Nearly 50 years ago geneticist James Neel famously proposed that “thrifty genes” were important contributors to the rising prevalence of diabetes . Such geThough intuitively appealing, the theory has often been challenged. Perhaps the strongest objection has been that there's little evidence our ancestors faced frequent, high-mortality famines that would have selected for thrifty genotypes . RecentlI believe the boom times' selection of genotypes prone to nutrition-cued accelerated development is having an especially problematic effect today because of widespread childhood overnutrition. Accelerated development, which enhanced reproductive success in the past, now has a pro-aging effect with rapidly growing costs. Indeed, when viewed through the lens of the antagonistic pleiotropy theory of aging , this efNeel's proposal that past famines reduced survival of individuals lacking thrifty genotypes has been invoked to explain, among other things, epidemic rates of type 2 diabetes that developed among various new-world populations after their adoption of western diets and lifestyles, such as the Central Pacific's Nauru Islanders ,9. HowevFixing this weak link in Neel's original argument, thrifty-gene proponents, notably Prentice and colleagues, have emphasized the high selective intensity engendered by differences in fertility . NutritiHomo ancestors are, in essence, “colonizing apes” whose global spread was likely aided by such a tendency , fertility and earlier menarche .  has a pro-growth effect and thus may play a central role in accelerating development and reproduction . When inCaenorhabditis elegans have shown that worms' lin-28 gene, homologous to LIN28B, controls the rate of progression through larval stages to adult cuticle formation , an important intracellular energy sensor and growth regulator, also helps regulate pubertal timing and reproductive function . InhibitOf special interest here is the fact that mTOR and AMPK are deeply implicated in aging. Studies in many species have shown that calorie restriction can extend lifespan, and this anti-aging effect appears to be critically dependent on suppression of the conserved TOR pathway, which integrates nutrition and growth-factor signals to regulate protein synthesis, cell growth and proliferation, autophagy, and other cellular functions ,65-67. RAging) that metformin treatment is more effective in prolonging life span in female mice when started early in life [AMPK helps regulate energy balance, insulin signaling, fatty-acid oxidation, and mitochondrial biogenesis ,71,72. A in life .These intersecting lines of evidence suggest that chronic activation of mTOR (concomitant with low AMPK activity) by early overnutrition plays a central role in eliciting the spendthrift phenotype. As Blagosklonny has observed, once a developmental program is switched on, it isn't necessarily switched off, even if its continuation is harmful later in life . ElaboraI believe that the spendthrift phenotype represents an exaggerated version of Blagosklonny's “quasi-programmed aging,” and that an important source of this exaggeration is the collision of modern lifestyles with genotypes that were favored during past periods of plenty. To slightly amend an analogy used by Blagosklonny, the spendthrift phenotype is like a car without brakes whose gas pedal has been floored since it was put on the road.My hypothesis has several testable implications. For instance, it suggests that early-menarche loci associated with high BMI are underrepresented in centenarians.The hypothesis also suggests that health risks associated with early overnutrition aren't simply consequences of obesity. As noted above, increased adiposity is one of the spendthrift phenotype's co-selected traits, and to some extent the obesity associated with the phenotype is secondary to, and potentiated by, activation of mTOR, IIS and other pro-growth pathways enlisted to accelerate growth, sexual maturation and reproduction. The pro-aging effect of chronically activating these pathways from an early age probably increases the age-associated risk of many adult diseases in tandem with overweight and obesity, including ones whose tie with adiposity has only recently become apparent, such as dementia . ConsistIf the hypothesis is correct, efforts to address childhood obesity would most effectively lower future health risks if they include focused efforts to interrupt early metabolic programming that elicits the spendthrift phenotype. For example, preventing rapid weight gain in infancy, as well as maternal overnutrition during pregnancy, may suppress the program during a critical window of time in early life, lessening its pro-aging effects more effectively than later interventions. Similarly, metformin's puberty-delaying effect suggests that it may be particularly effective at reducing the phenotype's adult health risks—its wider use in pediatric medicine probably makes sense. Better understanding of genes and pathways underlying the phenotype's pattern of fast development should suggest other drug targets that could yield highly effective preventive medicines.


During the end-Permian mass extinction, marine ecosystems suffered a major drop in diversity, which was maintained throughout the Early Triassic until delayed recovery during the Middle Triassic. This depressed diversity in the Early Triassic correlates with multiple major perturbations to the global carbon cycle, interpreted as either intrinsic ecosystem or external palaeoenvironmental effects. In contrast, the terrestrial record of extinction and recovery is less clear; the effects and magnitude of the end-Permian extinction on non-marine vertebrates are particularly controversial. We use specimen-level data from southern Africa and Russia to investigate the palaeodiversity dynamics of non-marine tetrapods across the Permo-Triassic boundary by analysing sample-standardized generic richness, evenness and relative abundance. In addition, we investigate the potential effects of sampling, geological and taxonomic biases on these data. Our analyses demonstrate that non-marine tetrapods were severely affected by the end-Permian mass extinction, and that these assemblages did not begin to recover until the Middle Triassic. These data are congruent with those from land plants and marine invertebrates. Furthermore, they are consistent with the idea that unstable low-diversity post-extinction ecosystems were subject to boom–bust cycles, reflected in multiple Early Triassic perturbations of the carbon cycle. ThAlthough well investigated in the marine fossil record, one of the major outstanding questions about the end-Permian mass extinction is how it affected terrestrial ecosystems. There is little doubt that a synchronous extinction event occurred on land , but howData are equally limited for non-marine vertebrates across the Permo-Triassic boundary. A number of studies have investigated the first and last appearances of individual lineages across the boundary –32, broaThe goal of our study is to elucidate the effect of the end-Permian extinction on terrestrial ecosystems, focusing on non-marine vertebrate faunas. By using specimen-level data, we ask what is the pattern of vertebrate richness, relative abundance and evenness across the Permo-Triassic boundary? Are these data consistent with an unstable ecosystem recorded by the Early Triassic chaotic carbon interval , a hypot2.(a)To investigate non-marine vertebrate palaeoecology across the Permo-Triassic boundary, we chose two published regional specimen-level datasets identified to genus. These data are ideal because they allow for easy sample-size standardization (i.e. by number of specimens), lessen problems with long-distance stratigraphic correlation and allow identification of any regional differences in patterns . Our main dataset comes from the Karoo Basin of southern Africa  and inclet al. [et al. [The temporal bins for our analyses were biostratigraphic zones identified by the original authors ,37. The et al.  for Russet al.  and Mund [et al. .(b)nalytic Rarefaction v. 1.3 [figure 3a; see the electronic supplementary material), but could not rarefy these data because of low sample sizes, and the fact that unequal relative abundance between clades might bias results. Evenness of assemblages (figure 3b) and accounted for sample-size differences by calculating confidence intervals using the method of Moore et al. [We investigated raw generic richness for each temporal bin  irrespective of sample size. These data were also rarefied using An v. 1.3  to standemblages  was calce et al.  (see theLystrosaurus Assemblage Zone (AZ) data using recent publications  and compared it with the original Dicynodon AZ data . This constitutes a conservative test, because recent taxonomic revisions and newly described taxa should increase the generic richness of both time bins [Lystrosaurus AZ) should bias our data against finding a major extinction signal. If, despite this bias, we find strong evidence for extinction, it will show that our data are robust to taxonomic biases. These revised Lystrosaurus AZ data were only used for this taxonomic bias test , and are not represented in figures To investigate possible taxonomic biases in our data, we revised the ime bins ,48. TherGeological and sampling biases of palaeoecological data are a pervasive concern. Sample-size differences across temporal bins were accounted for through rarefaction, but we also evaluated other potential biases within our data. The amount of outcrop area that fossils are collected from can influence observed diversity; the greater the expanse of outcrop, the more likely one is to find additional specimens and taxa. We fitted a linear regression to the total outcrop and productive outcrop area estimates of King  versus tδ13Ccarb record from shallow marine carbonates in South China, which spans the latest Permian through Middle Triassic [δ13Corg record from non-marine siliciclastic strata at Graphite Peak, Antarctica, which spans the latest Permian through early Middle Triassic (Anisian) [δ13Corg record from the Karoo Basin that crosses the Permo-Triassic boundary [To investigate how non-marine vertebrate palaeoecology across the Permo-Triassic interval relates to the global carbon cycle, we make comparisons with two main isotopic datasets: a Triassic ; and a δAnisian)  (figure boundary  (figure 3.Lystrosaurus and Cynognathus AZs) have much lower generic richness than the Late Permian Cistecephalus and Dicynodon AZs, even when accounting for sample size .Evenness of these vertebrate assemblages shows similar patterns. Both in southern Africa and Russia, evenness declines across the Permo-Triassic boundary, but recovers by the early Middle Triassic . In Russa), with the caveat that these values cannot be sample-standardized because of low total sample sizes. These data display clearly the extinction of gorgonopsian synapsids across the Permo-Triassic boundary. Dicynodonts show a major decline in richness across the boundary, whereas the decline in parareptiles and therocephalian synapsids is more gradual and extends into the Middle Triassic . Both temnospondyl amphibians and archosauromorphs display a distinct increase in generic richness across the Permo-Triassic boundary, and this increase continues into the Middle Triassic (figure 3a).Clade-specific generic richness for southern Africa also displays clear effects from the end-Permian mass extinction a, with tb). Dicynodont abundance (relative to other tetrapod clades) does not change, probably because the latest Permian Dicynodon AZ and earliest Triassic Lystrosaurus AZ are dominated by their eponymous dicynodont genera; however, Dicynodon AZ dicynodont evenness may actually be significantly higher based on an ongoing taxonomic revision of Dicynodon [Lystrosaurus AZ and Middle Triassic Cynognathus AZ. During this interval, dicynodont relative abundance decreases dramatically, from approximately 75 to approximately 20 per cent of specimens (b). In contrast, temnospondyl and cynodont abundance more than doubles; these two clades comprise more than 60 per cent of all specimens in the Middle Triassic Cynognathus AZ (b).The trends in relative abundance of individual clades for southern Africa are very different from those of richness. There are slight to moderate increases in the abundance of temnospondyls and parareptiles across the Permo-Triassic boundary, and similarly modest decreases in the relative abundance of gorgonopsians (which go extinct) and therocephalians, but all of these are either not statistically significant or only weakly so b. Dicynoicynodon . The majpecimens b. In conathus AZ b.4.