comparison to control. No evidence of heterogeneity was observed for these results even if (i) characteristics of participants, (ii) inclusion criteria and (iii) study designs differed among studies. For example, (i) included participants were not necessarily interested in smoking cessation. Furthermore, participants were not necessarily from the same ethnicity, although mixing ethnicities in genetic studies could lead to population stratification. The majority of studies did not report HWE calculation. (ii) Inclusion criteria differed in number of CPD and none of the studies met the criterion of nicotine addiction, often defined as smoking more than 10 CPD for at least 12 months The reason why some results of the pooled-analysis were significant and others were not remains open to interpretation. The impact of genetic notification is likely to vary with individual characteristics , with the distribution of high and low genetic risk notification, with the way in which the genetic notification is done , the intervention in the control group, and the length of the follow-up. Due to the low number of included studies, it was not possible to stratify the pooled-analyses by time of follow-up . Stratification of the results before and after 6 months follow-up should be also of interest. However, after 6 months there were only 2 studies available for genetic notification versus control In a recent meta-analysis the same primary outcome was studied, but there were differences in the method that was used Genetic notification did not influence intention-to-quit smoking, except in one study reporting motivation to quit at one week follow-up Most studies were testing only one single gene to determine smoking-related disease risk. This posed also ethical questions because of the uncertainty of disease risk which is enhanced when using only single gene test in common diseases.Genetic notification is one possible intervention among others. At the individual level, the most well-known one are the pharmaceutical interventions . However, nicotine dependence is not only a physical dependence but also a behavioral and a psychological dependence. Consequently, interventions might take into account these 3 types of dependence . Other interventions are also available at the household level (e.g. smoke-free home and partner support) and the society level . As the evidence for benefit of these interventions is strong and well-established, it is incumbent upon genetics to demonstrate additional benefit Regarding the pooled-analysis, the most important limitation was the low number of included studies, which did not allow us to determine whether the risk varied with particular conditions . Moreover, adjusted RRs were rarely presented in the included studies, which prevented control for confounding factors. Another limitation was that interventions in the control groups were not similar in the different studies included in the pooled-analysis: two studies had a control group without any intervention Finally, limitations are also due to the heterogeneity between the included studies. This is due to the diversity in the inclusion criteria. For example, the mean number of CPD ranged from 15.5 The results from this study suggest that genetic notification of smoking-related disease risk could have a positive impact on smoking cessation, particularly in short-term follow-up. To determine the possible implications for practice, further research of the impact of genetic notification on smoking cessation is needed. There is also need to investigate the cost-effectiveness of this intervention. Studies should (i) focus on smokers that want to quit smoking, (ii) focus on population of regular smokers by level of severity of nicotine addiction, (iii) use combination of genetic tests for a single or multiple smoking-related diseases, (iv) standardize different concepts  to minimize the heterogeneity and risk of bias between studies.Table S1Quality assessment of studies included in the systematic review of genetic notification. Quality assessment of the studies included in the systematic review and the pooled-analysis.+ Yes; - No or not reported; ? unclear;/Not applicable; 1No explanation on how the randomization was processed; 2Significant difference in desire to quit at baseline (but not corrected for multiple testing); 3Not even try to confirm biochemically the smoking status.(DOC)Click here for additional data file.PubMed research S1PubMed research. PubMed research used regarding the article selection.(DOC)Click here for additional data file.PRISMA Flow Diagram S1PRISMA flow diagram. PRISMA 2009 flow diagram regarding the article selection.(DOC)Click here for additional data file.PRISMA Checklist S1PRISMA Checklist. PRISMA 2009 checklist regarding the pooled-analysis data and position in the manuscript.(DOC)Click here for additional data file.


This coding strategy has been verified so far in visual and auditory cortices of monkeys at low frequencies [In the quencies ,2. Here quencies ,2, the eWe then examined whether the pure spiking activity contributes in the information manifested in the high frequency PoF components. The temporal precision of spikes was quantified using a novel measure, information in the phase of the band-bass filtered spike trains. In all depths, the spikes lacked any temporal-resolution larger than 30 Hz Figure . We conc


LA also increases nerve conductance, improves diabetes-induced polyneuropathies, and remediates the age-associated cognitive decline in canines. Furthermore, LA significantly improves hypertriglyceridemia and glucose handling. From our pre-clinical research we have found that LA primarily influences three areas of health: cognition, stress response, and lipids. This study examines the effects of LA on human subjects in components of each of the three aforementioned areas of health.Lipoic acid (LA) shows promise as a beneficial micronutrient in improving health, particularly in the elderly. Clinical and This study utilized acute treatments with LA supplements as well as chronic supplementation in an 8 week, double-blind placebo-controlled cross-over trial in human volunteers. The subjects were grouped into young (18-45 years) or elderly (~79 years), and all subjects were administered the R-enantiomer of LA in the form of oral supplements. This was a small pilot study with 2-6 subjects in each experiment.Results from the backward letter span and paired associates cognitive tests indicate that LA improved verbal episodic memory in the elderly, but did not improve short-term memory in the young or elderly. Chronic LA altered stress response systems as indicated by a transient increase in salivary cortisol and aldehyde dehydrogenase 3A1. Chronic LA also increased the amount of cholesterol taken up by high-density lipoproteins, particularly in the elderly subjects.LA may be useful as a complementary nutraceutical agent to improve cholesterol efflux and select memory processes, particularly in the elderly. Continuous supplementation with LA induces a transient catabolic state, as well as a transient stress response. Thus, LA may function as a hormetic agent by inducing an initial stress that primes the system to efficiently respond to future toxicological insult.


Mnemiopsis leidyi.An important question in experimental embryology is to understand how the developmental potential responsible for the generation of distinct cell types is spatially segregated over developmental time. Classical embryological work showed that ctenophores, a group of gelatinous marine invertebrates that arose early in animal evolution, display a highly stereotyped pattern of early development and a precocious specification of blastomere fates. Here we investigate the role of autonomous cell specification and the developmental timing of two distinct ctenophore cell types (motile compound comb-plate-like cilia and light-emitting photocytes) in embryos of the lobate ctenophore, Mnemiopsis, 9 h after fertilization, comb plate cilia differentiate into derivatives of the E lineage, while the bioluminescent capability begins in derivatives of the M lineage. Arresting cleavage with cytochalasin B at the 1-, 2- or 4-cell stage does not result in blastomere death; however, no visible differentiation of the comb-plate-like cilia or bioluminescence was observed. Cleavage arrest at the 8- or 16-cell stage, in contrast, results in the expression of both differentiation products. Fate-mapping experiments indicate that only the lineages of cells that normally express these markers in an autonomous fashion during normal development express these traits in cleavage-arrested 8- and 16-cell stage embryos. Lineages that form comb plates in a non-autonomous fashion (derivatives of the M lineage) do not. Timed actinomycin D and puromycin treatments show that transcription and translation are required for comb formation and suggest that the segregated material might be necessary for activation of the appropriate genes. Interestingly, even in the absence of cytokinesis, differentiation markers appear to be activated at the correct times. Treatments with a DNA synthesis inhibitor, aphidicolin, show that the number of nuclear divisions, and perhaps the DNA to cytoplasmic ratio, are critical for the appearance of lineage-specific differentiation.In Our work corroborates previous studies demonstrating that the cleavage program is causally involved in the spatial segregation and/or activation of factors that give rise to distinct cell types in ctenophore development. These factors are segregated independently to the appropriate lineage at the 8- and the 16-cell stages and have features of a clock, such that comb-plate-like cilia and light-emitting photoproteins appear at roughly the same developmental time in cleavage-arrested embryos as they do in untreated embryos. Nuclear division, which possibly affects DNA-cytoplasmic ratios, appears to be important in the timing of differentiation markers. Evidence suggests that the 60-cell stage, just prior to gastrulation, is the time of zygotic gene activation. Such cleavage-clock-regulated phenomena appear to be widespread amongst the Metazoa and these cellular and molecular developmental mechanisms probably evolved early in metazoan evolution. Ctenophores are a monophyletic group of seemingly simple marine animals with distinct features, such as unique comb rows, body symmetry and stereotypic cleavage program-11. The Mnemiopsis leidyi, generate distinct light-producing photocytes during development, which migrate to locations subjacent to each of the eight comb rows. Genome sequencing of Mnemiopsis has revealed two genomic clusters containing ten distinct copies of photoproteins closely resembling the luciferase-type photoproteins found at the base of the Metazoa. In situ hybridization studies have shown that at least subsets of these mRNAs are expressed in photocytes prior to when these embryos are bioluminescent[Many ctenophores, including the lobate ctenophore inescent. Thus, tMnemiopsis leidyi, embryos are generally fertilized at the time of spawning and