Network in Epigenetic Epidemiology to encourage transgenerational research in human cohorts.Mammalian experiments provide clear evidence of male line transgenerational effects on health and development from paternal or ancestral early-life exposures such as diet or stress. The few human observational studies to date suggest  transgenerational effects exist that cannot easily be attributed to cultural and/or genetic inheritance. Here we summarise relevant studies, drawing attention to exposure sensitive periods in early life and sex differences in transmission and offspring outcomes. Thus, variation, or changes, in the parental/ancestral environment may influence phenotypic variation for better or worse in the next generation(s), and so contribute to common, non-communicable disease risk including sex differences. We argue that life-course epidemiology should be reframed to include exposures from previous generations, keeping an open mind as to the mechanisms that transmit this information to offspring. Finally, we discuss animal experiments, including the role of epigenetic inheritance and non-coding RNAs, in terms of what lessons can be learnt for designing and interpreting human studies. This review was developed initially as a position paper by the multidisciplinary  The focus of public health is increasingly on chronic disorders, such as diabetes, cardiovascular disease, mental health and cancer manifesting in adulthood. Unlike monogenic (Mendelian) disorders or infectious disease, where the known primary cause permits an aetiological classification and rational approach to management, common diseases are generally multifactorial in origin with diverse genetic and environmental determinants. This contributes to the highly heterogeneous nature of such conditions, which remain an aetiological and management challenge. A multidisciplinary perspective, which includes genetic analysis, is recognised as the most appropriate approach to studying such conditions.For many years, parental and ancestral experience has been regarded as contributing only indirectly across generations. Clearly, such ‘cultural transmission’, which includes parental nurturing behaviour and social patterning of all kinds, does play an important role in shaping the phenotype/traits of progeny. This is an area of active research integrating information on social circumstances, skills, health and mortality, sometimes across three generations of the same lineage.5In contrast, biological inheritance of phenotype from one generation to the next is usually regarded as resulting from transmission of genetic material (DNA) from both parents, plus ‘maternal effects’ either (i) carried within the egg cytoplasm , (ii) experienced in utero, primarily through the transplacental passage of nutrients, metabolic signals and toxins, or (iii) experienced early postnatally, via breast feeding and/or the exchange of microbiota. Additionally, the maternal genotype also contributes to the early nutritional environment of progeny through its influence on maternal metabolism during pregnancy. This is best exemplified in studies on insulin sensing and fetal growth.Until recently, paternal exposures prior to conception (other than those known to induce DNA mutations) were assumed to have little or no effect on offspring development, primarily due to the lack of a plausible biological mechanism. So ingrained was this view that confirming a lack of paternal exposure effect has been promoted as a useful ‘control’ for social confounders in the study of maternal exposure effects.In light of the emerging evidence for both maternal and paternal transmission of non-genetic, non-cultural effects to progeny, consideration must be given to extend traditional epidemiological methodology to include not only early-life paternal and maternal exposures, but also ancestral exposure data. The consequences of such transmission are potentially profound, particularly in relation to past assumptions underpinning much of epidemiological and genetic analyses. In transgenerational epidemiology, assuming cultural inheritance automatically when results make genetic inheritance implausible (and vice versa) is no longer acceptable.intergenerational, multigenerational and transgenerational to mean specific routes and numbers of generations of transmission,Although some have adopted the terms what exposure at which life stage in parents, grandparents or distant ancestors is associated with a measurable phenotypic outcome in the offspring or subsequent generations? We suggest this observational research is best done in a setting where the effects of social patterning can be accounted for, thereby distinguishing cultural inheritance from a more direct biological transmission across generations. Defining exposure-sensitive periods associated with transgenerational effects will be highly informative in this regard.A priority for transgenerational epidemiology is to describe the range of measurable exposures and outcomes mediating transgenerational effects in humans. That is, exposure is key, not the founder, phenotype. In fact, there need not be a measureable phenotype in the exposed (or intermediate) generation. Similarly, phenotypic outcomes should not only be assumed to be detrimental. Although the usual research design in transgenerational epidemiology is to test associations between parental or ancestral exposure and adverse outcomes in unexposed offspring, transgenerational responses/effects can also be protective ‘adaptations’, potentially lessening the risk of adverse outcomes in successive exposed generations. The latter has been demonstrated in a remarkable male line rat experiment where ancestral carbon tetrachloride-induced liver damage led to heritable, epigenetically mediated, reprogramming of hepatic healing, such that exposure in the third generation did not produce liver cirrhosis.22Unlike much of the research in the field to date, it is important that future transgenerational epidemiology should not be restricted to the inheritance of acquired characteristics, or ‘phenotypic transmission’ to the next generation. The In summary, a transgenerational perspective is needed if we are to better understand the determinants of major public health problems and adequately assess the value and feasibility of potential interventions. Studies in the field should not be predicated upon a particular mode of transmission from one generation to the next and should make no assumptions about the relative contributions of cultural, genetic or other molecular mechanisms of inheritance. We need to keep an open mind and design appropriate experiments for testing each potential mode of transmission independently.not included.Below we summarise human studies by type of exposure and route  of transmission. Studies of exposures around the time of conceptiongrandfather's food supply in mid-childhood, whereas the mortality rate of the women studied was associated solely with the paternal grandmother's food supply. The mortality rate ratio point estimates of grandsons were 1.67 or 0.65 depending on good or poor food supply of the paternal grandfather during the slow growth period (compared with grandsons of grandfathers with moderate food supply). For granddaughters, the comparable figures were 2.13 and 0.72 for good or poor food supply of the paternal grandmother. This intriguing pattern of sex-specific association persisted when the grandchild's early life circumstances were taken into account.Compelling historical findings of paternal transgenerational effects in humans come from the Överkalix population in northern SwedenHuman studies of ancestral famine or food supplements during pregnancy on fetal growth of the grandchildren have been recently reviewed.38onset of paternal smoking on offspring outcomes (study child), using the Avon Longitudinal Study of Parents and Children (ALSPAC), was designed to specifically test the Överkalix hypothesis that mid-childhood was an exposure-sensitive period with respect to transgenerational effects. The initial offspring outcomes were those relevant to the Swedish results such as birth characteristics and body mass index (BMI) at ages 7 and 9 years. ALSPAC data, adjusted for paternal smoking at conception, showed that the earlier the father started smoking, the greater the BMI at 9 years of sons, but not daughters. The greatest association was found in sons of men who commenced smoking before the age of 11.The study of the age of , but no allowance was made for parity.An ALSPAC study of non-smoking ALSPAC mothers, who were exposed in utero to maternal smoking, showed their sons (but not daughters) to be, on average, 61 g bigger at birth compared with babies where neither the study mother or grandmother smoked.In addition to studies examining anthropometric measures, a few studies have also examined the transgenerational effects of smoking on asthma. One much-quoted study published in 2005 indicated that the risk of childhood asthma was not only influenced by prenatal maternal smoking, but also by the exposure of the mother in utero to her own mother's smoking.Areca catechu) in CD1 mice indicated that paternal exposure transmitted an increased risk of hyperglycaemia and obesity to non-betel-fed first-generation offspring, especially males.paan) chewing prior to offspring conception. Chewing usually began around 18 years  and interestingly, a similar association to that found in mice was observed, with a dose-dependent association of paternal betel-quid use with early metabolic syndrome in the adult offspring who had never chewed betel-quid themselves.46An experimental study of betel nut ; transmission of this information between generations; and phenotypic change in (unexposed) offspring/descendants, potentially associated with fetal programmingAs noted above , transmiIrrespective of the exposure type and timing, a prerequisite for transgenerational effects beyond the second generation is the involvement of germline transmission, which may occur in several ways. First, by directly impacting on the developing primordial germ cells early in utero, as has been reported in mice.59Caenorhabditis elegans), initial transmission down the female line can ‘spread’ to encompass paternal transmission in subsequent generations.61Epigenetic events are an essential mediator of cellular differentiation and therefore development in multicellular organisms. However, the underlying dynamism and responsiveness to subtle subcellular cues that are a hallmark of epigenetic change during development also make the developing epigenetic profile susceptible to external influence, with the potential to ‘program’ the underlying genome towards an altered phenotype in later life.In mammals, data demonstrating the erasure of nearly all epigenetic marks twice during the life course  have been difficult to reconcile with an epigenetic mode of transgenerational effects. Nevertheless, for decades it has been known that mammals have a set of imprinted genes; those that are selectively silenced in either the maternal or paternal germline during gamete formation, such that only one allele is expression following fertilisation. Genomic imprinting establishes the principle of