(a)Palaeoecological metrics like those reported above can correlate with a number of geological biases and other non-biotic controls. Thus, it is extremely important to investigate possible non-biological signals in any dataset; these primarily consist of sampling and geological biases, along with errors and other limitations among the original specimen data.At the most basic level, sample size is a major control of diversity; greater sample size increases the potential to sample more taxa and clades. Conversely, low sample size can amplify stochastic effects, whereby a random draw is not representative of the total fossil assemblage; emerging methodological alternatives may help ameliorate this problem in the near future. For total generic richness, rarefaction minimizes unequal sample sizes across different temporal bins . The cona–c). In contrast, when the Dicynodon AZ is removed from the comparison between number of specimens and outcrop area, the relationship becomes highly significant . This is because the Dicynodon AZ is particularly fossiliferous for its available outcrop area, with over 2500 specimens in our dataset (R2 = 0.1962) between bin duration and richness in southern Africa, but this is contrary to our prediction that longer time bins should have higher richness, and there is no significant correlation in the Russian dataset . Both of these biases are ameliorated by our rarefaction of the data, which has a weakly inverse correlation, contrary to our prediction that they would be directly correlated .Geological outcrop area is one major bias of palaeoecological datasets, because more widespread outcrop affords the opportunity to discover more specimens and taxa. Comparison of the available outcrop area for each southern African temporal bin with raw and rarefied richness for each bin indicates that there is a very weak to no correlation, and a moderate correlation with number of specimens . The only major change in clade-specific richness is that parareptiles no longer show a major drop across the Permo-Triassic boundary, in agreement with recent discoveries [Our conservative test of revising the coveries ; these ccoveries .Our analyses are conducted at the genus level because specimen datasets identified to species are not currently available. This means that our analysis could differentially underestimate diversity because some genera are monospecific, whereas others include a great many species. At present, there is no way to correct for this, other than to note that many Permo-Triassic genera are monospecific, and those that contain many species are currently being revised to include one or few species . This lib), though richness is somewhat affected by outcrop area, and both rarefied richness and evenness are moderately affected by taxonomic issues. Raw total and clade-specific generic richness (figures 3a) are sensitive to nearly all of the potential biases described above; thus, we remain fairly conservative in our interpretation of the trends in these data.To summarize the inherent limitations of our data, we regard our analyses of rarefied generic richness, evenness and clade relative abundance to be the most robust to various biases figures  and 3b, 3 figures  and 3a aTropidostoma AZ in Africa and Malokinelskaya svita in Russia as the immediate after-effects of the end-Guadalupian extinction event b), in agreement with analyses of ecological guilds [figure 3a), though these data are extremely sensitive to the biases outlined above. In summary, we argue that patterns in richness, evenness and relative abundance from both southern Africa and Russia all point to a major end-Permian extinction event in non-marine tetrapod communities, which did not recover until 5–8 million years later, during the Middle Triassic. Furthermore, the composition of these communities was permanently altered, with the extinction of gorgonopsians, decline in therocephalians, dicynodonts and parareptiles, and rise of temnospondyls, cynodonts and archosauromorphs . The abundant Early Triassic procolophonid parareptile Procolophon in southern Africa may have also acted in a similar manner. The low-evenness Early Triassic Lystrosaurus AZ, dominated by Lystrosaurus and Procolophon, is consistent with other terrestrial disaster taxa such as the lycopsid macrofossil Pleuromeia  [Lunatisporites and Striatoabieites [Prior to full recovery, these post-extinction Early Triassic ecosystems were dominated by a few very abundant forms that are considered ‘disaster taxa’, which took advantage of widespread ecological vacancies. This is exemplified in southern Africa by the extremely common dicynodont al range ,61; the boundary b. The abrelates) –23,62 anabieites . Togetheabieites .Our analysis is particularly novel in investigating evenness, which provides some striking patterns when compared with sample-standardized richness. It appears that in both southern Africa and Russia, evenness recovered to near-pre-extinction levels well before richness did . This prIn addition to the occurrence of disaster taxa, richness and evenness patterns from marine invertebrates and non-marine plants also match well with our new data from non-marine vertebrates. Though global analyses of land plant diversity show only a weak to moderate drop in diversity across the Permo-Triassic boundary ,65, high(c)2 output from flood basalt eruption, and subsequent atmospheric CO2 consumption by weathering of these basalts [The low Early Triassic richness and delayed Middle Triassic recovery of marine and terrestrial ecosystems (including vertebrates) have been attributed to extrinsic effects such as volcanic activity and/or further greenhouse warming ,14, beca basalts . HoweverOur non-marine tetrapod data are fully consistent with the chaotic carbon hypothesis. The prevalence of disaster taxa, low richness and low evenness in the Early Triassic, along with the low diversity of producers (i.e. plants), would have made this unstable ecosystem susceptible to boom–bust cycles and further extinctions ,64. This


Eutherian mammals and saurischian dinosaurs both evolved lineages of huge terrestrial herbivores. Although significantly more saurischian dinosaurs were giants than eutherians, the long bones of both taxa scale similarly and suggest that locomotion was dynamically similar. However, articular cartilage is thin in eutherian mammals but thick in saurischian dinosaurs, differences that could have contributed to, or limited, how frequently gigantism evolved. Therefore, we tested the hypothesis that sub-articular bone, which supports the articular cartilage, changes shape in different ways between terrestrial mammals and dinosaurs with increasing size. Our sample consisted of giant mammal and reptile taxa  plus erect and non-erect outgroups with thin and thick articular cartilage. Our results show that eutherian mammal sub-articular shape becomes narrow with well-defined surface features as size increases. In contrast, this region in saurischian dinosaurs expands and remains gently convex with increasing size. Similar trends were observed in non-erect outgroup taxa , showing that the trends we report are posture-independent. These differences support our hypothesis that sub-articular shape scales differently between eutherian mammals and saurischian dinosaurs. Our results show that articular cartilage thickness and sub-articular shape are correlated. In mammals, joints become ever more congruent and thinner with increasing size, whereas archosaur joints remained both congruent and thick, especially in sauropods. We suggest that gigantism occurs less frequently in mammals, in part, because joints composed of thin articular cartilage can only become so congruent before stress cannot be effectively alleviated. In contrast, frequent gigantism in saurischian dinosaurs may be explained, in part, by joints with thick articular cartilage that can deform across large areas with increasing load. Paraceratherium, respectively Paraceratherium  of secondary centers of ossification in the chondroepiphysis. This difference has significant effects on the shape of the articular cartilage, its thickness, and how it is loaded. In eutherian mammals, the chondroepiphysis develops atop a well-organized, dense layer of sub-articular bone Regardless of the mechanism of how epiphyseal bone centers form, these differences in articular cartilage thickness and composition between archosaur and mammal long bones should result in distinct sub-articular shape scaling patterns between these clades. The combined region of calcified cartilage and bone underlying the articular cartilage is known as subchondral bone For our study, we chose to test for shape changes in the subchondral surfaces in the humerus and femur because these elements are the most often compared bones in dinosaur and mammal studies. Moreover, these two elements both provide a majority of the mechanical support to their associated limbs, and the largest muscles that control locomotor movements of the entire limb insert or originate on these bones ShantungosaurusOrycteropus because, despite its burrowing habits, it is a terrestrial forager. The highly-derived limb morphologies of ungulates  have been shown to scale in unique ways that often effect the interpretation of allometric scaling patterns across larger mammal samples e.g., We examined 232 humeri and 224 femora across a range of archosaurian and mammalian taxa  and S2. Hyrax and Orycteropus comprised successive outgroup taxa to the giant proboscideans, whereas Tapirus and felid taxa formed successive Laurasiatherian outgroups for the ceratotherians. For Sauropoda, the sauropodomorphs Plateosaurus and Massospondylus, as well as the non-avian theropod Allosaurus and the avian theropod Numida were selected as successively distant outgroups. Numida meleagris was selected because of its predominantly terrestrial habits and because we had access to numerous wild  individuals from a previous morphometric analysis Ornithorhynchus and Tachyglossus for eutherian mammals, Alligator mississippiensis for saurischian dinosaurs). We chose these taxa specifically to test if posture influenced subchondral shape scaling in much the same way it does for overall long bone dimensions. In other