The development of genomic tests is one of the most significant technological advances in medical testing in recent decades. As these tests become increasingly available, so does the need for a pragmatic framework to evaluate the evidence base and evidence gaps in order to facilitate informed decision-making. In this article we describe such a framework that can provide a common language and benchmarks for different stakeholders of genomic testing. Each stakeholder can use this framework to specify their respective thresholds for decision-making, depending on their perspective and particular needs. This framework is applicable across a broad range of test applications and can be helpful in the application and communication of a regulatory science for genomic testing. Our framework builds upon existing work and incorporates principles familiar to researchers involved in medical testing (both diagnostic and prognostic) generally, as well as those involved in genomic testing. This framework is organized around six phases in the development of genomic tests beginning with marker identification and ending with population impact, and highlights the important knowledge gaps that need to be filled in establishing the clinical relevance of a test. Our framework focuses on the clinical appropriateness of the four main dimensions of test research questions  rather than prescribing a hierarchy of study designs that should be used to address each phase. The development of genetic and genomic tests is one of the most significant technological advances in medical testing in recent decades. A growing interest in “personalized” or “precision” medicine, commercial interests seeking return on investment, and limited regulatory oversight in many countries has resulted in increased availability of genomic tests. This environment has created a number of issues to determining the most appropriate point in time to adopt a new test in clinical practice which cannot be determined solely by availability, marketing, or regulatory approval.Medical testing, in general, lags behind therapeutics in the understanding and application of rigorous evidence-based evaluation, and has particularly lacked a focus on patient outcomes. Because medical testing primarily provides information that may change patient management which indirectly affects patient outcomes, the direct impact on patient health can be difficult to assess. However, not only are diagnostic issues a greater source of medical errors and safety concerns than therapeutics, diagnostic testing also directs the majority of health care expenditure . A focusDespite the importance of medical testing in patient management, the profit margins for the development of new tests are often low, compared to new pharmaceuticals, so there may be little incentive for diagnostic test developers to support clinical testing beyond that required for regulatory approval . GeneratThere have been a number of models or frameworks designed to specifically structure the evaluation of genomic test development , a particular clinical context , or one particular aspect or phase of evaluation . We believe that a single, inclusive framework would provide a common language across different stakeholders  despite their varying perspectives and needs. Second, frameworks such as the translational research models that specify phases of research from bench to bedside do not describe in sufficient detail the evidence needed within each phase of its development. From experience, we have found that general models lack specificity, are therefore difficult to operationalize and subsequently inconsistently applied. Third, some categories of frameworks are extremely comprehensive (involving detailed checklists or series of questions), and thus are better suited for identifying all possible research that might inform use of a genomic test without consideration of which research is necessary (and sufficient) before adoption in any specific clinical scenario. Together, these issues point to the critical need for a consistent and comprehensible framework for the efficient development and dissemination of genomic tests in order to facilitate informed decision-making.Our aim is to describe a framework for assessing the evidence base for genomic tests  that builds upon existing published frameworks for evaluating genomic tests, without necessarily specifying any particular threshold of evidence for regulatory approval or clinical implementation. We anticipate this framework will be helpful in communicating where evidence for a particular diagnostic or prognostic test is missing, and inform the types of research needed before test implementation.The proposed framework specifies six phases in the development of genomic tests and focuses on the important knowledge gaps that must be addressed before the tests can achieve clinical relevance Figure . Like soThe phases of development are laid out from the "ground up" to illustrate the cumulative aspect of knowledge development that relies on solid foundations of pre-clinical evidence and proceeds through successively more applicable clinical studies Figure . AlthougThis phase encompasses the vast majority of genomic research, including agnostic genome-wide association studies (GWAS) and candidate gene approaches. An online catalog of GWAS published findings, for example, includes over 1000 publications and over 5000 disease-single nucleotide polymorphism (SNP) associations to date . MaximumThis phase also includes the early development of promising molecular biomarkers into genomic assays. There are multiple technical test aspects affecting assay development, including the selection of variant(s) to be evaluated, selection of a technology or platform for genotyping, and determination of sample and sample handling conditions. This phase should establish the initial analytic validity of the chosen assay that includes the basic technical performance of the assay . Ideally, this phase would result in a (well-defined) assay suitable for possible application in a specific disease or health condition that could be assessed in subsequent steps.A test’s initial clinical test performance is almost always examined in highly selected populations that are not representative of the intended population the test is to be used on. For example, tests may be conducted in persons with known disease compared to healthy controls, or in persons at very high risk for having the outcome(s) of interest (higher pre-test probability than the intended patients). For diagnostic tests, test performance is generally compared to a reference standard assay using measures such as sensitivity, specificity and predictive values. The approaches used to measure initial test performance, however, rely on the current state and accuracy of the science. For example, the diagnostic accuracy of a new genomic assay to measure cystic fibrosis transmembrane conductance regulator (CFTR) genotype would be compared with a functional assay, such as the sweat test, which is still the current standard for diagnosing cystic fibrosis . The swePhase 1 also includes ongoing refinement of the assay’s technical performance. It is often necessary to refine the assay based on initial test performance by characterizing sample handling conditions or pre-analytic variables that result in superior performance of the test. For example, there may be changes in requirements for micro-dissection to isolate tumor tissue from contamination by normal tissue for a test of somatic mutations, or changes in the buffer conditions to stabilize a sample during transfer to a reference laboratory. This iterative process of initial clinical validation and assay refinement can sometimes lead to significant changes in the assay. As such, it is important for test development research to be explicit about the similarities and differences in iterative “versions” of assays. Fecal Deoxyribonucleic acid (DNA) testing, for example, has undergone multiple iterations during its development as a potential screening tool for colorectal cancer. As a result, the currently available assay compared to previously test versions (different biomarkers and different technologies) , which hAlthough, studies in this phase often use the same outcome measures as studies in Phase 1 , they establish the performance of the test in populations in which the test is intended to be used. The differences in study design and population between Phase 1 and Phase 2 often lead to differences in test performance . In the While previous phases focus solely on the diagnostic or prognostic ability of these tests, research in Phase 3 addresses the clinical impact and net benefit  to patients from using the test compared to not using the test. Outcomes for phase 3 can include the impact of testing on patient management and treatment decisions  and subsequent patient health outcomes . Established diagnostic accuracy or risk prediction  does not necessarily translate into improved health outcomes . This lack of effect on health outcomes may occur because a test may provide information which does not (or cannot) lead to changes in treatment or management options. Even if management options exist, they may not be effective in improving health outcomes. Alternatively a genomic test may identify a genotype with incomplete or variable penetrance, or the harms (or other tradeoffs) of testing outweigh or mitigate the clinical benefits.One well known example of genomic testing with established clinical utility is BRCA1/2 testing . There has been significant research in evaluating patient outcomes from bilateral prophylactic mastectomy to prevent breast cancer in carriers of BRCA 1/2 mutations, demonstrating improved survival and quality of life . In contResearch in this phase aims to answer the question of whether the test result provides added clinical value above and beyond other clinical information already available, or compared to existing tests. Whilst genomic tests can be novel tests for conditions for which no tests currently exist, it is always important to consider the existing standard of care: whether the new test performs better than any existing tests, or whether the new test has equal diagnostic/prognostic ability, but is less invasive or less expensive. For example, clinicians need to know whether an assay for cytochrome P450 (CYP) 2C19 variants is superior to existing platelet functioning testing in predicting response to clopidogrel in persons with acute coronary syndrome (or undergoing percutaneous coronary intervention). This issue of “comparative effectiveness” is paramount for genomic tests designed for risk prediction or prognosis. Ten new biomarkers have recently been identified that are strongly associated with breast cancer risk in older women at the genome-wide significance level . HoweverThe final phase of this framework evaluates the new test’s population impacts, including its implications for the family, community, or society as a whole . For example, an accurate method has been developed to determine fetal gender by detection of the sex-determining region Y (SRY) genotypes in maternal blood during the first trimester, which should only be present if the fetus is male . Even ifThis framework provides a common language and benchmarks that can be used by diverse stakeholders. This is important because while test developers may have expertise in particular genetic tests, these tests are likely to be evaluated increasingly by those with less-specialized knowledge as a test advances through phases of development. As such, we have incorporated general principles familiar to those doing research in medical testing generally (diagnostic and prognostic) as well as those doing research in genomic testing . We have also tried to use shared terminology and concepts that correlate with both medical testing and pharmaceutical development.This framework does not prescribe a hierarchy of study designs that should be used to answer the questions in each phase because the risk of bias within any given study (considering both design and implementation) is more important than the type of study per se. Multiple study designs may be possible within each of our proposed phases. Instead, our framework focuses on the