cleavages are unipolar and holoblastic  towards the aboral pole and one M macromere towards the oral pole[1, e2 and e3) at the aboral pole and an E macromere at the oral pole[Ctenophore development is highly stereotypic and unique within the animal kingdom,4,5. In  Figure [4-6,10,ral pole. E blastral pole. The micral pole.1 micromeres give rise to the comb plate cilia[1 micromere’s contributions to the formation of comb plates  to block cytokinesis (but not nuclear division), we show that the formation of differentiation markers associated with each of these cell types appears only after they have segregated into their own distinct lineages and they are never co-expressed in the same cells. Furthermore, using inhibitors of protein synthesis, transcription and DNA synthesis, we provide evidence for the existence of a cleavage clock that is based on the number of rounds of DNA synthesis (and possibly controlled by the nuclear-to-cytoplasmic ratio), which determines the temporal appearance of differentiation products in cleavage-arrested embryos.Here, we investigate the role of the cleavage program in the segregation and expression of the developmental potential of two distinct cell types (motile comb-plate-like cilia and light-emitting photocytes) during the development of the ctenophore Mnemiopsis leidyi adults were collected from the National Oceanic and Atmospheric Administration jetty and Eel Pond in Woods Hole, MA, during June and July and from 13660 Deering Bay Dr, Coral Gables, FL 33158, during December. Adults were brought into the lab and induced to spawn as described previously[eviously. Eggs weAfter the embryos were collected, they were transferred by pipet to 24-well culture dishes for drug treatments. The following drugs were used: cytochalasin B , actinomycin D , puromycin  and aphidicolin . Stock solutions of cytochalasin (1 mg/ml), actinomycin (1 mg/ml) and aphidicolin (1 mg/ml) were made up in dimethyl sulfoxide, with aliquots stored at -20°C. A puromycin stock solution (12.5 mg/ml) was made up in distilled water and stored at -20°C. Freshly thawed aliquots were used in each experiment. Working solutions were made by diluting stock solutions with filtered seawater. Approximately 1 ml of solution was added to each well containing embryos.The embryos were fixed for antibody staining in 4% paraformaldehyde and 0.02% glutaraldehyde, as previously described by Pang and Martindale. FollowiAfter the embryos were collected, the vitelline membranes were mechanically removed using sharpened forceps. The de-membranated embryos were allowed to develop to the correct cleavage stage prior to injection with DiI,  a lipophilic membrane stain that diffuses laterally to stain the entire cell, as described in. FollowiMnemiopsis development was recorded at three images per minute using DIC . Visualization of bioluminescence was performed on an Axio Scope, using the GFP filter  to stimulate photoprotein emission and with 0.5 to 1 sec exposure times with an AxioCam Mr to capture faint signals. Prior to visualization, an embryo was kept undisturbed in darkness for 10 to 20 min to prevent activation and depletion of the photoprotein.The embryos were scored for the presence or absence of comb cells using a Stereo Discovery  or Axio Scope  under transmitted light. Live comb plate cells were imaged using time-lapse microscopy with time intervals of five to ten images per second. Normal Confocal imaging after immunohistochemical staining was performed using a Zeiss 710 confocal microscope. Images were processed using Zen software  and Volocity  to create 3D image reconstructions of confocal sections.Mnemiopsis development is shown in Figure Cytochalasin B inhibits the polymerization of actin and thus prevents cytokinesis. During embryonic development, individual cleavages and specific developmental stages can be arrested by cytochalasin B, and thus the developmental potential of cells that were present at the time of arrest can be analyzed. We used cytochalasin B on zygotes, the 2-, 4-, 8- and 16-cell stages and at later time points up to the 60-cell stage . An overview of Mnemiopsis embryos led to the immediate arrest of cytokinesis. If the embryo was undergoing cell division at the time of treatment, the cleavage furrow immediately began to regress and the blastomeres rounded up  before arresting them at the 16-cell stage , were the cells producing the comb-plate-like cilia labeled , with 10 μg/ml of actinomycin D, an inhibitor of transcription,29. EmbrIt is interesting to note that embryos that were treated at the 1-, 2-, 4-, 8- and 16-cell stages continued to divide normally and arrested only prior to gastrulation at about the 60-cell stage. Embryos that were treated with actinomycin D at the 32- or 60-cell stage divided only a few more times and then became arrested . Comb foCells producing ctene-like cilia and photocytes are formed at the same time in control embryos and in embryos treated with cytochalasin B at the 8-cell stage or later, suggesting that the embryo has a molecular timing mechanism that operates in the absence of cytokinesis. Because nuclear divisions appear to proceed normally in embryos treated with cytochalasin B Figure , the numMnemiopsis leidyi embryos, treated embryos do not undergo apoptosis, but rather nuclear divisions continue and give rise to multinucleated cells. Cytochalasin B was used to study the segregation of developmental potential in Mnemiopsis by Freeman[We used cytochalasin B to inhibit cytokinesis in early ctenophore embryos to examine how developmental potential is partitioned into two distinct cell lineages: those giving rise to comb plates and those forming the photocytes. When cytokinesis is arrested in early  Freeman. Freeman Freeman inhibite Freeman. However Freeman.1 micromeres at the 16-cell state. These are the same cell lineages that autonomously generate comb-plate-like cilia when isolated from the rest of the embryo[1 micromere descendants also have the capacity to make comb-plate-like cilia[1 micromeres require inductive signals first from the e11 or e12 micromeres and later from endomesodermal cells derived from E and M macromeres before they form combs[1 micromeres require additional inductive signals from endomesodermal cells for comb formation, since no combs were formed by m1 micromeres in cleavage-arrested embryos. This supports the finding that the presence of e1 micromeres is not sufficient to induce comb formation in m1 micromeres[Surprisingly, as we demonstrate here, if cytokinesis is arrested at the 8-cell stage or later and is permitted to remain arrested for the following hours of development, comb-plate-like cilia and the photoprotein-mediated bioluminescence characteristic of photocytes form at the correct time in development compared with control embryos. Lineage-tracing experiments reveal that comb-plate-like cells are derived from E cells at the 8-cell stage and from the eke cilia but M lirm combs,20. Our cromeres,20.Interestingly, neither comb-plate-like cilia nor bioluminescence was detected in cleavage-arrested zygotes or 2- or 4-cell stage embryos, suggesting that the segregation of distinct E and M lineages is crucial for the expression of the lineage-specific differentiation products. These results suggest that factors that are required for comb-plate and photocyte determination are already present and localized at the 8- and 16-cell stages and may have mutually exclusive activity.One of the fundamental processes of development is the localization of factors that are required to establish cell polarity, break symmetry and drive the asymmetric specification of cell fates. Experimental embryologists have provided evidence for the segregation of factors that determine specific cell fates in a diverse variety of different organisms (examples are summarized in-34). In Clytia hemisphaerica [In ctenophore embryos, all evidence suggests that the asymmetrical localization of developmental potential is actively segregated by the cleavage process itself. Each of the early cell divisions leads to a definite asymmetric cell fate. The site of first cleavage gives rise to the oral-aboral axis. Interesegarium). Freemanegarium) showed tegarium),54. In cegarium)). Starfiegarium),57. Baseegarium). HoweverIn ctenophores, the second division gives rise to the anal axis,15,19. TCaenorhabditis elegans, the sperm-derived centrosome, a complex of several proteins that acts as the microtubule-organizing center, breaks the symmetry of the oocyte and sets off a series of events that relies on maternally deposited proteins and eventually leads to the establishment of the anterior-posterior, dorsal-ventral and left-right body axes[Ilyanassa[Mnemiopsis, where distinct developmental fates are decided at each of the early divisions.Although we have little information on the cell biological nature of the segregation process in ctenophores, centrosomes have been shown to be a causal factor in the segregation of factors that specify cell fates in other systems. In ody axes,59. A suIlyanassa. These mIlyanassa. ExperimIlyanassa,61. It wMnemiopsis remains unknown.Ctenophore development is rapid, with cleavages occurring every 15 to 20 min. The asymmetrical localization of proteins or mRNAs into distinct lineages during each division could be a simple way to distribute components quickly so as to distinguish different cell fates. Freeman showed tDrosophila neuroblast division[C. elegans development[In other systems, RNAs, proteins, protein complexes or a mix of these are the developmental determinants that are distributed unevenly during development and which break the symmetries. A well-known example is the localization of the protein Dishevelled – a key player of the Wnt/ β-catenin pathway – to one side of an unfertilized egg. This is one of the first symmetry-breaking events in many bilaterians,62,63. Odivision and the elopment.Oskar mRNA, which is one of the key components used to establish polarity in the Drosophila oocyte during Drosophila oogenesis[ASH1 in budding yeast, bicoid in Drosophila embryos, Vg1 in Xenopus oocytes and CamKIIa in distal dendrites in mammalian neurons .Future experiments will be needed to reveal the nature of the developmental determinants that are segregated during early ctenophore development.Mnemiopsis embryos, which are cleavage arrested at the 8-cell stage or later, subsequently occurs at the same time as in untreated control embryos. Furthermore, our results show that comb-plate formation is blocked if DNA replication is inhibited by aphidicolin in addition to the inhibition of cytokinesis by cytochalasin B. This indicates that the number of nuclear division cycles or the amount of DNA (the nuclear-to-cytoplasmic ratio) appear to be important factors for comb-cell differentiation and not simply the amount of time that has elapsed since the third cleavage. As previously shown, after the second and/or third cleavage is blocked, Mnemiopsis embryos skip the respective cleavages and continue in a timely manner with their subsequent cleavage program, suggesting that a timing system determines the orientation of each division[The regulation of growth and differentiation are key to developmental processes. Moreover, the timing of developmental events is a particularly important aspect of development. We showed that the differentiation of comb cells in division. Once thdivision. Since tdivision concludeCherax destructor, for example, all ectoteloblasts undergo exactly 15 rounds of asymmetric cell division and give birth to ectodermal blast cells, which undergo two distinct divisions each[Embryos that undergo exact numbers of cell divisions are known from a broad range of developmental systems; however, little is understood about the underlying mechanisms-75. A stons each.Fundamental experiments by Whittaker showed that ascidian embryos developed muscle-specific acetylcholinesterase and brain pigment cell tyrosinase in specific blastomeres even in cleavage-arrested embryos and concluded that specific positional information is differentially segregated during early development. Since tet al.