High-resolution asymmetric structure of a Fab-virus complex reveals overlap with the receptor binding site.

Canine parvovirus is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site has contributed to cross-species transmission, giving rise to closely related variants. It has been shown that Mab 14 strongly binds and neutralizes canine but not feline parvovirus, suggesting this antigenic site also controls species-specific receptor binding. To visualize the conformational epitope at high resolution, we solved the cryogenic electron microscopy (cryo-EM) structure of the Fab-virus complex. We also created custom software, Icosahedral Subparticle Extraction and Correlated Classification, to solve a Fab-virus complex with only a few Fab bound per capsid and visualize local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex map. Our results identified the antigenic epitope that had significant overlap with the receptor binding site, and the structures revealed that binding of Fab induced conformational changes to the virus. We were also able to assign the order and position of attached Fabs to allow assessment of complementarity between the Fabs bound to different positions. This approach therefore provides a method for using cryo-EM to investigate complementarity of antibody binding.

An Overview of Blood Flow Restriction Physiology and Clinical Considerations.

ABSTRACT: The utilization of blood flow restriction has garnished considerable attention due to its widespread application and benefits that include strength enhancement, muscle hypertrophy, and increased level of function for specific populations. Blood flow restriction induces a hypoxic environment within a muscle group, initiating a metabolic cascade that stimulates muscle protein synthesis, altered gene regulation of muscle satellite cells, and increased muscle fiber recruitment, ultimately resulting in improved strength and endurance. When using blood flow restriction, consideration of the individual patient, occlusion pressure, cuff width, and cuff size are paramount. Blood flow restriction has been proven to be a consistently safe and effective tool for augmenting rehabilitative regimens for the upper and lower extremity.

Potential Rediscovery of a Distinct Slow Loris Nycticebus (Mammalia: Strepsirrhini: Lorisidae) from Pulau Tioman.

Several slow loris (Nycticebus) sightings have occurred on the island of Pulau Tioman, Peninsular Malaysia, from 2011 to 2018. Records discussed here represent the first confirmed sightings and photographic evidence of Nycticebus on Tioman since its discovery in 1915, refuting presumptions that the Tioman slow loris is extinct. Although originally considered a subspecies of the Sunda slow loris (Nycticebus coucang), several morphological characteristics apparent in all observed individuals, including the white interocular stripe, rufous colouration and pale dorsal stripe, are similar to the Philippine slow loris (Nycticebus menagensis). Further, the broad snout and ears may be unique to this population and suggest that the population may be distinct. I, therefore, recommend that future studies consider the taxonomic status of remote and isolated Nycticebus populations given the possibility that they may represent distinct and unrecognised taxa.

Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release.

The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the three-dimensional (3-D) structures of DWV capsids isolated from infected honey bees, including the immature procapsid, the genome-filled virion, the putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the 5-fold vertices. The 5-fold spikes had an open flower-like conformation for the procapsid and genome-filled capsids, whereas the putative A-particle and empty capsids that had released the genome had a closed tube-like spike conformation. Between the two conformations, the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that the genome may escape from a 5-fold vertex to initiate infection. Finally, the structures illustrate that, similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release. IMPORTANCE: Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allowed the first glimpse into the conformational changes that may be required during transmission and genome release for DWV.

Evolution and Cryo-electron Microscopy Capsid Structure of a North American Bat Adenovirus and Its Relationship to Other Mastadenoviruses.

