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Vagal afferents to the gastrointestinal tract are crucial for the regulation of food intake, signaling negative feedback that contributes to satiation and positive feedback that produces appetition and reward. Vagal afferents to the small intestinal mucosa contribute to this regulation by sensing luminal stimuli and reporting this information to the brain. These afferents respond to mechanical, chemical, thermal, pH, and osmolar stimuli, as well as to bacterial products and immunogens. Surprisingly, little is known about how these stimuli are transduced by vagal mucosal afferents or how their transduction is organized among these afferents' terminals. Furthermore, the effects of stimulus concentration ranges or physiological stimuli on vagal activity have not been examined for some of these stimuli. Also, detection of luminal stimuli has rarely been examined in rodents, which are most frequently used for studying small intestinal innervation. Here we review what is known about stimulus detection by vagal mucosal afferents and illustrate the complexity of this detection using nutrients as an exemplar. The accepted model proposes that nutrients bind to taste receptors on enteroendocrine cells (EECs), which excite them, causing the release of hormones that stimulate vagal mucosal afferents. However, evidence reviewed here suggests that although this model accounts for many aspects of vagal signaling about nutrients, it cannot account for all aspects. A major goal of this review is therefore to evaluate what is known about nutrient absorption and detection and, based on this evaluation, identify candidate mucosal cells and structures that could cooperate with EECs and vagal mucosal afferents in stimulus detection.

Ecosystems globally are under threat from ongoing anthropogenic environmental change. Effective conservation management requires more thorough biodiversity surveys that can reveal system-level patterns and that can be applied rapidly across space and time. Using modern ecological models and community science, we integrate environmental DNA and Earth observations to produce a time snapshot of regional biodiversity patterns and provide multi-scalar community-level characterization. We collected 278 samples in spring 2017 from coastal, shrub, and lowland forest sites in California, a complex ecosystem and biodiversity hotspot. We recovered 16,118 taxonomic entries from eDNA analyses and compiled associated traditional observations and environmental data to assess how well they predicted alpha, beta, and zeta diversity. We found that local habitat classification was diagnostic of community composition and distinct communities and organisms in different kingdoms are predicted by different environmental variables. Nonetheless, gradient forest models of 915 families recovered by eDNA analysis and using BIOCLIM variables, Sentinel-2 satellite data, human impact, and topographical features as predictors, explained 35% of the variance in community turnover. Elevation, sand percentage, and photosynthetic activities (NDVI32) were the top predictors. In addition to this signal of environmental filtering, we found a positive relationship between environmentally predicted families and their numbers of biotic interactions, suggesting environmental change could have a disproportionate effect on community networks. Together, these analyses show that coupling eDNA with environmental predictors including remote sensing data has capacity to test proposed Essential Biodiversity Variables and create new landscape biodiversity baselines that span the tree of life., (© 2021 by the Ecological Society of America.)

Vagal afferents form the primary gut-to-brain neural axis, communicating signals that regulate gastrointestinal (GI) function and promote satiation, appetition and reward. Neurotrophin-4 (NT-4) is essential for the survival of vagal smooth muscle afferents of the small intestine, but not the stomach. Here we took advantage of near-complete labeling of GI vagal mucosal afferents in Nav1.8cre-Rosa26tdTomato transgenic mice to determine whether these afferents depend on NT-4 for survival. We quantified the density and distribution of vagal afferent terminals in the stomach and small intestine mucosa and their central terminals in the solitary tract nucleus (NTS) and area postrema in NT-4 knockout (KO) and control mice. NT-4KO mice exhibited a 75% reduction in vagal afferent terminals in proximal duodenal villi and a 55% decrease in the distal ileum, whereas, those in the stomach glands remained intact. Vagal crypt afferents were also reduced in some regions of the small intestine, but to a lesser degree. Surprisingly, NT-4KO mice exhibited an increase in labeled terminals in the medial NTS. These findings, combined with previous results, suggest NT-4 is essential for survival of a large proportion of all classes of vagal afferents that innervate the small intestine, but not those that supply the stomach. Thus, NT-4KO mice could be valuable for distinguishing gastric and intestinal vagal afferent regulation of GI function and feeding. The apparent plasticity of central vagal afferent terminals - an increase in their density - could have compensated for loss of peripheral terminals by maintaining near-normal levels of satiety signaling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The impact of proton-pump inhibitor (PPI) therapy on subsequent hemorrhage and mortality after variceal hemorrhage is unclear., Aim: Evaluate the associations of PPI use with upper gastrointestinal bleeding (UGIB) and death within 30 days of undergoing esophageal variceal band ligation (EBL) separately in inpatient and outpatient settings., Methods: Retrospective review of cirrhotic patients with variceal hemorrhage who underwent EBL between 2005 and 2018. Endoscopic findings, PPI use at admission (inpatients only), PPI use at discharge (inpatients and outpatients), and adverse outcomes data (liver transplant, UGIB, transjugular intrahepatic portosystemic shunt, and death within 30 days of discharge or death during hospitalization) were reviewed., Results: A total of 446 patients (164 inpatients, 282 outpatients) were included. The most commonly observed outcomes were death within 30 days of discharge in inpatients (12.8%), UGIB within 30 days of discharge in inpatients (21.3%), and UGIB within 30 days of discharge in outpatients (8.5%). For inpatients, prescription of PPI at discharge was associated with a lower risk of bleeding within 30 days (odds ratio: 0.30, P = 0.025) and death within 30 days (odds ratio = 0.16, P = 0.002). No other significant associations of PPI with death or UGIB were reported., Conclusion: Post-EBL PPI therapy is associated with reduced risk of bleeding and death within 30 days after variceal hemorrhage in hospitalized patients.