transitions in the four main dimensions of test research questions  as the main organizing principle to categorize the hierarchy of evidence for test development/performance across the “phases” of research Figure . Phases Finally, our proposed framework incorporates different clinical roles of genetic tests across the multiple phases of evaluation. As such, it avoids the “silo” approach of having multiple different frameworks for each different role of tests and highlights shared research aims across different test roles. For example, demonstrating an association between genetic variant and clinical outcome of interest is common to all potential future uses of genetic tests in Phase 0. In contrast, in Phase 1 the key outcomes for a genetic test depend on its intended use as a screening/diagnostic test versus prognostic or pharmacogenomic test. In Phase 3, studies of different clinical applications may differ in both study population and outcomes. For example, a study of BRCA1/2 testing for risk assessment (prediction) in asymptomatic women might evaluate how well the test classifies a woman’s risk of developing breast cancer over a specific time period, which could modify screening recommendations or prophylactic management strategies. A study of the same test’s use in determining appropriate treatment options in women who already have breast cancer, on the other hand, would focus on the test's ability to distinguish between those who did and didn’t respond to specific treatments . HighligWe have synthesized across many existing models-7,17-19 As genomic testing is increasingly disseminated into clinical practice, clinicians and their patients will encounter these tests on a regular basis. As a result, we anticipate the framework will be useful in communicating to clinicians and their patients, what evidence is available for a genomic tests and/or what evidence would be needed before considering using it for routine clinical use. Through using the framework it should be quite apparent that, for example, the latest media report of a new gene-disease association is only at the start of the evidence-gathering (Phase 0), and thus deserves little of the precious clinical time available for determining current patient care options.Our framework also allows different stakeholders to specify different thresholds for decision-making, depending on their perspective and particular needs . Regardless of where different stakeholder may choose to set their threshold, the framework usefully organizes evidence into two main stages: pre-clinical (Phases 0 and 1) where a test may be in various phases of development but not ready for clinical adoption and clinical phases (Phases 2 through 5), whereby increasing certainty of clinical impact is investigated. It is important to articulate these initial pre-clinical Phases 0 and 1 in a genomic testing framework, because often discovery research  or early/initial validity testing  pushes the early adoption of genomic testing into clinical practice. Although Phase 2 research may establish clinical validity in the relevant patient populations it does not translate directly into improved patient outcomes and therefore may be an adequate threshold for some stakeholders , but not others .Medical laboratory testing regulation is much more limited than, for example, the regulation of new drugs. In both the United States (US) and Europe, regulation is based heavily on technical performance and demonstration of some clinical relevance, but not on patient outcomes -44. In tOur proposed framework could help unify perspectives and shared understanding in the same way that the four phases of drug development allow a clearly understood benchmarking process for approval and usage of new pharmaceuticals. If this framework were incorporated into the FDA's developing “evidence-based regulatory science” approach, regulatory agencies could be explicit on what an appropriate level of evidence  might be for a given assay and what constitutes a similar enough (vs. new/different) assay. Other stakeholders  could similarly reflect upon and be explicit about their threshold of evidence before uptake, as different bodies will inevitably have different perspectives.Our suggested framework is intended to facilitate ongoing discussion and developmental activities among manufacturers, test researchers, systematic reviewers, regulators, and other policy-makers, as well as to facilitate understanding among clinicians and patients. We believe our framework is broad enough to be applied to a wide variety of genomic testing, however, we realize that the complexities and rapid advances within genomics (next generation sequencing) and related fields  will necessitate re-evaluating and tailoring our framework over time. If our effort reflects a useful synthesis of existing frameworking efforts to date, it may allow more consistency in definitions for terms and concepts going forward and provide a platform for future collaborative efforts.st century, holding out the promise of revolutionizing medicine towards a more personalized approach. However, we believe that in order to achieve their potential, and to avoid inappropriate use, genomic tests need to be much more broadly understood, and dialogue between different types of users needs to be facilitated. Our proposed framework improves upon the efforts of others, and offers unique features, including specifying in more detail the clinical questions and changes in research focus that accompany the clinical development of a genomic test. If this framework is applied generally, it will help users understand the state of the science for a given genomic application and to articulate their own clinical thresholds for evidence that may be required for a test to be adopted. This, in turn, could reduce confusion, minimize the possibility for inappropriate use, and enhance innovation.The field of genomics is one of the potentially most important developments in health care in the 21ACCE: Analytic validity, Clinical validity, Clinical utility, Ethical/legal/social issues; AUC: Area under the curve; CFTR: Cystic fibrosis transmembrane conductance regulator; CLIA: Clinical Laboratory Improvement Amendments; CYP: Cytochrome P450; DNA: Deoxyribonucleic acid; FDA: U.S. Food and Drug Administration; FVL: Factor V leiden; GWAS: Genome-wide association study; KRAS: Kirsten rat sarcoma oncogene; LDT: Laboratory-developed tests; SACGT: Secretary's Advisory Committee on Genetic Testing; SNP: Single nucleotide polymorphism; SRY: Sex-determining region Y; US: United States; VTE: Venous thromboembolism.The authors declare that they have no competing interests.JSL, Designed and developed the framework with examples and drafted the article, and worked on final revisions; MT, designed and developed the framework with examples and drafted the article, and worked on final revisions; KAB, developed the framework with examples, revised the article for intellectual content; MAP, revised the article for intellectual content; CH, revised the article for intellectual content; EPW, Developed the initial idea for the framework, developed the framework with examples, revised the article for intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6947/12/117/prepubPDF; Glossary of terms. Key terms used are defined.Click here for fileSearch strategy and article flow. Describes the search method we used to conduct our abbreviated systematic review. The number of abstracts and articles reviewed throughout the process are also indicated.Click here for fileExisting frameworks used to evaluate the evidence base for genomic testing. The main frameworks used in the field of genomics are identified and described in detail.Click here for file


The thyroid gland exerts a major control on the physiology of the whole body, and it raises fundamental questions about molecular physiological processes. Moreover, an increased proliferation of thyroid cells is associated with several pathologies, and several mechanisms may be involved. Thyroid disorders are very common, affecting millions of people. These include hypothyroidism, hyperthyroidism, thyroid nodules, thyroid cancer, and so forth, but they are also associated with other nonthyroid disorders. This special issue is devoted to illustrate the particular richness of current investigations in the field of thyroid function and growth regulation under normal and abnormal conditions.Role of estrogen in thyroid function and growth regulation.” Although thyroid gland function is mainly under the control of pituitary TSH in normal conditions, other factors may also play a role in this process. Thyroid disease is more common in women than in men. Tania Weber Furlanetto and Ana Paula Santin reviewed the direct effects of estrogens on thyroid function and growth regulation in the paper titled “Thyroid cancer is the most common endocrine malignancy, and its incidence has significantly risen in the last decades in the world. The knowledge how thyroid cancer develops is expanding rapidly. The sequential acquisition of mutations which arise as a consequence of damage to the genome is required in order to transform a normal cell into a malignant one. The understanding of the process of thyroid carcinogenesis at the molecular level will improve not only the diagnostic but also the treatment of this pathology.Recent advances in molecular diagnosis of thyroid cancer.” Thyroid-specific transcription factors regulate thyroid-specific gene expression and organogenesis. Their possible role in thyroid cancer as well as in the maintenance and/or activity of stem cells is discussed by Shioko Kimura in the paper titled “Thyroid-specific transcription factors and their roles in thyroid cancer.” MicroRNAs (miRNAs) are short ribonucleic acid molecules (around 22 nucleotides) found in eukaryotic cells. miRNAs are posttranscriptional regulators that bind to complementary sequences on messenger RNA transcripts inducing the translational repression or messenger RNA degradation. Several miRNAs have been found to have links with some types of cancer. Francesca Marini, Ettore Luzi, and Maria Luisa Brandi reviewed the role of miRNAs in thyroid cancer development in the paper titled “MicroRNA role in thyroid cancer development.” Growth factors play a role in thyroid proliferation and function, while EGF acts as a mitogen for thyroid cells inhibiting also thyroid differentiation. TGF-β is a potent inhibitor of thyroid cell growth. However, in some transformed thyroid cells this inhibition is lost. The role of TGF-β and EGF on thyroid carcinogenesis and the crosstalk between these growth factors are discussed by Gabriella Mincione Maria Carmela Di Marcantonio, Chiara Tarantelli, Sonia D'Inzeo, Arianna Nicolussi, Francesco Nardi, Caterina Francesca Donini, and Anna Coppa in the paper titled “β1 effects on thyroid function.EGF and TGF-”Ioannis Legakis and Konstantinos Syrigos describe the molecular events associated with the progression and dedifferentiation of thyroid carcinoma in the paper titled “Association between vitiligo and thyroid autoimmunity.” Grave's disease and Hashimoto's thyroiditis are the two main types of autoimmune thyroid disease. Occasionally they are also associated with other autoimmune diseases. Emina Kasumagic-Halilovic, Asja Prohic, Begler Begovic, and Nermina Ovcina-Kurtovic bring additional support to the existence of a significant association between vitiligo and thyroid autoimmunity in the paper titled “Thyroid Functions and Bipolar Affective Disorder).Although some authors have found an association between an abnormal thyroid condition and bipolar disorder, little is known about the implication of the hypothalamo-pituitary-thyroid in neuropsychological deficits. Subho Chakrabarti review the last findings on this topic including genetic and neuroimaging investigations (Guillermo JuvenalGuillermo JuvenalDaniel ChristopheDaniel ChristophePierre RogerPierre RogerMario PisarevMario Pisarev