[Xenopus development, where zygotic transcription is initiated after 11 to 12 rounds of cell division[C. elegans, the timing of gut marker gene expression only depends on an early period of DNA synthesis until the 8-cell stage when the gut is clonally established[A number of counting mechanisms have been suggested for this phenomenon. Based on the observation that genomic DNA is highly methylated in zygotes and gradually demethylated during development, which tends to de-repress transcriptional activity in mammals, Kataokaet al. suggesteet al.. Anotherdivision. The reqdivision. In contablished. Subsequablished.Future studies are required to unravel the mechanism that regulates the timing of cell differentiation in ctenophores, whether this is the ratio of DNA to cytoplasm, DNA methylation, protein degradation, telomeric alterations, a combination of different factors or an entirely different mechanism that is currently unknown.Xenopus[Early developmental events are regulated by gene products, which may be provided maternally. The actXenopus and has Xenopus. It is eXenopus,86.Clytia hemisphaerica, zygotic transcription starts at the late blastula to early gastrula stage[Nematostella vectensis exhibits an upregulation of gene expression around 10 hpf[Other than bilaterians, the onset of zygotic transcription has only been described in cnidarians. In la stage,87. The d 10 hpf, at a tid 10 hpf.Mnemiopsis.We used actinomycin D to inhibit transcription and we showed that treatment prior to the 60-cell stage did not visibly affect development until the animal reached the 60-cell stage, suggesting that maternally loaded transcripts are sufficient to support the early cleavage program up to this stage but not subsequent development or differentiation. These results suggest that the onset of zygotic transcription starts at about the 60-cell stage, just prior to gastrulation. The rapid early development of ctenophores also suggests that there may be little time for the transcription of new messages until the cell cycle began to slow down. Future experiments using labeled nucleotides will help to determine the precise onset of zygotic transcription in The transition from maternal to zygotic transcription is most likely an ancient feature in animal evolution and might be tightly linked with the evolution of multicellularity and sexual reproduction. If ctenophores are the sister group of all metazoans, as suggested by recent studies,91, one A phylogenetic comparison of maternally loaded proteins and the molecular regulation of the maternal-to-zygotic transition in bilaterian and non-bilaterian taxa might provide further insight into the evolution of sexual reproduction and life history.Mnemiopsis leidyi show differentiation of selected cell fates even when cytokinesis is arrested using cytochalasin B during the course of early embryogenesis[1 micromeres, and not any of the M or m1 lineages. Likewise, bioluminescence is only observed in the M lineage and specifically in M macromeres. In addition, we showed that developmental determinants that are required to generate motile comb-plate-like cilia and photoprotein expression are already present and localized at the 8-cell stage and further localize to the appropriate cells in the subsequent cell divisions. Further studies are required to unravel the nature of the developmental determinants and which mechanisms are used to segregate them. The timed inhibition of transcription and translation with actinomycin D and puromycin, respectively, shows that both transcription and translation are required for comb cells to form, suggesting that not all the necessary factors are maternally deposited. While these inhibitor studies are not definitive gene-specific approaches, they do provide insight into the potential nature of materials that are differentially segregated into different embryonic lineages. Finally, we presented evidence that suggests that zygotic transcription in Mnemiopsis begins around the 60-cell stage, just before the onset of gastrulation.As in other animals with a mosaic development, embryos of the comb jelly ogenesis,103,104.hpf: hours post fertilization; MZT: Maternal-to-zygotic transition; PBS: Phosphate-buffered saline; PBT: PBS plus 0.2% Triton; DIC: Differential interference contrast; AO: Apical organ.The authors declare that they have no competing interests.AHLF, KP, JQH and MQM conceived the study and analyzed the data. AHLF and KP conducted and analyzed the drug treatments and produced the figures. KP analyzed the immunohistochemistry, produced the confocal micrographs and recorded the time-lapse images. JQH and MQM performed the microinjection. KP, JQH and MQM imaged the injected embryos. AHLF wrote the first draft of the manuscript. All other authors helped to complete and edit the manuscript. All authors read and approved the final manuscript.Mnemiopsis leidyi.Normal embryonic development of  DIC microscopy time-lapse movie of developing Mnemiopsis embryos beginning with a zygote until 12 hpf. The aboral side is up. The animal shown is the same as in Figure 2A-P.Click here for fileMnemiopsis embryo after cytochalasin B treatment.Regression of the cleavage furrow in a  Cytochalasin B was added during the first cleavage of a Mnemiopsis zygote. DIC microscopy time-lapse showing the ingression of the cleavage furrow. Once cytochalasin B has been added, cleavage stops and the cleavage furrow regresses. The animal shown is the same as in Figure 3A-D.Click here for fileCleavage-arrested embryo with beating cilia at 2 comb cells. DIC microscopy movie showing the beating cilia on comb-cells in a cleavage-arrested embryo. The embryo shown is the same one as in figure 5A and 5A'.Click here for fileClose-up on one a comb cell with beating cilia in a cleavage-arrested embryo. DIC microscopy movie showing the beating cilia on comb-cells in a cleavage-arrested embryo. The embryo shown is the same one as in figure 5B and 5B'.Click here for fileCleavage-arrested embryo with beating cilia at a comb cells. DIC microscopy movie showing the beating cilia on comb-cells in a cleavage-arrested embryo. The embryo shown is the same one as in figure 5C and 5C'.Click here for fileControl embryo at around 10-12 hpf. DIC microscopy movie showing the beating cilia of the comb cells, which are well visible on both sides of the embryo. The embryo shown is the same one as in figure 5G.Click here for fileCleavage-arrested embryo with beating cilia and labeled e1-cell lineage. DIC microscopy movie in combination with fluorescent light showing an embryo in which the e1-cells were DiI labeled at the 16-cell stage. The embryo was arrested with cytochalasin B at the 32-cell stage. Please note that only labeled cells bear cilia. The embryo shown is the same one as in Figure 6E.Click here for fileBioluminescence in photocytes of a control embryos. Light microscopy movie showing light emission from the photocytes that was stimulated by a brief illumination with fluorescent light. The photocytes are well visible on either side of the embryo. The animal shown is the same one as in Figure 7B.Click here for fileBioluminescence in photocytes of a cleavage arrested embryos. Light microscopy movie showing light emission from the photocytes that was stimulated by a brief illumination with fluorescent light. Those cells that contain the bioluminescent protein are photocytes. The embryo shown is the same one as in Figure 7C.Click here for file


Dental caries in children is unevenly distributed within populations with a higher burden in low socio-economy groups. Thus, tools are needed to allocate resources and establish evidence-based programs that meet the needs of those at risk. The aim of the study was to apply a novel concept for presenting epidemiological data based on caries risk in the region of Halland in southwest Sweden, using geo-maps.® ArcGIS system.The study population consisted of 46,536 individuals between 3-19 years of age (75% of the eligible population) from whom caries data were reported in 2010. Reported dmfs/DMFS>0 for an individual was considered as the primary caries outcome. Each study individual was geo-coded with respect to his/her residence parish. A parish-specific relative risk (RR) was calculated as the observed-to-expected ratio, where the expected number of individuals with dmfs/DMFS>0 was obtained from the age- and sex-specific caries (dmfs/DMFS>0) rates for the total study population. Smoothed caries risk geo-maps, along with corresponding statistical certainty geo-maps, were produced by using the free software Rapid Inquiry Facility and the ESRIThe geo-maps of preschool children (3-6 years), schoolchildren (7-11 years) and adolescents (12-19 years) displayed obvious geographical variations in caries risk, albeit most marked among the preschoolers. Among the preschool children the smoothed relative risk (SmRR) varied from 0.33 to 2.37 in different parishes. With increasing age, the contrasts seemed to diminish although the gross geographical risk pattern persisted also among the adolescents (SmRR range 0.75-1.20).Geo-maps based on caries risk may provide a novel option to allocate resources and tailor supportive and preventive measures within regions with sections of the population with relatively high caries rates. In spite of the global decline in childhood caries, widening inequalities in oral health exist between social classes and certain minority ethnic groups ,2. In thThe Halland region has approximately 70,000 inhabitants below the age of 20 years and the vast majority is listed as regular patients at the Public Dental Service that provides free dental care between 1 and 19 years with recall intervals varying from 3 to 24 months depending on the individual need. Data on the experience of manifest (dentin) caries is registered according to the WHO-criteria  and annu® ArcGIS system . Each study individual was geo-coded with respect to his/her residence area (parish). Figure The Halland region consists of six municipalities that are subdivided into 66 parishes. Geo-maps were produced by using the ESRIin×i racross the