Objective:  The aim of this study was to report the average tibial plateau angle (TPA) from a large sample of dogs and to determine if breed or gonadectomy had a significant association with abnormal TPA., Study Design:  This was a retrospective case study., Materials and Methods:  Medical records from 2006 to 2015 were reviewed for dogs that were diagnosed with cranial cruciate ligament (CrCL) rupture and underwent dynamic surgical stabilization. Signalment, examination findings, preoperative TPA and surgical report were reviewed., Results:  A total of 3,249 dogs underwent CrCL stabilization surgery from 2006 to 2015. Of the 3,249 dogs, 3,054 dogs met the inclusion criteria for a total of 3,922 stifles to be evaluated. The average preoperative TPA was 29° ± 3.7°. Average breed-specific preoperative TPA ranged from 27° to 35.1°, with four breeds having significantly lesser TPA and four breeds significantly greater TPA than the overall mean of dogs. Neutered dogs had a higher TPA of 28.87° ± 3.8° than non-neutered dogs with an average TPA of 26.88° ± 4.0°, p  < 0.001. There was not a significant difference in average TPA between females and males, regardless of the neuter status., Conclusion and Clinical Relevance:  Our reported preoperative TPA in a large sample of dogs is higher than what has been previously published. In reviewing 3,922 stifles, the average preoperative TPA was 29°. We report the average preoperative TPA in specific breeds and report that neutered dogs have a significantly higher TPA than intact dogs. This updated information may guide clinicians in management decisions regarding treatment for CrCL rupture., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Vagal afferents innervating the small intestinal mucosa regulate feeding, gastrointestinal (GI) digestive, and immune functions. Their anatomical-functional characterization has been impeded by the inability to selectively label and manipulate them. Na v 1.8-Cre-tdTomato mice label 80% of nodose and dorsal root ganglia neurons. Here, the origin of these neuron's terminals and their distribution in the small intestinal mucosa were examined by quantitatively comparing tdTomato-labeled innervation in nonoperated (control), subdiaphragmatic vagotomy (VAGX), and sham-operated mice. Control mice exhibited a large proximal-to-distal decrease and a moderate mesentery-to-antimesentery decrease in villus innervation. VAGX reduced this innervation to a greater degree proximally (91-93%) than distally (65-72%), resulting in flat proximal-distal distributions. Therefore, estimates of vagal villus afferent distributions (control minus VAGX) paralleled control distributions, but were slightly reduced in magnitude. Compared with villus afferents, crypt innervation exhibited a muted proximal-to-distal decrease in control mice and a smaller loss after VAGX (45-48%). Sham-operated mice exhibited similar distributions of villus and crypt afferents as control mice, suggesting surgery did not contribute to the effects of VAGX. Most crypt and villus afferent terminals along the entire proximal-distal small intestinal axis had similar morphology to those previously reported in the proximal duodenum, but the density of terminal branches varied. Our findings suggest the majority of small intestinal mucosal innervation labeled in Na v 1.8-Cre-tdTomato mice is vagal in origin. Therefore, these mice will be valuable for studying vagal mucosal afferent morphology, interactions with other GI elements, plasticity, and function., (© 2019 Wiley Periodicals, Inc.)

Empirical investigations of trauma and post-traumatic stress disorder (PTSD) in individuals with autism spectrum disorder (ASD) are lacking despite indications of increased risk for exposure to potentially traumatic events in this population. Research on the treatment of traumatic stress psychopathology in ASD is even more limited and suggests a critical need for guidance in the area of ASD-specific treatment adaptations. The current paper provides preliminary recommendations for adapting current evidenced-based, trauma-specific interventions, specifically trauma-focused cognitive behavioral therapy (TF-CBT), for individuals with ASD based on well-established and evidence-based practices for working with this population. These adaptations highlight the need to incorporate treatment goals related to ASD core symptoms and associated characteristics during treatment targeting traumatic stress symptoms. Future directions are discussed, including the development of instruments measuring trauma reactions in ASD, empirical investigations of modified trauma interventions for children with ASD to evaluate effectiveness, and collaboration between professionals specializing in ASD and trauma/PTSD to advance research and facilitate effective care for this community., Competing Interests: DISCLOSURES All authors report no financial interests or potential conflicts of interest.

Motivation: Linkage disequilibrium (LD) measures the correlation between genetic loci and is highly informative for association mapping and population genetics. As many studies rely on called genotypes for estimating LD, their results can be affected by data uncertainty, especially when employing a low read depth sequencing strategy. Furthermore, there is a manifest lack of tools for the analysis of large-scale, low-depth and short-read sequencing data from non-model organisms with limited sample sizes., Results: ngsLD addresses these issues by estimating LD directly from genotype likelihoods in a fast, reliable and user-friendly implementation. This method makes use of the full information available from sequencing data and provides accurate estimates of linkage disequilibrium patterns compared with approaches based on genotype calling. We conducted a case study to investigate how LD decays over physical distance in two avian species., Availability and Implementation: The methods presented in this work were implemented in C/C and are freely available for non-commercial use from https://github.com/fgvieira/ngsLD., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Computational epidemiology seeks to develop computational methods to study the distribution and determinants of health-related states or events (including disease), and the application of this study to the control of diseases and other health problems. Recent advances in computing and data sciences have led to the development of innovative modeling environments to support this important goal. The datasets used to drive the dynamic models as well as the data produced by these models presents unique challenges owing to their size, heterogeneity and diversity. These datasets form the basis of effective and easy to use decision support and analytical environments. As a result, it is important to develop scalable data management systems to store, manage and integrate these datasets. In this paper, we develop EpiK -a knowledge base that facilitates the development of decision support and analytical environments to support epidemic science. An important goal is to develop a framework that links the input as well as output datasets to facilitate effective spatio-temporal and social reasoning that is critical in planning and intervention analysis before and during an epidemic. The data management framework links modeling workflow data and its metadata using a controlled vocabulary. The metadata captures information about storage, the mapping between the linked model and the physical layout, and relationships to support services. EpiK is designed to support agent-based modeling and analytics frameworks-aggregate models can be seen as special cases and are thus supported. We use semantic web technologies to create a representation of the datasets that encapsulates both the location and the schema heterogeneity. The choice of RDF as a representation language is motivated by the diversity and growth of the datasets that need to be integrated. A query bank is developed-the queries capture a broad range of questions that can be posed and answered during a typical case study pertaining to disease outbreaks. The queries are constructed using SPARQL Protocol and RDF Query Language (SPARQL) over the EpiK. EpiK can hide schema and location heterogeneity while efficiently supporting queries that span the computational epidemiology modeling pipeline: from model construction to simulation output. We show that the performance of benchmark queries varies significantly with respect to the choice of hardware underlying the database and resource description framework (RDF) engine., Competing Interests: Competing interestsThe authors declare that they have no conflict of interest., (© Springer International Publishing AG 2017.)

