These aggregations create dense and species-rich faunal “hot-spots”, attracting a diverse assemblage of local consumers that utilise this seasonal resource. The starlings nested primarily in poison-dart trees (Antiaris toxicaria) near the rainforest-woodland boundary. Surveys underneath these colonies revealed that bird-derived nutrients massively increased densities of soil invertebrates and mammals (primarily wild pigs) beneath trees, year-round. Flying invertebrates, nocturnal birds, reptiles, and amphibians congregated beneath the trees when starlings were nesting (the wet-season). Diurnal birds (primarily cockatoos and bush turkeys) aggregated beneath the trees during the dry-season to utilise residual nutrients when the starlings were not nesting. The abundance of several taxa was considerably higher (to > 1000-fold) under colony trees than under nearby trees. The system strikingly resembles utilisation of bird nesting colonies by predators in other parts of the world but this spectacular system has never been described, emphasizing the continuing need for detailed natural-history studies in tropical Australia.Large numbers of metallic starlings  Crocodylus niloticus) gathering at points in rivers where wildebeest cross, or grizzly bears (Ursus arctos ssp.) congregating at salmon spawning sites [In most ecosystems, the spatial and temporal distribution of resources drives faunal distributions , 2. All ng sites , 7. Thesng sites , 8. Massng sites , 8, 7, 9Aplonis metallica). As in communally-nesting African sociable weavers (Philetairus socius) and Peruvian yellow-rumped caciques (Cacicus cela), the high biomass of birds attracts diverse predators [Despite their spectacular nature and high public profile, these systems sometimes are poorly studied. We describe aggregations of wildlife that gather to exploit seasonally available resources .Metallic starlings (hereafter referred to as “starlings”) are small glossy-black birds , from northeastern Australia (Queensland), New Guinea, the Moluccas, and Solomon Islands . These b2 area of semi-deciduous notophyll vine forest, at the extreme northern tip of Cape York Peninsula in Queensland, Australia , starlings begin nesting around mid-November. They remain at the trees for the summer monsoon, during which time a pair of starlings raises up to three broods before leaving at the beginning of April . We defined the “nesting season” as 11 November to 1 April (mean timing of nesting at our study site), and the remainder of the year as the “off-season”. We collected data between November 2013 and April 2015, which consisted of two nesting seasons and the intervening off-season when no starlings were present at the colony trees.We initially located colony trees from ground level using sight and bird sound (a colony can be heard up to 300 m away), but also systematically surveyed the Lockerbie Scrub from a helicopter. If a colony tree was located, its location was recorded on a hand-held GPS unit (Garmin 60SCx). We then walked to each tree to confirm whether or not a colony was present, and estimated tree heights, identified species, and estimated number of nests in the colony. We were confident that we located most of the starling nesting colonies in the Lockerbie Scrub during our surveys.We overlaid colony tree locations onto Regional Ecosystem maps for Queensland in QGIS (version 1.8.0) to determine the broad vegetation types at each colony tree , 19). ToWe used four methods to quantify the abundance and diversity of fauna present at starling colonies throughout the year. To test whether faunal concentrations beneath colony trees were related to the presence of the bird colony, we also surveyed an identical number of nearby trees that were not used by starlings. For each colony tree, we selected a “random” tree 100–200 m away, and similar to the colony tree in height, girth and, if possible, species. Our surveys recorded animals in the “dead zone” . We placed the soil in a Tullgren funnel beneath a heat-lamp overnight, and counted invertebrates the next morning. We then placed the soil in a glass dish and visually located and counted remaining invertebrates.We randomly selected four colony trees and four random trees and surveyed them for flying and soil-dwelling invertebrates. To record flying invertebrates we stapled an 80 x 4 cm length of SanliFor one-year (January to December 2014) we surveyed six of the logistically most accessible colony trees in our study area, and their corresponding random trees, using Moultrie Panoramic 150™ infrared trail cameras. We deployed cameras with a 150° field of view on tree trunks (40 cm above ground) to provide an unobstructed view of either the colony tree or the random tree. This method recorded all animals within the 10 x 14 m clear area beneath the colony trees, as well as a corresponding area under random trees. To survey multiple trees each month, we deployed cameras at a tree for 7 to 10 days. After this period, we removed the cameras, changed their batteries, and deployed them at another site. This way most trees were surveyed every month. Cameras were set to motion detection, with a minimum of 5 minutes between successive shots . Many animals may have spent longer than five minutes under the starling colony, so we included the number of photos taken per day as a covariate in our analyses.Between November 2013 and April 2015 we surveyed a subset of colony trees and nearby random trees for nocturnal animals. In the first survey year we visited 12 colonies (and their associated random trees) one to five times per week. During our second survey year, we regularly visited only eight colony trees and their associated random trees. We conducted surveys on foot (1930–2230 h) and searched the 10 x 14 m area of ground (and the immediate surrounding vegetation) underneath each tree for 1–3 minutes with the aid of a head-torch. We conducted surveys during the nesting-season and the off-season .We used analysis of variance (ANOVA) to determine whether colony trees were distributed closer to rainforest edges than would be expected by chance. We square-root transformed the distance of colony tree locations and randomly generated point distances from the rainforest edge to meet assumptions of normality and homogeneity of variance. Because our faunal counts of all taxa contained a high proportion of zeros , we analyzed count data using generalized linear models with negative binomial distributions and log-link functions . To contF1,126 = 6.63, P = 0.01; Antiaris toxicaria 71%); milky pines (Alstonia scholaris 19%); and Morton Bay ash (Corymbia tesselaris 10%). Colony trees were always tall , with smooth bark, large trunks and branches separated from the surrounding canopy  and during the nesting season  and pigs  comprised most (93%) animals recorded under trees , especially during the off season .Over 880 camera-trap days  we identified more than 95,000 animals representing 23 species . Most (9er trees . Cameraser trees . For thiRhinella marina) (73%) and snakes of several species (22%) comprised most nocturnal survey records. Mammals were primarily pigs (N = 101) and Cape York melomys  but with small numbers of other species  and were more common during the off-season than the nesting season  and 22 were rufous owls (Ninox rufa). Birds were more common in the off-season than the nesting season . Although total counts were lower in the nesting season, presence was higher , with high counts only at the colony trees and only during the nesting season  and white-lipped tree frogs . Like reptiles, anurans were far more abundant beneath colony trees than random trees, especially during the nesting season . The same was not true for birds, which were recorded in greater numbers beneath trees with more nests, and this effect was more pronounced during the nesting season than the off season . Of the nocturnal animals, only the number of reptiles observed at trees in the nesting season was positively correlated with the number of nests in the colony . All other p-values > 0.05.The number of diurnal mammals beneath colony trees was not affected by the number of starling nests in the tree , soil-dwelling invertebrates are abundant, even outside the nesting season  during the time when starlings are present. This pattern holds true for flying insects, nocturnally-active amphibians, reptiles, and birds. Most of these species obtain food directly via starlings’ activities. Especially after heavy rain, windy conditions cause nests (and entire tree-limbs) to fall to the ground. Predators  feed on fallen starling eggs and nestlings, and invertebrates found beneath the trees . More geWhy are faunal concentrations so high beneath these trees compared to the rest of our study area? One plausible explanation is that the position of colony trees near the rainforest edge allows utilization of the resource by taxa inhabiting rainforest and woodland habitats. However, most of the taxa utilizing the trees are habitat generalists, and the concentrations observed are too great to be explained by subtle differences in habitat-specific faunal abundance. Furthermore, we did not survey all trees potentially suitable for starlings (because we cannot assume we know what attributes the birds require) and thus colony site location may simply be an artifact of tree distribution rather than because starlings actively select ecotonal areas for their colony sitesA more likely explanation is the ubiquitous nature of spatial and temporal variability in resource availability  . Most spLarus novaehollandiae) on small islands support dense populations of predatory tiger snakes (Notechis scutatus: [Aptenodytes patagonicus) and seals  likewise support dense populations of marine predators [Philetairus socius) in southern Africa and yellow-rumped caciques (Cacicus cela) in Peru are utilised by snakes and primates [2 beneath colony trees). To our knowledge, this system represents one of the highest-biomass  and most diverse faunal aggregations in the world . Coredators , 29. Marredators . For pasprimates , 11. AltStarling colonies in northern Australia provide an ideal system for asking ecological and evolutionary questions. Future research could usefully explore how and why starlings choose their nesting sites, how animals locate colony trees, or how far they travel to reach them. The high concentrations of animals around starling colonies suggest that there are fitness benefits associated with utilization of this nutrient subsidy. Plausibly, starling colonies increase the number of animals that can be supported by the habitat, which also makes these trees disproportionately important for conservation . High coS1 FileCaption: Raw camera trap data used in the analyses for this study.