age- and sex-strata i, where i ndenotes the stratum-specific number of study individuals residing in the parish and i rdenotes the corresponding caries rate observed in the total study population. The computations of the RRs were performed using the free software Rapid Inquiry Facility . Along with each caries risk geo-map, we provide the corresponding statistical certainty geo-map. A posterior probability of a parish-specific relative risk above one given the data, denoted Pr(RR>1|data), was obtained by the Bayesian approach. A parish with data yielding strong statistical evidence of an elevated caries risk, more precisely Pr(RR>1|data) > 0.95, was colored red in the certainty geo-map. By contrast, a parish with evidently low caries risk, Pr(RR<1|data) > 0.95, was colored green. Analogously, parish-specific 90% credibility intervals for the relative risk were obtained; and each parish with a 90% credibility interval that covers 1 was colored yellow in the certainty geo-map indicating a weaker statistical evidence for a high or low relative risk in such parishes.S) between the SmRRs and the proportions with post-secondary educational level among the residents (considered as a group-level indicator of socio-economy) across the 66 parishes.We addressed geographical co-variations between caries risk and residents'' level of education by calculating Spearman''s correlations , but not for schoolchildren  and adolescents .The proportion of children with no obvious decay at various ages and the cumulative burden of caries in the region, expressed as mean dmfs/DMFS, are shown in Table It is generally recognized that GIS may play an essential role in helping public health organizations understand population health and make decisions . The metFrom an international perspective, it should be pointed out that the overall cumulative caries burden in the present study population was very low Table  21]. Al. Al21]. Another interesting example was the demonstrated age-related impact of the smoothed relative risk which may imply that a relatively larger proportion of the preventive efforts should be steered towards the youngest age groups. Among the preschoolers, the SmRRs captured the geographical risk pattern of early childhood caries. There are several documented examples of successful programs for infant feeding and early fluoride exposure that have reduced inequalities in oral health in preschool children in a cost effective way . In the In summary, geo-maps based on caries risk may provide a novel option to allocate resources and tailor supportive and preventive measures within regions with sections of the population with relatively high caries rates.The authors declare that they have no competing interests.All of the listed authors contributed to the conduct of the study. US and ST analyzed and interpreted the data and drafted the manuscript. KE provided technical and administrative support and AH performed the computer work. All authors approved the final version of this manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/11/26/prepub


The existence of the teneurin C-terminal associated peptides (TCAP) was reported in 2004 after screening a rainbow trout hypothalamic cDNA for corticotropin-releasing factor (CRF)-related homologs. In vertebrates, there are four TCAP paralogs, where each peptide is associated with a teneurin transmembrane protein. The TCAPs are 40 or 41 amino acids in length and possess less than 20% residue identity with the CRF family of paralogs. Orthologs of TCAP are found in all metazoans with the possible exception of poriferans and cnidarians. Recent evidence indicates that TCAP and the teneurins may have been introduced into the Metazoa via horizontal gene transfer from prokaryotes into a basal protistan. Thus, the origin of the TCAPs likely predated that of the CRF family. In the mammalian brain, TCAP-1 is transcribed independently from teneurin-1. Moreover, TCAP-1 acts on neurons by a CRF-receptor independent signal transduction pathway to regulate cellular cytoskeletal function to stimulate cell activity. Administration of synthetic TCAP-1 to rodents inhibits a number of CRF- and stress-associated behaviors via a hypothalamic–pituitary–adrenal (HPA) axis-independent mechanism. Corticotropin-releasing factor (CRF) is part of an evolutionary conserved peptide family integral to the regulation of stress-associated behavior and physiology in metazoans. The long period of time during which CRF-like peptides have persisted suggests that the CRF response has been evolutionarily advantageous. Early-evolving gene systems integral to the survival of an organism are evolutionarily selected for, and because of this, may give rise to additional paralogous lineages via gene, genomic, and chromosomal duplication events. In addition, such early evolving systems will likely become associated with newer evolving molecular and physiological systems. Thus, because of the evolutionary age of the CRF system, it is likely that it is modulated, in turn, by even earlier evolving systems.In situ experiments in Sprague-Dawley rats show that TCAP is highly expressed in all hippocampal subregions, central amygdala (CeA), basolateral amygdala (BLA), and various hypothalamic nuclei regions that are known to express CRF receptors , the CRF family consists of four paralogous peptides that are the result of two rounds of genome duplications in early evolution and has been previously described in detail  axis   was expressed mainly in the limbic system, hypothalamus and cerebellum .in vitro screening of most of the receptors in this family did not show any significant binding or activation . However, gene microarray profiling indicated a similarity to neurotrophic factor activation and a relationship with the dystroglycan complex  similar to the family of GPCRs related to the CRF receptors. However,1 activation in the CA3 mediates dendritic retraction by inducing destabilization of f-actin and the cell adhesion molecule nectin-3  also mediates outgrowth of neuronal processes through the Rho GTPase Rac1 (Swinny and Valentino, 1 mediated changes in cytoskeletal re-organization-like synaptic plasticity within stress modulating brain areas that express both CRF1 and TCAP-1.Within multiple areas of the mouse brain, CRF induces stress-related cytoskeletal changes. CRF1 receptor. The observed TCAP-1 modulatory effects on CRF behaviors may then be mediated through TCAP-1 specific signaling that oppose CRF mediated effects on cellular cytoskeletal organization within brain areas that co-express TCAP-1 and CRF. This in turn manifests into modulation of CRF-mediated stress behaviors in brain regions that co-express CRF and TCAP-1.Current data suggests that TCAP-1 acts as a separate and distinct peptide modulating system, which is involved in cytoskeletal-associated synaptic plasticity. From an evolutionary perspective, it is perhaps not surprising that TCAP-1 acts independently of CRF signaling. The teneurins and TCAP evolved early in metazoan history, whereas the CRF family of peptides and their role in integrating energy metabolism and the perception of stressors evolved later after evolution of vertebrates (Lovejoy and Jahan, Dr. D. A. Lovejoy is a co-founder of Protagenic Therapeutics, Inc. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Skeletal muscle adapts its mass as consequence of physical activity, metabolism and hormones. Catabolic conditions or inactivity induce signaling pathways that regulate the process of muscle loss. Muscle atrophy in adult tissue occurs when protein degradation rates exceed protein synthesis. Two major protein degradation pathways, the ubiquitin-proteasome and the autophagy-lysosome systems, are activated during muscle atrophy and variably contribute to the loss of muscle mass. These degradation systems are controlled by a transcription dependent program that modulates the expression of rate-limiting enzymes of these proteolytic systems. The transcription factors FoxO, which are negatively regulated by Insulin-Akt pathway, and NF-κB, which is activated by inflammatory cytokines, were the first to be identified as critical for the atrophy process. In the last years a variety of pathways and transcription factors have been found to be involved in regulation of atrophy. This review will focus on the last progress in ubiquitin-proteasome and autophagy-lysosome systems and their involvement in muscle atrophy. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting. Muscle atrophy involves the shrinkage of myofibers due to a net loss of proteins, organelles and cytoplasm. Acute muscle atrophy, as occurs in many pathological conditions, is due to hyperactivation of the cellular main degradation pathways, including the ubiquitin-proteasome system and the autophagy-lysosome pathways. Recent studies have highlighted a complex scenario whereby these catabolic signaling modulate one another at different levels, and are also connected at various levels to biosynthetic pathways ((1)A transcriptional dependent upregulation of the atrophy-related gene at mRNA level does not always match with an increase of protein level. The field of muscle wasting should consider the fact that transcriptional-dependent induction of several atrophy related genes is required to replenish the enzymes/proteins that are lost during the enhanced protein breakdown. Importantly, the muscle-specific atrophy-related ubiquitin ligases atrogin1/MAFbx and MuRF1 similarly to other E3s, undergo auto-ubiquitination . It is r(2)A decrease of protein synthesis can not be considered alone as the mechanism of atrophy. The size of a postmitotic cell results from the balance between protein synthesis and degradation. Indeed, in condition of protein synthesis inhibition the total protein content of the cell is affected by protein’ half-life that depends on basal protein breakdown. Therefore, paradoxically in situations of decreased protein synthesis the cell size ultimately depends more on protein breakdown than in circumstances of normal protein synthesis.