A novel, reverse-phase-shifting, thermoreversible foaming hydrogel containing antibiotics for the treatment of thermal burns in a swine model - A pilot study.

BACKGROUND: This study compared a novel topical hydrogel burn dressing (CI-PRJ012) to standard of care (silver sulfadiazine) and to untreated control in a swine thermal burn model, to assess for wound healing properties both in the presence and absence of concomitant bacterial inoculation. METHODS: Eight equal burn wounds were created on six Yorkshire swine. Half the wounds were randomized to post-burn bacterial inoculation. Wounds were subsequently randomized to three treatments groups: no intervention, CI-PRJ012, or silver sulfadiazine cream. At study end, a blinded pathologist evaluated wounds for necrosis and bacterial colonization. RESULTS: When comparing CI-PRJ012 and silver sulfadiazine cream to no treatment, both agents significantly reduced the amount of necrosis and bacteria at 7 days after wound creation (p < 0.01, independently for both). Further, CI-PRJ012 was found to be significantly better than silver sulfadiazine (p < 0.02) in reducing bacterial colonization. For wound necrosis, no significant difference was found between silver sulfadiazine cream and CI-PRJ012 (p = 0.33). CONCLUSIONS: CI-PRJ012 decreases necrosis and bacterial colonization compared to no treatment in a swine model. CI-PRJ012 appeared to perform comparably to silver sulfadiazine. CI-PRJ012, which is easily removed with the application of room-temperature water, may provide clinical advantages over silver sulfadiazine.

Structural insight into an Arl1-ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin.

Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1-Gea2 complex also binds and activates the phosphatidylserine flippase Drs2 and these functions may be related, although the underlying molecular mechanism is unclear. Here we report high-resolution cryo-EM structures of the full-length Gea2 and the Arl1-Gea2 complex. Gea2 is a large protein with 1459 residues and is composed of six domains (DCB, HUS, SEC7, HDS1-3). We show that Gea2 assembles a stable dimer via an extensive interface involving hydrophobic and electrostatic interactions in the DCB and HUS region. Contrary to the previous report on a Gea2 homolog in which Arl1 binds to the dimerization surface of the DCB domain, implying a disrupted dimer upon Arl1 binding, we find that Arl1 binds to the outside surface of the Gea2 DCB domain, leaving the Gea2 dimer intact. The interaction between Arl1 and Gea2 involves the classic FWY aromatic residue triad as well as two Arl1-specific residues. We show that key mutations that disrupt the Arl1-Gea2 interaction abrogate Imh1 Golgi association. This work clarifies the Arl1-Gea2 interaction and improves our understanding of molecular events in the membrane trafficking.

Flipping the script: Advances in understanding how and why P4-ATPases flip lipid across membranes.

Type IV P-type ATPases (P4-ATPases) are a family of transmembrane enzymes that translocate lipid substrates from the outer to the inner leaflet of biological membranes and thus create an asymmetrical distribution of lipids within membranes. On the cellular level, this asymmetry is essential for maintaining the integrity and functionality of biological membranes, creating platforms for signaling events and facilitating vesicular trafficking. On the organismal level, this asymmetry has been shown to be important in maintaining blood homeostasis, liver metabolism, neural development, and the immune response. Indeed, dysregulation of P4-ATPases has been linked to several diseases; including anemia, cholestasis, neurological disease, and several cancers. This review will discuss the evolutionary transition of P4-ATPases from cation pumps to lipid flippases, the new lipid substrates that have been discovered, the significant advances that have been achieved in recent years regarding the structural mechanisms underlying the recognition and flipping of specific lipids across biological membranes, and the consequences of P4-ATPase dysfunction on cellular and physiological functions. Additionally, we emphasize the requirement for additional research to comprehensively understand the involvement of flippases in cellular physiology and disease and to explore their potential as targets for therapeutics in treating a variety of illnesses. The discussion in this review will primarily focus on the budding yeast, C. elegans, and mammalian P4-ATPases.

A Novel Peritoneal Packing Method for Management of Hyperkalemia During Acute Kidney Injury in Trauma.

