Mother-to-child transmission (MTCT) of HIV has been eliminated from the developed world with the introduction of multi-drug antiretroviral (md-ARV) regimens for the prevention of MTCT (PMTCT); but remains the major cause of HIV infection among sub-Saharan African children. This study compares two service delivery models of PMTCT interventions and documents the lessons learned and the challenges encountered during the transition from single-dose nevirapine (sd-nvp) to md-ARV regimens in a resource-limited setting.stand-alone site) versus sites providing PMTCT as well as antiretroviral therapy (ART) (full package site). CD4 cell count screening, enrolment into ART services and the initiation of md-ARV regimens during pregnancy, including dual (zidovudine [AZT] +sd-nvp) prophylaxis and highly active antiretroviral therapy (HAART) were analysed.Program data collected from 32 clinical sites was used to describe trends and compare the performance  of sites providing PMTCT as a stand-alone service  = 1.3; 95% confidence interval (CI): 1.1-1.4). Enrolment of HIV+ pregnant women in ART services was almost twice as likely at full package sites than at stand-alone sites . However, no significant differences were detected between the two models of care in providing md-ARV .From July 2006 to December 2008, 1,622 pregnant women tested HIV positive (HIV+) during antenatal care (ANC). CD4 cell count screening during pregnancy increased from 60% to 70%, and the initiation of md-ARV regimens increased from 35.5% to 97% during this period. In 2008, women attending ANC at Full package sites offer the most efficient model for providing immunological assessment and enrolment into care and treatment of HIV+ pregnant women. Strengthening the capacity of stand-alone PMTCT sites to achieve the same objectives is paramount.All sites successfully transitioned from sd-nvp to md-ARV regimens for PMTCT.  Mother-to-child transmission (MTCT) of HIV remains the major route of pediatric HIV infection in sub-Saharan Africa, where over 90% of the 2.1 million children living with HIV reside . Since 2Rwanda has a generalized HIV epidemic, 3.6% of women aged 15-49 are living with HIV according to the Demographic and Health Survey conducted in 2005 . It is eSince 2004, the International Center for AIDS Care and Treatment programs (ICAP) of Columbia University, Mailman School of Public Health , supports the implementation of HIV prevention, care and treatment programs in Rwanda including capacity building of staff, on-site mentoring, renovation of infrastructure, provision of equipment including laboratory machines, technical support to monitoring and evaluation, and operational research. In July 2006, ICAP supported the implementation of PMTCT programs at five health facilities (sites), including four district hospitals (DHs) and one health center (HC), and expanded to 32 sites (four DHs and 28 HCs) by December 2008 in the Western Province (Kibuye and Gisenyi regions) and Kigali, the capital city.PMTCT services are routinely implemented within MCH services in Rwanda. Opt-out HIV counseling and testing (CT) is routinely provided in the ANC by trained nurses using sequential rapid HIV tests following the national algorithm, with results available the same-day . Partner"full package" sites where PMTCT and ART services were both located on the same premises and "stand-alone" sites where only PMTCT services were available on-site. Full package and stand-alone sites were authorized to provide md-ARV regimens to pregnant women. However, at the time of writing this report, both stand-alone and full package sites were providing dual ARV and the sd-NVP regimens, but only full package sites offered 'therapeutic' HAART and 'short-course' HAART , to receive a TRACnet ID prior to CD4 testing requisition. In addition, because most CD4 blood samples originated from patients in ART services, pregnant women were therefore asked to come back on a different day, generally within a week after receipt of an HIV positive test result, as this allowed for common batching of blood samples from patients in ART services.In 2006, of the five PMTCT sites, only one had the equipment required to measure CD4 cell counts , the other four sites processed CD4 samples at the nearest laboratory possessing the necessary equipment. CD4 testing requisition was initially only performed for patients within ART clinics since a unique patient identifier (TRACnet number), only provided in the ART clinic, was required by the lab technician before blood could be taken. All pregnant women identified in PMTCT had to be referred to the ART clinic, within the same premises or via a referral  during pregnancy, number of women who initiated short-course HAART (sc-HAART) for prophylaxis during pregnancy, number of women who initiated dual ARV (AZT/NVP) prophylaxis during pregnancy, number of women who received sd-nvp only (sd-NVP) for PMTCT prophylaxis during pregnancy. These indicators are routinely collected from site registers, and reported monthly to district health offices for compilation and transmission to the central level. In January 2007, the national program revised the PMTCT program monitoring tools, including registers and monthly summary forms, adding new indicators for CD4 cell count assessments and enrolment in HIV care and treatment programs and HAART initiation. We reported changes over time for each indicator, and using chi-square tests of association with two-sided p-values, we compared the performance of the program across each indicator for the period 2007 - 2008, when comprehensive PMTCT program data was available.We collected the following routine PMTCT program indicators from the program monitoring database for the period July 2006 through December 2008: full package vs. stand-alone) across key program indicators using relative risks (RR) with associated 95% confidence intervals (CI) for the year 2008 only. The aggregated format of our data did not allow for controlling the effect of clustering across sites. In our analysis, we defined the following ARV regimens options as "more efficacious" ARV regimens . Data analysis was conducted using SAS version 9.1 . The data used for this analysis were aggregated site level indicators routinely collected by the national PMTCT program, and therefore did not contain any patient identifier. This secondary data analysis received approval from the Columbia University Institutional Review Board (IRB) and was also granted the non-research status  by the Centers for Disease Control and Prevention (CDC), Atlanta, USA.Finally, we compared the two model of service delivery  pregnant women were screened for their CD4 cell count during pregnancy, with the proportion increasing from 60% in 2006 to 70% in 2007 and 2008. Additional indicators collected in 2007-2008 showed that 96% of 1,268 women assessed for their CD4 cell count returned to the health facility to retrieve their results and 54% of 1,381 HIV+ women identified from July 2007 through December 2008 were enrolled in ART services. CD4 cell count screening remained at 70% in 2007 and 2008 (p = 0.18), however, the percentage of women receiving their CD4 test results and enrolling in HIV care and treatment programs increased significantly, from 92-98% (p < 0.001) and 41-60% (p = 0.04), respectively (Table 3), and 83% initiated HAART during pregnancy. The proportion of eligible women initiating HAART declined slightly from 86% in 2007 to 79% in 2008 (p = 0.12).Between July 2006 and December 2008, 71% of HIV+ pregnant women received an ARV regimen for PMTCT. The proportion of HIV+ pregnant women initiating more efficacious md-ARV regimens increased from 35.5% in 2006 to 97% in 2008 and women receiving sd-nvp decreased from 64.5% to 3% during the same time-period  and providing more efficacious ARV regimens .From January through December 2008, CD4 assessment of HIV+ pregnant women differed across the models of service delivery with women attending full package vs. stand-alone) within a 30-month period. To our knowledge, this is one of the first reports to comprehensively document the transition from a traditional sd-nvp regimen to more effective ARV regimens for PMTCT in a resource-limited setting. Despite PMTCT service decentralization to health centers with limited resources and capacities, addressing health systems bottlenecks at site and district levels led to increased numbers of HIV+ women undergoing an immunologic assessment during pregnancy, and improved identification of pregnant women who were eligible for therapy and subsequently initiated on HAART in both full package and stand-alone sites. However, more effort is needed to ensure effective CD4+ cell count assessments and the enrolment of HIV+ women in ART services in stand-alone sites. The finding of such studies will be useful in the design of strategies for implementing the 2009 WHO recommendations into national PMTCT programs.Until recently, most PMTCT programs still relied on the administration of sd-nvp to mothers and babies but md-ARV regimens have now been implemented. This report documents the successful implementation of md-ARV regimens, including HAART, for HIV+ pregnant women with advanced disease in 32 PMTCT sites in Rwanda. The transition from sd-nvp to more efficacious regimens occurred independently of the model of service delivery  and who received their results (up to 98% in 2008) significantly increased following the introduction of changes to the CD4 testing system at both the site and district levels. One major innovation was the same-day point-of-care blood sample taken from HIV+ pregnant women in ANC clinics. The importance of same-day CD4 cell count testing was also highlighted in two studies from South Africa reporting an uptake rate of over 97% for CD4 cell count screening during pregnancy ,11. Alth3. This is two-fold lower than the 55% reported in a study in the Western Cape, South Africa,[full package sites were more likely to undergo CD4 testing, women who received testing and were determined to be eligible were just as likely to initiate HAART in stand-alone sites as in full package sites in 2008, suggesting that the changes that were introduced had a positive impact, as summarized in Figure We found that approximately one quarter of pregnant women were eligible for HAART, based on CD4 cell counts ≤ 350 cells/mmh Africa, suggestiIn our report, the uptake of HAART among women identified as eligible was found to be higher than the 51% reported in five sites in the Western Cape  and the full package sites may be prompted to actively enroll women found to be eligible for HAART in the ART program, given that HAART initiation could be done in the same facility. We could hypothesize therefore, that making HAART available in stand-alone sites and accessible for eligible pregnant women may provide a motivation for providers to undertake CD4 cell count screening and enroll HIV+ and eligible pregnant women into ART services. By doing so, it would further reduce the burden on pregnant women who were asked to travel, in some cases, long distances to reach the nearest ART site.Health care providers in stand-alone sites to the nearest full package site, coupled with the availability of doctors from the DH who usually visit full package sites on a weekly basis for HAART initiation among new HIV+ patients. As PMTCT services continue to be scaled-up at the district level, the "flying doctors" approach might not be cost-effective and sustainable in resource limited settings. Task-shifting HAART initiation to nurses is therefore necessary and this has been successful performed in various settings [We noted, however, that the two models of service delivery did not differ with regard to HAART initiation among eligible pregnant women. This results from the effective system for referral of eligible pregnant women from settings -24. RwanSome key factors for an effective transition from sd-nvp to md-ARV regimens include among others the capacity building and regular mentoring of nurses, the routine assessment of the quality of care provided by nurses during the transition to md-ARV regimens, the full integration of PMTCT care components into MCH services, the strengthening of the CD4 testing capacity at district level, the reorganizing of services at site level with an emphasis on effective linkages between PMTCT and ART services, and the high level commitment and leadership of the Rwandan government's to improving maternal and child survival, including virtual elimination of pediatric HIV infection.The analysis carried out in this study was subject to some limitations. A recent analysis of routine PMTCT data in South Africa concluded that there were major weaknesses in the completeness and accuracy of this data which precluded its use in the tracking of process performance of facilities, however,stand-alone sites, and task-shifting is necessary to ensure the timely initiation and follow-up of HAART among pregnant women. These findings provide valuable insight which can be applied to the design of effective service delivery models for the transition from sd-nvp to md-ARV within national PMTCT programs in resource limited settings, accelerating progress towards virtual elimination of MTCT of HIV by 2015 [This report has demonstrated that regardless of the model of PMTCT service delivery, it is feasible to transition from sd-nvp regimen to more effective md-ARV regimen in a resource limited, public sector PMTCT program. However, more efforts are needed to ensure effective CD4+ cell count assessment and the enrolment of HIV+ women in ART services in  by 2015 .The authors declare that they have no competing interests.LT, FT, RC, VM, GT, EM, SK, PM, EK, RS, EJA contributed to the design of the program and the development of the concept for this manuscript. LT, FT, RC wrote the first draft of the manuscript. SK reviewed the laboratory section of the methodology. LT, RC, FT, EJA conducted data analysis and interpretation. And all the authors reviewed the final manuscript.At the time of this report, Landry Tsague was the Clinical Director at the International Center for AIDS Care and Treatment Program (ICAP)/Columbia University, Kigali, Rwanda. He is now with UNICEF in Rwanda.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/753/prepub


