Multi-Omics Resolves a Sharp Disease-State Shift between Mild and Moderate COVID-19.

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.

POGZ promotes homology-directed DNA repair in an HP1-dependent manner.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.

The Australian New Zealand Consortium in Children, Adolescents, and Young Adults Oncofertility action plan.

International and national oncofertility networks, including the US-led Oncofertility Consortium, FertiProtekt, and the Danish Network, have played pivotal roles in advancing the discipline of oncofertility over the last decade. Many other countries lack a shared approach to pediatric oncofertility health service delivery. This study aims to describe baseline oncofertility practices at Australian New Zealand Children's Haematology/Oncology Group centers in 2019-2021, describe binational priorities for care, and propose a 5-year action plan for best practice to be implemented by the newly formed Australian New Zealand Consortium in Children, Adolescents, and Young Adults (CAYA) Oncofertility (ANZCO).

LIN28 expression and function in medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current treatment modalities are not completely effective and can lead to severe neurological and cognitive adverse effects. In addition to urgently needing better treatment approaches, new diagnostic and prognostic biomarkers are required to improve the therapy outcomes of MB patients. The RNA-binding proteins, LIN28A and LIN28B, are known to regulate invasive phenotypes in many different cancer types. However, the expression and function of these proteins in MB had not been studied to date. This study identified the expression of LIN28A and LIN28B in MB patient samples and cell lines and assessed the effect of LIN28 inhibition on MB cell growth, metabolism and stemness. LIN28B expression was significantly upregulated in MB tissues compared to normal brain tissues. This upregulation, which was not observed in other brain tumors, was specific for the aggressive MB subgroups and correlated with patient survival and metastasis rates. Functionally, pharmacological inhibition of LIN28 activity concentration-dependently reduced LIN28B expression, as well as the growth of D283 MB cells. While LIN28 inhibition did not affect the levels of intracellular ATP, it reduced the expression of the stemness marker CD133 in D283 cells and the sphere formation of CHLA-01R cells. LIN28B, which is highly expressed in the human cerebellum during the first few months after birth, subsequently decreased with age. The results of this study highlight the potential of LIN28B as a diagnostic and prognostic marker for MB and open the possibility to utilize LIN28 as a pharmacological target to suppress MB cell growth and stemness.

FAIMS Enhances the Detection of PTM Crosstalk Sites.

Protein post-translational modifications (PTMs) enable cells to rapidly change in response to biological stimuli. With hundreds of different PTMs, understanding these control mechanisms is complex. To date, efforts have focused on investigating the effect of a single PTM on protein function. Yet, many proteins contain multiple PTMs. Moreover, one PTM can alter the prevalence of another, a phenomenon termed PTM crosstalk. Understanding PTM crosstalk is critical; however, its detection is challenging since PTMs occur substoichiometrically. Here, we develop an enrichment-free, label-free proteomics method that utilizes high-field asymmetric ion mobility spectrometry (FAIMS) to enhance the detection of PTM crosstalk. We show that by searching for multiple combinations of dynamic PTMs on peptide sequences, a 6-fold increase in candidate PTM crosstalk sites is identified compared with that of standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflows. Additionally, by cycling through FAIMS compensation voltages within a single LC-FAIMS-MS/MS run, we show that our LC-FAIMS-MS/MS workflow can increase multi-PTM-containing peptide identifications without additional increases in run times. With 159 novel candidate crosstalk sites identified, we envisage LC-FAIMS-MS/MS to play an important role in expanding the repertoire of multi-PTM identifications. Moreover, it is only by detecting PTM crosstalk that we can "see" the full picture of how proteins are regulated.

SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice.

DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1-, and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision-making process during DSB repair.

COVID-19 vaccination in children and adolescents aged 5 years and older undergoing treatment for cancer and non-malignant haematological conditions: Australian and New Zealand Children's Haematology/Oncology Group consensus statement.

