The Dilute domain in Canoe is not essential for linking cell junctions to the cytoskeleton but supports morphogenesis robustness.

Robust linkage between adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The Drosophila multidomain protein Canoe and its mammalian homolog afadin are crucial for this, as in their absence many events of morphogenesis fail. To define the mechanism of action for Canoe, we are taking it apart. Canoe has five folded protein domains and a long intrinsically disordered region. The largest is the Dilute domain, which is shared by Canoe and myosin V. To define the roles of this domain in Canoe, we combined biochemical, genetic and cell biological assays. AlphaFold was used to predict its structure, providing similarities and contrasts with Myosin V. Biochemical data suggested one potential shared function - the ability to dimerize. We generated Canoe mutants with the Dilute domain deleted (CnoΔDIL). Surprisingly, they were viable and fertile. CnoΔDIL localized to adherens junctions and was enriched at junctions under tension. However, when its dose was reduced, CnoΔDIL did not provide fully wild-type function. Furthermore, canoeΔDIL mutants had defects in the orchestrated cell rearrangements of eye development. This reveals the robustness of junction-cytoskeletal connections during morphogenesis and highlights the power of natural selection to maintain protein structure.

H3K27ac HiChIP in prostate cell lines identifies risk genes for prostate cancer susceptibility.

Genome-wide association studies (GWASs) have identified more than 200 prostate cancer (PrCa) risk regions, which provide potential insights into causal mechanisms. Multiple lines of evidence show that a significant proportion of PrCa risk can be explained by germline causal variants that dysregulate nearby target genes in prostate-relevant tissues, thus altering disease risk. The traditional approach to explore this hypothesis has been correlating GWAS variants with steady-state transcript levels, referred to as expression quantitative trait loci (eQTLs). In this work, we assess the utility of chromosome conformation capture (3C) coupled with immunoprecipitation (HiChIP) to identify target genes for PrCa GWAS risk loci. We find that interactome data confirm previously reported PrCa target genes identified through GWAS/eQTL overlap (e.g., MLPH). Interestingly, HiChIP identifies links between PrCa GWAS variants and genes well-known to play a role in prostate cancer biology (e.g., AR) that are not detected by eQTL-based methods. HiChIP predicted enhancer elements at the AR and NKX3-1 prostate cancer risk loci, and both were experimentally confirmed to regulate expression of the corresponding genes through CRISPR interference (CRISPRi) perturbation in LNCaP cells. Our results demonstrate that looping data harbor additional information beyond eQTLs and expand the number of PrCa GWAS loci that can be linked to candidate susceptibility genes.

Localizing Components of Shared Transethnic Genetic Architecture of Complex Traits from GWAS Summary Data.

Despite strong transethnic genetic correlations reported in the literature for many complex traits, the non-transferability of polygenic risk scores across populations suggests the presence of population-specific components of genetic architecture. We propose an approach that models GWAS summary data for one trait in two populations to estimate genome-wide proportions of population-specific/shared causal SNPs. In simulations across various genetic architectures, we show that our approach yields approximately unbiased estimates with in-sample LD and slight upward-bias with out-of-sample LD. We analyze nine complex traits in individuals of East Asian and European ancestry, restricting to common SNPs (MAF > 5%), and find that most common causal SNPs are shared by both populations. Using the genome-wide estimates as priors in an empirical Bayes framework, we perform fine-mapping and observe that high-posterior SNPs (for both the population-specific and shared causal configurations) have highly correlated effects in East Asians and Europeans. In population-specific GWAS risk regions, we observe a 2.8× enrichment of shared high-posterior SNPs, suggesting that population-specific GWAS risk regions harbor shared causal SNPs that are undetected in the other GWASs due to differences in LD, allele frequencies, and/or sample size. Finally, we report enrichments of shared high-posterior SNPs in 53 tissue-specific functional categories and find evidence that SNP-heritability enrichments are driven largely by many low-effect common SNPs.

A Randomized Prospective Trial Comparing Repository Corticotropin Injection and Intravenous Methylprednisolone for Neuroprotection in Acute Optic Neuritis.