A. glycines on upper (i.e. vigorous-growing) versus lower (i.e. slow-growing) soybean nodes and under contrasting plant ages. In a second experiment, we measured aphid dispersion in response to predation. Bottom-up and top-down controls had additive effects on A. glycines population growth. Plant age and within-plant quality had significant bottom-up effects on aphid size and population growth. However, top-down control was the dominant force suppressing aphid population growth, and completely counteracted bottom-up effects at the plant and within-plant scales. The intensity of predation was higher on upper than lower soybean nodes, and resulted in a non-consumptive reduction in aphid population growth because most of the surviving aphids were located on lower plant nodes, where rates of increase were reduced. No effects of predation on aphid dispersal among plants were detected, suggesting an absence of predator avoidance behavior by A. glycines. Our results revealed significant non-consumptive predator impacts on aphids due to the asymmetric intensity of predation at the within-plant scale, suggesting that low numbers of predators are highly effective at suppressing aphid populations.There is increasing evidence that top-down controls have strong non-consumptive effects on herbivore populations. However, little is known about how these non-consumptive effects relate to bottom-up influences. Using a series of field trials, we tested how changes in top-down and bottom-up controls at the within-plant scale interact to increase herbivore suppression. In the first experiment, we manipulated access of natural populations of predators (primarily lady beetles) to controlled numbers of  Herbivores are regulated by a combination of top-down and bottom-up forces, but the relative strengths of these forces vary among communities Top-down control of herbivores is usually attributed to consumption by predators Aphis glycines Matsumura (Hemiptera: Aphididae) towards more mature leaves in response to predation A. glycines. Therefore, we hypothesize that 1) vigorous-growing plant parts support higher A. glycines population growth rates, and 2) predation on vigorous-growing plant parts results in non-consumptive reductions in A. glycines population growth rates by differentially removing individuals with the highest reproductive potential. Here we report on field manipulations of predator access to the upper (i.e. vigorous-growing) and lower (i.e. slow-growing) nodes of soybean (Glycine max L.) plants using partial and whole-plant exclusion cages. In addition, we accounted for potential confounding effects of plant age on node quality by adjusting planting dates to have contrasting plant phenologies in two of the three trials reported here. By manipulating these treatments in a factorial design, we tested: 1) bottom-up effects of upper versus lower plant nodes, 2) top-down effects on upper versus lower plant nodes on aphid population growth rates, and 3) the relative strength of top-down versus bottom-up controls on aphid population growth rates. In addition, we conducted a separate field experiment to test 4) whether top-down impacts resulting from predator avoidance behaviors increase aphid dispersal.Although progress has been made in investigating the relative strength of top-down and bottom-up controls on herbivores, few studies have used manipulative experiments in agroecosystems but see . Such stet al. Three field trials were conducted at the University of Minnesota Outreach, Research and Education (UMore) Park near Rosemount, MN, from 19 July to 24 August 2007. In order to have plants with different ages available simultaneously, replicated plots of soybean  were established prior to experiments on 4 May, 29 May, and 26 June. Plots (10×20 m) were 8 rows wide, and planted in a randomized complete block design with one replicate of each planting date per block; ground was left fallow between plots to facilitate access. Two plant age treatments (“old” versus “young” treatments) were compared simultaneously in trials 1 and 2, whereas no young plants were available for the last trial. All plants used were at the reproductive stage (R) and were classified using the system described in Ritchie Each trial compared two levels of predation  and locations on the plant (“lower” versus “upper” nodes), resulting in four treatment combinations: a) no predation , b) lower node predation , c) upper node predation , and d) full predation  . All trerd or 4th node, and supported externally by tomato cage frames −ln(N0))/t, where N0 = initial number of aphids, Nt = number of aphids at time t, and t = duration of the experiment (in days). Aphid growth rate was calculated separately for upper and lower nodes to test for within-plant bottom-up effects, and for the whole plant to contrast bottom-up and top-down effects, on overall aphid population growth.High Larger adult aphid size has been shown to be an indirect indicator of favorable plant quality and higher reproduction Finally, we compared the results of our experimental manipulations with naturally occurring aphid and predator populations from the same plots. Three to ten haphazardly selected plants were sampled in each plot at weekly intervals, counting the total number of aphids per plant (July) or estimating this count using a three node sampling unit (August) A. glycines among soybean plants in a field at UMore Park, MN, during June 2008. We caged a 1-m row of soybean using a 1×1×1 m cage, which consisted of a polyvinyl chloride (PVC) frame covered primarily by no-see-um netting with a 15-cm wide band of coarser netting (2 mm) on the upper part of all cage walls to allow for alate emigration  in 10 randomly-selected field cages with the remaining 10 left as untreated controls. After 24 h, we recounted the number of aphids present on each plant. Since we had to remove the lady beetles from the cages to perform the aphid counts, we replaced them with freshly field-collected adult H. axyridis and Coccinella septempunctata L. (3∶2 ratio), as the latter species became more readily available. After the first 24 h of predation, we observed no impacts of the lady beetle treatment on aphid redistribution  upper node predation treatments decreased the proportion of aphids on the upper nodes below 0.5; 2) lower node predation treatments increased the proportion on the upper nodes above 0.5; and 3) the combination or absence of both resulted in a proportion differing from 0.5  and percentage (arcsine-transformed) of aphids on each of the three groups of five plants  using a one-way ANOVA with predator treatment  as a fixed effect. Means were separated using Tukey Honest Significant Difference test . A similar trend was found for late instar nymphs  but differences were only marginally significant .Larger adult aphids were collected on the upper than the lower nodes , with significant effects of plant age in trial 2, indicating that upper nodes had higher quality for aphid growth , Table 1A. glycines population growth. Moreover, effects of plant age that occurred without predators could not be detected with upper node predation , or were all but eliminated under full predation .Upper node predation (−LP+UP) suppressed aphid populations in all trials, reducing rates of increase from 27 to 130% (67±20% mean ± SE), compared to the predator exclusion treatment −LP−UP; , Table 1A. glycines varied significantly with sampling date , reaching outbreak levels above the economic injury level of 674 aphids/plant during the first 5 weeks of the study , nor PA×sampling date interaction  affected aphid populations. Similarly, on 16 August planting date did not affect field population size  1%), followed by damsel bugs , spiders (Araneae 10.9%), minute pirate bugs  5.1%), brown lacewings  and green lacewings . Predator populations were significantly higher during the second week of the experiment , but after that they declined . Plant age did not affect predator populations on 16 August . During the 24 h following, predation by lady beetles was negligible: no difference in the number of aphids was detected between lady beetle treatments on the release plants ; or colonized plants on the left ; or right . After five more days lady beetle predation reduced aphid density by 13- to 21-fold in comparison with controls on release plants , and colonized plants  than older plants (i.e. full pod and beginning seed stages), supporting predictions of the plant vigor hypothesis, and previous studies (e.g. Myzus persicae (Sulzer) and Aphis fabae Scopoli (Homoptera: Aphididae), resulting in a bottom-up controlled decline in field populations as the season progressed Our results demonstrate that a combination of top-down and bottom-up factors governs the population increase of ies e.g. . For exaA. glycines, both at the whole- and within-plant scales, diluting differences in population growth due to plant age and reversing the within-plant relative abundance of aphids. Although A. glycines responded in accordance to the plant vigor hypothesis in the absence of predation, strong top-down factors exerted the most significant control of aphid populations in our experimental plants, confirming previous studies Top-down control completely counteracted bottom-up control of The strong top-down control observed in our experimental plants was not equally effective at suppressing aphid populations naturally occurring in the field plots. Several factors can explain this mismatch, including effects of aphid immigration, and potential spill-over of predators from un-manipulated to experimental plants, resulting in artificially high predator: prey ratios. In a trial conducted two weeks before this study, the field plots received a massive immigration of alate aphids. In a separate contribution A. glycines in experiments conducted both during years with high A second hypothesis explaining the discrepancy between natural and manipulated plants is that the large number of aphids naturally occurring in our plots attracted a large number of natural enemies, resulting in artificially higher predator: prey ratios in our experimental plants and stronger top-down control in comparison with un-manipulated plants. However, several lines of evidence suggest that the strong top-down control observed in our experiment was not merely an artifact of potential “spill over” of predators. First, previous studies manipulating predation have shown strong impacts of predators on A. glycines system, previous studies have utilized similar cages to demonstrate the impact of natural enemies on aphid populations The use of cages to manipulate natural enemies has the potential to affect micro-environmental conditions for the plants, herbivores, and predators involved in the study. In the A. glycines, despite differences