Didelphis virginiana) and coyotes (Canis latrans). Hunting on adjoining lands was the second strongest predictor of occupancy for three mammal species, negatively influencing black bears (Ursus americanus) and bobcats (Lynx rufus), while positively influencing raccoons (Procyon lotor). Modeling also indicated an avoidance of high trail use areas by bears and proclivity towards high use areas by red fox (Vulpes vulpes). Roads had the lowest predictive power on species occupancy within the corridor and were only significant for deer. The occupancy models stress the importance of compounding direct and indirect anthropogenic influences operating at the regional level. Scientists and managers should consider these human impacts and their potential combined influence on wildlife persistence when assessing optimal habitat or considering management actions.Anthropogenic effects on wildlife are typically assessed at the local level, but it is often difficult to extrapolate to larger spatial extents. Macro-level occupancy studies are one way to assess impacts of multiple disturbance factors that might vary over different geographic extents. Here we assess anthropogenic effects on occupancy and distribution for several mammal species within the Appalachian Trail (AT), a forest corridor that extends across a broad section of the eastern United States. Utilizing camera traps and a large volunteer network of citizen scientists, we were able to sample 447 sites along a 1024 km section of the AT to assess the effects of available habitat, hunting, recreation, and roads on eight mammal species. Occupancy modeling revealed the importance of available forest to all species except opossums ( As the distribution of species shift due to recent global and local environmental changes e.g., , there iTo study anthropogenic effects on mammal occurrence at a landscape level, we chose to focus on the Appalachian forest ecosystem Environmental variation between samples, and across landscapes, creates a major challenge to studying anthropogenic influences on species distributions Beyond the habitat-based limitations of most macro-level studies, logistical and financial challenges also often hinder researchers’ ability to achieve sufficient sample sizes. Such studies require large amounts of labor and technological equity, resources that typically limit studies to smaller extents than the science would dictate. Financial, technical and human resource constraints can be met through the use of a citizen-run camera trapping methodology. Citizen science methods have been highly successful in understanding ecological processes occurring at broad geographic extents Utilizing local volunteers in this macro-level spatial study of mammal occupancy, we address the question: Do localized human activities have predictive power across the broader landscape for determining occupancy of mammals within the Appalachian Trail corridor? In answering this question, we hope to identify region-wide threats to mammal persistence.This study took place along a section of the Appalachian National Scenic Trail (AT), a protected 300 m corridor that stretches 3625 km from Maine to Georgia and is administered by the National Park Service. Though the AT corridor itself is mostly forested, it is surrounded by a mosaic of agricultural, residential, and industrial development We focused efforts along a 1024 km section of the AT from Pennsylvania to North Carolina . Data weThe Appalachian Trail study area was divided into 2 km segments, and sample points (UTM coordinates) were randomly selected within each segment. Selected points were separated by ≥1 km and placed 50–500 meters from the trail. Land ownership of each segment was determined and permissions were obtained from the 8 public agencies involved along this segment of trail. We excluded 76 of 512 segments (15%) following consultation with NPS staff based on difficulty of access across private lands or presence of sensitive plant species.Trail clubs responsible for each section of trail were identified and, through our partners at the Appalachian Trail Conservancy (ATC), club members were recruited to form survey teams. Two Virginia Naturalist Clubs and one local chapter of the Sierra Club also participated in volunteer recruitment. Volunteers  participated in training sessions prior to each field season. They were instructed on camera placement, maintenance, file management, and sampling protocol. Teams were assigned multiple sample points and given enough cameras to complete their assignment over a 7-month (April 1– October 30) period. Cameras were left at each location for an average of 30 days. Each team was free to organize placement and movement of cameras, but remained in periodic contact with the authors and ATC staff for assistance.Infra-red, remote-trip camera units  were used to record presence/absence of mammal species at each sample point. Volunteer teams were given UTM coordinates and instructed to place the camera <100 m from that location and >50 m from the trail. Cameras were set on trees at knee height (<0.5 m), oriented parallel with the slope, and the viewing area was cleared of obstructions. One ml of scent lure  was used 1 meter in front of the camera to slow animal movement and compensate for slow camera trigger times. Digital flash cards and batteries were switched at the end of each sampling session and cameras were moved to the next survey site. Volunteers recorded location (UTM), dates, scent and lure type. When possible, cameras were checked and lure was reapplied mid-month. A National Park Service website was created for volunteers to enter data and, through the website, digital pictures on the flash cards were uploaded to an ftp server. All volunteers recorded a picture each time they setup or checked the camera to ensure its continued operation. If the camera failed to trigger, the last recorded picture was considered the last operational day [LOOSESR]All uploaded photos were reviewed and subjectively graded (0–3) for quality based on orientation of camera. Sites with improper placements , camera malfunctions, insufficient survey time (<15 days), or incomplete data collected by volunteers were discarded. Species were identified and presence/absence data was recorded for each day in a detection history matrix following the approach of MacKenzie et al. We extracted from our GIS coverage environmental covariates that may be important for determining occupancy and detection probabilities. These covariates are listed in The modeling program PRESENCE was used to estimate occupancy (psi) and detection (p) probabilities for each species of interest (v. 2.2) A detection history for each site was created using the results from the repeated site surveys over the course of the three years given the aims of this study. Due to the nature of camera trapping data and the immense amount of data received, the detection history was consolidated prior to analysis to increase the statistical power of the models a priori models examined, we used a two-step model building method based on the Cormack-Jolly-Seber data type PRESENCE was then used to estimate the influence of the four sampling covariates  on site occupancy for each species. Multi-season modeling was used to account for colonization or extinction occurring between the sampling seasons. While utilizing this type of modeling allowed for the incorporation of colonization and extinction parameters in our models, we did not attempt to predict these parameters using covariates. The goal of this study is to determine the best predictors of site occupancy, not inter-season dynamics. Due to the strong correlation between the different scales of forest cover, each percent forest covariate was investigated independently in separate models. PRESENCE was also used to determine the effect of the 5 detection covariates on detection probabilities for each species . To limiA sampling effort of 18 807 camera days was conducted across 447 sites. Nineteen meso and large mammal species were detected and identified, along with 5 small mammal taxa, individuals from which we were unable to confidently identify to species . For theLynx rufus) showed a strong positive relationship and red fox (Vulpes vulpes), raccoons (Procyon lotor), and gray fox (Urocyon cinereoargenteusa strong negative association with the amount of deciduous forest within a 10 km radius of each site. Black bears (Ursus americanus) and white-tailed deer (Odocoileus virginianus) demonstrated a positive relationship with the amount of deciduous forest within 5 km. Forest cover was not an important predictor for opossums (Didelphis virginiana) or coyotes (Canis latrans) occupancy.Given lack of spatial autocorrelation, we determined top models for each species and summed model weights for each covariate with results listed in The presence of hunting on adjoining lands was the second most common factor selected by the models. Black bear and bobcat were negatively influenced by the presence of hunting in adjoining lands. The occupancy estimate of only one meso-predator, the raccoon, was positively affected by hunting in these adjoining areas.The third strongest predictor for mammal occupancy was the amount of recreational trail use surrounding each sample point. Occupancy for bears was negatively influenced by high trail use. Red fox was the only species positively influenced by high trail use. The influence of roads was represented in the top models for most species, but was only a heavily weighted variable for deer occupancy models. Deer occurrence demonstrated a negative correlation with distance to road.Detection probabilities for all species were affected by all the detection covariates. The summer season (June–Sept.) had the strongest positive influence on detection for bears, raccoon, and deer. Red and gray fox both showed a negative relationship with the summer season, with higher detection probabilities during the non-summer season. Lure type showed a positive effect on all species, with Pro’s Choice lure increasing detections for all species but red fox and opossum. The reapplication of lure during the mid-month check significantly increased detection probabilities for bears, bobcats, and white-tailed deer, but decreased detection probabilities for gray fox. Camera type had an influence on all species except for coyote and red fox, with Cuddeback brand cameras having a positive association with detection. Setup quality was consistently the top variable for most species and its impact was relative to the body size. For smaller mammals  cameras set too high had a negative influence on detection probabilities, while for larger animals  lower set cameras had a negative impact. Bobcat detections were not significantly influenced by camera setup.Using the AT as a mega-transect, we were able to model the relationship between species occupancy and multiple anthropogenic influences. Macro-level distribution studies have primarily focused on factors visible through readily available remote data (i.e. satellite) and have shown strong relationships between such factors and species distributions While much of the upland Appalachian oak forest has experienced significant expansion in the past 100 years, there has been significant habitat loss at lower elevations. Nearly 83 percent of the oak forest in this region has been altered, and large patches of intact habitat are limited primarily to upland public lands Our results also demonstrate the impact of forest loss is species-specific. Forest loss negatively affected the occurrence of larger carnivores such as bears and bobcats, while positively affecting many meso-carnivores such as fox, and raccoon. Two species, opossum and coyote, were not drastically impacted by change in forest cover. Not only did our results show variable response to forest cover, but also in the spatial scale at which species responded to forest loss, a trend demonstrated in other studies e.g., . All ourHunting has a direct effect on wildlife populations and their distributions. Wildlife management of game species is based on the premise that populations can be regulated by public hunting e.g., . Our resTrail use is highly variable along the AT, with heavy trail use generally occurring on sections of trail that are in close proximity to large population centers, provide easy access, and/or occur in popular state and national parks The ecological effects of roads have been well documented. Roads can have both negative and positive effects on species occurrence and may lead to increasing habitat