Since the first description of adenoviruses in bats in 2006, a number of micro- and megabat species in Europe, Africa, and Asia have been shown to carry a wide diversity of adenoviruses. Here, we report on the evolutionary, biological, and structural characterization of a novel bat adenovirus (BtAdV) recovered from a Rafinesque's big-eared bat (Corynorhinus rafinesquii) in Kentucky, USA, which is the first adenovirus isolated from North American bats. This virus (BtAdV 250-A) exhibits a close phylogenetic relationship with Canine mastadenovirus A (CAdV A), as previously observed with other BtAdVs. To further investigate the relationships between BtAdVs and CAdVs, we conducted mass spectrometric analysis and single-particle cryo-electron microscopy reconstructions of the BtAdV 250-A capsid and also analyzed the in vitro host ranges of both viruses. Our results demonstrate that BtAdV 250-A represents a new mastadenovirus species that, in contrast to CAdV, has a unique capsid morphology that contains more prominent extensions of protein IX and can replicate efficiently in a phylogenetically diverse range of species. These findings, in addition to the recognition that both the genetic diversity of BtAdVs and the number of different bat species from disparate geographic regions infected with BtAdVs appears to be extensive, tentatively suggest that bats may have served as a potential reservoir for the cross-species transfer of adenoviruses to other hosts, as theorized for CAdV. IMPORTANCE: Although many adenoviruses are host specific and likely codiverged with their hosts over millions of years, other adenoviruses appear to have emerged through successful cross-species transmission events on more recent time scales. The wide geographic distribution and genetic diversity of adenoviruses in bats and their close phylogenetic relationship to Canine mastadenovirus A (CAdV A) has raised important questions about how CAdV A, and possibly other mammalian adenoviruses, may have emerged. Although most adenoviruses tend to cause limited disease in their natural hosts, CAdV A is unusual in that it may cause high morbidity and sometimes fatal infections in immunocompetent hosts and is thus an important pathogen of carnivores. Here, we performed a comparative evolutionary and structural study of representative bat and canine adenoviruses to better understand the relationship between these two viral groups.

Extracorporeal cellular therapy (ELAD) in severe alcoholic hepatitis: A multinational, prospective, controlled, randomized trial.

Severe alcoholic hepatitis (sAH) is associated with a poor prognosis. There is no proven effective treatment for sAH, which is why early transplantation has been increasingly discussed. Hepatoblastoma-derived C3A cells express anti-inflammatory proteins and growth factors and were tested in an extracorporeal cellular therapy (ELAD) study to establish their effect on survival for subjects with sAH. Adults with sAH, bilirubin ≥8 mg/dL, Maddrey's discriminant function ≥ 32, and Model for End-Stage Liver Disease (MELD) score ≤ 35 were randomized to receive standard of care (SOC) only or 3-5 days of continuous ELAD treatment plus SOC. After a minimum follow-up of 91 days, overall survival (OS) was assessed by using a Kaplan-Meier survival analysis. A total of 203 subjects were enrolled (96 ELAD and 107 SOC) at 40 sites worldwide. Comparison of baseline characteristics showed no significant differences between groups and within subgroups. There was no significant difference in serious adverse events between the 2 groups. In an analysis of the intent-to-treat population, there was no difference in OS (51.0% versus 49.5%). The study failed its primary and secondary end point in a population with sAH and with a MELD ranging from 18 to 35 and no upper age limit. In the prespecified analysis of subjects with MELD < 28 (n = 120), ELAD was associated with a trend toward higher OS at 91 days (68.6% versus 53.6%; P = .08). Regression analysis identified high creatinine and international normalized ratio, but not bilirubin, as the MELD components predicting negative outcomes with ELAD. A new trial investigating a potential benefit of ELAD in younger subjects with sufficient renal function and less severe coagulopathy has been initiated. Liver Transplantation 24 380-393 2018 AASLD.

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE: Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time.

Ex Vivo Porcine Organ Perfusion Models as a Suitable Platform for Translational Transplant Research.

In transplantation surgery, extending the criteria for organ donation to include organs that may have otherwise been previously discarded has provided the impetus to improve organ preservation. The traditional method of cold static storage (CS) has been tried and tested and is suitable for organs meeting standard criteria donation. Ex vivo machine perfusion is, however, associated with evidence suggesting that it may be better than CS alone and may allow for organ donation criteria to be extended. Much of our knowledge of organ preservation is derived from animal studies. We review ex vivo porcine organ perfusion models and discuss the relevance to the field of transplantation surgery. Following a systematic literature search, only articles that reported on experimental studies with focus on any aspect(s) of ex vivo and porcine perfusion of organs yet limited to the context of organ transplantation surgery were included. The database search and inclusion/exclusion criteria identified 22 journal articles. All 22 articles discussed ex vivo porcine organ perfusion within the context of transplant preservation surgery: 8 liver, 3 kidney, 3 lung, 2 pancreas/islet, 4 discussed a combined liver-kidney multiorgan model, 1 small bowel, and 1 cardiac perfusion model systems. The ex vivo porcine perfusion model is a suitable, reliable, and safe translational research model. It has advantages to investigate organ preservation techniques in a reproducible fashion in order to improve our understanding and has implications to extend the criteria for organ donation.