Mutations in the brain-derived neurotrophic factor (BDNF) gene are associated with human obesity, and BDNF has potent inhibitory effects on eating and body weight. Little is known about the effects of energy balance manipulations on BDNF protein in the hypothalamus, though this brain region is critical for regulation of feeding and body weight and has high levels of BDNF. Here we investigated the effects of negative and positive energy status on BDNF protein levels in the arcuate (ARC), paraventricular, and ventromedial (VMH) hypothalamic nuclei and the ectorhinal cortex. To achieve this, mice were food deprived for 48 h or fed a Western diet (WD), a restricted amount of WD, or chow for 6 h, 48 h, 1 wk, or 3 wk. BDNF protein levels were estimated as the number of neurons in each brain region that exhibited BDNF-like immunoreactivity. Food deprivation decreased BDNF protein (and mRNA) expression in the ARC compared with fed mice (32%). In contrast, 1 wk of WD consumption increased BDNF protein expression in the VMH compared with chow or restricted WD feeding (40%) and, unexpectedly, increased BDNF protein in the ectorhinal cortex (20%). Furthermore, of the diet conditions and durations tested, only 1 wk of WD consumption was associated with both hyperphagia and excess weight, suggesting that effects of one or both contributed to the changes in BDNF levels. The decrease in ARC BDNF may support increased feeding in food-deprived mice, whereas the increase in the VMH may moderate overeating in WD-fed mice., (Copyright © 2017 the American Physiological Society.)

Brain-derived neurotrophic factor (BDNF) is produced by developing and mature gastrointestinal (GI) tissues that are heavily innervated by autonomic neurons and may therefore control their development or function. To begin investigating this hypothesis, we compared the morphology, distribution, and density of intraganglionic laminar endings (IGLEs), the predominant vagal GI afferent, in mice with reduced intestinal BDNF (INT-BDNF(-/-)) and controls. Contrary to expectations of reduced development, IGLE density and longitudinal axon bundle number in the intestine of INT-BDNF(-/-) mice were increased, but stomach IGLEs were normal. INT-BDNF(-/-) mice also exhibited increased vagal sensory neuron numbers, suggesting that their survival was enhanced. To determine whether increased intestinal IGLE density or other changes to gut innervation in INT-BDNF(-/-) mice altered feeding behavior, meal pattern and microstructural analyses were performed. INT-BDNF(-/-) mice ate meals of much shorter duration than controls, resulting in reduced meal size. Increased suppression of feeding in INT-BDNF(-/-) mice during the late phase of a scheduled meal suggested that increased satiation signaling contributed to reduced meal duration and size. Furthermore, INT-BDNF(-/-) mice demonstrated increases in total daily intermeal interval and satiety ratio, suggesting that satiety signaling was augmented. Compensatory responses maintained normal daily food intake and body weight in INT-BDNF(-/-) mice. These findings suggest a target organ-derived neurotrophin suppresses development of that organ's sensory innervation and sensory neuron survival and demonstrate a role for BDNF produced by peripheral tissues in short-term controls of feeding, likely through its regulation of development or function of gut innervation, possibly including augmented intestinal IGLE innervation., (Copyright © 2014 the authors 0270-6474/14/3410379-15$15.00/0.)

Many common human mesenchymal tumors, including gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS), feature myogenic differentiation. Here we report that intragenic deletion of the dystrophin-encoding and muscular dystrophy-associated DMD gene is a frequent mechanism by which myogenic tumors progress to high-grade, lethal sarcomas. Dystrophin is expressed in the non-neoplastic and benign counterparts of GIST, RMS and LMS tumors, and DMD deletions inactivate larger dystrophin isoforms, including 427-kDa dystrophin, while preserving the expression of an essential 71-kDa isoform. Dystrophin inhibits myogenic sarcoma cell migration, invasion, anchorage independence and invadopodia formation, and dystrophin inactivation was found in 96%, 100% and 62% of metastatic GIST, embryonal RMS and LMS samples, respectively. These findings validate dystrophin as a tumor suppressor and likely anti-metastatic factor, suggesting that therapies in development for muscular dystrophies may also have relevance in the treatment of cancer.

Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics.

Purpose: Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets., Experimental Design: We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML., Results: Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34(+) cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis., Conclusions: Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease., (©2013 AACR)

A large proportion of vagal afferents are dependent on neurotrophin-3 (NT-3) for survival. NT-3 is expressed in developing gastrointestinal (GI) smooth muscle, a tissue densely innervated by vagal mechanoreceptors, and thus could regulate their survival. We genetically ablated NT-3 from developing GI smooth muscle and examined the pattern of loss of NT-3 expression in the GI tract and whether this loss altered vagal afferent signaling or feeding behavior. Meal-induced c-Fos activation was reduced in the solitary tract nucleus and area postrema in mice with a smooth muscle-specific NT-3 knockout (SM-NT-3(KO)) compared with controls, suggesting a decrease in vagal afferent signaling. Daily food intake and body weight of SM-NT-3(KO) mice and controls were similar. Meal pattern analysis revealed that mutants, however, had increases in average and total daily meal duration compared with controls. Mutants maintained normal meal size by decreasing eating rate compared with controls. Although microstructural analysis did not reveal a decrease in the rate of decay of eating in SM-NT-3(KO) mice, they ate continuously during the 30-min meal, whereas controls terminated feeding after 22 min. This led to a 74% increase in first daily meal size of SM-NT-3(KO) mice compared with controls. The increases in meal duration and first meal size of SM-NT-3(KO) mice are consistent with reduced satiation signaling by vagal afferents. This is the first demonstration of a role for GI NT-3 in short-term controls of feeding, most likely involving effects on development of vagal GI afferents that regulate satiation.