INTRODUCTION: The Australian Technical Advisory Group on Immunisation and New Zealand Ministry of Health recommend all children aged ≥ 5 years receive either of the two mRNA COVID-19 vaccines: Comirnaty (Pfizer), available in both Australia and New Zealand, or Spikevax (Moderna), available in Australia only. Both vaccines are efficacious and safe in the general population, including children. Children and adolescents undergoing treatment for cancer and immunosuppressive therapy for non-malignant haematological conditions are particularly vulnerable, with an increased risk of severe or fatal COVID-19. There remains a paucity of data regarding the immune response to COVID-19 vaccines in immunosuppressed paediatric populations, with data suggestive of reduced immunogenicity of the vaccine in immunocompromised adults. RECOMMENDATIONS: Considering the safety profile of mRNA COVID-19 vaccines and the increased risk of severe COVID-19 in immunocompromised children and adolescents, COVID-19 vaccination is strongly recommended for this at-risk population. We provide a number of recommendations regarding COVID-19 vaccination in this population where immunosuppressive, chemotherapeutic and/or targeted biological agents are used. These include the timing of vaccination in patients undergoing active treatment, management of specific situations where vaccination is contraindicated or recommended under special precautions, and additional vaccination recommendations for severely immunocompromised patients. Finally, we stress the importance of upcoming clinical trials to identify the safest and most efficacious vaccination regimen for this population. CHANGES IN MANAGEMENT AS A RESULT OF THIS STATEMENT: This consensus statement provides recommendations for COVID-19 vaccination in children and adolescents aged ≥ 5 years with cancer and immunocompromising non-malignant haematological conditions, based on evidence, national and international guidelines and expert opinion. ENDORSED BY: The Australian and New Zealand Children's Haematology/Oncology Group.

The Australia and New Zealand Cardio-Oncology Registry: evaluation of chemotherapy-related cardiotoxicity in a national cohort of paediatric cancer patients.

Cancer therapy related cardiac dysfunction (CTRCD) is an area of increasing focus, particularly during the survivorship period, for paediatric, adolescent and adult cancer survivors. With the advent of immunotherapy and targeted therapy, there is a new set of mechanisms from which paediatric and young adult patients with cancer may suffer cardiovascular injury. Furthermore, cardiovascular disease is the leading cause of morbidity and mortality in the survivorship period. The recently established Australian Cardio-Oncology Registry is the largest and only population-based cardiotoxicity database of paediatric and adolescent and young adult oncology patients in the world, and the first paediatric registry that will document cardiotoxicity caused by chemotherapy and novel targeted therapies using a prospective approach. The database is designed for comprehensive data collection and evaluation of the Australian practice in terms of diagnosis and management of CTRCD. Using the Australian Cardio-Oncology Registry critical clinical information will be collected regarding predisposing factors for the development of CTRCD, the rate of subclinical left ventricular dysfunction and transition to overt heart failure, further research into protectant molecules against cardiac dysfunction and aid in the discovery of which genetic variants predispose to CTRCD. A health economic arm of the study will assess the cost/benefit of both the registry and cardio-oncology clinical implementation. Finally, an imaging arm will establish if exercise cardiac magnetic resonance imaging and VO2 max testing is a more sensitive predictor of cardiac reserve in paediatric and adolescent and young adult oncology patients exposed to cardiac toxic therapies.

Effect of Vagotomy and Sympathectomy on the Feeding Responses Evoked by Intra-Aortic Cholecystokinin-8 in Adult Male Sprague Dawley Rats.

The vagus nerve and the celiaco-mesenteric ganglia (CMG) are required for reduction of meal size (MS) and prolongation of the intermeal interval (IMI) by intraperitoneal (ip) sulfated cholecystokinin-8 (CCK-8). However, recently we have shown that the gut regulates these responses. Therefore, reevaluating the role of the vagus and the CMG in the feeding responses evoked by CCK is necessary because the gut contains the highest concentration of enteric, vagal and splanchnic afferents and CCK-A receptors, which are required for reduction of food intake by this peptide, compared to other abdominal organs. To address this necessity, we injected sulfated CCK-8 (0, 0.1, 0.5, 1 and 3 nmol/kg) in the aorta, near the gastrointestinal sites of action of the peptide, in three groups of free-feeding rats (n = 10 rats per group), subdiaphragmatic vagotomy (VGX), celiaco-mesenteric ganglionectomy (CMGX) and sham-operated, and recorded seven feeding responses. In the sham group, CCK-8 reduced MS (normal chow), prolonged the intermeal interval (IMI, time between first and second meals), increased satiety ratio (SR, IMI/MS), shortened duration of first meal, reduced total (24 hrs) food intake and reduced number of meals relative to saline vehicle. Vagotomy attenuated all of the previous responses except IMI length and SR, and CMGX attenuated all of those responses. In conclusion, the feeding responses evoked by sulfated CCK-8 require, independently, the vagus nerve and the CMG.

The Vagus Nerve and the Celiaco-mesenteric Ganglia Participate in the Feeding Responses Evoked by Non-sulfated Cholecystokinin-8 in Male Sprague Dawley Rats.