fragmentation, mortality, behavioral modification, and resource availability Our findings show that localized human activities strongly affect mammal occupancy across broad landscapes. Such findings provide an important bridge between occupancy studies at smaller spatial extents Our results also indicate that the compounding effects that anthropogenic factors have on wildlife cannot be ignored. While models including only roads, trail use, or hunting were never among the top model set for any species, these covariates were often represented in top multivariate models and did contribute to predictive power of overall occupancy. For example, a model representing hunting alone did not predict bobcat occupancy well (ΔAIC = 4.40), however, when considered in concert with forest cover, hunting was a meaningful predictor for this species (Akaike weight = 0.23). Minor contributions to occupancy may not appear important when assessed individually, but can exert meaningful influence when considered cumulatively The citizen science-based camera trapping protocol employed in this study proved highly successful for generating macro-level occupancy and local environmental data of particularly high quantity and quality. The methodology employed in this study opens up new and burgeoning possibilities for asking large scale wildlife questions that otherwise may prove impossible to answer due to logistical constraints. Our success in using this methodology not only shows the promise of such approaches for continued monitoring within the AT corridor, but for other broader extent occupancy studies around the world. Employing numerous volunteers probably affected detection probabilities for most species due to variation in camera setup quality and ability to reapply lures mid-survey, but the development of software to successfully account for these effects on detections (i.e. PRESENCE) greatly enhances the potential of both citizen science and camera trapping methodologies in macro-level studies.A final and essential point is that the results of this study contribute to management of the Appalachian Trail corridor. The Appalachian Trail is the single most important corridor across the eastern United States. If it is to serve as a corridor between public lands for important wildlife, we must understand the attributes of an effective corridor and how to measure and monitor these attributes. While movement and connectivity were not assessed, occupancy modeling was able to identify influences on wildlife persistence within the corridor. As suggested in our results, protecting current forest habitat and encouraging continued reforestation and land acquisition would be extremely beneficial to a number of mammal species. For certain target species, hunting should be monitored closely and regulations should be adaptable in areas where occupancy might otherwise be low due to the effects of recreation and/or roads. Managers of these wildlife species should consider these factors on adjoining lands when setting management teams and actions along the AT.


Xenopus embryos provide a rich source of pluripotent cells that can be differentiated into functional organs. Since the molecular principles of vertebrate organogenesis appear to be conserved between Xenopus and mammals, this system can provide useful guidelines for the directional manipulation of human embryonic stem cells. Pluripotent Xenopus cells can be easily isolated from the animal pole of blastula stage Xenopus embryos. These so called “animal cap” cells represent prospective ectodermal cells, but give rise to endodermal, mesodermal and neuro-ectodermal derivatives if treated with the appropriate factors. These factors include evolutionary conserved modulators of the key developmental signal transduction pathways that can be supplied either by mRNA microinjection or direct application of recombinant proteins. This relatively simple system has added to our understanding of pancreas, liver, kidney, eye and heart development. In particular, recent studies have used animal cap cells to generate ectopic eyes and hearts, setting the stage for future work aimed at programming pluripotent cells for regenerative medicine. Amphibians have traditionally been used as model systems for vertebrate embryogenesis. They are particularly suited for the study of early embryonic patterning and induction events. In the 1920s, Hans Spemann and Hilde Mangold demonstrated that transplantation of a small piece of dorsal mesoderm (the “Spemann organizer”) into the ventral epidermis of a recipient embryo results in the formation of a complete secondary body axis [Xenopus embryo. If ACC are cultured without further manipulation they develop into “atypical epidermis”. Combination of ACC with vegetal cells generates dorsal, intermediate or ventral mesodermal tissue, depending on whether the vegetal fragment was taken from the dorsal or ventral side of the embryo [in situ hybridization based techniques. During the late 1960s, Peter Nieuwkoop and colleagues discovered that ectodermal cells can differentiate into mesodermal tissue if combined with prospective endodermal cells of the vegetal pole . These se embryo . Subseque embryo , or by ae embryo ,8,9. Allin vivo organogenesis. Second, programmed ACC were transplanted into Xenopus embryos where they generated organ specific cell types or functional organs . Thus, the discovery of the ACC as a source for pluripotent precursor cells, which can be directed to differentiate into a broad variety of specialized cell types, has paved the road for their use in the generation of ectopic, functional organs in vivo.Taking the use of the ACC system a step further, various higher order organotypic structures were generated employing a two-step protocol. First, ACC were programmed using different concentrations/combinations of regulatory molecules as outlined above. While the programming protocols utilized in these studies were mostly empirical, others studies have aimed to recapitulate the genetic program that controls t review ). Strikia)ACC can be exposed to extracellular signaling molecules like growth factors or chemical agents affecting signal transduction. Recombinant growth factors can be applied for short intervals or throughout the incubation time. Alternatively, growth factors can be produced by the ACC themselves. To this end, fertilized embryos are injected with mRNA or DNA coding for the respective growth factor prior to dissection of the ACC.b)etc. can also be supplied by microinjection of the corresponding mRNAs. As a variation of this procedure, microinjected explants can be sandwiched with naïve ACC, allowing for the distinction of cell-autonomous and non-cell-autonomous effects.Intracellularly active proteins, such as transcription factors, signal transduction proteins, c)The developmental potential of specific tissues can be analyzed by heterotopic “Sandwich” assays combining ACC with tissue explants from the same or different embryonic sources.d)Manipulated ACC can be transplanted into recipient embryos to analyze if they can form functional ectopic organs.The pluripotent cells of the blastocoel roof, the ACC, are organized in several ectodermal cell layers, normally fated to develop into epidermis. They can be cultivated in simple saline solution for up to one or two weeks , correspThese different experimental protocols have not only been applied to the analysis of tissue/organ formation, but quite extensively also to problems relating to signal transduction mechanisms, cell adhesion phenomena and more. In addition to the activities employed in the pioneering studies mentioned above, various other factors, including secreted modulators and transcription factors, have successfully been used to direct ACC development. As outlined below, activin and retinoic acid, as well as the BMP-inhibitor noggin, have proven particularly useful to generate a broad range of tissue types from ACC. Furthermore, transcription factors can not only be used to generate a germ layer specific ground state (such as VegT for the endoderm), but combinations of transcription factors can drive organ specific development with high efficiency and specificity. An example of this is the generation of functional eyes by the eye field transcription factors. These and further examples of use of the ACC to induce different organs will be summarized in the following chapter.in vivo signaling situation [Xenopus embryo microinjection, Afelik et al. [The vertebrate pancreas and liver are organs that play a key role in energy metabolism and homeostasis. They both originate from the anterior foregut endoderm, where the pancreas derives from a dorsal and a ventral anlage. The liver and ventral pancreas originate from a common population of precursor cells. High levels of FGF and BMP signaling from the adjacent mesodermal tissue induce the liver specific gene program and suppress pancreas specification. Additionally, retinoic acid (RA) signaling has a conserved, essential role for pancreas development . The fiituation ,15,16 . Proin vitro .Xenopus embryos. It is a derivative of the intermediate mesoderm and requires inducing signals to specify the pronephric anlagen during early gastrula stages of development. At tailbud stage, the developing pronephros becomes morphologically distinguishable and turns into a functional secretory organ (reviewed in [The pronephros constitutes the primary secretory organ of iewed in ).in vitro. Histological analysis showed that ACC treated with a combination of activin and RA developed into pronephric tubules at a high frequency [Xenopus embryos, which had undergone bilateral pronephrectomy, suggests that they develop into functional secretory units. Although pronephrectomized embryos exhibited edema formation at high frequency and died after 7–9 days of surgery, transplanted animals formed less edema and had an increased survival rate [It was again Asashima and colleagues who first reported that pronephric structures can be generated from ACC requency . With threquency ,25,26. Sval rate . Xenopus embryos. The programmed ACC or the intermediate mesoderm cultured in isolation did not reveal any significant indication of pronephros formation; however, their combination (“Holtfreter sandwich”) resulted in the formation of pronephric structures at high frequency. This procedure takes in vitro organ formation a step further by mimicking aspects of the often very complex tissue interactions.A recent, elegant study, using a “sandwich” explant system including programmed ACC, identified a prime candidate signaling molecule for pronephros induction . ACC werXenopus, heart development is initiated at the onset of gastrulation and requires an interplay of signals from the dorsal lip and the underlying endoderm. Initially, two patches of the mesoderm on the dorsal side of the embryo are specified towards cardiac fate. During gastrulation, these heart progenitors move dorso-anteriorly and continue to migrate ventrally at neurula stages. After meeting at the ventral midline, they fuse and form the linear heart tube, which then undergoes the looping and remodeling processes of cardiac morphogenesis.The development of the heart is highly conserved among vertebrates. In Xenopus provides a unique combination of straightforward gain- and loss-of-function analyses with a remarkable ability to heal after microsurgery, which has enabled Xenopus to become one of the favorite model systems for studying cardiogenesis [in vitro by treating dorsal lip explants with suramin, a polyanionic compound [Xenopus explants as valuable tools for the molecular analysis of cardiac induction and differentiation.Analyzing the precise molecular events that determine cardiac fate and ultimately formation of the heart is a topic of intense ongoing research. ogenesis ,30. In acompound . Transplcompound . These ein vitro-generated myocardial cells were functional, activin-treated ACC reaggregates were implanted into the posterior abdomen of neurula stage control embryos. Ectopic beating hearts were generated  and the neural patterning gene otx2, differentiate into all retinal cell classes and form a neuronal network necessary for vision . Using al embryo A–D, indil embryo E–J. ThesTaken together, these data show that