Introduction: Vagal gastrointestinal (GI) afferents do not appear to contribute to long-term controls of feeding, despite downstream connections that could support such a role. This view is largely attributable to a lack of evidence for long-term effects, especially the failure of vagal afferent lesions to produce hyperphagia or obesity., Aims: Here, the possibility is evaluated that "side effects" of vagal lesion methods resulting largely from complexities of vagal organization would probably suppress long-term effects. Criteria based on knowledge of vagal organization were utilized to critique and compare vagal lesion methods and to interpret their effects on GI function, feeding and body weight., Results and Conclusions: This analysis suggested that it was premature to eliminate a long-term vagal GI afferent role based on the effects of these lesions and highlighted aspects of vagal organization that must be addressed to reduce the problematic side effects of vagal lesions. The potential of "genetic" lesions that alter vagal sensory development to address these aspects, examination of the feasibility of this approach, and the properties of brain-derived neurotrophic factor (BDNF) that made it an attractive candidate for application of this approach are described. BDNF knockout from GI smooth muscle unexpectedly demonstrated substantial overeating and weight gain associated with increased meal size and frequency. The decay of eating rate during a scheduled meal was also reduced. However, meal-induced c-Fos activation was increased in the dorsal motor nucleus of the vagus, suggesting that the effect on eating rate was due to augmentation of GI reflexes by vagal afferents or other neural systems.

Global-heterozygous and brain-specific homozygous knockouts (KOs) of brain-derived neurotrophic factor (BDNF) cause late- and early-onset obesity, respectively, both involving hyperphagia. Little is known about the mechanism underlying this hyperphagia or whether BDNF loss from peripheral tissues could contribute to overeating. Since global-homozygous BDNF-KO is perinatal lethal, a BDNF-KO that spared sufficient brainstem BDNF to support normal health was utilized to begin to address these issues. Meal pattern and microstructure analyses suggested overeating of BDNF-KO mice was mediated by deficits in both satiation and satiety that resulted in increased meal size and frequency and implicated a reduction of vagal signaling from the gut to the brain. Meal-induced c-Fos activation in the nucleus of the solitary tract, a more direct measure of vagal afferent signaling, however, was not decreased in BDNF-KO mice, and thus was not consistent with a vagal afferent role. Interestingly though, meal-induced c-Fos activation was increased in the dorsal motor nucleus of the vagus nerve (DMV) of BDNF-KO mice. This could imply that augmentation of vago-vagal digestive reflexes occurred (e.g., accommodation), which would support increased meal size and possibly increased meal number by reducing the increase in intragastric pressure produced by a given amount of ingesta. Additionally, vagal sensory neuron number in BDNF-KO mice was altered in a manner consistent with the increased meal-induced activation of the DMV. These results suggest reduced BDNF causes satiety and satiation deficits that support hyperphagia, possibly involving augmentation of vago-vagal reflexes mediated by central pathways or vagal afferents regulated by BDNF levels., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Abnormal perinatal nutrition (APN) results in a predisposition to develop obesity and the metabolic syndrome and thus may contribute to the prevalence of these disorders. Obesity, including that which develops in organisms exposed to APN, has been associated with increased meal size. Vagal afferents of the gastrointestinal (GI) tract contribute to regulation of meal size by transmitting satiation signals from gut-to-brain. Consequently, APN could increase meal size by altering this signaling, possibly through changes in expression of factors that control vagal afferent development or function. Here two studies that addressed these possibilities are reviewed. First, meal patterns, meal microstructure, and the structure and density of vagal afferents that innervate the intestine were examined in mice that experienced early postnatal overnutrition (EPO). These studies provided little evidence for EPO effects on vagal afferents as it did not alter meal size or vagal afferent density or structure. However, these mice exhibited modest hyperphagia due to a satiety deficit. In parallel, the possibility that brain-derived neurotrophic factor (BDNF) could mediate APN effects on vagal afferent development was investigated. Brain-derived neurotrophic factor was a strong candidate because APN alters BDNF levels in some tissues and BDNF knockout disrupts development of vagal sensory innervation of the GI tract. Surprisingly, smooth muscle-specific BDNF knockout resulted in early-onset obesity and hyperphagia due to increases in meal size and frequency. Microstructure analysis revealed decreased decay of intake rate during a meal in knockouts, suggesting that the loss of vagal negative feedback contributed to their increase in meal size. However, meal-induced c-Fos activation within the dorsal vagal complex suggested this effect could be due to augmentation of vago-vagal reflexes. A model is proposed to explain how high-fat diet consumption produces increased obesity in organisms exposed to APN, and may be required to reveal effects of EPO on vagal function.

Unlabelled: The relative timing of genetic alterations that contribute to follicular lymphoma remains unknown. We analyzed a donor-recipient pair who both developed grade 2/3A follicular lymphoma 7 years after allogeneic transplantation and donor lymphocyte infusions. Both patients harbored identical BCL2/IGH rearrangements also present in 1 in 2,000 cells in the donor lymphocyte infusion, and the same V(D)J rearrangement, which underwent somatic hypermutation both before and after clonal divergence. Exome sequencing of both follicular lymphomas identified 15 shared mutations, of which 14 (including alterations in EP300 and KLHL6) were recovered from the donor lymphocyte infusion by ultra-deep sequencing (average read coverage, 361,723), indicating acquisition at least 7 years before clinical presentation. Six additional mutations were present in only one follicular lymphoma and not the donor lymphocyte infusion, including an ARID1A premature stop, indicating later acquisition during clonal divergence. Thus, ultrasensitive sequencing can map clonal evolution within rare subpopulations during human lymphomagenesis in vivo., Significance: For the first time, we define the molecular ontogeny of follicular lymphoma during clonal evolution in vivo. By using ultrasensitive mutation detection, we mapped the time-course of somatic alterations after passage of a malignant ancestor by hematopoietic cell transplantation.