words, by including taxa with homologous epiphyseal joints but non-parasagittal postures, we could test how much subchondral shape scaling was influenced by long bone orientation. It should be added that as our goal was to track subchondral bone shape scaling associated with gigantism in eutherian mammals and non-avian saurischian dinosaurs, our outgroup taxa were not selected to be an exhaustive phylogenetic sample of all possible variants. In both samples, the outgroup taxa served primarily as smaller-bodied specimens that helped us constrain and root our analysis of subchondral scaling trends in mammalian and dinosaurian giants in a broader context.To place our samples within a broad evolutionary and postural context, we examined additional outgroups within and outside our “giant” lineages. For clarity, we are using the term “outgroup” here to mean taxa successively distant from the giant taxa, not in a strict phylogenetic sense. For Afrotheria, Permission was obtained to examine specimens at the following institutions: AMNH, American Museum of Natural History, USA; BP, Bernard Price Institute for Palaeontological Research, University of Witwatersrand, Johannesburg, South Africa; BYU, Brigham Young University, USA; CM, Carnegie Museum of Natural History, USA; DNM, Dinosaur National Monument, USA; FMNH, Field Museum of Natural History, USA; MWC, Museum of Western Colorado, USA; OMNH, Oklahoma Museum of Natural History, USA; SAM, Iziko South African Museum of Natural History, Cape Town, South Africa; SMNS, Stuttgart Museum of Natural History, Germany; UMNH, Utah Museum of Natural History, USA; USNM, United States Museum of Natural History (Smithsonian), USA; WIU, Western Illinois University, USA; YPM, Yale Peabody Museum, USA. See Length measurements were made with digital calipers, tree calipers, or metric tape to the nearest millimeter. The measurements were subsequently log10 transformed to normalize their distribution Deinotherium and the titanosaur sauropod taxa , is often used in geometric morphometrics because it represents a pure geometric scaling variable with mathematical independence from shape Next, regression of partial warps onto log-transformed humerus and femur length was used to determine how subchondral bone shape scaled with size. Multiple regression analyses of the partial warps on log10-transformed humerus and femur length were used to determine how well the data fit a linear model, generating multiple rp<.05 based on Goodall’s F-test) in partial warp scores from the predicted mean indicates humerus or femur subchondral allometry (significant shape change). Goodall’s F-test, a statistic designed specifically for geometric morphometric regression 2) which, following Monteiro 2 value, the greater influence size has on shape. A multivariate regression analysis using Wilk’s λ was used to determine if the coefficient of determination and deformation grids reflected a significant allometric trend. This statistical analysis was run in the TPSRegr program.Multivariate regressions of the partial warps on log10-transformed humerus and femur length were used to test for allometry and to produce thin-plate splines deformations. Unlike linear regression, geometric morphometric (shape) allometry is not reported as a slope value. Instead, against a null model of isometry , a significant, multivariate difference  remain informative and indicative of differences in its adaptive role in parlaying dynamic locomotor stresses absorbed through the overlying chondroepiphysis.Why did we use a two-dimensional analysis instead of a three-dimensional analysis? Undoubtedly, the capture and analysis of three-dimensional shape would further enhance our analysis and assist us in interpreting patterns of subchondral bone shape. However, a number of challenges prevented such an approach. First and most significantly, the data collected in this study span a period of over 10 years during which time cost-effective and portable three-dimensional scanning technologies for acquiring large bone geometries have only recently started to become available. Had we access to these technologies ten years prior, we would have utilized them, as we plan to utilize such approaches in future studies. Second, our main goal in this study was to quantify whether or not there were significant differences in the scaling patterns of surface morphology between eutherian mammal and saurischian dinosaur long bones, and whether such differences were correlated with known differences in articular cartilage properties. Again, our goal was not to realistically recreate joint surfaces or establish precise measures of joint articulation, nor do we propose how the three-dimensional shape of the subchondral bone is used to reconstruct joint geometry. Our selection of the humerus and femur furthers our goal: these are long bones in which a significant portion of the subarticular surfaces can be reliably captured and interpreted in two dimensions Paraceratherium) have the most robust humeri among the eutherian taxa, whereas proboscideans and Paraceratherium have narrower, more gracile humeri. Perhaps not surprisingly, all felids plot negatively along PRIN 1, reflecting their relatively gracile humeral morphology. Curiously, among all the eutherian mammals in the sample, whereas significant differences in shape exist, variation in humerus morphology is relatively limited. This is reflected in the KW test for posture that showed a significant difference between monotremes (non-erect) and the eutherians (erect) in the sample  and distal ends  as well as at their midshaft . Conversely, specimens plotting negatively along the PRIN 1 axis show distinct narrowing proximally , distally , and at midshaft  . A MANOVhis axis . The femf PRIN 1 . Moreovef PRIN 1 . As withf PRIN 1 , but agaRegression of the partial warps onto log10 transformed femur length indicates that size and shape are well-correlated , but thaAlligator humerus sample (archosaur sample hereafter) shows a pattern different from that reported for the mammals. As with the mammal sample, the PRIN 1 axis describes expansion and robustness: specimens that plot positively along this axis show expanded proximal  and distal  ends as well as a lengthening and more medially oriented deltopectoral crest   . A MANOV2, 3, 4) , and sublligator . Confirmlligator . Howeverlear why ; Table 3Regression of the partial warps on log10 transformed humerus length shows that size and shape are indeed well-correlated, with size-related shape changes accounting for nearly 30% of the shape variation ! SurprisAlligator, theropods, and basal sauropodomorphs  and distal (landmarks 4 and 5) expansion . Subsequdomorphs . This shdomorphs .The regression of the partial warps onto log10 transformed femur length again shows that size and shape are well-correlated, and that over 23% of the shape variation in the sample is correlated with size . As withOur results show that despite differences in posture and phylogeny, there is a clear and significant signal in the data related to subchondral shape scaling. For our mammal sample, whereas many variations in shape occur among the different clades sampled and between non-erect and parasagittal postures, only minor changes to subchondral shape occur with increasing size  and 4. TCould the use of bipedal taxa in the saurischian sample  affect the scaling patterns we report, especially for the humerus? Whereas as this is certainly possible, our results for these archosaurs show no particular deviation among bipeds or quadrupeds with increasing size see . MoreoveOur results suggest that subchondral shape scaling patterns reflect relative articular cartilage thickness. For mammals articular cartilage remains relatively thin across all size ranges (a few millimeters in thickness), even for elephants Numdia), no significant difference between subchondral bone shape and articular cartilage shape were reported in larger individuals and adults in vivo orientations. Thus, thinner articular cartilage would appear to be associated with a subchondral bone shape displaying well-developed surfaces, condyles, and convexity. In contrast, as shown by Bonnan et al. How do these data support our suggestion that subchondral shape scaling patterns reflect articular cartilage thickness? Bonnan and colleagues Paraceratherium, maximum known femur lengths of approximately 1.7 m and 1.2 m, respectively The inescapable conclusion from our data is that saurischian dinosaurs frequently achieved gigantism while retaining joints comprised of relatively thick articular cartilage, a claim that bolsters recent work and similar suggestions by Holliday and colleagues It is well-established that bones change their shapes to best resist dynamic loads It has been demonstrated that when articular cartilage is thin, stress is best dissipated by forming more and more congruent joints The picture that emerges for archosaurs and saurischian dinosaurs in particular is a bit more complex. Certainly, although articular cartilage was significantly thicker than in mammals, there is no doubt that the articular surfaces themselves formed congruent joints. In fact, that the thick, cartilaginous articular cartilages articulated in congruent ways has been well-documented What would be the advantage of retaining thick articular cartilage at large size? There are certainly disadvantages arising from thick articular cartilage, including the increased likelihood of “ploughing” and shearing We speculate that these differences in articular cartilage thickness are related to aspects of joint deceleration when the limb is planted on the ground during locomotion. In mammals, highly congruent joints with thin articular cartilage ensure that the bones involved are already in close contact, diminishing the force of deceleration . In contOur results also highlight an intriguing postural signal that differs between eutherian mammals and saurischian dinosaurs. The deltopectoral crest of the humerus serves as a region of insertion for the deltoid and pectoral musculature Overall, our results indicate that the subchondral shapes of the humerus and femur scale differently between eutherian mammals and saurischian dinosaurs. These differences in subchondral shape are inferred to be correlated with cartilage thickness and its ability to transfer stress to the subchondral bone. For mammals, the narrowing of the subchondral regions and the development of distinct surfaces with increasing size appear to be correlated with thin articular cartilage and congruent joints. As archosaur size increases, subchondral surfaces expand and become gently convex, trends which strongly suggest the retention of thick articular cartilage. Scaling of subchondral shapes in non-erect taxa with thin (monotreme) and thick  articular cartilages support these associations. Articular cartilage deformation, or lack thereof, may have contributed in significant ways to the attainment and frequency of gigantism in saurischian dinosaurs and eutherian mammals.Dermochelys coriacea) is heavily invested with cartilage canals and a blood supply Many questions remain, chief among them being what factors would allow the articular cartilage of the largest terrestrial vertebrates to remain viable but pliable. Thick articular cartilage in other reptiles such as leatherback sea turtles Click here for additional data file.Table S2Archosaur specimens utilized in the study.(DOCX)Click here for additional data file.


