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Supplementary Table S6. ChIP-sequencing datasets used in the present study.

Supplementary Figure S1. mRNA levels of PRC2 components in CML and normal samples used in this study. Supplementary Figure S2. Effect of TKI treatment on mRNA levels of PRC2 components on CML cells in vitro. Supplementary Figure S3. Analysis of promoters reprogrammed for H3K27me3 levels at promoters. Supplementary Figure S4. Relationship between changes in levels of H3K27me3 and changes in mRNA expression at H3K27me3 targets. Supplementary Figure S5. EZH2i treatment of CML and normal CD34+ cells. Supplementary Figure S6. EZH2i treatment of CML CD34+CD38- cells (LSCs). Supplementary Figure S7. Validation of EZH2 shRNAs in a CML cell line. Supplementary Figure S8. The effect of treatment of CML or normal CD34+ cells with EZH2i (GSK343; 1000 nM) on the global mRNA levels of H3K27me3 and H3K27me1 targets. Supplementary Figure S9. Both imatinib and EZH2i selectively target upregulation of H3K27me3 target genes. Supplementary Figure S10. Nilotinib, dasatinib and EZH2i selectively target upregulation of H3K27me3 target genes. Supplementary Figure S11. mRNA expression levels for H3K27me3 target genes in HSC and LSCs and their behaviour after TKI treatment. Supplementary Figure S12. mRNA expression levels for H3K27me3 target genes in HPC and LPCs (â‰^ CML CD34+) and their behaviour after EZH2i treatment. Supplementary Figure S13. Primary CML cells engrafted in NSG mice treated in vivo with EZH2i and TKI, alone and in combination. Supplementary Figure S14. Validation of mRNA expression changes determined by Affymetrix GeneChip analysis (E-MTAB-2581, Affymetrix HuGe 1.0 ST). Supplementary Figure S15. PRC2 mis-regulation in the three CML patient samples used for Affymetrix GeneChIP and ChIP-sequencing in this study. Supplementary Figure S16. Determining thresholds for identifying H3K27me3 target genes. Supplementary Figure S17. Validation of seqMiner(3) promoter classifications.

Supplementary Table S2 from Inactivation of HOXA Genes by Hypermethylation in Myeloid and Lymphoid Malignancy is Frequent and Associated with Poor Prognosis

Purpose: The HOX genes comprise a large family of homeodomain-containing transcription factors, present in four separate clusters, which are key regulators of embryonic development, hematopoietic differentiation, and leukemogenesis. We aimed to study the role of DNA methylation as an inducer of HOX gene silencing in leukemia.Experimental Design: Three hundred and seventy-eight samples of myeloid and lymphoid leukemia were quantitatively analyzed (by COBRA analysis and pyrosequencing of bisulfite-modified DNA) for methylation of eight HOXA and HOXB cluster genes. The biological significance of the methylation identified was studied by expression analysis and through re-expression of HOXA5 in a chronic myeloid leukemia (CML) blast crisis cell line model.Results: Here, we identify frequent hypermethylation and gene inactivation of HOXA and HOXB cluster genes in leukemia. In particular, hypermethylation of HOXA4 and HOXA5 was frequently observed (26-79%) in all types of leukemias studied. HOXA6 hypermethylation was predominantly restricted to lymphoid malignancies, whereas hypermethylation of other HOXA and HOXB genes was only observed in childhood leukemia. HOX gene methylation exhibited clear correlations with important clinical variables, most notably in CML, in which hypermethylation of both HOXA5 (P = 0.00002) and HOXA4 (P = 0.006) was strongly correlated with progression to blast crisis. Furthermore, re-expression of HOXA5 in CML blast crisis cells resulted in the induction of markers of granulocytic differentiation.Conclusion: We propose that in addition to the oncogenic role of some HOX family members, other HOX genes are frequent targets for gene inactivation and normally play suppressor roles in leukemia development.

Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRα

Accumulation of circular RNAs (circRNAs) during aging occurs on a genome-wide level for multiple organisms, but its significance is unknown. Generating circRNA loss-of-function mutants is difficult because the vast majority of these RNAs are comprised of exons shared with protein-coding mRNAs. In Caenorhabditis elegans, most circRNAs were previously found to accumulate during aging. Two of the most abundant, age-accumulating circRNAs are generated from exon 4 of the crh-1 gene (circ-crh-1). Here, we found that the biogenesis of circ-crh-1 was regulated by the double-stranded RNA-binding protein ADR-1. We identified Reverse Complementary Match (RCM) sequences in introns flanking circ-crh-1. Using CRISPR-Cas9, we deleted the downstream RCM and found that this completely eliminated expression of the circRNA without affecting linear mRNA expression from the crh-1 gene. Remarkably, worms lacking circ-crh-1 exhibited a significantly longer mean lifespan. Lifespan was partially restored to wild type by expression of circ-crh-1 in neural tissues. Widespread transcriptome alterations in circ-crh-1 mutants were identified using RNA-Seq. Moving forward, intronic RCM deletion using CRISPR should be a widely applicable method to identify lifespan-regulating circRNAs in C. elegans.

The gastrointestinal (GI) tract in mammals is comprised of dozens of cell types with varied functions, structures, and histological locations that respond in a myriad of ways to epigenetic and genetic factors, environmental cues, diet, and microbiota. The homeostatic functioning of these cells contained within this complex organ system has been shown to be highly regulated by the effect of microRNAs (miRNA). Multiple efforts have uncovered that these miRNAs are often tightly influential in either the suppression or overexpression of inflammatory, apoptotic, and differentiation-related genes and proteins in a variety of cell types in colorectal cancer (CRC). The early detection of CRC and other GI cancers can be difficult, attributable to the invasive nature of prophylactic colonoscopies. Additionally, the levels of miRNAs associated with CRC in biofluids can be contradictory and, therefore, must be considered in the context of other inhibiting competitive endogenous RNAs (ceRNA) such as lncRNAs and circRNAs. There is now a high demand for disease treatments and noninvasive screenings such as testing for bloodborne or fecal miRNAs and their inhibitors/targets. The breadth of this review encompasses current literature on well-established CRC-related miRNAs and the possibilities for their use as biomarkers in the diagnoses of this potentially fatal GI cancer.

Background: The E-wave propagation index (EPI) is a measure of apical blood flow and has been proposed as a risk marker of left ventricular thrombus (LVT) formation. We sought to investigate the association between EPI and LVT in patients with heart failure with reduced ejection fraction (HFrEF). Methods: This was a retrospective cohort study of patients with HFrEF. Follow-up echocardiograms were investigated for development of LVT. EPI was measured at baseline investigation as the velocity time integral of the transmitral E-wave divided by the left ventricular length. Logistic regression was used to investigate the association between EPI and LVT. Multivariable adjustments were made for age, significant mitral regurgitation, apical aneurysm, prior myocardial infarction, and left ventricular ejection fraction (LVEF). Results: Of 785 patients, 24 (3%) developed LVT. Mean age was 66 years, 73% were men, and LVEF was 28%. Those who developed LVT had significantly lower EPI (1.12 vs. 1.36, p=0.02). EPI was significantly associated with LVT in univariable logistic regression (OR=1.16 (1.03-1.31), per 0.1 decrease) (figure). These findings were unchanged after multivariable adjustments (OR=1.15 (1.00-1.31), per 0.1 decrease). The EPI provided an area under the curve of 0.68 with an optimal cutoff of 1.2. This cutoff had a sensitivity of 75%, specificity of 60%, positive predictive value of 6%, and negative predictive value of 99%. A low EPI was associated with a more than 4-fold increased risk of LVT (OR=4.56 (1.79-11.62), p=0.001). Conclusion: Decreasing EPI is associated with a higher risk of LVT. While a low EPI poses a markedly increased risk of LVT, a high EPI may be used to rule out LVT.

Background Videogames are emerging as increasingly popular tools for training complex skills, in part due to their potential for improving the transfer of learning to changing demands. One caveat is that training outcomes are primarily influenced by a learner’s capability and willingness to engage in adaptive learning processes. The present study examined the role of epistemic curiosity as a predisposition for seeking information and resolving gaps in knowledge. Method Using a game-based learning environment with strong cognitive and perceptual-motor demands, we tested the effects of interest- and deprivation-type epistemic curiosity on performance during skill acquisition and adaptation to unforeseen change. A task-change paradigm and piecewise latent trajectory model disentangled the effects on performance trajectories corresponding to skill acquisition and adaptation. Results Interest-type epistemic curiosity, but not deprivation-type, was positively related to baseline performance, even after controlling for cognitive ability and videogame experience. Moreover, the positive effect of interest-type epistemic curiosity on post-change performance (i.e., adaptation) was fully mediated by pre-change performance. Discussion These findings indicate that interest-type epistemic curiosity is particularly important in the earlier phases of learning, but explains little variability in adaptive performance beyond its direct influence on performance acquired prior to change. This research speaks to the roles of individual differences in cognition and motivation during game-based learning.

