Modeling cellular responses to serum and vitamin D in microgravity using a human kidney microphysiological system.

The microgravity environment aboard the International Space Station (ISS) provides a unique stressor that can help understand underlying cellular and molecular drivers of pathological changes observed in astronauts with the ultimate goals of developing strategies to enable long- term spaceflight and better treatment of diseases on Earth. We used this unique environment to evaluate the effects of microgravity on kidney proximal tubule epithelial cell (PTEC) response to serum exposure and vitamin D biotransformation capacity. To test if microgravity alters the pathologic response of the proximal tubule to serum exposure, we treated PTECs cultured in a microphysiological system (PT-MPS) with human serum and measured biomarkers of toxicity and inflammation (KIM-1 and IL-6) and conducted global transcriptomics via RNAseq on cells undergoing flight (microgravity) and respective controls (ground). Given the profound bone loss observed in microgravity and PTECs produce the active form of vitamin D, we treated 3D cultured PTECs with 25(OH)D3 (vitamin D) and monitored vitamin D metabolite formation, conducted global transcriptomics via RNAseq, and evaluated transcript expression of CYP27B1, CYP24A1, or CYP3A5 in PTECs undergoing flight (microgravity) and respective ground controls. We demonstrated that microgravity neither altered PTEC metabolism of vitamin D nor did it induce a unique response of PTECs to human serum, suggesting that these fundamental biochemical pathways in the kidney proximal tubule are not significantly altered by short-term exposure to microgravity. Given the prospect of extended spaceflight, more study is needed to determine if these responses are consistent with extended (>6 months) exposure to microgravity.

Endothelial dysfunction is dampened by early administration of fresh frozen plasma in a rodent burn shock model.

BACKGROUND: Endothelial dysfunction has been implicated in the pathogenesis of burn shock affecting patients with large thermal injury. In response to injury, glycocalyx components like Syndecan-1 (SDC-1) are shed into circulation and have been used as markers of endothelial damage. Previous work in our lab has shown plasma inclusive resuscitation (PIR) with fresh frozen plasma (FFP) ameliorates endothelial damage. However, there remains a paucity of information regarding optimal timing and dosing of PIR as well as organ specific endothelial responses to shock. We aimed to examine the impact of PIR on endothelial dysfunction using clinically-translatable timing and dosing. METHODS: Spraque-Dawley rats were used to create thermal burns. Rats were subjected to 40% TBSA scald burns and were resuscitated with LR only, LR + albumin and LR + early 1 ml boluses of FFP at 0,2,4 and 8 hours post-injury. A late group also received LR + FFP starting at hour 10 post-injury. SDC-1 levels were quantified by ELISA and qRT-PCR analysis characterized transcription of glycocalyx components and inflammatory cytokines in the lung and spleen. Evan's blue dye (EBD) was used to quantify amount of vascular leakage. RESULTS: LR + early FFP reduced EBD extravasation when compared to LR only groups while late FFP did not. When comparing LR only vs LR + early FFP, SDC-1 levels were reduced in the LR + early FFP group at hour 8, 12 and 24 (5.23 vs. 2.07, p < 0.001, 4.49 vs. 2.05, p < 0.01, 3.82 vs 2.08, p < 0.05, respectively). LR only groups had upregulation of Exostosin-1 (EXT-1) and SDC-1 in the lung compared to LR + early FFP groups (p < 0.01 and p < 0.05) as well as upregulation of cytokines IL-10 and IFN-Ƴ (p < 0.001 and p < 0.001). CONCLUSIONS: Early administration of LR + FFP reduces the magnitude of SDC-1 shedding and dampens the cytokine response to injury. The upregulation of glycocalyx components as a response to endothelial injury is also decreased in the lung and spleen by LR + early FFP administration.

Development of a kidney microphysiological system hardware platform for microgravity studies.