INTRODUCTION: United States Military operations in resource limited areas are increasing. Furthermore, future peer or near-peer conflicts will require caring for larger numbers of casualties with limited resources. In this setting, traditional renal replacement therapy is not feasible and novel methods are required to address severe acute kidney injury in austere environments lacking definitive therapies. Here, we describe experiments designed to determine the efficacy of a novel peritoneal packing material (Potassium Binding Pack-PBP, CytoSorbents INC) for the acute management of severe hyperkalemia. MATERIALS AND METHODS: Male swine (52 ±1 kg) were nephrectomized via midline laparotomy under a plane of anesthesia and randomized into one of two experimental groups (PBP & CON). Exogenous potassium was infused to achieve a serum potassium level of 7.5 mEq/L. Novel potassium absorbing packs (PBP) or sham packs (CON) were placed in the right and left upper quadrants, and the right and left paracolic gutters of the abdomen to simulate four-quadrant packing (n = 6, n = 5, respectively). Two liters of peritoneal dialysis fluid was instilled into the abdomen and temporary closure performed. Animals were observed for 12 hours. Serum and peritoneal fluid (dialysate) potassium levels were sampled at T = 15, 30, 60 min, and Q60min thereafter. Animals were humanely euthanized at the end of the observation period. RESULTS: Baseline characteristics were similar between groups. Pairwise analysis showed that serum potassium concentrations were significantly lower in the PBP group compared to CON at T = 540 and T = 720 (P = 0.006 and P = 0.015, respectively). Potassium concentrations were significantly lower in dialysate of the PBP group compared to CON at all time points after T = 15 (T = 30, P = 0.017; T = 60 through T = 720, P < 0.001). CONCLUSIONS: This is the first demonstration of an effective technology for the management of hyperkalemia in trauma in the absence of standard of care; renal replacement therapy. We identified that PBP was able to consistently maintain a concentration gradient between dialysate in the peritoneum and system potassium concentration throughout the experiment. Furthermore, systemic potassium concentrations were reduced in a clinically relevant manner in the PBP group compared to CON. This suggests that peritoneal packing technology for the management of metabolic disturbances in trauma has potential for clinical application. These results are preliminary and should be interpreted with caution.

ATP10A deficiency results in male-specific infertility in mice.

Over 8% of couples worldwide are affected by infertility and nearly half of these cases are due to male-specific issues where the underlying cause is often unknown. Therefore, discovery of new genetic factors contributing to male-specific infertility in model organisms can enhance our understanding of the etiology of this disorder. Here we show that murine ATP10A, a phospholipid flippase, is highly expressed in male reproductive organs, specifically the testes and vas deferens. Therefore, we tested the influence of ATP10A on reproduction by examining fertility of Atp10A knockout mice. Our findings reveal that Atp10A deficiency leads to male-specific infertility, but does not perturb fertility in the females. The Atp10A deficient male mice exhibit smaller testes, reduced sperm count (oligozoospermia) and lower sperm motility (asthenozoospermia). Additionally, Atp10A deficient mice display testes and vas deferens histopathological abnormalities, as well as altered total and relative amounts of hormones associated with the hypothalamic-pituitary-gonadal axis. Surprisingly, circulating testosterone is elevated 2-fold in the Atp10A knockout mice while luteinizing hormone, follicle stimulating hormone, and inhibin B levels were not significantly different from WT littermates. The knockout mice also exhibit elevated levels of gonadotropin receptors and alterations to ERK, p38 MAPK, Akt, and cPLA2-dependent signaling in the testes. Atp10A was knocked out in the C57BL/6J background, which also carries an inactivating nonsense mutation in the closely related lipid flippase, Atp10D. We have corrected the Atp10D nonsense mutation using CRISPR/Cas9 and determined that loss of Atp10A alone is sufficient to cause infertility in male mice. Collectively, these findings highlight the critical role of ATP10A in male fertility in mice and provide valuable insights into the underlying molecular mechanisms.

Deficiency of the lipid flippase ATP10A causes diet-induced dyslipidemia in female mice.

Genetic association studies have linked ATP10A and closely related type IV P-type ATPases (P4-ATPases) to insulin resistance and vascular complications, such as atherosclerosis. ATP10A translocates phosphatidylcholine and glucosylceramide across cell membranes, and these lipids or their metabolites play important roles in signal transduction pathways regulating metabolism. However, the influence of ATP10A on lipid metabolism in mice has not been explored. Here, we generated gene-specific Atp10A knockout mice and show that Atp10A-/- mice fed a high-fat diet did not gain excess weight relative to wild-type littermates. However, Atp10A-/- mice displayed female-specific dyslipidemia characterized by elevated plasma triglycerides, free fatty acids and cholesterol, as well as altered VLDL and HDL properties. We also observed increased circulating levels of several sphingolipid species along with reduced levels of eicosanoids and bile acids. The Atp10A-/- mice also displayed hepatic insulin resistance without perturbations to whole-body glucose homeostasis. Thus, ATP10A has a sex-specific role in regulating plasma lipid composition and maintaining hepatic liver insulin sensitivity in mice.