The enterovirus 71 procapsid binds neutralizing antibodies and rescues virus infection in vitro.

UNLABELLED: Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in young children. Although several vaccines are currently in clinical trials, no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71-infected cells: an expanded empty capsid, sometimes called a procapsid, and the infectious virus. Specifically, an immunodominant epitope of EV71 that maps to the virus canyon is structurally different in the procapsid and virus. This structure-function study shows that the procapsid can sequester antibodies, thus enhancing EV71 infection in vitro. The results presented here suggest that, due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics. IMPORTANCE: In a picornavirus infection, both an infectious and a noninfectious empty capsid, sometimes referred to as a procapsid, are produced. It was novel to discover that the procapsid form of EV71 was expanded and antigenically distinct from the infectious virus. Previously, it had been supposed that this empty capsid was an off-pathway dead end or at best served for storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much noninfectious capsid. Here, we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus, the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics.

The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy.

UNLABELLED: The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers. IMPORTANCE: Human papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

A cryo-electron microscopy study identifies the complete H16.V5 epitope and reveals global conformational changes initiated by binding of the neutralizing antibody fragment.

UNLABELLED: Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid-Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called "invading-arm" structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE: Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and "invading-arm" structures. This study advances the understanding of the neutralization mechanism used by H16.V5.

Current principles and practice in autologous intraportal islet transplantation: a meta-analysis of the technical considerations.

OBJECTIVES: Autologous islet transplantation (IAT) following pancreatectomy is now a recognized, albeit highly specialized procedure carried out in a small number of centers worldwide. Current clinical principles and best practice with emphasis on examining the technical aspects of surgery in centers with significant IAT experience are reviewed. METHODS: Literature search for studies discussing any technical aspect of pancreatectomy with intraportal IAT was included. RESULTS: Thirty-five papers were included; all were single-center case series. The indications, surgical approach to pancreatectomy with IAT, islet yield, static pancreas preservation prior to islet digestion, portal vein access, absolute islet infusion volumes, and portal venous pressure changes during transfusion evaluated. CONCLUSIONS: IAT is considered a "last resort" when alternative approaches have been exhausted. Pre-morbid histology and prior surgical drainage adversely influence islet yields and may influence the clinical decision to perform pancreatectomy and IAT. Following pancreas digestion, absolute numbers of islets recovered and smaller islet size predict rates of insulin independence following IAT. Islet volumes and portal venous pressure changes are important factors for the development of complications. Surgical access for IAT includes intra-operative, immediate or delayed infusion via an "exteriorized" vein, and radiological percutaneous approaches. Delayed infusion can be combined with pancreas preservation techniques prior to islet isolation.

The enterovirus 71 A-particle forms a gateway to allow genome release: a cryoEM study of picornavirus uncoating.

Since its discovery in 1969, enterovirus 71 (EV71) has emerged as a serious worldwide health threat. This human pathogen of the picornavirus family causes hand, foot, and mouth disease, and also has the capacity to invade the central nervous system to cause severe disease and death. Upon binding to a host receptor on the cell surface, the virus begins a two-step uncoating process, first forming an expanded, altered "A-particle", which is primed for genome release. In a second step after endocytosis, an unknown trigger leads to RNA expulsion, generating an intact, empty capsid. Cryo-electron microscopy reconstructions of these two capsid states provide insight into the mechanics of genome release. The EV71 A-particle capsid interacts with the genome near the icosahedral two-fold axis of symmetry, which opens to the external environment via a channel ∼10 Šin diameter that is lined with patches of negatively charged residues. After the EV71 genome has been released, the two-fold channel shrinks, though the overall capsid dimensions are conserved. These structural characteristics identify the two-fold channel as the site where a gateway forms and regulates the process of genome release.