BACKGROUND: Repository corticotrophin injection (RCI, Acthar Gel) and intravenous methylprednisolone (IVMP) improve the rate but not the extent of visual recovery following acute optic neuritis. RCI has adrenal-stimulating and melanocortin receptor-stimulating properties that may endow it with unique anti-inflammatory properties relative to IVMP. METHODS: Individuals with acute optic neuritis of less than 2 weeks duration were prospectively enrolled and randomized 1:1 to receive either RCI or IVMP. Peripapillary retinal nerve fiber layer (pRNFL) and ganglion cell plus inner plexiform layer thickness (GC + IPL) were serially evaluated by OCT. In addition, patient-reported outcomes (PROs) for changes in fatigue, mood, visual function, depression, and quality of life (QOL) were measured, and high and low contrast visual acuity were recorded. RESULTS: Thirty-seven subjects were enrolled (19 RCI; 18 IVMP); the average time from symptom to treatment was 8.8 days. At 6 months, there was no difference in the primary outcome: loss of average pRNFL thickness in the affected eye (RCI vs IVMP: -13.1 vs -11.7 µm, P = 0.88) 6 months after randomization. Additional outcomes also showed no difference between treatment groups: 6-month attenuation of GC + IPL thickness (RCI vs IVMP: -13.8 vs -12.0 µm, P = 0.58) and frequency of pRNFL swelling at 1 month (RCI vs IVMP: 63% vs 72%, P = 0.73) and 3 months (RCI vs IVMP: 26% vs 31%, P = 0.99). Both treatments resulted in improvement in visual function and PROs. CONCLUSIONS: Treatment of acute optic neuritis with RCI or IVMP produced no clinically meaningful differences in optic nerve structure or visual function.

Hexagonal patterning of the Drosophila eye.

A complex network of transcription factor interactions propagates across the larval eye disc to establish columns of evenly-spaced R8 precursor cells, the founding cells of Drosophila ommatidia. After the recruitment of additional photoreceptors to each ommatidium, the surrounding cells are organized into their stereotypical pattern during pupal development. These support cells - comprised of pigment and cone cells - are patterned to encapsulate the photoreceptors and separate ommatidia with an hexagonal honeycomb lattice. Since the proteins and processes essential for correct eye patterning are conserved, elucidating how these function and change during Drosophila eye patterning can substantially advance our understanding of transcription factor and signaling networks, cytoskeletal structures, adhesion complexes, and the biophysical properties of complex tissues during their morphogenesis. Our understanding of many of these aspects of Drosophila eye patterning is largely descriptive. Many important questions, especially relating to the regulation and integration of cellular events, remain.

Estimation of regional polygenicity from GWAS provides insights into the genetic architecture of complex traits.

The number of variants that have a non-zero effect on a trait (i.e. polygenicity) is a fundamental parameter in the study of the genetic architecture of a complex trait. Although many previous studies have investigated polygenicity at a genome-wide scale, a detailed understanding of how polygenicity varies across genomic regions is currently lacking. In this work, we propose an accurate and scalable statistical framework to estimate regional polygenicity for a complex trait. We show that our approach yields approximately unbiased estimates of regional polygenicity in simulations across a wide-range of various genetic architectures. We then partition the polygenicity of anthropometric and blood pressure traits across 6-Mb genomic regions (N = 290K, UK Biobank) and observe that all analyzed traits are highly polygenic: over one-third of regions harbor at least one causal variant for each of the traits analyzed. Additionally, we observe wide variation in regional polygenicity: on average across all traits, 48.9% of regions contain at least 5 causal SNPs, 5.44% of regions contain at least 50 causal SNPs. Finally, we find that heritability is proportional to polygenicity at the regional level, which is consistent with the hypothesis that heritability enrichments are largely driven by the variation in the number of causal SNPs.

Assessing the Effects of the COVID-19 Pandemic on Nontraditional Students' Mental Health and Well-Being.

The current study sought to measure how the COVID-19 pandemic affected the mental health and well-being of college students, particularly nontraditional students. Participants (n = 321) completed a series of surveys assessing their level of depression, anxiety, sleep disturbances, insomnia, and well-being. Participants also indicated their nontraditional student characteristics, level of resilience, and additional life stressors due to the pandemic. Statistical analyses found that participants reported higher levels of depression, anxiety, sleep disturbances, and insomnia, with corresponding lower levels of well-being across all students, compared with prepandemic levels. Results showed that while nontraditional students indicated an increased number of life stressors during the pandemic compared with their traditional peers, nontraditional students also demonstrated higher levels of resilience. Nontraditional students appear to be more successful at managing stressful life events due to the increased resilience that comes with age and experience, which can better prepare them to persevere and overcome challenges.

Mask, a component of the Hippo pathway, is required for Drosophila eye morphogenesis.