(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Small trials suggest that levosimendan is associated with a favorable outcome in patients undergoing cardiac surgery. However, recently published larger-scale trials did not provide evidence for a similar benefit from levosimendan. We performed a meta-analysis to assess the survival benefits of levosimendan in patients undergoing cardiac surgery and to investigate its effects in subgroups of patients with preoperative low-ejection fraction (EF).We identified randomized clinical trials through 20 April 2017 that investigated levosimendan therapy versus control in patients undergoing cardiac surgery. Individual patient data from each study were compiled. Meta-analyses were performed for primary outcomes, secondary outcomes and serious adverse events, and subgroup analyses according to the preoperative EF of enrolled patients were also conducted. The risk of bias was assessed using the Cochrane risk-of-bias tool.p = 0.02) and reduced the risk of death in single-center trials  and in subgroup trials of inferior quality ; however, in multicenter and in high-quality subgroup-analysis trials, no significant difference in mortality was observed between patients who received levosimendan therapy and controls (p > 0.05). However, in high-quality subgroup trials, levosimendan therapy was associated with reduced mortality in patients in a preoperative low-EF subgroup . Similarly, only patients in the preoperative low-EF subgroup benefited in terms of reduced risk of renal replacement therapy . Furthermore, levosimendan therapy was associated with a significant reduction in intensive care unit (ICU) length of stay .Seventeen studies involving a total of 2756 patients were included. Levosimendan therapy was associated with a significant reduction in 30-day mortality  contains supplementary material, which is available to authorized users. Many patients with advanced stages of cardiac disease need cardiac surgery, which can lead to severe left ventricular dysfunction and, in particular, postoperative low cardiac output syndrome (LCOS) . These cLevosimendan is an inotropic agent that enhances myocardial contractility without increasing myocardial oxygen demand in patients with low cardiac systolic dysfunction . ClinicaThe objective of this meta-analysis was to assess the survival benefits of levosimendan in patients undergoing cardiac surgery. Previous meta-analyses showed that patients with low ejection fraction (EF) benefit more from levosimendan therapy than patients with normal EF , 8. TherType of study: randomized controlled clinical trialsPopulation: patients undergoing cardiac surgeryIntervention: patients receiving intravenous levosimendanPrimary outcomes: 30-day mortality or in-hospital mortality.Secondary outcomes: requiring renal replacement therapy, the duration of mechanical ventilation, and intensive care unit (ICU) length of stay.Serious adverse events: the occurrence of postoperative arrhythmia and hypotension.The following outcomes were includedWe included trials with the following features:We conducted a search of the Medline, Elsevier, Cochrane , Web of Science and ClinicalTrials.gov databases for studies investigating the perioperative use of levosimendan in patients undergoing cardiac surgery. When searching each database, the term “levosimendan” was combined with the Cochrane highly sensitive search strategy for identifying randomized trials . SearcheTwo reviewers independently screened abstracts and titles to determine whether the studies met the inclusion criteria. The full texts of the articles were then reviewed independently in accordance with the inclusion and exclusion criteria. Studies that reported randomized clinical trials of the preoperative or postoperative administration of levosimendan in patients undergoing cardiac surgery were included. “low-EF studies” were defined as studies that limited their analysis to patients with a preoperative EF ≦40% or enrolled patients with a preoperative mean EF ≦40%. The remaining studies were designated “preserved-EF studies” . Any disTwo authors independently extracted the data using a standardized data extraction protocol. Any disagreements between the two reviewers were resolved through discussion, after which consensus was reached. Some means and standard deviations of the patients’ duration of mechanical ventilation and ICU length of stay data were estimated according to the method described by Hozo . InformaTwo reviewers independently completed a risk of bias assessment following the instructions of the Cochrane Collaboration tool for systematic reviews of interventions . The iteWe perform a trial sequential analysis (TSA) to prevent the risk of random error from being increased by repeated updates. We used TSA-adjusted random-effects modes to pool results from the included studies for primary outcomes. A one-sided TSA was conducted to maintain a 5% risk of type I error and 80% power. Furthermore, we used the estimated function to calculate the required information size.I2 test. Any obvious heterogeneity was predefined as p < 0.05 using the Mantel-Haenszel chi-square test or I2 > 50%. Furthermore, publication bias was assessed using funnel plot techniques.For the meta-analysis, data from the included studies were analyzed using Review Manager , and pooled risk ratios for dichotomous data and mean differences for continuous data with 95% CIs were calculated. The statistical heterogeneity of the data was quantified using the Mantel-Haenszel chi-square test and the We identified a total of 581 titles and abstracts after the primary search. After screening the abstracts, 553 articles were found to be repeated or non-relevant and were therefore excluded. The remaining 28 articles were retrieved for an eligibility assessment, as the result of which 11 studies were deemed ineligible and were therefore excluded. Seventeen studies with a total of 2756 patients were included in the final analysis , allocation concealment was assessed in 12 studies 75%), blinding of participants was assessed in 13 studies 76%), blinding of outcome assessors was assessed in 13 studies (76%), incomplete outcome data was assessed in one study (6%) and selective outcome reporting was assessed in all studies (100%). Thirteen trials [%, blindi%, blindi%, allocaA sensitivity analysis of TSA including all trials revealed that the diversity-adjusted information size was 2739 patients. The cumulative z-curve crossed the conventional boundary for benefit and the trial sequential monitoring boundary for benefit but did not cross the estimated information size boundary . The result showed a significant reduction in the overall risk of death following the levosimendan intervention . Levosimendan significantly reduced 30-day mortality  but not in-hospital mortality . However, in multicenter trial subgroups, there was no significant difference in mortality between patients who received levosimendan therapy and those who did not receive levosimendan  . However, levosimendan therapy did not reduce the risk of death in high-quality trials  . However, no benefit was observed in the preserved-EF subgroup  . Subgroup analysis indicated that the benefit was confined to the low-EF studies  . There was a significant reduction in the preserved-EF subgroup  and in the low-EF subgroup   . Subgroup analysis indicated that the adverse effect was confined to the low-EF studies   reported data on postoperative atrial fibrillation. Pooled analysis of all studies and subgroup analyses showed no significant impact on the risk of postoperative atrial fibrillation Additional file 2:The effect of levosimendan on duration of ICU stay in patients undergoing cardiac surgery. (PNG 13 kb)Additional file 3:The effect of levosimendan on postoperative atrial fibrillation in patients undergoing cardiac surgery. (PNG 13 kb)Additional file 4:The effect of levosimendan on postoperative hypotension in patients undergoing cardiac surgery. (PNG 9 kb)


Plasmodium knowlesi threatens elimination efforts in southeast Asia. Changes in land use are thought to be driving the rise in reported P knowlesi cases, but the role of individual-level factors is unclear. To address this knowledge gap we assessed human and environmental factors associated with zoonotic knowlesi malaria risk.The emergence of human malaria due to the monkey parasite P knowlesi cases and controls, and between P knowlesi and human-only Plasmodium spp malaria cases.We did this population-based case-control study over a 2 year period in the state of Sabah in Malaysia. We enrolled cases with microscopy-positive, PCR-confirmed malaria who presented to two primary referral hospitals serving the adjacent districts of Kudat and Kota Marudu. We randomly selected three malaria-negative community controls per case, who were matched by village within 2 weeks of case detection. We obtained questionnaire data on demographics, behaviour, and residential malaria risk factors, and we also assessed glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. We used conditional logistic regression models to evaluate exposure risk between P knowlesi malaria mono-infection and 91 cases with other Plasmodium spp infection. We enrolled 953 matched controls, including 683 matched to P knowlesi cases and 270 matched to non-P knowlesi cases. Age 15 years or older , male gender , plantation work , sleeping outside , travel , being aware of the presence of monkeys in the past 4 weeks , and having open eaves or gaps in walls  were independently associated with increased risk of symptomatic P knowlesi infection. Farming occupation , clearing vegetation , and having long grass around the house  increased risk for P knowlesi infection but not other Plasmodium spp infection. G6PD deficiency seemed to be protective against P knowlesi , as did residual insecticide spraying of household walls , with the presence of young sparse forest  and rice paddy around the house  also associated with decreased risk.From Dec 5, 2012, to Jan 30, 2015, we screened 414 patients and subsequently enrolled 229 cases with P knowlesi transmission could be targeted in future public health interventions.Adult men working in agricultural areas were at highest risk of knowlesi malaria, although peri-domestic transmission also occurrs. Human behavioural factors associated with United Kingdom Medical Research Council, Natural Environment Research Council, Economic and Social Research Council, and Biotechnology and Biosciences Research Council. Plasmodium knowlesi has emerged as a widespread cause of zoonotic human malaria in southeast Asia.P knowlesi symptomatic infections in Sarawak in Malaysian Borneo in 2004.P knowlesi cases have now been reported from all countries in southeast Asia, with the exception of Laos,Macaca fascicularis and Macaca nemestrina) and Anopheles leucosphyrus group vectors.P knowlesi is indistinguishable from Plasmodium malariae,Plasmodium falciparum and Plasmodium vivax, suggesting that its true incidence continues to be underestimated.7Plasmodium species confirmation.P falciparum and P vivax case numbers to a point where elimination of these species by 2020 is considered realistic.P knowlesi is now the most common cause of malaria in Malaysia12Most notably, Malaysia has reported marked increases in knowlesi malaria incidence since 2004—a trend that cannot solely be explained by improved molecular P knowlesi in endemic areas are thought to be predominantly related to changes in human land use,The drivers of the emergence of In this population-based case-control study we examined factors associated with the risk of acquiring zoonotic knowlesi malaria in adults and children presenting to two primary referral hospitals serving the adjacent districts of Kudat and Kota Marudu in Sabah, east Malaysia . The stuThe government referral hospitals provide free admission and malaria treatment and are accessible from all villages in the catchment area. National public health guidelines mandate microscopic malaria screening for all patients presenting to government facilities with fever and notification of all positive cases, thus increasing the likelihood of enrolling all symptomatic knowlesi malaria cases seeking care.Plasmodium species infection by microscopy were enrolled if they had a documented fever or history of fever in the past 48 h, had resided in the study catchment area in the previous 3 weeks, and had not been enrolled previously. We randomly selected controls from the case’s village within 2 weeks of case detection using updated Malaysian government village and household population data, and corroboration from health workers and village heads. To minimise selection bias, absent controls were revisited up to three times before an alternative household or individual was selected. We repeated the same selection process until three controls had been enrolled for each case. Inclusion criteria for controls were a negative blood slide for malaria, no history of fever in the preceding 48 h, not previously having been a case, not previously having been a control nor living in the same household as a control, and living at least 200 m away from the case household. We obtained informed consent for both cases and controls.In brief, patients positive for The selection criteria for controls were designed to allow the evaluation of micro-epidemiological human factors while minimising confounding effects from village-level forest cover, elevation, rainfall, and