There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.

Training on micro- and nano- computed tomography (CT) scanners has been traditionally conducted via extensive practice on the instrument. This entails presence of an instructor to guide through the training procedure, until reasonable experience is attained. Modern tomographic instruments being expensive to maintain, the operational costs escalates with increasing number of training conducted. In a pioneering approach, the technical know-how to operate such equipment has been partly imparted via virtual reality environment running on the Second Life grid. The experimentation has indicated a reduction of the total training time. The authors hope that in the long run, such techniques will aid in significant reduction of instruction time and costs associated with training.

AML patients under the age of 60 whose blasts harbor a FLT3 internal tandem duplication (ITD) mutation have a higher relapse rate and inferior survival compared to those without this mutation. To determine if FLT3ITD also carries a negative prognostic impact in older adults receiving therapies commonly used in this age group, we retrospectively analyzed outcomes of patients ≥60 years with CN-AML according to FLT3 mutation status. We identified 91 newly diagnosed CN-AML patients, 55 with wild-type FLT3 and 36 with FLT3ITD. Of the 91 patients, 36 received supportive care and/or experimental therapies while the remaining 55 received induction chemotherapy, followed by allogeneic SCT in 17 of these patients. Based on univariate analysis, advanced age at diagnosis was significantly associated with shorter overall survival (OS) (p<.0001) while intensive therapies were associated with improved OS (p<.0001). In a multivariate analysis that accounted for type of treatment, patient age, gender, and WBC count, FLT3ITD was significantly associated with shorter OS compared to wtFLT3 [p=.001; hazard ratio (HR)=2.23; 95% CI: 1.35-3.70]. Our data support the negative prognostic impact of FLT3ITD in older adults with CN-AML., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The vagus nerve is the major neural connection between the gastrointestinal tract and the central nervous system. During fetal development, axons from the cell bodies of the nodose ganglia and the dorsal motor nucleus grow into the gut to find their enteric targets, providing the vagal sensory and motor innervations respectively. Vagal sensory and motor axons innervate selective targets, suggesting a role for guidance cues in the establishment of the normal pattern of enteric vagal innervation., Purpose: This review explores known molecular mechanisms that guide vagal innervation in the gastrointestinal tract. Guidance and growth factors, such as netrin-1 and its receptor, deleted in colorectal cancer, extracellular matrix molecules, such as laminin-111, and members of the neurotrophin family of molecules, such as brain-derived neurotrophic factor have been identified as mediating the guidance of vagal axons to the fetal mouse gut. In addition to increasing our understanding of the development of enteric innervation, studies of vagal development may also reveal clinically relevant insights into the underlying mechanisms of vago-vagal communication with the gastrointestinal tract., (© 2011 Blackwell Publishing Ltd.)

Cigarette smoking has been a well-established risk factor of lung cancer for decades. How smoking contributes to tumorigenesis in the lung remains not fully understood. Here we report the results of a genome-wide study of DNA copy number and smoking pack-years in a large collection of nonsmall-cell lung cancer (NSCLC) tumors. Genome-wide analyses of DNA copy number and pack-years of cigarette smoking were performed on 264 NSCLC tumors, which were divided into discovery and validation sets. The copy number-smoking associations were investigated in three scales: whole-genome, chromosome/arm, and focal regions. We found that heavy cigarette smokers (>60 pack-years) have significantly more copy number gains than non- or light smokers (≤60 pack-years) (P = 2.46 × 10(-4)), especially in 8q and 12q. Copy number losses tend to occur away from genes in non/light smokers (P = 5.15 × 10(-5)) but not in heavy smokers (P = 0.52). Focal copy number analyses showed that there are strong associations of copy number and cigarette smoking pack-years in 12q23 (P = 9.69 × 10(-10)) where IGF1 (insulin-like growth factor 1) is located. All of the above analyses were tested in the discovery set and confirmed in the validation set. DNA double-strand break assays using human bronchial epithelial cell lines treated with cigarette smoke condensate were also performed, and indicated that cigarette smoke condensate leads to genome instability in human bronchial epithelial cells. We conclude that cigarette smoking leads to more copy number alterations, which may be mediated by the genome instability.

PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy, making phosphatidylinositol 3-kinase (PI3K) a target for cancer therapy. Despite the promise of targeted therapy, resistance often develops, leading to treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CA(H1047R). Notably, most PIK3CA(H1047R)-driven mammary tumors recurred after PIK3CA(H1047R) inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of Met or Myc (also known as c-Met and c-Myc, respectively). Whereas Met amplification led to tumor survival dependent on activation of endogenous PI3K, tumors with Myc amplification became independent of the PI3K pathway. Functional analyses showed that Myc contributed to oncogene independence and resistance to PI3K inhibition. Notably, PIK3CA mutations and c-MYC elevation co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.

The incorporation of rituximab into various regimens has improved depth of response in Waldenstrom macroglobulinaemia (WM), though the impact of achieving better responses remains to be determined. We examined response depth on progression-free survival (PFS) in 159 rituximab-naïve WM patients who received rituximab-based therapy. The median follow-up was 33·5 months, and categorical responses were as follows: complete response (CR, 8·8%); very good partial response (VGPR, 13·2%); partial response (50%); minor response (18·9%); Non-Responders (8·8%). Sequencing for polymorphic variants of FCGR2A, FCGR2B, and FCGR3A was performed, and impact on response depth determined. Achievement of better categorical responses was incrementally associated with improved PFS (P < 0·0001). No separation was observed between CR and VGPR, and attainment of at least a VGPR was associated with improved time-to-progression. Neither age, serum IgM, haematocrit, platelet count, serum β(2) microglobulin, WM International Prognostic Scoring System score, and treatment group predicted for CR/VGPR. Polymorphisms at FCGR3A-48 and -158 were associated with improved categorical responses, particularly attainment of CR/VGPR (P ≤ 0·03). The attainment of CR/VGPR was associated with significantly longer PFS in rituximab-naïve WM patients undergoing rituximab-based therapy, and was predicted by polymorphisms in FCGR3A., (© 2011 Blackwell Publishing Ltd.)