Determining the physiological effects of microgravity on the human kidney is limited to relatively insensitive tests of biofluids (blood and urine) that do not return abnormal results until more than 50% of kidney function is lost. We have developed an "organ on chip" microphysiological model of the human kidney proximal tubule (PT-MPS) that can recapitulate many kidney functions and disease states and could play a critical role in determining mechanisms of early kidney dysfunction in microgravity. However, the ground-based PT-MPS system is incompatible with spaceflight as it requires a large pneumatic system coupled to a cell incubator for perfusion and intensive hand-on manipulation. Herein, we report the hardware engineering and performance of the Kidney Chip Perfusion Platform (KCPP), a small, advanced, semi-autonomous hardware platform to support kidney microphysiological model experiments in microgravity. The KCPP is composed of five components, the kidney MPS, the MPS housing and valve block, media cassettes, fixative cassettes, and the programable precision syringe pump. The system has been deployed twice to the ISSNL (aboard CRS-17 and CRS-22). From each set of ISSNL experiments and ground-based controls, we were able to recover PT-MPS effluent for biomarker analysis and RNA suitable for transcriptomics analysis demonstrating the usability and functionality of the KCPP.

Human Enteroid Monolayers: A Novel, Functionally-Stable Model for Investigating Oral Drug Disposition.

To further the development of an in vitro model which faithfully recapitulates drug disposition of orally administered drugs, we investigated the utility of human enteroid monolayers to simultaneously assess intestinal drug absorption and first-pass metabolism processes. We cultured human enteroid monolayers from three donors, derived via biopsies containing duodenal stem cells that were propagated and then differentiated atop permeable Transwell® inserts, and confirmed transformation into a largely enterocyte population via RNA-seq analysis and immunocytochemical (ICC) assays. Proper cell morphology was assessed and confirmed via bright field microscopy and ICC imaging of tight junction proteins and other apically and basolaterally localized proteins. Enteroid monolayer barrier integrity was demonstrated by elevated transepithelial electrical resistance (TEER) that stabilized after 10 days in culture and persisted for 42 days. These results were corroborated by low paracellular transport probe permeability at 7 and 21 days in culture. The activity of a prominent drug metabolizing enzyme, CYP3A, was confirmed at 7, 21, and 42 days culture under basal, 1α,25(OH)2 vitamin D3-induced, and 6',7'-dihydroxybergamottin-inhibited conditions. The duration of these experiments is particularly noteworthy, as this is the first study assessing drug metabolizing enzymes and transporters (DMET) expression/function for enteroids cultured for greater than 12 days. The sum of these results suggests enteroid monolayers are a promising ex vivo model to investigate and quantitatively predict an orally administered drug's intestinal absorption and/or metabolism. Significance Statement This study presents a novel ex vivo model of the human intestine, human intestinal organoid (enteroid) monolayers, that maintain barrier function and metabolic functionality for up to 42-days in culture. The incorporation of both barrier integrity and metabolic function over an extended period within the same model is an advancement over historically used in vitro systems, which either lack one or both of these attributes or have limited viability.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1ß appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Identification of prognostic biomarkers for antibiotic associated nephrotoxicity in cystic fibrosis.

BACKGROUND: Our objective was to discover novel urinary biomarkers of antibiotic-associated nephrotoxicity using an ex-vivo human microphysiological system (MPS) and to translate these findings to a prospectively enrolled cystic fibrosis (CF) population receiving aminoglycosides and/or polymyxin E (colistin) for a pulmonary exacerbation. METHODS: We populated the MPS with primary human kidney proximal tubule epithelial cells (PTECs) from three donors and modeled nephrotoxin injury through exposure to 50 µg/mL polymyxin E for 72 h. We analyzed gene transcriptional responses by RNAseq and tested MPS effluents. We translated candidate biomarkers to a CF cohort via analysis of urine collected prior to, during and two weeks after antibiotics and patients were followed for a median of 3 years after antibiotic use. RESULTS: Polymyxin E treatment resulted in a statistically significant increase in the pro-apoptotic Fas gene relative to control in RNAseq of MPS: fold-change = 1.63, FDR q-value = 7.29 × 10-5. Effluent analysis demonstrated an acute rise of soluble Fas (sFas) concentrations that correlated with cellular injury. In 16 patients with CF, urinary sFas concentrations were significantly elevated during antibiotic treatment, regardless of development of AKI. Over a median of three years of follow up, we identified seven cases of incident chronic kidney disease (CKD). Urinary sFas concentrations during antibiotic treatment were significantly associated with subsequent development of incident CKD (unadjusted relative risk = 2.02 per doubling of urinary sFas, 95 % CI = 1.40, 2.90, p < 0.001). CONCLUSIONS: Using an ex-vivo MPS, we identified a novel biomarker of proximal tubule epithelial cell injury, sFas, and translated these findings to a clinical cohort of patients with CF.

