Human astrovirus capsid protein releases a membrane lytic peptide upon trypsin maturation.

The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus-host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro. IMPORTANCE Human astroviruses (HAstVs) are an understudied family of viruses that cause mild gastroenteritis but have recent cases associated with a more severe neural pathogenesis. Many important elements of the HAstV life cycle are not well understood, and further elucidating them can help understand the various forms of HAstV pathogenesis. In this study, we utilized an in vitro liposome-based assay to describe and characterize a previously unreported lipid disruption activity. This activity is dependent on the protease cleavage of key sites in HAstV capsid core and can be controlled by site-directed mutagenesis. Our group observed this activity in multiple strains of HAstV and in multiple lipid conditions, indicating this may be a conserved activity across the AstV family. The discovery of this function provides insight into HAstV cellular entry, pathogenesis, and a possible target for future therapeutics.

First-principles based Monte Carlo modeling of the magnetization of oxygen-deficient Fe-substituted SrTiO3.

Transition-metal (TM) substituted SrTiO3 has attracted much attention because its magnetism and/or ferroelectricity can be tuned via cation substitution, point defects, strain and/or oxygen deficiency. For example, Goto et al. [Phys. Rev. Applied, 7, 024006 (2017)] reported the magnetization of SrTi1-xFexO3-δ (STF) grown under different oxygen pressures and on various substrates. Here, we use hybrid density functional theory to calculate the effects of different oxygen vacancy (VO) states in STF on the magnetization for a variety of Fe cation arrangements. The magnetic states of the cations associated with the VO ground-states for x = {0.125, 0.25} are used within a Monte Carlo model for collinear magnetism to simulate the spontaneous magnetization. Our model captures several experimental features of STF, i.e., an increase in magnetization for small δ up to a maximum of ∼0.35µB per formula unit at an intermediate number of vacancies, with a slower decrease in magnetization with an increasing number of vacancies. Our approach gives insight into the relation between vacancy concentration and the oxygen pressure required to maximize the magnetization.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer.

The endoplasmic reticulum (ER) is composed of flattened sheets and interconnected tubules that extend throughout the cytosol and makes physical contact with all other cytoplasmic organelles. This cytoplasmic distribution requires continuous remodeling. These discrete ER morphologies require specialized proteins that drive and maintain membrane curvature. The GTPase atlastin is required for homotypic fusion of ER tubules. All atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal cytosolic tail. Here, we examined several Drosophila-human atlastin chimeras to identify functional domains of human atlastin-1 in vitro Although all chimeras could hydrolyze GTP, only chimeras containing the human C-terminal tail, hydrophobic segments, or both could fuse membranes in vitro We also determined that co-reconstitution of atlastin with reticulon does not influence GTPase activity or membrane fusion. Finally, we found that both human and Drosophila atlastin hydrophobic membrane anchors do not span the membrane, but rather form two intramembrane hairpin loops. The topology of these hairpins remains static during membrane fusion and does not appear to play an active role in lipid mixing.

Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

The Atlastin C-terminal tail is an amphipathic helix that perturbs the bilayer structure during endoplasmic reticulum homotypic fusion.

Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.

Repercusión ecocardiográfica del estado de hidratación en los pacientes en diálisis / Echocardiographic impact of hydration status in dialysis patients

Introducción: La enfermedad cardiovascular es la principal causa de muerte en los pacientes con enfermedad renal crónica. La hipertrofia ventricular izquierda (HVI) es la manifestación más frecuente y está relacionada con la hipertensión arterial y la hiperhidratación. El objetivo del presente trabajo es estratificar a los pacientes en diálisis según el estado de hidratación y valorar las posibles alteraciones ecocardiográficas en los distintos grupos. Métodos: Realizamos un estudio transversal de 117 pacientes, 65 en hemodiálisis (HD) y 52 en diálisis peritoneal (DP). Las exploraciones realizadas fueron: bioimpedancia multifrecuencia con el sistema BCM-Body Composition Monitor de Freesenius, ecocardiografía transtorácica y analítica de sangre. Definimos hiperhidratación cuando el cociente volumen extracelular-volumen corporal total (ECW/TBW) normalizado para edad y sexo es > 2,5% de la desviación estándar. Resultados: Los pacientes en HD están pre-HD (67,1%) más hiperhidratados de forma significativa que los de DP (46,1%), presentando casi la mitad de la población hiperhidratada hipertensión arterial; tras la sesión de HD se consigue un mejor control del estado de hidratación (26,1%). Los pacientes en DP presentan con más frecuencia cifras de tensión arterial alta y/o llevan tratamiento antihipertensivo (DP 76,9 vs. HD 49,2%). La HVI es más frecuente en los pacientes en HD e hiperhidratados, siendo la más prevalente la HVI excéntrica. Los pacientes hiperhidratados presentan cifras superiores, de forma significativa, del IVAI (volumen de aurícula izquierda indexada por superficie corporal, la IMVI (masa ventricular izquierda indexada) y el cociente sobrehidratación-agua extracelular. Conclusiones: La bioimpedancia es una técnica que nos permite detectar un gran número de pacientes hiperhidratados. Al estudiar las alteraciones ecocardiográficas en los pacientes en diálisis encontramos una alta correlación entre el estado de hidratación por ECW/TBW normalizado para edad y sexo, y el IVAI e IMVI (AU)