transgenerational epigenetic inheritance—the gene is active or silent depending on epigenetic marks placed in the parental generation that survive erasure as they pass to the offspring.64–72Recent mouse experiments have also reported a small percentage of loci that escape demethylation at the second phase during primordial gonadal cell differentiation in the early embryo. These are largely associated with repeat sequences, often adjacent to transposable DNA elements.80et al noted a behavioural sensitivity to acetophenone in the next two generations of animals, despite having no exposure to this odour. This was associated with structural changes in the olfactory nervous system accompanying the fear conditioning and distinct hypomethylation of the Olfr151 gene (known to be activated by acetophenone) in sperm of conditioned F0 males and F1-naive offspring.Of particular note is a recent study in mice that provides a plausible mechanism for transgenerational inheritance of environmental information, linking behavioural, neuroanatomical and epigenetic processes. Using an olfactory fear conditioning protocol, wherein an odour (acetophenone) is paired with a mild foot-shock, Dias Another recent study in a non-genetic prediabetes mouse model provides further compelling evidence for gametic transmission of environmentally induced epigenetic change.81There is evidence for a role of sperm-derived RNAs in mediating paternal transgenerational effects, with several classes of RNA recently identified in sperm that are likely to be essential factors in male fertility, including many Y-linked ncRNAs.83The specific RNA content of sperm has been assessed in response to specific environmental exposures. Diet-induced paternal obesity in mice alters microRNA (miRNA) content and sperm methylation status in male mice in association with altered metabolic function in two subsequent generations of progeny.84MLH1 germline epimutation analysis. Originally thought to be solely due to inheritance of a ‘pure’ epigenetic variant (DNA hypermethylation) across generations,cis (regions on the same chromosome) and trans (different chromosome) to mediate the effects of environmental exposures on the neonatal epigenetic profile in humans.Genetic variation will clearly impact to a variable extent on the apparent heritability of some epigenetic marks as evidenced by multigenerational familial studiesAs described above, environmental exposures can potentially initiate transgenerational effects through altered miRNA in sperm, but some aspects of the phenotype can be transmitted to later generations in the absence of the original miRNA change.94Non-genetic biological transmission between generations, of which epigenetic hypotheses are an exemplar, spell out how exposures in one generation may lead to effects on health, behaviour or other personal characteristics in later generations. To date human studies of transgenerational responses have ranged from historical analysis of the Överkalix cohorts for which biological samples are not available  to the contemporary birth cohort ALSPAC where samples are available on the mother and offspring, plus limited paternal samples. The collection of blood or tissue samples is often seen as the next step to further our knowledge about transgenerational mechanisms in humans. Discoveries of epigenetic and other mechanisms in cells may indeed further our understanding of the aetiological relations. However, this does not mean that large population studies without direct information from human blood or tissue are redundant. On the contrary, epigenetic hypotheses that predict that a specific exposure in one generation will give rise to a specific outcome in a later generation(s) could and should be tested in large-scale population studies that hold information about such exposures and outcomes. Conventional non-experimental epidemiological studies can still provide an effective means to explain and potentially prevent (adverse) transgenerational responses. Public health interventions may be applied at many different entry points in the aetiological chain of events. Often one and the same condition may be causally attributed to different aetiological antecedents in the web of causation dependent on whether the focus of the study is primarily preventive, predictive, therapeutic or, more generally, explanatory.96particular mechanistic theory, although studies over three generations with biological samples are likely to be informative in this respect. However, it will be important to pay particular attention to the parental/ancestral exposures, both in terms of dose and timing of the exposure in relation to the person's stage of development—ideally with prospectively collected information. Standardisation of outcome measures across cohorts is important for many epidemiological studies, including transgenerational studies, and is being actively addressed.There are many birth cohorts and other general population samples worldwide , and curMany cohorts are establishing DNA banks, but it is also worth bearing in mind that neonatal blood spots (Guthrie cards), collected and stored for many decades in some countries, can be a valuable resource for capturing neonatal epigenome and other data.


Alzheimer’s disease (AD) is recognized to have a long presymptomatic period, during which there is progressive accumulation of molecular pathology, followed by inexorable neuronal damage. The ability to identify presymptomatic individuals with evidence of neurodegenerative change, to stage their disease, and to track progressive changes will be important for early diagnosis and for prevention trials. Despite recent advances, particularly in magnetic resonance imaging, our ability to identify early neurodegenerative changes reliably is limited. The development of diffusion-weighted magnetic resonance imaging, which is sensitive to microstructural changes not visible with conventional volumetric techniques, has led to a number of diffusion imaging studies in AD; these have largely focused on white matter changes. However, in AD cerebral grey matter is affected very early, with pathological studies suggesting that grey matter changes predate those in white matter. In this article we review the growing number of studies that assess grey matter diffusivity changes in AD. Although use of the technique is still at a relatively early stage, results so far have been promising. Initial studies identified changes in diffusion measures in the hippocampi of patients with mild cognitive impairment, which predated macroscopic volume loss, with positive predictive value for progression to AD dementia. More recent studies have identified abnormalities in multiple neocortical areas (particularly the posterior cingulate) at various stages of disease progression. Studies of patients who carry genetic mutations predisposing to autosomal dominant familial AD have shown cortical and subcortical grey matter diffusivity changes several years before the expected onset of the first clinical symptoms. The technique is not without potential methodological difficulties, especially relating to partial volume effects, although recent advances appear to be reducing such issues. Going forward, further utilization of grey matter diffusion measurements in AD may improve our understanding with regards to the timing and nature of the earliest presymptomatic neurodegenerative changes. This imaging technique may also be useful in comparing and contrasting subtle variations in different disease subgroups, and as a sensitive outcome measure for presymptomatic clinical trials in AD and other neurodegenerative diseases. There is growing evidence in Alzheimer’s disease (AD) of a long latency period, with pathological changes beginning decades before symptom onset , 2. DuriSignificant advances in AD biomarkers and neuroimaging measures, in particular, have been made over the past two decades. These measures have helped improve both our understanding of the disease and our ability to detect and monitor pathological changes in research and clinical settings. The earliest magnetic resonance imaging (MRI) studies focused on macroscopic brain changes, with the best established symptomatic and late presymptomatic marker being hippocampal atrophy –9. HowevIn recent years, there have been great advances in diffusion-weighted imaging . The keyThe initial focus of diffusion-weighted MRI in neurodegeneration research was on the study of white matter tract integrity, using the diffusion tensor model . This waA growing body of literature is emerging that describes the use of diffusion-weighted MRI to detect microscopic grey matter changes in a number of neurodegenerative disorders. Indeed in one such condition - Creutzfeldt-Jakob disease (CJD) - detection of diffusivity changes in grey matter has now become the gold standard in clinical diagnostic practice , 26. AlzMore recently diffusion-weighted imaging of grey matter in AD has been successfully used in a number of different studies. The majority of studies have used the metric of mean diffusivity (MD), which assesses the average degree of diffusion in all directions, as opposed to quantifying directionality. It has been argued that, compared with fractional anisotropy, MD lends itself better to the assessment of cortical and subcortical grey matter, where net diffusion may not be expected to conform to any one specific direction . As cellA literature search was conducted on PubMed in October 2014. We aimed to identify all articles that used diffusion-weighted MRI to investigate grey matter diffusivity changes in AD. The search [Alzheimer’s] AND [DTI OR diffusion] AND [grey matter OR cortex] was applied. The title and abstract of all articles identified by the search were assessed for suitability for inclusion in the review. For all suitable articles from the initial search, the titles and abstracts of articles in their reference lists were reviewed, and any additional suitable articles also included.et al. [et al. [Early studies of grey matter diffusion assessed MD in the hippocampi of patients with amnestic mild cognitive impairment (MCI), and compared the imaging results of those who did and did not go on to progress to clinical AD , 18. In  [et al. ) amnesti [et al. –31 went et al. [Douaud et al.  used an et al. [Following the finding of early hippocampal diffusivity changes, the focus has broadened to other cortical areas. Rose et al.  used whoet al. . A furthet al. . A signiet al. .Diffusion analysis of multiple cortical areas has demonstrated some value in differentiation of different types of dementia . ComparePatients with earlier stages of AD have also shown widespread patterns of change. One study  of 20 paAlthough rare, autosomal dominant familial AD (FAD) shares many aspects, both molecularly and clinically, with sporadic AD . StudyinPSEN1) gene mutation carriers (n = 10) found significant differences in hippocampal diffusivity compared with controls [A region of interest study of presenilin 1 . This reduction in presymptomatic MD was also associated with marginally increased cortical thickness. Although unexpected, and requiring further replication in larger studies, the finding of a presymptomatic fall in MD is very