A Pan1/End3/Sla1 complex links Arp2/3-mediated actin assembly to sites of clathrin-mediated endocytosis.

More than 60 highly conserved proteins appear sequentially at sites of clathrin-mediated endocytosis in yeast and mammals. The yeast Eps15-related proteins Pan1 and End3 and the CIN85-related protein Sla1 are known to interact with each other in vitro, and they all appear after endocytic-site initiation but before endocytic actin assembly, which facilitates membrane invagination/scission. Here we used live-cell imaging in parallel with genetics and biochemistry to explore comprehensively the dynamic interactions and functions of Pan1, End3, and Sla1. Our results indicate that Pan1 and End3 associate in a stable manner and appear at endocytic sites before Sla1. The End3 C-terminus is necessary and sufficient for its cortical localization via interaction with Pan1, whereas the End3 N-terminus plays a crucial role in Sla1 recruitment. We systematically examined the dynamic behaviors of endocytic proteins in cells in which Pan1 and End3 were simultaneously eliminated, using the auxin-inducible degron system. The results lead us to propose that endocytic-site initiation and actin assembly are separable processes linked by a Pan1/End3/Sla1 complex. Finally, our study provides mechanistic insights into how Pan1 and End3 function with Sla1 to coordinate cargo capture with actin assembly

Oxytocin Attenuates the Stress-Induced Reinstatement of Alcohol-Seeking in Male Rats: Role of the Central Amygdala

Factors such as stress and anxiety often contribute to alcohol-dependent behavior and can trigger a relapse of alcohol addiction and use. Therefore, it is important to investigate potential pharmacological interventions that may alleviate the influence of stress on addiction-related behaviors. Previous studies have demonstrated that the neuropeptide oxytocin has promising anxiolytic potential in mammals and may offer a pharmacological target to diminish the emotional impact on reinstatement of alcohol-seeking. The purpose of the present study was to investigate the effect of oxytocin on stress-induced alcohol relapse and identify a neural structure mediating this effect through the use of an ethanol self-administration and yohimbine-induced reinstatement paradigm. While yohimbine administration resulted in the reinstatement of ethanol-seeking behavior, the concurrent administration of yohimbine and oxytocin attenuated this effect, suggesting that oxytocin may disrupt stress-induced ethanol-seeking behavior. The central amygdala (CeA) is a structure that drives emotional responses and robustly expresses oxytocin receptors. Intra-CeA oxytocin similarly attenuated the yohimbine-induced reinstatement of ethanol-seeking behavior. These results demonstrate that oxytocin has the potential to attenuate stress-induced relapse into ethanol-seeking behavior, and that this mechanism occurs specifically within the central amygdala

Repurposing Albendazole: new potential as a chemotherapeutic agent with preferential activity against HPV-negative head and neck squamous cell cancer.

Albendazole is an anti-helminthic drug that has been shown to exhibit anti-cancer properties, however its activity in head and neck squamous cell cancer (HNSCC) was unknown. Using a series of in vitro assays, we assessed the ability of albendazole to inhibit proliferation in 20 HNSCC cell lines across a range of albendazole doses (1 nM-10 μM). Cell lines that responded to treatment were further examined for cell death, inhibition of migration and cell cycle arrest. Thirteen of fourteen human papillomavirus-negative HNSCC cell lines responded to albendazole, with an average IC50 of 152 nM. In contrast, only 3 of 6 human papillomavirus-positive HNSCC cell lines responded. Albendazole treatment resulted in apoptosis, inhibition of cell migration, cell cycle arrest in the G2/M phase and altered tubulin distribution. Normal control cells were not measurably affected by any dose tested. This study indicates that albendazole acts to inhibit the proliferation of human papillomavirus-negative HNSCC cell lines and thus warrants further study as a potential chemotherapeutic agent for patients suffering from head and neck cancer

Grading of recommendations, assessment, development and evaluations concept 7: issues and insights linking guideline recommendations to trustworthy essential medicine lists