pluripotent ACC can form diverse retinal cell classes and even functional eyes, if treated with the appropriate combination of factors. Thus, these studies open the way to characterize the role of various intrinsic and extrinsic factors and their combinations for their ability to direct pluripotent ACC to a retinal fate. Future challenges will be to determine how to maintain cultures of retinal precursor cells and to analyze if they could be used for retinal repair of damaged or diseased eye tissue. Xenopus ACC system has already a long and successful tradition, but it is expected that it will further benefit from recent genetic and genomic advances. The use of mutant and transgenic Xenopus lines, which are increasingly available, remain to be exploited in this context. For example, the generation of transgenes carrying inducible versions of transcription factors would allow temporal control of their activation. Thus, more refined, multistep differentiation protocols could be developed. Once Xenopus lines with mutations in disease-associated genes are available, in vitro organ generation from such animals may be suitable for drug development. Moreover, advanced technologies that are based upon genomic sequence information, like microarray analysis and proteomics, are now readily available. Organ-specific tissues generated from manipulated ACC making use of wild-type as well as mutant Xenopus lines hold the promise of the discovery of novel genes and a more complete definition of the gene networks that govern organ development in vertebrates.The Several of the experiments outlined here were designed with the strategy to recapitulate key regulatory steps in organ development as they are known from embryological studies, others were equally successful using a more empirical approach. The most impressive example for the latter scenario is provided by the pioneering studies of the Asashima group, where different concentrations of RA in combination with activin were found to be sufficient to generate a whole variety of different organs and tissues . These iXenopus ACC define a source of pluripotent vertebrate precursor cells; their versatility is comparable to other populations of pluripotent precursor cells, such as mammalian IPS cells. A hope that is connected to the IPS system is that these pluripotent cells could be used to engineer patient-specific tissue or even organs for regenerative medicine. In general, the underlying molecular principles in vertebrate organogenesis appear to be highly conserved. Therefore, protocols established in the very robust—and therefore easy to handle—ACC system can serve as guidelines for the directional manipulation of mammalian embryonic stem cells.Xenopus animal cap explants makes us optimistic for another 50 years of groundbreaking insights into the secrets of organ development.Thus, looking back on half a century of exciting research with


Antisocial and criminal behaviors are multifactorial traits whose interpretation relies on multiple disciplines. Since these interpretations may have social, moral and legal implications, a constant review of the evidence is necessary before any scientific claim is considered as truth. A recent study proposed that men with wider faces relative to facial height (fWHR) are more likely to develop unethical behaviour mediated by a psychological sense of power. This research was based on reports suggesting that sexual dimorphism and selection would be responsible for a correlation between fWHR and aggression. Here we show that 4,960 individuals from 94 modern human populations belonging to a vast array of genetic and cultural contexts do not display significant amounts of fWHR sexual dimorphism. Further analyses using populations with associated ethnographical records as well as samples of male prisoners of the Mexico City Federal Penitentiary condemned by crimes of variable level of inter-personal aggression  did not show significant evidence, suggesting that populations/individuals with higher levels of bellicosity, aggressive behaviour, or power-mediated behaviour display greater fWHR. Finally, a regression analysis of fWHR on individual's fitness showed no significant correlation between this facial trait and reproductive success. Overall, our results suggest that facial attributes are poor predictors of aggressive behaviour, or at least, that sexual selection was weak enough to leave a signal on patterns of between- and within-sex and population facial variation. Since the form of the head is a good predictor of brain shape, it follows from this perspective that an individual's morality could be predicted by its head shape. This research program attained its maximum splendour during the mid-19th century under the label of phrenology, and was revitalized during early 20th century positivism as an attempt to solve criminological cases. The most prominent defender of this school was Cesare Lombroso, who argued that criminals are distinguished from non-criminals by a set of physical anomalies, reminiscent of primitive, ancestral human stages Since the work published by Gall in 1835 The advent of the population genetics paradigm provided a new scenario for the analysis of human evolutionary and developmental patterns of craniofacial variation. Along with research on the socio-cultural variability of human behaviours performed from the last half of the 20th century, it has been demonstrated that there is no straightforward connection between behaviour and physical appearance However, a handful of recent articles Some recent papers have demonstrated that fWHR is not dimorphic on a sample of Turkish university students Even when the papers detailed above present contradictory evidence regarding the alleged adaptive nature of fWHR variation, it is still necessary to test this adaptive hypothesis on worldwide, cross-cultural populations, to measure fWHR sexual dimorphism on groups displaying differing levels of interpersonal violence, and to evaluate putative associations among male fitness and fWHR. Cross-cultural work, particularly in traditional societies, will be especially useful to this end To maximize the number of sampled populations and to achieve large sample sizes, we based our analysis on previously published databases that involve several types of raw data, including craniofacial measurements, 2D and 3D craniofacial landmark coordinates taken on dried skulls. We verified that for each database a good estimator of fWHR could be computed. Note that all of these methods to estimate fWHR are superior to that based on non oriented photographs Databases were analyzed separately because of subtle differences in measurement or landmark definitions. Nevertheless, all the databases matched with the general definition of fWHR (the ratio of bizygomatic breadth to nasion-prostion height). Sample composition and specific details of each database are provided on For each database, a variable amount of indices were computed according to traditional formulae see . These iA global estimation of sexual dimorphism was computed as the ratio among the male to the female average value for each index. Statistical significance of sexual dimorphism for each index on each database was evaluated by t-tests for independent samples. Then, the significance of sexual dimorphism was evaluated at the population level, within each database. To analyze the apportionment of population variation across the different indices, male and female Fst values for each index were also computed Specific subsets of populations were selected to compare fWHR's sexual dimorphism on populations for which ethnographic records of within-group interpersonal violence To further assess differences among groups with varying levels of interpersonal violence we performed an additional analysis based on the Mexico database, directly testing for differences on fWHR scores between samples of male prisoners of the Mexico City Federal Penitentiary condemned by crimes of variable level of inter-personal aggression  and a comparative sample of random, non-condemned individuals belonging to the same urban population Finally, we regressed and correlated male fWHR scores on a measure of reproductive fitness using a pedigreed sample from the Austrian population of Hallstatt, which furnishes a unique chance to compute quantitative genetic parameters for skull shape P = 0.05, excepting for the Hallstat sample, showing significant dimorphism. Furthermore, only seven out of 89 comparisons (7.86%) yielded fWHR values significantly greater in males than in females  Previous studies assessing the relationship between fWHR and aggressiveness suggest that fWHR itself is not responsible for the behavioural phenotype, but a co-variable of another trait, such as testosterone level However, a growing number of studies demonstrate that fWHR is not a dimorphic trait Our results demonstrate that fWHR is a poor predictor of aggressive behaviour, or, at least, that sexual selection was weak enough to leave a signal on patterns of between and within sex and population human facial variation. More complex models of sexual selection Our further results concerning fWHR dimorphism comparisons on groups with ethnographically documented variable levels of within-group interpersonal violence, and fWHR comparison in individuals subjected to prosecution decisions based on different levels of violence with non-prosecuted individuals also underscore that fWHR is not significantly associated with aggressive behaviour. Despite caution is needed with general classifications of society types and corresponding estimations of interpersonal violence, this type of information can be used to test whether and how fWHR varies in populations with variable influence of the socio-cultural factors controlling and preventing the more extreme forms of violence To fully understand the inherent variation of fWHR in human populations, it must be recalled that fWHR is an attribute of the skull, which is a complex structure with a pervasive pattern of morphological integration that constraints its evolution along lines or planes of least evolutionary resistance To sum up, we argue that, even when localized, low-scale studies may suggest that fWHR is a trait shaped by sexual selection, a population genetics approach to fWHR variation on worldwide cross-cultural modern human populations clearly show that this trait does not present any of the expected signals of past sexual selection operating during the course of human evolution. A likely explanation for these results is that behavioural repertoires are so plastic that socio-cultural environments These kinds of results are at odds with adaptive explanations, and demonstrate how socio-cultural aspects are determinants of great amounts of the behavioural repertoire.Furthermore, we suggest that analyses made on particular, highly localized samples and focused on the correlation among physical attributes and specific behaviours should incorporate and control for specific measurements of socio-cultural context in order to provide a more realistic approach to the complexity of behavioural manifestations. For instance, analyses made on different populations Since the alleged existence of significant, statistically demonstrated and scientifically based relationship among facial attributes and behavioural traits involving morality and ethics can have social effects , we suggest that future analyses aimed to detect relationships among facial attributes and behaviour must be reinforced by cross-cultural controls, longitudinal samples, and a solid background on population genetics.Figure S1Box and whisker plots of fWHR depicting inter-sexual differences across the populations of each database. a) Howells database; b) Pucciarelli database, c) 2D Geometric Morphometric database, d) 3D Geometric Morphometric database, e) Patagonian groups database. Square: median; box: 25%–75%; whisker: minimum-maximum values. Orange: females, blue: males. Populations showing significantly greater male fWHR (after t-test for independent samples) are marked with grey boxes.(TIF)Click here for additional data file.Table S1Sample details for each database, including Pinker's sociocultural classifications.(XLSX)Click here for additional data file.