seasonality.Plasmodium spp infection.Plasmodium species case confirmation. All female cases of childbearing age underwent pregnancy testing and pregnancy status for controls was self-reported. We entered data into electronic case record forms using Pendragon Forms VI software.Cases were classified according to PCR-confirmed P knowlesi cases were required to reject the null hypothesis that there is no difference between cases and controls , with 80% power and an α of 0·05.19With three controls per case, assuming a probability of exposure among controls of 0·1, and the relative risk of acquiring knowlesi malaria in exposed subjects relative to unexposed subjects of 0·3, a minimum of 210 The primary analysis of categorical exposure variables compared knowlesi malaria cases and controls as per protocol. We compared odds ratios (OR) and 95% CIs for exposure risk, calculated by the Mantel-Haenszel method. We included variables with a p value less than 0·2 on univariate analysis in the multivariate analysis within associated subgroups using conditional logistic regression models, including adjustment for age and gender. Variables with p values less than 0·2 then proceeded into the final multivariate model with adjusted odds ratios (aOR), followed by further reduction of predictor variables through a process of model-fitting using Bayesian information criterion (BIC) and backward step-wise exclusion of variables with p values greater than 0·05.Plasmodium species combined (zoonotic vs non-zoonotic transmission). We assessed non-P knowlesi cases with matched controls with a similar approach to the primary univariate analysis, then made comparisons between P knowlesi and non-P knowlesi models for each exposure variable using Wald’s test.The secondary outcome compared knowlesi malaria cases with cases from other Data were analysed with Stata 12.P knowlesi mono-infection and 91 with other Plasmodium species infection. 953 controls were enrolled, including 683 people matched to P knowlesi cases and 270 matched to non-P knowlesi cases. The P knowlesi cases had only two matched controls, due to the low number of households in these villages. One non-P knowlesi case had only one control enrolled and another non-P knowlesi case only had two controls enrolled because there were few households nearby.From Dec 5, 2012, to Jan 30, 2015, 414 patients with microscopy-positive malaria were screened for eligibility at the district sites . We exclThe exposure variables used for the univariate analysis and any missing data are shown in the P knowlesi . 47 (27%) of 174 male knowlesi cases, and 17 (31%) of 55 female knowlesi cases identified as farmers, including individuals with other primary occupations. Occupations involving work that occurs predominantly inside or around the household or other buildings, such as shopkeeping, traditional female household duties, and studying, were associated with a lower risk of malaria on univariate but not multivariate analysis. Specific occupational activities, such as palm-oil plantation work  or clearing vegetation or forest in the previous month  were independently associated with increased risk.Identifying as a farmer was associated with an increased risk of symptomatic infection with Sleeping outside was an independent acquisition risk factor , as was a history of recent travel , although specifically sleeping outside in the forest or plantation during a trip were not significant risk factors on multivariate analysis. Residual insecticide spraying of household walls was the only malaria-prevention activity to reduce acquisition risk in the model . Use of a mosquito bednet remained unassociated with protection after controlling for other variables. Interaction with monkeys, predominantly long-tailed macaques (144 [97%] of 148 cases reporting exposure to monkeys), was associated with increased acquisition risk, although higher frequency of observing monkeys did not remain significant . A histoYoung or sparse regenerating forest was the most commonly reported vegetation type surrounding knowlesi malaria case households (163 [72%] of 225 households) and was associated with decreased malaria risk , as was rice paddy . By contrast, the presence of long grass around the area of the household was associated with increased acquisition risk . The only household construction factor to remain in the final model was the presence of open roof eaves, large gaps, or both in the walls, which increased acquisition risk , whereas bamboo walls and floors did not remain significantly associated.P knowlesi malaria are shown in the Plasmodium spp , but a higher risk of P knowlesi . Walking to work or school was also associated with increased risk of knowlesi malaria acquisition compared with non-knowlesi malaria. A history of palm-oil plantation work in the preceding 3 weeks gave a similarly increased risk of both types of malaria. Clearing vegetation or forest in the past month  was associated with an increase in knowlesi malaria risk  but not non-knowlesi malaria. Sleeping outside posed a similarly high malaria risk for knowlesi and non-knowlesi malaria, as did a history of travel. Differences existed between the malaria types in terms of type of vegetation in the immediate vicinity of the house, with young sparse forest not having a protective benefit for non-P knowlesi cases , as seen in the P knowlesi group . Having a fruit tree plantation near the house was associated with decreased risk of non-knowlesi malaria , but not knowlesi malaria . Malaria prevention activities inside the household did not seem to be protective for non-P knowlesi cases, including residual insecticide spraying or mosquito bednet use.Adjusted odds ratios for exposure variables associated with non-P knowlesi infection, our key findings support the most common risk profile for knowlesi malaria, as consisting of adult men working as traditional subsistence farmers, especially those with a history of recent palm-oil plantation work. The highest individual transmission risk was at the forest-edge, including in people with a history of clearing vegetation. This finding suggests that recent ecological changes affecting the human–vegetation interface are associated with spatially heterogeneous P knowlesi infection,P knowlesi cases, and although agricultural or other activities away from the household gave the greatest risk in these groups, we also identified peri-domestic transmission risk in the immediate vicinity of the household. Strong associations with overnight travel and sleeping outdoors emphasise the importance of population movement on zoonotic knowlesi malaria transmission. Walking to and from work also seemed to increase risk compared with human-only Plasmodium species transmission. Overall, these findings support previously reported data from neighbouring SarawakAnopheles balabacensis,P knowlesi between proximal canopy-dwelling macaques and ground-dwelling humans.21In this first, comprehensive assessment of the factors associated with acquiring symptomatic Plasmodium species,M fascicularis,P knowlesi.Plasmodium species in the area,Individual intrinsic factors seemed to provide protection against knowlesi malaria. In particular, phenotypic G6PD enzyme deficiency was associated with decreased risk of symptomatic infection, as seen for human-only P knowlesi also occurred in or around the household, as evidenced by a small number of cases, including young children, with no history of work or travel away from the house. Additionally, household factors associated with increased exposure to mosquitoes such as open eaves and gaps , the protective benefit of residual insecticide spraying on household walls, and a positive association between risk of infection and presence of monkeys in the vicinity of house and garden all suggest some degree of peri-domestic transmission. This notion is consistent with entomological findings from a subset of participants in this study, with A balabacensis being found both inside households, and more commonly (five times more) outdoors in peri-domestic areas, as well as being more abundant around case households than control households.P knowlesi between human and macaque hosts in other areas of Malaysia show no evidence of such transmission.P knowlesi acquisition, consistent with findings from the same study area describing earlier peak biting times of A balabacensis in the early evening from 1800 h to 2000 h as mosquitoes adapt to human bednet use later in the night, with biting mainly occurring outdoors.Transmission of P knowlesi village-level case incidence that was associated with proximate forest cover and historical forest loss.P knowlesi vector sporozoite rates and parity are highest in areas of Sabah with intact forest, increased vector abundance has been reported in villagesP knowlesi such as farming, palm-oil plantation work, and clearing vegetation predominantly required a short journey from the household or village. This was in contrast with human-only Plasmodium spp, which was not associated with these risk factors, despite both zoonotic and human-only malaria species being transmitted by the same vector in this area.P knowlesi group than in the human-only Plasmodium species cases, which was potentially related to closer proximity to macaques through their foraging behaviour. However, evidence suggests that the density of long-tailed macaques in Sabah is higher in areas of intact forest than in fragmented areas of oil-palm, rubber, acacia, or coconut plantations.28Evolutionary understanding of emerging vector-borne diseases highlights the role of land use and deforestation, with resultant changes in ecological habitats affecting vector bionomics that favour increased blood feeding on humans.P knowlesi infections who did not seek the free government provided treatment. However, the wide age range and geographical distribution of the cases suggests that this potential bias did not have a major effect. It has also been shown that asymptomatic submicroscopic P knowlesi infections can occur,P knowlesi acquisition.Limitations of the study include recall bias, given that knowlesi cases who were aware of their diagnosis were potentially more likely to recall seeing monkeys or describe childhood malaria episodes. This study only enrolled symptomatic malaria cases presenting to the catchment area hospitals, and it is likely that there were people with symptomatic but spontaneously resolving P knowlesi infection, the results of this study would seem to be generalisable to other rural forested and agricultural areas within this region with a similar profile of cultural activities, large-scale plantation or forestry-related work, subsistence farming, and recent land use change; consistent with descriptions of P knowlesi infection in Sumatra, Indonesia.P knowlesi infection prevalence, complexity of land cover change, and the densities and bionomics of both primate hosts and different anopheline vectors at fine spatial scales to reliably estimate risk of potential spread or establishment of transmission in human hosts.Despite the variation in patterns of P knowlesi infection, although peri-domestic transmission also occurred. Individual-level factors affecting zoonotic P knowlesi transmission in established endemic areas are potential targets in future public health interventions, which can be designed to reflect the realities of population expansion, wildlife sustainability, and agricultural development, along with ongoing promotion of conventional malaria prevention activities.Risk of knowlesi malaria is associated with a range of human interactions in farm, forest, and village environments where macaque hosts and mosquito vectors are present. Adult men working in agricultural areas had the highest risk of symptomatic Supplemental Data