interesting. It may suggest the presence of more than one pathological process affecting diffusion imaging changes: the presymptomatic reduction in MD may indicate an inflammatory response to amyloid accumulation (Fig. The above studies detected ion Fig. , occurriMeasuring molecular diffusion in grey matter structures is not without pitfalls. An important issue is the potential for results to be biased by partial volume effects , 35. ThiInvestigators have addressed the issue of potential partial voluming in a number of ways. Accurate segmentation, ensuring the region does not include any CSF, is important , 31, 33.A methodological development that offers potential benefits for the measurement of grey matter diffusivity is the recent advent of multiband MRI . By simuAn additional factor to consider if grey matter diffusion is to enter widespread clinical use is the significant time and effort required to segment regions of interest manually. Grey matter diffusivity analysis would ideally be automated . Such meWhilst detection of grey matter diffusivity changes in AD does show promise as a potential early biomarker, the number and size of the studies performed to date is relatively limited. Therefore, further replication of results, ideally in large multicentre cohorts, will be very important.One interesting and potentially useful direction of investigation would be to develop a cortical 'signature' of microstructural change for early AD, with the aim of identifying a characteristic pattern of specific cortical regions that undergo the earliest changes. This approach has already been employed successfully for macrostructural measurements in AD .Diffusion imaging may prove powerful in detecting differences in underlying disease mechanisms, with one study having already demonstrated significant differences between different underlying neurodegenerative pathologies . FurtherA further area of study that may improve sensitivity to the earliest neurodegenerative changes in AD would be the measurement of grey matter diffusivity across serial MRI scans in order to assess longitudinal change. Measurement of within-individual changes avoids between-individual variability and may be a more sensitive and relevant marker of pathological change than a single assessment . Trackinin vitro investigation to replicate and ascertain the likely underlying mechanism.The finding that MD initially decreases in the presymptomatic stage of FAD , 39, priin vivo microstructural changes in the brain; such changes are likely to predate the macrostructural atrophy that characterizes neurodegenerative disorders such as AD. The most striking example of this is in CJD where diffusion imaging of grey matter has proven to be a remarkably sensitive and specific marker of disease. Diffusion changes in grey matter in other, less rapidly progressive disorders have been relatively understudied, but are increasingly of interest in AD given that the earliest pathological changes appear in grey matter in this disease and the growing acceptance of a long prodromal period when molecular pathology accumulates and yet cerebral atrophy can be difficult to detect.Diffusion imaging provides a means of assessing Grey matter diffusivity has shown initial promise in the detection of early presymptomatic changes, as well as in prediction of conversion from MCI to AD, and in the differentiation of different dementia subtypes. Grey matter structures commonly implicated, including the hippocampus and posterior cingulate, have been identified as parts of a network vulnerable to early AD pathological changes. Measuring diffusion changes in the grey matter 'nodes' of these networks may provide information that is complementary to the more studied changes in white matter. However, whilst initial results appear positive, current literature is relatively limited, with findings to date focusing on cross-sectional group changes only. It is therefore true that a note of caution is required when looking to the future. That said, magnetic resonance diffusion acquisition capabilities and analytical techniques are advancing rapidly. These advances offer the prospect of grey matter diffusivity adding to our understanding of the evolution of early changes in AD and other neurodegenerative disorders - and contributing to clinical diagnosis or providing inclusion or outcome measures for trials.


Obesity and its associated medical conditions continue to increase and add significant burden to patients, as well as health-care systems, worldwide. Bariatric surgery is the most effective treatment for severe obesity and its comorbidities, and resolution of diabetes is weight loss-independent in the case of some operations. Although these weight-independent effects are frequently described clinically, the mechanisms behind them are not well understood and remain an intense area of focus in the growing field of metabolic and bariatric surgery. Perceptions of the mechanisms responsible for the beneficial metabolic effects of metabolic/bariatric operations have shifted from being mostly restrictive and malabsorption over the last 10 to 15 years to being more neuro-hormonal in origin. In this review, we describe recent basic and clinical findings of the major clinical procedures  as well as other experimental procedures  that recapitulate many of the metabolic effects of these complex operations in a simpler fashion. As the role of bile acids and the gut microbiome on metabolism is becoming increasingly well described, their potential roles in these improvements following metabolic surgery are becoming better appreciated. Bile acid and gut microbiome changes, in light of recent developments, are discussed in the context of these surgical procedures, as well as their implications for future study. Medical therapy consisting of intensive lifestyle modification  fails to maintain significant long-term weight loss. Although medical intervention can lead to modest weight loss in select patients7, and its usage continues to increase with the increasing prevalence of obesity and metabolic disease. Early studies from Pories and colleagues8 and others10 in gastric bypass patients described diabetes resolving almost immediately after surgery. Even though this effect was described more than 30 years ago11, its complex underlying mechanisms remain an intense research focus. Multiple reviews and meta-analyses have confirmed a diabetes resolution rate of approximately 80%14 and also provide evidence that operating in patients with a body mass index of less than 35 kg/m2 may be warranted as well for non-obese diabetics19. Overall, the benefits of bariatric and metabolic surgery continue to be better described, particularly the decreases in cardiovascular disease and cancer mortality21. With the alleviation of diabetes and other comorbidities, it is not surprising that bariatric surgery also exhibits cost savings compared with chronic medical treatment of these diseases24.Metabolic and bariatric surgery  is recognized as the most effective treatment for obesity and its associated comorbidities, such as type 2 diabetes25.Our aim herein is not to focus on the anatomic differences of the particular operations per se but instead to highlight the discoveries and new questions each procedure has provided over about the last 5 years. The field of metabolic and bariatric surgery has a rich history, although our understanding of how these operations lead to their beneficial effects has significantly changed over the last 10 to 15 years. Operations that were originally intended to produce weight loss through combinations of gastric restriction or malabsorption (or both) clearly have metabolic benefits that are independent from either one of these previously long-held beliefs of their mechanism of action. The historical bile diversion and ileal interposition operations are scientifically in vogue once again and are helping to examine the complex role of bile acids in metabolic regulation. We have focused on the insights from the most popular procedures clinically and experimentally, including gastric banding, vertical sleeve gastrectomy (VSG), Roux-en-Y gastric bypass (RYGB), biliopancreatic diversion (BPD), ileal interposition, and bile diversion. Importantly, we have emphasized many of the species-specific changes that must be considered when translating findings to a clinical context. The role of bile acids and the gut microbiome and their potential interaction is discussed. For a comprehensive discussion of the rapidly growing field of metabolic and bariatric surgery, we direct the reader to this excellent review26. This decline is due to the relative ineffectiveness of banding for long-term weight loss and reduced comorbidity compared to other bariatric procedures. Consistent with this trend and clinical findings, gastric banding has been modeled in rodents29 and has been shown to be less effective for long-term weight loss or improvements in glucose tolerance compared with other procedures  that have additional hormonal or malabsorptive characteristics or both greater and more durable effects. In rodent models, gastric banding is purely restrictive and does not confer any additional benefits beyond restriction of food intake33. Banding exerts a temporary weight loss that is compensated for over the course of several weeks27. The delayed resolution of diabetes or other metabolic comorbid conditions is greater with, and attributable to, weight loss secondary to decreased food intake and not direct neuro-hormonal effects35.The contribution of gastric restriction to the efficacy of bariatric surgery is an area that has been well studied clinically and experimentally. The adjustable gastric band  and gast36. The science behind the balloon is similar to gastric banding in that the balloons are meant to simulate a false sense of gastric distention and promote satiety even after consumption of a minuscule amount of food. Again, similar to the banding procedure, these devices in theory are purely restrictive; however, as they become more popularly used in Europe and the US, further investigation into the potential changes in hormonal or metabolic effects can be studied38.Similarly, gastric balloons have recently been approved in the US, although they have been used in Europe for over a decadeWithout a doubt the most exciting advancements in the field of metabolic and bariatric surgery over the last decade have been the identification of mechanisms that have challenged the long-held beliefs that “bariatric” surgical procedures induce weight loss purely through a combination of gastric restriction or nutrient malabsorption, or both. Neural, hormonal, and other nutrient signaling pathways that have previously been unrecognized may be mediating many of the metabolic benefits of these surgical procedures. We examine three of these operations to help highlight these novel and alternative mechanisms in the following section.26. However, whether VSG provides similar remission to obesity and diabetes long-term has yet to be determined. The ongoing STAMPEDE trial 4, the first prospective randomized clinical trial designed to compare both VSG and RYGB to intensive lifestyle modification alone, is not