Post-discharge support is key to effective treatment for hospitalized smokers. The few hospitals that systematically address tobacco refer smokers via fax to tobacco quitlines, yet few smokers enroll. “Warm handoff”  is used in some cases of tobacco screening and brief intervention (SBI) to link patients with treatment, but little data exists on process or outcomes. We present pilot outcomes and early implementation data on an ongoing randomized controlled trial (RCT). Recruitment began in July 1, 2011. The purpose of the trial (Enhancing Quitline Utilization by Inpatients [EQUIP]) is to determine the effectiveness and cost-effectiveness of warm handoff versus fax referral for linking hospitalized smokers with tobacco quitlines. The EQUIP trial is a two-arm RCT funded by the National Institutes of Health  in which smokers who wish to quit permanently after discharge are randomized to either fax referral  or warm handoff . Outcomes, including costs, are assessed at one, six, and 12 months following baseline. We hypothesize that warm handoff will outperform fax referral in terms of enrollment in services, cessation, and cost-effectiveness. This study explores what alcohol and other drug trials have done to strengthen handoffs and the evidence for efficacy; how EQUIP compares with AOD handoffs, and what pitfalls might be expected; and whether tobacco SBI and referral to treatment would or could be incorporated into alcohol and other drug SBI and referral to treatment.


Biological invasions can transform our understanding of how the interplay of historical isolation and contemporary (human-aided) dispersal affects the structure of intraspecific diversity in functional traits, and in turn, how changes in functional traits affect other scales of biological organization such as communities and ecosystems. Because biological invasions frequently involve the admixture of previously isolated lineages as a result of human-aided dispersal, studies of invasive populations can reveal how admixture results in novel genotypes and shifts in functional trait variation within populations. Further, because invasive species can be ecosystem engineers within invaded ecosystems, admixture-induced shifts in the functional traits of invaders can affect the composition of native biodiversity and alter the flow of resources through the system. Thus, invasions represent promising yet under-investigated examples of how the effects of short-term evolutionary changes can cascade across biological scales of diversity. Here, we propose a conceptual framework that admixture between divergent source populations during biological invasions can reorganize the genetic variation underlying key functional traits, leading to shifts in the mean and variance of functional traits within invasive populations. Changes in the mean or variance of key traits can initiate new ecological feedback mechanisms that result in a critical transition from a native ecosystem to a novel invasive ecosystem. We illustrate the application of this framework with reference to a well-studied plant model system in invasion biology and show how a combination of quantitative genetic experiments, functional trait studies, whole ecosystem field studies and modeling can be used to explore the dynamics predicted to trigger these critical transitions. It is increasingly clear that interactions between ecological and evolutionary processes have the potential to affect biodiversity at multiple scales of organization   and invasive (I) species):In which, BN indicates native species biomass and BI invasive species biomass. Further, a is the rate at which nutrients are replenished, S the soil nutrient availability in the absence of plants, qR,i the nitrogen content of plant species i, and ρ the bulk density of the soil and lRoot the plant rooting depth. Plant material intercepts light, meaning that light availability decreases with increasing plant biomass density. Because this process occurs on a much faster timescale than the processes mentioned above, a quasi-steady state can be assumed .Previous research suggests that novel, invasive genotypes such as those produced by admixture may undergo a shift in biomass production and carbon:nitrogen ratios in leaf tissue  will decompose slower, higher quality litter decomposes faster.in which If litter dynamics are included, feedbacks can arise: competitive interactions between the native and invasive plant species then determine the quantity and quality of litter formed, which in turns modifies environmental conditions , which feeds back to the competitive ability of both species  caused by admixture, and possibly also post-admixture phenotypic selection, shifts the trait distribution of the invasive population past the predicted tipping point. The proposed framework can be used to analyze how admixture-induced changes in the mean or variance of key functional traits would affect ecosystem stability.Phalaris arundinacea in North American wetlands, Eppinga et al.  Phalaris genotypes in the system increases. This can initiate a positive litter feedback, as reduced competition from native species may outweigh the negative effect of litter accumulation on the growth of invasive Phalaris genotypes. Model analyses suggest that this positive litter feedback may be initiated above a certain threshold in the litter:biomass ratio i is the probability of occupation of the cell at position  by genotype i in the next timestep, C is the total number of genotypes and species in the community. Further, the summations over x′ and y′ indicate the size of the assumed interaction neighborhood (here we use a discrete neighborhood (Moore)). By including genotypes that vary in their C:N ratio in the simulations as is expected post-admixture, we can investigate how genetically determined variation in this key trait (leaf C:N ratio) affects the potential spatial spread of Phalaris and triggers a transition.in which Phalaris arundinacea. Therefore, we simulate competition between a phenotypically diverse Phalaris population and a native species along a nutrient supply gradient. To parameterize the model, we include 10 genotypes spanning the range of C:N ratios observed in natural populations of invasive Phalaris that are the products of admixture between divergent native range sources  can be planted into plots with the idea that the resulting ecosystem properties can be tested pre- and post-introduction. Such data would provide a mechanistic understanding of the role that genotypic variation and even genotype-specific traits in an invasive population have on ecosystem properties. While these experiments would no doubt be challenging to conduct, requiring many replicate gardens or mesocosms to capture the effects of genotypic variation, they are feasible, especially for plants that can be clonally propagated for experimentation. Further, while these types of experiments would likely involve a complex series of predictors and hypothesized feedback relationships, the relationships could be synthesized using tools such as structural equation modeling (SEM) that combines path analysis and factor analysis to compare specific hypotheses about how different variables in the system interrelate Grace .We have outlined a synthetic framework that incorporates model predictions and experimental approaches to determining how variance in trait expression during invasion may lead to ecosystem transformation. Our study has indicated that the enhanced trait variation found in introduced populations of invasive species brought about through admixture can ultimately alter the diversity and structure of natural communities, reduce ecosystem resilience, and ultimately be responsible for abrupt shifts in ecosystem states. Moreover, studying the extreme trait variance that results from admixed genotypes post-introduction can enhance our understanding of how intraspecific diversity in functional traits can alter ecosystem function and ultimately lead to a better understanding of how the combined effects of both ecological and evolutionary dynamics interact in natural populations. A comprehensive approach that combines theoretical modeling, quantitative genetics, and manipulative field studies will lead to a deeper understanding of how human-aided dispersal of populations to new ranges can be a key step in the ecological and evolutionary processes that lead to critical transitions to a new ecosystem state.


