Evolutionary and phenotypic characterization of two spike mutations in European lineage 20E of SARS-CoV-2.

We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro, and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutrali- zation by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multi- ple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investi- gated was consistent with the lack of advantage observed in the clinical and ex- perimental data

The meaning of negation in the second language classroom: evidence from 'any'

This article brings together an experimental study involving L2 knowledge of negation in English and an analysis of how English language textbooks treat negation, in order to consider whether textbook explanations of negation could better exploit linguistic insights into negation. We focus on the English negative polarity item any, whose distribution is contingent on negation, whether through the explicit negator not or through lexical semantic negators (e.g. hardly). Our experiment compares Chinese-speaking learners with existing data from Arabic-speaking learners, finding lower accuracy on any with lexical semantic negators in both groups. Our textbook analysis reveals an approach to negation that is limited to form, focusing on the explicit negator not without explicit treatment of other types of negation. We propose that emphasizing the meaning of negation, with explicit treatment of the full range of negative forms could facilitate more complete acquisition across a range of grammatical properties where negation plays a role

Rendimiento de trébol rojo (Trifolium pratense L.) a diferente edad de cosecha

Objective: The objective of this research was to obtain the optimum cutting moment of the red clover by varying the cutting frequency in the spring season. Design/methodology/approach: The treatments were cuts at different ages (eight weeks) in the spring season. The variables studied were: forage accumulation, botanical and morphological composition, foliar area per stem and photosynthesis. The statistical analysis of the data obtained was performed with the GLM procedure of the SAS statistical package. The results obtained in yield of dry matter showed an increase as the age of regrowth advanced, in week 8, 5013 kg MS ha-1 total were obtained, resulting in the maximum value registered. There was a direct positive relationship between the mass of green leaves, rate of photosynthesis, leaf area per stem and leaf: stem ratio, with the best values ??recorded at the fourth week of regrowth. The morphological component that contributes most to forage yield was the stem with an average of 55% in all the weeks except the fourth. Limitations on study/implications: After the fourth week the unwanted structural characteristics increase. Findings/conclusions: It is concluded that the yield increases as the evaluation time passes and it is recommended to harvest the red clover in the fourth week of regrowth.Objetivo: El objetivo de esta investigación fue obtener el momento óptimo de corte del trébol rojo variando la frecuencia de corte en la estación de primavera. Diseño/metodología/aproximación: Los tratamientos fueron los cortes a diferente edad (ocho semanas) en la estación primavera. Las variables estudiadas fueron: acumulación de forraje, composición botánica y morfológica, área foliar por tallo y fotosíntesis. El análisis estadístico de los datos obtenidos se realizó con el procedimiento GLM del paquete estadístico SAS. Resultados: Los resultados obtenidos en rendimiento de materia seca mostraron un aumento conforme avanzo la edad de rebrote, en la semana 8 se obtuvieron 5,013 kg MS ha-1 total que resulto el máximo valor registrado. Existió una relación positiva directa entre la masa de hojas verdes, tasa de fotosíntesis, área foliar por tallo y relación hoja:tallo, registrándose los mejores valores a la cuarta semana de rebrote. El componente morfológico que más contribuye al rendimiento de forraje fue el tallo con un promedio de 55 % en todas las semanas excepto la cuarta. Limitaciones del estudio/implicaciones: Después de la cuarta semana aumentan características estructurales no deseadas. Hallazgos/conclusiones: Se concluye que el rendimiento se incrementa conforme pasa el tiempo de evaluación y se recomienda cosechar el trébol rojo en la cuarta semana de rebrote

The T cell differentiation landscape is shaped by tumour mutations in lung cancer

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours and whether this affects patient outcomes is unknown. Here, we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets with strong phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states was associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC

Land conflict in peri-urban areas: Exploring the effects of land reform on informal settlement in Mexico

Peri-urban areas are often subject to intensive construction, through both formal and informal processes. As land transitions from rural to urban status, different land tenure and administration systems may come into conflict, leading to disputes, contestation and, in some cases, violence. However, little is known about the precise causes of peri-urban land conflict. In Mexico, peri-urban growth has historically proceeded peacefully, owing to the control exerted by a corporatist system of government, and the political use of land tenure regularisation. However, the effects of land reforms on transactions at the peri-urban fringe, in the context of wider processes of liberalisation, may be increasing vulnerability to conflict over land. This paper explores these issues through a case study of an irregular settlement on the peri-urban fringe of the provincial Mexican city of Xalapa, where contestations over informally developed land have escalated into violent encounters between groups of settlers and the state. The findings show that vulnerability to conflict in peri-urban areas can be attributed to the interaction of macro-level processes with local-level factors, including diverse claims, overlapping legal and governance frameworks and, critically, local power relations

N-3 PUFA Supplementation Triggers PPAR-α Activation and PPAR-α/NF-κB Interaction: Anti-Inflammatory Implications in Liver Ischemia-Reperfusion Injury

Dietary supplementation with the n-3 polyunsaturated fatty acids (n-3 PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to rats preconditions the liver against ischemia-reperfusion (IR) injury, with reduction of the enhanced nuclear factor-κB (NF-κB) functionality occurring in the early phase of IR injury, and recovery of IR-induced pro-inflammatory cytokine response. The aim of the present study was to test the hypothesis that liver preconditioning by n-3 PUFA is exerted through peroxisone proliferator-activated receptor α (PPAR-α) activation and interference with NF-κB activation. For this purpose we evaluated the formation of PPAR-α/NF-κBp65 complexes in relation to changes in PPAR-α activation, IκB-α phosphorylation and serum levels and expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in a model of hepatic IR-injury (1 h of ischemia and 20 h of reperfusion) or sham laparotomy (controls) in male Sprague Dawley rats. Animals were previously supplemented for 7 days with encapsulated fish oil (General Nutrition Corp., Pittsburg, PA) or isovolumetric amounts of saline (controls). Normalization of IR-altered parameters of liver injury (serum transaminases and liver morphology) was achieved by dietary n-3 PUFA supplementation. EPA and DHA suppression of the early IR-induced NF-κB activation was paralleled by generation of PPAR-α/NF-κBp65 complexes, in concomitance with normalization of the IR-induced IκB-α phosphorylation. PPAR-α activation by n-3 PUFA was evidenced by enhancement in the expression of the PPAR-α-regulated Acyl-CoA oxidase (Acox) and Carnitine-Palmitoyl-CoA transferase I (CPT-I) genes. Consistent with these findings, normalization of IR-induced expression and serum levels of NF-κB-controlled cytokines IL-lβ and TNF-α was observed at 20 h of reperfusion. Taken together, these findings point to an antagonistic effect of PPAR-α on NF-κB-controlled transcription of pro-inflammatory mediators. This effect is associated with the formation of PPAR-α/NF-κBp65 complexes and enhanced cytosolic IκB-α stability, as major preconditioning mechanisms induced by n-3 PUFA supplementation against IR liver injury

Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- And Rab8-Dependent and recycling endosome-independent

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the m1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells. © 2014 Bertuccio et al