Background: Protein needs for athletes are likely higher than those for the general population. However, athletes may perceive their protein needs to be excessively high. The purpose of this research was to compare collegiate athletes' perceived protein needs and measured protein intake to the recommended protein intake (RDI) for healthy adults (i.e. 0.8 g/kg/d) and to the maximum beneficial level for strength-trained athletes (i.e. 2.0 g/kg/day)., Methods: Perceived protein needs were quantified in 42 strength-trained collegiate male athletes by using a survey that asked the athletes to provide their perception about protein needs in specific quantitative terms (i.e. g/kg/d). Perceived protein needs were also determined by having the athletes select a daylong menu that they perceived to have adequate protein content from a collection of 5 isoenergetic menus, which differed in terms of protein content. Actual protein intake was quantified using 3-day food records and nutrient analysis. Single sample t-tests were used to compare protein intake and perceived protein needs to 0.8 g/kg/day and 2.0 g/kg/day., Results: When asked to provide, in quantitative terms, protein needs for athletes, 67% of the athletes indicated "do not know." Of the remaining 33% of athletes, all gave values greater than 2.0 g/kg/d (mean 21.5 ± 11.2 g/kg/d, p = 0.14 vs. 2.0 g/kg/d). Based on the menu selection method for determining perceived protein needs, the athletes indicated that their protein needs were 2.4 ± 0.2 g/kg/d, which was greater than the RDI for protein (p < 0.0001) and tended to be greater than the maximally beneficial protein intake of 2.0 g/kg/d (p = 0.13). Measured protein intake was 2.0 ± 0.1 g/kg/d, which was greater than the RDI (p < 0.0001) but not different from the maximally beneficial protein intake of 2.0 g/kg/d (p = 0.84)., Conclusions: Male collegiate athletes recognize that their protein needs are higher than that of the general population and consume significantly more protein than recommended in the RDI. However, it also appears that athletes are not aware of objective recommendations for protein intake and may perceive their needs to be excessively high. This study highlights the need for nutrition education in collegiate athletes, in particular nutrition education on macronutrient distribution and protein needs.

Early postnatal overnutrition results in a predisposition to develop obesity due in part to hypothalamic and sympathetic dysfunction. Potential involvement of another major regulatory system component--the vagus nerve--has not been examined. Moreover, feeding disturbances have rarely been investigated prior to development of obesity when confounds due to obesity are minimized. To examine these issues, litters were culled on the day of birth to create small litters (SL; overnutrition), or normal size litters (NL; normal nutrition). Body weight, fat pad weight, meal patterns, and vagal sensory duodenal innervation were compared between SL and NL adult mice prior to development of obesity. Meal patterns were studied 18 h/day for 3 weeks using a balanced diet. Then vagal mechanoreceptors were labeled using anterograde transport of wheatgerm agglutinin-horseradish peroxidase injected into the nodose ganglion and their density and morphology were examined. Between postnatal day 1 and weaning, body weight of SL mice was greater than for NL mice. By young adulthood it was similar in both groups, whereas SL fat pad weight was greater in males, suggesting postnatal overnutrition produced a predisposition to obesity. SL mice exhibited increased food intake, decreased satiety ratio, and increased first meal rate (following mild food deprivation) compared to NL mice, suggesting postnatal overnutrition disrupted satiety. The density and structure of intestinal IGLEs appeared similar in SL and NL mice. Thus, although a vagal role cannot be excluded, our meal parameter and anatomical findings provided no evidence for significant postnatal overnutrition effects on vagal gastrointestinal afferents., (Copyright 2010 Elsevier Inc. All rights reserved.)

Vagal sensory neurons are dependent on neurotrophins for survival during development. Here, the contribution of brain-derived neurotrophic factor (BDNF) to survival and other aspects of gastric vagal afferent development was investigated. Post-mortem anterograde tracing with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate (DiI) was used to label selectively vagal projections to the stomach on postnatal days (P) 0, 3, 4, and 6 in wild types and heterozygous or homozygous BDNF mutants. Sampling sites distributed throughout the ventral stomach wall were scanned with a confocal microscope, and vagal axon bundles, single axons, putative mechanoreceptor precursors (intraganglionic laminar endings, IGLEs; intramuscular arrays, IMAs), and efferent terminals were quantified. Also, myenteric neurons, which are innervated by IGLEs, were stained with cuprolinic blue and counted. Quantitative comparisons across wild-type stomach compartments demonstrated that the adult distribution of IMAs was not present at P0 but began to form by P3-6. Among all the quantified elements, at P0, only IGLE density was significantly different in homozygous mutants compared with wild types, exhibiting a 50% reduction. Also, antrum innervation appeared disorganized, and some putative IMA precursors had truncated telodendria. At P3-6, the effect on IGLEs had recovered, the disorganization of antrum innervation had partially recovered, and some IMA telodendria were still truncated. The present results suggest that gastric IGLEs are among the vagal sensory neurons dependent on BDNF for survival or axon guidance. Alternatively, BDNF deficiency may delay gastric IGLE development. Also, BDNF may contribute to IMA differentiation and patterning of antral vagal innervation., ((c) 2010 Wiley-Liss, Inc.)