Thirty years since the discovery of HIV, the HIV pandemic in sub-Saharan Africa accounts for more than two thirds of the world’s HIV infections. Southern Africa remains the region most severely affected by the epidemic. Women continue to bear the brunt of the epidemic with young women infected almost ten years earlier compared to their male counterparts. Epidemiological evidence suggests unacceptably high HIV prevalence and incidence rates among women. A multitude of factors increase women’s vulnerability to HIV acquisition, including, biological, behavioral, socioeconomic, cultural and structural risks. There is no magic bullet and behavior alone is unlikely to change the course of the epidemic. Considerable progress has been made in biomedical, behavioral and structural strategies for HIV prevention with attendant challenges of developing appropriate HIV prevention packages which take into consideration the socioeconomic and cultural context of women in society at large. Sub-Saharan Africa (SSA) remains the region most affected by the HIV epidemic. Almost three quarters (69%) of the 23.5 million people infected worldwide reside in this region . Most coet al. substantiated these results in the 2013 Human Sciences Research Council (HSRC) household survey which showed that being young and unmarried increased the risk of HIV acquisition among South African women [In SSA, women bear the brunt of the HIV epidemic . In 2011an women .The disproportionate impact of the HIV epidemic on women can be attributable to several factors including biological, social, behavioral, cultural, economic and structural. In SSA a combination of these factors has led to the disparate increase in HIV infection rates among women compared to their male counterparts .et al.[Neisseria Gonorrhea, Chlamydia Trachomatis, Syphilis, and Trichomonas Vaginalis) at 4 clinical trials sites namely Durban and Hlabisa (South Africa), Lusaka (Zambia) and Moshi (Tanzania). The study showed that women at the South African sites were 3 times more likely to acquire an STI than participants at the Tanzanian and Zambian sites. Studies on HIV/STI “hot spots” among women in communities in the greater Durban area in the province of KwaZulu-Natal (South Africa) theorize an overlap of prevalence and incidence of these infections, suggesting increased risk of HIV acquisition in STI/HIV “hot spot” areas within these communities [Women are at a greater physiological risk of contracting HIV than men. This is in part because women have a greater mucosal surface area exposed to pathogens and infectious fluid for longer periods during sexual intercourse and are likely to face increased tissue injury. Young women are at particularly high risk due to cervical ectopy which facilitates greater exposure of target cells to trauma and pathogens in the vagina . It has et al. determinmunities .Hormones such as progesterone are reported to be playing a role in a woman’s biological vulnerability to HIV infection. Observational evidence suggests that progesterone containing injectable contraception depot medroxyprogesterone acetate (DMPA) may be putting women at higher risk of HIV acquisition -23. TherStudies in Uganda, Rwanda and Zimbabwe have shown that pregnant women are at a higher risk of HIV infection than lactating or other women, possibly due to physiological changes that a woman undergoes during pregnancy . High leSupporting evidence suggests that women have a window of vulnerability approximately seven to ten days after ovulation in their menstrual cycle in which the potential for viral infectivity in the female reproductive tract is increased. This is due to the suppressing influence of sex hormones on the innate, humoral and cell-mediated immune systems. This takes place in the upper and lower female reproductive tract, and overlaps with the upregulation of co-receptors for HIV uptake and the recruitment of potentially infectable cells .For women in SSA, probabilities of HIV exposure, acquisition and transmission is influenced by a range of factors such as infectiousness of the male partner according to the stage of the disease, or presence of ulcers in the male partner in the event of unprotected sexual intercourse. At a broader societal level, several factors influence the scale and rate of the epidemic spread such as overall HIV and STI prevalence, sexual practices, marriage and other cultural norms including structural factors not in control of the individual.The dominant patriarchal culture and society in Africa exacerbates women’s inferiority and their disparate health status. Here, the needs and desires of women are not considered significant and often women play no part in sexual decision making, nor are they allowed to express their sexuality ,32. TherIn countries such as Malawi, Kenya, Zambia, and Botswana, it is widely believed that a widow becomes unclean after her late husband’s burial ceremonies. In cleansing rituals, an unprotected sexual act is believed to purify the recipient through the semen entering the woman’s body . CommuniThe practice of “dry sex” is reportedly high in some southern African countries . This prVirginity testing, , is now widely regarded as a human rights abuse. Although virginity testing was a common traditional practice among unmarried couples in South Africa and Uganda, it was, until recently, no longer commonplace. However, in an attempt to promote abstinence among girls, this practice has seen resurgence . A conceIn many African countries, young girls are encouraged or forced to marry older men ,38 whichIn the last two decades, nearly all SSA countries have faced slowing economic growth which has influenced spending on social services . This haPoverty is another driving force of HIV transmission in women . EvidencTransactional sex (sex for money) is common in SSA. Many women engage in sex work which is defined as the provision of sexual services in exchange for money, goods, or other benefits . GloballIt is well documented that behavioral change can lead to a decrease in HIV acquisition . These iConcurrent partnerships are powerful transmitters of HIV in the community . ConcurrThe vulnerability to HIV infection increases by engaging in unprotected anal sex by 13 fold compared to oral sex . This isAlcohol abuse in much of Africa is characterized by irregular episodes of heavy drinking, frequently in the form of weekend bingeing . These dMany structural vulnerabilities place women at risk including gender inequality and gender based violence (GBV) , migratiGender norms promote multiple concurrent sexual partners for men while women are expected to be monogamous and unquestioning of their partner’s behavior ,65. HoweViolence against women is a global problem and a well-recognized risk factor for HIV and other STIs ,63,69. IDiscrimination is often based on gender, race/ethnicity, sexual orientation, and HIV status . Stigma Migration trends in Africa vary in terms of their spatial, temporal and social characteristics. Many individuals migrate due to better job prospects or are forced to migrate due to political instability, war and famine . WorldwiUrbanization has also fuelled the rapid spread of HIV. In SSA, urban HIV prevalence is higher than rural populations . UrbanizGiven the magnitude of women’s vulnerability to HIV acquisition as described above, it is not surprising that there is a worldwide call to address the high HIV infection rates reported among women in SSA and elsewhere. Biomedical, behavioural and structural interventions have been investigated to reduce women’s vulnerability to HIV acquisition but also HIV incidence in general.Male condoms in general remain the most effective method of HIV prevention. However condom use or negotiation thereof is not always in the woman’s hands. Although female condoms are available, the use still requires negotiation. Thus female-initiated prevention methods have been investigated.Microbicides are products with anti-HIV properties developed in numerous formulations for prevention of HIV. Microbicides were initially developed for vaginal application but are now also tested for rectal application . Over thSuccess with research among HIV discordant couples showed a decrease in HIV acquisition when a negative partner in a dicordant partnership was provided with oral pre-exposure prophylaxis (PrEP). Partners taking oral tenofovir or tenofovir plus emtricitibine (Truvada) as prophlaxis had a reduced rate of HIV acquisition 67% (95% CI 44 to 81) and 75% (95% CI 55 to 87) respectively . The higTreatment with ARV for prevention (TasP) has been reported to be highly efficacious (96%) among both men and women in a discordant couple relationship where the positive partner was provided ARV treatment to reduce HIV transmission to the negative partner . TheforeHIV counseling and testing is recognized as a critical component for all HIV prevention interventions. Testing provides the opportunity to identify people who are HIV positive and in need for treatment and care and can also identify people who are HIV negative and referred for HIV prevention and care. Regular HIV testing will be critical for prevention interventions using PrEP. HIV counseling and testing not only allows women, couples, and families to learn their HIV status and obtain personalized risk reduction counseling and further care appropriate to their status, but can also assist communities in addressing stigma and discrimination associated with HIV/ AIDS ,88.Peer education interventions in developing countries have shown a significant increase in HIV knowledge and increased condom use. However, these programs have shown only a moderate improvement of behavioral outcomes, with no significant effect on biological outcomes . Kenyan It is widely known that women who have an early sexual debut participate in risky sexual behavior, such as having multiple partners, higher STI incidence and decreased condom and contraceptive use ,52. HighConcurrent sexual partnerships are recognized as being significantly responsible for the transmission of STIs, particularly heterosexual HIV transmission. Evidence implies that concurrent partnerships can increase the size of an HIV epidemic, the speed at which it infects a population and its persistence within a population . Uganda Structural interventions attempt to change the social norms that worsen HIV vulnerability. The South African IMAGE project sought to decrease gender based HIV vulnerabilities such as sexual violence, women’s lack of knowledge about HIV and its transmission and women’s economic dependency on men. The study partnered with a local microfinance institution to permit women to participate in microenterprises, while providing them with HIV education and a forum to discuss action against gender-based violence. These women improved their household wellbeing and empowerment, in addition to the significant reduction in gender-based violence .Microfinance programs attempt to ease poverty by offering access to credit, savings or business skills. In South Africa, a study using an integrated curriculum of gender equity, anti-violence work and HIV/AIDS education together with an existing microfinance program demonstrated higher levels of HIV-related communication by women in addition to a 55% reduction in domestic violence. Women in this study were less likely to have had unprotected sex at their last sex act and more likely to have accessed HIV Counselling and Testing . In MalaEducation reduces risk of HIV infection, boosts self-esteem, and provides alternatives for earning a livelihood . CommuniPolicy structural factors play a major role in HIV transmission and Uganda is the best example of successful policy changes and the link to decreased HIV transmission. The Ugandan government committed itself to extensive training of trainers, mass media, health education campaigns, countrywide messaging and district mobilization causing changes in sexual behavior, social norms and HIV prevalence . ResearcConvincing evidence from biological, epidemiological, sociocultural and structural challenges identify women’s vulnerability to HIV infection in SSA. The evidence shows that women in SSA are at higher risk of HIV infection compared to their male counterparts. Through gender power dynamics, women are embedded in relationships which increase their risk even more. Other than personal behavior, several other issues such as structural influences are identified which are beyond women's control and are likely to affect women globally. Given the unacceptably high prevalence and incidence rates of HIV in young women in southern Africa, every effort to mitigate this risk is urgently needed with a combination of biomedical, behavioural and structural interventions targeted not only to the women but their sexual partner(s) as well. Recent advances in the HIV prevention field need to be stepped up by implementing appropriate HIV prevention packages taking into consideration the cultural environment, biological, socio-economic and structural risks facing women.The authors declare that they have no competing interests.Both GR and BD contributed to the drafting of the manuscript. Both authors read and approved the final manuscript.