Eptesicus fuscus) as an initial investigation into using metabolomics for age determination. Samples were collected from captive, known-aged big brown bats (Eptesicus fuscus) from 1 to over 14 years of age: these two ages represent age groups separated by approximately 75% of the known natural lifespan of this taxon. Results showed 41 metabolites differentiated young (n = 22) and elderly (n = 6) Eptesicus. Significant differences in metabolites between young and elderly bats were associated with tryptophan metabolism and incomplete protein digestion. Results support further exploration of the physiological mechanisms bats employ to achieve exceptional longevity.Vespertilionid bats  live 3–10 times longer than other mammals of an equivalent body size. At present, nothing is known of how bat fecal metabolic profiles shift with age in any taxa. This study established the feasibility of using a non-invasive, fecal metabolomics approach to examine age-related differences in the fecal metabolome of young and elderly adult big brown bats ( Myotis and Eptesicus) of the vespertilionid family, have a remarkable lifespan of 19–40+ years, a range that is 3–10 times longer than the expected longevity of size-comparable, non-volant mammals , Human Metabolome Database , Human Statistical analyses  includedEptesicus, ranging from one to over fourteen years of age. To identify age-associated differences in metabolic profiles, the animals were divided into two different age groups: the “young” group included 22 animals aged 1–2 years, while the “elderly” group contained 6 individuals all 14 years of age and older. The final metabolite dataset contained 2003 entities  in the elderly metabolome , Table 2+, Na+; ). Compar+, Na+; , which i+, Na+; .Eptesicus. This finding was striking as some of these compounds are known to contribute to longevity and play key roles in immune function including cancer and inflammation [Of the candidate metabolites, four were functionally associated with metabolism of the amino acid tryptophan ; ), an antEptesicus potentially indicate the onset of glycation. Although multiple studies showed modifications associated with mitochondrial function [Beyond skatole and peptides, still other compounds  potentiafunction ,20, resufunction ). Presenfunction . PresencEptesicus fuscus). Analyses detected 41 metabolites that demonstrated significant increases with age in Eptesicus. Currently, our analyses lack a larger cohort of elderly animals as well as middle-aged samples which, unfortunately, impedes understanding of how these metabolites do or do not fluctuate across the lifespan in Eptesicus. Future studies will target larger sample sizes of young, middle, and elderly age groups to better assess how fecal metabolites vary with age and better understand the range of individual variation within these animals. Beyond offering the first age-comparative study of fecal metabolomics in bats, our results also provide new insights into how bats maintain physiological health into old age.The exceptional longevity of many bat species has piqued the interest of researchers aiming to elucidate the physiological adaptations contributing to these extended lifespans ,19,21. TAlthough other tissues and fluids are available for metabolomics analysis , feces were the focus of this pilot analysis as they offer a reliable and non-invasive means for sample collection that may ultimately be useful for data collection in wild populations. We utilized databases containing identities for over 200,000 metabolites to analyze our fecal samples and 41 metabolites that demonstrated age-associated differences between young and elderly bats. While urine is commonly utilized in conjunction with fecal analyses, the authors found collection of uncontaminated urine to be a major obstacle. Future assays will also address how circulating levels of identified metabolites in blood correlate to levels found in bat fecal samples.Nycticeius humeralis) and little brown bats (Myotis lucifugus)) have increased HSP levels compared to shorter-lived rodents and marsupials [Progressive deterioration of protein homeostasis contributes to age-related functional decline and adversely affects longevity. The cellular mechanisms of translation, folding and clearance cooperatively regulate protein production and stability as well as the refolding or removal of damaged products . With agrsupials . These ersupials  and incrrsupials . Our resEptesicus.The fecal microbiome is an ecosystem of over 1000 species of bacteria which undergo complex interactions that ultimately influence the physiology and health of their host. Metabolites formed during the bacterial metabolism of the essential amino acid tryptophan (TRP) play key roles in the molecular regulation of host behavior, neuronal activity, immune activity, longevity and extend the healthspan ,80. WithThe most common naturally occurring analog of indole is skatole , also kTryptophan is most commonly metabolized via the kynurenine pathway , which gThe first step of the kynurenine pathway, the breaking of the indole ring of L-tryptophan, is regulated by the enzymatic activities of indoleamine 2, 3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase . NormalIn contrast, inhibition of IDO promotes immune cell health and activity . InhibitEptesicus fuscus). Results establish the feasibility of this metabolomics approach and lay the foundation for future studies to expand our knowledge of age-related changes in bat fecal metabolic profiles and further develop fecal metabolomics as a means of age determination in bats. Our metabolomics approach, utilizing LC-QTOF-MS, identified at least 40 metabolites that were significantly increased in fecal samples of elderly Eptesicus over 14 years of age. While most of the candidate metabolites identified have known physiological significance in mammals Click here for additional data file.


A single PGCC formed tumors in immunodeficient mice. PGCCs are also the key contributors to the cellular atypia and associate with the malignant grade of tumors. PGCCs have the properties of cancer stem cells and produce daughter cells via asymmetric cell division. Compared with diploid cancer cells, these daughter cells express less epithelial markers and acquire mesenchymal phenotype with importance in cancer development and progression. Tumor budding is generally recognized to correlate with a high recurrence rate, lymph node metastasis, chemoresistance, and poor prognosis of colorectal cancers (CRCs) and is a good indicator to predict the metastasis and aggressiveness in CRCs. Micropapillary pattern is a special morphologic pattern and also associates with tumor metastasis and poor prognosis. There are similar morphologic features and molecular phenotypes among tumor budding, micropapillary carcinoma pattern, and PGCCs with their budding daughter cells and all of them show strong ability of tumor invasion and migration. In this review, we discuss the cancer stem cell properties of PGCCs, the molecular mechanisms of their regulation, and the relationships with tumor budding and micropapillary pattern in CRCs. We previously reported that polyploid giant cancer cells (PGCGs) induced by CoCl Colorectal cancer (CRC) is one of the most common malignant tumors and its incidence ranks the third of malignant tumors . The metNormal human cells contain 46 chromosomes, but tumors cells contain abnormal numbers  of chromosomes, with cell-to-cell variability. Structural abnormalities of chromosomes such as inversions, deletions, duplications, and translocations are commonly observed in cancer cells but are rare in normal cells . The celTumor budding is similar to the morphological features of micropapillary pattern –20. BaseCancer stem cells, often referred to as tumor-initiating or tumor-propagating cells , 23, arePGCCs are often considered as the senescent cells. PGCCs in tumors have not attracted major attention due to lack of extensive study and their poorly understood biology . Actuall2) [We recently reported that PGCCs can be purified from human ovarian and breast cancer cells lines and primary human ovarian tumors with the use of chemical hypoxic mimetic, cobalt chloride (CoCl2) , 36–38, 2) . A singl2) –41. ThesPGCCs generate daughter cells via asymmetric cell division which is a hallmark of stem cells. In multicellular eukaryotes, mitosis is the recognized process for somatic cell division, ensuring the accurate separation of genetic material , 43. AsyPrognosis of CRC patients has been associated with several morphological features including the infiltration depth, tumor cell differentiation, and lymph node or distant metastasis. These morphological features are the primary parameters for prognostic evaluation and tumor staging of CRCs in clinics . HoweverSeveral large studies tried to establish the criteria of clinical significances of tumor budding in CRCs and confirmed that tumor budding correlates with the lymph node or distant metastasis and associates with patient prognosis. Hase et al. studied 663 patients with CRCs; they divided patients into two groups according to the number of buddings including none or mild group and moderate or severe group . They foThe morphologic characteristics of tumor budding reflect the loss of adhesive epithelial phenotype in cancer cells and are accompanied by the metastasis of cancer –73. ResuEMT is involved in many physiological processes including mesoderm formation and neural tube formation and pathological processes including wound healing and organ fibrosis , 76. It 2 and paclitaxel. When cancer cell lines recovered from the treatment of CoCl2 and paclitaxel, PGCCs could produce daughter cells via asymmetric cell division. Western blot analysis was performed to confirm that cytokeratin (AE1/AE3) expression was lower in the daughter cells than in cancer cells without treatment. The increased expression of mesenchymal markers such as vimentin was evident in daughter cells [2 and the control cells. Results showed that a panel of stem cell-regulating factors and EMT-related proteins were upregulated in PGCCs [EMT has also been demonstrated to play an essential role during the formation of cancer stem cells . Previouer cells , 15, 21.β-catenin and is an important component of β-catenin degradation complex. Tumor budding was characterized by increased expression for both β-catenin and p16 (cyclin-dependent kinase inhibitor 2A) [β-Catenin is important for linkage of E-cadherins to the cytoskeleton [β-catenin subcellular localization. When β-catenin translocates from the cytoplasm to the nucleus, epithelial adhesion was reduced and migratory potential increased. Loss of Ep-CAM of CRC cell membrane is highly correlated with tumor budding, cancer grade, and local recurrence [Jass et al. reported that APC (adenomatous polyposis coli) mutation was much less frequent in sporadic microsatellite instability-high (MSI-H) cancers than in MSI-low or microsatellite stable cancers . APC canitor 2A) . β-Catenskeleton , 81. Theskeleton , 83. Furskeleton . Loss ofcurrence .Micropapillary pattern was first reported in breast cancer, in which a small cluster of tumor cells appear in tumor tissue and there are no vessels and stromal cells in the middle of tumor cluster . MicropaPGCCs appear in most of micropapillary carcinoma patterns and tumor budding. In some micropapillary patterns, the absence of PGCCs may be due to the five-micrometer thickness for slide processing. According to the morphological observations in CRCs with large sample, we speculate that tumor budding and micropapillary pattern may have the same origin, which derive from PGCCs with their budding daughter cells. We previously reported that single PGCCs which appeared after paclitaxel treatment from an invasive breast cancer cell line MCF-7 formed cancer organotypic structure (COS) including glandular, vessel-like, and papilla-like structures in vitro . The papThere is a close association among tumor differentiation, tumor budding, PGCCs, and micropapillary carcinoma pattern. Lv et al. also reported that single stromal PGCCs with their budding daughter cells were often associated with tumor metastasis in OSCs . The numCRCs with tumor budding were identified to respond to chemoradiotherapy poorly and have adverse prognosis. Tumor budding is mostly unreported in daily diagnostic practice due to the lack of a standardized evaluating system. Karamitopoulou et al. suggested a method using 10 high-power fields to assess tumor budding at the invasive front. Their opinion showed that using 10 high-power fields to evaluate tumor budding had independent prognostic value and could show good interobserver consistency . Jass etβ-catenin signaling pathway which may provide clinicians with new hints to treat tumor with high-grade malignancy through targeting these proteins involved in tumor budding and PGCCs formation.Since tumor budding is related to the prognosis of CRC patients, the standardized evaluating index including tumor budding and PGCCs could improve the grading of CRCs. Thus, it is worth paying more attention to the relationships between PGCCs with their newly budding daughter cells and tumor budding. The molecular mechanisms of tumor budding are involved in the expression of EMT-related proteins and Wnt/