Neutrophil Extracellular Traps Are Induced by Coronavirus 2019 Disease-Positive Patient Plasma and Persist Longitudinally: A Possible Link to Endothelial Dysfunction as Measured by Syndecan-1.

Background: Neutrophil extracellular trap (NET) formation is a mechanism that neutrophils possess to respond to host infection or inflammation. However, dysregulation of NETosis has been implicated in many disease processes. Although the exact mechanisms of their involvement remain largely unknown, this study aimed to elucidate NET formation over the time course of coronavirus disease 2019 (COVID-19) infection and their possible role in endothelial injury. Patients and Methods: Plasma samples from COVID-19-positive patients were obtained at six timepoints during hospitalization. Neutrophils were extracted from healthy donors and treated with COVID-19-positive patient plasma. Myeloperoxidase (MPO) assay was used to assess for NETosis. Syndecan-1 (SDC-1) enzyme-linked immunosorbent assay (ELISA) was run using the same samples. Immunocytochemistry allowed for further quantification of NETosis byproducts MPO and citrullinated histone 3 (CitH3). The receiver operating characteristic (ROC) curve discriminated between admission levels of SDC-1 and MPO in predicting 30-day mortality and need for ventilator support. Results: Sixty-three patients with COVID-19 were analyzed. Myeloperoxidase was upregulated at day 3, 7, and 14 (p = 0.0087, p = 0.0144, p = 0.0421). Syndecan-1 levels were elevated at day 7 and 14 (p = 0.0188, p = 0.0026). Neutrophils treated with day 3, 7, and 14 plasma expressed increased levels of MPO (p < 0.001). Immunocytochemistry showed neutrophils treated with day 3, 7, and 14 plasma expressed higher levels of MPO (p < 0.001) and higher levels of CitH3 when treated with day 7 and 14 plasma (p < 0.01 and p < 0.05). Admission SDC-1 and MPO levels were found to be independent predictors of 30-day mortality and need for ventilator support. Conclusions: Neutrophil dysregulation can be detrimental to the host. Our study shows that COVID-19 plasma induces substantial amounts of NET formation that persists over the course of the disease. Patients also exhibit increased SDC-1 levels that implicate endothelial injury in the pathogenesis of COVID-19 infection. Furthermore, MPO and SDC-1 plasma levels are predictive of poor outcomes.

Incorporating Uremic Solute-mediated Inhibition of OAT1/3 Improves PBPK Prediction of Tenofovir Renal and Systemic Disposition in Patients with Severe Kidney Disease.

BACKGROUND: Dose modification of renally secreted drugs in patients with chronic kidney disease (CKD) has relied on serum creatinine concentration as a biomarker to estimate glomerular filtration (GFR) under the assumption that filtration and secretion decline in parallel. A discrepancy between actual renal clearance and predicted renal clearance based on GFR alone is observed in severe CKD patients with tenofovir, a compound secreted by renal OAT1/3. Uremic solutes that inhibit OAT1/3 may play a role in this divergence. METHODS: To examine the impact of transporter inhibition by uremic solutes on tenofovir renal clearance, we determined the inhibitory potential of uremic solutes hippuric acid, indoxyl sulfate, and p-cresol sulfate. The inhibition parameters (IC50) were incorporated into a previously validated mechanistic kidney model; simulated renal clearance and plasma PK profile were compared to data from clinical studies. RESULTS: Without the incorporation of uremic solute inhibition, the PBPK model failed to capture the observed data with an absolute average fold error (AAFE) > 2. However, when the inhibition of renal uptake transporters and uptake transporters in the slow distribution tissues were included, the AAFE value was within the pre-defined twofold model acceptance criterion, demonstrating successful model extrapolation to CKD patients. CONCLUSION: A PBPK model that incorporates inhibition by uremic solutes has potential to better predict renal clearance and systemic disposition of secreted drugs in patients with CKD. Ongoing research is warranted to determine if the model can be expanded to include other OAT1/3 substrate drugs and to evaluate how these findings can be translated to clinical guidance for drug selection and dose optimization in patients with CKD.