Type IV P-Type ATPases: Recent Updates in Cancer Development, Progression, and Treatment.

Adaptations of cancer cells for survival are remarkable. One of the most significant properties of cancer cells to prevent the immune system response and resist chemotherapy is the altered lipid metabolism and resulting irregular cell membrane composition. The phospholipid distribution in the plasma membrane of normal animal cells is distinctly asymmetric. Lipid flippases are a family of enzymes regulating membrane asymmetry, and the main class of flippases are type IV P-type ATPases (P4-ATPases). Alteration in the function of flippases results in changes to membrane organization. For some lipids, such as phosphatidylserine, the changes are so drastic that they are considered cancer biomarkers. This review will analyze and discuss recent publications highlighting the role that P4-ATPases play in the development and progression of various cancer types, as well as prospects of targeting P4-ATPases for anti-cancer treatment.

Deficiency of the lipid flippase ATP10A causes diet-induced dyslipidemia in female mice.

Genetic association studies have linked ATP10A and closely related type IV P-type ATPases (P4-ATPases) to insulin resistance and vascular complications, such as atherosclerosis. ATP10A translocates phosphatidylcholine and glucosylceramide across cell membranes, and these lipids or their metabolites play important roles in signal transduction pathways regulating metabolism. However, the influence of ATP10A on lipid metabolism in mice has not been explored. Here, we generated gene-specific Atp10A knockout mice and show that Atp10A-/- mice fed a high-fat diet did not gain excess weight relative to wild-type littermates. However, Atp10A-/- mice displayed female-specific dyslipidemia characterized by elevated plasma triglycerides, free fatty acids and cholesterol, as well as altered VLDL and HDL properties. We also observed increased circulating levels of several sphingolipid species along with reduced levels of eicosanoids and bile acids. The Atp10A-/- mice also displayed hepatic insulin resistance without perturbations to whole-body glucose homeostasis. Thus, ATP10A has a sex-specific role in regulating plasma lipid composition and maintaining hepatic liver insulin sensitivity in mice.

The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

An interaction between ß'-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling.

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how ß'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both ß'-COP propeller domains are required to bind Glo3. An acidic patch on ß'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or ß'-COP abrogate the interaction in vitro, and loss of the ß'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the ß'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where ß'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.

Quantification of Golgi Protein Mislocalization to the Budding Yeast Vacuole.

The localization of proteins to the Golgi complex is a dynamic process requiring sorting signals in the cytosolic domains of resident Golgi proteins and retrograde vesicular trafficking. Disruptions in these signals or in the retrograde pathways often lead to mislocalization of Golgi proteins to the vacuole in budding yeast. The extent of vacuolar mislocalization can be quantified through colocalization of GFP-tagged Golgi proteins with fluorescent dyes that mark either the vacuole limiting membrane or the vacuole lumen. Manders' colocalization coefficient (MCC) is a useful tool for quantifying the degree of colocalization. However, the dilution of fluorescence signal intensity that occurs when GFP-tagged Golgi proteins mislocalize to the much larger vacuole is problematic for thresholding the images prior to calculating the MCC. In this chapter, we describe the use of Multi-Otsu thresholding in ImageJ to quantify the degree of GFP-tagged protein mislocalization to the vacuole. Furthermore, these methods can be applied to other colocalization events within the cell.

Thermoreversible Reverse-Phase-Shift Foam for Treatment of Noncompressible Torso Hemorrhage, a Safety Trial in a Porcine Model.