(3)A decrease of nuclear turnover or of the satellite cell pool can not explain muscle atrophy in adulthood.The myonuclear domain is not constant in various conditions including growth and atrophy. Recent evidences underlie that myonuclear turnover is negligible in adulthood after postnatal growth . MoreoveSkeletal muscle mass and muscle fiber size may vary according to physiological and pathological conditions. An increase in muscle mass and fiber size, i.e. muscle growth or hypertrophy, occurs during development, in response to mechanical overload and/or anabolic hormonal stimulation. During development, the growth of skeletal muscle, like the mass of any other tissue, depends on protein and cellular turnover. In adulthood, the regulation of muscle mass and fiber size essentially reflects pathways . The res2Muscle atrophy involves the shrinkage of myofibers due to a net loss of proteins, organelles and cytoplasm. The ubiquitin-proteasome system and the autophagy-lysosome pathway are the degradation systems involved in this process.2.1From the discovery that FoxO transcription factors coordinate the activation of the ubiquitin proteasome and of autophagy-lysosome pathways in atrophying muscles , a greatSeveral years ago autophagy-lysosome system was described to be activated in muscle cells during catabolic conditions . HoweverMitochondria regulation is important to preserve muscle function and for the regulation of general metabolism and indeed, this organelle changes morphology and number as consequence of muscle activity. The mitochondrial network can be remodeled by the fusion/fission proteins, the mitochondrial shaping machinery, and by the selective removal of small mitochondria via autophagy, a process named mitophagy  Fig. 1)Fig. 1). C. elegans homolog of EPG-5 plays a critical role in the autophagy process  enzyme that generates PI3-phosphate from PI, while Vps15 is a regulatory subunit that is required for Vps34 activity. Therefore, Vps34 and Vps15 are obligate partners that form the core for the association of different proteins to generate distinct complexes that regulate autophagosome biogenesis and endosome trafficking. The beclin1/Vps34/Vps15/Atg14L complex is required for autophagosome biogenesis and indeed drugs that block Vps34 activity prevents autophagosome formation in cell culture. The role of Vps15 in adult muscle has been recently investigated by a loss of function approach. Surprisingly, muscle-specific Vps15 knockout mice showed normal LC3 lipidation and autophagosome formation but impaired autophagosome lysosome fusion . Importa process  Fig. 1)C. elegan process . The pre process . These d process . Interes2.2In the ubiquitin-proteasome system, proteins are targeted for degradation by the 26S proteasome through covalent attachment of a chain of ubiquitin molecules. The ubiquitin ligase enzyme, or E3, binds the protein substrate and catalyzes the movement of the ubiquitin from the E2 enzyme to the substrate. This is the rate-limiting step of the ubiquitination process, which affects the subsequent proteasome-dependent degradation. Once the protein is ubiquitinated it is docked to the proteasome for degradation, unless the polyubiquitin chain is removed by the de-ubiquitinating enzymes. Among the different E3s, only a few have been found to regulate atrophy process and to be transcriptionally induced in atrophying muscle.The first to be identified were Atrogin-1/MAFbx and MuRF1. These two E3s are specifically expressed in striated and smooth muscles . AtroginSince two ubiqutin ligases cannot effort for the degradation of all the sarcomeric and soluble proteins, additional E3s are involved in muscle loss. Specific ubiquitin-ligases may be involved in different models of muscle wasting and at different stages of the atrophy process. For instance, the HECT domain ubiquitin ligase Nedd4-1 has been reported to be upregulated mainly during disuse. Indeed deletion of the Nedd4-1 gene specifically in skeletal muscle results in partial protection from muscle atrophy in denervated type II fibers. However, Nedd4-1 knockout mice have smaller muscles, suggesting that this E3 may play additional roles during myogenesis or in the control of protein synthesis . RecentlAnother E3 ubiquitin ligase found to play a critical role in atrophy is TRAF6 , which mMul1 is a mitochondrial ubiquitin ligase that plays an important role in mitochondrial network remodeling. Mul1 is upregulated in catabolic conditions, such as fasting or denervation, by the FoxO family of transcription factors and causes mitochondrial fragmentation and removal via autophagy . ImportaAnother ligase is CHIP, which regulates ubiquitination and lysosomal-dependent degradation of filamin C, a muscle protein found in the Z-line . FilaminFbxo40 knockout mice display bigger muscle fibers  are ubiquitin-specific proteases (USPs). So far, only two (USP14 and USP19) have been found to be upregulated in atrophying muscles . Knockin2.3+-adenosine triphosphatase ATPase (v-ATPase) recruiting mTORC1 to the lysosomes. This mTOR localization initiates the amino acid signaling and protein synthesis (Synthesis and degradation of proteins are two processes that are intimately connected. Indeed, most of the above mentioned systems are regulated by pathways that impinge on both synthesis and degradation, so that when protein synthesis is induced degradation is suppressed and vice versa . This coynthesis . Concomiynthesis . Activatynthesis . These dThe mechanisms controlling muscle mass have attracted increasing interest in the scientific community due to their potential to address various clinical problems, such as aging, the prognosis of many diseases, quality of life and sports medicine. The results of recent research offer new and exciting perspectives to the field, that will hopefully identify new therapeutic targets and drugs.Several potentially interesting therapeutic targets have already been identified, although an effective drug that can counteract muscle wasting is not yet clinically available. The most interesting targets belong to the anabolic pathways and the ubiquitin-proteasome system. The Akt signaling and its downstream targets seem to be at the intersection of several different pathways, including β-adrenergic signaling, myostatin and JunB. Moreover, the IGF1-Akt axis is unique in that it controls both protein synthesis and protein degradation. Therefore, IGF-1 analogs might be extremely useful for counteracting muscle loss and weakness. However, the same pathway plays major roles in other biological processes, including cell survival, and in other contexts it can promote tumorigenesis. Thus, the development of a new generation of IGF1 mimetics that specifically activate the Akt pathway in skeletal muscle is a goal for the field. It is important to underline that prolonged inhibition of protein degradation can have a major impact on protein quality control and muscle performance. Such agents could have major drawbacks, such as promoting the accumulation of misfolded or aggregate-prone proteins .Recent data identified SGK1 as an important inhibitor of FoxOs and of muscle atrophy. By studying the mechanisms that protect squirrel from muscle wasting during hibernation it was found that SGK1 and not Akt was the critical factor that blocked FoxO activity and muscle atrophy . The facIn fact promoting protein synthesis without acting on protein breakdown may be not sufficient to prevent muscle atrophy and weakness. Indeed in a recent study it was analyzed the role of TSC1 in muscle growth and atrophy by generating muscle-specific TSC1 knockout mice. These animals showed an activation of TORC1 pathway and an increase of protein synthesis but surprisingly, glycolytic muscles were atrophic due to an hyperactivation of FoxO3-dependent atrophy program . Therefotibialis anterior muscles and cultured myotubes induced by FoxO3 overexpression, dexamethasone treatment or starvation. In these conditions, JunB prevents the activation of atrogin-1 and partially of MuRF-1, thereby reducing the increase in overall protein degradation induced by activated FoxO3. Further analysis revealed that JunB does not inhibit FoxO3-mediated activation of the autophagy-lysosome system, but only ubiquitin-proteasome degradation, by inhibiting atrogin-1 and MuRF-1 induction during catabolic conditions. In fact, JunB directly binds FoxO3, thereby preventing its recruitment to the promoters of key atrogenes. Moreover, JunB overexpression is sufficient to induce dramatic hypertrophy of myotubes and of adult muscle. These hypertrophic changes depend on increased protein synthesis, without affecting the basal rate of protein degradation.An alternative approach would consider the development of drugs that activate negative modulators of FoxO transcription factors such as JunB , or PGC-Recently the hormone ghrelin was found to prevent muscle wasting . GhrelinBeta-adrenergic agonists such as clenbuterol are considered pro-growth and anti-atrophic drugs. Most effects of clenbuterol are mediated by activating Akt-mTOR signaling , so the The last category of drug targets is represented by the proteasome system. Proteasome inhibitors have been successfully used to block atrophy in different animal models . However


Primary effusion lymphoma (PEL) is a rare KSHV/HHV8-associated high-grade non-Hodgkin's lymphoma (NHL) of B-cell origin, characterized by serous effusions in body cavities. Most patients are HIV-infected men with severe immunosuppression and other HHV8-associated diseases such as Kaposi's sarcoma (KS). The prognosis for those infected is poor, with a median survival of less than 6 months in most cohorts. Sustained complete remission is rare. High-dose chemotherapy regimens are used to improve remission rate and survival. The aim of the present study was to compare the drug sensitivity pattern of the available primary effusion (body cavity based) lymphoma-derived cell lines in order to find additional, potentially effective drugs that are not included in current chemotherapy treatment protocols.We have analyzed 11 cell lines against 27 frequently used cytostatic drugs in short term (3 days) survival assays using automated high throughput confocal microscopy.in vitro used and clinically achieved drug concentration, Vinorelbine, Paclitaxel, Epirubicin and Daunorubicin were the most effective drugs.All cell lines showed a distinct, individual drug sensitivity pattern. Considering the We suggest that inclusion of the above drugs into PEL chemotherapy protocols may be justified. The heterogeneity in the drug response pattern however indicated that assay-guided individualized therapy might be required to optimize therapeutic response. Human herpesvirus 8 (HHV8) or Kaposi sarcoma herpesvirus (KSHV) is the probable causative agent of two distinct lymphoproliferative disorders: primary effusion lymphoma (PEL) and the plasma cell variant of multicentric Castleman disease (MCD) in addition to Kaposi sarcoma (KS) .Primary effusion lymphoma (PEL), or alternatively: body cavity lymphoma is a non-Hodgkin's lymphoma (NHL) of B-cell origin that develops predominantly in the serous body cavities . The lymConversely, PEL cells generally express various markers associated with activation, including CD30 (approximately 75% of cases), CD38, CD71 and the epithelial membrane antigen. Moreover, PEL cells express several plasma cell markers including CD138, VS38c and MUM-1/IRF4 [The prognosis of PEL is poor, as the median survival in the previously published series does not exceed 3 months -10.Given its rarity, however, there are very few longitudinal observational series of patients with PEL and no large prospective trials have ever defined optimal treatment strategies .Prior to the introduction of antiretroviral therapy, the therapeutic results were unsatisfactory in cohorts of HIV+ patients, despite the use of aggressive polychemotherapy regimens including anthracyclines. The significant improvement in the prognosis of AIDS-related lymphomas observed in the antiretroviral therapy era also applies to the PEL setting.In addition, the routine use of growth factors, such as the