We have shown that non-sulfated cholecystokinin-8 (NS CCK-8) reduces food intake in adult male Sprague Dawley rats by activating cholecystokinin-B receptor (CCK-BR). Here, we tested the hypothesis that the vagus nerve and the celiaco-mesenteric ganglia may play a role in this reduction. The hypothesis stems from the following facts. The vagus and the celiaco-mesenteric ganglia contain NS CCK-8, they express and have binding sites for CCK-BR, NS CCK-8 activates CCK-BR on afferent vagal and sympathetic fibers and the two structures link the gastrointestinal tract to central feeding nuclei in the brain, which also contain the peptide and CCK-BR. To test this hypothesis, three groups of free-feeding rats, vagotomy (VGX), celiaco-mesenteric ganglionectomy (CMGX) and sham-operated, received NS CCK-8 (0, 0.5 and 1 nmol/kg) intraperitoneally prior to the onset of the dark cycle and various feeding behaviors were recorded. We found that in sham-operated rats both doses of NS CCK-8 reduced meal size (MS), prolonged the intermeal interval (IMI, time between first and second meal), increased satiety ratio (SR = IMI/MS), reduced 24-h food intake and reduced the number of meals relative to saline control. In the VGX and the CMGX groups, all of the previous responses were attenuated. Consistent with our hypothesis, the findings of the current work suggest a role for the vagus nerve and the celiaco-mesenteric ganglia in the feeding responses evoked by NS CCK-8.

Osteosarcoma in a Child Below 2 Years of Age: Case Report and Review of the Literature.

BACKGROUND: Osteosarcoma in children below the age of 5 is extremely rare. OBSERVATION: We report on a previously well 14-month-old male infant, who presented with a reluctance to weight-bear on his right leg and had an associated limp. Plain imaging and a magnetic resonance imaging scan demonstrated a lytic lesion in the right distal femur. An open surgical biopsy confirmed the diagnosis of osteosarcoma. There was no significant family history of cancer and genetic screening for Li-Fraumeni syndrome was negative. CONCLUSIONS: This case highlights the importance of timely consideration of osteosarcoma in an infant, when the clinical presentation and medical imaging are consistent with that diagnosis.

LAR protein tyrosine phosphatase regulates focal adhesions through CDK1.

Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton- and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix.

Allosteric Inhibitor of KRas Identified Using a Barcoded Assay Microchip Platform.

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC50) of ∼24 µM.

Environmental and genetic determinants of transcriptional plasticity in Chinook salmon.

Variation in gene transcription is widely believed to be the mechanistic basis of phenotypically plastic traits; however, comparatively little is known about the inheritance patterns of transcriptional variation that would allow us to predict its response to selection. In addition, acclimation to different environmental conditions influences acute transcriptional responses to stress and it is unclear if these effects are heritable. To address these gaps in knowledge, we assayed levels of messenger RNA for 14 candidate genes at rest and in response to a 24-h confinement stress for 72 half-sib families of Chinook salmon reared in two different environments (hatchery and semi-natural stream channel). We observed extensive plasticity for mRNA levels of metabolic and stress response genes and demonstrated that mRNA level plasticity due to rearing environment affects mRNA level plasticity in response to stress. These effects have important implications for natural populations experiencing multiple stressors. We identified genotype-by-environment interactions for mRNA levels that were dominated by maternal effects; however, mRNA level response to challenge also exhibited a non-additive genetic basis. Our results indicate that while plasticity for mRNA levels can evolve, predicting the outcome of selection will be difficult. The inconsistency in genetic architecture among treatment groups suggests there is considerable cryptic genetic variation for gene expression.

Quantifying receptor trafficking and colocalization with confocal microscopy.

Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocytosis. Unbiased and robust image analysis workflows are required for the identification, and study, of aberrant trafficking. After a brief review of related strategies, identifying both good and bad practice, custom workflows for the analysis of live cell 3D time-lapse data are presented. Strategies for data pre-processing, including denoising and background subtraction are considered. We use a 3D level set protocol to accurately segment cells using only the signal from fluorescently labelled receptor. A protocol for the quantification of changes to subcellular receptor distribution over time is then presented. As an example, ligand stimulated trafficking of epidermal growth factor receptor (EGFR) is shown to be significantly reduced in both AG1478 and Dynasore treated cells. Protocols for the quantitative analysis of colocalization between receptor and endosomes are also introduced, including strategies for signal isolation and statistical testing. By calculating the Manders and Pearson coefficients, both co-occurrence and correlation can be assessed. A statistically significant decrease in the level of ligand induced co-occurrence between EGFR and rab5 positive endosomes is demonstrated for both the AG1478 and Dynasore treated cells relative to a control. Finally, a strategy for the visualisation of co-occurrence is presented, which provides an unbiased alternative to colour overlays.

Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling.

Inference of Low and High-Grade Glioma Gene Regulatory Networks Delineates the Role of Rnd3 in Establishing Multiple Hallmarks of Cancer.

Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.

The Human Immunodeficiency Virus Endemic: Maintaining Disease Transmission in At-Risk Urban Areas.

OBJECTIVES: A study of network relationships, geographic contiguity, and risk behavior was designed to test the hypothesis that all 3 are required to maintain endemicity of human immunodeficiency virus (HIV) in at-risk urban communities. Specifically, a highly interactive network, close geographic proximity, and compound risk (multiple high-risk activities with multiple partners) would be required. METHODS: We enrolled 927 participants from two contiguous geographic areas in Atlanta, GA: a higher-risk area and lower-risk area, as measured by history of HIV reporting. We began by enrolling 30 "seeds" (15 in each area) who were comparable in their demographic and behavioral characteristics, and constructed 30 networks using a chain-link design. We assessed each individual's geographic range; measured the network characteristics of those in the higher and lower-risk areas; and measured compound risk as the presence of two or more (of 6) major risks for HIV. RESULTS: Among participants in the higher-risk area, the frequency of compound risk was 15%, compared with 5% in the lower-risk area. Geographic cohesion in the higher-risk group was substantially higher than that in the lower-risk group, based on comparison of geographic distance and social distance, and on the extent of overlap of personal geographic range. The networks in the 2 areas were similar: both areas show highly interactive networks with similar degree distributions, and most measures of network attributes were virtually the same. CONCLUSIONS: Our original hypothesis was supported in part. The higher and lower-risk groups differed appreciably with regard to risk and geographic cohesion, but were substantially the same with regard to network properties. These results suggest that a "minimum" network configuration may be required for maintenance of endemic transmission, but a particular prevalence level may be determined by factors related to risk, geography, and possibly other factors.

Peptide Tyrosine Tyrosine 3-36 Reduces Meal Size and Activates the Enteric Neurons in Male Sprague-Dawley Rats.

BACKGROUND: Peptide tyrosine tyrosine 3-36 (peptide YY 3-36 or PYY 3-36) reduces food intake by unknown site(s). AIM: To test the hypothesis that the gastrointestinal tract contains sites of action regulating meal size (MS) and intermeal interval (IMI) length by PYY 3-36. METHODS: Peptide YY 3-36 (0, 1, 5, 10 and 20 nmol/kg) was injected in the aorta, the artery that supplies the gastrointestinal tract, prior to the onset of the dark cycle in free feeding male Sprague-Dawley rats and food intake was measured. Then, PYY 3-36 (25 nmol/kg) was injected intraperitoneally in these rats and Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) was quantified in the small intestinal enteric neurons, both myenteric and submucosal, and the dorsal vagal complex (DVC) of the hindbrain. RESULTS: PYY 3-36 reduced first MS, decreased IMI length, shortened duration of first meal and increased Fos-LI in enteric and DVC neurons. However, PYY 3-36 failed to change the size of the second meal, satiety ratio, latency to first meal, number of meals and 24 h intake relative to saline control. CONCLUSION: The gastrointestinal tract may contain sites of action regulating MS reduction by PYY 3-36.

Infusion of exogenous cholecystokinin-8, gastrin releasing peptide-29 and their combination reduce body weight in diet-induced obese male rats.

We hypothesized that exogenous gastrin releasing peptide-29 (GRP-29), cholecystokinin-8 (CCK-8) and their combination reduce body weight (BW). To test this hypothesis, BW was measured in four groups of diet-induced obese (DIO) male rats infused in the aorta (close to the junctions of the celiac and cranial mesenteric arteries) with saline, CCK-8 (0.5 nmol/kg), GRP-29 (0.5 nmol/kg) and CCK-8+GRP-29 (0.5 nmol/kg each) once daily for a total of 23 days. We found that CCK-8, GRP-29 and CCK-8+GRP-29 reduce BW relative to saline control. In conclusion, CCK-8, GRP-29 and their combination reduce BW in the DIO rat model. If infused near their gastrointestinal sites of action CCK-8, GRP-29 and their combination may have a role in regulating BW.