Molecular Mechanisms of Organic Anion Transporting Polypeptide-Mediated Organic Anion Clearance at the Blood-Cerebrospinal Fluid Barrier.

The blood-cerebrospinal fluid barrier (BCSFB), formed by the choroid plexus epithelial (CPE) cells, plays an active role in removing drugs and metabolic wastes from the brain. Recent functional studies in isolated mouse choroid plexus (CP) tissues suggested the presence of organic anion transporting polypeptides (OATPs, encoded by SLCOs) at the apical membrane of BCSFB, which may clear large organic anions from the cerebrospinal fluid (CSF). However, the specific OATP isoform involved is unclear. Using quantitative fluorescence imaging, we showed that the fluorescent anions sulforhodamine 101 (SR101), fluorescein methotrexate (FL-MTX), and 8-fluorescein-cAMP (fluo-cAMP) are actively transported from the CSF to the subepithelial space in CP tissues isolated from wild-type mice. In contrast, transepithelial transport of these compounds across the CPE cells was abolished in Oatp1a/1b-/- mice due to impaired apical uptake. Using transporter-expressing cell lines, SR101, FL-MTX, and fluo-cAMP were additionally shown to be transported by mouse OATP1A5 and its human counterpart OATP1A2. Kinetic analysis showed that estrone-3-sulfate and SR101 are transported by OATP1A2 and OATP1A5 with similar Michaelis-Menten constants (Km). Immunofluorescence staining further revealed the presence of OATP1A2 protein in human CP tissues. Together, our results suggest that large organic anions in the CSF are actively transported into CPE cells by apical OATP1A2 (OATP1A5 in mice), then subsequently effluxed into the blood by basolateral multidrug resistance-associated proteins (MRPs). As OATP1A2 transports a wide array of endogenous compounds and xenobiotics, the presence of this transporter at the BCSFB may imply a novel clearance route for drugs and neurohormones from the CSF. SIGNIFICANCE STATEMENT: Drug transporters at the blood-cerebrospinal fluid (CSF) barrier play an important but understudied role in brain drug disposition. This study revealed a functional contribution of rodent organic anion transporting polypeptide (OATP) 1A5 towards the CSF clearance of organic anions and suggested a similar role for OATP1A2 in humans. Delineating the molecular mechanisms governing CSF organic anion clearance may help to improve the prediction of central nervous system (CNS) pharmacokinetics and identify drug candidates with favorable CNS pharmacokinetic properties.

Examining Obesity and Its Association With Burn Injury: A Secondary Analysis of the Transfusion Requirement in Burn Care Evaluation Study.

INTRODUCTION: Literature examining the connection between obesity and burn injuries is limited. This study is a secondary analysis of a multicenter trial data set to investigate the association between burn outcomes and obesity following severe burn injury. MATERIALS AND METHODS: Body mass index (BMI) was used to stratify patients as normal weight (NW; BMI 18.5-25), all obese (AO; any BMI>30), obese I (OI; BMI 30-34.9), obese II (OII; BMI 35-39.9), or obese III (OIII; BMI>40). The primary outcome examined was mortality. Secondary outcomes included hospital length of stay (LOS), number of transfusions, injury scores, infection occurrences, number of operations, ventilator days, intensive care unit LOS, and days to wound healing. RESULTS: Of 335 patients included for study, 130 were obese. Median total body surface area (TBSA) was 31%, 77 patients (23%) had inhalation injury and 41 patients died. Inhalation injury was higher in OIII than NW (42.1% versus 20%, P = 0.03). Blood stream infections (BSI) were higher in OI versus NW (0.72 versus 0.33, P = 0.03). Total operations, ventilator days, days to wound healing, multiorgan dysfunction score, Acute Physiology and Chronic Health Evaluationscore, hospital LOS, and intensive care unit LOS were not significantly affected by BMI classification. Mortality was not significantly different between obesity groups. Kaplan-Meier survival curves did not significantly differ between the groups (χ2 = 0.025, P = 0.87). Multiple logistic regression identified age, TBSA, and full thickness burn as significant independent predictors (P < 0.05) of mortality; however, BMI classification itself was not predictive of mortality. CONCLUSIONS: No significant association between obesity and mortality was seen after burn injury. Age, TBSA, and percent full- thickness burn were independent predictors of mortality after burn injury, while BMI classification was not.