The mammalian intestine contains many different cell types but is comprised of 2 main cell types: epithelial cells and smooth muscle cells. Recent in vivo and in vitro evidence has revealed that various alterations to the DNA methylation apparatus within both of these cell types can result in a variety of cellular phenotypes including modified differentiation status, apoptosis, and uncontrolled growth. Methyl groups added to cytosines in regulatory genomic regions typically act to repress associated gene transcription. Aberrant DNA methylation patterns are often found in cells with abnormal growth/differentiation patterns, including those cells involved in burdensome intestinal pathologies including inflammatory bowel diseases and intestinal pseudo-obstructions. The altered methylation patterns being observed in various cell cultures and DNA methyltransferase knockout models indicate an influential connection between DNA methylation and gastrointestinal cells’ development and their response to environmental signaling. As these modified DNA methylation levels are found in a number of pathological gastrointestinal conditions, further investigations into uncovering the causative nature, and controlled regulation, of this epigenetic modification is of great interest.

Maturity-Onset Diabetes of the Young (MODY) is a rare form of diabetes which affects between 1% and 5% of diagnosed diabetes cases. Clinical characterizations of MODY include onset of diabetes at an early age (before the age of 30), autosomal dominant inheritance pattern, impaired glucose-induced secretion of insulin, and hyperglycemia. Presently, 14 MODY subtypes have been identified. Within these subtypes are several mutations which contribute to the different MODY phenotypes. Despite the identification of these 14 subtypes, MODY is often misdiagnosed as type 1 or type 2 diabetes mellitus due to an overlap in clinical features, high cost and limited availability of genetic testing, and unfamiliarity with MODY outside of the medical profession. The primary aim of this review is to investigate the genetic characterization of the MODY subtypes. Additionally, this review will elucidate the link between the genetics, function, and clinical manifestations of MODY in each of the 14 subtypes. In providing this knowledge, we hope to assist in the accurate diagnosis of MODY patients and, subsequently, in ensuring they receive appropriate treatment.

Sand and gravel mining creates novel ecosystems along the Platte, Loup, and Elkhorn rivers in Nebraska, USA. Piping plovers and least terns are state and/or federally threatened and endangered species, respectively, that nest and raise young at these sites and their derivatives. Despite hosting relatively large numbers of piping plovers and least terns for decades, an important question that has largely gone unaddressed is whether the industry that has produced these novel ecosystems is stable and will continue to produce habitat consistently in the future. We evaluated how the number, size, and spatial distribution of different site types hosting different numbers of nesting plovers and terns have changed over time and how current trends in the number of different site types will affect future habitat using a multi‐state modeling approach. Overall area and total number of sites declined during the period 1993–2020. More important, one site type, traditional mines, are being replaced by another site type, modern mines, which host lower numbers of nests of both species. The difference between these two site types is primarily how waste sand is stored. Traditional mines store waste sand in spoil piles or plumes along the edge of a lake created by the mining process, forming relatively large expanses of nesting habitat used by both species. Modern mines store waste sand in limited quantities along the edge of the lake but also in piles away from the lake. Traditional mines also differ from modern mines in that they are routinely converted to housing developments with intermediate transition sites that host the largest number of nests for brief periods. Based on the previous 28 yr of decline, traditional mines and their productive derivatives are projected to continue to decline, thereby further reducing overall nesting habitat. Piping plovers and least terns are expected to nest in our study sites for the foreseeable future, but overall numbers are expected to be smaller than what has been observed in previous decades. Local declines in our study area will have local and regional implications for the recovery and management of these two species of conservation concern.