in quality between the top and the bottom of the plant. Previous studies on other species have shown aphid dispersion after exposure to predation A. glycines relocation in response to predation at the scale of groups of plants, and did not find any response by aphids, despite strong predation by lady beetles and moderate predation in sham cages. Butler and O’Neil A. glycines against O. insidiosus, that involved a sticky substance and a potential alarm pheromone, but the defenses resulted in very limited escape behavior. Thus, our results combined with previous studies suggest that dispersal for A. glycines under lady beetle predation is not an important defense.At least two different mechanisms can explain the change in aphid within-plant relative abundance in response to predation. First, within-plant redistribution can be the result of aphid dispersal A. glycines revealed more predation on the upper-third of the plant than on the lower nodes A second mechanism that explains aphid within-plant distributions follows from patterns resulting in higher rates of predation on upper nodes of the plants. Direct field observations of predation on A. glycines. Population dynamics are significantly affected by mortality of individuals with the higher reproductive value A. glycines sizes (adults or nearly adults), which presented the highest potential for aphid population increase. Using a detailed demographic model, the authors demonstrated that lower numbers of parasitoids were required to control aphid populations due to this differential removal of the individuals with the highest reproductive value. Similarly, in our study we observed the highest impact of predation on the upper nodes of the plant, which supported larger aphids and higher rates of population increase. Therefore, our results suggest that this asymmetric pressure on the top of the plant allows relatively low numbers of predators to control aphid populations. Furthermore, this non-consumptive impact of predators provide a mechanistic explanation to previous field studies that found low numbers of predators suppressing A. glycines populations The asymmetric impact of top-down controls at the within-plant scale results in non-consumptive effects on aphid population growth that explain the strength of predator impacts on Uroleucon ambrosiae (Thomas) results in almost complete exclusion of aphids from tall host plants, restricting the population to shorter plants that have low quality. Similarly, Moon and Stiling Pissonotus quadripustulatus suffers higher impact of parasitism by Anagrus parasitoids on high quality green stems than on low quality woody stems. Our results, together with these previous studies, support the theoretical prediction that under strong top-down controls, herbivores will find advantage in the use of refuges, in which a trade-off of lower fecundity or longevity is compensated by a reduction of predation Non-consumptive impacts of predators, including trait-mediated effects on prey behavior, change of prey life history parameters, and selectivity on prey life stages File S1Comparison of aphid growth rates in predator exclusion cages with and without restriction on aphid movement.(DOC)Click here for additional data file.File S2Comparison of aphid growth rate in predator exclusion cages with single versus double mesh cage treatments.(DOC)Click here for additional data file.


Prostate stem cells are thought to be responsible for generation of all prostate epithelial cells and for tissue maintenance. The lineage relationship between basal and luminal cells in the prostate is not well clarified. We developed a mouse model to trace cell fate and a mouse model with a slowly cycling cell label to provide insight into this question. The results obtained indicate that putative mouse prostate stem cells are likely to reside in the basal layer. The prolonged use of androgen deprivation therapy is associated with a decline in responsiveness to treatment of prostate cancer treatment, an effect that generally results in a poor clinical outcome. Thus, a focus on the origin of cells capable of developing the androgen-independent tumor mass subsequent to hormonal therapy is of interest.in vitro [ Normal prostatic epithelium is comprised of a stratified structure with secretory luminal cells that are androgen-responsive and an interspersed minor population of highly proliferative, androgen-receptor- (AR-) negative basal cells. These AR-negative cells are thought to give rise to AR-positive luminal cells . As detein vitro . Other min vitro –5.  The traditional model of prostate epithelial differentiation proposes that adult prostate stem cells are contained within the basal layer and give rise to progenitor/stem cells and androgen-independent, transit-amplifying cells, which can differentiate into luminal cells. This concept is supported by the isolation of intermediate cells, which express both luminal and basal makers, keratin 5 and keratin 18, and by the differentiation of these cells down the luminal lineage . In animAs presently reported, two approaches were used to determine the lineage-derived fate of basal cells: (1) tracking that allows lineage-specific tagging of the keratin 14-expressing population of cells throughout prostate development in adult mice, and (2) a keratin 5 H2BGFP-label retaining model demonstrating that slowly cycling cells are represented in only a subfraction of keratin 5-expressing cells after hormone/castration manipulation. These cells have the capacity to give rise to more differentiated luminal cells. Results with these models, which suggest that the putative adult stem cells, which have a slowly-cycling feature, most likely reside in the basal layer and are positive for keratin 5 and keratin 14, clarify some of the basic aspects of the biology of the rodent prostate gland.tam transgenic mice (STOCK Tg (KRT14-cre/Esr1)20Efu/J), LacZ reporter mice (B6.129S4-Gt (ROSA)26Sortm1Sor/J), and EGFP reporter mice 26Sortm2Sho/J) were purchased from the Jackson Laboratory, Bar Harbor, ME, USA. The two transgenic mouse lines were crossed, and double transgenic markers of Cre and LacZ were confirmed by PCR with genomic DNA isolated from their progenies. Tamoxifen (Sigma-Aldrich) was prepared at 100 mg/mL in ethanol, heated to 60°C to dissolve the powder, and then diluted 10 times with sunflower oil aided by 2 min vortex plus sonication for an additional 30 min. Tamoxifen was used to induce Cre activity in gaining its access to the nuclear compartment from the cytoplasm. The administration to young (3 weeks old or younger), double-transgenic mice, of 0.5 mg tamoxifen was accomplished by intraperitoneal injections daily for 5 days. The double-transgenic mice were sacrificed after two cycles of prostate involution (2 weeks) and regeneration by castration and by supplementation (for 2-3 weeks) with testosterone pellets . The prostates were removed, and frozen sections from dorsal and ventral glands were obtained. The presence of β-galactosidase was demonstrated with X-gal by use of a commercial kit (Invitrogen).Keratin 14-CreERTransgenic mice expressing a Tet-OFF tTA regulatory transactivator under the control of a keratin 5 promoter were generously provided by Adam Glick . These mice were bred to TRETight-H2B/GFP transgenic mice that we created in the NCI-Frederick animal facility. Expression of H2B/GFP is naturally turned on in the double-transgenic mice (keratin 5-tTA:TRETight-H2B/GFP) following development and can be turned off in the basal epithelium of the prostate following the application of doxycycline. A wash-out period is required to identify the label-retaining cells. The prostate epithelial cells that are still H2B/GFP+ after the wash-out period mark the label-retaining cells, which, because of their slowly cycling feature, should represent prostate epithelial stem cells. To ensure the observation of those rare putative stem cells resistant to label dilution due to cell turnover, this wash-out period continued throughout the multiple cycling (over 10) of the prostate epithelium between regression and expansion by hormonal manipulation. After the label-retaining assay of the murine prostate was complete, the animals were anesthetized, and the prostates were removed for histologic analysis.The hormonal manipulation consisted of alternating 2-3 week periods of androgen deprivation and restoration. This manipulation results in regression of the prostate epithelium to a residual basal layer (androgen deprivation) followed by expansion to a multilayered epithelium (androgen restoration). Hormonal deprivation was accomplished by castration, and androgen restoration was achieved by use of implantable testosterone pellets , which maintained a serum level of 4 ng/mL. The β-galactosidase staining kit was used according to the manufactures suggested procedure to demonstrate the activity of LacZ. Briefly, thawed cryosections of mouse prostates were placed in the fixative solution for 10 min. After washing, the samples were incubated at 37°C with a solution containing X-gal, and the blue cells were assessed within 2 h. The slides were counterstained with nuclear fast red solution. μm mouse tissue cryosections. Briefly, the thawed sections were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature and, after washing with PBS, blocked with 10% donkey whole serum in PBS. After washing, cells were incubated sequentially with primary antibody rabbit polyclonal anti-mouse keratin 5  and Alexa Fluor-594 or 546-conjugated donkey-anti-rabbit secondary antibody (Invitrogen), 4′,6-diamidino-2-phenylindole (Sigma) with washing after each step, and mounting medium (Vector). Rabbit anti-mouse keratin 18 (clone LE61) was a gift from E.B. Lane . Standard direct immunofluorescence was performed similar to the above procedures with EGFP detection, except that the only antibody used was Alexa Fluor 594 conjugated rabbit anti-GFP (Invitrogen). The images were acquired and analyzed with a Zeiss Axio fluorescence imaging system (Carl Zeiss MicroImaging Inc.).For marker expression, standard indirect immunofluorescence staining was performed on 6  Analysis of keratin expression during prostate development has been useful in distinguishing undifferentiated basal cells and, during tissue regeneration, the more differentiated luminal cells. To determine if luminal cells, which constitute the major population 90%) in mouse prostate epithelia, are derived from basal cells during injury repair, the lineage tracing Cre/loxP system was utilized to track cells expressing keratin 14  and their differentiated progenies in the mouse prostate. These transgenic mice contain the tamoxifen-inducible Cre gene (CreER) under the control of the keratin 14 promoter. To visualize the developmental and regenerative pattern of keratin 14 expressing-cells, keratin 14-CreER mice were crossed with ROSA 26-LacZ mice. ROSA 26 is a universal promoter, in this case driving a LacZ gene preceded by a floxed stop sequence [0% in mou Before being sacrificed, tamoxifen-treated keratin 14-CreER/ROSA-LacZ mice underwent 2 cycles of androgen manipulation by castration and testosterone supplementation . Experim The accepted paradigm for prostate development is that all epithelial cells are derived from a common embryonic precursor that expresses keratin 5 . Experim Keratin 5 expression was tracked through multiple cycles of androgen manipulation, by castration of young mice and by injection of testosterone pellets into adult mice . Keratinin vitro and in vivo; murine models have been used predominately. In contrast to human prostate, which has a complete prostate tissue unit with a continuous basal and luminal layer, the mouse prostate has four major lobe pairs and is further divided into numerous ducts. There is no clear basal layer in the ducts. Results from previous investigations utilizing the label-retaining assay suggest that the proximal region of the mouse prostate is more likely to contain adult prostate stem cells that express basal markers and that the distal region of the duct contains more differentiated luminal cells, with expression of luminal makers [ Stem cell biology and tumor biology are intimately related. An understanding of the function of prostate stem cells in the lineage-associated differentiation responsible for maintaining tissue integrity has potential application to prostate regeneration and tumorigenesis. Previous attempts to define these lineage-derived cells have utilized assays performed l makers , 12. In l makers , 14. Nev To identify stem/progenitor cells capable of generating multiple lineages, we utilized a multidirected approach. First, we were interested in identifying cells within the basal layer and determining which basal-derived cells were capable of luminal differentiation. Keratin 14 is a specific marker of basal cells. As established with the mouse model of tamoxifen-induced, label-retaining keratin 14-CreER/ROSA-LacZ, the initial keratin 14-mediated LacZ expression was eventually restored in the luminal cells, as opposed to basal cells in the distal and intermediate regions. However, within the proximal region, keratin 14 expression was primarily expressed in the basal layer. Based on these findings, we propose that, during prostate development, especially in the scenario of tissue injury, luminal cells are derived from the basal layer. However, since most, if not all, of the basal cells retained keratin 14 expression, these cells are likely to be the progenitor cells, not the stem cells, within the basal layer that give rise to keratin 14-expressing luminal cells. Thus, our results agree with other reports that the basal layer is the origin of more differentiated luminal cells.  