Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies

Physiological and Behavioral Changes of Water Buffalo in Hot and Cold Systems: Review

This review's objective is to provide information on the mechanisms that buffaloes express during the thermoregulation process. Generally, the water buffalo is associated with warm and tropical climates. In these systems, the combination of high temperature, relative humidity, and radiation cause different physiological and behavioral changes, particularly during the summer months. Wallowing behavior in water or mud promotes heat dissipation through physical mechanisms, such as conduction, convection, and radiation. Furthermore, the provision of natural or artificial shades contributes to thermoregulation and maintains homeostasis. In production systems in cold climates, the wallowing behavior is inhibited by the water temperature, so it is important to keep the animals protected in stables to avoid the cold winds and rapid drops in temperature, causing increased illness pneumonia and sometimes death. Finally, in cold conditions, the animals require an appropriate diet since the use of energy is distributed mainly for the production of heat. Thus, heat stress and cold stress generates relevant problems in health, welfare, and productivity in water buffaloes. A comprehensive assessment of the severity of the resulting problems associated with thermal stress and specialty in cold stress in water buffaloes is necessary so far, and there's very little information about it in this species

New insights into interactions between the nucleotide-binding domain of CFTR and keratin 8

The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for cystic fibrosis patients bearing the ΔF508 mutation. Here we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1. This article is protected by copyright. All rights reserved

Structure of the benthonic macroinvertebrate assemblage of an urban lagoon in the Mesopotamic Pampa

El objetivo de este trabajo fue estudiar la estructura del ensamblaje de macroinvertebrados bentónicos de la laguna del Parque Unzuéy discutir sobre su calidad de agua. El área de estudio se encuentra en la ciudad de Gualeguaychú (Entre Ríos, Argentina), sobre la margen izquierda del río homónimo. La laguna tiene un área de 2750 m2y una profundidad máxima de 1,70 m. Durante 2016-2017, se muestreó en 3 puntos (n=10), se midieron parámetros in situ y se recolectaron muestras de bentos, se caracterizaron los sedimentos y los macroinvertebrados fueron identificados hasta el menor niveltaxonómico posible y se los agrupó en grupos funcionales alimenticios (GFA). Se calculó la diversidad, ladiversidad máxima esperada (Hmáx) y los números de Hill. No se hallaron diferencias estadísticamente significativas (p<0,05) entre los puntos de muestreo y los parámetros in situ; sin embargo, respecto a los macroinvertebrados, los puntos 1 y 3 presentaron mayor similitud que estos con el punto 2, posiblemente debido a las diferencias en el sustrato. Se colectaron 1726 individuos distribuidos en 13 taxones: Oligochaeta (1), Hirudinea (1), Bivalvia (4), Gastropoda (3), Amphipoda (1) e Insecta (3), los cuales son característicos de ambientes con abundante materia orgánica. La diversidad fue baja y coincidió con la Hmáx solo en el 23% de los casos, el número de especies efectivas varió de 1-5 y los taxones dominantes representaron el 40% de la riqueza. Respecto a los GFA, la abundancia se distribuyó en: colector-recolector, colector-filtrador y depredador y, la biomasa, en raspador y colector-filtrador. La laguna del Parque Unzué evidenció efectos de contaminación orgánica durante todo el período de muestreo reflejado en la estructura y composición del ensamblaje de macroinvertebrados.The aim of this work was to study the structure of the benthonic macroinvertebrate assemblages in the Parque Unzué lagoon and to discuss about its water quality. The area of study is located in Gualeguaychú city (Entre Ríos, Argentina), on the left margin from Gualeguaychú river. The lagoon has an area of 2 750 m2 and a maximum depth of 1.70 m. During 2016-2017, some samples were taken at 3 points (n = 10), in situ parameters were measured and benthic samples were collected, sediments were characterized and macroinvertebrates were identified into the lowest possible taxonomic level and grouped into functional groups food (GFA). Richness (S) and diversity were calculated and the macroinvertebrates were classified into functional feeding group (FFG). No significant differences were found (p <0.05) between sample sites and in situ parameters, however, with respect to macroinvertebrates, the sample sites 1 and 3 were more similar than those with site 2. Diversity, maximum diversity (Hmax) and Hill’s numbers were calculated. No statistically significant differences (p <0.05) were found between the sampling points and the in situparameters; however, with respect to macroinvertebrates, points 1 and 3 presented greater similarity than these with point 2, possibly due to differences in the substrate. 1726 individuals distributed in 13 taxa: Oligochaeta (1), Hirudinea (1), Bivalvia (4), Gastropoda (3), Amphipoda (1), and Insecta (3). All of these are characteristic of large amounts of organic matter environments. Diversity was low, this coincided with maximum diversity on 23 % of the cases, and the dominant taxa represent 40 % of richness. The FFG abundance was distributed in gathering-collectors, filtering-collectors and predators and, the biomass, in scrapers, and filtering-collectors. The lagoon from Parque Unzué showed effects of organic pollution during the whole sampling period reflected in the structure and composition of the macroinvertebrate assemblage.Fil: Crettaz Minaglia, Melina Celeste. Universidad Autónoma de Entre Ríos. Facultad de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gianello, Diamela. Universidad Autónoma de Entre Ríos. Facultad de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Roldan, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Autónoma de Entre Ríos. Facultad de Ciencia y Tecnología; ArgentinaFil: Chaves, Eduardo Alberto. Universidad Autónoma de Entre Ríos. Facultad de Ciencia y Tecnología; ArgentinaFil: Aguer, Irene. Universidad Autónoma de Entre Ríos. Facultad de Ciencia y Tecnología; ArgentinaFil: Juárez, Ricardo Ariel. Universidad Autónoma de Entre Ríos. Facultad de Ciencia y Tecnología; Argentin