A strain-specific epitope of enterovirus 71 identified by cryo-electron microscopy of the complex with fab from neutralizing antibody.

Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody.

Structures of the procapsid and mature virion of enterovirus 71 strain 1095.

Enterovirus 71 (EV71) is an important emerging human pathogen with a global distribution and presents a disease pattern resembling poliomyelitis with seasonal epidemics that include cases of severe neurological complications, such as acute flaccid paralysis. EV71 is a member of the Picornaviridae family, which consists of icosahedral, nonenveloped, single-stranded RNA viruses. Here we report structures derived from X-ray crystallography and cryoelectron microscopy (cryo-EM) for the 1095 strain of EV71, including a putative precursor in virus assembly, the procapsid, and the mature virus capsid. The cryo-EM map of the procapsid provides new structural information on portions of the capsid proteins VP0 and VP1 that are disordered in the higher-resolution crystal structures. Our structures solved from virus particles in solution are largely in agreement with those from prior X-ray crystallographic studies; however, we observe small but significant structural differences for the 1095 procapsid compared to a structure solved in a previous study (X. Wang, W. Peng, J. Ren, Z. Hu, J. Xu, Z. Lou, X. Li, W. Yin, X. Shen, C. Porta, T. S. Walter, G. Evans, D. Axford, R. Owen, D. J. Rowlands, J. Wang, D. I. Stuart, E. E. Fry, and Z. Rao, Nat. Struct. Mol. Biol. 19:424-429, 2012) for a different strain of EV71. For both EV71 strains, the procapsid is significantly larger in diameter than the mature capsid, unlike in any other picornavirus. Nonetheless, our results demonstrate that picornavirus capsid expansion is possible without RNA encapsidation and that picornavirus assembly may involve an inward radial collapse of the procapsid to yield the native virion.

Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells.

The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3C(pro)) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3C(pro) can be toxic for cells. The expression level of 3C(pro) activity has now been reduced relative to the P1-2A, and the effect on the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells has been determined. Using a vaccinia-virus-based transient expression system, P1-2A (from serotypes O and A) and 3C(pro) were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3C(pro) expression dependent on a mutant FMDV internal ribosome entry site (IRES) (designated P1-2A-mIRES-3C). The effects of using a mutant 3C(pro) with reduced catalytic activity or using two different mutant IRES elements (the wt GNRA tetraloop sequence GCGA converted, in the cDNA, to GAGA or GTTA) were analysed. For both serotypes, the P1-2A-mIRES-3C construct containing the inefficient GTTA mutant IRES produced the highest amount of processed capsid proteins. These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD-dependent integrin αvß6, a cellular receptor for FMDV, and are recognized appropriately in serotype-specific antigen ELISAs.

Secretion of genome-free hepatitis B virus--single strand blocking model for virion morphogenesis of para-retrovirus.

As a para-retrovirus, hepatitis B virus (HBV) is an enveloped virus with a double-stranded (DS) DNA genome that is replicated by reverse transcription of an RNA intermediate, the pregenomic RNA or pgRNA. HBV assembly begins with the formation of an "immature" nucleocapsid (NC) incorporating pgRNA, which is converted via reverse transcription within the maturing NC to the DS DNA genome. Only the mature, DS DNA-containing NCs are enveloped and secreted as virions whereas immature NCs containing RNA or single-stranded (SS) DNA are not enveloped. The current model for selective virion morphogenesis postulates that accumulation of DS DNA within the NC induces a "maturation signal" that, in turn, triggers its envelopment and secretion. However, we have found, by careful quantification of viral DNA and NCs in HBV virions secreted in vitro and in vivo, that the vast majority of HBV virions (over 90%) contained no DNA at all, indicating that NCs with no genome were enveloped and secreted as empty virions (i.e., enveloped NCs with no DNA). Furthermore, viral mutants bearing mutations precluding any DNA synthesis secreted exclusively empty virions. Thus, viral DNA synthesis is not required for HBV virion morphogenesis. On the other hand, NCs containing RNA or SS DNA were excluded from virion formation. The secretion of DS DNA-containing as well as empty virions on one hand, and the lack of secretion of virions containing single-stranded (SS) DNA or RNA on the other, prompted us to propose an alternative, "Single Strand Blocking" model to explain selective HBV morphogenesis whereby SS nucleic acid within the NC negatively regulates NC envelopment, which is relieved upon second strand DNA synthesis.