INTRODUCTION: Noncompressible torso hemorrhage is the leading cause of exsanguination on the battlefield. A self-expanding, intraperitoneal deployed, thermoreversible foam has been developed that can be easily administered by a medic in austere settings to temporarily tamponade noncompressible torso hemorrhage. The purpose of this study was to assess the long-term safety and physical characteristics of using Fast Onset Abdominal Management (FOAM; Critical Innovations LLC) in swine. MATERIALS AND METHODS: Yorkshire swine (40-60 kg) were sedated, intubated, and placed on ventilatory support. An external jugular catheter was placed for sampling of blood. Continuous heart rate, temperature, saturation of peripheral oxygen, end-tidal carbon dioxide, and peak airway pressures were monitored for a 4-hour period after intervention (i.e., FOAM agent injection or a sham introducer without agent delivery). The FOAM agent was injected to obtain an intra-abdominal pressure of 60 mmHg for at least 10 minutes. After 4 hours, the animals were removed from ventilatory support and returned to their housing for a period of 7-14 days. Group size analysis was not performed, as this was a descriptive safety study. Blood samples were obtained at baseline and at 1-hour post-intervention and then on days 1, 3, 7, and 14. Euthanasia, necropsy, and harvesting of samples for histologic analysis (from kidneys, terminal ilium, liver, pancreas, stomach, spleen, and lungs) were performed upon expiration. Histologic scoring for evidence of ischemia, necrosis, and abdominal compartment sequela was blinded and reported by semi-quantitative scale (range 0-4; 0 = no change, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked). Oregon Health & Science University's Institutional Animal Care and Use Committee, as well as the U.S. Army Animal Care and Use Review Office, approved this protocol before the initiation of experiments (respectively, protocol numbers IP00003591 and MT180006.e002). RESULTS: Five animals met a priori inclusion criteria, and all of these survived to their scheduled endpoints. Two animals received sham injections of the FOAM agent (one euthanized on day 7 and one on day 14), and three animals received FOAM agent injections (one euthanized on day 7 and two on day 14). A transitory increase in creatinine and lactate was detected during the first day in the FOAM injected swine but resolved by day 3. No FOAM agent was observed in the peritoneal cavity upon necropsy at day 7 or 14. Histologic data revealed no clinically relevant differences in any organ system between intervention and control animals upon sacrifice at day 7 or 14. CONCLUSIONS: This study describes the characteristics, survival, and histological analysis of using FOAM in a porcine model. In our study, FOAM reached the desired intra-abdominal pressure endpoint while not significantly altering basic hematologic parameters, except for transient elevations of creatinine and lactate on day 1. Furthermore, there was no clinical or histological relevant evidence of ischemia, necrosis, or intra-abdominal compartment syndrome. These results provide strong support for the safety of the FOAM device and will support the design of further regulatory studies in swine and humans.

Development of a Novel Epidural Hemorrhage Model in Swine.

INTRODUCTION: Traumatic brain injury is a major public health concern. Among patients with severe traumatic brain injury, epidural hemorrhage is known to swiftly lead to brain herniation and death unless there is emergent neurosurgical intervention. However, immediate neurosurgeon availability is frequently a problem outside of level I trauma centers. In this context, the authors desired to test a novel device for the emergent management of life-threatening epidural hemorrhage. A review of existing animal models determined that all were inadequate for this purpose, as they were found to be either inappropriate or obsolete. Here, we describe the development of a new epidural hemorrhage model in swine (Sus scrofa, 18-26 kg) ideal for translational device testing. MATERIALS AND METHODS: Vascular access was achieved using an ultrasound-guided percutaneous Seldinger catheter-over-wire technique with 5 Fr catheters placed in the bilateral carotid arteries, for continuous blood pressure and to allow for withdrawal of blood for creation of epidural hemorrhage. To simulate an actively bleeding and life-threatening epidural hemorrhage, unadulterated autologous blood was infused from the vascular access point into the epidural space. To be useful for this application and clinical scenario, brain death needed to occur after the planned intervention time but before the end of the protocol period (if no intervention took place). An iterative approach to model development determined that this could be achieved with an initial infusion rate of 1.0 mL/min, slowed to 0.5 mL/min after the first 10 minutes, paired with an intervention time at 15 minutes. All experiments were performed at Oregon Health & Science University, an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility. Oregon Health & Science University's Institutional Animal Care and Use Committee, as well as the United States Army Animal Care and Use Review Office, reviewed and approved this protocol before the initiation of experiments (respectively, protocol numbers IP00002901 and 18116010.e001). RESULTS: The final developed model allows for the infusion of a known volume of autologous, unadulterated blood directly into the epidural space, without the use of a balloon or other restricting membranes, and is rapidly fatal in the absence of intervention. CONCLUSIONS: This animal model is the first to mirror the expected clinical course of epidural hemorrhage in a physiologically relevant manner, while allowing translational testing of emergency devices. This model successfully allowed the initial testing of a novel interventional device for the emergent management of epidural hemorrhage that was designed for use in the absence of traditional neurosurgical capabilities.