This approach circumvents the additional time and cost of generating HLA-specific data and capitalizes on the increasing accessibility and affordability of massively parallel sequencing.The human leukocyte antigen (HLA) is key to many aspects of human physiology and medicine. All current sequence-based HLA typing methodologies are targeted approaches requiring the amplification of specific HLA gene segments. Whole genome, exome and transcriptome shotgun sequencing can generate prodigious data but due to the complexity of HLA loci these data have not been immediately informative regarding HLA genotype. We describe HLAminer, a computational method for identifying HLA alleles directly from shotgun sequence datasets ( The widespread uptake of large-scale genome, exome and transcriptome shotgun sequencing approaches for biomedical research, and now for clinical use, prompted us to explore the utility of these types of NGS data sets for HLA typing. The need has been for a solution to the problem of managing the many millions of short sequence reads NGS technologies produce, managing the many thousands of reference allele sequences, and integrating all of these data in a manner that maximally informs HLA content. Here we present a method for HLA allele prediction from next-generation shotgun sequence datasets. We focus on data generated from the Illumina platform, from which most sequence data are currently derived worldwide. Importantly, HLA allele assignments from shotgun datasets can not be derived from standard alignment-based interpretive methods for the simple reason that the extant genome reference sequences [Due to its central role in adaptive immunity, human leukocyte antigen (HLA) is implicated in wide ranging areas of medicine, from infectious disease and vaccinology to cancer, autoimmunity, aging and regenerative and transplantation medicine -7. The Hequences ,11 on whequences -15.Written informed consent was obtained from all donors and samples were collected following assessment of tissue specimens by a pathologist according to standardized operating procedures, immediately following surgical resection. Library construction and Illumina sequencing were performed as previously described for RNA-Seq  and wholExome capture libraries were prepared using the SureSelect system (Agilent) according to the manufacturer's instructions. Approximately 30 million  and 120 million  100-nucleotide exon capture paired-end sequence reads were generated from three ovarian cancer patients whose HLA alleles were verified by PCR-based methods. Verification of HLA allele predictions was accomplished by PCR amplification of exons 2 and 3 from HLA-I A, B and C, followed by capillary sequencing as previously described [HLA coding DNA sequence (CDS) and genomic sequence databases from release 3.3.0 and 3.4.0 were obtained, respectively, from . HLA-I eex-equo best hits and tracking HLA sequences that, reciprocally, best identify each contig. For each putative HLA, a score HLA Sis calculated as the sum of score computed for each contig aligning to it. Individual contig scores factor in the contig depth of coverage, length and percent sequence identity, such that a score reflects the number of bases aligned to a particular HLA allele. A reciprocal best hit where a given HLA aligns best to a given contig doubles the score for the identified HLA sequence:HLA CDS or genomic sequences from IMGT/HLA (sequence targets) are read by TASR (default options used with -i 1), creating a hash table of every possible 15-nucleotide word (k-mers) encountered. NGS data sets are interrogated for the presence of one of these k-mers in 5' (on either strand) and candidate reads recruited. Recruited reads seed the assembly in a manner analogous to that of SSAKE . Only seP that a contig characterizes one or another HLA type is mutually exclusive such that:For any given contig, the probability of characterizing a single HLA allele by chance is equal to the inverse proportion of HLA sequences in the sequence database. And since shorter contigs may not capture sufficient bases to characterize any one type unambiguously, the probability x, EvalxHLA, is calculated as:The expect value  of each computationally determined HLAsince individual contig probabilities and reciprocal best hits are independent events. A short list of HLA allele groups  and protein coding alleles , sorted by decreasing score, are catalogued for each major HLA gene. When separate contigs characterize the same types, only the types that overlap are reported, unless the non-overlapping ones are characterized by additional, distinct contig(s). In addition, we summarize ambiguous HLA alleles using the P designation, when applicable.In separate experiments, we removed HLA CDS, exonic regions and genes from 15K randomly selected Ensembl  transcriWe define the sensitivity as a proportion, that is, the number of HLA allele groups or protein coding alleles detected over the sum of distinct groups or protein coding alleles randomly chosen for the simulation or predicted by PCR, when applicable. The ambiguity rate is the proportion of all ambiguous predictions per total allele groups or protein coding alleles predicted. Ambiguous predictions arise when HLA allele groups or protein coding gene names differ despite having an identical score and probability. The specificity is defined as a proportion of number of groups or alleles predicted accurately divided by the total number of groups or alleles detected, respectively.HLA class I alleles were predicted directly from the RNA-Seq data as described . Brieflyde novo assembly of sequence reads prior to alignment  and substituting HLA-I A, B and C sequences with two randomly chosen alleles of HLA-I A, B and C. From these modified references we generated faux sequence reads. For each sequence format, 20 such data sets were generated and these were queried in triplicate, yielding a total of 360 allele predictions per condition tested. The sensitivity, specificity and ambiguity of HLA class I allele prediction was evaluated by comparing the highest-scoring HLAminer predictions to the randomly selected alleles. By ambiguity we mean the prediction of multiple, equally probably alleles.HLA nomenclature  defines the digits immediately following the asterisk as the allele group  and the next set of digits  as the individual protein coding allele . FurtherOverall, from simulation, RNA-Seq datasets provided the greatest utility for HLA prediction. This may be due, in part, to lower representation in RNA-Seq data of off-target regions, such as the minor HLA class I genes, pseudogenes, and HLA class II genes, compared to genome or exome data, where these regions would be expected to have approximately equal representation as the class I alleles of interest, A, B and C. The stark contrast in HLAminer predictions derived from RNA-Seq compared to WGS or exome capture highlights intrinsic properties of these datasets and their value for computational HLA predictions. Functional HLA-I alleles are expressed on all nucleated cells, and despite possible amplification biases in the RNA-Seq library construction protocol, the high abundance of HLA-I transcripts is such that relatively low depth of sequencing is needed for robust predictions (approximately 5 million). On the other hand, non-functional (null) HLA alleles that are present in the genome  can confound HLA prediction from WGS or exome capture data, since the functional alleles and null alleles are equally represented in these data types. HLAminer has the functionality to report predictions from null alleles, if desired.We explored further the effects of read length (up to 150 nucleotides) and sequencing errors (up to 3%) with RNA-Seq data. Not unexpectedly, performance improved with increasing read length and decreasing base error Table . Reads wNext, we evaluated the performance of HLAminer with real shotgun datasets, including RNA-Seq data from 16 CRC libraries , WGS and RNA-Seq from four lymphoma libraries  and exonHLAminer can evaluate reads by direct alignment Figure , right. Regardless of input data, HLAminer predictions for HLA allele groups (two-digit resolution) are more robust than for HLA protein-coding alleles (four-digit resolution) (Figure HLAminer is the implementation of a strategy for automated HLA typing directly from NGS data sets. It is a fundamentally different approach compared to conventional methods that all rely on first amplifying HLA genes. The identification of allelic variants from an individual NGS data set by simple sequence search or alignment is precluded by the complexity of the locus, the massive allelic diversity in the population and limitations of short sequence reads to adequately capture these variations. The option of typing, retrospectively, existing cohorts for which NGS data have already been generated is enabling, particularly for large community resource projects -15. In tIt is recognized that certain HLA allele combinations are common in certain populations, presumably due to linkage disequilibrium . For exaCDS: coding DNA sequence; CRC: colorectal cancer; HLA: human leukocyte antigen; NGS: next-generation sequencing; PCR: polymerase chain reaction; WGS: whole genome shotgun.The authors declare that they have no competing interests.RLW and RAH designed the research; DJF extracted RNA and constructed the sequencing libraries; SM and GC verified predicted HLA calls by conventional sequencing; MC provided ovarian cancer exon capture sequence datasets; RM coordinated the sequencing; RLW and RAH analyzed the data and made the figures; RLW and RAH wrote the manuscript. All authors read and approved the final manuscript.


Until now, insomnia has not been much of interest in epidemiological neighbourhood studies, although literature provides evidence enough for insomnia-related mechanisms being potentially dependent on neighbourhood contexts. Besides, studies have shown differences in sleep along individual social characteristics that might render residents more vulnerable to neighbourhood contextual exposures. Given the role of exposure duration and changes in the relationship between neighbourhoods and health, we studied associations of neighbourhood unemployment and months under residential turnover with insomnia by covering ten years of residential history of nearly 3,000 urban residents in the Ruhr Area, Germany.Individual data were retrieved from the Heinz Nixdorf Recall Study, a population-based study of randomly chosen participants from adjacent cities, which contains self-rated insomnia symptoms and individual social characteristics. Participants’ residential addresses were retrospectively assessed using public registries. We built individually derived exposure measures informing about mean neighbourhood unemployment rates and months under high residential turnover. These measures were major predictors in multivariate logistic regressions modelling the association between social neighbourhood characteristics and insomnia in the whole sample and subgroups defined by low income, low education, social isolation, and change of residence. Traffic-related noise, age, gender, economic activity, and education were considered as covariates.Nearly 12 per cent of the participants complained about insomnia. Associations of neighbourhood unemployment with insomnia were more consistent than those of residential turnover in the whole sample . In low-income and socially isolated participants, neighbourhood unemployment odds of reporting insomnia were particularly elevated . Less educated participants displayed relatively high odds of reporting insomnia throughout all upper neighbourhood unemployment exposure categories. Change of residence weakened associations, whereas undisrupted exposure sharpened them by trend.Our findings hint at multiple stressors being effective in both the neighbourhood context and individual resident, possibly reflecting precarious life situations undermining residents’ sleep and health chances. Moreover, our results suggest a temporal dependency in the association between neighbourhood and insomnia. Urban health research in social epidemiology suggests that social neighbourhood characteristics are involved in urban residents’ developing chronic diseases. A growing body of evidence supports causal pathways associating contextual features with a variety of outcomes such as cardiovascular and -metabolic diseases, depression or unhealthy lifestyles. In spitFurther exploration of this subject is desirable, since poor sleep affects major physiological and psychological functions, particularly in the elderly-9. InsomWe conceive macro-social inequalities to be the cause of socio-spatially unequal quantities and qualities of contextual stressors and resources, and population compositions in the urban realm-14. In tneighbourhood unemployment and residential turnover. Despite the sleep-related