Amphipathic Motifs Regulate N-BAR Protein Endophilin B1 Auto-inhibition and Drive Membrane Remodeling.

Membrane remodeling is a common theme in a variety of cellular processes. Here, we investigated membrane remodeling N-BAR protein endophilin B1, a critical player in diverse intracellular trafficking events, including mitochondrial and Golgi fission, and apoptosis. We find that endophilin B1 assembles into helical scaffolds on membranes, and that both membrane binding and assembly are driven by interactions between N-terminal helix H0 and the lipid bilayer. Furthermore, we find that endophilin B1 membrane remodeling is auto-inhibited and identify direct SH3 domain-H0 interactions as the underlying mechanism. Our results indicate that lipid composition plays a role in dictating endophilin B1 activity. Taken together, this study provides insight into a poorly understood N-BAR protein family member and highlights molecular mechanisms that may be general for the regulation of membrane remodeling. Our work suggests that interplay between membrane lipids and membrane interacting proteins facilitates spatial and temporal coordination of membrane remodeling.

Operation Blood Rain: The Effect of Airdrop on Fresh Whole Blood.

BACKGROUND: Administration of fresh whole blood (FWB) is a life-saving treatment that prolongs life until definitive surgical intervention can be performed; however, collecting FWB is a time-consuming and resource-intensive process. Furthermore, it may be difficult to collect sufficient FWB to treat critically wounded patients or multiple-hemorrhaging casualties. This study describes the effect of airdrop on FWB and explores the possibility of using airdrop to deliver FWB to combat medics treating casualties in the prehospital setting when FDA-approved, cold-stored blood products are not readily available and timely casualty evacuation (CASEVAC) is not feasible. METHODS: Four units of FWB were collected from volunteer donors and loaded into a blood cooler that was dropped from a fixed-wing aircraft under a standard airdrop training bundle (SATB) parachute. A control group of 4 units of FWB was stored in a blood cooler that was not dropped. Baseline and postintervention laboratory samples were measured in both airdropped and control units, including full blood counts, prothrombin time/partial thromboplastin time/international normalized ratio (PT/PTT/INR), pH, lactate, potassium, indirect bilirubin, glucose, fibrinogen, lactate dehydrogenase, and peripheral blood smears. RESULTS: The blood cooler, cooling bags, and all 4 FWB units did not sustain any damage from the airdrop. There was no evidence of hemolysis. All airdropped blood met parameters for transfusion per the Whole Blood Transfusion Clinical Practice Guideline of the Joint Trauma System (JTS). CONCLUSIONS: Airdrop of FWB in a blood cooler with a SATB parachute may be a viable way of delivering blood products to combat medics treating hemorrhaging patients in the prehospital setting, although further research is needed to fully validate the safety of this method of FWB delivery.

Real-time analysis of living animals and rapid screening of human fluid samples using remote sampling electrospray ionization mass spectrometry.

Real-time and in-situ mass-spectrometry analyses of living animal and biological sample were performed using a novel remote sampling electrospray ionization (RS-ESI) probe. Unlike conventional ESI, in which injection or syringe loading is required for sample introduction, the RS-ESI probe ionizes the samples when the sampling capillary is in contact with the sample. As the sampling capillary is electrically held at ground potential, the safety of the animal and operator is assured. The liquid sample is aspirated to the ESI emitter at the other end of the capillary by the Venturi effect. Subsequently, the electrospray is generated when a high voltage is applied to the counter electrode placed inside the ion source chamber. The probe unit is attached to the mass spectrometer with a long flexible tube and its position can be freely manipulated during the analysis. In this report, we demonstrate a real-time analysis of a living mouse liver and an automatic analysis of 138 serum samples using this new technique.