Impact of the first wave of the SARS-CoV-2 pandemic on the outcome of neurosurgical patients: A nationwide study in Spain

Objective To assess the effect of the first wave of the SARS-CoV-2 pandemic on the outcome of neurosurgical patients in Spain. Settings The initial flood of COVID-19 patients overwhelmed an unprepared healthcare system. Different measures were taken to deal with this overburden. The effect of these measures on neurosurgical patients, as well as the effect of COVID-19 itself, has not been thoroughly studied. Participants This was a multicentre, nationwide, observational retrospective study of patients who underwent any neurosurgical operation from March to July 2020. Interventions An exploratory factorial analysis was performed to select the most relevant variables of the sample. Primary and secondary outcome measures Univariate and multivariate analyses were performed to identify independent predictors of mortality and postoperative SARS-CoV-2 infection. Results Sixteen hospitals registered 1677 operated patients. The overall mortality was 6.4%, and 2.9% (44 patients) suffered a perioperative SARS-CoV-2 infection. Of those infections, 24 were diagnosed postoperatively. Age (OR 1.05), perioperative SARS-CoV-2 infection (OR 4.7), community COVID-19 incidence (cases/10 5 people/week) (OR 1.006), postoperative neurological worsening (OR 5.9), postoperative need for airway support (OR 5.38), ASA grade =3 (OR 2.5) and preoperative GCS 3-8 (OR 2.82) were independently associated with mortality. For SARS-CoV-2 postoperative infection, screening swab test <72 hours preoperatively (OR 0.76), community COVID-19 incidence (cases/10 5 people/week) (OR 1.011), preoperative cognitive impairment (OR 2.784), postoperative sepsis (OR 3.807) and an absence of postoperative complications (OR 0.188) were independently associated. Conclusions Perioperative SARS-CoV-2 infection in neurosurgical patients was associated with an increase in mortality by almost fivefold. Community COVID-19 incidence (cases/10 5 people/week) was a statistically independent predictor of mortality. Trial registration number CEIM 20/217

Maqui (Aristotelia chilensis [Mol.] Stuntz) morphological and phenolic traits associated with forests type and latitudinal gradient in natural populations of Patagonia Argentina