knowledge gap in the field of neighbourhood research, literature provides us with sufficient evidence to examine associations between these two neighbourhood exposures and the presence of insomnia.In this explorative work, we studied two neighbourhood contextual characteristics reflecting an array of residential psychosocial and physical stressors which might trigger adverse behavioural, psychological and physiological changes and thereby induce insomnia: Neighbourhood unemployment represents neighbourhood or area socioeconomic status[c status and is sc status,6,18,19.Residential turnover in urban neighbourhoods – as opposed to residential stability[tability – might tability. The dyntability. Recent tability,24. Thistability,25. PoteSummarising, neighbourhood unemployment and residential turnover might precondition a vast range of intermediate sleep-related stressors in urban residential contexts. For this reason, the study focus on these two neighbourhood exposures might serve as a suitable starting point for an epidemiological exploration into the development of chronic ‘neighbourhood insomnia’.Further clarification is also needed when it comes to individual social characteristics influencing residents’ capabilities to deal with stressors inscribed in social neighbourhood characteristics. If individual social characteristics do not adequately buffer neighbourhood stressors, psychosocial stress might become chronic because of the inevitably chronic nature of residential neighbourhood exposures. Vulnerabilities for sleep complaints may occur in socio-economically disadvantaged individuals in terms of education and income, for example-29: As aIn these theoretical considerations, time is an inherent factor in the interplay of social-structural, intermediate and proximal stressors and resources across life-courses, since it takes time for social networks and ties to develop, social engagements and practices in urban neighbourhood collectives to grow, access to resources to be worked out, contextual stressors to be effective, and finally, psychological and physiological pathways to insomnia and health to evolve. From this time-related perspective, individual changes of residence may interfere with these complex mechanisms laid out here: If changes of residence disrupt neighbourhood relationships as well as the resources and stressors conveyed by them, residential ‘moves’ might be used to avoid contextual stressors in the current neighbourhood. Conversely, residents stuck in their place have longer durations of exposure to negative (or positive) neighbourhood stressors. Besides, they might be even more affected by their neighbourhoods, as neighbourhood unemployment and residential turnover could be particularly burdensome if there is no realistic escape from them. Up to now, insomnia studies concerned with social neighbourhood characteristics rarely consider exposure duration and change of residence during the observation period in their exposure assessments. Figure Against this background, we specify our research questions focussing on neighbourhood unemployment and residential turnover as major exposures as follows:1. Are residents with chronic exposure to higher levels of neighbourhood unemployment and residential turnover more likely to report insomnia compared to those with lower exposure levels?2. Are residents with relatively low income, low education, and social isolation especially vulnerable to higher levels of chronic neighbourhood exposures?3. Does individual change of residence influence the associations between neighbourhood exposures and insomnia?Individual data are derived from the Heinz Nixdorf Recall (HNR) Study, a population-based longitudinal study of 4814 randomly chosen participants (45–75 years of age) from three adjacent cities in the German Ruhr Area, Mülheim, Essen and Bochum,32. BeloThis study relies on individual level data gathered at the baseline medical examination of the HNR study (2000–2003). After giving written consent in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS) 1993, participants were interviewed face-to-face by trained personnel using computer-assisted questionnaires. Moreover, a sound medical examination including anamnestic interviews, blood samples, ECG, blood pressure measurement and many other examinations was conducted. The study was approved by the institutional review boards of the Ethical Commission of the Medical Faculty of the University of Duisburg-Essen and a quality management according to standard industrial norms (DIN EN ISO 9001: 2000) was implemented by TÜV Med Rheinland. Recruitment and potential sources of bias have been already evaluated and published elsewhere.In order to build chronic exposure measures, participants’ residential histories were assessed retrospectively up to ten years before the baseline examination date using public registries administrated by the municipal authorities. Any changes of residence of people moving into, within or out of the respective city area are recorded in these registries. As no residential history data could be acquired for the city of Bochum, the sample had to be restricted to participants from the two neighbouring cities of Mülheim and Essen (N = 3033). We excluded participants with an incomplete history of residence to ensure a comparable exposure duration during the longitudinal observation period for all subjects . Incompleteness of residential history mainly resulted from participants who had moved into the study cities after the beginning of the observation period or, vice versa, left the region during the period (N = 160). Except for a slightly younger age, a higher unemployment rate, and, naturally, more changes of residence, the population with incomplete residential data did not differ from the main sample.In sleep epidemiology, there is still no universally applied standard definition for insomnia. In thisThe addresses of residency during the observation period were linked to a predefined set of neighbourhood characteristics in order to estimate the individual exposure to area-based contextual stressors. Thus, addresses were not directly linked to the individual dataset to ensure data security. Neighbourhood data were retrieved from the municipal departments in charge of statistics and urban research in Mülheim and Essen and refers to 78 statistical units with an average of 11,747 residents. In part, these statistical units reflect grown structures and histories of former self-administered places. Other units have been created and reshaped for political reasons, aiming to make municipal administration and distribution of public means more efficient.Two indicators were applied for the present analyses whose data availability and operationalisation should be generally accessible and replicable for standard small-scale area monitoring.First, considering neighbourhood unemployment as a chronic conditional frame for the evolution of sleep-related residential stressors . Data from 1990 was assigned to each month until December 1994, of 1995 until December 1999 and of 2000 until the last participants’ examination date in 2003 . Aside from socioeconomic disadvantages, social isolation as a third individual social characteristic was determined. In previous analyses on social inequality, social networks and social support in the HNR study population, the socAll three individual social characteristics were weakly, but significantly correlated.non-movers and those who had changed address at least once as movers. In comparing the estimates for these two contrasting groups, the impact of potential exposure discontinuities on the hypothesised neighbourhood associations should be figured out. For reasons of data security, we were confined to creating ‘exposure sum’ measures, only (see neighbourhood exposure measurement above). Therefore, it was not possible to rate whether a move was related to a significant change in neighbourhood exposures.Furthermore, analyses were stratified by change of residence. Participants who had been living at the same address for the whole observation period were classified as As background variables, we considered determinants which might not only have fostered the development of insomnia symptoms , which exceeds the average night-time level of 40 dB (A) recommended by the Night Noise Guidelines of the World Health Organisation for Europe[We included years living under high exposure to traffic-related noise during the night as a physical stressor. Noise data is based on modelling according to the EU-Directive 2002/49/EG for the year 2006, mapped as isophone areas assigned to the participants’ addresses. We defined a relatively high exposure by a cut-off point of Lr Europe. Above tNext, we considered individual social characteristics, i.e. age (continuous), gender, and current economic activity . Following from age-related sleep changes and susceptibilities to sleep-disrupting chronic diseases, age is an important background characteristic for insomnia studies. Also, women are more prone to impaired sleep quality and insomnia than men. Economic activity structures daily activity patterns, sleep-wake-rhythms, time under neighbourhood exposure, and regular access to social resources other than those available in the neighbourhood. In the analyses for the whole sample, non-movers, and movers, we also adjusted for education as described above. In the low-income and socially isolated groups, we did not additionally account for education because the highly educated reference group (≥18 years) was completely missing among the insomniacs in both these subgroups.First, distributions of individual social characteristics, changes of residence, and social neighbourhood characteristics were compared between insomniacs and non-insomniacs for the whole sample and for the defined subgroups. Relationships between neighbourhood unemployment and residential turnover were explored both at the city and individual level by non-parametric correlations.Apart from descriptive statistics, block-wise multivariate logistic regression analyses with insomnia as the dependent variable were conducted. Neighbourhood unemployment and residential turnover acted as major independent variables in separate regression models adjusted for relevant covariates. Since neighbourhood characteristics were operationalised as individual level means and months of exposure at the home address , spatial clustering was not accounted for. However, multi-level modelling was applied for the subgroup of non-movers, yielding results no different from one-level logistic regression models. This is why main analyses were based on one-level multivariate logistic regressions, so that comparability of models between all the subgroups including movers was ensured.In subgroup analyses, we employed the same statistical procedures in order to recognise modification of associations between social neighbourhood characteristics and insomnia by individual social characteristics.Further, stratification was performed in order to compare the strength of associations between the neighbourhood contextual characteristics and insomnia in participants without and with change of residence which were hypothesised to increase or decrease vulnerability.The main analyses were conducted in SPSS, versions 17 and 19, the multi-level modelling was done in Stata, version 11.2.Nearly 12 per cent of the participants reported having insomnia at least two times a week . Observation numbers were too small to yield reliable results, though.Based on an approach of multiple stressors, we investigated if social neighbourhood characteristics such as unemployment and residential turnover were associated with insomnia and whether these associations were modified by individual social characteristics and change of residence.In the whole sample, we observed a statistically significant association between neighbourhood unemployment and insomnia. If neighbourhood unemployment encompasses multiple contextual stressors, this finding could hint at precarious situations in regard to psychosocial and other urban stressors influencing residents’ sleep quality in deprived neighbourhoods. Our study region is the Ruhr Area, a formerly highly industrialised area which has experienced massive deindustrialisation during the past decades. Long-term unemployment, financial strain and associated disorder and stigmatisation certainly affect both individuals and neighbourhood communities. Sleep is termed an adaptive behaviour,6 and associal isolation hypothesis adopted by others[Despite its weakness, the association between months lived under high residential turnover and insomnia might still indicate the existence of neighbourhood influences which result in negative emotional states detrimental to sleep , personal habits  and personal characteristics such as hearing loss. However, the tendency of insomniacs having endured