Background: Child maltreatment is a major public issue in the United States, yet most children affected by abuse or neglect never engage in evidence-based practices (EBP) for child mental health. Children’s Advocacy Centers (CACs’) are uniquely situated to serve as Family Navigators who connect children impacted by maltreatment to appropriate EBPs. In fact, the CAC position of Victim Advocate mirrors the Mental Health Family Navigator national initiative.Methods: We developed, implemented, and will evaluate web-based and consultative training for Victim Advocates to enhance early engagement in services (E3 training). The interactive web-based training will embed key targets of knowledge and skills related to family engagement, trauma, and EBP services. Participating CACs will be randomized to E3 webinar-based training, E3 webinar plus consultation, or delayed training. The project will test the E3 training’s impact on key mechanisms of change (e.g., knowledge, skills) to improve rates of screening, referral, and access to EBP services. The feasibility of implementing the training program, and differential impact and costs by level of training will be examined.Discussion: The overarching goal of this project is to test a training that is readily implemented through CACs and examine the mechanisms for improving early engagement and, ultimately, child and adolescent mental health outcomes. Results and cost findings will be used to plan a large-scale comprehensive, mixed-methods Hybrid Type II effectiveness-implementation and cost-effectiveness trial of Family Navigator E3 training. If outcomes are positive, considerable infrastructure exists to support the scale-up and sustainability of E3 training nationwide, by embedding the training in national CAC training protocols.Trial registration: NCT04221633Date and version identifier: March 25 2021; Vers. 1.0 (original)

Background & Aims Interstitial cells of Cajal (ICCs) and pancreatic β cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. Methods We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. Results miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of β cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Kruppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. Conclusions miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.

Background: Child maltreatment is a major public issue in the United States, yet most children affected by abuse or neglect never engage in evidence-based practices (EBP) for child mental health. Children’s Advocacy Centers (CACs’) are uniquely situated to serve as Family Navigators who connect children impacted by maltreatment to appropriate EBPs. In fact, the CAC position of Victim Advocate mirrors the Mental Health Family Navigator national initiative. Methods: We will develop, implement, and evaluate web-based and consultative training for Victim Advocates to enhance early engagement in services (E3 training). The interactive web-based training will embed key targets of knowledge and skills related to family engagement, trauma, and EBP services. Participating CACs will be randomized to E3 webinar-based training, E3 webinar plus consultation, or delayed training. The project will test the E3 training’s impact on key mechanisms of change (e.g., knowledge, skills) to improve rates of screening, referral, and access to EBP services. The feasibility of implementing the training program, and differential impact and costs by level of training will be examined.Discussion: The overarching goal of this project is to test a training that is readily implemented through CACs and examine the mechanisms for improving early engagement and, ultimately, child and adolescent mental health outcomes. Results and cost findings will be used to plan a large-scale comprehensive, mixed-methods Hybrid Type II effectiveness-implementation and cost-effectiveness trial of Family Navigator E3 training. If outcomes are positive, considerable infrastructure exists to support the scale-up and sustainability of E3 training nationwide, by embedding the training in national CAC training protocols.Trial registration: NCT04221633

The purpose of this laboratory study involving repeated measures of emotion as 214 undergraduates (58.4% male) learned a complex video game was to address the need for empirical research on dynamic personality constructs by examining how two aspects of affect variability-spin and pulse-explain variance in skill acquisition and adaptive performance. Spin refers to within-person fluctuations in affect pleasantness and activation potential. Pulse refers to within-person fluctuations in affect intensity. Despite research showing high affect variability reflects a personality profile of heighted reactivity to emotionally charged events and poor adjustment, little empirical research has examined their relationships with behavioral outcomes, much less aspects of skilled performance. Compared to traditional measures of personality, which yield weak effects for predicting acquisition and adaptive performance, measures of affect variability hold considerable promise because they, like performance, reflect dynamic within-person phenomena. Accordingly, the main question addressed by this study was whether spin and pulse incrementally explain acquisition and adaptive performance beyond Big Five measures of personality. In general, we expected harmful, incremental effects for both spin and pulse, and hypothesized two mechanisms for these harmful effects: (1) by undermining effort and (2) by undermining the effort-performance relationship. Using a task-change paradigm and discontinuous growth modeling that disentangled adaptation from acquisition, results showed that affect variability, independent of the Big Five, produced harmful effects via both hypothesized mechanisms. Participants higher in affect spin and pulse showed less sustained effort across performance sessions and exhibited lower performance. Furthermore, the harmful effects of spin and pulse were stronger in adaptation compared to acquisition, with pulse showing stronger direct effects on performance during adaptation and spin moderating the effort-performance relationship such that effort was only beneficial during adaptation for those lower in spin. In light of these results, one might question the common advice "keep calm and carry on," which may not be viable for persons high in affect variability. Accordingly, results are discussed in terms of the need to better understand the specific mediating processes by which high affect variability undermines success across a variety of learning and performance contexts.