Multiple lines of evidence support the hypothesis that stem cells within the basal layer of prostates in noncastrated and castrated animals are responsible for tubule-forming capacity and give rise to multipotent progenitor cells within the basal layer. By use of an artificial tissue injury and repair process, the presence of living keratin 5-positive stem cells was tracked in keratin 5-tTA-TRE-H2BGFP mice, with the hypothesis that the luminal lineage is derived from a basal lineage. Furthermore, the slowly cycling cells that retain the H2BGFP label after multiple cycles of castration/hormone replacement would represent the population of adult prostate stem cells. The keratin 5 GFP-positive cells were only a minor fraction of the cells. These GFP-positive, keratin 5-expressing cells also lacked coexpression of luminal marker, keratin 18. This suggests that all other cells, including transit-amplifying cells and possibly some progenitor/stem cells entering into the cell cycle, which lose H2BGFP expression, are diluted out. Since keratin 5 is a marker of basal cells, the results indicate that these cells are basal cells, or at least that they are derived from the basal layer. +/keratin 18− cells are prostate stem cells, since it is impossible to determine distant lineage with this approach. Our evidence does suggest that these cells are basal-restricted, slowly cycling progenitor cells. Further evidence is required to characterize this population of cells.  With keratin 5 as a marker, others have shown that the proximal cells of mouse prostatic ducts are slow cycling, have a high proliferative potential, and give rise to large, branched glandular structures that produce prostatic secretory products . Neverth We present strong evidence that the basal-derived lineage is responsible for prostate development and tissue regeneration, but there are indications that luminal cells retain regenerative capacity after animals are castrated. The most recent is a report of experiments involving use of a PSA-CreERT2-based genetic lineage tracing system. These results demonstrate that, in a mouse model, preexisting luminal epithelial cells survive and proliferate during cycles of regression and regrowth of adult prostate . Althougin immunodeficient mice [ The present findings have implications in initiation of prostate cancer. Basal cells from primary benign human prostate tissue, with the cooperative effects of AKT, ERG, and AR, recapitulate the histological and molecular features of human prostate cancer, with loss of basal cells and expansion of luminal cells expressing PSA and alpha-methylacyl-CoA racemase ent mice , 17. In ent mice , and the In summary, with the use a mouse model for tracking cell fates and a mouse label-retaining assay, we determined that, in the prostate, luminal cells are derived from a basal lineage and that slowly cycling cells, which may represent adult prostate stem cells, reside in the basal cell compartment. These observations have implications for the concept of epithelial cell lineage in the developing human prostate and for identification of cancer stem/initiating cells.


We sought to evaluate the test characteristics of the acute cardiac ischemia time-insensitive predictive instrument (ACI-TIPI) in relation to 30-day major adverse cardiac events (MACE) among patients who presented to the Emergency Department with symptoms suggestive of an acute coronary syndrome. We then examined the test characteristics of various dichotomous ACI-TIPI cut points.We prospectively recruited a cohort of Emergency Department (ED) patients with acute chest pain at two urban university hospitals between June and September 2006. Upon enrollment, baseline demographics and cardiac risk factors were collected. An electrocardiogram (ECG) was performed and analyzed with the built-in ACI-TIPI multiple regression model software. An ACI-TIPI probability score was recorded for each patient. Diagnostic test characteristics of ACI-TIPI for MACE , percutaneous coronary intervention, coronary artery bypass grafting, and all-cause mortality) within 30 days were determined.p < 0.01). An ACI-TIPI score of ≥ 20 had 100% sensitivity (95% CI 82-100), 100% negative predictive value (95% CI 86-100), and 21% specificity (14-31%).Of 144 patients enrolled , 19 (13%) patients suffered MACE within 30 days. Receiver-operating characteristics (ROC) for ACI-TIPI yielded a c-statistic of 0.69 (95% CI 0.59-0.80, These preliminary results suggest that, while ACI-TIPI has limited discriminatory value for MACE overall, a score of < 20 may have 30-day prognostic utility to allow for safe outpatient management in patients with acute chest pain. Over 6 million patients undergo evaluation for chest pain in the United States each year ,2. A larThe acute cardiac ischemia time-insensitive predictive instrument (ACI-TIPI) was developed as a means to risk-stratify patients in real time ,23. SubsThe ability of an ACI-TIPI cut point to identify patients at very low risk of myocardial infarction (less than 2% at 45 days) has been studied in patients admitted to the hospital or chest pain observation units ,22,25,29A prospective cohort study of patients with acute chest pain was conducted at two urban university emergency departments, each with over 55,000 visits per year. Both institutions are staffed 24 hours a day with board-certified emergency doctors. The study protocol was approved by each hospital's institutional review board, and all patients provided written informed consent.We enrolled consecutive ED patients with symptoms consistent with myocardial ischemia during periods of research assistant availability, on weekdays during business hours (9 a.m. to 5 p.m.), between June and September 2006. All patients were evaluated by a board-certified emergency physician and received standard treatment irrespective of their participation in the study. We included all patients with chest pain or symptoms consistent with myocardial ischemia . All patients had a single troponin measurement as part of their workup. Because this was an observational study, there was variability among treating physicians regarding the decision to measure serial biomarkers and obtain functional cardiac studies. We excluded patients with obvious alternative diagnoses who did not receive any workup for myocardial ischemia, as well as patients with ST elevations on their initial electrocardiogram, as we did not want the research protocol to interrupt expeditious transfer to the catheterization suite. Assuming a baseline 20% prevalence of the predictor (ACI-TIPI cut point) and a 5% MACE rate, we calculated the need to include 156 patients to have 80% power and 5% alpha to show a two-fold risk difference.Patients meeting the enrollment criteria above were approached by trained research assistants. After providing written informed consent, a standardized data collection instrument was completed by the research assistant. This instrument assessed baseline demographics, traditional cardiac risk factors , and current medication use. These data were recorded and entered into the study database.Upon enrollment, patients had an ECG performed, which was analyzed with the built-in ACI-TIPI multiple regression model software. To do this, a research assistant acquired and entered the following data required for the ACI-TIPI program: the patient's age and sex, the presence of chest or left arm pain, and whether these symptoms were their primary complaint. Based on ECG evidence of pathologic Q waves and ST- and T-wave changes, the software calculated a probability score for myocardial infarction, reported as a percentage, which represents the probability of acute ischemia. Exact numerical values for these log-odds ratios are reported elsewhere .The diagnosis of myocardial infarction was based upon European Society of Cardiology/American College of Cardiology (ESC/ACC) guidelines as a typical rise and fall of troponin T  or troponin I  in the setting of ischemic symptoms or characteristic ECG changes such as pathologic Q waves or ST depression .The primary outcome was any MACE within 30 days of evaluation, including these events during the index visit. MACE was defined as any one of the following: non-ST elevation myocardial infarction (NSTEMI) as defined by ESC/ACC criteria , percuta2 and Student's t tests were used to compare categorical and continuous variables, respectively. All p-values were two-tailed with a value less than 0.05 considered significant. Area under the receiver-operating characteristics curves (AUCs) of ACI-TIPI to predict MACE were plotted, to assess overall discrimination and to define sensitivity and specificity for various cut points of the ACI-TIPI score . A dichotomous cut point was chosen as the ACI-TIPI score that achieved a sensitivity and negative predictive value (NPV) of 100%. Data were analyzed using SPSS version 14.0 .We calculated sensitivities, odds ratios (ORs), and 95% confidence intervals. Descriptive statistics were used for baseline characteristics. χWe enrolled 144 patients who met the inclusion criteria. Eight patients (5.5%) were lost to follow-up at 30 days, yielding 136 patients with complete data. The clinical characteristics of patients with and without MACE are summarized in Table An ACI-TIPI score above 20% was associated with a 20% increase in the odds of having a MACE within 30 days, while an ACI-TIPI score above 30% was associated with a 13 times greater odds of having a MACE , given our MACE rate of 13%, we would have needed 43 MACEs. To accomplish these this number of events would require approximately twice the number of patients enrolled in this study. A larger study is needed to validate these findings. Second, enrollment of patients only during the hours of research assistant availability may have introduced an inclusion bias into our sample. Due to the broad constellation of complaints that could be interpreted as possible ACS, we were unable to retrospectively identify the overall number of patients who met our enrollment criteria and were not enrolled. However, the baseline characteristics of our study population reveal a quite heterogeneous sample with respect to cardiac risk factors. Our MACE rate was 13.8%, which is similar to prior studies of undifferentiated chest pain cohorts . Third, These preliminary results suggest that ACI-TIPI is useful for 30-day prediction of MACE. When used in the setting of ED patients with acute chest pain, a dichotomous cut point of less than 20% may aid identification of patients amenable for outpatient management. A prospective cohort study with a larger sample size is warranted to confirm these findings.The authors declare that they have no competing interests.JI participated in the design of the study, provided oversight for the data collection, performed the 30-day follow-up data collection, and drafted the manuscript. AM participated in the design of the study, provided oversight of the 30-day follow-up data, performed the statistical analyses, and edited the manuscript. UH participated in the study design and edited the manuscript. VN participated in the design of the study, provided oversight of the primary data collection, and edited the manuscript. EG and SN performed the primary data collection. JSB conceived the original study design and edited the manuscript. All authors read and approved the final manuscript.