designed to make direct comparisons between VSG and RYGB. Regardless, the data from the STAMPEDE trial should give us rough insight into how well VSG compares to intensive lifestyle intervention and medical therapy in the long term.The vertical sleeve gastrectomy is a surgical procedure that decreases gastric volume by approximately 70% with excision of a large portion of stomach along the greater curvature . As ment39, lactating female rats that had previously received a VSG or sham operation were both able to increase their food intake in response to lactation without any evidence of food intake restriction in the VSG rats that were lactating—one of the most energy-demanding processes physiologically when dams routinely double or even triple their food intake40. Initially, VSG was thought to work by excision of the ghrelin-producing portion of the stomach41. Indeed, circulating ghrelin concentrations are significantly decreased up to a year after VSG42. Interestingly, even in the absence of intestinal rearrangement, VSG is associated with increased secretion of the distal intestinal hormones GLP-1 and peptide YY (PYY)45. Studies using rodent models of VSG have examined the contribution of these hormonal changes and other mechanisms to VSG efficacy. Mice with genetic deletion of ghrelin or the GLP-1 receptor continue to show weight loss following VSG47, suggesting that isolated changes in these hormones cannot explain the efficacy of the procedure. Unlike ghrelin and GLP-1 receptor knockout mice, though, mice deficient in the bile acid receptor FXR (farnesoid X receptor) have completely abrogated effects of VSG while on a high-fat diet48, thus implicating FXR as a major target of VSG. Consistent with this bile acid receptor dependency, VSG is associated with increased plasma bile acid concentrations in the mouse49, but whether the same holds true in humans is not year clear50. Stefater and colleagues25 examined the effects of VSG compared with diet-induced obesity, pair-fed, or chow-fed control rats. In that study, total bile acids were increased by VSG or pair-feeding by unknown mechanisms, which began to approach the higher concentrations of total bile acids reported in the chow-fed controls. How FXR signaling is altered by bariatric surgery and what other pathways may be affected by VSG remain unclear, although a number of other VSG-related effects require further study, including changes to taste preference similar to RYGB as well as changes in intestinal triglyceride metabolism54.Aside from its clinical usage, VSG has been described in multiple rodent studies and has several interesting physiologic effects that cannot be explained by restriction alone. In fact, data have directly challenged the notion that VSG involves any intrinsic gastric restriction at all. In studies by Grayson and colleagues57. Compared with RYGB patients, BPD patients tend to have more nutritional and gastrointestinal (GI) side effects58, which may indicate a more severe malabsorptive phenotype. Regardless, both operations produce improvements in diabetes/insulin resistance62. Several studies have attempted to determine the magnitude of malabsorption following these procedures clinically and experimentally. In terms of macronutrient malabsorption, animal studies suggest a higher degree of malabsorption in RYGB/BPD models, which could contribute to significant weight loss64. However, this degree of malabsorption is not consistently observed in practice, and several clinical studies show frank macronutrient malabsorption, minimal macronutrient malabsorption, or no macronutrient malabsorption at all68. Both nutrient and macronutrient malabsorption appear to be much more easily observed in the BPD models compared with RYGB69. Collectively, macronutrient malabsorption appears to play a much greater role in the case of BPD than RYGB in the weight loss observed in these patients clinically. However, the complex intestinal rearrangement, altered nutrient absorption, and physical separation of biliopancreatic secretions from nutrients in these operations may alter the intestinal nutrient milieu to explain what drives many of the hormonal and histologic changes70 observed in these procedures.The RYGB  and BPD 71, although the identity of such a factor remains elusive73. Two studies examined the foregut hypothesis directly in RYGB patients with gastrostomy tubes in the gastric remnant75. Hansen and colleagues74 found that reintroduction of nutrients into the excluded foregut in the first 6 weeks after RYGB did not reverse improvement in glucose responses, whereas Pournaras and colleagues75 found reversal between 9 and 24 months after RYGB. Differences between these two studies could be related to early-versus-late post-operative effects or possible enteral feeding prior to testing in the latter study. Animal studies have also had mixed results, showing that duodenal nutrient exclusion is associated with villous hyperplasia but that improvements in glucose tolerance, weight loss, or incretins are mixed and may depend heavily on strain/genetic background79. Conversely, the “hindgut hypothesis” posits that the more rapid delivery of nutrients to the hindgut stimulates either neural or hormonal factors (or both) that lead to the metabolic benefits and weight loss. Within the first month after surgery, concomitant increases in postprandial GLP-1 and insulin secretion and an enhanced incretin effect are observed and have positioned GLP-1 as the prime mediator of improved glucose homeostasis after the procedure. Recent data using GLP-1 receptor antagonists have challenged a dominant role for GLP-1. Although these data indicate that the increased GLP-1 contributes to the increased insulin secretion, this does not translate into improved glucose homeostasis80. It is somewhat unfortunate that these opposing theories arose, because the field has come to appreciate that there are likely components of each that could potentially be at work following RYGB and BPD.Given the anatomic changes with RYGB and BPD, two opposing hypotheses—referred to as the “foregut hypothesis” and the “hindgut hypothesis”—arose in the field. The foregut hypothesis posits that an unknown factor—neural or hormonal or both—originates from the bypassed intestinal region in RYGB/BPD that promotes insulin resistance. Thus, bypass of that segment is associated with metabolic improvements. There is considerable evidence in support of this hypothesis, proposed as early as the 1980s by Scopinaro and colleagues81. A working hypothesis in rats is that Roux limb hypertrophy, secondary to energetic demands on the jejunum, results in increased nutrient uptake following RYGB, making the Roux limb a significant contributor to glucose homeostasis. This is evident by increased basolateral glucose uptake of the Roux limb in the post-absorptive state with corresponding changes in glucose transporters on immunohistologic analysis82. Consistent with this intestinal hypertrophy hypothesis, other procedures have also shown changes in intestinal histology and increased L-cell populations with concomitant changes in L-cell hormonal responses 84. However, histologic changes also occur in the common channel  that could represent hypertrophy secondary to undigested luminal nutrients85.Aside from foregut bypass, a number of anatomic/histologic changes are associated with the intestinal rearrangement that can be potentially linked to the metabolic changes. Cell proliferation and villous surface area are increased in the Roux limb of RYGB in humans, as are expression levels of genes suggestive of increased energy demands88. These findings appear to reflect changes in central reward pathways, but whether this reflects direct central effects or actions of peripheral signals needs to be determined91. Additionally, similar neural sensing mechanisms operating via the vagus nerve have been implicated in luminal nutrient sensing, which adds another layer of complexity between the neural and hormonal regulation that is changed by RYGB and potentially other bariatric operations. Further studies are needed to identify these mechanisms, as the role of the vagus nerve in these effects remains unclear94.Besides the improvements in insulin sensitivity/glucose homeostasis and weight loss observed with RYGB, nutrient signaling within the GI tract to alter taste or other metabolic processes is significantly changed by these operations. Several studies have demonstrated altered taste preference for lipid or glucose solutions in humans and animals after RYGB97. Within the first month after surgery, improvements in hepatic insulin sensitivity are evident99, indicating an important contribution of the liver in mediating the weight loss-independent effects of RYGB. On the other hand, an improvement in peripheral insulin sensitivity, which mediates glucose disposal after a meal, occurs later and is related to the ensuing weight loss101. It has been debated whether an increase in energy expenditure is responsible for weight loss post-operatively in these patients. Recent evidence suggests that resting energy expenditure is increased in mice102 as well as rats104 following RYGB. Unlike animal data, human studies using appropriate methodology  do not support any increases in energy expenditure following RYGB109. In fact, the massive weight loss of RYGB occurs in the setting of metabolic adaptation (decreased energy demands greater than expected for the degree of weight loss), suggesting the contribution of hormonal or neuronal factors (or both) in the anorectic effect110. The effects of RYGB on energy expenditure appear to be contrary to those observed in BPD, which Scopinaro and colleagues have reported111. Overall, when examining the metabolic and body weight changes observed both acutely and chronically by RYGB and comparing those with BPD, researchers are tempted to speculate that the BPD operation represents an extreme physiologic response. A response of this magnitude tends to make sense given the apparent effects of BPD on energy expenditure, malabsorption, and long-term body weight, which are more easily observed with that procedure compared with RYGB.Although most of the focus has been placed on the intestines, considerable evidence indicates that negative energy balance may also have beneficial metabolic effects after RYGB. In the immediate post-operative period, significant caloric restriction contributes to the early improvements in glucose metabolism112 first identified that ileal interposition surprisingly led to decreased food intake in obese rats, which the authors attributed to an “over-stimulated ileum”. In most cases, however, these changes in food intake or body weight are either negligible or modest and this may explain why they are not uniformly detectable—indicating potential importance of genetic background  or feeding/housing methods in rodent studies116. Moreover, these changes appear to be heavily dependent on the length of the ileal segment interposed; longer interposed segments have more robust metabolic effects that could account for variability of findings117. Consistent with this over-stimulated ileal hypothesis, the ileal interposition has profound effects on glucose homeostasis