Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopy

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Most retroviruses enter their host cells by fusing the viral envelope with the plasma membrane. Although the protein machinery promoting fusion has been characterized extensively, the dynamics of the process are largely unknown. Results We generated human immunodeficiency virus-1 (HIV-1) particles pseudotyped with the envelope (Env) protein of ecotropic murine leukemia virus eMLV to study retrovirus entry at the plasma membrane using live-cell microscopy. This Env protein mediates highly efficient pH independent fusion at the cell surface and can be functionally tagged with a fluorescent protein. To detect fusion events, double labeled particles carrying one fluorophor in Env and the other in the matrix (MA) domain of Gag were generated and characterized. Fusion events were defined as loss of Env signal after virus-cell contact. Single particle tracking of >20,000 individual traces in two color channels recorded 28 events of color separation, where particles lost the Env protein, with the MA layer remaining stable at least for a short period. Fourty-five events were detected where both colors were lost simultaneously. Importantly, the first type of event was never observed when particles were pseudotyped with a non-fusogenic Env. Conclusion These results reveal rapid retroviral fusion at the plasma membrane and permit studies of the immediate post-fusion events.Published versio

Origen de la casiterita detrítica en los aluviones recientes de Tirados de la Vega-Golpejas (Salamanca)

9 páginas, 6 figuras, 5 tablas.[ES] En el sector Tirados de la Vega-Golpejas han sido detectadas dos anomalías de casiterita en los aluviones del Regato de los Lentiscos y en otros existentes al sur de Tirados de la Vega. El estudio granométrico y morfométrico de éstas casiteritas. contrastado con el de la casiterita procedente del granito de Golpejas. permite asegurar que la anomalía del Regato de los Lentiscos se debe a la dispersión de la casiterita de aquel granito. mientras que la de Tirados de la Vega procede de filones estanníferos. La aplicación del análisis factoral corrobora este hecho y permite diferenciar las casiteritas que proceden de rocas graníticas de aquellas que vienen de filones estanníferos. Según esto se puede indicar que la capacidad de migración de la casiterita desde el yacimiento estannífero de Golpejas es de unos 3 Kms.[EN] Two cassiterite anomalies have been detected in the Regato de los Lentisco and Tirados de la Vega alluviums, in the Golpejas area (Salamanca). The granometric and morphometric parameters of these cassiterites have been compared with those shown by the cassiterite crystal ocurrying in the Golpejas granite. The comparison between the two groups of data allow us to conclude that the anomaly observed in the Regato de los Lentistos has been originated by the disperssion of the cassiterite from the primary deposit of Golpejas. On the contrary, the anomaly ocurring in the alloviums to the south of Tirados de la Vega shows a different origin, and is coming from stanniferous veins. The factor anylisis corroborates the differentiation mentionated above, and indicates a migration capacite of about 3 kms. from the stannifeous deposits of Golpejas for the alluvial cassiterite.Peer reviewe

Chemogenetic Control of Nanobodies

Engineered nanobody allows reversible control of activity in cells through the binding of small molecules.We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules

Structure-Aided Design of Novel Inhibitors of HIV Protease Based on a Benzodiazepine Scaffold

HIV protease is a primary target for the design of virostatics. Screening of libraries of non-peptide low molecular weight compounds led to the identification of several new compounds that inhibit HIV PR in the low micromolar range. X-ray structure of the complex of one of them, a dibenzo­[<i>b</i>,<i>e</i>]­[1,4]­diazepinone derivative, showed that two molecules of the inhibitor bind to the PR active site. Covalent linkage of two molecules of such a compound by a two-carbon linker led to a decrease of the inhibition constant of the resulting compound by 3 orders of magnitude. Molecular modeling shows that these dimeric inhibitors form two crucial hydrogen bonds to the catalytic aspartates that are responsible for their improved activity compared to the monomeric parental building blocks. Dibenzo­[<i>b</i>,<i>e</i>]­[1,4]­diazepinone analogues might represent a potential new class of HIV PIs

Pooled RT-qPCR testing for SARS-CoV-2 surveillance in schools-a cluster randomised trial

Background: The extent to which children and adolescents contribute to SARS-CoV-2 transmission remains not fully understood. Novel high-capacity testing methods may provide real-time epidemiological data in educational settings helping to establish a rational approach to prevent and minimize SARS-CoV-2 transmission. We investigated whether pooling of samples for SARS-CoV-2 detection by RT-qPCR is a sensitive and feasible high-capacity diagnostic strategy for surveillance of SARS-CoV-2 infections in schools. Methods: In this study, students and school staff of 14 educational facilities in Germany were tested sequentially between November 9 and December 23, 2020, two or three times per week for at least three consecutive weeks. Participants were randomized for evaluation of two different age adjusted swab sampling methods (oropharyngeal swabs or buccal swabs compared to saliva swabs using a 'lolli method'). Swabs were collected and pooled for SARS-CoV-2 RT-qPCR. Individuals of positive pooled tests were retested by RTqPCR the same or the following day. Positive individuals were quarantined while the SARS-CoV-2 negative individuals remained in class with continued pooled RT-qPCR surveillance. The study is registered with the German Clinical Trials register (registration number: DRKS00023911). Findings: 5,537 individuals were eligible and 3970 participants were enroled and included in the analysis. In students, a total of 21,978 swabs were taken and combined in 2218 pooled RT-qPCR tests. We detected 41 positive pooled tests (1 cent 8%) leading to 36 SARS-CoV-2 cases among students which could be identified by individual re-testing. The cumulative 3-week incidence for primary schools was 564/100,000 (6/1064, additionally 1 infection detected in week 4) and 1249/100,000 (29/2322) for secondary schools. In secondary schools, there was no difference in the number of SARS-CoV-2 positive students identified from pooled oropharyngeal swabs compared to those identified from pooled saliva samples (lolli method) (14 vs. 15 cases; 1.3% vs. 1.3%; OR 1.1; 95%-CI 0.5-2.5). A single secondary school accounted for 17 of 36 cases (47%) indicating a high burden of asymptomatic prevalent SARS-CoV-2 cases in the respective school and community. Interpretation: In educational settings, SARS-CoV-2 screening by RT-qPCR-based pooled testing with easily obtainable saliva samples is a feasible method to detect incident cases and observe transmission dynamics. Funding: Federal Ministry of education and research (BMBF; Project B-FAST in NaFoUniMedCovid19; registration number: 01KX2021). (c) 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/