In a recent study, Farnsworth et al. (2017) used distributions of nest initiation dates drawn mostly from human-created, off-channel habitats and a model of emergent sandbar habitat to evaluate the hypothesis that least terns (Sternula antillarum) and piping plovers (Charadrius melodus) are physiologically adapted to initiate nests concurrent with the cessation of spring river flow rises on two sections of the Platte River, Nebraska. The study by Farnsworth et al. (2017) has several shortcomings which bring into question the authors' principal assertion that interior least tern and piping plovers are not adapted to occupying and nesting on river sandbars on the Platte River system. We identify these shortcomings and provide information, which, we suggest, would change their conclusions if incorporated. Linked Article: https://doi.org/10.1002/ece3.4097.

Birds are altering the phenology of critical life history events, including migration, in response to the effects of global climate change. Whooping Cranes (Grus americana) are one of the most critically endangered birds in the world. Their remnant population, referred to as the Aransas-Wood Buffalo Population, numbers between 300–400 individuals and migrates between the U.S. Gulf of Mexico coast and north-central Canada twice each year. Previous analyses suggested Whooping Crane migration was temporally constant in spring and fall. New analyses of observations spanning 1942–2016 show Whooping Crane migration is now occurring earlier in spring by approximately 22 days and later in fall by approximately 21 days. Spring temperatures have also increased in the migration corridor during the same period; however, there is no apparent temperature pattern during the fall. In spring, earlier migrating Whooping Cranes stopped over for longer periods of time compared to late-migrating cranes. This result may be partially explained by single Whooping Cranes migrating with earlier migrating Sandhill Cranes (Antigone canadensis). These results have important conservation implications as the timing of Whooping Crane migration and availability of the habitat and foraging resources, including those associated with agriculture, on which this species relies will be increasingly affected by climate change.

Background & Aims Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine [5-HT]), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiologic roles of 5-HT deficiency in gastric motility in mice and humans. Methods We developed an inducible, EC cell–specific Tph1CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell–depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell–depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG). Results Tph1-tdTom mice showed EC cells that were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate. Conclusions The Tph1CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiologic mechanism of 5-HT deficiency in IG.

West Nile virus (WNV) is an introduced pathogen, transmitted by mosquitos, that spread across North America following its arrival there in 1999. Birds host the virus, but consequences of the disease to bird species have been variable. A small number of avian species are especially susceptible to WNV, experience high mortality rates when infected, and have shown regional declines apparently because of the disease. Other species have seemingly been unaffected. Transmission of WNV is associated with climate, with higher incidence of transmission in dry areas with warm winters. The north-central United States is an area that exhibits clines in temperature and precipitation, and in this area changes in species abundance due to WNV have not been closely examined. We used Christmas Bird Count (CBC) data to investigate changes in winter abundance of selected species before and after the arrival of WNV in the Great Plains. After arrival of WNV, average estimated abundances of Black-billed Magpie (Pica hudsonia) were significantly lower than projected abundances across much of the Great Plains. Black-capped Chickadee (Poecile atricapillus) abundances reached their lowest counts in portions of the Great Plains immediately after the arrival of WNV and experienced overall negative annual declines from 1988 to 2017. Two other species that were examined did not experience changes in abundance across the study area. Abundances of Black-billed Magpies and Black-capped Chickadees have declined over the past 30 years in the Great Plains, and WNV has likely played a major role in recent declines of magpies throughout the study area.

Transcriptome data on the quantitative numbers of transcriptional variants expressed in primary cells offer essential clues into specific cellular functions and biological processes. We have previously collected transcriptomes from primary smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and PDGFRα+ cells (fibroblast-like cells) isolated from murine jejunal and colonic smooth muscle and/or mucosal tissues as well as transcriptomes from the associated tissues (jejunal smooth muscle, colonic smooth muscle, and colonic mucosa). In this study, we have built the Smooth Muscle Transcriptome Browser (SMTB), https://med.unr.edu/physio/transcriptome, a web-based, graphical user interface that offers genetic references and expression profiles of all transcripts expressed at both the cellular (SMC, ICC, and PDGFRα+ cells) and tissue level (smooth muscle and mucosal tissue). This browser brings new insights into the cellular and biological functions of the cell types in gastrointestinal smooth muscle biology.