Hippo signaling is an important regulator of tissue size, but it also has a lesser-known role in tissue morphogenesis. Here we use the Drosophila pupal eye to explore the role of the Hippo effector Yki and its cofactor Mask in morphogenesis. We found that Mask is required for the correct distribution and accumulation of adherens junctions and appropriate organization of the cytoskeleton. Accordingly, disrupting mask expression led to severe mis-patterning and similar defects were observed when yki was reduced or in response to ectopic wts. Further, the patterning defects generated by reducing mask expression were modified by Hippo pathway activity. RNA-sequencing revealed a requirement for Mask for appropriate expression of numerous genes during eye morphogenesis. These included genes implicated in cell adhesion and cytoskeletal organization, a comprehensive set of genes that promote cell survival, and numerous signal transduction genes. To validate our transcriptome analyses, we then considered two loci that were modified by Mask activity: FER and Vinc, which have established roles in regulating adhesion. Modulating the expression of either locus modified mask mis-patterning and adhesion phenotypes. Further, expression of FER and Vinc was modified by Yki. It is well-established that the Hippo pathway is responsive to changes in cell adhesion and the cytoskeleton, but our data indicate that Hippo signaling also regulates these structures.

Tyrosine Phosphorylation of CD2AP Affects Stability of the Slit Diaphragm Complex.

BACKGROUND: CD2-associated protein (CD2AP), a slit diaphragm-associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a "stabilizer" of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. METHODS: We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in Cd2apKD zebrafish and in Drosophila cindrRNAi mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. RESULTS: We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo. We demonstrated that tyrosine in position Y10 of the SH3-1 domain of CD2AP is indispensable for CD2AP function in vivo. We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. CONCLUSIONS: Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.

Timely Venous Thromboembolism Prophylaxis in Trauma: A Team Approach to Process Improvement.

Venous thromboembolism is a significant complication in trauma. Multisystem injury, advancing age, surgery, and blood transfusion all contribute to the risk of venous thromboembolism in trauma patients. Our Level I trauma center was identified as an outlier with compliance in timely venous thromboembolism prophylaxis in the Michigan Trauma Quality Improvement Program, a statewide collaborative for improving trauma care. The purpose of this study was to provide an evaluation of a performance improvement project to increase the timely administration of venous thromboembolism prophylaxis in admitted trauma patients. Using a Plan-Do-Study-Act method of quality improvement, we initiated a focused, goal-directed team approach that emphasized education, tracking, and feedback. This approach resulted in improved and sustained compliance rates. Resolute focus, audit, and feedback moved our center from a low- to high-performing center for timely venous thromboembolism prophylaxis.

JNK is antagonized to ensure the correct number of interommatidial cells pattern the Drosophila retina.

Apoptosis is crucial during the morphogenesis of most organs and tissues, and is utilized for tissues to achieve their proper size, shape and patterning. Many signaling pathways contribute to the precise regulation of apoptosis. Here we show that Jun N-terminal Kinase (JNK) activity contributes to the coordinated removal of interommatidial cells via apoptosis in the Drosophila pupal retina. This is consistent with previous findings that JNK activity promotes apoptosis in other epithelia. However, we found that JNK activity is repressed by Cindr (the CIN85 and CD2AP ortholog) in order to promote cell survival. Reducing the amount of Cindr resulted in ectopic cell death. Increased expression of the Drosophila JNK basket in the setting of reduced cindr expression was found to result in even more severe apoptosis, whilst ectopic death was found to be reduced if retinas were heterozygous for basket. Hence Cindr is required to properly restrict JNK-mediated apoptosis in the pupal eye, resulting in the correct number of interommatidial cells. A lack of precise control over developmental apoptosis can lead to improper tissue morphogenesis.

A unifying framework for joint trait analysis under a non-infinitesimal model.

Motivation: A large proportion of risk regions identified by genome-wide association studies (GWAS) are shared across multiple diseases and traits. Understanding whether this clustering is due to sharing of causal variants or chance colocalization can provide insights into shared etiology of complex traits and diseases. Results: In this work, we propose a flexible, unifying framework to quantify the overlap between a pair of traits called UNITY (Unifying Non-Infinitesimal Trait analYsis). We formulate a Bayesian generative model that relates the overlap between pairs of traits to GWAS summary statistic data under a non-infinitesimal genetic architecture underlying each trait. We propose a Metropolis-Hastings sampler to compute the posterior density of the genetic overlap parameters in this model. We validate our method through comprehensive simulations and analyze summary statistics from height and body mass index GWAS to show that it produces estimates consistent with the known genetic makeup of both traits. Availability and implementation: The UNITY software is made freely available to the research community at: https://github.com/bogdanlab/UNITY. Supplementary information: Supplementary data are available at Bioinformatics online.

An automated machine learning-based model predicts postoperative mortality using readily-extractable preoperative electronic health record data.