Caenorhabditis elegans providing a new and powerful strategy to delineate the molecular pathways that lead to metabolic disorder. The C. elegans intestine is our model system where genetics, molecular biology, and cell biology are used to identify and understand genes required in fat metabolism. Thus far, we have found an important role of C. elegans KLF in FA biosynthesis, mitochondrial proliferation, lipid secretion, and β-oxidation. The mechanism by which KLF controls these events in lipid metabolism is unknown. We have recently observed that C. elegans KLF-3 selectively acts on insulin components to regulate insulin pathway activity. There are many factors that control energy homeostasis and defects in this control system are implicated in the pathogenesis of human obesity and diabetes. In this review we are discussing a role of KLF in human metabolic regulation.Lipid metabolic disorder is a critical risk factor for metabolic syndrome, triggering debilitating diseases like obesity and diabetes. Both obesity and diabetes are the epicenter of important medical issues, representing a major international public health threat. Accumulation of fat in adipose tissue, muscles and liver and/or the defects in their ability to metabolize fatty acids, results in insulin resistance. This triggers an early pathogenesis of type 2 diabetes (T2D). In mammals, lipid metabolism involves several organs, including the brain, adipose tissue, muscles, liver, and gut. These organs are part of complex homeostatic system and communicate through hormones, neurons and metabolites. Our study dissects the importance of mammalian Krüppel-like factors in over all energy homeostasis. Factors controlling energy metabolism are conserved between mammals and  Mammals possess the unique ability to store excess fat in the form of triglycerides (TG), the primary component of adipose tissue. Due to its homeostatic nature, energy storage is tightly regulated through many signaling pathways that integrate fat deposition with its mobilization and utilization throughout the body. Given the number of steps that are collectively associated with energy storage and consumption underpins the importance of understanding the entire process of nutrient uptake, storage and its distribution. This knowledge is also important because deregulation in any step of this metabolic pathway can lead to metabolic disorders such as diabetes, obesity, atherosclerosis, and accelerated aging. Obesity or being overweight is an issue of great concern given that much of the increase in this condition is driven by changes in nutrient uptake, and its distribution. A concern relating to obesity is that it raises the risk for many chronic and potentially life-threatening illnesses, including diabetes, and heart disease. Fat storage is central to whole body energy homeostasis that involves multiple organs including the brain, adipose, muscle, liver, and gut. Obesity has been linked to diabetes and therefore, the term diabesity was coined to describe the intimate link between diabetes and obesity. A potentially unifying view is that insulin resistance, which arises from an excess of body fat, links T2D to obesity. Consistent with this view it has been observed that fat deposition in insulin effector cells  decreases their sensitivity to insulin, causing insulin resistance. Fat accumulation in nonadipose tissues promotes lipotoxicity. This toxicity can diminish or impair the ability of pancreatic β-cells to secrete sufficient insulin leading to an array of metabolic changes characteristic of insulin resistant and T2D. The primary genetic, environmental and other metabolic factors responsible for insulin resistance are not completely understood. Insulin is a central regulator of both energy homeostasis and glucose metabolism and has a prominent role in liver, muscle, and fat cells.The increased incidence of obesity and diabetes represents the biggest public health problems facing the world today. In the United States about two-thirds of the populations are now either overweight or obese. According to the World Health Organization (WHO), the obesity rates have more than doubled worldwide since 1980. The numbers are just as staggering for diabetes. Many years of genetic, molecular and biochemical studies have provided evidence that energy homeostasis is maintained by the combined action of hormonal, metabolic, physiological, genetics, and neuronal system that regulate appetite and food intake. The neuronal network of the brain plays a critical role in the control of overall processes of energy homeostasis. This network is somewhat similar to a molecular circuit board which has remarkable ability to organize varieties of circuit relationships between signaling proteins. This collection of circuits can sense signals from notable array of hormones, nutrients and neural inputs. Recent studies by Schwartz and his colleague. These hCaenorhabditis elegans offers a relevant model to elucidate the mechanism of metabolic regulation that matches the available mouse model.Leptin is secreted from adipose tissue, suggesting that adipose tissue is not only the site for fat storage, but it can also serve as an active endocrine organ with the capacity to control energy homoeostasis. Adipose tissues communicate with the brain and peripheral tissues by secreting hormones and adipokines-5. TheseCaenorhabditis elegans model, its genomic databases, and the accessibility of mutants associated with fat metabolism in order to understand the molecular and genetic mechanism of metabolic regulation in human. Because of its important role in mammalian lipid and glucose metabolism[C. elegans model. Many of the regulatory genes involved in C. elegans fat metabolism are involved in adipocyte biology. This relatively simple organism provides a powerful system to study the genes with critical roles in human metabolic diseases.We take advantage of the availability of tabolism-17, we cC/ebpα promoter, whereas a decrease in KLF3 prevents differentiation[There is an elaborate network of adipogenic transcription factors which include transcriptional activators, co-activators and repressors that coordinate the expression of essential proteins involved in the formation of mature fat cells through several important signaling pathways. On one hand these factors initiate and regulate the transcriptional cascade during development of new fat cells. These same factors also regulate the expression of essential metabolic enzymes, signaling components, and adipokines in the mature fat cell. By controlling these events, these factors maintain the differentiated state by allowing fat cells to perform their functions. The human KLF family has 17 members, which are expressed in a variety of cell types-20. Seventiation showed tntiation observedntiation,23. Whilntiation, it alsontiation,26. Humantiation. Thus KLntiation. This inntiation,29. The Food intake is not only influenced by physiologic signals for hunger and satiation but it can also be stimulated or inhibited by internal signaling systems in order to regulate the energy stores. At present, we do not know much about the detailed mechanisms that control our desire to eat. Even though, some interesting hypothesis can be drawn on the connections between, foods, the level of neurotransmitter and metabolic interaction in producing signals to the liver or brain Figure. AccordiConsiderable interest in the physiology of food intake was generated more than half a century ago when Kennedy suggesteFood-intake involves neuronal, visual, hormone receptors, and many other nutritional signaling mechanisms. Hormones and neural signals maintain energy homeostasis by regulating the size of food intake. These signals are received, transmitted and integrated by CNS, which involves many neural circuits and transmitters Figure. Many siThe nervous system acting as a central command and control system coordinates both metabolism and behaviors. Insulin receptors and insulin signaling proteins are broadly distributed throughout CNS. Both hormone receptors are expressed in neuronal cells in the brain-41 and tThe discovery of leptin in 1994 brought about an entirely new physiological system with direct implications for the pathophysiology of human obesity and has helped develop treatments for obesity as well as other metabolic conditions, such as diabetes . ob/ob mice are obese and have neuroendocrine abnormalities similar to those of starvation, suggesting that leptin may be involved in the physiology of starvation. Preventing the starvation-induced fall in leptin with exogenous leptin considerably reduces the changes in gonadal, adrenal and thyroid axes in male mice, and prevents the starvation-induced delay in ovulation in female mice. In contrast, leptin repletion during this period of starvation has little or no effect on body weight[The physiological effects of leptin are mediated through the hypothalamus, the expression site of OB-R. OB-R isy weight, suggestC. elegans to study the role of KLF in the control of factors that play important role in mammalian metabolic regulation. There are only three KLF members  in C. elegans genome[C. elegans klf-1 is an essential regulator of lipid metabolism and its loss of function affects lipid metabolism leading to fat accumulation and increased cell death[C. elegans klf-3(ok1975) accumulates large fat droplets rich in neutral lipids in intestine; the lipid accumulation is associated with increase in TG levels[klf-3 with these two processes. KLF-3 genetically interacts with the genes encoding enzymes involved in FA β-oxidation in mitochondria or peroxisome and FA synthesis in cytosol, namely acyl-CoA synthetase (acs-1 and acs-2), acyl-CoA oxidase (F08A8.1 and F08A8.2), and stearoyl-CoA desaturase [Klf-3 maintains the balance of saturated and monounsaturated FAs by regulating the expression of several Δ9 desaturases, which in turn catalyze the biosynthesis of monounsaturated C16:1 and C18:1 FAs from saturated C16:0 and C18:0 FAs. Although additional factors appear to be necessary it is likely that the fat phenotype of the klf-3 mutant results from an aberrant ratio of saturated to monounsaturated FAs. Whether this observation is valid for the role of mammalian KLFs in these processes is not clear. Although the role of mammalian KLFs in lipid transport system has not been fully explored, it is interesting to note that mutation in klf-3 reduces expression of C. elegans dsc-4 and/or vit genes, the orthologues of mammalian MTP and apoB respectively. Both MTP and apoB are essential for mammalian lipoprotein assembly and transport. Mutation in both dsc-4 (qm182) and vit-5 (ok3239), results in high fat accumulation in worm intestine. The recognition of dsc-4 and all vit genes as potential targets of KLF-3 regulation prompted us to explore their genetic interactions, given the direct link to lipoprotein assembly and secretion pathway. We analyzed dsc-4; klf-3 as well as vit-5; klf-3 double mutants to study klf-3 interactions with these genes. We noticed that the klf-3; dsc-4 double mutant