Endothelial damage occurs early after inhalation injury as measured by increased syndecan-1 levels.

Inhalation injury is a significant cause of morbidity and mortality in the burn patient population. However, the pathogenesis of inhalation injury and its potential involvement in burn shock is not well understood. Preclinical studies have shown endothelial injury, as measured by syndecan-1 (SDC-1) levels, to be involved in the increased vascular permeability seen in shock states. Furthermore, the lung has been identified as a site of significant SDC-1 shedding. Here we aim to characterize the contribution of endotheliopathy caused by inhalation alone in a swine model. When comparing injured animals, the fold change of circulating SDC-1 levels from preinjury was significantly higher at 2, 4, and 6 hours postinjury (P = .0045, P = .0017, and P < .001, respectively). When comparing control animals, the fold change of SDC-1 from preinjury was not significant at any timepoint. When comparing injured animals versus controls, the fold change of SDC-1 injured animals was significantly greater at 2, 4, 6, and 18 hours (P = .004, P = .03, P < .001, and P = .03, respectively). Histological sections showed higher lung injury severity compared to control uninjured lungs (0.56 vs 0.38, P < .001). This novel animal model shows significant increases in SDC-1 levels that provide evidence for the connection between smoke inhalation injury and endothelial injury. Further understanding of the mechanisms underlying inhalation injury and its contribution to shock physiology may aid in development of early, more targeted therapies.

Double trouble: a case of bilateral multiple epidermal inclusion cysts after reduction mammaplasty.

Epidermal inclusion cysts (EIC) of the breast are uncommon and can occur after previous surgery or trauma. Here we discuss a case of massive bilateral multiple EIC of the breast presenting seven years after reduction mammaplasty. This report highlights the importance of accurate diagnosis and management of this rare condition.

Feasibility and Acceptance of a Teletrauma Surgery Consult Service to Rural and Community Hospitals: A Pilot Study.

BACKGROUND: Many trauma patients currently transferred from rural and community hospitals (RCH) to Level I trauma centers (LITC) for trauma surgery evaluation may instead be appropriate for immediate discharge or admission to the local facility after evaluation by a trauma and acute care surgery (TACS) surgeon. Unnecessary use of resources occurs with current practice. We aimed to demonstrate the feasibility and acceptance of a teletrauma surgery consultation service between LITC and RCH. STUDY DESIGN: LITC TACS surgeons provided telehealth consults on trauma patients from 3 local RCHs. After consultation, appropriate patients were transferred to LITC; selected patients remained at or were discharged from RCH. Participating TACS surgeons and RCH physicians were surveyed. RESULTS: A total of 28 patients met inclusion criteria during the 5-month pilot phase, with 7 excluded due to workflow issues. The mean ± SD age was 63 ± 17 years. Of 21 patients, 7 had intracranial hemorrhage; 12 had rib fractures. The mean ± SD Injury Severity Score was 8.1 ± 4.0). A total of 6 patients were discharged from RCH, 4 admitted to RCH hospitalist service, 2 transferred to a LITC emergency room, and 9 transferred to LITC as direct admission. There was one 30-day readmission and no missed injuries or complications, or deaths. RCH providers were highly satisfied with the teletrauma surgery consultation service, TACS surgeons, and equipment used. Mental demand and effort of consulting TACS surgeons decreased significantly as the consult number increased. CONCLUSIONS: Teletrauma surgery consultation involving 3 RCH within our system is feasible and acceptable. A total of 10 transfers and 19 emergency department visits were avoided. There was favorable acceptance by RCH providers and TACS surgeons.

Impact of microgravity on a three-dimensional microphysiologic culture of the human kidney proximal tubule epithelium: cell response to serum and vitamin D.