Objectives: Guidelines and essential medicine lists (EMLs) bear similarities and differences in the process that lead to decisions. Access to essential medicines is central to achieve universal health coverage. The World Health Organization (WHO) EML has guided prioritization of essential medicines globally for nearly 50 years, and national EMLs (NEMLs) exist in over 130 countries. Guideline and EML decisions, at WHO or national levels, are not always coordinated and aligned. We sought to explore challenges, and potential solutions, for decision-making to support trustworthy medicine selection for EMLs from a Grading of Recommendations, Assessment, Development and Evaluations (GRADE) Working Group perspective. We primarily focus on the WHO EML; however, our findings may be applicable to NEML decisions as well. Study Design and Setting: We identified key challenges in connecting the EML to health guidelines by involving a broad group of stakeholders and assessing case studies including real applications to the WHO EML, South Africa NEML, and a multiple sclerosis guideline connected to a WHO EML application for multiple sclerosis treatments. To address challenges, we utilized the results of a survey and feedback from the stakeholders, and iteratively met as a project group. We drafted a conceptual framework of challenges and potential solutions. We presented a summary of the results for feedback to all attendees of the GRADE Working Group meetings in November 2022 (approximately 120 people) and in May 2023 (approximately 100 people) before finalizing the framework. Results: We prioritized issues and insights/solutions that addressed the connections between the EML and health guidelines. Our suggested solutions include early planning alignment of guideline groups and EMLs, considering shared participation to strengthen linkage, further clarity on price/cost considerations, and using explicit shared criteria to make guideline and EML decisions. We also provide recommendations to strengthen the connection between WHO EML and NEMLs including through contextualization methods. Conclusion: This GRADE concept article, jointly developed by key stakeholders from the guidelines and EMLs field, identified key conceptual issues and potential solutions to support the continued advancement of trustworthy EMLs. Adopting structured decision criteria that can be linked to guideline recommendations bears the potential to advance health equity and gaps in availability of essential medicines within and between countries

Lestaurtinib is a potent inhibitor of anaplastic thyroid cancer cell line models

Anaplastic thyroid cancer (ATC) is a rare and lethal human malignancy with no known effective therapies in the majority of cases. Despite the use of conventional treatments such as chemotherapy, radiation and surgical resection, this disease remains almost universally fatal. In the present study, we identified the JAK2 inhibitor Lestaurtinib as a potent compound when testing against 13 ATC cell lines. Lestaurtinib demonstrated a potent antiproliferative effect in vitro at nanomolar concentrations. Furthermore, Lestaurtinib impeded cell migration and the ability to form colonies from single cells using scratch-wound and colony formation assays, respectively. Flow cytometry was used for cell cycle analysis following drug treatment and demonstrated arrest at the G2/M phase of the cell cycle, indicative of a cytostatic effect. In vivo studies using the chick chorioallantoic membrane xenograft models demonstrated that treatment with Lestaurtinib resulted in a significant decrease in endpoint tumor volume and vascularity using power Doppler ultrasound imaging. Overall, this study provides evidence that Lestaurtinib is a potent antiproliferative agent with potential antiangiogenic activity that warrants further investigation as a targeted therapy for ATC

Chemerin Suppresses Breast Cancer Growth by Recruiting Immune Effector Cells Into the Tumor Microenvironment

Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of neoplastic cells, impacting tumorigenesis and tumor progression. Correlations between the number of effector immune cells present in a tumor and clinical outcomes in many human tumors, including breast, have been widely described. Current immunotherapies utilizing checkpoint inhibitors or co-stimulatory molecule agonists aim to activate effector immune cells. However, tumors often lack adequate effector cell numbers within the TME, resulting in suboptimal responses to these agents. Chemerin (RARRES2) is a leukocyte chemoattractant widely expressed in many tissues and is known to recruit innate leukocytes. CMKLR1 is a chemotactic cellular receptor for chemerin and is expressed on subsets of dendritic cells, NK cells, and macrophages. We have previously shown that chemerin acts as a tumor suppressive cytokine in mouse melanoma models by recruiting innate immune defenses into the TME. Chemerin/RARRES2 is down-regulated in many tumors, including breast, compared to normal tissue counterparts. Here, using a syngeneic orthotopic EMT6 breast carcinoma model, we show that forced overexpression of chemerin by tumor cells results in significant recruitment of NK cells and T cells within the TME. While chemerin secretion by EMT6 cells did not alter their phenotypic behavior in vitro, it did significantly suppress tumor growth in vivo. To define the cellular effectors required for this anti-tumor phenotype, we depleted NK cells or CD8+ T cells and found that either cell type is required for chemerin-dependent suppression of EMT6 tumor growth. Finally, we show significantly reduced levels of RARRES2 mRNA in human breast cancer samples compared to matched normal tissues. Thus, for the first time we have shown that increasing chemerin expression within the breast carcinoma TME can suppress growth by recruitment of NK and T cells, thereby supporting this approach as a promising immunotherapeutic strategy