have twice as much fat as individual mutants. Perhaps a combination of defect in lipoprotein secretion and reduced β-oxidation might explain higher fat in double mutants. Thus both dsc-4 and klf-3 genes are likely to act together or in succession to promote lipoprotein secretion. Eliminating vit-5 gene activity in klf-3 mutant does not however, alter fat content in klf-3; vit-5 double mutant, implying that both genes function in the same pathway (un published).We are using s genome-60. We hll death. RecentlG levels,59 and dC. elegans worm is one of the few invertebrates in which the physiology of food intake has been extensively studied. The worm feeds on bacteria and takes up nutrients through pharyngeal pumping. Thus the starved animals pump faster when re-exposed to food than well-fed animals. The C. elegans moves around on bacterial lawn but the speed and pattern of its movement are modulated by starvation. The mutation in tryptophan hydroxylase gene tph-1 (the key enzyme for serotonin biosynthesis)[C. elegans klf in worm feeding behavior and relate our findings to previous feeding studies, we analyzed klf-3 (ok1975) mutant worm. We found a profound increase in the mRNA levels of tph-1 gene in klf-3 (ok1975) mutant suggesting that klf-3 may negatively control the expression of tph-1. However, in contrast to tph-1 mutant, the pharyngeal pumping as well as food-intake is normal in klf-3 mutant[klf-3 and tph-1 interacts during food intake and/or pharyngeal pumping. The potential relationship between klf-3 and tph-1 genes merits further investigation.Apart from vertebrate models, such as mouse and rat, the nthesis) results nthesis). It is inthesis). To inve3 mutant. CurrentCaenorhabditis elegans passes through four larval stages; L1, L2, L3, and L4 before reaching adulthood. During development, C. elegans exhibits food seeking behavior and hunts for easy to eat food. If there is no food available, then animals can go through an alternative developmental sequence in which a resistant 'dauer' larval form is produced at the L2 to L3 molt. Dauers can survive extreme conditions (desiccation and lack of food) for long periods until conditions improve and food becomes available, at which time they will molt and become normal adults[C. elegans uses its mechanosensory system for navigation. Quiescent behavior in C. elegans has been equated to satiety in mammals[C. elegans is ready to eat when it senses the presence of good food, the animal starts eating and stops moving, except for short foraging excursions forwards and backwards. After a period of fasting, the worm begins feeding again during which a sense of calmness is induced resulting in gradual inhibition of food intake and movement[C. elegans L4 animals from a synchronized population of klf-3 (ok1975) mutant animals and compared with the same developmental stage of wild-type (N2 strain). We found that mutant worms feed as wild-type but retained more fat than wild-type[klf-3 (ok1975) mutant animals fed on regular worm diet, E. coli OP50 had prolong resting period (period of lethargus) than the wild-type animals. C. elegans is accompanied by four larval stages[klf-3 in worm’s quiescence behavior? If so, what is the mode of action of klf-3 in this regulatory function? We hypothesize that prolonged quiescence could be secondary to the buildup of fat storage, given that the loss-of-function of KLF-3 would primarily affect the functions of the intestine. The intestines of worms are major endocrine systems and tissues engaged in nutrient sensing and energy metabolism[l adults. C. eleg mammals. The C. movement. The seqmovement. Previoumovement. We examild-type. We alsol stages that arel stages. Althougtabolism. Many stThe history of insulin discovery is very interesting. Frederick Banting, J. R. Macleod, Charles Best, and Bertram Collin were the major players in the insulin project. In the A series of neuropeptides  and neurotransmitters  in the hypothalamus have emerged as key participant in behavioral, physiological and metabolic responses. The neurons expressing these neuropeptides interact with each other and with insulin, leptin and with several other molecules to regulate energy uptake and its utilization. There aC. elegans provides an important research tool to these kinds of studies because this animal comprises 302 neurons that make up the nervous system and its neural mechanism is responsible for its chemotaxis, thermotaxis and mechanotransduction behavior. Apart from neuronal circuits, there are several key chemical transmitters including serotonin, acetylcholine, glutamate, and octopamine that control feeding in C. elegans. In addition, there are several neuropeptides, including insulin-like peptides[C. elegans. The worm nervous system also regulates fat storage both in conjunction with and independent of feeding pathways and its neuronal network coordinates the actions of this animal in transmitting signals between different parts of its body.The peptides that areC. elegans orthologue; however, the insulin signaling is highly conserved between worm and human. The C. elegans genome has predicted approximately 40 insulin-like genes[C. elegans because of its role in lipid metabolism and aging. Perhaps the most direct evidence of a major role for insulin signaling in C. elegans came from the finding that mutation in the daf-2 insulin receptor-like gene induced metabolic changes similar to that in mammalian metabolic control by the insulin receptor[daf-2 insulin receptor mutant, the neuroendocrine signaling defects results in increased fat in the intestine[The human leptin has no apparent ke genes-81. The receptor. The funntestine.klf-3 (ok1975) mutant suggested that klf-3 is key regulator of fat metabolism and its mutation disturbs fat storage-associated signal transduction. To understand how klf-3 regulates insulin signaling genes, we first performed microarray analysis of klf-3 mutant. On the basis of microarray data, we selected 16 potential klf-3 target genes to confirm their expression profiles using RT-PCR , receptor (daf-2) and downstream effector, the FOXO-family transcription factor (daf-16). Likewise, tph-1, not tub-1 was notably increased  pathway. The DAF-2 pathway regulates metabolism, development and aging independently of one another. A lack of daf-2 activity causes an extension in life-span, increased fat storage and constitutive arrest at the dauer stage[Daf-16 has no obvious function during reproductive development and its mutation daf-16(mgDf50) does not alter fat accumulation or cause dauer arrest, while a strong functional loss of daf-16 completely suppresses the mutant phenotype of daf-2[klf-3 (ok1975) and daf-2(e1391) mutants accumulate extra fat. Thus to study their genetic interaction, we created klf-3; daf-2 double mutant and assayed for fat deposition in the mutant animals. We found that animals bearing both klf-3 and daf-2 mutations accumulate large but similar amounts of fat as individual mutants (data not shown) suggesting that they are targeting the same molecular pathway of fat control.The research conducted in our own laboratory on se tph-1, and tub-1[tub-1. We founer stage. Daf-16  of daf-2. Both klklf-3 created a profound effect on TGF-β-mediated pathway as expression of several genes function in this pathway was significantly altered, linking klf-3 with TGF-β-mediated pathway. Studies in C. elegans have shown that DAF-7 binds DAF-1 and DAF-4 type I and type II TGF-β receptors to promote reproductive growth[daf-14 acts as a transducer of the DAF-7/TGF-β-mediated signal in this process. In daf-2, daf-7, and tph-1 deficient worms, metabolism is shifted towards fat accumulation[C. elegans KLF-3 has essential functions required for metabolic homeostasis: not only it regulates fat storage but intersect insulin signaling. Mutation in klf-3 disrupts its regulatory roles and underlies the chronic pathologic effects of fat accumulation on the endocrine function of intestine. Such a link in the worm opens up a window to look into parallel disease states in human diabetes, i.e., obesity-conditioned insulin resistance and lipotoxicity-induced β-cell failure.Deletion of e growth,86 and dmulation,82,84. TIn conclusion, it is clear that there are many factors that play their unique role in fat metabolism. Dysfunction of any of these factors results in fat accumulation in various organs, such as adipose tissue, muscles and liver. There is also complex interaction between food intake, environmental factors, and genetics. Laboratory and clinical data have undoubtedly explained some of the patho-physiology, yet there are many gaps in the inclusive understanding of human metabolic disorder. Appetite regulates body weight by affecting and limiting food intake. Various other biological parameters, that are sensitive to food intake however, are not fairly known; further investigation is therefore critical in identifying the mechanism that controls food intake. Characterizing genetic and epigenetic changes, significant to lipid metabolism, is a big task. It is important however to know about these genetic changes so that to understand the mechanism of human metabolic disorder. In this regard, the completion of genome sequence for model organisms offers a unique opportunity to identify the gene conservation among organisms. It permits the analysis of genetic changes associated with human metabolic disorder. Although the clinical application of model organisms to humans remains a challenge, it may guide further efforts to explore metabolic disorder and its relation to obesity and diabetes. It may also provide insights to the molecular basis of obesity and diabetes, ultimately aiding treatments efforts of such diseases.The authors declare that they have no competing interests.klf-3 and drafted the manuscript, YW designed the figures, RP, KC, WC, FM, and RG participated in writing and editing the manuscript. All authors read and approved the final manuscript.SH carried out studies on