The microgravity environment aboard the International Space Station (ISS) provides a unique stressor that can help understand underlying cellular and molecular drivers of pathological changes observed in astronauts with the ultimate goals of developing strategies to enable long-term spaceflight and better treatment of diseases on Earth. We used this unique environment to evaluate the effects of microgravity on kidney proximal tubule epithelial cell (PTEC) response to serum exposure and vitamin D biotransformation capacity. To test if microgravity alters the pathologic response of the proximal tubule to serum exposure, we treated PTECs cultured in a microphysiological system (PT-MPS) with human serum and measured biomarkers of toxicity and inflammation (KIM-1 and IL-6) and conducted global transcriptomics via RNAseq on cells undergoing flight (microgravity) and respective controls (ground). We also treated 3D cultured PTECs with 25(OH)D3 (vitamin D) and monitored vitamin D metabolite formation, conducted global transcriptomics via RNAseq, and evaluated transcript expression of CYP27B1, CYP24A1, or CYP3A5 in PTECs undergoing flight (microgravity) and respective ground controls. We demonstrated that microgravity neither altered PTEC metabolism of vitamin D nor did it induce a unique response of PTECs to human serum, suggesting that these fundamental biochemical pathways in the kidney proximal tubule are not significantly altered by short-term exposure to microgravity. Given the prospect of extended spaceflight, more study is needed to determine if these responses are consistent with extended (> 6 month) exposure to microgravity.

Development of a Kidney Microphysiological System Hardware Platform for Microgravity Studies.

Study of the physiological effects of microgravity on humans is limited to non-invasive testing of astronauts. Microphysiological models of human organs recapitulate many functions and disease states. Here we describe the development of an advanced, semi-autonomous hardware platform to support kidney microphysiological model experiments in microgravity.

Spatial Volume Necessary to Perform Open Appendectomy in a Spacecraft.

BACKGROUND: With the increase in crewed commercial spaceflight and expeditions to the Moon and Mars, the risk of critical surgical problems and need for procedures increases. Appendicitis and appendectomy are the most common surgical pathology and procedure performed, respectively. The habitable volume of current spacecraft ranges from 4 m³ (Soyuz) to 425 m³ (International Space Station). We investigated the minimum volume required to perform an appendectomy and compared that to habitable spacecraft volumes.METHODS: The axes of a simulated operating room were marked and cameras placed to capture movements. An expert surgeon, chief surgical resident, junior surgical resident, and a nonsurgeon physician each performed a Focused Assessment with Sonography for Trauma and an appendectomy on a simulated patient. Dimensions and volume needed were collected and compared using unpaired t-tests.RESULTS: Mean volume (± SD) needed was 3.83 m³ ± 0.47 m³ for standing and 3.68 m³ ± 0.49 m³ for kneeling (P = 0.638). Minimal volume needed was 3.20 m³ for standing and 3.26 m³ for kneeling. Minimal theoretical volume was 2.99 m³ for standing and 2.87 m³ for kneeling.DISCUSSION: The unencumbered volume needed for an appendectomy is between 2.87 m³ and 4.3 m³. It may be technically feasible to perform an open appendectomy inside the smallest of currently operating spacecraft, at 4 m³ (Soyuz-MS). Space vessels operating without rapid evacuation to Earth will need to consider this volume for potential surgical emergencies. Additional investigation on microgravity and standardization of procedures for novices must be completed.Kamine TH, Siu M, Kramer K, Kelly E, Alouidor R, Fernandez G, Levin D. Spatial volume necessary to perform open appendectomy in a spacecraft. Aerosp Med Hum Perform. 2022; 93(10):760-763.

Time Cost of Provider Skill: A Pilot Study of Medical Officer Occupied Time By Knowledge, Skill, and Ability Level.