granulocyte colony-stimulating factor (G-CSF), to avoid prolonged periods of neutropenia resulting from chemotherapy is standard practice for all AIDS-related lymphoma (ARL) patients.Despite the improvement in therapeutical strategies during the last few years, there is no evidence of a cure for PEL patients with conventional systemic chemotherapy addressed to aggressive NHL. The suggested benefit of high-dose Methotrexate in association with CHOP -like regimens is negatively balanced by the hampered toxicity of Methotrexate in the presence of serous effusions .Novel approaches for body cavity lymphoma therapy outside traditional chemotherapy have been suggested as well . These iin vitro [Another approach may be to target NF-κB through the use of proteasome inhibition with drugs, such as Bortezomib that induces apoptosis of PEL cell lines in vitro .In the present study we have investigated 11 different primary effusion (body cavity based) lymphoma-derived cell lines to compare the drug sensitivity pattern, in order to find new potentially successful chemotherapy agents, that are not used in current treatment protocols.The following primary effusion (body cavity based) lymphoma-derived cell lines were used in the present study. CRO-AP/2, CRO-AP/5, CRO-AP/6, BC-2, BC-3 were established from pleural effusion, CRO-AP/3, HBL-6, BC-3, BCBL-1, JSC-1 were established from ascites fluid and BCP-1 from peripheral blood.2. Cultures were fed twice weekly with the above-mentioned medium; maintained at ca. 0.5 × 106 cells/ml. All cell lines were examined daily in their culture vessels under an inverted microscope. Absence of mycoplasma contamination was routinely assessed using staining with Hoechst 33258.Body-cavity cell lines were cultured in IMDM (Sigma), supplemented with 20% heat-inactivated (at 56°C for 45 min) fetal calf serum , 100 IU/ml penicillin (Sigma), 100 μg/ml streptomycin (Sigma) and 2 mM L-glutamine (Sigma). Cell lines were grown at 37°C in the presence of 5% COIn vitro drug resistance of body-cavity cell lines were assessed using a 3-day cell culture on microtiter plates. 27 drugs  values of the individual drugs. For this comparison Quotient of Area Under Curve values (QAUC72 hr) were determined by the following formula:To calculate the relationship between the in vivo AUC72 hr corresponds to the area under curve value achieved in patients under a 72 hours period. The in vivo AUC72 hr was established from the clinical dose and half-time using the standard trapezoidal rule calculation. The in vivo AUC72 hr data is summarized in the seventh column of Table in vivo halftime data are available at the Swedish pharmacological website http://fass.se. A QAUC72 hr value higher than 1 indicates that the in vitro drug concentration is higher than the one used in the clinical practice. If this value is 1, it means that the in vitro concentration corresponds to the clinically achieved in vivo concentration.The in vitro survival assays. The clinical origin and viral status of the individual lines is summarized in Figure We have tested the drug sensitivity patterns of the body cavity lymphoma lines in short term, The summarized cell survival data is shown in Figure Most of the lines were sensitive for sixteen of the 27 drugs where sensitivity was defined as less than 50% mean survival at any of the drug dilutions (effective drugs). If more than half of the cells were alive even at the highest concentration than the drug was considered to be ineffective.We found that the sixteen effective drugs against body-cavity lymphoma were the following in the order of effectiveness: Dactinomycin, Chlorambucil, Paclitaxel, Vinorelbine, Epirubicin, Docetaxel, Daunorubicin, Vinblastin, Doxorubicin, Etoposide, Gemcitabine, Asparaginase, Fluorouracil, Topotecan, Vincristine and Methotrexate.Most body-cavity lymphoma lines were resistant to Oxaliplatin, Bleomycin, 6-Mercaptopurine, Hydroxyurea, Cladribine, Carboplatin, Bortezomib, Cytosine-arabinosid, Prednisolone, Mitomycin and Fludarabin. Although the Oxaliplatin, Cisplatin and Prednisolone drugs were not effective against any of the body-cavity lymphoma lines these drugs show concentration-dependent growth-inhibitory effect on other cell lines or primary tumors in parallel experiments (data not shown) at the same concentration as used in this paper .In order to identify possible co-segregation of the sensitivity patterns of the individual drugs as well as to systematically compare all the lines with each other, we have carried out unsupervised two-dimensional hierarchical clustering of the simplified drug sensitivity data using the Cluster 3.0 program for MacOS X. The results were visualized using the program TreeView . The senin vivo concentrations. In order to analyze the data in relation to the pharmacokinetic behavior of the given drugs we have plotted the mean survival values as the function of the Quotient of the Area Under Curve (QAUC72 hr) values of the particular drugs. The QAUC72 hr values of a drug were created by dividing the calculated in vitro AUC72 hr values by the in vivo achievable AUC values . As shown in Figure 72 hr (close to 1).Absolute drug sensitivity values have relatively little clinical relevance if they are not correlated with clinically achievable 72 hr axis shows that Daunorubicin, Epirubicin, Paclitaxel and Vinorelbine were the most effective drugs . Moreover most body cavity lymphoma lines were sensitive to these drugs. Importantly, Doxorubicin, the only antracyclin drug that is currently included in chemotherapy protocols against body cavity lymphomas showed a rather heterogeneous effect. Two of the eleven lines were highly sensitive for Doxorubicin whereas two were completely resistant at the maximum drug concentration that we could reach in the current assay (QAUC = 0.11). The two lines (JSC-1 and BC-3) were, in general, the least sensitive for chemotherapeutic drugs, however were still sensitive for Daunorubicin, Epirubicin and Vinorelbine.Plotting the mean cell survival for each individual drug against a common QAUCin vitro assay. The calculation of the QAUC72 hr value for the corresponding Dactinomycin concentration however revealed that the concentration that was required for the high killing effect is higher than the levels that are realistically achievable in a patient.Dactinomycin showed the highest killing efficiency in the present 72 hr values, a relative increase in the number of surviving cells was observed as compared to non-treated controls. The survival was above 100% in case of all the 11 lines suggesting that low dose Carboplatin protected from spontaneous cell death.When treating the body-cavity lymphoma cells with Carboplatin at low QAUCIn vitro growing cell lines are the closest model systems available today for studying the biological features of body cavity lymphomas. The cell lines that were used in the present study represent a variety of different origin. The investigation included cell lines established from ascites fluid, pleural effusion or from the peripheral blood of PEL patients. Despite their different origin, the body-cavity lymphoma lines showed a remarkably similar sensitivity pattern for a number of drugs. Only one cell line was highly resistant for most of the drugs (JSC1) whereas two cell lines (BC1 and BCBL-1) showed increased overall sensitivity to most of the drugs.The presented data suggests that, for a number of cytostatic drugs the body cavity lymphoma cell lines share a common cytotoxic drug sensitivity profile. These profiles show no obvious correlation with the biological or clinical features of the lymphomas. Clustering of the drug sensitivity data revealed that the profiles are independent of the EBV status, anatomical localization of the lesion, the age the patient or the rapidity of the progression of the disease. The two cell lines (BCP-1 and BC-3) that arouse from HIV negative patients showed relatively low drug sensitivity.The current treatment alternative is a combination of Methotrexate with CHOP -regimes. The present data showed that the cell lines exhibit varying sensitivity to Methotrexate and Vincristine and are completely resistant to Prednisolone.in vivo.Cyclophosphamide or Ifosphamide were not tested on the body cavity lymphoma lines, because both of these compounds are prodrugs that have to be converted into active metabolites by the liver in vitro [It has been reported that the proteasome inhibitor Bortezomib induces apoptosis of the cell lines BCBL-1 and BCP-1 in vitro . In this72 hr values. Epirubicin required tenfold lower concentration than the in vivo achievable concentration to kill more than 80% of the cells for most of the lines. Epirubicin is primarily used against breast and ovarian cancer, gastric cancer, lung cancer and lymphomas, but has not yet been tested against body cavity lymphomas [In the present study body cavity lymphoma lines showed considerable sensitivity for anti-microtubule drugs and anthracyclins. Importantly all lines were sensitive to Epirubicin and Vinorelbine even at low QAUCymphomas .In summary, the analysis of drug sensitivity profiles of the available body cavity lymphoma lines against 27 commonly used drugs revealed considerable heterogeneity in drug response. Four drugs, namely, Daunorubicin, Epirubicin, Paclitaxel and Vinorelbine showed uniformly high efficiency on the cell lines. These drugs are not yet included in the current chemotherapy protocols of body cavity lymphomas. The heterogeneity of drug response also suggests that optimal care of the lymphoma patients would include the determination of drug sensitivity patterns of the primary tumor samples and that these patients would benefit from assay guided individualized therapy.We suggest that inclusion of the above drugs into PEL chemotherapy protocols may be justified. The heterogeneity in the drug response pattern however indicated that assay guided individualized therapy might be required to optimize therapeutic response.The authors declare that they have no competing interests.in vitro drug sensitivity assays. EF was responsible for measuring the plates using the automated laser confocal fluorescent microscope. LS and EF wrote the computer programs QuantCapture 4.0 and QuantCount 5.0. LM and HS analysed and interpreted the data. SE made comparable the in vitro results with the in vivo data. JK, LG, AC together with the other authors have been involved in the planning of the experimental details, and the drafting and critical reading of the manuscript. All authors read and approved the final manuscript.The project was conceived and designed by LS. The experiments were mainly carried out and/or coordinated by ÖR. LLK, AG and LM took part in cell culturing, and preparation of the microtiter plates for The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/441/prepub