Causes of phenotypic variation are fundamental to evolutionary ecology because they influence the traits acted upon by natural selection. One such cause of phenotypic variation is a maternal effect, which is the influence of the environment experienced by a female (and her corresponding phenotype) on the phenotype of her offspring (independent of the offspring’s genotype). While maternal effects are well documented, the longevity and fitness impact of these effects remains unclear because it is difficult to follow free-living individuals through their reproductive lifetimes. For long-lived species, it has been suggested that maternal effects are masked by environmental variables acting on offspring in years following the period of dependence. Our objective was to use indirect measures of maternal condition to determine if maternal effects have long-lasting influences on male offspring in two species of cervid. Because antlers are sexually selected, we used measures of antler size at time of death, 1.5–21.5 years after gestation to investigate maternal effects. We quantified antler size of 11,000 male elk and mule deer born throughout the intermountain western US (6 states) over nearly 30 years. Maternal condition during development was estimated indirectly using a suite of abiotic variables known to influence condition of cervids . Antler size of male cervids was significantly associated with our indirect measure of maternal condition during gestation and lactation. Assuming the correctness of our indirect measure, our findings demonstrate that antler size is a sexually selected trait that is influenced–into adulthood–by maternal condition. This link emphasizes the importance of considering inherited environmental effects when interpreting population dynamics or examining reproductive success of long-lived organisms. Causes of phenotypic variation are fundamental to evolutionary ecology because they influence the traits acted upon by natural selection in utero or during dependency By definition, maternal effects are “the influence of the maternally provided environment on the phenotype of her offspring” Odocoileus virginianus) through 2.5 years Bison bison; 4 years of maternal effects) Rangifer tarandus; 18 months) Ovis canadensis; body mass at 5 years was influenced by maternal effects) (Cervus elaphus) on the Isle of Rum  have offered a thorough examination of the impacts of maternal effects While maternal effects are well documented, the longevity and impact of these effects remains unclear as most available data evaluate maternal effects only through the first 1–4 years of life. For example, Schultz and Johnson found maternal effects influenced body mass of white-tailed deer  generally have greater birth masses and survival rates through their first summer Ovis aries) mothers exhibit lighter birth masses and lower survival. This association illustrates that the lower survival of these offspring is likely not a result of lesser maternal experience, but is instead a result of decreased maternal condition in yearling mothers. Conversely, the survival of young white-tailed deer increases with maternal age (likely related to both condition and experience) Ungulate populations may provide a model system to study maternal effects because individuals are relatively long-lived and short-term maternal effects are well established Odocoileus hemionus], and elk [Cervus canadensis]). Additionally, climate factors are known to influence survival probability in mule deer and elk The limitation of using ungulates (or any other long-lived species) to assess the influence of maternal effects on offspring is that it is difficult to follow free-living individuals and obtain measures of condition or quantify life history characters. Indirect assessment of maternal condition may provide supporting evidence for long-lasting maternal effects. Abiotic variables , for example, are strongly correlated with female condition Our objective was to use indirect measures to determine if maternal condition has long-lasting influences on a sexually selected character of male cervids. More specifically, we examined the relative influence of climate variables during year of birth  on antler size. If climate variables from the year of birth have an effect on adult antler size at time of harvest, then maternal effects are long-lasting for these two species and are not entirely masked by environmental variables acting on offspring in the years following dependence. Conversely, if climate variables from year-of-birth do not affect antler size at time of harvest, then we would conclude that maternal effects do not exist or are masked by environmental variables in the years between the period of dependence and adulthood for long-lived species.We collected data on annual climatic conditions during the year of birth of offspring  and male antler size from two species, the American elk and the mule deer, across 20 free-range sites in the Intermountain United States. Sites were located in Colorado, Montana, Nevada, New Mexico, Utah, and Wyoming . These sWe removed teeth from individuals following harvest and determined age by counting annuli in cementum We paired each sample site with a national climatic data center (NCDC) weather station. We selected weather stations based on the station’s proximity and elevation relative to the site where sampling occurred. We chose the nearest station to the site if that station had collected data across all years data were available from the associated site. If the nearest station lacked data for some of the years, we selected the nearest station with similar temperature, precipitation, and snowfall averages to the station with insufficient data. If two stations were similar distances from the site, we selected the station nearer in elevation.We obtained both winter and spring/summer weather data for each site from its corresponding NCDC weather station. Weather variables included the average temperature, precipitation, and snowfall for October through July for the year of harvest and the year of birth of each individual. In a few cases where these data were not available, we used the 40-year (1970–2010) average value for that site and variable to ensure that the missing data did not bias our analysis (imputation occurred for less than 1.5% of climate observations obtained from NCDC weather stations). From these data, we developed synthetic climate variables to minimize the number of parameters in our models. We determined a monthly z-score iw) a priori models for both elk and mule deer. These models described hypotheses about variation in antler size as a function of location, age, and weather influences  did not exist within individual models. We again judged relative support for these final models based on the minimization of Akaike’s Information Criterion iw) We used model selection to determine relative support for biologically plausible, linear, mixed-effects models  of antler size where site was treated as a random effect. First, we modeled antler size as a function of year-of-birth or year-of-harvest influences  and 3. WWe collected age and antler measurements from 4,548 mule deer . ResultsWe collected age and antler measurements from 5,295 elk . ResultsOur results indicate that year-of-birth environmental conditions influence the adult antler size at time of harvest for both elk and mule deer. Because environmental conditions (particularly during winter) often dictate an individual’s phenotypic quality  Using climate indices as an indicator of maternal condition results in a proxy for average maternal condition across a population. Using this surrogate  limits our ability to detect individual variation  and make inference about the effects of varying habitat quality, health of individual females, or other causes of varying female condition. However, based on current understanding of abiotic factors that influence condition, all individuals in a population should, on average, be either negatively or positively affected by climatic variation, albeit to different extents. Therefore, inter-annual variation in climate should be an acceptable surrogate for condition. Further, there was a significant effect of our proxy even though we could not account for variation among individual females . We recognize that through selective harvest our samples may potentially be biased toward individuals that grow larger antlers earlier in life. However, this potential bias should be consistent among locations and across the study period, and therefore unlikely to influence our conclusions.As our data were collected on a broad temporal and spatial scale, we conclude that our results are applicable to elk and mule deer populations across the western United States. It is important however to consider the drastically different climates found in this region when interpreting results. For example, our analysis of elk antler size indicates that increased snowfall (October – July) during the year of birth is positively correlated with adult antler size. While this seems counter-intuitive for many western climates, water is likely the limiting factor in the desert systems of the southwest After detecting a maternally affected trait, the next logical step is to examine its evolutionary or biological consequences. Variance attributed to maternal effects  may disappear during development through compensatory growth Capreolus capreolus). Among other things, this discrepancy may be the result of location, sample size, or the species of ungulate in question. Vanpe et al. In conjunction with support for the maternal effects hypothesis, our analysis indicates that year-of-harvest environmental conditions have a significant impact on the antler growth, and therefore reproductive success of elk and mule deer. This is contradictory to recent findings by Vanpe et al. Our results do not support the assertion that environmental factors might mask any maternal influence on the reproductive success of male offspring These findings are consistent with those from previous examinations of ungulates; specifically that the effect of maternal condition lasts into adulthood and has the potential to influence reproductive success. The body mass of bighorn sheep ewes is associated with offspring mass at 4 months, which in turn is correlated with an individual’s subsequent adult (4 years) body mass Indirect measures have a history of being used to look for maternal effects on offspring