BACKGROUND: Rapid, preoperative identification of patients with the highest risk for medical complications is necessary to ensure that limited infrastructure and human resources are directed towards those most likely to benefit. Existing risk scores either lack specificity at the patient level or utilise the American Society of Anesthesiologists (ASA) physical status classification, which requires a clinician to review the chart. METHODS: We report on the use of machine learning algorithms, specifically random forests, to create a fully automated score that predicts postoperative in-hospital mortality based solely on structured data available at the time of surgery. Electronic health record data from 53 097 surgical patients (2.01% mortality rate) who underwent general anaesthesia between April 1, 2013 and December 10, 2018 in a large US academic medical centre were used to extract 58 preoperative features. RESULTS: Using a random forest classifier we found that automatically obtained preoperative features (area under the curve [AUC] of 0.932, 95% confidence interval [CI] 0.910-0.951) outperforms Preoperative Score to Predict Postoperative Mortality (POSPOM) scores (AUC of 0.660, 95% CI 0.598-0.722), Charlson comorbidity scores (AUC of 0.742, 95% CI 0.658-0.812), and ASA physical status (AUC of 0.866, 95% CI 0.829-0.897). Including the ASA physical status with the preoperative features achieves an AUC of 0.936 (95% CI 0.917-0.955). CONCLUSIONS: This automated score outperforms the ASA physical status score, the Charlson comorbidity score, and the POSPOM score for predicting in-hospital mortality. Additionally, we integrate this score with a previously published postoperative score to demonstrate the extent to which patient risk changes during the perioperative period.

Improved methods for multi-trait fine mapping of pleiotropic risk loci.

MOTIVATION: Genome-wide association studies (GWAS) have identified thousands of regions in the genome that contain genetic variants that increase risk for complex traits and diseases. However, the variants uncovered in GWAS are typically not biologically causal, but rather, correlated to the true causal variant through linkage disequilibrium (LD). To discern the true causal variant(s), a variety of statistical fine-mapping methods have been proposed to prioritize variants for functional validation. RESULTS: In this work we introduce a new approach, fastPAINTOR, that leverages evidence across correlated traits, as well as functional annotation data, to improve fine-mapping accuracy at pleiotropic risk loci. To improve computational efficiency, we describe an new importance sampling scheme to perform model inference. First, we demonstrate in simulations that by leveraging functional annotation data, fastPAINTOR increases fine-mapping resolution relative to existing methods. Next, we show that jointly modeling pleiotropic risk regions improves fine-mapping resolution compared to standard single trait and pleiotropic fine mapping strategies. We report a reduction in the number of SNPs required for follow-up in order to capture 90% of the causal variants from 23 SNPs per locus using a single trait to 12 SNPs when fine-mapping two traits simultaneously. Finally, we analyze summary association data from a large-scale GWAS of lipids and show that these improvements are largely sustained in real data. AVAILABILITY AND IMPLEMENTATION: The fastPAINTOR framework is implemented in the PAINTOR v3.0 package which is publicly available to the research community http://bogdan.bioinformatics.ucla.edu/software/paintor CONTACT: gkichaev@ucla.eduSupplementary information: Supplementary data are available at Bioinformatics online.

The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer.

MAGI-1 Interacts with Nephrin to Maintain Slit Diaphragm Structure through Enhanced Rap1 Activation in Podocytes.

MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.

Moderate-to-severe obstructive sleep apnea is associated with telomere lengthening.

Obstructive sleep apnea (OSA) is associated with cardiometabolic diseases. Telomere shortening is linked to hypertension, diabetes mellitus, and cardiovascular diseases. Because these conditions are highly prevalent in OSA, we hypothesized that telomere length (TL) would be reduced in OSA patients. We identified 106 OSA and 104 non-OSA subjects who underwent polysomnography evaluation. Quantitative PCR was used to measure telomere length in genomic DNA isolated from peripheral blood samples. The association between OSA and TL was determined using unadjusted and adjusted linear models. There was no difference in TL between the OSA and non-OSA (control) group. However, we observed a J-shaped relationship between TL and OSA severity: the longest TL in moderate-to-severe OSA [4,918 ± 230 (SD) bp] and the shortest TL in mild OSA (4,735 ± 145 bp). Mean TL in moderate-to-severe OSA was significantly longer than in the control group after adjustment for age, sex, body mass index, hypertension, dyslipidemia, and depression (ß = 96.0, 95% confidence interval: 15.4-176.6, P = 0.020). In conclusion, moderate-to-severe OSA is associated with telomere lengthening. Our findings support the idea that changes in TL are not unidirectional processes, such that telomere shortening occurs with age and disease but may be prolonged in moderate-to-severe OSA.NEW & NOTEWORTHY Here, we show that moderate-to-severe obstructive sleep apnea is associated with longer telomeres, independent of age and cardiovascular risk factors, challenging the hypothesis that telomere shortening is a unidirectional process related to age/disease. A better understanding of the mechanisms underlying telomere dynamics may identify targets for therapeutic intervention in cardiovascular aging/other chronic diseases.