as well as GI hormone secretion and bile acid metabolism similar to RYGB, BPD, and VSG. Weight-independent improvements in glucose homeostasis in rats are secondary to improved skeletal muscle glucose uptake and insulin signaling via Akt113. These benefits persist in monogenic and polygenic rat models of obesity or diabetes, or both114. The interposition procedure is also associated with increased expression and secretion of ileal hormones 116 that correspond to increased enteroendocrine cell numbers118.The ileal interposition , also pr116, the mechanism of which remains unclear. However, both basal and nutrient-stimulated bile acid concentrations are a common finding after bariatric operations. In this regard, bile acids are recognized players in the regulation of glucose and lipid metabolism through the FXR and TGR5 receptors, respectively. It is tempting to speculate that activation of those receptors could improve glucose tolerance and enhance insulin sensitivity.Aside from ileal hormones, ileal interposition causes marked elevations in circulating bile acids119 suggested that the beneficial effects of ileal interposition come from nutrient or bile exposure of the interposed ileal segment at much higher concentrations, thus overwhelming the transposed segment and subsequently causing compensatory changes to the “neo-ileum”. In rats, following ileal interposition the apical sodium bile acid transporter is decreased (~95%) in the interposed ileal segment, although the cytosolic transporter is increased. In those same animals, however, the most distal segments of intestine  had robust increases in expression of these transporters119.The mechanisms responsible for the observed elevations in plasma bile acid concentrations following ileal interposition remain unclear. Strader and colleagues121 for surgical management of hypercholesterolemia. The proposed mechanism of the procedure was to prevent bile salts from mixing with intestinal contents prior to reaching the colon, and thus cholesterol (and other lipid) absorption would be significantly less. The bile diversion procedure paralleled the development of the jejunal-ileal and ileal bypasses pioneered by Buchwald and Gebhard122 at the University of Minnesota and Scott and colleagues123 at Vanderbilt University, and this culminated in the Program of Surgical Control of Hyperlipidemias (POSCH) trial124. From these studies, as one would expect, there were tremendous improvements in total cholesterol levels that were attributed to the lack of bile-nutrient mixing and thus retarded lipid absorption.Bile diversion  was an e126. Thus, more recent studies have revitalized the potential uses for bile diversion as a model to dissect the mechanisms at work following bariatric surgery because of its similarities to other bariatric procedures. Bile diversion, similar to RYGB or BPD, limits bile-nutrient mixing until a more distal point in the intestine. Unlike the RYGB or BPD, though, there is no biliopancreatic limb for these secretions to flow; they are completely diverted by cannula or anastomosis directly to the intestinal segment of interest maintaining unaltered alimentary flow. Coincidentally, it was noted that glucose tolerance was improved even in the absence of weight loss in dogs after bile diversion127. Manfredini and colleagues128 replicated these observations and described improvements in oral and intravenous glucose tolerance in the absence of any changes in insulin secretion. However, a recent report of bile diversion in lean rats has shown conflicting results on whether insulin secretion is altered, and this requires further study129.At the time bile diversion was developed, bile acids had not yet been recognized as the metabolic hormones that we know today129. Similarly, in a high fat-fed model, Kohli and colleagues130 have shown that bile diversion to the jejunum produces weight loss compared with sham/control rats. These beneficial changes are recapitulated with oral bile acid administration and appear to be mediated by alleviating endoplasmic reticulum stress. Moreover, bile diversion in rats was associated with increased number and length of villi, similar to procedures like RYGB, BPD, and ileal interposition131. Each of the bile diversion models is associated with increased circulating bile acid concentrations, which appear to be necessary for the metabolic effects since administration of bile acid sequestrants  normalizes plasma bile acid concentrations and abolishes the metabolic improvements. These findings are consistent with our recent studies using a gallbladder to ileum anastomosis in the mouse, which compared it alongside RYGB in a mouse model of diet-induced obesity. Similar to the findings in rats, bile diversion in mice is associated with striking improvements in glucose tolerance, insulin sensitivity, normalization of blood lipids, and complete resolution of hepatic steatosis63. These changes are secondary to a degree of malabsorption of dietary lipid in the mice; however, they are also observed with marked increases in total circulating bile acids—specific bile acids that are implicated in metabolic signaling through bile acids receptors 132.Regardless of whether insulin secretion is altered, there is improved insulin responsiveness and decreased fasting glucose after bile diversion suggestive of a change in insulin sensitivity in lean rats138. Whether the changes in circulating bile acid concentrations lead to changes in known downstream metabolic effectors like fibroblast growth factor 19  is still under investigation139. The changes in bile acid metabolism and circulating concentrations appear to be dynamic following RYGB133 and this may explain why some investigators observe increases in the metabolically beneficial hormone FGF19 and some do not142. From a mechanistic perspective, the fact that the ileal interposition and bile diversion procedures recapitulate these bile acid elevations is quite intriguing and implies that either absence of bile in the proximal intestine or overabundance in the distal intestine may contribute the metabolic effects observed. The changes in bile acids appear to be mediated in part by increases in bile acid synthesis through increased expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis. However, there are also concurrent changes in bile acid transporter proteins in the liver and the ileum63 that would also be expected to increase the circulating total bile acid pool. Paradoxically, bile acid synthesis is elevated in the face of increased SHP and decreased FXR expression in the livers of these animals. These changes in SHP and FXR expression are inconsistent with our current understanding of hepatic bile acid synthesis143. Regardless of how bile acid concentrations are increased following the ileal interposition and bile diversion procedures, the molecular mechanisms of how the increased abundance of bile acids may alter glucose homeostasis is currently unknown. Evidence suggests that these effects are mediated via FXR or TGR5 signaling (or both) in the beta-cell or in the enterocytes themselves (or in both)145, but further studies need to focus on clarifying these mechanisms and their physiologic importance. These bile acid-mediated effects on glucose and lipid metabolism have therapeutic implications on diabetes irrespectively of changes on body weight and are a focus of current investigation.The contributions of bile acids in the metabolic effects following bariatric surgery are at the forefront of current investigation. There is considerable evidence that bile acid concentrations are increased following bariatric surgical procedures—both clinical and experimental—and the most robust clinical changes are observed in RYGB and BPD146. Similarly, the opposite effect can be demonstrated in obese mice with stool from RYGB mouse donors, which demonstrates the transferability of at least some of the metabolic benefits of gastric bypass147. There has been significant evidence that the gut microbiota is altered following bariatric surgery, although the mechanisms of these changes and the potential contribution they make in the metabolic benefits post-operatively are unknown. Changes in the microbiota have been examined in mouse models of sleeve gastrectomy48 as well as biliary diversion63, and these procedures show what appear to be beneficial changes. Interestingly, le Roux and colleagues148 have recently shown that the gut microbiota alter the pattern of adipose tissue deposition. The interaction between the gut microbiota and bile acid metabolism is complex, and particular bile acids are antibacterial and likely affect the gut microbiota. In contrast, the gut microbiota are the major source of bile acid diversity, chemically transforming endogenously produced bile acids to a number of different chemical species that likely have varying potencies at bile acid receptors. At this time, it is not clear whether these changes in microbiota are a cause or an effect of the metabolic improvements and weight loss observed following bariatric surgery, but the potential for altering the microbiome as a treatment for obesity or diabetes (or both) continues to emerge149.Perhaps one of the most intriguing observations of this century thus far has been that fecal transfer of gut microbiota from obese donors causes weight gain in lean recipients150. Scientifically, while much progress has been made regarding the weight loss-independent metabolic effects of bariatric surgery, the complexity of the system continues to provide challenges. The field is progressing from a “GLP-1-centric view” of bariatric surgery to encompass the importance of early benefits of caloric restriction and non-traditional regulators of metabolism. The changes in bile acid metabolism with bariatric surgery will continue to drive the study of these procedures and the mechanisms in animal models—especially as the bile acid field continues to advance our understanding of these potent metabolic regulators. Lastly, the interaction of the bile acid milieu and the gut microbiome cannot be ignored. The gut microbiome field continues to grow and become more intervention-driven, as opposed to descriptive, and will continue to help identify the changes underlying the metabolic benefits of bariatric and metabolic surgery. These continue to be exciting times for metabolic and bariatric surgical research, and future studies examining the contributions of bile acids and microbiota both clinically and experimentally may lead to more effective treatments, and perhaps new interventional procedures for obesity and diabetes.From a clinical perspective, prospective clinical trials are needed to directly compare not only durability of weight loss but also resolution of other obesity comorbidities  after VSG and RYGB