White-tailed deer are a culturally and economically important game species in North America, especially in South Texas. The recent discovery of chronic wasting disease (CWD) in captive deer facilities in Texas has increased concern about the potential emergence of CWD in free-ranging deer. The concern is exacerbated because much of the South Texas region is a semi-arid environment with variable rainfall, where precipitation is strongly correlated with fawn recruitment. Further, the marginally productive rangelands, in combination with erratic fawn recruitment, results in populations that are frequently density-independent, and thus sensitive to additive mortality. It is unknown how a deer population in semi-arid regions would respond to the presence of CWD. We used long-term empirical datasets from a lightly harvested  population in conjunction with 3 prevalence growth rates from CWD afflicted areas  via a multi-stage partially deterministic model to simulate a deer population for 25 years under four scenarios: 1) without CWD and without harvest, 2) with CWD and without harvest, 3) with CWD and male harvest only, and 4) with CWD and harvest of both sexes. The modeled populations without CWD and without harvest averaged a 1.43% annual increase over 25 years; incorporation of 2% annual harvest of both sexes resulted in a stable population. The model with slowest CWD prevalence rate growth  without harvest resulted in stable populations but the addition of 1% harvest resulted in population declines. Further, the male age structure in CWD models became skewed to younger age classes. We incorporated fawn:doe ratios from three CWD afflicted areas in Wisconsin and Wyoming into the model with 0.26% annual increase in prevalence and populations did not begin to decline until ~10%, ~16%, and ~26% of deer were harvested annually. Deer populations in variable environments rely on high adult survivorship to buffer the low and erratic fawn recruitment rates. The increase in additive mortality rates for adults via CWD negatively impacted simulated population trends to the extent that hunter opportunity would be greatly reduced. Our results improve understanding of the potential influences of CWD on deer populations in semi-arid environments with implications for deer managers, disease ecologists, and policy makers. Chronic-wasting disease (CWD), a transmissible spongiform encephalopathy, has had a large impact on the management of afflicted cervid populations in the United States and Canada –5. DiseaOdocoileus virginanus) in Texas tested positive for CWD [In June 2015, the first white-tailed deer  within the South Texas region to parametrize our population models. Deer were harvested conservatively; 1–4% of the total estimated population were harvested annually. Although supplemental nutrition can increase deer productivity in semi-arid environments ,47, we dWe used a 20-year dataset of fawn:doe ratios and population counts collected from helicopter surveys  during SWe developed a multi-stage deterministic matrix model  that repBecause variable rainfall and fawn recruitment rates influence population structure, we input fawn:doe ratios in the order of year observed during our model validation efforts. Fecundity, defined as the fawn:doe ratio, was equal among all female age classes except for fawns (none). Although there is evidence that older females are more productive than younger females , we usedHarvest occurred in the reference population and the number of harvested deer was known. We calculated the proportion of males and females that were harvested based on the annual deer counts corrected for visibility bias. The proportion of deer harvested, in order of year observed, was used to remove deer from the population following the birth pulse . The disSex- and age-specific survival rates were obtained from field studies in South Texas . SurvivaBefore conducting simulations, we ensured that our models produced realistic results by comparing trends between observed and modeled population trajectories. We compared model output to both annual estimates of population size and a 3-year moving average to smooth out variation in the year-to-year estimates ,54.During 1996–2015, observed fawn:doe ratios were highly variable  and coyotes (Canis latrans) exist in South Texas and may selectively prey on infected deer [2 [CWD mortality was modeled to be additive because of the density-independent nature of deer dynamics in South Texas . It is possible that CWD mortality would be compensatory because of predation , deer-veted deer ,75; howe deer [2 ). Coyote deer [2  but prim deer [2 ,81, whic deer [2 . Deer-ve deer [2 . However deer [2 ,84. The If our hypothetical CWD-afflicted population was able to sustain itself, perhaps during a period of favorable rainfall and increased fawn:doe ratios ,85,86, tDeclines in our simulated CWD populations with only 1% female harvest suggest that a slight increase in additive mortality rates would be unsustainable for deer populations in semi-arid regions. This finding is not unexpected because deer managers have long known that harvest rates in native rangelands, where deer population density-independence occurs frequently, need to be conservative to prevent population declines due to the additive nature of harvest ,90. The We acknowledge that our relatively simplistic population model contains both process variation  and sampling variation (variation in measured parameters) which could lead to bias in population projections ,101. HowChronic wasting disease is a slow-spreading disease that may take years or decades to result in detectable prevalence rates . ModelinS1 File(TXT)Click here for additional data file.


A 76 year-old man with a past medical history of type 2 Diabetes, stage 3 chronic kidneydisease and previous multivessel coronary artery bypass surgery 10 years before wasadmitted with an acute coronary syndrome. Coronary angiogram showed an occludedsaphenous vein graft (SVG) to first diagonal. After restoring flow by means of thrombusaspiration, optical coherence tomography (OCT) runs were required. Localized and widelypatent segments of the graft showed a clear three-layer configuration, suggestive of SVGfocal arterialization. Proximally, predominantly red thrombus determined a criticalstenosis and a thin cap fibroatheroma was evident in an arterialized segment of thegraft contiguous to the thrombotic lesion. Conversely, the rest of the SVG kept itsvenous appearance and had three different areas of stenosis .Since the early 70´s, there has been multiple efforts to describe and understand factorsdetermining SVG patency. Remodeling - mainly through intimal hyperplasia and further neoatherosclerosis - is responsible for most of the late graft loss.The present case adds to what is previously known by showing distinct segments ofthree-layered vein graft wall, and also that SVG "arterialization" can be a focal andheterogeneous process throughout the vessel wall. Furthermore, it was a vulnerableplaque from this remodeled segment the one that probably harbored the thromboticcomplication.Given its superior image resolution, OCT can significantly contribute to ourunderstanding of vein graft failures.


Resuscitative endovascular balloon occlusion of the aorta (REBOA) is an emergency procedure to manage severe hemorrhagic shock from torso injury but can cause severe ischemia of the lower extremities. However, lower extremity ischemia occurring as a complication of REBOA has been rarely reported. We describe the severe lower extremity ischemia caused by REBOA with a 12-Fr sheath in a small-built patient.The patient was a 16-year-old male who developed severe hemorrhagic shock due to abdominal blunt trauma. Following REBOA with a 12-Fr sheath on the right femoral artery, an emergency laparotomy and angiography to control the hemorrhage were performed. Twenty-eight hours after admission, suspecting lower extremity ischemia and compartment syndrome, we removed the sheath with a manual maneuver and performed fasciotomy. The limb ischemia was thus partially resolved. However, amputation was necessary because of ischemic necrosis on day 32. Our patient was physically small, and the diameter of his femoral artery on the contralateral site of sheath placement was also small. Therefore, disproportion of the sheath and femoral artery sizes may have caused the ischemic complication.Our experience highlights the importance of appropriate size selection for the sheath in line with the target vessel. We also recommend postoperative monitoring of limb perfusion in such cases with the use of near-infrared spectroscopy to facilitate the early detection of ischemia. Resuscitative endovascular balloon occlusion of the aorta (REBOA) is an emergency procedure for the management of severe hemorrhagic shock caused by torso injury. This intervention can cause severe ischemia of the lower extremities . HoweverA previously healthy 16-year-old male with blunt abdominal injury sustained in a road traffic accident was referred to our department. On arrival, he was in severe hemorrhagic shock, and his vital signs were as follows: heart rate 160 bpm, blood pressure unmeasurable, respiratory rate 27/min, and Glasgow Coma Scale E3V4M6. His chest X-ray revealed no apparent hemothorax and pneumothorax, and pelvic X-ray revealed no unstable fracture of the pelvic ring. Focused assessment with sonography for trauma was positive; thus, we suspected intra-abdominal hemorrhage. The arterial blood gas assessment showed metabolic acidosis, and the laboratory test on admission revealed coagulopathy Table . SuspectWe describe the severe ischemic complication of the lower extremities associated with sheath placement for REBOA. Potential ischemic complications of REBOA include acute kidney injury, vascular injury, bowel ischemia, spinal cord ischemia, and lower extremity ischemia . HoweverWe believe that the use of large-sized sheaths for REBOA is a critical risk factor for lower extremity ischemia. In a prospective REBOA case registry, 74 % 34/46) of all REBOA interventions were performed using a 12- or 14-Fr sheath [4/46 of aWe believe that decreasing the duration of sheath placement and aorta occlusion may also help avert lower extremity ischemia. The reported median duration of sheath placement in a study was 28 18–45) h, which is equal to the duration in our case (28 h)  h, which, and theMonitoring of lower extremity perfusion is essential to detect ischemia. We relied on visual examination and Doppler study for this purpose; however, we could not avoid the ischemic complication as both these methods lack objectivity and are prone to inter-observer variability. This probably contributed to the delay in the detection of ischemia. Near-infrared spectroscopy (NIRS) is reported as a good monitoring tool to detect lower extremity ischemia and compartment syndrome , 8. Use We described severe lower extremity ischemia occurring as a complication of REBOA using 12-Fr sheath in a small-built patient. We highlighted the importance of appropriate size selection for the sheath in line with the target vessel. We also recommend postoperative monitoring of limb perfusion in such cases with the use of NIRS to facilitate early detection of ischemia.