Lactobacillus jensenii TL2937 in particular. This review article emphasizes the cellular and molecular interactions of immunobiotics with IECs and immune cells through TLRs and their application for improving animal and human health.The intestinal mucosa plays a critical role in the host’s interactions with innocuous commensal microbiota and invading pathogenic microorganisms. Intestinal epithelial cells (IECs) and gut associated immune cells recognize the bacterial components via pattern-recognition receptors (PRRs) and are responsible for maintaining tolerance to the large communities of resident luminal bacteria while being also able to mount inflammatory responses against pathogens. Toll-like receptors (TLRs) are a major class of PRRs that are present on IECs and immune cells which are involved in the induction of both tolerance and inflammation. A growing body of experimental and clinical evidence supports the therapeutic and preventive application of probiotics for several gastrointestinal inflammatory disorders in which TLRs exert a significant role. This review aims to summarize the current knowledge of the beneficial effects of probiotic microorganisms with the capacity to modulate the immune system (immunobiotics) in the regulation of intestinal inflammation in pigs, which are very important as both livestock and human model. Especially we discuss the role of TLRs, their signaling pathways, and their negative regulators in both the inflammatory intestinal injury and the beneficial effects of immunobiotics in general, and  The mammalian gastrointestinal tract harbors trillions of beneficial commensal bacteria, a population composed of at least 1,000–5,000 species . In factMammals have an evolutionary partnership with the microbiota that is critical for host defense. In the gastrointestinal tract, part of the local immune response is aimed at maintaining a peaceful coexistence with the resident microbiota. Abundant experimental and clinical data support the idea that commensals residing in the gastrointestinal tract can calibrate both innate and adaptive responses , 4. UniqIn recent years, the study of microbe-intestinal cell interactions has unraveled several molecular mechanisms and cellular pathways, showing that these interactions play a crucial role in the regulation of several immunological functions in the gut. Moreover, better understanding of the host-microbe interactions in the gut has provided new opportunities for preventing and treating a number of inflammatory disorders such as the use of specific probiotic strains to beneficially modulate the intestinal immune system. Probiotic bacteria that are able to modulate the immune system (immunobiotics) are demonstrably beneficial for treating a variety of mucosal disorders, including inflammatory diseases .Escherichia coli, Salmonella spp., and Clostridium perfringens  and immune cells through TLRs and their application for improving animal health and also human’s because the pigs are expected to be a better human model than rodents.Various nutritional approaches for optimizing the weaning transition and minimizing gut inflammation and enteric diseases have been tested in the past decade. Among the novel dietary strategies investigated that are focused on improving gut health in pigs, prebiotics and probiotics are clear nutritional options. This review aims to summarize the current knowledge of the beneficial effects of probiotic microorganisms with the capacity to modulate the immune system (immunobiotics) in the regulation of intestinal inflammation in pigs. We discuss the role of toll-like receptors (TLRs), their signaling pathways, and their negative regulators in both the inflammatory intestinal injury and the beneficial effects of immunobiotics in general, and Toll-like receptor-4 is expressed by epithelial and immune cells and might play a role in the intestinal mucosal host defense against Gram-negative bacteria. However, since many body surfaces are colonized by the physiological microflora, activation of epithelial TLR4 must be tightly controlled to avoid unintended stimulation and mucosal inflammation.Upon recognition of its cognate ligand, TLR4 dimerizes and initiates a signaling cascade that leads to the activation of a pro-inflammatory response Figure . Ligand Various negative regulatory mechanisms have evolved to attenuate TLR signaling and maintain the immune balance. At least six levels of negative regulation have been discovered so far , 15: (i)Toll-like receptor signaling in IECs and immune cells has been shown to be involved in three important mechanisms that are crucial for maintaining a healthy epithelial barrier: (i) epithelial cell proliferation and maintenance of tight junctions; (ii) expression of antimicrobial factors; and (iii) modulation of immune responses [reviewed in Ref. ]. In a hBacteroides fragilis is a unique TLR2 ligand found in the human microbiome, which orchestrates anti-inflammatory immune responses that ameliorate diseases mediated by the immune system. This polysaccharide is ingested by intestinal DCs, which then stimulate responses of Foxp3+ Treg cells  in mediating non-inflammatory immune responses to the microbiota, challenging the paradigm that PRRs have evolved solely to recognize and respond to pathogens. MyD88-deficient mice are more susceptible to DSS-induced colitis, suggesting that commensal bacteria may be directly recognized by TLRs under steady-state conditions to mediate host-protective responses . To correg cells . Intereseg cells . TLR2-del et al.  identifient mice . Furtherent mice . Transfeent mice . Convers colitis . TherefoB. thetaiotaomicron that attenuates pro-inflammatory cytokine expression in IECs by promoting nuclear export of the NF-κB subunit RelA through a PPARγ-dependent pathway has been reported , ensure that chronic inflammatory and potentially destructive TLR responses to MAMPs do not occur . In thisreported . Furtherreported .In addition to TLRs, other PRRs have been involved in the anti-inflammatory effects of gut microbiota. The peptidoglycan recognition protein (PGRP) family is involved in the regulation of commensal microbiota in mice. Mice deficient in any one of the four PGRPs harbor a microbiota that promote increased sensitivity to DSS-induced colitis . Indeed,Several studies have shown that immunobiotics can beneficially modulate the PRRs-mediated inflammatory response in the gut by modulating the functions of IECs and APCs , 31.S. typhimurium-induced IL-8 gene expression and secretion by IECs in an NF-κB-dependent manner (Lactobacillus rhamnosus GG counteracts the enterotoxigenic Escherichia coli (ETEC)-induced up-regulation of IL-1β and TNF-α and the down-regulation of TGF-β1 expression, consequently blocking cytokine deregulation  of the bacterium. L. acidophilus with mutated SlpA fails to induce Th2 polarization of the DCs, and instead, promotes IL-12p70, TNF-α, and IL-1 production , resulting in reduced lymphocyte recruitment in an ileitis model . The soluble factor(s) present in supernatants effectively induced apoptosis of immune cells. These were mainly soluble heat-stable proteins. For immune cells, pre-treatment with the supernatant significantly promoted apoptosis via a mitochondrial pathway. The supernatant also inhibited the release of LPS-induced pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 by immune cells  when is cleaved by intestinal proteases. It is characterized by the abundance of serine and threonine residues within its sequence. STp is encoded in one of the main extracellular proteins produced by such species, which includes some probiotic strains. In vitro studies using DCs from human peripheral blood showed that STp increased the production of regulatory IL-10 in healthy controls. In addition, T cells stimulated with STp-pulsed DCs decreased the production of pro-inflammatory IFNs and increased anti-inflammatory IL-10 production, suggesting that these T cells acquired an immunoregulatory phenotype  isolated from weaning pigs was able to reduce E. coli and anaerobe counts in the gut, and decrease diarrhea. Additionally, lactobacilli treatment significantly improved average daily feed intake of pigs compared to controls during the first 2 weeks after weaning and the average daily gain , and histological and physiological modifications in intestines. Another recent study evaluated the effect of the co-administration of B. subtilis RJGP16 and Lactobacillus salivarius B1 on intestinal immunity in piglets  . Studies control . InteresL. jensenii TL2937, the effect of this strain on the expression of the negative TLR regulators in PIE cells was evaluated. The expression of SIGIRR, Tollip, A20, Bcl-3, MKP-1, and IRAK-M was studied, and it was found that MKP-1, A20, and Bcl-3 mRNA expression was up-regulated in PIE cells stimulated with L. jensenii TL2937 . In swine, the most frequent marker expressed on DCs and macrophages is CD172a. Additionally, CD11R1 is considered to be a marker that is specifically and differentially expressed on porcine DCs, but not on macrophages  involved in the immunoregulatory effects of the TL2937 strain, the expression of negative regulators of TLRs in porcine APCs was also evaluated. Of the six regulators tested, SIGIRR, A20, and IRAK-M mRNA expression was up-regulated in CD172aytokines . The LPSytokines . As descytokines , 72. Notytokines . IRAK-M ytokines . Therefoin vitro models may be overly simplified and may not account for the effect of cell-cell interactions in a complex organic microenvironment, completely changing the resulting response. As mentioned before, IECs express a broad range of factors that may influence intestinal APCs and lymphocytes  L. jensenii TL2937, through its capacity of stimulating the production of immunoregulatory factors such as TGF-β in EICs, would indirectly increase the expression of Bcl-3 and the production of IL-10 in CD172a+ APCs reinforcing its effects in these cells. Then, L. jensenii TL2937 would functionally modulated IECs and APCs to improve resistance against infections and avoid unproductive inflammation. In fact, experiments using ETEC challenge, clearly demonstrated that the TL2937 strain is able to induce protection against inflammatory damage and improve immunity at the same time (Figure L. jensenii TL2937 help to maintain intestinal tolerance and improve the development of appropriate protective and controlled immune responses. Then, L. jensenii TL2937 has a great potential to be used as a pig probiotic feed. In addition, accumulation of empirical data in pigs may increase the probiotic use in human because the pigs are also expected for development as human model.The scientific research into probiotic mode of actions has come to age and has shown how probiotics are able to induce beneficial changes in the host. Our research work allows us to propose a complete view of the cellular and molecular mechanisms involved in the immunoregulatory effects of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to evaluate clinicopathologic factors that could possibly affect the outcome of patients with triple negative breast cancer and subsequently build a prognostic model to predict patients’ outcome.We retrospectively analyzed clinicopathologic characteristics and outcome of 504 patients diagnosed with triple-negative invasive ductal breast cancer. 