DNA methylation is a key epigenetic modification that can regulate gene expression. Genomic DNA hypomethylation is commonly found in many gastrointestinal (GI) diseases. Dysregulated gene expression in GI smooth muscle cells (GI-SMCs) can lead to motility disorders. However, the consequences of genomic DNA hypomethylation within GI-SMCs are still elusive. Utilizing a Cre-lox murine model, we have generated SMC-restricted DNA methyltransferase 1 (Dnmt1) knockout (KO) mice and analyzed the effects of Dnmt1 deficiency. Dnmt1-KO pups are born smaller than their wild-type littermates, have shortened GI tracts, and lose peristaltic movement due to loss of the tunica muscularis in their intestine, causing massive intestinal dilation, and death around postnatal day 21. Within smooth muscle tissue, significant CpG hypomethylation occurs across the genome at promoters, introns, and exons. Additionally, there is a marked loss of differentiated SMC markers (Srf, Myh11, miR-133, miR-143/145), an increase in pro-apoptotic markers (Nr4a1, Gadd45g), loss of cellular connectivity, and an accumulation of coated vesicles within SMC. Interestingly, we observed consistent abnormal expression patterns of enzymes involved in DNA methylation between both Dnmt1-KO mice and diseased human GI tissue. These data demonstrate that DNA hypomethylation in embryonic SMC, via congenital Dnmt1 deficiency, contributes to massive dysregulation of gene expression and is lethal to GI-SMC. These results suggest that Dnmt1 has a necessary role in the embryonic, primary development process of SMC with consistent patterns being found in human GI diseased tissue.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFRα(+), and neuronal cells during the development of intestinal obstruction.

Piping plovers (Charadrius melodus) are shorebirds federally protected by the U.S. Endangered Species Act that often nest on beaches in proximity to human recreation. We evaluated whether awareness...

Humans have altered nearly every natural disturbance regime on the planet through climate and land-use change, and in many instances, these processes may have interacting effects. For example, projected shifts in temperature and precipitation will likely influence disturbance regimes already affected by anthropogenic fire suppression or river impoundments. Understanding how disturbance-dependent species respond to complex and interacting environmental changes is important for conservation efforts. Using field-based demographic and movement rates, we conducted a metapopulation viability analysis for piping plovers (Charadrius melodus), a threatened disturbance-dependent species, along the Missouri and Platte rivers in the Great Plains of North America. Our aim was to better understand current and projected future metapopulation dynamics given that natural disturbances (flooding or high-flow events) have been greatly reduced by river impoundments and that climate change could further alter the disturbance regime. Although metapopulation abundance has been substantially reduced under the current suppressed disturbance regime (high-flow return interval similar to 20 yr), it could grow if the frequency of high-flow events increases as predicted under likely climate change scenarios. We found that a four-year return interval would maximize metapopulation abundance, and all subpopulations in the metapopulation would act as sources at a return interval of 15 yr or less. Regardless of disturbance frequency, the presence of even a small, stable source subpopulation buffered the metapopulation and sustained a low metapopulation extinction risk. Therefore, climate change could have positive effects in ecosystems where disturbances have been anthropogenically suppressed when climatic shifts move disturbance regimes toward more historical patterns. Furthermore, stable source populations, even if unintentionally maintained through anthropogenic activities, may be critical for the persistence of metapopulations of early-successional species under both suppressed disturbance regimes and disturbance regimes where climate change has further altered disturbance frequency or scope. Nebraska Environmental Trust Nebraska State Wildlife Grant Program Nebraska Wildlife Conservation Fund U.S. Army Corps of Engineers U.S. Fish and Wildlife Service (USFWS) USFWS North Atlantic Landscape Conservation Cooperative Virginia Tech Funding was provided by the Nebraska Environmental Trust, the Nebraska State Wildlife Grant Program, the Nebraska Wildlife Conservation Fund, the U.S. Army Corps of Engineers, the U.S. Fish and Wildlife Service (USFWS), the USFWS North Atlantic Landscape Conservation Cooperative, and Virginia Tech. We thank C. Aron, K. Brennan, R. Cobb, K. Crane, E. Dowd-Stukel, C. Huber, K. Kreil, C. Kruse, G. Pavelka, G. Wagner, W. Werkmeister, S. Wilson, L. Yager, and cooperators from the National Park Service, U.S. Fish and Wildlife Service, South Dakota Department of Game, Fish, and Parks, Nebraska Game and Parks Commission, and the Missouri River Institute for support throughout the project. We would like to thank sand and gravel mining companies and lakeshore housing development communities for access to property. We acknowledge the tireless efforts of our many technicians from 2005 to 2013. We also thank five anonymous reviewers, T. Simmons, and C. McGowan (USGS) for their comments on earlier drafts of this manuscript. This work was conducted under Institutional Animal Care and Use Committee permits 877 and 14-003, and U.S. Fish and Wildlife Service Threatened and Endangered Species Permits TE070027-0 and TE103272-3. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