Vagal gastrointestinal (GI) afferents are essential for the regulation of eating, body weight, and digestion. However, their functional organization and the way that this develops are poorly understood. Neurotrophin-3 (NT-3) is crucial for the survival of vagal sensory neurons and is expressed in the developing GI tract, possibly contributing to their survival and to other aspects of vagal afferent development. The identification of the functions of this peripheral NT-3 thus requires a detailed understanding of the localization and timing of its expression in the developing GI tract. We have studied embryos and neonates expressing the lacZ reporter gene from the NT-3 locus and found that NT-3 is expressed predominantly in the smooth muscle of the outer GI wall of the stomach, intestines, and associated blood vessels and in the stomach lamina propria and esophageal epithelium. NT-3 expression has been detected in the mesenchyme of the GI wall by embryonic day 12.5 (E12.5) and becomes restricted to smooth muscle and lamina propria by E15.5, whereas its expression in blood vessels and esophageal epithelium is first observed at E15.5. Expression in most tissues is maintained at least until postnatal day 4. The lack of colocalization of beta-galactosidase and markers for myenteric ganglion cell types suggests that NT-3 is not expressed in these ganglia. Therefore, NT-3 expression in the GI tract is largely restricted to smooth muscle at ages when vagal axons grow into the GI tract, and when vagal mechanoreceptors form in smooth muscle, consistent with its role in these processes and in vagal sensory neuron survival.

Contributors to increased obesity in children may include perinatal under- or overnutrition. Humans and rodents raised under these conditions develop obesity, which like obesities of other etiologies has been associated with increased meal size. Since vagal sensory innervation of the gastrointestinal (GI) tract transmits satiation signals that regulate meal size, one mechanism through which abnormal perinatal nutrition could increase meal size is by altering vagal development, possibly by causing changes in the expression of factors that control it. Therefore, we have begun to characterize development of vagal innervation of the GI tract and the expression patterns and functions of the genes involved in this process. Important events in development of mouse vagal GI innervation occurred between midgestation and the second postnatal week, suggesting they could be vulnerable to effects of abnormal nutrition pre- or postnatally. One gene investigated was brain- derived neurotrophic factor (BDNF), which regulates survival of a subpopulation of vagal sensory neurons. BDNF was expressed in some developing stomach wall tissues innervated by vagal afferents. At birth, mice deficient in BDNF exhibited a 50% reduction of putative intraganglionic laminar ending mechanoreceptor precursors, and a 50% increase in axons that had exited fiber bundles. Additionally, BDNF was required for patterning of individual axons and fiber bundles in the antrum and differentiation of intramuscular array mechanoreceptors in the forestomach. It will be important to determine whether abnormal perinatal environments alter development of vagal sensory innervation of the GI tract, involving effects on expression of BDNF, or other factors regulating vagal development.

To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach's plexus were encountered.

The presence of valine (V) at position 158 of FcgammaRllla (CD16) is known to improve clinical response to rituximab in indolent non-Hodgkin lymphoma (NHL). Little is known about the basic mechanisms for this observation. We examined natural killer (NK) cells from healthy donors representing the FcgammaRIIIa-158 polymorphic subgroups (V/V, V/F, and F/F) for gene transcript and cell surface CD16 expression, rituximab binding, and rituximab-dependent NK cell-mediated cytotoxicity. We observed higher levels of FcgammaRIIIa transcripts among individuals with the FcgammaRIIIa-158 V/V versus V/F or F/F genotype (P < .001); increased cell surface CD16 expression by quantitative flow cytometry on NK cells from individuals expressing at least one valine at FcgammaRIIIa-158 versus F/F (P = .029); as well as augmented rituximab binding and rituximab-mediated, antibody-dependent cellular cytotoxicity (ADCC). These results suggest that individuals expressing at least one valine at FcgammaRIIIa-158 might, in part, have better clinical outcomes due to increased CD16 expression, rituximab binding, and rituximab-mediated ADCC.

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.

Background: The definition of human MHC class I haplotypes through association of HLA-A, HLA-Cw and HLA-B has been used to analyze ethnicity, population migrations and disease association., Results: Here, we present HLA-E allele haplotype association and population linkage disequilibrium (LD) analysis within the ~1.3 Mb bounded by HLA-B/Cw and HLA-A to increase the resolution of identified class I haplotypes. Through local breakdown of LD, we inferred ancestral recombination points both upstream and downstream of HLA-E contributing to alternative block structures within previously identified haplotypes. Through single nucleotide polymorphism (SNP) analysis of the MHC region, we also confirmed the essential genetic fixity, previously inferred by MHC allele analysis, of three conserved extended haplotypes (CEHs), and we demonstrated that commercially-available SNP analysis can be used in the MHC to help define CEHs and CEH fragments., Conclusion: We conclude that to generate high-resolution maps for relating MHC haplotypes to disease susceptibility, both SNP and MHC allele analysis must be conducted as complementary techniques.

Background: Neuroblastomas are characterized by chromosomal alterations with biological and clinical significance. We analyzed paired blood and primary tumor samples from 22 children with high-risk neuroblastoma for loss of heterozygosity (LOH) and DNA copy number change using the Affymetrix 10K single nucleotide polymorphism (SNP) array., Findings: Multiple areas of LOH and copy number gain were seen. The most commonly observed area of LOH was on chromosome arm 11q (15/22 samples; 68%). Chromosome 11q LOH was highly associated with occurrence of chromosome 3p LOH: 9 of the 15 samples with 11q LOH had concomitant 3p LOH (P = 0.016). Chromosome 1p LOH was seen in one-third of cases. LOH events on chromosomes 11q and 1p were generally accompanied by copy number loss, indicating hemizygous deletion within these regions. The one exception was on chromosome 11p, where LOH in all four cases was accompanied by normal copy number or diploidy, implying uniparental disomy. Gain of copy number was most frequently observed on chromosome arm 17q (21/22 samples; 95%) and was associated with allelic imbalance in six samples. Amplification of MYCN was also noted, and also amplification of a second gene, ALK, in a single case., Conclusions: This analysis demonstrates the power of SNP arrays for high-resolution determination of LOH and DNA copy number change in neuroblastoma, a tumor in which specific allelic changes drive clinical outcome and selection of therapy.

