The Sec1-Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B.

Loss-of-function variants of vacuolar protein sorting proteins VPS33B and VPS16B (VIPAS39) are causative for arthrogryposis, renal dysfunction, and cholestasis syndrome, where early lethality of patients indicates that VPS33B and VPS16B play essential cellular roles. VPS33B is a member of the Sec1-Munc18 protein family and thought to facilitate vesicular fusion via interaction with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, like its paralog VPS33A in the homotypic fusion and vacuole sorting complex. VPS33B and VPS16B are known to associate, but little is known about the composition, structure, or function of the VPS33B-VPS16B complex. We show here that human VPS33B-VPS16B is a high molecular weight complex, which we expressed in yeast to perform structural, composition, and stability analysis. Circular dichroism data indicate VPS33B-VPS16B has a well-folded α-helical secondary structure, and size-exclusion chromatography-multiangle light scattering revealed a molecular weight of ∼315 kDa. Quantitative immunoblotting indicated a VPS33B:VPS16B ratio of 2:3. Expression of arthrogryposis, renal dysfunction, and cholestasis syndrome-causing VPS33B missense variants showed L30P disrupts complex formation but not S243F or H344D. Truncated VPS16B (amino acids 143 to 316) was sufficient to form a complex with VPS33B. Small-angle X-ray scattering and negative-staining EM revealed a two-lobed shape for VPS33B-VPS16B. Avidin tagging indicated that each lobe contains a VPS33B molecule, and they are oriented in opposite directions. We propose a structure for VPS33B-VPS16B that allows the VPS33B at each end to interact with separate SNARE bundles and/or SNAREpins, plus associated membrane components. These observations reveal the only known potentially bidirectional Sec1-Munc18 protein complex.

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.

The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis.

Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis.

Surgical Teams' Attitudes About Surgical Safety and the Surgical Safety Checklist at 10 Years: A Multinational Survey.

To assess health care professionals' attitudes on the Surgical Safety Checklist ("the Checklist") in resource-rich health systems and provide insights on strategies for optimizing Checklist use. Background: In use for over a decade, the Checklist is a safety instrument aimed at improving operating room communication, teamwork, and evidence-based safety practices. Methods: An online survey was sent to surgeons, nurses, and anesthesiologists in 5 high-income countries (Canada, the United States, the United Kingdom, Australia, and New Zealand). Survey results were analyzed using SPSS. Results: A total of 2032 health care professionals completed the survey. Of these respondents, 47.6% were nurses, 70.5% were women, 65.1% were from the United States, and 50.0% had 20 years of experience or more in their role. Most respondents felt the Checklist positively impacted patient safety (70.9%), team communication (73.1%), and teamwork (58.9%). Only 50.3% of respondents were satisfied their team's use of the Checklist, and only 47.5% reported team members stopping to fully participate in the process. More nurses lacked confidence regarding their role in the Checklist process than surgeons and anesthesiologists combined (8.9% vs 4.3%). Fewer surgeons and anesthesiologists than nurses felt they received adequate training on the Checklist's use (57.8% vs 76.7%). Conclusions: While most respondents perceive the Checklist as enhancing patient safety, not all surgical team members are actively engaging with its use. To enhance buy-in and meaningful use of the Checklist, health systems should provide more training on the Checklist with respect to its purpose and strengthening teamwork.

Deriving literature-based benchmarks for pediatric appendectomy and cholecystectomy complications from national databases in high-income countries: A systematic review and meta-analysis.

BACKGROUND: Health systems must identify preventable adverse outcomes to improve surgical safety. We conducted a systematic review to determine national rates of postoperative complications associated with two common pediatric surgery operations in High-Income Countries (HICs). METHODS: National database studies of complication rates associated with pediatric appendectomies and cholecystectomies (2000-2016) in Canada, the US, and the UK were included. Outcomes included mortality, length of hospital stay (LOS), and other surgical complications. Outcome data were extracted and comparisons made between countries and databases. RESULTS: Thirty-three papers met inclusion criteria (1 Canadian, 1 UK, and 4 US Databases). Mean LOS was 3.00 (±1.42) days and 3.44 (±1.55) days for appendectomy and cholecystectomy, respectively. Mortality was 0.06% after appendectomy and 0.24% after cholecystectomy. Readmission and reoperation rates were 6.79% and 0.32% for appendectomy, and 1.37% and 0.71% for cholecystectomy. For appendectomies, LOS was shorter in Canadian and UK studies compared to US studies, and mortality and readmission rates were lower (OR 0.46 95%CI 0.23 to 0.93, OR 3.63 to 3.77 95%CI) in UK studies compared to US studies. CONCLUSIONS: Outcomes after pediatric appendectomy and cholecystectomy are good but vary between HICs. Understanding national outcomes and intercountry differences is essential in developing health system approaches to pediatric surgical safety. LEVEL OF EVIDENCE: II.

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.

Safety and efficacy of tranexamic acid in bleeding paediatric trauma patients: a systematic review protocol.

INTRODUCTION: Trauma is the leading cause of death among children aged 1-18. Studies indicate that better control of bleeding could potentially prevent 10-20% of trauma-related deaths. The antifibrinolytic agent tranexamic acid (TxA) has shown promise in haemorrhage control in adult trauma patients. However, information on the potential benefits of TxA in children remains sparse. This review proposes to evaluate the current uses, benefits and adverse effects of TxA in the bleeding paediatric trauma population. METHODS AND ANALYSIS: A structured search of bibliographic databases (eg, MEDLINE, EMBASE, PubMed, CINAHL, Cochrane CENTRAL) has been undertaken to retrieve randomised controlled trials and cohort studies that describe the use of TxA in paediatric trauma patients. To ensure that all relevant data were captured, the search did not contain any restrictions on language or publication time. After deduplication, citations will be screened independently by 2 authors, and selected for inclusion based on prespecified criteria. Data extraction and risk of bias assessment will be performed independently and in duplicate. Meta-analytic methods will be employed wherever appropriate. ETHICS AND DISSEMINATION: This study will not involve primary data collection, and formal ethical approval will therefore not be required. The findings of this study will be disseminated through a peer-reviewed publication and at relevant conference meetings. TRIAL REGISTRATION NUMBER: CRD42016038023.

Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome.

Next-generation RNA sequence analysis of platelets from an individual with autosomal recessive gray platelet syndrome (GPS, MIM139090) detected abnormal transcript reads, including intron retention, mapping to NBEAL2 (encoding neurobeachin-like 2). Genomic DNA sequencing confirmed mutations in NBEAL2 as the genetic cause of GPS. NBEAL2 encodes a protein containing a BEACH domain that is predicted to be involved in vesicular trafficking and may be critical for the development of platelet α-granules.