Thyroid fine-needle aspiration biopsy results are cytologically indeterminate in 15-30% of cases. When these nodules undergo diagnostic surgery, approximately three-quarters are histologically benign. These unnecessary surgeries diminish quality of life, generate complications, and increase healthcare costs. The Afirma gene expression classifier (GEC) is validated to pre-operatively identify cytologically indeterminate nodules likely to be truly benign so that surgery can be avoided. Its performance is supported by robust multicenter prospective and blinded clinical validation studies, and supported by extensive independent clinical utility publications which show a marked reduction in surgery among patients with benign Afirma GEC results. To rule-out cancer and avoid unnecessary diagnostic surgery, Afirma’s quality and depth of validation stand alone. The accuracy of a benign result is the negative predictive value (NPV). Afirma achieves an NPV ≥94% among cytologically indeterminate nodules (Bethesda III or IV). Thirteen clinical utility studies describing 1468 GEC benign patients demonstrate that few Afirma GEC benign nodules undergo surgery, including after 3 years of follow-up. With a specificity of 52%, over half of the truly benign nodules with indeterminate cytology receive a benign GEC result. High test sensitivity is critical to safely rule out cancer. The Afirma GEC’s 90% sensitivity means that regardless of the pre-test risk of malignancy, 90% of all malignant nodules are GEC suspicious. The Afirma GEC has transformed patient care. Where the majority of cytologically indeterminate patients were once operated to determine if the nodule was benign or malignant, now nearly half of these surgeries can be avoided. Prior to the adoption of thyroid nodule fine-needle aspiration biopsy (FNAB), thyroid nodules were regularly referred for diagnostic surgery because of their 5-15% risk of malignancy (ROM) . Still, A test with a high sensitivity and high negative predictive value (NPV) is able to rule-out cancer ,12. TestThe Afirma GEC is based on the measurement of messenger ribonucleic acid (mRNA) expression. There are several diagnostic advantages to using RNA instead of other approaches such as DNA mutations or microRNA expression. Unlike cancers whose cytology is Bethesda V or VI, cancers that are cytologically indeterminate (Bethesda III and IV) typically lack the most common genomic abnormality of differentiated thyroid cancer: BRAFV600E mutation. In its absence, the most common classic mutation amongst cytologically indeterminate cancers are RAS mutations, but these are found in the minority and are also found in benign nodules. As benign nodules outnumber malignant nodules 4:1 among nodules with indeterminate cytology, the PPV of RAS mutations is poor in a number of studies ,19,20,21While there are only approximately 23,000 known protein-coding DNA genes , each ofGene expression classifiers quantitatively evaluate the relative expression levels of multiple genes that comprise the genomic signature of the interrogated tissue. In the development of Afirma GEC, instead of discriminately relying on genes previously identified in the literature, analysis of the whole genome (transcriptome) was used to identify candidate genes, and support vector machine learning methods were used to develop the classifier algorithm ,25. The Physicians find risk of cancer associated with a cytological benign FNAB diagnosis to be low enough to defer surgery in the vast majority of such patients. A 6-8% risk of cancer among operated cytology benign nodules has been described ,28,29,30The unique and often overlooked strength of this prospective, multicenter, and blinded validation design is that it supports generalizability of the results. Prospective and multicenter study designs reduce selection bias and better represent what is likely to occur in real-world practice. The 3.789 patients were prospectively consented and enrolled in the trial before undergoing FNAB at 49 study sites across the country, including academic and community practices, which provides confidence in the external validity of the findings. Strong internal validity was demonstrated when no differences were found between the final validation cohort of 265 patients compared to the full prospective and consecutive total enrollment cohort in patient age, gender, cancer risk factors, or nodule size. As investigators were blinded to the Afirma GEC result, the test result did not influence which patients underwent surgery. These important study design elements  support the internal and external validity of the study, and provide confidence in the broader generalizability of the study findings to a physician’s own clinical practice . In contOverall, the ROM for a thyroid nodule with Bethesda categories III and IV indeterminate cytology with an Afirma GEC benign classifier result is about 5% (1-NPV). This risk is comparable to the 6-8% cancer risk for an operated thyroid nodule with a benign cytology diagnosis ,28,29,30While clinical validation demonstrates the test’s ability to accurately predict the diagnosis, clinical utility measures the test’s impact on real-world patient management decisions and impact on net health outcomes . FourteeTwo cohorts of 2667  and 2040As noted above, clinical experience/clinical utility studies serve an important role in the chain of evidence regarding the effectiveness and value of a test. These allow medical centers and community practices to describe the impact the GEC has had in their management setting. As more long-term follow-up data becomes available, these important studies will allow researchers to model the overall GEC impact on reducing unnecessary surgeries nationally.It is important to note, however that most GEC benign patients in the clinical series reported to date did not undergo surgery, consistent with the purpose of the test ,49,50,51Additionally, clinical experience series may differ from properly performed prospective validation studies as the former may not report on a consecutive cohort of tested patients from the catchment area, but rather report only on patients who come to their attention through a variety of referral patterns. Thus, the cohort described may not reflect how the test works in the intended use population. Another limitation of clinical experience studies is that when Afirma GEC suspicious nodules are unoperated then test sensitivity among the operated cases is likely to be reduced . More imThe accuracy of an Afirma GEC benign call (NPV) remains high amongst Hürthle cell cytology, although the rate of benign calls is lower. Hürthle cell cytology has been a challenge for molecular diagnostics. Performance can be increased by removing these samples from clinical testing , but thiPhysicians collect two extra FNAB passes for potential molecular testing with the Afirma GEC on every FNAB they perform, or have on-site rapid cytological assessment so that the GEC can be collected on every patient with indeterminate cytology during one patient visit . This paIt is well known that cytologically indeterminate nodules may not be categorized as indeterminate if they undergo a repeat FNAB . While tThe PPV and NPV are determined by the pre-test ROM. To practice personalized medicine, it is important to consider the individual patient’s pre-test risk. The patient’s pre-test ROM includes their individual features  and the interpreting cytologist’s thresholds to utilize cytology indeterminate categories. Ignoring this step of personalized care and assuming that every patient at a practice or institution has the same pre-test risk ignores important medical information.The 2015 ATA guidelines allow for either hemithyroidectomy or near-total/total thyroidectomy for thyroid malignancy 1-4 cm in size without gross extra-thyroidal extension or clinical evidence of lymph node metastases . Thus, mThe 2015 ATA guideline emphasizes ultrasound characteristics to predict the nodule’s ROM . Afirma The 2015 ATA guidelines do not provide recommendations on the follow-up of cytologically indeterminate nodules that are Afirma GEC benign . Angell An independent cost-effectiveness study found no difference in the number of missed cancers between paradigms with and without the Afirma GEC in a Markov model employing 10,000 Monte Carlo simulations of the expected range of probabilities for different potential outcomes . HoweverLee et al.  modeled Additional limitations of the 3 cost-effectiveness studies described above include that none consider indirect costs due to time lost from work and impacted responsibilities of daily living as a result of surgery and its recovery. Neither Lee et al.  nor Wu eV600EBRAF point mutation classifier, and a cassette for MTC. Additional cassettes automatically run with every Afirma GEC test screen for parathyroid tissue  , there is value to a test that can identify malignancy (a rule-in test) only when it alters clinical care to the benefit of the patient. Clinical care can be altered by enhancing the rationale for surgery, and more directly by altering the extent of surgical care . The Afiignancy) , and metMTC is frequently a cytological challenge to diagnose, and the field has had attention recently drawn to the low sensitivity of FNAB for the specific diagnosis of MTC ,80. MTC BRAF are by far the most common genomic abnormality associated with papillary thyroid carcinomas, and nearly all are V600EBRAF mutations . In a consecutive cohort of 7,066 de-identified FNABs, 3,187 samples were benign by Afirma GEC, of which none were Afirma BRAF positive  for Afirma BRAF than for qPCR (24.5%), a further advantage of using RNA in FNAB small sample biopsies. In the US, the V600EBRAF classifier is an option on Afirma GEC suspicious nodules, and Bethesda V or VI nodules without Afirma GEC.Point mutations in utations . The Afi28 genes . Compare28 genes . When cu nodules ,86. Unli nodules ,19,20,21positive . Thus, tCytologically indeterminate nodules have historically been referred for surgery given that their ROM was above the typical threshold of ~5% for physicians to consider clinical observation in lieu of diagnostic surgery. Molecular diagnostic testing of these nodules has rapidly become accepted. Current guidelines include that molecular testing may be used among Bethesda III/IV nodules to add additional information about the nodule’s ROM. The 2015 ATA guideline reviews the molecular testing landscape, and voices caution over tests supported only by single center and unblinded validation data, and those with no published clinical utility data to demonstrate a change in clinical care and patient benefit as a result of the test. The Afirma GEC is the only molecular test supported by multicenter, prospective, and blinded validation data, and the only test supported by published clinical utility data demonstrating a dramatic reduction in diagnostic surgery for patients with benign Afirma GEC results. Nearly 1 out of every 2 Afirma GEC tests performed yields a molecularly benign result, and >80% of patients with a benign GEC result remain unoperated 3 years after the biopsy in real-world experience. Reducing unnecessary diagnostic surgery improves patient safety, reduces healthcare costs, and improves patient quality of life.