Background: Neuronal cells respond to changes in intracellular calcium ([Ca2+]i) by affecting both the abundance and architecture of specific mRNAs. Although calcium-induced transcription and transcript variation have both been recognized as important sources of gene regulation, the interplay between these two phenomena has not been evaluated on a genome-wide scale., Results: Here, we show that exon-centric microarrays can be used to resolve the [Ca2+]i-modulated gene expression response into transcript-level and exon-level regulation. Global assessments of affected transcripts reveal modulation within distinct functional gene categories. We find that transcripts containing calcium-modulated exons exhibit enrichment for calcium ion binding, calmodulin binding, plasma membrane associated, and metabolic proteins. Additionally, we uncover instances of regulated exon use in potassium channels, neuroendocrine secretory proteins and metabolic enzymes, and demonstrate that regulated changes in exon expression give rise to distinct transcript variants., Conclusion: Our findings connect extracellular stimuli to specific exon behavior, and suggest that changes in transcript and exon abundance are reflective of a coordinated gene expression response to elevated [Ca2+]i. The technology we describe here lends itself readily to the resolution of stimulus-induced gene expression at both the transcript and exon levels.

The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.

Activating mutations in NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). Here, we report the analysis for activating NOTCH1 mutations in a large number of acute myeloid leukemia (AML) primary samples and cell lines. We found activating mutations in NOTCH1 in a single M0 primary AML sample, in three (ML1, ML2 and CTV-1) out of 23 AML cell lines and in the diagnostic (myeloid) and relapsed (T-lymphoid) clones in a patient with lineage switch leukemia. Importantly, the ML1 and ML2 AML cell lines are derived from an AML relapse in a patient initially diagnosed with T-ALL. Overall, these results demonstrate that activating mutations in NOTCH1 are mostly restricted to T-ALL and are rare in AMLs. The presence of NOTCH1 mutations in myeloid and T-lymphoid clones in lineage switch leukemias establishes the common clonal origin of the diagnostic and relapse blast populations and suggests a stem cell origin of NOTCH1 mutations during the molecular pathogenesis of these tumors.

Sensory innervation of the gastrointestinal (GI) tract by the vagus nerve plays important roles in regulation of GI function and feeding behavior. This innervation is composed of a large number of sensory pathways, each arising from a different population of sensory receptors. Progress in understanding the functions of these pathways has been impeded by their close association with vagal efferent, sympathetic, and enteric systems, which makes it difficult to selectively label or manipulate them. We suggest that a genetic approach may overcome these barriers. To illustrate the potential value of this strategy, as well as to gain insights into its application, investigations of CNS pathways and peripheral tissues involved in energy balance that benefited from the use of gene manipulations are reviewed. Next, our studies examining the feasibility of using mutations of developmental genes for manipulating individual vagal afferent pathways are reviewed. These experiments characterized mechanoreceptor morphology, density and distribution, and feeding patterns in four viable mutant mouse strains. In each strain a single population of vagal mechanoreceptors innervating the muscle wall of the GI tract was altered, and was associated with selective effects on feeding patterns, thus supporting the feasibility of this strategy. However, two limitations of this approach must be addressed for it to achieve its full potential. First, mutation effects in tissues outside the GI tract can contribute to changes in GI function or feeding. Additionally, knockouts of developmental genes are often lethal, preventing analysis of mature innervation and ingestive behavior. To address these issues, we propose to develop conditional gene knockouts restricted to specific GI tract tissues. Two genes of interest are brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), which are essential for vagal afferent development. Creating conditional knockouts of these genes requires knowledge of their GI tract expression during development, which little is known about. Preliminary investigation revealed that during development BDNF and NT-3 are each expressed in several GI tract regions, and that their expression patterns overlap in some tissues, but are distinct in others. Importantly, GI tissues that express BDNF or NT-3 are innervated by vagal afferents, and expression of these neurotrophins occurs during the periods of axon invasion and receptor formation, consistent with roles for BDNF or NT-3 in these processes and in receptor survival. These results provide a basis for targeting BDNF or NT-3 knockouts to specific GI tract tissues, and potentially altering vagal afferent innervation only in that tissue (e.g., smooth muscle vs. mucosa). Conditional BDNF or NT-3 knockouts that are successful in selectively altering a vagal GI afferent pathway will be valuable for developing an understanding of that pathway's roles in GI function and food intake.

Neurotrophin-4 (NT-4) deficient mice exhibit substantial loss of intestinal vagal afferent innervation and short-term deficits in feeding behavior, suggesting reduced satiation. However, they do not show long-term changes in feeding or body weight because of compensatory behaviors. The present study examined whether high-fat hyperphagia induction would overcome compensation and reveal long-term effects associated with the reduced vagal sensory innervation of NT-4 mutants. First, modifications of a feeding schedule previously developed in rats were examined in wild-type mice to identify the regimen most effective at producing hyperphagia. The most successful schedule, which was run for 26 days, included access to a 43%-fat diet and pelleted chow every other day and access to only powdered chow on the alternate days. On high-fat access days mice consumed 25% more calories than mice with continuous daily access to the same high-fat diet and pelleted chow. This feeding regimen also induced hyperphagia in NT-4 deficient mice and their wild-type controls: on high-fat exposure days mutants consumed 35% more calories relative to continuous-access mutants, and wild types ate 25% more than continuous-access wild types. Moreover, on high-fat access days the alternating NT-4 mutants significantly increased caloric intake by 9% compared to alternating wild types. Thus, high-fat hyperphagia appeared to override compensation, permitting short-term changes in meal consumption by mutants that accrued into long-term changes in total daily food intake. This raises the possibility that intestinal vagal sensory innervation contributes to long-term, as well as to short-term regulation of food intake.