Lipid Transport by Candida albicans Dnf2 Is Required for Hyphal Growth and Virulence.

Candida albicans is a common cause of human mucosal yeast infections, and invasive candidiasis can be fatal. Antifungal medications are limited, but those targeting the pathogen cell wall or plasma membrane have been effective. Therefore, virulence factors controlling membrane biogenesis are potential targets for drug development. P4-ATPases contribute to membrane biogenesis by selecting and transporting specific lipids from the extracellular leaflet to the cytoplasmic leaflet of the bilayer to generate lipid asymmetry. A subset of heterodimeric P4-ATPases, including Dnf1-Lem3 and Dnf2-Lem3 from Saccharomyces cerevisiae, transport phosphatidylcholine (PC), phosphatidylethanolamine (PE), and the sphingolipid glucosylceramide (GlcCer). GlcCer is a critical lipid for Candida albicans polarized growth and virulence, but the role of GlcCer transporters in virulence has not been explored. Here, we show that the Candida albicans Dnf2 (CaDnf2) requires association with CaLem3 to form a functional transporter and flip fluorescent derivatives of GlcCer, PC, and PE across the plasma membrane. Mutation of conserved substrate-selective residues in the membrane domain strongly abrogates GlcCer transport and partially disrupts PC transport by CaDnf2. Candida strains harboring dnf2-null alleles (dnf2ΔΔ) or point mutations that disrupt substrate recognition exhibit defects in yeast-to-hypha growth transition, filamentous growth, and virulence in systemically infected mice. The influence of CaDNF1 deletion on the morphological phenotypes is negligible, although the dnf1ΔΔ dnf2ΔΔ strain was less virulent than the dnf2ΔΔ strain. These results indicate that the transport of GlcCer and/or PC by plasma membrane P4-ATPases is important for the pathogenicity of Candida albicans.

Ubiquitination drives COPI priming and Golgi SNARE localization.

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae. The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of ß'-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

An orthologous gene coevolution network provides insight into eukaryotic cellular and genomic structure and function.

The evolutionary rates of functionally related genes often covary. We present a gene coevolution network inferred from examining nearly 3 million orthologous gene pairs from 332 budding yeast species spanning ~400 million years of evolution. Network modules provide insight into cellular and genomic structure and function. Examination of the phenotypic impact of network perturbation using deletion mutant data from the baker's yeast Saccharomyces cerevisiae, which were obtained from previously published studies, suggests that fitness in diverse environments is affected by orthologous gene neighborhood and connectivity. Mapping the network onto the chromosomes of S. cerevisiae and Candida albicans revealed that coevolving orthologous genes are not physically clustered in either species; rather, they are often located on different chromosomes or far apart on the same chromosome. The coevolution network captures the hierarchy of cellular structure and function, provides a roadmap for genotype-to-phenotype discovery, and portrays the genome as a linked ensemble of genes.

AP-3 shows off its flexibility for the cryo-EM camera.

The tetrameric adaptor protein AP-3 is critical for the transport of proteins to lysosomes and lysosome-related organelles. The structures of homologous adaptors AP-1 and AP-2 have revealed a closed-to-open conformational change upon membrane recruitment and phosphoinositide binding. Recently, Schoppe et al. reported the first cryo-EM structures of AP-3 from budding yeast and described remarkably flexible solution structures that are all in the open conformation. The apparent lack of a closed conformational state, the first such description in the literature, allows AP-3 to be more reliant on cargo interaction for its initial membrane recruitment compared with AP-1.

Structural basis of the P4B ATPase lipid flippase activity.

P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory ß-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a ß-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1's ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.

Lewy Body Dementia Association's Industry Advisory Council: proceedings of the second annual meeting.

In 2019, the Lewy Body Dementia Association formed an Industry Advisory Council to bring together a collaborative group of stakeholders with the goal of accelerating clinical research into Lewy body dementia treatments. At the second annual meeting of the Industry Advisory Council, held virtually on June 18, 2020, the key members presented ongoing and planned efforts toward the council's goals. The meeting also featured a discussion about the effects of the COVID-19 pandemic on Lewy body dementia clinical research, lessons learned from that experience, and how those lessons can be applied to the design and conduct of future clinical trials. This report provides a brief summary of the meeting proceedings with a focus on efforts to improve and adapt future Lewy body dementia clinical research.