noise for a longer cumulative time might still point to noise effects adding up to psychosocial stress associated with high neighbourhood unemployment and residential turnover.Even if we cannot infer Ruhr Area-specific combinations of stressors from our crude contextual data basis and even if we cannot uncover specific biopsychosocial mechanisms from our analyses, it is plausible to partially ascribe insomnia as well as sleep-adverse behaviour and psychosomatic factors to neighbourhood-related living conditions.Our approach of multiply effective stressors was further confirmed by our subgroup analyses, with each individual social characteristic suggesting a dimension of potential ‘stress’ on its own. We may assume that maintaining a high sleep quality while being exposed to an adverse urban neighbourhood context could be hindered by disadvantages in income, educational attainment and social isolation. Urban societies are shaped by market exchange and price regulations, regimes of civil rights and redistributional welfare, reciprocally social resources in social networks as well as their interdependencies. If individual social characteristics constitute a vulnerable position in relation to these realms, they consequently account for locally unequal exposures to stressors and resources, health capabilities, including psychosocial constructs like self-efficacy, attitudes towards health and collective lifestyles, and health outcomes . By contrast, we could not detect any effects of neighbourhood contextual characteristics on insomnia in movers. First of all, this finding supports our hypothesis of time shaping the relationship between neighbourhood and health, i.e. that an enduring exposure is more likely to negatively impact sleep quality. Moreover, in participants familiar with changing their socio-spatial networks, relations and resources as a consequence of their own residential mobility, the neighbourhood context might be of less importance for well-being and health. What is more, this finding presses us to explore reasons, patterns and effects of change of residence in the future. In order to assess the ‘harmfulness’ of social segregation for residents’ social exclusion, the capabilities of the ‘moving’ households are for urban planners and sociologists decisive to know. In view of the relatively high percentage of employed movers .Alongside the need to consider life-course trajectories, epidemiology has become interested in residential history approaches and modelling cumulative exposures,56. We tModelling of long term neighbourhood exposures has become rather sophisticated, using multiple-year measures, indicesFrom 2000 onwards, German cities have been growing more and more fragmented, but public socio-spatial monitoring has been much improved, partly as a reaction to this development of social segregation. Prospectively, downward spirals wearing away neighbourhood resources may become more and more traceable as selective population dynamics are developing at a quicker pace. For this reason, our study continues to assess neighbourhood contextual exposures during follow-up, creating a longitudinal design at both individual and neighbourhood level, testing more precise neighbourhood exposure accumulation measures, and exploring the relationship between neighbourhood unemployment and residential turnover in an enlarged database. In this way, we might be able to identify critical neighbourhood windows in the ongoing process of aging, because growing economic and physical inactivity in daily life are thought to enhance susceptibilities.Our findings hint at multiple stressors being effective in both the neighbourhood context and individual resident, possibly reflecting precarious life situations undermining residents’ sleep and health chances. Moreover, our results suggest a temporal dependency in the association between neighbourhood and insomnia.as well as subgroups defined by specific individual social characteristics throughout the city and in specific neighbourhoods , thoughThe authors declare that they have no competing interests.NR was responsible for the statistical analyses and drafting and writing the manuscript. ND and JSch supervised the statistical analyses and helped to interpret the results as well as to revise the manuscript. ND and BH coordinated neighbourhood contextual and noise data acquisition, NR, KF, and AV prepared and manipulated these data and designed contextual exposure measures. ND and BH were head of the residential history approach. ND and SW participated in conducting the HNR study. ND, JSch, BH, AS, and SW critically reviewed the manuscript. RE and JS are principal investigators of the HNR study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/810/prepub


General transcription is required for the growth and survival of all living cells. However, tumor cells require extraordinary levels of transcription, including the transcription of ribosomal RNA genes by RNA polymerase I (RNPI) and mRNA by RNA polymerase II (RNPII). In fact, cancer cells have mutations that directly enhance transcription and are frequently required for cancer transformation. For example, the recent discovery that MYC enhances the transcription of the majority genes in the genome correlates with the fact that several transcription interfering drugs preferentially kill cancer cells. In recent years, advances in the mechanistic studies of the basal transcription machinery and the discovery of drugs that interfere with multiple components of transcription are being used to combat cancer. For example, drugs such as triptolide that targets the general transcription factors TFIIH and JQ1 to inhibit BRD4 are administered to target the high proliferative rate of cancer cells. Given the importance of finding new strategies to preferentially sensitize tumor cells, this review primarily focuses on several transcription inhibitory drugs to demonstrate that the basal transcription machinery constitutes a potential target for the design of novel cancer drugs. We highlight the drugs’ mechanisms for interfering with tumor cell survival, their importance in cancer treatment and the challenges of clinical application. Cells require transcription for basic processes, such as survival, cell growth and differentiation. Transformation highly correlates with enhanced transcription of oncogenes and other transcription factors in cancer cells . For manInterestingly, several studies in various models have demonstrated that oncogenically transformed cells are more susceptible to apoptosis in response to transcriptional inhibition, suggesting that transcriptional inhibition has direct cytotoxic effects in malignant cells. In mouse lymphoma models, tumor cells were more sensitive to apoptosis than wild-type cells after treatment with an inhibitor of RNPI (RNA polymerase I) transcription , and simIn this review, we briefly describe transcriptional machinery components and several transcription inhibitory drugs. We discuss how these drugs interfere with survival of tumor cells as well as the challenges and limitations for clinical application.The basal transcription machinery, a central component of general transcription, consists of several universal components that are required for promoter interaction, thereby achieving efficient and regulated transcription. Several components of these transcriptional complexes exhibit enzymatic activity, which can potentially be inhibited by newly designed drugs Figure . In addiThe rRNA genes are located in the nucleolus and are transcribed by RNPI in eukaryotic cells. rRNA genes are transcribed during the S and G2 phases of the cell cycle; however, transcription is repressed during mitosis and is slowly recovered in G1 . SelectiThe main component of the basal transcription machinery is RNPII, which is composed of 12 subunits  and requTFIID and SAGA (Spt–Ada–Gcn5–acetyltransferase) interact with the promoter and participate in the recruitment of the pre-initiation complex (PIC). SAGA specifically activates gene transcription in response to environmental stress -13. AfteMediator is a multisubunit complex that cooperatively binds with RNPII and a subset of general factors during an intermediate step of PIC formation. Mediator is a transducer of the signals that link the activators with the general transcription machinery, thereby activating transcription ,17. HoweTransformed cells require active transcription for proliferation and survival. Certain oncogenes, ribosomal genes and components of the transcriptional machinery are overexpressed in tumor cells to maintain proliferation ,26,27. Fin vivo. Additionally, cancer cells can modulate their transcriptome and physiology to enhance survival and proliferation under stress conditions. This notion was demonstrated for heat shock factor 1 (HSF1), which drives a transcriptional program different from the heat shock supporting oncogenic processes, including protein folding, stress response, cell cycle and signaling genes, to maintain highly malignant human cancers [Interestingly, the depletion of Xist RNA, involved in mammalian X-chromosome inactivation, induces hematological cancer in mice due to the increased expression of X-specific transcripts that are potentially associated with carcinogenesis . In this cancers . These fDrugs that potentially target the basal transcription machinery components could preferentially affect highly proliferative cells. Various components targeted by drugs include cyclin-dependent kinases (CDKs), RNA polymerases or components of associated transcriptional complexes.Several CDKs that participate in RNPII transcription are targets for global transcription inhibition. CDK7 is a component of the basal transcription factor TFIIH that phosphorylates Ser 5 and 7 in the C-terminal domain (CTD) heptapeptide repeat sequence of the large subunit of RNPII, which is important for promoter escape and the recruitment of the mRNA processing machinery during transcription. CDK7 is a target of global transcription inhibition ,15. Addiki of 3 nM. In addition, flavopiridol inhibits CDK8 with a ki of 18 nM [The most commonly used transcription inhibition drugs are flavopiridol and DRB. The flavone flavopiridol is a potent CDK9 inhibitor with a of 18 nM ,64. In cof 18 nM ,65. In fof 18 nM . DRB , similar to flavopiridol. In fact, CDKI-71 induces apoptosis by downregulating the anti-apoptotic factor Mcl-1 in various cancer cell lines with minimal effects in normal fibroblasts and B and T-cells. Interestingly, CDKI-71 triggers apoptosis in several cancer cell lines with heterogeneous genetic backgrounds, including Rb and p53 mutations, suggesting that cell death is p53-independent [Other novel compounds exhibit promising antitumor activity and display lower toxicities compared with traditional inhibitors. CDKI-71 is a novel CDK9 inhibitor that displays a high affinity for CDK9 cytosineECyd) is a cytidine analog that exhibits potent cytotoxic and anti-tumor properties against solid tumors. TAS-106’s main mechanism of cytotoxicity is inhibition of RNPI-, II- and III-mediated RNA synthesis, thereby inducing apoptosis . TAS-106Recently, a study reported that BMH-21, a compound that is a potent p53 activator and DNA intercalator at GC rich regions, which are abundant in the rRNA genes promoter, induces the degradation of the RPA194 subunit of RNPI, the largest RNPI subunit . As a coDrosophila, triptolide phenocopies mutations in TFIIH subunits, inducing apoptosis in the wing discs. Interestingly, apoptosis is enhanced in p53-deficient cells by JNK pathway activation [Transcription can be disrupted via targeting of associated transcriptional complex components. Triptolide is a diterpene triepoxide that covalently binds to the XPB subunit of TFIIH and inhibits its ATPase activity. This action disrupts the opening of double-stranded DNA for RNPII transcription and repair as well as RNPI transcription ,80-82. Itivation . This fitivation . Therefotivation . IntriguBRD4 inhibition by JQ1 is an emerging and relevant target for the treatment of various cancers. JQ1 is a thieno-triazolo-1,4-diazepine that displaces BET bromodomains from chromatin through competitive binding to the acetyl-lysine recognition pocket, preventing BRD4 reader activity . JQ1 redCX-5461 is another potent and selective drug that inhibits rRNA transcription by targeting RNPI basal machinery. CX-5461 specifically inhibits rRNA synthesis by directly preventing the interaction between the SL1 complex and rDNA. CX-5461 does not affect RNPII transcription and DNA replication . InteresOne key of successful therapy is the identification of critical nodes in the oncogenic network that can be inhibited to promote tumor growth cessation by apoptosis, differentiation, necrosis or senescence. Several findings suggest that cancer cells require high transcription rates and harbor mutations or genetic backgrounds that favor enhanced