Platelet-derived growth factor receptor alpha (PDGFRα)+ cells are distributed into distinct morphological groups within the serosal, muscular, and submucosal layers as well as the myenteric and deep muscular plexi. PDGFRα+ cells directly interact with interstitial cells of Cajal (ICC) and smooth muscle cells (SMC) in gastrointestinal smooth muscle tissue. These three cell types, SMC, ICC, and PDGFRα+ cells (SIP cells), form an electrical syncytium, which dynamically regulates gastrointestinal motility. We have previously reported the transcriptomes of SMC and ICC. To complete the SIP cell transcriptome project, we obtained transcriptome data from jejunal and colonic PDGFRα+ cells. The PDGFRα+ cell transcriptome data were added to the Smooth Muscle Genome Browser that we previously built for the genome-scale gene expression data of ICC and SMC. This browser provides a comprehensive reference for all transcripts expressed in SIP cells. By analyzing the transcriptomes, we have identified a unique set of PDGFRα+ cell signature genes, growth factors, transcription factors, epigenetic enzymes/regulators, receptors, protein kinases/phosphatases, and ion channels/transporters. We demonstrated that the low voltage-dependent T-type Ca2+ channel Cacna1g gene was particularly expressed in PDGFRα+ cells in the intestinal serosal layer in mice. Expression of this gene was significantly induced in the hyperplasic PDGFRα+ cells of obstructed small intestine in mice. This gene was also over-expressed in colorectal cancer, Crohn's disease, and diverticulitis in human patients. Taken together, our data suggest that Cacna1g exclusively expressed in serosal PDGFRα+ cells is a new pathological marker for gastrointestinal diseases.

This study examined the microbial diversity and community assembly of oral microbiota in periodontal health and disease and after nonsurgical periodontal treatment. The V4 region of 16S rRNA gene from DNA of 238 saliva and subgingival samples of 21 healthy and 48 diseased subjects was amplified and sequenced. Among 1979 OTUs identified, 28 were overabundant in diseased plaque. Six of these taxa were also overabundant in diseased saliva. Twelve OTUs were overabundant in healthy plaque. There was a trend for disease-associated taxa to decrease and health-associated taxa to increase after treatment with notable variations among individual sites. Network analysis revealed modularity of the microbial communities and identified several health- and disease-specific modules. Ecological drift was a major factor that governed community turnovers in both plaque and saliva. Dispersal limitation and homogeneous selection affected the community assembly in plaque, with the additional contribution of homogenizing dispersal for plaque within individuals. Homogeneous selection and dispersal limitation played important roles, respectively, in healthy saliva and diseased pre-treatment saliva between individuals. Our results revealed distinctions in both taxa and assembly processes of oral microbiota between periodontal health and disease. Furthermore, the community assembly analysis has identified potentially effective approaches for managing periodontitis.

Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC), which serve as slow-wave electrical pacemakers for gastrointestinal (GI) smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (http://med.unr.edu/physio/transcriptome) based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.