Previous research found a high prevalence of depression, along with chronic illnesses and disabilities, among older ED patients. This study examined the relationship between depressive symptom severity and the number of ED visits among low-income homebound older adults who participated in a randomized controlled trial of telehealth problem-solving therapy (PST).The number of and reasons for ED visits were collected from the study participants (n=121 at baseline) at all assessment points—baseline and 12- and 24-week follow-ups. Depressive symptoms were measured with the 24-item Hamilton Rating Scale for Depression (HAMD). All multivariable analyses examining the relationships between ED visits and depressive symptoms were conducted using zero-inflated Poisson regression models.Of the participants, 67.7% used the ED at least once and 61% of the visitors made at least one return visit during the approximately 12-month period. Body pain  was the most common reason. The ED visit frequency at baseline and at follow-up was significantly positively associated with the HAMD scores at the assessment points. The ED visit frequency at follow-up, controlling for the ED visits at baseline, was also significantly associated with the HAMD score change since baseline.The ED visit rate was much higher than those reported in other studies. Better education on self-management of chronic conditions, depression screening by primary care physicians and ED, and depression treatment that includes symptom management and problem-solving skills may be important to reduce ED visits among medically ill, low-income homebound adults.ClinicalTrials.gov Identifier: NCT00903019 Older adults in North America use emergency departments (EDs) at a higher rate than younger adults -6. PreviCompared to their nondepressed peers, depressed older adults tend to use both ambulatory and inpatient healthcare services at a significantly higher rate even after adjusting for chronic medical illness, and their high rates of ED visits and return visits have been well documented ,11,15-18Despite the high volume of extant research on depressed older adults’ ED visits, few studies examined the longitudinal relationship between depressive symptoms and the frequency of ED visits. The primary aim of the present study was to examine (1) whether depressive symptom severity was associated with the frequency of ED visits at two different points of time among low-income, depressed homebound older adults aged 50 years and older; and (2) whether changes in depressive symptom severity overtime was associated with the changes in the frequency of ED visits and return visits. The older adults’ self-reported reasons for their ED visits were also presented. The study subjects were residents of central Texas in the United States who participated in a randomized controlled trial (RCT) of a short-term psychotherapy—telehealth-delivered or in-person problem-solving therapy (PST) compared to telephone support call—for low-income homebound older adults with moderately severe or severe depressive symptoms at baseline. The longitudinal data on the changes, or lack thereof, in the study subjects’ depressive symptoms and ED visits during the study period offered a great opportunity to examine longitudinal relationship between depressive symptoms and the frequency of ED visits among these older adults who were largely underrepresented in previous research on older adults’ ED use.According to the 2011 United States census data, of 40 million non-institutionalized adults age 65 years and older, 23.5% (9.2 million) had ambulatory disability/activity limitation and about 10% were considered homebound -23. The Employing Andersen’s behavioral model of health services use  as the chomebound adults in this study referred to older individuals who, due to medical conditions and/or mobility-affecting impairments, cannot freely leave their home and who require help in doing so. Case managers at a large Meals on Wheels program and other social service agencies serving low-income, homebound adults age 50 years and older in central Texas referred to the project those who scored 10 or higher on the PHQ-9  still a problem?” Included conditions were arthritis, hypertension, diabetes, heart disease, stroke, lung disease, cancer, and kidney disease. Only conditions that were still problems were counted. Pain frequency at baseline was measured with two questions: (1) Have you had chronic pain in any part of your body during the past six months? and (2) How would you rate the frequency of your pain on a 10-point scale, with 1 being very infrequently (like once a week or less often) and 10 being all the time? Those who reported having no pain were assigned a score of 0.All analyses were performed using SAS v. 9.2 . Prior to assessing bivariate associations between the frequency of ED visits and participants’ predisposing, enabling, and need characteristics, the probability distributions of the outcomes were assessed. Due to non-normal distributions, robust regression, which is robust to heteroscedastic error and outliers, was used for all group comparisons using SAS PROC ROBUSTREG. H1 and H2 were tested using zero-inflated Poisson regression models using SAS PROC GENMOD, with the frequency of ED visits at baseline and at 24-week follow-up, respectively, as the dependent variable, and with predisposing, enabling, and need factors  evaluated as covariates. H3 was also tested using zero-inflated Poisson regression analysis, with the ED visits at 24-week follow-up as the dependent variable and the difference between the 24-week follow-up HAMD score and the baseline HAMD score as the predictor variable, controlling for the baseline HAMD score and other covariates. Prior to implementing the models, we assessed possible overdispersion of the count outcome data by testing whether the negative binomial dispersion parameter was significantly different from zero. Because it was not, we used a Poisson, rather than a negative binomial, distribution to model the count outcome variables.Because there were a large number of variables relative to the sample size and all the variables could potentially be included in both the logistic and count portions of the model, a variable reduction strategy was employed. Guidelines provided by Hosmer and Lemeshow  were useTable n = 37) reported 1 visit, 21.7% (n = 15) reported 2 visits, and 39.1% reported 3–9 visits; thus, almost 61% made return visits. At 24-week follow-up, 49.6% reported no ED visit since baseline, 16.5% reported 1 visit, 5.8% reported 2 visits, and 7.5% reported 3–8 visits. Of the 36 participants who had any ER visit, 55.6% (n = 20) had only 1 visit, 19.4% (n = 7) had 2 visits, and 25% (n = 9) had 3–8 visits. The total number of ED visits reported was 170 (for an average of 2.5 visits per visitor) at baseline and 88 (for an average of 2.4 visits per visitor) at 24-week follow-up. The baseline HAMD scores were not normally distributed according to the Shapiro-Wilk test ; thus, comparisons between ED visits and HAMD scores were assessed using robust regression. The HAMD scores for those who had had 3+ ED visits at baseline were significantly higher than the scores of those who had had no visits , those who had had one visit , and those who had two visits . On the other hand, the HAMD scores for those who had had 3+ ED visits at 24-week follow-up were not different from the HAMD scores for those who had had no visits , those who had had one visit , and those that had two visits .Table 2 = 3.73, p = .053), but the HAMD scores were significantly higher than those who had reported ED visits only at baseline  and those who had used ED only during the follow-up . Reduction in HAMD scores since baseline did not differ between ED users at both baseline and follow-up and those who had not used ED  or those who had had ED visits only during the follow-up .Table Table 2 = 5.97, p = .015). Gender, being Black, Medicaid coverage, number of chronic medical conditions, and pain frequency were not significant correlates of the baseline ED visit frequency.Preliminary analyses of covariates in the logistic portion of the zero-inflated Poisson model for baseline ED visits indicated that Hispanic status, family support, and the number of medical conditions met the inclusion criteria based on the decision rules described above. The same analyses for the zero-inflated Poisson model for ED visits at 24-week follow-up indicated that Hispanic status, being in the 60–69 age group, and the number of medical conditions met the inclusion criteria based on the decision rules described above. Table The ED visit frequency reported at follow-up was significantly positively associated with the follow-up HAMD score, female gender, 50–59 years of age, and pain frequency and it was marginally significantly associated with being in the 60–69 age group. The ED visit frequency at follow-up was also significantly associated with the HAMD score change since baseline, with lower level of reduction or higher level of increase in HAMD scores predicting higher ED visit frequency.Depression is a serious problem among older adults who are homebound due to disability -27. Low-This study found that 67.7% of the subjects (who provided data at both baseline and follow-up) used the ED at least once during the approximately 12-month study period and that 61% of the visitors made at least one return visit during the 12-month period. These are much higher visit and return visit rates than those reported in other studies [The study findings support H1 and H2 in that the ED visit frequency was significantly associated with higher depressive symptoms at both baseline and at 24-week follow-up. As noted ,20, deprContrary to the findings of some previous studies, this study did not find the number of medical conditions (and level of disability) to be a significant correlate of the ED visit. The discrepancy between the previous and the present study findings is likely due to the greater homogeneity of our subjects compared with those in the previous study samples with respect to their physical and functional health and their economic status. Although pain was the most frequently cited reason for making an ED visit, pain frequency was associated with making an ED visit only at follow-up, controlling for predisposing, enabling, and other need factors. Hispanics were more likely than participants of other ethnicities to use the ED at baseline but not at follow-up, which may indicate that their level of self-care knowledge and skills was lower than that of the other participants at baseline. For example, one study  of low-iThe study had a few limitations. First, the self-reported ED visit data were not validated against the subjects’ medical records. Even with possible underreports due to a recall or response bias in self-reports, however, the ED visit rate was much higher than those reported in other studies. Likewise, the reasons for ED use were not validated against the discharge diagnoses. Although self-reported reasons are important in exploring the users’ perception of their need for an ED visit, discharge diagnoses would have helped us understand the true nature of their conditions that brought them to the ED. Second, we chose the 24-item HAMD cutoff score of 15 that was not validated. As described previously, in choosing the cutoff score, we used an empirical approach based on research data from (1) a study of home healthcare patients with major depression that found the mean baseline 24-item HAMD score to be about 15 ; and (2)Despite these limitations, the study has the following clinical implications: First, repeat ED users who tend to visit the ED for the same reason over time need to be provided with better education on self-management of their chronic conditions, taking into consideration their level of health literacy. Second, given the significant relationship between higher depressive symptoms and ED visit frequency, PCPs and EDs may use ED visits as a cue for depression screening. Third, instituting depression treatment that includes symptom management and problem-solving skills may be important to reduce ED visits among medically ill, homebound adults. Fourth, effective pain management is likely to reduce the need for ED visits among low-income homebound older adults. Clinicians need to educate these older adults about effective pain management strategies and the possible relationship between pain and depressive symptoms. Fifth, further research is needed to identify factors contributing to higher ED use by those who are under 70 years.ED: Emergency department; RCT: Randomized controlled trial; HAMD: Hamilton Rating Scale for Depression; PCP: Primary care physician.The authors declare that they have no competing interests.All authors contributed to the drafting of this version of the manuscript and agree to submission. NGC, MLB, and MEK: conception and design of the study. NGC and CNM: statistical analysis. NGC, CNM; MLB, and MEK: interpretation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/12/233/prepub