transcription to sustain growth. Several MLL translocations and components of the SEC complex, including P-TEFb, AF9 and ELL, have been described in various leukemias, suggesting that transcription is misregulated in leukemias . LikewisThe use of selective inhibitors for the transcriptional machinery offers advantages as these agents are considered less genotoxic to non-tumor cell populations compared with commonly used chemotherapy or radiation. This observation is important because a transcription inhibitor that specifically kills cancer cells may have a reduced incidence of secondary tumors compared with genotoxic agents that also affect normal cells. For example, chronic exposure to cisplatin leads to the development of resistance in patients and mouse models, and it has been demonstrated that enhanced damage repair contributes to tumor progression .Initially, transcription-based therapies were primarily used to inhibit specific oncogenes or key genes required for tumor growth and survival. However, the use of these drugs was limited because the heterogeneity of some tumors allowed for the survival of cancer cell populations. For example, some tumors contain large hypoxic regions, which are generally are resistant to chemotherapy and radiation. Reduced MYC levels have been demonstrated in colon cancer cell lines. Reduced MYC expression is associated with survival because MYC is overexpressed and induces apoptosis in regions with poor energy supply . In contIn addition, transcriptional inhibition also sensitizes stem cell populations that are generally chemoresistant. CDK8 and CDK9 are required for the maintenance of undifferentiated states of tumor embryonic stem cells ,92. CDK8On the other hand, the lack of knowledge regarding the regulation of certain genes could generate different outcomes for the same treatment in clinical trials. For example, MYC is overexpressed in several tumors; however, MYC is surprisingly involved in the repression of integrins, which are required for breast cancer cell invasion and motility. This finding suggests that MYC inhibition is contraindicated in certain tumors . A gene Regardless of the nature of tumorigenic signal, the transcription of certain oncogenic events can be disrupted by transcription inhibition, and therefore the transcriptional consequences cannot be predicted. Several recent findings suggest mechanisms involved in sensitization, which will be described in the following section.Normal and tumor cells require transcription for survival. So, how does transcription inhibition lead to apoptosis in cancer cells? Transcription inhibition induces apoptosis by four possible mechanisms: altering the balance of apoptotic and anti-apoptotic factors to favor apoptosis, activating p53 and promoting its translocation to mitochondria, inhibiting DNA replication and promoting the accumulation of aberrant proteins in the nucleus . SupportDrosophila, the ectopic expression of various genes can be suppressed in a background that is partially deficient in transcription or by the pharmacological inhibition of transcription without affecting the proper expression of host genes [In st genes ,97. Simist genes , or oncost genes ,59. Addist genes  or MYC ist genes . In addist genes ,100. Trast genes . In factst genes . Thus, icis structures on DNA called super-enhancers have been identified in genes required for cell identity and involved in cancer [How does an inhibitor of general transcription selectively exert its effects on certain genes? Studies suggest that the high levels of expression by transgenes or gain of function mutations are dependent on their promoter, and these proteins are affected when transcription is inhibited or compromised. Moreover, n cancer ,103,104 n cancer . Given tIn summary, transcription inhibition can interrupt transcriptional programs directed by key oncogenes or disrupt favorable growth conditions associated with the overexpression of non-oncogenes that contribute to survival and tumor progression.Transcription inhibition potentiates apoptosis and other types of cell death in tumor cells. However, studies have demonstrated differential responses to various drugs in a variety of cell lines and tumors. This differential sensitization might be dependent on a variety of factors, such as genetic background, tissue type or tumor heterogeneity. Thus, drugs must be evaluated experimentally to determine clinical efficacy.Genetic background is a key factor responsible for the variable outcomes of chemotherapy. Triptolide treatment in two prostate cancer cell lines revealed that LNCaP cells (androgen dependent) are more sensitive to triptolide-induced apoptosis than PC-3 cells (androgen independent) . These rIn addition to genetic background, differences in the chemotherapy responses are dependent on the tissue type. For example, CX5461-treated solid tumor cancer cell lines display autophagy and senescence, not apoptosis. In contrast, CX5461 induces p53-dependent apoptotic cell death in hematological malignancies. However, no correlation exists between nucleolar stress and p53 status in solid tumor cell lines, indicating that the stress response is p53-independent . In hemaAnother interesting point to consider is unselectivity. In this review, we describe the transcription inhibitory activities of many drugs, but only a few drugs exhibit specific effects on transcription given that most drugs target several proteins. For example, most CDK inhibitory drugs exhibit a high affinity for numerous CDKs that are not involved in transcription and proteins that are not cyclins. This unselectivity does not distinguish the target’s transcriptional activities from its additional functions. Unselectivity might cause side effects given the association with unspecific partners. Moreover, cytotoxicity may be induced by agents that generate DNA damage, such as flavopiridol . Thus, tin vivo models [The targeting of transcriptional machinery represents an interesting mechanism to downregulate the key oncogenes driving tumorigenesis. However, transcription is an essential process that occurs in all living cells. Therefore, this strategy has several limitations. Whole organisms are comprised of several tissues and cell populations with different sensitivities to transcriptional perturbation. Specifically, the BRD4 inhibitor JQ1 disrupts cell specific factors in ESCs involved in pluripotency . Therefoo models , suggestThe downregulation of certain proteins involved in transcription results in tumor suppression. For example, the downregulation of the MED-19 subunit of mediator inhibits cell growth and migration in tongue cancer , and CDKThe efficacy of transcription inhibitory drugs can be enhanced when combined with other treatments. Triptolide has been combined with several agents to significantly increase the apoptosis in several tumors using lower doses of the agents without increasing the side effects of chemotherapy . JQ1 comGene expression profiling of tumors can aid in the identification of molecular signatures that determine several tumor characteristics, such as proliferation rate, differentiation and metastatic capacity. The technique can also identify a possible spectrum of drugs that are effective for tumor growth inhibition as well as specific drug doses. In addition, possible drug synergies and the effects of genetic variation on clinical outcomes in patients can be predicted. For example, the BCR/ABL1-positive chronic myelogenous leukemia line K562 is resistant to JQ1; however, it may be sensitive to triptolide because triptolide may have a more penetrant effect on global transcription than JQ1 .Cancer cells require increased levels of transcription for growth and survival compared with normal cells. Cancer cells are more susceptible to alterations in gene expression. Various drugs that act as transcription inhibitors have been traditionally used in cancer treatment given that they preferentially reduce the global transcription of tumor cells. Thus, transcription inhibitors can potentially target the proteins that comprise the transcription machinery. As such, the transcription machinery is considered an ideal target for the design and improvement of drugs given that cancer cells are exquisitely sensitive to transcription inhibition and selectively affected by these drugs.CDKs are one of the most common targets, but additional proteins have been targeted. However, some of these drugs have an affinity for other targets not involved in transcription, thereby generating possible side effects. Although drugs that inhibit transcription have challenges in clinical application, these drugs can be improved based on new discoveries regarding their mechanisms of action and information from clinical trials.As previously demonstrated, the clinical application of drugs has several parameters that must be considered. However, future investigations on transcription mechanisms in eukaryotic cells will generate more knowledge that will aid in the design of novel drugs targeting new proteins and DNA with enhanced affinities and effects on cancer cells. However, improvements in the pharmacological properties of transcription inhibitors can increase efficacy and potentially reduce toxicity and side effects.In recent years, it has been established that the genomic information of a tumor will indicate specific treatments for each cancer patient. However, personalized therapy must take into account the fact that tumor cells evolve and genomes are heterogeneous. Therefore, the use of drugs with generalized effects will still be required. However, cancer stem cells are typically chemoresistant. Thus, additional studies on the effects of drugs that alter global transcription must be performed in cancer stem cells to determine which components of the basal transcription machinery preferentially affect cancer stem cells when depleted. It is important to consider whether the cancer type is susceptible to drugs that target the basal transcription machinery.Although eukaryotic transcription machinery mechanisms still require additional investigation, novel discoveries will offer new targets for the treatment of cancer in the near future.RNPI II, III: RNA polymerase I, II, III; TBP: TATA-binding protein; SL1: Selective factor 1; UBF: Upstream binding factor; TFII: Transcription factor II; SAGA: Spt–Ada–Gcn5–acetyltransferase; PIC: Pre-initiation complex; NER: Nucleotide excision repair; CTD: Carboxy-terminal domain; P-TEFb: Positive elongation factor b; CDK: Cyclin depending kinase; BRD4 3: Bromo domain containing protein 4, 3; HSF1: Heat shock factor 1; HCC: Hepatocellular carcinoma; JNK: June kinase; XPB XPD: Xeroderma pigmentosum,B or D; DSBs: Double strand breaks; MM: Multiple myeloma; ESC: Embryonic stem cells; AML: Acute myeloid leukemia; TGF: Transforming growth factor.The authors of this manuscript declare that they have no competing interests.CV, GC and MZ wrote the manuscript. All authors read and approved the final manuscript.


Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease worldwide , characterized by steatosis, necro-inflammation, and eventually fibrosis Brunt, .The NAFLD development is still unclear, however, it is now largely accepted that, beside to the genetic background, the increased consumption of obesogenic foods may have a role in the NAFLD pathogenesis. In particular, diets enriched in fat and fructose may be steatogenic in two ways: favoring the occurrence of systemic insulin resistance closed to a dangerous accumulation of free fatty acid (FFA) in the liver; causing deposition of visceral fat and consequent hepatic insulin resistance responsible for steatosis development  comprising the Toll-like receptors (TLRs), which are evolutionary conserved type I transmembrane glycoproteins. The TLRs are the immune sensors of PAMPs and/or DAMPs initiating a signaling cascade leading to the activation of pro-inflammatory genes. Notably, each TLR has selectivity for specific PAMPs or DAMPs and can also differ from their proper localization  (Eckburg et al., Lactobacillus rhamnosus strain GG reduced transaminase levels and antipeptidoglycan-polysaccharide antibodies, a surrogate test for SIBO evaluation (Vajro et al., Appropriate dietetic regimens and physical exercise may improve simple steatosis, even though this lifestyle approach is unable to recover NAFLD-associated liver damage. In fact, it is widely recognized that lifestyle modifications combined with a multi-targeted therapeutic approach against specific triggering factors could be more effective than mono-therapeutic approach in at least paediatric NAFLD (Alisi and Nobili, In conclusion, as several observations suggest a potential role of microbiota in NAFLD development, we believe that probiotics effects on gut flora associated to their excellent tolerability may represent promising therapeutic agents to revert NASH-related liver damage.