Low-magnification microwear techniques have been used effectively to infer diets within many unrelated mammalian orders, but the extent to which patterns are comparable among such different groups, including long extinct mammal lineages, is unknown. Microwear patterns between ecologically equivalent placental and marsupial mammals are found to be statistically indistinguishable, indicating that microwear can be used to infer diet across the mammals. Microwear data were compared to body size and molar shearing crest length in order to develop a system to distinguish the diet of mammals. Insectivores and carnivores were difficult to distinguish from herbivores using microwear alone, but combining microwear data with body size estimates and tooth morphology provides robust dietary inferences. This approach is a powerful tool for dietary assessment of fossils from extinct lineages and from museum specimens of living species where field study would be difficult owing to the animal’s behavior, habitat, or conservation status. Mammalian teeth, being both obviously relevant to feeding and well preserved in the fossil record, have been the focus of dietary reconstructions for generations 22Microwear damage to teeth is related to the incidental ingestion of hard particles  during mastication Both SEM and LDM techniques rely on observer measurements and therefore direct comparison of results between different methodologies and users can be difficult. Inter-observer error rates have been estimated at 5–6% in users counting from an SEM image 3D dental microwear texture analysis (DMTA) techniques have been developed to alleviate inter-observer bias by automating the recognition of microwear features Different microwear methodologies have different strengths and weaknesses and all have value depending upon the scientific questions being addressed. The goal of this study is to develop a suite of techniques useful for the assignment of very broad dietary categories across the mammals, irrespective of the animal’s lineage. LDM microscopy (direct counting through the lens) was chosen as the microwear methodology most suitable for this study due to the high throughput of samples required and the wide availability of LDM to researchers in a variety of fields. Although training in the method is required, no equipment is needed other than a stereomicroscope. LDM studies analyze a larger proportion of the total tooth surface than do DMTA techniques, giving more representative coverage of the tooth and making comparisons across a wide variety of mammalian lineages more feasible. While recognizing that this method allows differentiation of coarser dietary bins than does DMTA, LDM has been shown in numerous studies to discriminate reliably between broad herbivorous dietary categories  A study using LDM to distinguish a broader range of dietary categories  in the same analysis, however, has not yet been undertaken and complications arise when considering different types of foods that possess similar physical properties. For example, flesh-consuming carnivores and browsing ungulates have been found in some cases to have overlapping microwear patterns The two main strategies for oral processing (shearing and crushing) are also reflected in tooth shape Body size–as can be estimated from molar tooth size The goal of this study is to develop a widely applicable and widely available analytical protocol for the assignment of diet in mammals by augmenting LDM microwear with additional dietary metrics. Like microwear, neither body mass nor tooth morphology is diagnostic of diet when standing alone, but each can narrow the pool of potential dietary guilds to which a mammal might belong. Analytical techniques individually evaluating LDM microwear, body mass, and tooth morphology have been used effectively in numerous dietary studies, however these analyses are typically limited to comparisons within a single extant mammalian clade, and rarely used in combination with one another. It is unclear how consistent microwear patterns and shearing crest lengths should be in more distantly related lineages with similar diets but different jaw mechanics, and whether microwear and morphological analyses should be trusted in extinct lineages without close extant relatives. To fill this gap in current knowledge, an analytical protocol is developed for the assignment of broad dietary categories  in mammals using the combined information available from body mass, microwear, and tooth morphology. The extent to which these variables may be used as predictors of ecological niche in phylogenetically divergent lineages is tested by taking advantage of the frequent convergence in diet between extant placental (eutherian) and marsupial (metatherian) mammals despite at least 100 million years of separate evolution All of the animal species chosen for analysis were dietary specialists: browsers (leaves of trees and shrubs), grazers (grass and forbs), hard-object feeders , insectivores (cuticle-bearing), bone carnivores, or flesh carnivores. Dietary information was taken from the literature Casts of tooth crowns were made on-site at the Field Museum of Natural History  and the American Museum of Natural History . Following standard techniques in situ in occlusal view. Tooth length and width were measured in ImageJ (available at http://rsbweb.nih.gov/ij/), as was the total length of the shearing crests  on an Olympus SZX16 stereomicroscope. Microwear features tallied included number of small pits (sP), number of large pits (lP), number of fine scratches (fS), and number of coarse scratches (cS) (tS) and total number of pits (tP) were calculated by adding the two size classes together. Adjustment of the unidirectional light source provided a qualitative measure of feature depth: small features appear light on a dark background, while large features appear dark on a light background  . The totMost molar measurements were made on the M2 protocone, a shared morphological character among all mammals studied and a different area than is generally analyzed in LDM studies. Because of the great variety of tooth morphologies and chewing mechanisms encompassed by the animals in this study, choosing a single Phase I or Phase II facet for analysis . All count data were log-transformed to achieve homoscedasticity  and normality  prior to analysis. An independent-samples t-test (comparing two groups) or a univariate ANOVA (more than two groups) was coupled with a post-hoc least significant difference (LSD) test to determine the significance of individual variable pairings. To test for the combined influence of multiple variables, nested ANOVA (two variables) and Linear Discriminant Analysis (LDA) (more than two variables) were then performed using variables found to have discriminating power between dietary guilds. Post-hoc leave-one-out classification results were employed with LDA to evaluate the degree to which the resulting clusters could be distinguished. The significance of LDA was assessed using Wilks’ Lambda. Statistical tests were performed on both the global data set, which includes all sampled teeth, as well as a set comprised only of species with three or more individuals represented (referred to hereafter as the “limited data set”). The limited data set serves as a check on microwear variability sourcing from species represented by limited numbers of individuals, and yielded the same results as the global data set for all statistical tests performed  resulted in the “dietary triangle” typically observed in LDM analyses f, ScS, and tS. Pit frequencies, on the other hand, are generally higher in carnivores, hard-object feeders, and insectivores. The discriminatory power of LDM microwear alone as a diagnostic dietary feature begins to fail when considering animals feeding on items that can have similar physical properties, such as carnivores vs. browsers , or carnivores vs. insectivores . Insectivory, carnivory, and hard-object feeding are particularly problematic because of the variation involved  reveals separation between the three herbivorous guilds in dietary space (s = P0.892), while function 2 (24.9% of variance) is strongly correlated with coarse scratches (cS = 0.798). Hard-object feeders, which have a higher proportion of pits to scratches, have positive function 1 values while browsers and grazers tend to have more negative values. Grazers generally to have a higher proportion of coarse to fine scratches  than browsers do , although there is some overlap between these two guilds on function 2. The “dietary space” defined by this analysis is therefore hallmarked by increasing numbers of small pits on function 1 and increasing numbers of coarse scratches on function 2. This LDA test of the three herbivorous guilds resulted in defined clusters and a post-hoc leave-one-out correct assignment rate of 82.8% (limited data set 84.2%), however the same test including all five guilds had a correct assignment rate of only 57.2% (limited data set 57.1%) and substantial overlap between insectivores, carnivores and hard object feeding herbivores  for each individual by plotting total shearing crest length against the square root of first molar area (length×width) yielded significant differences between carnivores, hard-object feeders, and browsers/grazers of all orders, marsupials included . CarnivoEach of the three independently derived lines of evidence  is informative in different ways for the assignment of diet among the five broad feeding guilds discussed here. None of these techniques on its own is able to discriminate reliably between all five, however the complementary strengths in discriminatory power from all three analytical methods can be taken into account . Body maThe following of this algorithm results in correct assignment of dietary guild 92% of the time, determined by re-assigning diet to all specimens used in this study in the context of Low-magnification microscopy of dental microwear The specimens used in the analysis are drawn from a global sample of extant mammals, collected in different seasons and over several decades. Because microscopic damage records diet over a relatively short period of time The method presented here is a powerful tool for assigning a general dietary category to an animal with a truly unknown ecology. Specific dietary distinctions beyond the five categories discussed are outside the scope of this paper, although a variety of studies have used different microwear techniques such as DMTA to subdivide diet  on a much finer scale, which can include looking for evidence of fallback foods and even inter-individual differences Having been tested using extant mammals with known diets and demonstrated to be successful across the mammalian phylogeny, this workflow is also suitable for studies on the evolution and ecology of extinct mammals as well as museum specimens of living species where field study would be difficult owing to the animal’s behavior, habitat, or conservation status. Importantly for the wide application of this diagnostic approach, no significant difference was found between the LDM microwear patterns or SCS distributions of eutherians and metatherians belonging to the same feeding guilds. Marsupial and placental mammals, separated by more than 100 million years of evolution, have different chewing cycles and use their teeth in different ways Supporting Information S1Additional data tables and statistical analyses. This file contains specimen information and data used in the microwear/morphological analyses, as well as the results of statistical analysis of the limited data set.(DOCX)Click here for additional data file.