To assess the cross-sectional association between exercise capacity, gas exchange efficiency and endothelial function, as measured by flow-mediated dilation (FMD) and nitroglycerin-mediated dilation (NMD) of the brachial artery, in a large-scale population-based survey.2@AT), peak exercise (peakVO2) and ventilatory efficiency (VE vs. VCO2 slope and VE/VCO2@AT) were assessed on a breath-by-breath basis during incremental symptom-limited cardiopulmonary exercise. FMD and NMD measurements at rest were performed using standardised ultrasound techniques.The study population was comprised of 1416 volunteers 25 to 85 years old. Oxygen uptake at anaerobic threshold  in the overall population [VE vs. VCO2 slope -3.9 ]. These results remained robust after adjusting for all major influencing factors. Neither exercise capacity nor ventilatory efficiency was significantly associated with NMD.Multivariable logistic regression analyses revealed a significant association between FMD and ventilatory efficiency in current smokers but not in ex-smokers or non-smokers. There was no association between FMD and VOIn current smokers, FMD is significantly associated with ventilatory efficiency. This result may be interpreted as a potential clinical link between smoking and early pulmonary vasculopathy due to smoking. Endothelial dysfunction represents an early, subclinical stage of vascular dysfunction that precedes the development of atherosclerosis  and predDyspnoea, which is a symptomatic hallmark in patients with cardiovascular or pulmonary vascular diseases, can be quantified by gas exchange and ventilatory efficiency . An impaTherefore, this investigation aimed to assess the potential link between peripheral endothelial function and gas exchange in a large-scale population-based study called the Study of Health in Pomerania (SHIP). The major hypothesis tested was that FMD is related to exercise capacity and ventilatory efficiency in a sample representing a wide age range of the general population.The Study of Health in Pomerania (SHIP) is a population-based investigation in West Pomerania, a region in the northeastern part of Germany. The details of the study are given elsewhere ,25. In bWe offered all SHIP-1 participants the opportunity to take part in measurements of endothelial function (FMD and NMD), body plethysmography and CPET. Of the 3300 SHIP-1 participants, 1705 volunteered in both CPET and endothelial function determination. We excluded 278 subjects with non-readable ultrasound images and 11 subjects with missing data. The study population available for the present analyses consisted of 1416  volunteers. Table For sensitivity analyses, an apparently healthy population without factors possibly interfering with endothelial function and CPET was defined. For this purpose, subjects with the following characteristics were excluded (overlaps exist): past myocardial infarction, echocardiographic evidence of ventricular dysfunction or valvular disease, electrocardiographic signs of ischaemia, neuromuscular or musculoskeletal disorders based on neurological examination, malignancies, pulmonary diseases, chronic obstructive bronchitis, bronchial asthma, drugs against obstructive airway disease including inhaled steroids [(ATC) code R03], arterial hypertension according to the definition of the World Health Organization or the uSociodemographic and medical characteristics were assessed by computer-assisted personal interviews. Previous history of diseases was assessed based on self-reported physician's diagnosis. According to tobacco consumption, participants were categorised into current (one or more cigarettes per day), former, and non-smokers. Data on medication were collected using the anatomical therapeutic chemical (ATC) code . AntihypEndothelial function was assessed by FMD and endothelial-independent vasoregulation by NMD. Examinations were performed in a supine position by two observers. The subject's right arm was comfortably immobilised, and the brachial artery diameter was recorded 3-7 cm above the antecubital fossa using a 10-MHz linear array transducer ultrasound system . After the resting scan, a pneumatic cuff placed around the forearm 10 cm distal to the ultrasound location was inflated above a pressure of 220 mmHg for 5 min. Diameter measurements were repeated 60 s after cuff deflation. FMD was expressed as the post-occlusion brachial artery diameter corrected for baseline artery diameter (BAD) and as the ratio between brachial diameters before and after inflation of the pneumatic cuff. NMD was taken 3 min after sublingual administration of nitroglycerin (400 μg) in 1096 subjects (465 women). Examinations were performed and read by two observers. All ultrasound measurements in SHIP use strict quality management . IntrareCPET was performed with a physician in attendance according to a modified Jones protocol  using a 2 and VCO2 were acquired on a breath-by-breath basis and averaged over 10-second intervals. The peak oxygen uptake was defined as the highest 10-second average of VO2 in late exercise. The peak heart rate was averaged over that same period, and the peak O2 pulse was calculated as peak VO2 divided by peak heart rate. The peak respiratory exchange rate (RER) was calculated as the ratio of peak carbon dioxide output (VCO2) to peakVO2. The anaerobic threshold (AT) was determined according to Wasserman et al.  and -4.6 in apparently healthy volunteers  [VE vs. VCO2 slope -5.3 ]. In non- and ex-smokers FMD did not show any significant association with parameters of ventilatory efficiency (Table 2 ratio from rest to exercise at the anaerobic threshold was not significantly associated with FMD. All effects were consistently reproducible through all reported or diagnosed comorbidities. There were no detectable differences between women and men.In current smokers, FMD was inversely related to ventilatory efficiency Table . In curr2 or aerobic exercise capacity, as judged by VO2@AT. Thus, it has to be postulated that NO-dependent endothelial function plays a minor, unverifiable role in muscle endurance and exercise capacity as assessed within a symptom-limited CPET in healthy volunteers.In terms of this study's hypotheses, neither in smokers nor non-smokers did endothelial function reveal any association with peak exercise capacity as verified by peak VO2@AT and peak VO2 in FMD in young adults [Previous studies have suggested a potential interference of endothelial functioning with exercise capacity -6. Palmig adults , which ig adults ,6. Furthg adults , and trag adults . HoweverThis study shows that in current smokers, FMD is significantly correlated to ventilatory efficiency independent of sex and co-morbidities. This correlation is not verifiable in non- or ex-smokers. To the best of our knowledge, this is the first study describing a correlation between NO-dependent endothelial function and gas exchange efficiency and exercise capacity in a large-scale population-based study. Our previous work assessed the influence of smoking on exercise capacity and gas exchange efficiency in the same population-based study . In thatFinally, our study has limitations. The SHIP project, as a large-scale observational population-based study, was not designed to test the hypotheses that vascular abnormalities are related to ventilatory inefficiency in smokers. However, to the best of our knowledge, this is the first study to describe the interaction of endothelial function, exercise capacity and ventilatory efficiency in a large population sample. Because this study is based on individual volunteering, as in any population-based cross-sectional survey, we cannot fully rule out selection bias. We observed that CPET volunteers were younger than non-participants, which might have led to a healthier study population .Furthermore, due to ethical reasons the design of a population-based survey does not allow for histopathological investigations. Thus, the final proof of the hypotheses discussed here is pending.In conclusion, in a general adult population, peripheral NO-dependent vasodilation assessed by FMD was not associated with exercise capacity and was independent of coexisting diseases. A significant, inverse association between FMD and ventilatory efficiency did exist in smokers, whereas this association was not verifiable in non- or ex-smokers. In current smokers, a decreased FMD was associated with impaired ventilatory efficiency. This association may be interpreted as a potential link between smoking and early pulmonary vasculopathy due to smoking exposure.AT: Anaerobic threshold; ATC code: Anatomical-technical-chemical code; BAD: Brachial artery diameter; CPET: Cardiopulmonary exercise testing; ECG: Electrocardiogram; FMD: Flow-mediated dilation; IPAH: Idiopathic pulmonary arterial hypertension; NMD: Nitrogen-mediated dilation; NO: Nitrate oxide; peakVO2: Peak oxygen uptake; RER: Respiratory exchange rate; SHIP: Study of Health in Pomerania; VCO2: Carbon dioxide output; VE vs. VCO2 slope: Slope of the regression of minute ventilation to carbon dioxide output; VE/VCO2@AT: Minute ventilation to carbon dioxide ratio at anaerobic threshold; VO2: Oxygen uptake; VO2@AT: Oxygen uptake at anaerobic threshold.The authors declare that they have no competing interests.SG drafted the manuscript, made substantial contributions to the conception, design and acquisition of the data as well as the analysis and interpretation of the data and has given final approval for the version to be published. AO acted as one of the leading statisticians that analysed the data, made substantial contributions to the conception, design and acquisition of the data, was involved in drafting the manuscript and revising it critically for important intellectual content and has given final approval for the version to be published. CFO, SBF, KE, CS, RE, MD, HV and BK have made substantial contributions to the conception, design and acquisition of the data as well as the analysis and interpretation of the data, were involved in drafting the manuscript or revising it critically for important intellectual content and have given final approval for the version to be published.