Birds frequently interact with people when they occur in coupled human-ecological or anthropogenic environments, which makes the protection of legally protected species a challenge. Flight initiation distances (FIDs) are often used to inform development of appropriate buffer distances required for human exclusion zones used to protect birds nesting in anthropogenic landscapes. Piping Plovers (Charadrius melodus) are protected by the Endangered Species Act in the United States and often nest in areas used by humans. Studies evaluating Piping Plover FIDs are limited and implementation of exclusion zones has been inconsistent across the species’ range. We measured Piping Plover response and FIDs to naturally occurring stimuli on public beaches at Lake McConaughy, Nebraska, USA. Piping Plover FIDs differed most by stimulus class (vehicle, human, dog, human with dog), Julian day, and hour of day. Piping Plover FIDs were greatest for dog and human with dog compared to humans and vehicles. For all types of stimuli, Piping Plover FIDs decreased over time during the nesting season and increased slightly during each day. In the majority of instances in which Piping Plovers left their nests, return times to the nest were relatively short (less than three minutes). These results suggest Piping Plovers become habituated to the presence of human-related stimuli over the course of a nesting season, but other explanations such as parental investment and risk allocation cannot be excluded. Additional research and improved guidance regarding the implementation of exclusion zones is needed so managers can implement effective protection programs in anthropogenic landscapes. Distances de fuite de Pluviers siffleurs (Charadrius melodus) nicheurs en reaction au derangement humain RESUME. Souvent, les oiseaux interagissent avec les humains lorsqu'ils frequentent des environnements mixtes anthropiques-naturels ou anthropiques, rendant difficile la protection d'especes protegees legalement. On utilise couramment la distance de fuite (DF) afin de determiner une distance securitaire appropriee pour des zones d'exclusion humaine necessaires a la protection d'oiseaux qui nichent dans des paysages anthropiques. Les Pluviers siffleurs (Charadrius melodus) sont proteges en vertu de la Loi sur les especes en peril (Endangered Species Act) aux Etats-Unis et nichent regulierement dans des endroits aussi utilises par les humains. Les recherches visant a evaluer les DF des Pluviers siffleurs sont peu nombreuses et la mise en place de zones d'exclusion humaine a ete inegale dans l'ensemble de l'aire de repartition de cette espece. Nous avons mesure la reaction des pluviers et les DF en presence de stimuli naturels sur les plages publiques au lac McConaughy, au Nebraska, Etats-Unis. Les DF des pluviers ont surtout varie selon le type de stimuli (vehicule, humain, chien, humain avec chien), le jour julien et l'heure du jour. Les DF des pluviers etaient le plus elevees en presence de chiens et d'humains avec des chiens, comparativement a la presence d'humains et de vehicules. Pour tous les types de stimuli, les DF diminuaient au fil de la saison de nidification et augmentaient legerement en fonction de l'heure du jour. Dans la majorite des cas ou les pluviers ont quitte leur nid, le temps qu'ils ont mis a y retourner etait plutot court (moins de trois minutes). Nos resultats indiquent que les Pluviers siffleurs s'habituent a la presence de stimuli lies aux humains au cours d'une saison de nidification, mais d'autres explications, comme l'investissement parental et la prise en compte du risque, ne peuvent pas etre ecartees. Davantage de recherches et de meilleures directives en ce qui concerne la mise en place de zones d'exclusion humaine sont necessaires pour que les gestionnaires soient bien outilles au moment de la mise en oeuvre des programmes de protection efficaces dans les paysages anthropiques.

Background Many species are distributed as metapopulations in dynamic landscapes, where habitats change through space and time. Individuals locate habitat through dispersal, and the relationship between a species and landscape characteristics can have profound effects on population persistence. Despite the importance of connectivity in dynamic environments, few empirical studies have examined temporal variability in dispersal or its effect on metapopulation dynamics. In response to this knowledge gap, we studied the dispersal, demography, and viability of a metapopulation of an endangered, disturbance-dependent shorebird. We examined three subpopulations of piping plovers (Charadrius melodus) on the lower Platte and Missouri rivers from 2008–2013. High flow events from an upstream dam on the Missouri River in 2010 and 2011 allowed us to assess the effect of total habitat loss and the subsequent creation of new habitat associated with a large disturbance at one ‘natural’ study location. The other two sites within the metapopulation, which were maintained by anthropogenic activities (e.g., mining, development, habitat restoration), were largely unaffected by this disturbance, resulting in a controlled natural experiment. Results High flow events were associated with increased emigration, decreased immigration, and decreased survival in the subpopulation that experienced high flows. Following the high flow event, immigration into that subpopulation increased. Dispersal rates among subpopulations were negatively correlated with distance. The metapopulation had a low probability of extinction over 100 years (0 %) under the current disturbance interval and associated dispersal and survival rates. However, persistence depended on relatively stable, human-created habitats, not the dynamic, natural habitat (47.7 % extinction probability for this subpopulation). Conclusions We found that functional connectivity, as measured by the rate of dispersal among subpopulations, increased as a result of the high flow event in our study metapopulation. Plovers also increased reproductive output following this event. Although the study metapopulation had a low overall probability of extinction, metapopulation persistence depended on anthropogenically created habitats that provided a small but stable source of nesting habitat and dispersers through time. However, all subpopulations remained small, even if persistent, making them individually vulnerable to extinction through stochastic events. Given the highly dynamic nature of habitat availability in this system, maintaining several subpopulations within the metapopulation and stable sources of habitat will be critical, and this species will likely remain conservation-reliant. Electronic supplementary material The online version of this article (doi:10.1186/s40462-016-0072-y) contains supplementary material, which is available to authorized users.