185 patients enrolled between 2000 and 2002 were designated to the training set. The variables that had statistically significant correlation with prognosis were combined to build a model. The prognostic value of the model was further validated in the separate validation set containing 319 patients enrolled between 2003 and 2006. The median follow-up duration was 66 months. 174 patients experienced recurrence, and 111 patients died. Positivity for ≥4 lymph nodes, Cathepsin-D positivity, and Ki-67 index ≥20% were independent factors for DFS, while the lymph nodes status and Ki-67 index were the prognostic factors for OS. The prognostic model was established based on the sum of all three factors, where positivity for ≥4 lymph nodes, Cathepsin-D and Ki-67 index ≥20% would individually contribute 1 point to the risk score. The patients in the validation set were assigned to a low-risk group (0 and 1 point) and a high-risk group (2 and 3 points). The external validation analysis also demonstrated that our prognostic model provided the independent high predictive accuracy of recurrence.This model has a considerable clinical value in predicting recurrence, and will help clinicians to design an appropriate level of adjuvant treatment and schedule adequate appointments of surveillance visits.  Breast cancer remains the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide, despite the improvement of early screening and adjuvant treatment. It is a heterogeneous disease highly variable with respect to its biology, etiology, and treatment options. Molecular pathological characterization and gene expression profiling are useful for identifying breast cancer subtypes with different clinical characteristics and developing therapeutic options. DNA micA majority of TNBC is invasive ductal carcinoma of no special type, and the remaining is medullary carcinoma, invasive lobular, metaplastic carcinoma, etc. The triple negativity can occur in many histological subtypes of breast cancer, with possible implications on their pathogenesis, progression and prognosis,6. On thThe breast cancer patients with higher histologic grade, larger size, high ERK protein expression, low E-cadherin expression and Ki-67 staining may have a tendency toward local and visceral metastases,9–11. HoIn this study, we accessed several clinical and pathologic characteristics of 185 TNBC patients in order to identify additional prognostic markers that can identify tumors with more aggressive behavior and develop a prognostic model comprised of the significant biomarkers. We then validated the model in additional 319 patients in the same institution and demonstrated that the predictive model is successfully to discriminate the high-risk group of TNBC patients. Our proposed model provides the potential value that could be used to tailor treatment and surveillance strategies.A total of 504 eligible TNBC patients undertaking breast surgery between 2000 and 2006 at Shanghai Cancer Center were retrospectively analyzed. All patients had the following criteria in this study: (1) histologically confirmed mainly invasive ductal breast carcinoma, (2) a unilateral and non-inflammatory tumor, (3) status of ER, PR and HER-2 were available and negative, (4) patients had complete follow-up history, (5) the pathologic tissues were available for immunohistochemistry of other routine biomarkers in pathological department. Patient management was handled by the same department of surgeons, and the diagnosis was assessed by two senior pathologists. Conservative treatment and node resection along with radiotherapy, chemotherapy were applied according to current guidelines at that time, and all patients did not receive endocrine and trastuzumab treatment. The retrospective study was approved by the Ethics Committee of Shanghai Cancer Center. All patients gave their written informed consent before inclusion in this study.Tissue samples were fixed in formalin and embedded in paraffin. Hematoxylin and eosin stained slide from each tissue was examined microscopically to confirm the presence of tumor before biomarker evaluation. Immunohistochemistry was performed on 4-μm-thick deparaffinized sections on plus-coated slides, and immunohistochemical staining was carried out according to the manufacturer’s instructions. Endogenous peroxidase and biotin were blocked with 3% hydrogen peroxide and the Avidin-Biotin blocking kit , respectively. This was followed by incubation with primary antibodies for 60 min at room temperature, appropriate secondary antibodies , labeled streptavidin-horseradish-peroxidase , DAB+chromogen, and 0.2% osmium tetroxide , followed by counterstaining with light hematoxylin. Appropriate positive controls for each antibody and negative controls using species-matched immunoglobulin to replace the primary antibody were run with each batch. Positive tumor cells were quantified by at least 1,000 cells and expressed as percentage. Samples were independently evaluated by two trained pathologist in the blind procedure without knowing the patients’ background and clinical outcome. The cut-off for ER and PR positivity was 1% tumor cell with positive nuclear staining. HER-2 positivity was defined as a complete membrane staining in >10% of tumor cells. It was then divided into a qualitative scale from 0 to 3+, according to the criteria of a Hercep Test. Scores 0 and 1 were negative, and scores 2 and 3 were positive. The cutThe chi-square test was used to compare clinicopathological parameters or expression levels of molecules in patients between the two sets. Descriptive statistics were calculated to summarize patient characteristics, tumor size, and biomarker levels of surgical tumor samples. Survival results were last updated in October 2012. Disease-free survival (DFS) was defined as the elapsed time between the date of the first diagnosis and the date of the first relapse . Overall survival (OS) was defined as the length of time from the date of operation to death. Patients without events or death were censored at the last follow-up. Survival curves were established according to the Kaplan–Meier method. The log-rank test was used for univariate comparisons of survival endpoints. The Cox regression procedure was carried out to assess the relative influence of prognostic factors on DFS and OS. All tests were considered significant at two-sided P < 0.05. All analyses were carried out using SPSS 17.0 The median age of 504 women with TNBC was 51 years old (rang 23-85 years). All patients had received a curative operation of a mastectomy or a conservative surgery with axillary lymph node dissection in Shanghai Cancer Center between 2000 and 2006. 185 patients enrolled between 2000 and 2002 were included in the training set, while 319 patients recruited between 2003 and 2006 were designed in the validation set. The clinicopathological features of patients in the training set and validation set were shown in The median follow-up duration for the patients in the training set was 69.7 months; among them, 69 patients (37.3%) experienced a local or distant recurrence, and 40 patients (21.6%) died. We analyzed clinicopathologic characteristics of the patients using the Cox univariate survival analysis to identify potential predictors for DFS and OS . The uniBecause the lymph node status, Cathepsin-D and Ki-67 index were factors strongly associated with outcome of TNBC patients, we built up a prognostic model based on the summation of points scored by the features of these three factors in tumor tissues. For example, positive for ≥4 lymph nodes, positive for Cathepsin-D expression or Ki-67 index ≥20% would be individually recorded 1 point to the prognostic score. According to the prognostic score, patients were assigned to eight categories . Based oIn order to further validate the proposed prognostic model which is able to distinguish the TNBC patients with different outcomes, we tested the model using the survival data from the TNBC patients in the validation set. In this study, we evaluated the relationships between the clinical outcomes, clnicopathological characterics, and expression levels of prognostic biomarkers in patients with confirmed triple-negative breast cancer. Our analysis demonstrated that the combination of 3 prognostic factors, including positive for ≥4 lymph nodes , high Cathepsin-D expression and high Ki-67 index, would provide a strong prognostic power to differentiate the patients with worse outcome. Based on the score system consisted of local or distant metastasis in multiple lymph nodes, elevated Cathepsin-D expression and high Ki-67 proliferation index , we built up a mathematic prognostic model to distinguish the TNBC patients with the low survival rate (high-risk group) and the high survival rate (low-risk group). The proposed model demonstrates that the high-risk group has worse outcomethan the low-risk group in both training and validation sets. Our model would help select the subgroup of patients with high prevalence of local recurrence or distant metastasis. The cost-benefit ration and the selection of the most efficient design can be determined on the basis of the population selected using the model.Despite the fact that Cathepsin-D plays a critical role in a non-specific protein degradation in a markedly acidic environment of lysosomes, overexpression of Cathepsin-D in breast tumors is associated with the increased metastatic potential and the poor survival rate, in part by facilitaing cancer cell proliferation, fibroblast outgrowth, angiogenesis and metastasis. A recent study found that overexpression of Cathepsin-D in neuroblastoma cells attenuates doxorubicin-induced apoptosis through the activation of both the Akt-dependent pro-survival and the Bcl-2-dependent anti-apoptotic pathways, suggestKi-67 is a non-histone nuclear protein that is closely linked to proliferation cells. High Ki-67 expression is associated with a higher histologic grade, larger tumor size, the presence of axillary lymph nodal metastasis, and worse outcome–23. In nThe size of the primary tumor and the number of positive lymph nodes have an inverse relationship with prognosis and survival in breast cancer. The sixth edition of staging in breast cancer has included the nodal status as the most important prognostic factor. In large retrospective analysis, TNBC patients with N0/1 have better DFS than those with N2/3  . On contCurrently, the factors associated with the poor clinical outcomes in TNBC patients have been studied using gene expression profiles and more advanced genomic techniques. However, these approaches are not widely available. The application of therapeutic options and the assessment of the accompanied prognosis are dependent on the common tumor-related characteristics in patients. Based on the nodal status, Cathepsin-D expression and Ki-67 index in a group of patients as a training set, we built a faithful model that had considerable clinical value in predicting recurrence in another group of TNBC patients. This prognostic model can help clinicians to provide TNBC patients with appropriate adjuvant treatments and surveillance visits.