Introduction: Cardiovascular disease is the main cause of death in Chronic Kidney Disease patients. Left ventricular hypertrophy is the most common manifestation and it is linked to arterial hypertension and overhydration. The goal of this paper is to stratify dialyzed patients according to hydration status and to make an evaluation about the possible echocardiography alterations of the different groups. Methods: a transversal study was carried out with 117 patients: 65 were on hemodialysis and 52 on peritoneal dialysis. We performed the following tests: multifrequency bioimpedance with the BCM-Body Composition Freesenius’ Monitor system, transthoracic echocardiography, and blood tests. If ECW/TBW (extracellular water vs total body water) normalization ratio for age and gender was > 2.5% SD, the patient was considered overhydrated. Results: HD patients are significantly overhydrated before HD (67.1%) compared to DP patients (46.1%), and almost half of the overhydrated population presents arterial hypertension. However, after an HD session, a better control of the hydration status is reached (26.1%). DP patients frequently present high arterial pressure and/or are under antihypertensive treatment (DP 76.9% vs HD 49.2%). Left ventricular hypertrophy is much more common in HD overhydrated patients, eccentric LVH being more prevalent. Overhydrated patients present significantly high values of LAVI, ILVM, OH/ECW. Conclusions: Bioimpedance technique allows for the detection of a large number of overhydrated patients. Echocardiographic alterations in dialyzed patients show a high correlation between the hydration stage by ECW/TBW normalized ratio for age and gender and the LAVI and ILVM (AU)

Echocardiographic impact of hydration status in dialysis patients.

INTRODUCTION: Cardiovascular disease is the main cause of death in Chronic Kidney Disease patients. Left ventricular hypertrophy is the most common manifestation and it is linked to arterial hypertension and overhydration. The goal of this paper is to stratify dialyzed patients according to hydration status and to make an evaluation about the possible echocardiography alterations of the different groups. METHODS: A transversal study was carried out with 117 patients: 65 were on hemodialysis and 52 on peritoneal dialysis. We performed the following tests: multifrequency bioimpedance with the BCM-Body Composition Freesenius' Monitor system, transthoracic echocardiography, and blood tests. If ECW/TBW (extracellular water vs total body water) normalization ratio for age and gender was > 2.5% SD, the patient was considered overhydrated. RESULTS: HD patients are significantly overhydrated before HD (67.1%) compared to DP patients (46.1%), and almost half of the overhydrated population presents arterial hypertension. However, after an HD session, a better control of the hydration status is reached (26.1%). DP patients frequently present high arterial pressure and/or are under antihypertensive treatment (DP 76.9% vs HD 49.2%). Left ventricular hypertrophy is much more common in HD overhydrated patients, eccentric LVH being more prevalent. Overhydrated patients present significantly high values of LAVI, ILVM, OH/ECW. CONCLUSIONS: Bioimpedance technique allows for the detection of a large number of overhydrated patients. Echocardiographic alterations in dialyzed patients show a high correlation between the hydration stage by ECW/TBW normalized ratio for age and gender and the LAVI and ILVM.

Estimated Glomerular Filtration Rate in Short-Risk Stratification in Acute Myocardial Infarction.

BACKGROUND/AIMS: Renal dysfunction is associated with a higher risk of cardiovascular disease in patients with acute myocardial infarction (AMI). The aim of this study was to investigate the independent prognostic value of renal dysfunction and its incremental predictability risk after adjusting for well-known clinical factors in patients with AMI. METHODS: 751 consecutive patients with AMI admitted to the Coronary Care Unit (CCU) were included. Patients were grouped into 2 categories according to the baseline estimated glomerular filtration rate (eGFR) on admission (eGFR <60 vs. eGFR ≧60 ml/min/1.73 m2). C-reactive protein and white blood cell count (WBC) as well as clinical prognostic variables were assessed. The endpoint was mortality during CCU stay. The discriminatory power was estimated by the C-index. RESULTS: The patient group with an eGFR <60 ml/min/1.73 m2 was older, had more cardiovascular risk factors, a lower left ventricular ejection fraction and higher cardiovascular mortality during CCU stay (13 vs. 3%). Logistic regression analysis revealed the following predictors of mortality: degree of renal impairment (eGFR <60 ml/min/1.73 m2), hazard ratio (HR) = 2.2 (95% CI 1.1-4.3; p = 0.028); WBC >11,000 × 106/l, HR = 2.3 (95% CI 1.2-4.5; p = 0.017); Killip class on admission, HR = 3.8 (95% CI 1.7-8.5; p = 0.001), and New York Heart Association Functional Classification, HR = 3.6 (95% CI 1.7-7.4; p = 0.001). The adjusted C-index was 0.78 for baseline clinical variables and 0.84 for eGFR. CONCLUSIONS: In patients with AMI, decreased eGFR is an important prognostic factor for impaired cardiac function and mortality in the short-term follow-up. The eGFR may be reliably used in the risk stratification of patients with AMI.