This research topic presents speech as a natural, well-learned, multisensory communication signal, processed by multiple mechanisms. Reflecting the general status of the field, most articles focus on audiovisual speech perception and many utilize the McGurk effect, which arises when discrepant visual and auditory speech stimuli are presented  it was shown that attention (Alsius et al., Interactions between auditory and motor brain areas during auditory speech perception were also investigated. By using magnetoencephalography, Alho et al.  demonstrSomatosensory information can also influence speech perception. Ito et al.  used EEGThe current research topic shows that speech can be perceived via multiple senses and that speech perception relies on sophisticated unisensory, multisensory and sensorimotor mechanisms. Multisensory information can facilitate perception and learning of speech. Still, there is great variation in multisensory perception and integration in both typical and special populations at different ages, which should be studied further in the future.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is increasing interest in deploying Screening, Brief Intervention, and Referral to Treatment (SBIRT) practices in emergency departments (ED). However, the current literature is inconclusive on whether or not SBIRT practices are cost effective and cost beneficial. In order to answer this question, new analytical methods need to be developed.The objective of the following study was to pilot a new experimental methodology - modeling costs using a multilevel generalized linear model (GLM) - for studying the impact of SBIRT on healthcare utilization and costs.In the study, healthcare utilization and costs were quantified for patients who received emergency department (ED) services and participated in an SBIRT program entitled Safe Landing. The healthcare and utilization costs of patients who underwent the Safe Landing program were compared using a multilevel GLM to patients in three control groups where no SBIRT protocol was implemented.The study found that SBIRT was associated with 24% lower health care costs from the 12 months preceding the index ED visit to the 12 months following the index ED visit. This translates to approximately $2,600 per patient per year. This reduction in healthcare costs could be linked mainly to decreased inpatient use. The study found a reduction in inpatient claims and visits, suggesting that the reduction in health care costs was driven mainly by a decrease in inpatient visits.This study provides further support that SBIRT programs are a cost-effective and cost-beneficial approach to substance use disorder management. The study also contributes to important literature on the impact of SBIRT implemented in real world settings, rather than traditional randomized clinical trials.


To the Editor—As of February 2, 2015, 13 healthcare workers (HCWs) workingfor international organizations or volunteers who acquired Ebola Virus Disease (EVD) in WestAfrica have been medically evacuated, and another 12 were repatriated after sustaining asignificant exposure (frequently a contaminated needle stick),These isolation facilities were identified early in the course of the current epidemic asthose better equipped and prepared to safely manage EVD cases, being able to minimize theoccupational risk for HCWs.Actually, in 2009 the European Network for Highly Infectious Diseases project,This extensive preparation and training is necessary because performing an invasive procedurenormally represents a risk and this risk is higher when wearing a full gear with goggles andfacial respirators. Among specific factors possibly increasing the risk of contaminated sharpsinjuries, visibility, communication, and range of motion were found to affect this risk.High-efficiency particulate air respirators have a negative impact on each of thesevariables,Recent debate among medical experts is sharply divided over whether most patients in WestAfrica should, or can, be given intravenous hydration. Some have argued that more aggressivetreatment with IV fluids is medically possible and a moral obligation, and others havecounseled caution, saying that pushing too hard would put overworked doctors and nurses indanger. It is difficult to insert needles while wearing three pairs of gloves and foggygoggles: IVs must be monitored; drawing virus-laden blood for tests is dangerous; and patientssometimes yank needles out.The first recommendation by the Emergency Care Research Institute (ECRI) regardingequipment-related preparedness for Ebola is to use needle-stick prevention devices, to ensurethat all areas likely to be used for treating Ebola patients have a full complement of thosedevices, and to try out available devices to make sure their protective features can be safelyengaged when staff are double- or triple-gloved.HCW health and safety are of great concern in Europe and in the United States, and theyshould be even more a priority in epidemic areas. Where safety devices were introduced, withthe help and support of international programs for patient and HCW safety, no furtherneedle-stick injuries were reported in relation to the specific at-risk procedure.


Lung cancer is the leading cause of cancer-related death worldwide and surgical resection is the treatment of choice for early-stage non-small-cell lung cancer (NSCLC). However, in this subgroup of patients, survival can range from 10% to 60% in stage II (with involvement of lymph nodes — N1), and stage IIIA . Stage IA is the most favorable situation with very small tumors without lymph node metastasis. However, the risk of relapse is still rather unpredictable despite the many prognostic gene signatures that have been developed. In spite of the lack of commonality in many genes identified in published prognostic signatures, numerous gene expression signatures perform overlapping prognostic functions and may be able to provide uniform information on outcome in early NSCLC .To this end, Chen and Chen have reported a lung cancer prognostic index (LCPI) that includes the expression of seven genes and combines with age and stage. The LCPI permits separation of patients into three different risk groups  following surgery. Using this LCPI, patients classified as low risk of recurrence can be deemed as not needing any further treatment. For patients deemed to be at high risk, complete surgical resection is insufficient treatment and they will require further therapy. Intriguingly, for patients in low/intermediate risk groups adjuvant chemotherapy could be detrimental for survival .What lessons can be learned from this LCPI? NSCLC has become recognized as being a heterogeneous set of diseases  and, in There is a need for a cancer systems-biology approach that can help to build the optimal prognostic and predictive gene signatures. Chen and Chen have tried to resolve this challenge with the aid of ever increasing computing power. Although information regarding intergene regulatory relationships and their cascades is accumulating, the whole picture regarding gene regulatory circuitry remains elusive. Further research is to be encouraged, employing systems biology-based approaches to identify the altered regulatory circuitry involved in early NSCLC and decipher the web of interconnected genes that confer hallmark cancer capabilities . The onl


Reducing the burden of emerging and endemic infectious diseases on commercial livestock production systems will require the development of innovative technology platforms that enable information from diverse animal health resources to be collected, analyzed, and communicated in near real-time. In this paper, we review recent initiatives to leverage data routinely observed by farmers, production managers, veterinary practitioners, diagnostic laboratories, regulatory officials, and slaughterhouse inspectors for disease surveillance purposes. The most commonly identified challenges were (1) the lack of standardized systems for recording essential data elements within and between surveillance data streams, (2) the additional time required to collect data elements that are not routinely recorded by participants, (3) the concern over the sharing and use of business sensitive information with regulatory authorities and other data analysts, (4) the difficulty in developing sustainable incentives to maintain long-term program participation, and (5) the limitations in current methods for analyzing and reporting animal health information in a manner that facilitates actionable response. With the significant recent advances in information science, there are many opportunities to develop more sophisticated systems that meet national disease surveillance objectives, while still providing participants with valuable tools and feedback to manage routine animal health concerns. The recent outbreaks of porcine epidemic diarrhea virus (PEDV) in the United States swine industry  and SchmCurrent methods for detecting emerging infectious diseases in commercial livestock production systems rely heavily on individual veterinarians observing cases with overt clinical signs, pathognomonic lesions, or atypical presentations in the field and then notifying regulatory officials if there is reason to suspect an outbreak, and/or laboratory confirmation of disease and then notifying regulatory officials , 6. ThisIn commercial livestock production systems, the earliest indication that an infectious disease may have been introduced to a farm is often changes in animal health parameters such as feed intake, water intake, activity levels, production levels, reproductive performance, and mortality. This in combination with presence of clinical signs may prompt the farmer to seek advice from a veterinarian. During the farm visit, the veterinarian examines sick animals in the herd, generates a list of differential diagnoses, and then decides whether or not to submit samples for laboratory diagnostic testing. Positive test results may confirm the presence of a known infectious disease agent, while negative test results may indicate the presence of a novel or emerging pathogen. Over the course of this timeline, animals may be shipped to slaughter facilities where the carcasses are examined for lesions as part of routine food safety inspections. Animals that are sold to other farms or slaughter facilities through livestock markets may also be observed by regulatory officials for overt clinical signs of infectious disease as well as may be subjected to further disease-specific diagnostic laboratory testing as part of established national disease surveillance programs. Data collected at any point in this continuum can theoretically be monitored using automated outbreak detection algorithms. However, as highlighted in Figure This paper reviews the five primary data streams  that can be used to support infectious disease surveillance in commercial livestock production systems. Particular emphasis is placed on factors influencing data quality and coverage, methods to facilitate data collection in real-time, and insights from published emerging infectious disease surveillance initiatives. The challenges associated with data collection, standardization, analysis, and dissemination are also discussed along with opportunities to improve biosurveillance systems through innovative technology frameworks.Farmers and/or production managers observe animals in their herds or flocks for evidence of disease on a regular basis as part of providing routine husbandry care. The frequency of these observations can depend on many factors, such as the differences in management practices between commercial livestock species . Disease may initially manifest itself as reduced feed and water intake, decreased growth rates, decreased production levels, increased mortality rates, poor fertility, or abnormal behavior well before the appearance of overt clinical signs. With the intensification of commercial livestock production systems, there have been significant advances in developing automated systems for collecting production data to compensate for the decreased time spent observing individual animals in large herds or flocks . For exaGiven the low-economic value of most commercial livestock on an individual animal basis, farmers will often attempt to treat simple conditions identified through direct observation of sick animals or through alerts generated by