Discovery of a Peptoid-Based Nanoparticle Platform for Therapeutic mRNA Delivery via Diverse Library Clustering and Structural Parametrization

Nanoparticle-mediated mRNA delivery has emerged as a promising therapeutic modality, but its growth is still limited by the discovery and optimization of effective and well-tolerated delivery strategies. Lipid nanoparticles containing charged or ionizable lipids are an emerging standard for in vivo mRNA delivery, so creating facile, tunable strategies to synthesize these key lipid-like molecules is essential to advance the field. Here, we generate a library of N-substituted glycine oligomers, peptoids, and undertake a multistage down-selection process to identify lead candidate peptoids as the ionizable component in our Nutshell nanoparticle platform. First, we identify a promising peptoid structural motif by clustering a library of >200 molecules based on predicted physical properties and evaluate members of each cluster for reporter gene expression in vivo. Then, the lead peptoid motif is optimized using design of experiments methodology to explore variations on the charged and lipophilic portions of the peptoid, facilitating the discovery of trends between structural elements and nanoparticle properties. We further demonstrate that peptoid-based Nutshells leads to expression of therapeutically relevant levels of an anti-respiratory syncytial virus antibody in mice with minimal tolerability concerns or induced immune responses compared to benchmark ionizable lipid, DLin-MC3-DMA. Through this work, we present peptoid-based nanoparticles as a tunable delivery platform that can be optimized toward a range of therapeutic programs

Establishing a core outcome set for peritoneal dialysis : report of the SONG-PD (standardized outcomes in nephrology-peritoneal dialysis) consensus workshop

Outcomes reported in randomized controlled trials in peritoneal dialysis (PD) are diverse, are measured inconsistently, and may not be important to patients, families, and clinicians. The Standardized Outcomes in Nephrology-Peritoneal Dialysis (SONG-PD) initiative aims to establish a core outcome set for trials in PD based on the shared priorities of all stakeholders. We convened an international SONG-PD stakeholder consensus workshop in May 2018 in Vancouver, Canada. Nineteen patients/caregivers and 51 health professionals attended. Participants discussed core outcome domains and implementation in trials in PD. Four themes relating to the formation of core outcome domains were identified: life participation as a main goal of PD, impact of fatigue, empowerment for preparation and planning, and separation of contributing factors from core factors. Considerations for implementation were identified: standardizing patient-reported outcomes, requiring a validated and feasible measure, simplicity of binary outcomes, responsiveness to interventions, and using positive terminology. All stakeholders supported inclusion of PD-related infection, cardiovascular disease, mortality, technique survival, and life participation as the core outcome domains for PD

Inbred Mouse Populations Exhibit Intergenerational Changes in Intestinal Microbiota Composition and Function Following Introduction to a Facility

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Inbred mice are used to investigate many aspects of human physiology, including susceptibility to disease and response to therapies. Despite increasing evidence that the composition and function of the murine intestinal microbiota can substantially influence a broad range of experimental outcomes, relatively little is known about microbiome dynamics within experimental mouse populations. We investigated changes in the intestinal microbiome between C57BL/6J mice spanning six generations (assessed at generations 1, 2, 3, and 6), following their introduction to a stringently controlled facility. Fecal microbiota composition and function were assessed by 16S rRNA gene amplicon sequencing and liquid chromatography mass spectrometry, respectively. Significant divergence of the intestinal microbiota between founder and second generation mice, as well as continuing inter-generational variance, was observed. Bacterial taxa whose relative abundance changed significantly through time included Akkermansia, Turicibacter, and Bifidobacterium (p < 0.05), all of which are recognized as having the potential to substantially influence host physiology. Shifts in microbiota composition were mirrored by corresponding differences in the fecal metabolome (r = 0.57, p = 0.0001), with notable differences in levels of tryptophan pathway metabolites and amino acids, including glutamine, glutamate and aspartate. We related the magnitude of changes in the intestinal microbiota and metabolome characteristics during acclimation to those observed between populations housed in separate facilities, which differed in regards to husbandry, barrier conditions and dietary intake. The microbiome variance reported here has implications for experimental reproducibility, and as a consequence, experimental design and the interpretation of research outcomes across wide range of contexts