Creation of an Enhanced Recovery after Surgery (ERAS) Guideline for neonatal intestinal surgery patients: A knowledge synthesis and consensus generation approach and protocol study

Introduction: Enhanced Recovery After Surgery (ERAS) guidelines integrate evidence-based practices into multimodal care pathways designed to optimise patient recovery following surgery. The objective of this project is to create an ERAS protocol for neonatal abdominal surgery. The protocol will identify and attempt to bridge the gaps between current practices and best evidence. Our study is the first paediatric ERAS protocol endorsed by the International ERAS Society. Methods: A research team consisting of international clinical and family stakeholders as well as methodological experts have iteratively defined the scope of the protocol in addition to individual topic areas. A modified Delphi method was used to reach consensus. The second phase will include a series of knowledge syntheses involving a rapid review coupled with expert opinion. Potential protocol elements supported by synthesised evidence will be identified. The Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) system will be used to determine strength of recommendations and the quality of evidence. The third phase will involve creation of the protocol using a modified RAND/UCLA Appropriateness Method. Group consensus will be used to rate each element in relation to the quality of evidence supporting the recommendation and the appropriateness for guideline inclusion. This protocol will form the basis of a future implementation study. Ethics and dissemination: This study has been registered with the ERAS Society. Human ethics approval (REB 18-0579) is in place to engage patient families within protocol development. This research is to be published in peer-reviewed journals and will form the care standard for neonatal intestinal surgery

Characterization of VPS16B, a Novel Protein Involved in Platelet α-granule Biogenesis

Platelets are small, anucleate cells that fulfill a central role in hemostasis through their ability to adhere to sites of vessel injury, where they change shape, secrete molecules, and aggregate to stop blood leakages. Their secretory organelles - alpha (α)-granules, dense (δ)-granules and lysosomes - are critical in mediating the formation of a hemostatic plug, and interference with their biogenesis leads to bleeding disorders. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Studies in our laboratory have identified the only proteins known to date to be relevant for α-granule biogenesis: the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B. Mutations in VPS33B have been associated with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. ARC platelets are pale and agranular in appearance, and display a complete absence of α-granule structures and contents. Using a yeast two-hybrid screen, mass spectrometry, co-immunoprecipitation, and bioinformatics studies, VPS16B was identified as a VPS33B-binding protein. Platelets from a patient with ARC syndrome containing mutations in C14orf133 encoding VPS16B recapitulate the phenotype observed in VPS33B-null platelets. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B co-localized with markers of the trans-Golgi network, late endosomes and α-granules. Depletion of VPS16B in platelet progenitor cells - human iii megakaryocytes - resulted in absent α-granules, though α-granule proteins were still synthesized. Taken together, these data identify VPS16B as a novel molecule required for platelet α-granule biogenesis.Ph

α-granule biogenesis: from disease to discovery

Platelets are critical to hemostasis and thrombosis. Upon detecting injury, platelets show a range of responses including the release of protein cargo from α-granules. This cargo is synthesized by platelet precursor megakaryocytes or endocytosed by megakaryocytes and/or platelets. Insights into α-granule biogenesis have come from studies of hereditary conditions where these granules are immature, deficient or absent. Studies of Arthrogryposis, Renal dysfunction, and Cholestasis (ARC) syndrome identified the first proteins essential to α-granule biogenesis: VPS33B and VPS16B. VPS33B and VPS16B form a complex, and in the absence of either, platelets lack α-granules and the granule-specific membrane protein P-selectin. Gray Platelet Syndrome (GPS) platelets also lack conventionally recognizable α-granules, although P-selectin containing structures are present. GPS arises from mutations affecting NBEAL2. The GPS phenotype is more benign than ARC syndrome, but it can cause life-threatening bleeding, progressive thrombocytopenia, and myelofibrosis. We review the essential roles of VPS33B, VPS16B, and NBEAL2 in α-granule development. We also examine the existing data on their mechanisms of action, where many details remain poorly understood. VPS33B and VPS16B are ubiquitously expressed and ARC syndrome is a multisystem disorder that causes lethality early in life. Thus, VPS33B and VPS16B are clearly involved in other processes besides α-granule biogenesis. Studies of their involvement in vesicular trafficking and protein interactions are reviewed to gain insights into their roles in α-granule formation. NBEAL2 mutations primarily affect megakaryocytes and platelets, and while little is known about NBEAL2 function some insights can be gained from studies of related proteins, such as LYST