Ocypode spp.), a numerical response most strongly expressed by the largest size-class in the population, and likely due to aggregative behaviour in the short term. Consumption of carrion at the beach-dune interface was rapid and efficient, driven overwhelmingly by facultative avian scavengers. This guild of vertebrate scavengers comprises several species of birds of prey , crows and gulls, which reacted strongly to concentrations of fish carrion, creating hotspots of intense scavenging activity along the shoreline. Detection of carrion effects at several trophic levels suggests that feeding links arising from carcasses shape the architecture and dynamics of food webs at the land-ocean interface.Food webs near the interface of adjacent ecosystems are potentially subsidised by the flux of organic matter across system boundaries. Such subsidies, including carrion of marine provenance, are predicted to be instrumental on open-coast sandy shores where in situ productivity is low and boundaries are long and highly permeable to imports from the sea. We tested the effect of carrion supply on the structure of consumer dynamics in a beach-dune system using broad-scale, repeated additions of carcasses at the strandline of an exposed beach in eastern Australia. Carrion inputs increased the abundance of large invertebrate scavengers (ghost crabs,  These e deserts –6.Organic matter is imported into subsidised food webs as either plant detritus or as animal carcasses. Animal carcasses (i.e. carrion) are an abundant and widespread resource: many ecosystems contain large numbers of dead animals that have died from non-predation events ,8. Thesepar excellence, including the processing of marine carrion [Systems with large perimeter-to-area ratios  respond more strongly to trophic subsidies due to their greater propensity to receive inputs across their long boundaries . Similar carrion ,15.Material transfer across ecosystem boundaries, including the beach-dune interface, requires a vector to move matter – either biological or physical. On beaches, physical vectors that move marine matter onshore are primarily wind, currents, tides and waves . These pTheoretical expectations therefore are that food webs of open-coast sandy beaches contain scavengers that have evolved to be efficient consumers of stranded carrion resources. We aimed to test three aspects of this ecological context: i) trajectories of change in abundance of scavengers following altered availability of food resources, ii) capacity for carrion processing in the ecosystem, and iii) carrion as donor control of top-level consumers.The response of scavengers to carrion subsidies was tested experimentally on an exposed stretch of sandy beach on North Stradbroke Island, Australia . Plots were dispersed along the beach, with distances between plots randomised to fall within a range of 100 to 500 m . Treatments were randomly allocated to plots, with the constraint that no more than two consecutive plots of the same type were acceptable (before’ phase) and on 8 days after carrion had been added to treatment plots (i.e. ‘carrion pulse’ phase).The experiment comprised two phases: i) ‘BEFORE’, consisting of measurements of scavenger density over 4 weeks (01 to 30 March 2012) without any manipulation of carrion levels in either treatment or control plots, followed by ii) ‘CARRION PULSE’, representing one week (31 March to 7 April 2012) of experimental manipulation of fish carcass numbers in treatment plots. Densities of scavengers were measured on 7 days before the experimental augmentation of carrion  to treatment plots. Mullet was chosen for the experiment because, as carrion, they form part of the diet of ghost crabs and raptors, the primary invertebrate and vertebrate scavengers in the experiments [Carrion levels were manipulated repeatedly by adding whole fish carcasses  number and diameter of ghost crab burrows (see below), ii) number of fish carcasses remaining, and iii) presence of vertebrate scavengers  or their tracks.Ocypode spp.). Ghost crabs were chosen as the target scavenger species because they are the most abundant invertebrate scavengers on sandy beaches in the region, and are facultative scavengers with a catholic diet [The primary ecological response to carrion augmentation measured in the experiment was change in the density of ghost crabs , whistling kite  and brahminy kite ), as well as torresian crows (Corvusorru) and silver gulls . Although each of these species is known to consume carrion [Vulpesvulpes, Canidae) and lace monitors .During pilot studies, five species of avian scavengers were observed to consume fish carrion on the beach. There were three raptors  sampling to compute 95% highest posterior density intervals . For bur2 test [For the vertebrate scavengers, we extracted three complementary metrics of scavenging behaviour from the time-stamped, camera records: i) time to detection , ii) duration of scavenging bouts (i.e. time spent at the carcass during each distinct scavenging event), and iii) time of scavenging events over a 24h period . Mean values of detection time and duration were compared among species with ANOVA, followed by Tukey’s HSD post-hoc test when significant main effects were present ,51. Unif2 test .Carrion removal from the beach was near complete: of the 720 fish carcasses experimentally added to treatment plots over 8 days, 698, or 97%, were completely consumed , and these burrow diameters were on average 2.3 mm larger  mixed model proved most parsimonious, retaining all random and fixed effects , Table 2Vertebrate scavengers, dominated numerically and functionally by birds, reacted quickly and strongly to enhanced carrion availability , Table 4The avian scavengers displayed a remarkable aggregative response to carrion patches  and a few hours afterwards in the morning  increased numbers of scavengers in distinct aggregations at carcasses, 2) fast and near-complete removal of available fish carrion by scavengers, 3) intense consumption by a vertebrate scavenger guild that was numerically and functionally dominated by birds, and 4) fluxes of marine matter across the beach-dune interface with terrestrial scavengers as vectors.Carrion forms part of the material imported from the ocean that underpins food webs on sandy beaches . Two disOcypode spp.) have a catholic diet, locating carrion using chemoreceptors to detect low-molecular-weight compounds (e.g. hydrogen sulphide and putrescine) that emanate from rotting animal flesh [Ghost crabs  to capiThe aggregative response by the invertebrate scavengers was most pronounced in the largest crabs, which moved to experimental food patches in comparatively larger numbers than smaller crabs. Ghost crabs are prey for raptors , crows aZygiella x-notata) were more fecund when more prey was available on the seashore. Coastal populations of arctic foxes (Vulpeslagopus) also respond positively to marine subsidies [True reproductive responses to trophic subsidies in coastal areas have been recorded for both invertebrate and vertebrate consumers. For example, Spiller  demonstrubsidies . In our ubsidies . NeverthMany ecosystems contain unexpectedly large numbers of dead animals that have died from non-predation events, such as disease, malnutrition, exposure to extreme weather events, mass parasites, and accidents . BecauseGallus gallusdomesticus) carcasses placed in a tropical forest were consumed within 3 days [Mephitismephitis) fed on small bird carcasses at 66% removal efficiency [Marmosaelegans) scavenged 100% of the rodent carcasses that were experimentally added [Procyonlotor), grey foxes (Urocyoncinereoargenteus) and feral pigs (Sus scrofa) scavenged 65% of house mouse carrion in North American hardwood forests [Dicrostoxrichardsoni) were scavenged by red foxes (Vulpesvulpes) and arctic foxes (Vulpeslagopus) in one season [Carcass use (97% removed over 8 days) on sandy beaches rivals that of other ecosystems. Scavenging rates have been measured in several systems, generally reporting high proportions of carcasses removed. For example, 97% of brown chicken  in ways generally not possible for other species . SoaringBirds other than raptors and vultures can, however, also be important scavengers on a diversity of carcass types ,73. TorrVulpesvulpes) scavenging along the shoreline at night. We also recorded fox prints in treatment plots and tracks that ran parallel to the shore, in what may have been part of the fox’s territory, where the animal displays a nocturnal traversing behaviour [Marine carrion is an important dietary component in many carnivorous terrestrial mammals that feed on the seashore , subsidiehaviour . SignifiVaranusvarius) being attracted to fish carrion is interesting because there are few published records of terrestrial reptiles scavenging on beaches (but see Our record of a large carnivorous lizard (lace monitor,  but see ,80. Yet in situ productivity and receive marine input across long and open interfaces with the ocean [Cross-boundary exchanges are ubiquitous, representing donor-controlled subsidies of energy and nutrients that can modify the architecture and dynamics of recipient systems ,6. Thesehe ocean . In a sphe ocean ,84.Such spatial coupling is probable in the system studied here, chiefly because of the mobility of consumers across the interface region of interest. Generally, mobile organisms that cross boundaries and transfer material between systems can be important biological vectors ,86. All Birds are important vectors in the transport of marine nutrients to terrestrial systems . In our Our experiment showed unequivocally that increased carrion availability at the beach-dune interface results in quick and substantial aggregative responses of scavengers to carcasses. These trophic subsidy effects, resulting from imports of marine animal carcasses, are measurable at multiple levels of the food web. Terrestrial raptors, lace monitors, and foxes efficiently consumed virtually all marine necromass within a few days, demonstrating the potential for substantial ecotonal coupling in this system. Birds dominated the scavenging activity of the vertebrates, with a minor contribution by foxes and reptilian scavengers. High mobility of terrestrial raptors that concentrate along the shoreline and their efficient scavenging habits on marine carrion confer a capacity to transfer marine matter across the beach-dune interface, forming a biological vector linking ocean productivity with terrestrial food webs. An important aspect of these predicted energetic linkages across habitat boundaries is the spatial ambit of trophic cross-boundary effects. Thus, future work may involve tracking raptor species to determine the spatial extent of marine subsidies at the land-ocean interface.