We address the protein targets for guanosine action and its interaction with glutamatergic and adenosinergic systems and with calcium-activated potassium channels. We also discuss the intracellular mechanisms modulated by guanosine preventing oxidative damage, mitochondrial dysfunction, inflammatory burden and modulation of glutamate transport. New and exciting avenues for future investigation into the protective effects of guanosine include characterization of a selective guanosine receptor. A better understanding of the neuromodulatory action of guanosine will allow the development of therapeutic approach to brain diseases.Guanosine is a purine nucleoside with important functions in cell metabolism and a protective role in response to degenerative diseases or injury. The past decade has seen major advances in identifying the modulatory role of extracellular action of guanosine in the central nervous system (CNS). Evidence from rodent and cell models show a number of neurotrophic and neuroprotective effects of guanosine preventing deleterious consequences of seizures, spinal cord injury, pain, mood disorders and aging-related diseases, such as ischemia, Parkinson’s and Alzheimer’s diseases. The present review describes the findings of  Guanosine is an endogenous guanine nucleoside to which has been attributed several neuroprotective and neurotrophic effects. This review will describe the extracellular role of guanosine as an intercellular messenger in the Central Nervous System (CNS). We also introduce a brief historical overview of the purinergic system, centering in the guanine-based purinergic system.Purines are a class of aromatic organic molecules essential for all cells that include adenine- and guanine-derivatives nitrogenous bases, as nucleotides with one or more phosphates  and related metabolites such as adenosine, inosine, adenine, hypoxanthine and guanosine, guanine, xanthine and uric acid.Intracellular purines are primarily identified as structural constituents of nucleic acids, but they are also part of the structure of some coenzymes, and display roles as second messengers. Adenine-based nucleotides are well known for their fundamental intracellular role in the maintenance of energetic metabolism in the cells .The adenine-based purines effects in different cell types have unfolded the role of purines in cell signaling. Important biological functions of adenosine were initially unraveled by Drury & Szent-Giorgy , demonstSimilarly to adenine derivatives, guanine-based purines have been firstly identified by their intracellular effects as modulators of G-proteins function. Guanine-nucleotide regulatory proteins, GTP binding proteins, or simply G-proteins have been identified as central actors in the signal transduction field, by coupling transmembrane protein receptors to intracellular effectors . G-proteAs expected, guanine nucleotides and the nucleoside guanosine have also been shown to exert extracellular effects and a guanine-based purinergic system has been highlighted after innumerous findings demonstrating the extracellular actions of GTP, GMP and guanosine, most of them concerning CNS effects . In theiIn the present review, we will briefly discuss the guanine-based purinergic system and then we will focus on the extracellular effects of the guanine nucleoside guanosine.The extracellular effects of guanine-based purines or guanine derivatives have been primarily shown in the CNS, and these effects are relating to the modulation of the glutamatergic system, the main excitatory neurotransmission system in the brain . The neuBinding studies initially performed by Sharif and Roberts  and thenA seminal study from Souza and Ramirez  on guaniIn addition to modulating glutamate binding to its receptors, guanine derivatives modulate glutamate transport. Guanine derivatives are involved in the regulation of glutamate uptake into synaptic vesicles , suggestAdditional evidence of guanine-based purines importance in the extracellular space were obtained from studies showing that purine nucleosides are released after an ischemic injury and their levels are maintained elevated from 2 hours to 7 days . CultureExtracellular nucleotides are hydrolyzed by a family of ecto-nucleotidases associated to the cell surface, the ecto-nucleoside triphosphate (ecto-NTPase) family . The ectIn conclusion, major findings regarding the guanine-based purinergic system are: (i) guanine nucleotides displace the binding of glutamate and analogs to metabotropic and ionotropic glutamate receptors; (ii) guanine nucleotides present a competitive pattern of antagonistic interaction with glutamate receptors; (iii) the effects of guanine-based purines do not rely only on G-proteins interaction; (iv) guanine-based purines are present in the extracellular space and their released may be increased under certain harmful conditions; (v) extracellular guanine nucleotides may be hydrolyzed to guanosine and increase the extracellular level of this nucleoside after brain damage; (vi) guanine-based purines modulate glutamate transporters (located at synaptic vesicles and at cellular membrane) activity; (vii) guanosine modulates glutamate transporters activity although it is uncertain whether it directly interacts with glutamate transporters.in vivo and in in vitro experimental approaches and presents an update on guanosine effects and mechanisms of action as an intercellular messenger mainly in the CNS. Although the focus of this review is guanosine as a protective and trophic messenger in the CNS, some peripheral effects that help to further understand the mechanism of guanosine action are also discussed.This review discusses the extracellular roles of guanosine from in vivo models of brain disorders.Guanosine is a nucleoside that may act as a neuroprotective or retaliatory endogenous system. However to evaluate the effects of guanosine studies often use exogenous administration of this nucleoside. Hereafter, we will discuss the metabolism and distribution of exogenously administrated guanosine followed by the presentation of guanosine effects in in vivo breakdown of the administered guanosine. In fact, the enzyme purine nucleoside phosphorylase  and guanine deaminase (that converts irreversibly guanine to xanthine) were identified at brain membranes, and their activities may result in elevated levels of purines metabolites in the brain -guanosine concentration peaked about 30 minutes after injection. Further investigations on guanosine metabolism revealed that guanine was the major metabolic product in all sites, with over twice as much guanine compared to guanosine after 30 minutes [3H]guanosine, also given i.p. [3H]guanosine given via intramuscular was primarily found at animals kidney guanosine with total rat brain membranes preparations led to the identification of a single high affinity binding site for guanosine, with a dissociation constant (Kd) of 95.4 ± 11.9 nM and an apparent number of maximmal binding sites (Bmax) of 0.57 ± 0.03 pmol/mg protein. Both association and dissociation kinetics were rapid, which is characteristic of natural compounds binding to theirs receptors -guanosine binding to GPR23. Another meeting communication reported GPR23 expression and [3H]-guanosine binding in different brain areas and they found that cerebral cortex has the higher GPR23 expression and the maximal [3H]guanosine affinity binding site. Affinity binding site rank order for all tested areas was cortex > hippocampus > striatum > spinal cord [More recently, the receptor GPR23 is as a new candidate suggested for guanosine.GPR23 is one receptor for lysophosphatidic acid (LPA), identified as the LPA4 receptor. A communication from Di Liberto et al.,  reportednal cord . TogetheGuanosine ability to modulate glutamatergic system was demonstrated by different groups, as previously discussed. Guanine nucleotides  have been shown to act as antagonist of glutamate receptors  subjected to mitochondrial oxidative stress was abolished by both adenosine A1R and A2AR antagonists [1R mediates the neuroprotective effect of guanosine in hippocampal slices subjected to OGD, as DPCPX reversed guanosine-induced decrease in ROS formation and mitochondrial membrane potential, although DPCPX did not interfere with guanosine effect on glutamate uptake. The effect of guanosine of recovering glutamate uptake impairment caused by OGD was blocked by PTX (showing an interaction with a GPCR) and by the activation of A2AR by its agonist CGS21680. Thus, adenosine A2AR activation, but not the blockade with the antagonist ZM241385, inhibited guanosine-induced neuroprotective effect observed on cellular viability and glutamate uptake [1R blockade or A2AR activation can reverse guanosine-evoked neuroprotective effect, we hypothesized that guanosine effect may involve an interaction with A1R-A2AR oligomers, which are known to associate and interact in an antagonistic manner [1R-A2AR oligomers is currently under investigation in our laboratory.An ectively). We alsoe uptake . Since wc manner -155. A p+) channels activity on guanosine-induced effects was also assessed following the observation that guanosine was able to modulate K+ channels activity and expression. In cultured rat cortical astrocytes, chronic exposition (48h) to guanosine in high micromolar levels (500 µM) induced an increase in activity and expression of functional inward rectifier K+ channels [+ channels interaction, since charybdotoxin, an inhibitor of the large (big) conductance Ca2+-activated K+ channels (BK), abolished guanosine-induced increase on cellular viability in hippocampal slices subjected to OGD and in SH-SY5Y cells subjected to mitochondrial damage [+ channels, or apamin inhibitor of small conductance Ca2+-activated K+ (SK) channels, had no effect on guanosine-promoted neuroprotection, pointing to a selective effect on BK channels. BK inhibition also abolished guanosine effect of recovering glutamate uptake decrease in hippocampal slices subjected to OGD [Participation of potassium  channels [Whole cell patch clamp performed in HEK293 cells transiently transfected with the functional α-subunit of BK channels showed that guanosine promoted Kchannels , suggestIn conclusion, although there are reports claiming the existence of a selective guanosine receptor, several studies also demonstrated other putative interaction targets for guanosine, as glutamate transporters, adenosine receptors and potassium channels. To date, there is still puzzling information about guanosine selectivity to a protein target, what supports the idea that this nucleoside might act as a multi-target neuromodulator.MAPK pathways [Guanosine effect on modulation of glutamate transporter activity has an important neuroprotective mechanism against excitotoxicity events. To understand the mechanism by which guanosine stimulates glutamate transport, our group investigated the intracellular pathways related to the effect of guanosine in hippocampal slices subjected to glutamatergic excitotoxicity. We found that guanosine prevented glutamate release after glutamate challenge through activation of the phosphatidylinositol-3 protein kinase (PI3K) and protein kinase B (Akt) pathway . Additiopathways . Recent Guanosine-induced cell viability also activates several signaling pathways including PKC, protein kinase A (PKA), MAPK/ERK and PI3K to protect hippocampal slices subject to OGD . GuanosiMAPK and Jun Kinase (JNK) induced by 6-OHDA and increased Akt phosphorylation and anti-apoptotic Bcl-2 protein expression in SH-SY5Y cells [Guanosine also reduced p385Y cells .In summary, the PI3K/Akt pathway seems to be required for neuroprotection evoked by guanosine against ischemia , apoptosIn microglia cell cultures, guanosine reduces inflammation and expression of pro-inflammatory proteins, an effect associated with the activation of PI3K pathway . In hippin vivo studies do not explore the signaling pathways involved in guanosine mechanism of action. However, Bettio and coworkers [Most of oworkers  have demThe intracellular signaling pathways by which guanosine promotes trophic effects has also been