Exogenous glucagon-like peptide-1 acts in sites supplied by the cranial mesenteric artery to reduce meal size and prolong the intermeal interval in rats.

Three experiments were done to better assess the gastrointestinal (GI) site(s) of action of GLP-1 on food intake in rats. First, near-spontaneous nocturnal chow meal size (MS), intermeal intervals (IMI) length and satiety ratios (SR = MS/IMI) were measured after infusion of saline, 0.025 or 0.5 nmol/kg GLP-1 into the celiac artery (CA, supplying the stomach and upper duodenum), cranial mesenteric artery (CMA, supplying small and all of the large intestine except the rectum), femoral artery (FA, control) or portal vein (PV, control). Second, infusion of 0.5 nmol/kg GLP-1 was tested after pretreatment with the GLP-1 receptor (GLP-1R) antagonist exendin-4(3-39) via the same routes. Third, the regional distribution of GLP-1R in the rat GI tract was determined using rtPCR. CA, CMA and FA GLP-1 reduced first MS relative to saline, with the CMA route more effective than the others. Only CMA GLP-1 prolonged the IMI. None of the infusions affected second MS or later eating. CA and CMA GLP-1 increased the SR, with the CMA route more effective than the CA route. CMA exendin-4 (3-39) infusion reduced the effect of CMA GLP-1. Finally GLP-1R expression was found throughout the GI tract. The results suggest that exogenous GLP-1 acts in multiple GI sites to reduce feeding under our conditions and that GLP-1R in the area supplied by the CMA, i.e., the small and part of the large intestine, plays the leading role.

Planar cell polarity controls directional Notch signaling in the Drosophila leg.

The generation of functional structures during development requires tight spatial regulation of signaling pathways. Thus, in Drosophila legs, in which Notch pathway activity is required to specify joints, only cells distal to ligand-producing cells are capable of responding. Here, we show that the asymmetric distribution of planar cell polarity (PCP) proteins correlates with this spatial restriction of Notch activation. Frizzled and Dishevelled are enriched at distal sides of each cell and hence localize at the interface with ligand-expressing cells in the non-responding cells. Elimination of PCP gene function in cells proximal to ligand-expressing cells is sufficient to alleviate the repression, resulting in ectopic Notch activity and ectopic joint formation. Mutations that compromise a direct interaction between Dishevelled and Notch reduce the efficacy of repression. Likewise, increased Rab5 levels or dominant-negative Deltex can suppress the ectopic joints. Together, these results suggest that PCP coordinates the spatial activity of the Notch pathway by regulating endocytic trafficking of the receptor.

Celiac and the cranial mesenteric arteries supply gastrointestinal sites that regulate meal size and intermeal interval length via cholecystokinin-58 in male rats.

The site(s) of action that control meal size and intermeal interval (IMI) length by cholecystokinin-58 (CCK-58), the only detectable endocrine form of CCK in the rat, are not known. To test the hypothesis that the gastrointestinal tract may contain such sites, we infused low doses of CCK-58 (0.01, 0.05, 0.15 and 0.25nmol/kg) into the celiac artery (CA, supplying stomach and upper duodenum), the cranial mesenteric artery (CMA, supplying small and most of the large intestines), the femoral artery (FA, control) and the portal vein (PV, draining the gastrointestinal tract) prior to the onset of the dark cycle in freely fed male rats. We measured the first meal size (chow), second meal size, IMI and satiety ratio (SR, IMI/meal size). We found that (1) all doses of CCK-58 given in the CA and the highest dose given in the CMA reduced the first meal size, (2) all doses of CCK-58 given in the CA reduced the second meal size, (3) a CCK-58 dose of 0.15nmol/kg given in the CA and 0.15 and 0.25nmol/kg given in the CMA prolonged the IMI, (4) CCK-58 (0.05, 0.15, 0.25nmol/kg) given in the CA and 0.25nmol/kg given in the CMA increased the SR, and (5) CCK-58 given in the FA and PV had no effect on the meal size or intermeal interval. These results support our hypothesis that the gastrointestinal tract contains sites of action that regulate meal size and IMI length via CCK-58. The stomach and upper duodenum may contain sites regulating meal size, whereas the small intestine and part of the large intestine may contain sites regulating the IMI.