Maqui (Aristotelia chilensis [Mol.] Stuntz) is a native berry of the Patagonia Andean forests, which has one of the highest antioxidant levels currently known. This biochemical characteristic strongly depends on the phenolic profile and is highly influenced by the growing environment. The objective of this study was to characterize natural maqui populations in the immediate lee (Argentina) of the Northern Patagonian Andes, distributed in a latitudinal gradient and associated with coihue (Nothofagus dombeyi) and cypress (Austrocedrus chilensis) forests, humid and xeric environments, respectively. Twenty-five natural populations that spanned the geographical distribution of the species in Argentina, were identified. The sites were associated with two forests (coihue and cypress) and three latitudinal groups (north, center and south). Canopy cover and light interception were measured in the selected sites, as well as plant morphology, fruit yield components, phenolic profiles and oxygen radical absorbance capacity (ORAC) of mature berries. Maqui populations associated to cypress are exposed to greater incident total solar radiation and lesser rainfalls, and showed shorter plants with several main stems, multiple branching and smaller specific leaf area (SLA) as compared to those of coihue forest. Maqui populations associated to coihue presented an increase in the number of berries per bunch, and also in the accumulation of phenols and ORAC. The evaluation of the acclimation responses of the natural populations by different environments can contribute to the development of high-yield and quality maqui crops as source of bioactive compounds.Estación Experimental Agropecuaria BarilocheFil: Roldan, Cecilia Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Roldan, Cecilia Soledad. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; ArgentinaFil: Caballe, Gonzalo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Caballe, Gonzalo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; ArgentinaFil: Fontana, Ariel. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Viale, Maximiliano. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; ArgentinaFil: Berli, Federico. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentin

Mutation of the SP1 Sequence Impairs both Multimerization and Membrane-Binding Activities of Human Immunodeficiency Virus Type 1 Gag

The Gag protein of human immunodeficiency virus type 1 contains a 14-amino-acid region, termed SP1, between the capsid and downstream nucleocapsid sequences. Although SP1 is known to be indispensable for virus production, the mechanisms involved are mostly unclear. In this study, we demonstrate that an M368A mutation within SP1 severely diminished the ability of Gag to associate with cellular membranes. Although wild-type levels of membrane binding were restored to the M368A Gag by a second-site L20K mutation within matrix, the resultant Gag mutant L20K-M368A remained defective in virus production. This latter deficit was partially consequent to the binding of L20K-M368A Gag to nonraft membranes as opposed to raft association seen for wild-type Gag. Further analysis revealed that the majority of membrane-bound M368A Gag proteins were small oligomers, indicating a multimerization defect. In support of this observation, purified recombinant Gag derivatives containing the M368A mutation formed much lower amounts of high-molecular-weight complexes that were pelletable at 21,000 × g than did wild-type Gag. Based on the myristyl switch model, we propose that the M368A mutation inhibits Gag multimerization and, as a result, restricts the binding of Gag to cellular membranes

Characterization of a Putative α-Helix across the Capsid-SP1 Boundary That Is Critical for the Multimerization of Human Immunodeficiency Virus Type 1 Gag

A 14-amino-acid spacer peptide termed SP1 that separates the capsid (CA) and nucleocapsid (NC) sequences plays an active role in the assembly of human immunodeficiency virus type 1. This activity of SP1 involves its amino-terminal residues that, together with adjacent CA residues, constitute a putative α-helical structure spanning Gag residues from positions 359 to 371. In this study, we have determined that the virus assembly determinants within this putative α-helix were residues H359, K360, A361, L364, A367, and M368, of which K360 and A367 contribute to virus production to lesser extents. Notably, changes of the two basic amino acids H359 and K360 to arginine (R) impaired virus production, whereas mutations L364I and M368I, in contrast to L364A and M368A, generated near-wild-type levels of virus particles. This suggests that within Gag complexes, amino acids H359 and K360 are involved in stricter steric interactions than L364 and M368. Since L364 and M368 are separated by four residues and thus presumably located on the same side of the helical surface, they may initiate synergistic hydrophobic interactions to stabilize Gag association. Further analysis in the context of the protease-negative mutation D185H confirmed the key roles of amino acids H359, A361, L364, and M368 in virus assembly. Importantly, when transfected cells were subjected to Dounce homogenization and the cell lysates were treated by ultracentrifugation at 100,000 × g, Gag molecules containing each of the H359A, A361V, L364A, and M368A mutations were found mainly in the supernatant fraction (S100), whereas approximately 80% of wild-type Gag proteins were found in the pellet. Therefore, these four mutations must have prevented Gag from generating large complexes

Synergy-Based Small-Molecule Screen Using a Human Lung Epithelial Cell Line Yields ΔF508-CFTR Correctors That Augment VX-809 Maximal Efficacy

The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium–derived cell line (CFBE41o(−)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC(50) < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the “therapeutic ceiling” of first-generation correctors