scarcely investigated. Guanosine protected primary cerebellar neurons culture from hypoxia and promoted neurite outgrowth by activation of PCK-related kinase-1 (PRK1) . PRK1 isGuanosine promoted neural stem cells proliferation by stimulation of intracellular cAMP and phosphorylation of CREB (cAMP response element-binding protein). Moreover, guanosine treatment increased BDNF mRNA levels suggesting the involvement of cAMP/CREB in neurotrophic effects promoted by guanosine .In coculture of neuron and astrocytes pre-treated with guanosine, the inhibition of MAPK/ERK, CaMKII, PKC, PI3K or PKA blocked the reorganization of extracellular matrix proteins from a diffuse to a fibrillar matrix . FurtherTherefore, considering the neuroprotective and trophic effects promoted by guanosine, it is imperative to identify its membrane binding sites (putative receptor or selective target proteins) and understand the sequence of cell signaling pathways activation by guanosine in order to unravel the mechanisms triggered by this nucleoside. The studies discussed in this review add important information regarding the mechanism of action of guanosine, contributing for its potential use as a pharmacological strategy against neurodegenerative and neurotoxic events.per se has little effect on basal arterial blood pressure or renal blood flow and therefore it would be safer than adenosine for in vivo administrations. Nowadays, there is no effective treatment for ischemia in humans and the current available treatment, the tissue plasminogen activator (t-PA), is not effective for all patients [Guanosine presents beneficial effects in several rodent and cellular models of neurodegeneration, mainly brain diseases associated to glutamatergic system unbalance, as aging-related disorders, mood-related disorders and peripheral damages that affect the CNS. Additionally, studies describe guanosine as a safe drug once there is no evidence of toxicity after exogenous guanosine administration. Recently, an intravenous guanosine administration used to evaluate vascular cardiorenal effects showed that guanosine may increase adenosine release and promote anti-inflammatory vascular effects . Also inpatients . Resultspatients . Taken tin vivo [in vitro [Recently, statins have been also prescribed after ischemia onset in order to prevent vascular damage . Atorvasin vivo  and in vin vitro , similarin vitro . An addiin vitro . The guaRegarding purines metabolism, it has been suggested that drugs that facilitate the salvage pathway of purines recycling, as the inhibitor of xanthine oxidase activity allopurinol, may represent a clinical strategy in refractory epilepsy  and as aIn vivo human studies have shown higher levels of guanosine in patients suffering from chronic pain and it is correlated with pain severity [severity . In pregseverity . Taken t+ channels in neuronal cells, therefore orchestrating an integrated cellular symphony to promote neuroprotection.The question remains whether guanosine has (or not) a selective receptor and present the necessity of developing selective pharmacological tools to study and modulate the guanine-based purinergic system. Currently, several studies are addressing the possible targets to guanosine, considering the “orphan ligand” guanosine situation. If a guanosine receptor does exist , it woulGuanosine is an endogenous molecule that exerts neuroprotection in several disease models; however, its mechanisms of action are not well clear. Studies have proposed some guanosine receptors to explain the protective role of guanosine. However, guanosine may also act as a multi-target neuromodulator, because of its interaction with others systems, as adenosine receptors, glutamate transporters and potassium channels. It is feasible that guanosine composes an endogenous modulatory system integrating glutamatergic and purinergic transmission, gathering systems that are responsible for important plastic effects on the CNS.Finally, mechanistic studies on guanosine action are necessary to better define the guanine-based purinergic system. These evaluations will support the future direction of clinical investigations and evaluations of safety profile that will support the use of guanosine or drugs acting at purines metabolism as clinical strategies against neurodegeneration.


While the benefits of EVH include reducedwound complications and improved cosmetic outcome, an inferior patency rate of EVH-SVscompared to those harvested by open vein harvesting (OVH) has beenreported[3] ([4]. This decision was based on data from the Project ofEx-vivo Vein Graft Engineering via Transfection (PREVENT) IV trial, where EVH-SV graftsshowed higher failure rates than OVH grafts and, at 3 years, a higher death rate,myocardial infarction or revascularization compared to OVH grafts[5]. Originally, the PREVENT IVtrial was designed as a randomized controlled trial to assess the effectiveness andsafety of edifoligide on angiographic SV graft failure 12-18 months following CABG, aswell as the effect of edifoligide on major adverse cardiac events throughout 5 yearsafter CABG. Considering that the study by Lopes et al.[5] is a secondary analysis, theresults should be evaluated carefully and potential biases inherent to a non-randomizedstudy design should be considered. However, data published subsequently included morepatients and, although it was judged that "EVH did not show increased occlusion rates orincidences of reintervention, myocardial infarction or death for endoscopicallyharvested grafts...the Committee noted the importance of training and regular experiencefor any clinician doing this procedure" [4]. Simultaneously, the 2014 Guideline on MyocardialRevascularization of the European Society of Cardiology (ESC) and the EuropeanAssociation for Cardio-Thoracic Surgery (EACTS) acknowledged the lack of unequivocalevidence concerning patency rates of EVH-SVs and no inferiority in clinical outcomesassociated with EVH[6].The saphenous vein (SV) is the most commonly used conduit for coronary artery bypasssurgery (CABG)orted[3] .Previou[7] showed endothelial damage in EVH-SV grafts anddemonstrated that cellular metabolic activity, viability and membrane damage to theendothelium is less in OVH compared with EVH grafts. Also, using optical coherencetomography, Kiani & Poston[8] described marked damage to the adventitia of EVH-SVgrafts as well as regions of endothelial denudation and abnormalities within the intimaand deeper vessel layers, damage likely to affect graft patency.The unfavorable results regarding EVH-SV grafts are due to vascular damage inflictedwhere they are subjected to vascular trauma, including traction, adventitial stripping,and venous compression. Rousou et al.. This data is supported by a recentrandomized trial from the Swedish group, which compared no-touch SV patency to theradial artery, providing further evidence for the superiority of no-touch SVgrafts[11].This is important given the general consensus amongst cardiac surgeons that radialartery patency is superior and therefore the second conduit of choice. While theincidence of wound complications in patients receiving no-touch SV is higher than inthose receiving EVH grafts, they are similar to patients receiving OVH grafts and theirperformance is far superior, at ~90% vs. 50% long-term. Several studieshave reported SV graft failure rates of up to 10% to 20% after 1 year and an additional5% failure rate for each subsequent year with conventional OVH. When performing OVH, the recent 2014 ESC/EACTSGuidelines on myocardial revascularization support the use of the notouch technique toreduce graft injury and improve patency[6]. There is evidence that the improved patency ofno-touch SV over conventional OVH grafts is associated with a reduction in vasculardamage[15],maintaining normal vessel architecture, preservation of an intactendothelium and endotheliumderived NO[17], as well as preservationof the vasa vasorum and the SVs surrounding cushion of perivascularfat. The damage inflicted to the SV using both OVH and EVHaffects both short- and long-term graft performance. For example, reduced luminal nitricoxide availability, due to endothelial injury, leads to increased platelet aggregation,thrombus formation and early graft occlusion[12]. The results of the immunohistochemicalanalysis with CD34, iNOS and three isoforms of nitric oxide synthase in the human SVremoved conventionally showed an evident impairment of the endothelialfunction[22-24]. Damage to the outer layers of the SV, in particularthe adventitial vasa vasorum, reduces transmural blood flow, resultingin medial ischemia, conditions that have been shown to promote neointimal hyperplasiaand atherosclerosis, thereby affecting mid- and long-term graftpatency[19]. The cushion of perivascular fat that remains intactusing no-touch technique is an important source of adipocyte-derived vasodilatorfactors, suggested to play an important role in preventing venospasm at harvesting andpost implantation[20]. In addition, this pronounced outermost layer of fatprevents kinking of excessively long grafts and provides mechanical support thatprotects the vein once it is subjected to arterial hemodynamics at completion of distalanastomoses and removal of arterial clamps[18-21].At the time that EVH was described, an atraumatic, no-touch technique of harvesting theSV was introduced that, for up to 16 years, provides a superior graft with a patencyrate comparable to the internal thoracic artery (ITA)[3].EVH was recommended  to reduce wound-related complications, improvepatient satisfaction, and decrease postoperative pain, hospital length of stay, andoutpatient wound-management resources when compared with OVH[25]. While wound complicationsare reduced using EVH compared with OVH and no-touch-harvested SVs, graft performance ofEVH grafts may be inferior[25]. Early no-touch SV harvest site complications have beenreported to range from 11% to 18%[25], considerably higher when compared to EVH, althoughsimilar to conventional OVH[25]. Furthermore, the learning curve for EVH islonger[3],and although it has been suggested that EVH is a cost effective method for harvestingthe SV, the 2005 Consensus Statement of the International Society of Minimally InvasiveCardiothoracic Surgery (ISMICS) concluded there was inadequate cost-effectiveness datato allow recommendations on the resource implications of OVH vs. EVHtechniques[25]. Several recommendation statements and guidelines haveemphasized the importance of experience when performing EVH. Interestingly, although EVH may reducewound complications, the recent study by Te Kolste et al.[26] describes aspects of acutecompartment syndrome, a rare, but serious, limb-threatening condition that may occurafter CABG, especially following EVH. It is noteworthy that, over 10 years ago in theirexcellent review, Shuhaiber et al.[27] stated "In the operating room, tissue manipulationand the role of the surgeon or surgical assistant is quite essential. The no-touchtechnique of handling tissues during harvesting should be adopted in order to preservethe endothelial integrity and function."Considering the dramatic improvement reported for notouch SV grafts, it is surprisingthat this technique is limited to a few cardiac centers, mainly in Sweden and Braziland, at a rough estimate, amount to less than 1000 cases per year worldwide. The reasonsfor this low take up rate are unclear, but may be associated with senior surgeons'resistance to change and/or unwillingness to "retrain", rather surprising given therapid adoption of EVH to a present level of over 80% of all CABG in theUSAEVH is becoming popular in patients undergoing CABG in Brazil, a situation most likelyinfluenced by the low wound healing complications and improved cosmetic outcomeassociated with this technique, in addition to its favored use in the USA. Whenconsidering the extra cost and problems regarding EVH-SV graft performance as describedabove, should the patient be made aware of the potential benefits of no-touch SV grafts?Should the benefits of no-touch SV harvesting be discussed and should the patient begiven a choice?