automated monitoring systems themselves before calling the veterinarian for a farm visit . The sysA more recent study by Menéndez et al.  comparedx” for a single day or by a factor of “y” for two consecutive days based on values determined by a previous study in the Netherlands , and vaccinations as well as to diagnose and treat animals with clinical illnesses. The frequency of routine visits varies greatly between livestock operations with larger herds and herds using intensive management practices utilizing veterinary services more frequently due in part to the lower average cost of care per animal per visit . In addiMost regulatory authorities require veterinarians to maintain basic medical records with sufficient detail on the animal identification, history, clinical findings, diagnostics, and treatments so that another veterinarian could easily follow the case. Given the remote nature of food animal veterinary work, the majority of practitioners keep medical records in paper format only  and mustThe quality of data submitted by veterinarians is highly variable regardless of whether the surveillance program is voluntary or compulsory. In a retrospective study of data collected from seven veterinary practitioners participating in the Ontario swine veterinary-based surveillance (OSVS) pilot program , it was Detailed reviews on the design of recent voluntary veterinary practitioner-based surveillance initiatives can be found in other published sources , 6, 45. Laboratory diagnostic testing is frequently used in conjunction with clinical examinations to determine the underlying cause of disease problems in livestock herds. Samples may also be submitted routinely to diagnostic laboratories as part of national disease surveillance programs, herd health certification schemes, or pre-purchase/movement testing requirements. This data stream has become popular in syndromic surveillance research since most veterinary diagnostic laboratories maintain electronic laboratory information management systems (LIMS). However, the capability to electronically transfer data  varies greatly between LIMS systems used by the diagnostic laboratories that serve commercial livestock production systems, with LIMS systems used ranging from systems developed in-house to those developed by commercial vendors. Furthermore, many laboratories do not have the information technology (IT) resources and personnel available with expertise to implement this capability, nor have they been fully incentivized so as to have this capability be a mandatory requirement of LIMS systems used. As such, the overwhelmingly majority of laboratory diagnostic data being shared between laboratories and regulatory authorities is done via email and spreadsheets. Those LIMS systems that can be accessed remotely through secure connections to obtain animal health data in near real-time for further analysis most readily support the objectives of syndromic surveillance programs .Veterinarians are required to complete sample submission forms for diagnostic test requests, with the type of information requested on the forms including the date, owner identification, animal identification , relevant clinical and treatment history, specimen characteristics, and diagnostic tests requested. However, there are known issues with the quality and completeness of data recorded on these forms, which are currently primarily paper-based and manually entered into LIMS systems. The test requests can be classified into broad syndromic categories based on the clinical signs associated with the pathogen  and moniThe population coverage of laboratory submission data can be influenced by practitioner perceptions and experience  as well The likelihood of sample submission also appears to increase if the samples are convenient to collect and the farms are located in closer proximity to diagnostic laboratories  and if tSeveral research studies have reported using historical data from veterinary diagnostic laboratories to retrospectively identify disease outbreaks in livestock populations. Hyder et al.  scanned Livestock farmers routinely sell animals to maximize the returns on their available farm resources. This includes selling animals that have reached an appropriate market weight for slaughter, animals that are transferred to other livestock operations for further finishing or as breeding replacements, and animals that have been culled from the herd due to disease, poor performance, or surplus stock. Many countries require animals to be examined by an accredited veterinarian prior to shipment to verify that they are free from notifiable infectious diseases. The corresponding certificates of veterinary inspection may contain information on the shipper, receiver, livestock transport company, date of examination, date and purpose of the movement, animal identification , animal or herd disease status, and any relevant diagnostic testing results. A study by Portacci et al.  evaluateIn commercial poultry and swine production systems, farmers often have fixed contracts with other producers and slaughter facilities to transport animals directly between locations when they reach a specified age, weight, and/or production stage. The commercial beef and dairy industries are much less vertically integrated and livestock markets play a more important role in facilitating animal trade. In the United Kingdom, approximately 30% of cattle moved off agricultural holdings pass through livestock markets  with a rVan Metre et al.  piloted In most commercial livestock production systems, animals intended for consumption are subject to ante-mortem and post-mortem examination at slaughter facilities to identify diseases that may pose a risk to human health. The ante-mortem examination involves inspecting animals for abnormal respiration, behavior, gait, posture, discharge, swelling, and other external lesions that warrant segregated slaughter. Data loss may occur since animals with overt clinical signs of disease are not supposed to be transported to slaughter facilities. In the United Kingdom, it has been estimated that 18% of recorded cattle deaths occur on locations other than slaughter facilities . After sSeveral studies have highlighted that the rates of carcass condemnation at slaughter facilities can vary based on other non-biological and non-outbreak factors. For example, Alton et al.  found thSyndromic surveillance systems based on monitoring trends in condemnation rates have successfully been used to detect emerging spatio-temporal clusters in disease incidence. In evaluating historical data from Ontario swine slaughter facilities, Thomas-Bachli et al.  identifiFor syndromic surveillance systems to be useful in providing an early warning of emerging disease outbreaks, data must be collected and analyzed in near real-time. This can prove challenging given that the majority of farmers, veterinarians, laboratories, markets, and slaughter facilities still rely on paper-based recording systems to manually capture animal health data in the field. These data must subsequently be transferred into electronic databases, which can lead to significant delays before the data become centrally available for analysis. For example, in the Ontario Farm call Surveillance Project (OFSP), the average time from farm visit to report submission was 16 days for paper-based submission forms, 13 days for web-based submission forms, and 7 days for submissions through handheld mobile devices with the majority of participating veterinarians (72 out of 98) choosing to use paper-based submission forms . The OSVMuch of the data collected within commercial livestock production systems is considered business sensitive by farmers and veterinarians. In countries without national herd and animal identification programs, there is often reluctance to share identifying farm information with regulatory authorities due to concerns over how the data will be shared and its potential to negatively impact business interests , 70. A cThe lack of standardized systems for recording animal health events has been highlighted as a significant barrier to integrating data across multiple data streams and systems . Almost When implemented on a national scale, syndromic surveillance systems are expected to generate large volumes of heterogeneous data that become difficult to analyze using traditional statistical methods. Early changes in disease frequency can easily be masked by the greater natural variation in baseline disease levels observed in large populations . The mosMost prospective outbreak detection algorithms operate on the same basic principle that when the number of observed cases exceeds the number of expected cases by a specified amount, the system alerts the analyst to a potential emerging disease threat , 95. SetWith the exception of a few Scandinavian countries, the participation of farmers and veterinarians in syndromic surveillance initiatives has typically been on a voluntary basis. For that reason, many pilot projects have provided incentives such as direct financial compensation  or crediThe long-term success of syndromic surveillance programs in commercial livestock production systems hinges on being able to use innovative technology platforms to integrate animal health information from diverse data sources into a common operating picture where it can be used to support emerging infectious disease detection and decision-making as well as efforts to manage endemic diseases more cost-effectively at the farm and industry levels.A key step toward improving the quality and timeliness of animal health data collected through syndromic surveillance systems will be developing mobile technology platforms that allow participants to capture information electronically as part of their normal work routines . The VetAnother possible solution is to ensure that the different, but complementary information recorded by the various data streams can be linked through either herd or animal identification numbers. Glass-Kaastra et al. , for exaThe consistent use of herd and animal identification numbers in surveillance reports also facilitates the development of better statistical methods to detect emerging disease outbreaks. In countries where detailed information on livestock movements and farm locations is available through national computerized databases, it may be possible to strategically select space-time windows using network-based approaches to avoid the problem of using artificial boundaries to subset the large volumes of surveillance data. This process first involves reconstructing the contact network by creating links between farms that trade animals or are located in close proximity. These links may be weighted by the volume and frequency of animals traded in the case of movements or by the distance between farms in the case of spatial proximity. Various community detection algorithms can then be used to divide the population of farms into linked networks or communities based on the strength of connections between them , 106. ThSeveral methods have been proposed to reduce the error caused by setting arbitrary threshold values. Dórea et al.  developeSome of the basic principles from risk-based surveillance  may alsoWith the increasing sophistication of the technology platforms supporting syndromic surveillance efforts, it is also possible to provide participating farmers and veterinarians with customized tools to improve animal health management. This has been identified as an important incentive for continued participation . It has As highlighted by this review, there is still much to be learned about how data collected from farmers, veterinarians, diagnostic laboratories, markets, and slaughter facilities can be used to support infectious disease surveillance in commercial livestock production systems. Each data stream has its own unique challenges associated with achieving adequate specificity, timeliness, and population coverage. However, advances in IT are greatly expanding opportunities to collect and integrate animal health data in real-time for use in detecting emerging infectious disease outbreaks as well as for managing common endemic diseases more cost-effectively than traditional surveillance systems.MG conducted the literature review and drafted the manuscript. TB, KB, and LH provided scientific guidance on the manuscript structure and contents. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.