INTRODUCTION: On space missions one must consider the operating cost of the medical system on crew time. Medical Officer Occupied Time (MOOT) may vary significantly depending on provider skill. This pilot study assessed the MOOT Skill Effect (MOOTSkE).METHODS: An expert surgeon (ES), fifth year surgical resident (PGY5), second year surgical resident (PGY2), and an expert Emergency Physician (EP) with only 4 mo direct surgical training each performed two simulated appendectomies. The completion times for endotracheal intubation, appendectomy, and two subprocedures (multilayer tissue repair and single layer tissue repair) were recorded.RESULTS: The ES performed the appendectomy in 410 s, the PGY-5 in 498 s, the PGY-2 in 645 s, and the EP in 973 s on average. The PGY-2 and EP time difference was significant compared to the expert. The PGY-5 was not. The EP's time was significantly longer for the appendectomy and the multilayer repair than either surgical resident. For the single layer repair, only the EP-ES difference was significant. A single intubation attempt by the PGY-2 took 73 s while the EP averaged 27 s. The average recorded MOOTSkE between novice and expert was 2.5 (SD 0.34).DISCUSSION: This pilot study demonstrates MOOTSkE can be captured using simulated procedures. It showed the magnitude of the MOOTSkE is likely substantial, suggesting that a more highly trained provider may save substantial crew time. Limitations included small sample size, limited number of procedures, a simulation that may not reflect real world conditions, and suboptimal camera angles.Levin DR, Siu M, Kramer K, Kelly E, Alouidor R, Fernandez G, Kamine T. Time cost of provider skill: a pilot study of medical officer occupied time by knowledge, skill, and ability level. Aerosp Med Hum Perform. 2022; 93(11):816-821.

Optimal Timing of Cholecystectomy for Acute Cholecystitis: A Retrospective Cohort Study.

Background Laparoscopic cholecystectomy performed less than 72 hours from hospital admission for acute cholecystitis has shown to decrease hospital cost without an increase in length of stay (LOS). Very few studies have examined clinical and cost outcomes of performing cholecystectomy less than 24 hours from hospital admission. The aim of this study was to examine the cost and LOS of laparoscopic cholecystectomy performed on an early (less than 24 hours from admission) and late (more than 24 hours from hospital admission) basis. Methods We performed a retrospective observational study of 569 patients at Baystate Medical Center, Springfield, USA, who underwent urgent laparoscopic cholecystectomy for acute cholecystitis between January 1, 2018 and February 28, 2020. We evaluated preoperative/postoperative LOS, operative duration, hospital cost, and patient complications. Results 468 patients underwent urgent laparoscopic cholecystectomy for acute cholecystitis during our study period. Early cholecystectomy (less than 24 hours from admission) had an overall decreased LOS (43.6 hours versus 102.9 hours, p-value < 0.01) and decreased hospital cost ($23,736.70 versus $30,176.40, p-value < 0.01) compared to late cholecystectomy (more than 24 hours from admission). There was also a significantly higher rate of bile leak in patients who underwent surgery more than 24 hours from hospital admission compared to those who had surgery less than 24 hours from admission (5.9% versus 0.4%, p-value < 0.01). Additionally, those procedures performed greater than 24 hours from hospital admission were significantly more likely to be converted to an open procedure (6.9% versus 2.2%, p-value = 0.02).  Conclusion Urgent laparoscopic cholecystectomy performed within 24 hours of hospital admission for acute cholecystitis decreased hospital cost, LOS, and operative complications in our institution's patient population. Our data suggests that performing laparoscopic cholecystectomy within 24 hours of hospital admission would be beneficial from a patient and hospital standpoint.

American Helicopter Emergency Medical Service Pilots Report to Work Despite High Rates of Sleepiness.

OBJECTIVE: Previous studies on helicopter emergency medical service (HEMS) pilots found a positive correlation among fatigue, nodding off in flight, and accidents. We sought to quantify the amount of sleepiness in HEMS pilots using the Epworth Sleepiness Scale (ESS). METHODS: An anonymous survey was sent via the National EMS Pilots Association emergency medical services listserv including demographics, the ESS, and subjective effects of fatigue on flying. Statistical analyses were performed using the t-test and analysis of variance. RESULTS: Thirty-one surveys were returned. Twenty-one (65%) reported an ESS > 10, indicating excessive daytime sleepiness. Twelve (39%) reported nodding off in flight; 20 (65%) indicated that they should have refused to fly, but only 14 (45%) actually did. En route was the most likely phase of flight to be affected by fatigue (23 [74%]), whereas takeoff (2 [7%]) and landing (2 [7%]) were the least likely to be affected. CONCLUSION: Many HEMS pilots in this small study reported excessive daytime sleepiness. Most respondents indicated that they should have turned down a flight because of fatigue. More research is necessary to quantify the burden of fatigue among HEMS pilots.