In silico prediction of mutant HIV-1 proteases cleaving a target sequence

HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636 -- 1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidates for specificity and cleavability towards the target sequence

Hierarchy of neural organization in the embryonic spinal cord: Granger-causality graph analysis of in vivo calcium imaging data

The recent development of genetically encoded calcium indicators enables monitoring in vivo the activity of neuronal populations. Most analysis of these calcium transients relies on linear regression analysis based on the sensory stimulus applied or the behavior observed. To estimate the basic properties of the functional neural circuitry, we propose a network-based approach based on calcium imaging recorded at single cell resolution. Differently from previous analysis based on cross-correlation, we used Granger-causality estimates to infer activity propagation between the activities of different neurons. The resulting functional networks were then modeled as directed graphs and characterized in terms of connectivity and node centralities. We applied our approach to calcium transients recorded at low frequency (4 Hz) in ventral neurons of the zebrafish spinal cord at the embryonic stage when spontaneous coiling of the tail occurs. Our analysis on population calcium imaging data revealed a strong ipsilateral connectivity and a characteristic hierarchical organization of the network hubs that supported established propagation of activity from rostral to caudal spinal cord. Our method could be used for detecting functional defects in neuronal circuitry during development and pathological conditions

A Computational Methodology to Screen Activities of Enzyme Variants

We present a fast computational method to efficiently screen enzyme activity. In the presented method, the effect of mutations on the barrier height of an enzyme-catalysed reaction can be computed within 24 hours on roughly 10 processors. The methodology is based on the PM6 and MOZYME methods as implemented in MOPAC2009, and is tested on the first step of the amide hydrolysis reaction catalyzed by Candida Antarctica lipase B (CalB) enzyme. The barrier heights are estimated using adiabatic mapping and are shown to give barrier heights to within 3kcal/mol of B3LYP/6-31G(d)//RHF/3-21G results for a small model system. Relatively strict convergence criteria (0.5kcal/(mol{\AA})), long NDDO cutoff distances within the MOZYME method (15{\AA}) and single point evaluations using conventional PM6 are needed for reliable results. The generation of mutant structure and subsequent setup of the semiempirical calculations are automated so that the effect on barrier heights can be estimated for hundreds of mutants in a matter of weeks using high performance computing

Salmonellosis in wild birds and its relationship with the infection in finishing pigs

The potential relationship between Salmonella infection in wild birds and pigs was investigated. Feces from pigs, wild birds, and bird droppings or other environmental samples from 25 finishing farms were cultured for Salmonella isolation. In 17 (68%) farms Salmonella was isolated. Out of 57 Salmonella isolates found, 32 (56.1%) were Typhimurium. In 6 (24%) farms the same Salmonella serotype was isolated from samples from different origins and similar AR and PFGE patterns were found, which would support the existence of a transmission cycle of Salmonella infection between birds and pigs in this area. Preventing bird access to farm premises is highly recommended

Estudio serológico de la infección por Salmonella spp. en cerdos de engorde del Noreste de España y factores de riesgo asociados

The results of a serological survey carried out in northeastern Spain to estimate the seroprevalence to Salmonella spp. and to determine potential risk factors are presented. Sera were obtained from farms submitting serum samples to the Regional Diagnostic Laboratory (RDL) for the diagnosis of other infectious diseases included within official eradication/surveillance programs, and farm data collected through a questionnaire. Out of 6,182 pig sera (217 farms) analyzed 1,219 (19.7%) were positive (optical density, OD ≥ 40%). More than 70% of the herds presented ≥ 1 seropositive animal indicating that salmonellosis was widespread. In a multivariable logistic mixed regression model seroprevalence was associated with farms where birds were visible inside the fattening units (OR = 2.1; 95% CI: 1.3-3.2) or that shared workers with other pig farms (OR = 2.5; 95% CI: 1.4-4.7). Seropositivity also increased when farmers used footwear exclusive for the farm (OR = 3.1; 95% CI: 1.3-7.3) or pigs were fed mostly with pellets (OR = 1.7; 95% CI: 1.1-2.6). It was also higher during the fall and winter months. The presence of a changing room and shower in the farm was negatively associated with it (OR = 0.5; 95% CI: 0.3-0.8). Most risk factors could be mitigated through strict hygiene and biosecurity measures, but the high (> 40%) within-herd seroprevalence observed in many herds may challenge the capacity of intervention of animal health authorities.Se presentan los resultados de un estudio serológico realizado en explotaciones porcinas del Noreste de España para estimar la seroprevalencia frente a Salmonella spp. y determinar posibles factores de riesgo asociados. Los sueros se obtuvieron a partir de aquellos enviados por las explotaciones al Laboratorio Regional de Diagnóstico para el diagnóstico de otras enfermedades infecciosas sometidas a erradicación/vigilancia oficial, y los datos sobre las explotaciones se recogieron mediante un cuestionario. De un total de 6.182 sueros de cerdo (217 granjas) analizados 1.219 (19,7%) resultaron positivos (densidad óptica, DO ≥ 40%). Más del 70% de los rebaños presentaron al menos un animal positivo, indicando que la salmonelosis está ampliamente distribuida en la región. Según un modelo multivariable mixto de regresión logística, la seroprevalencia se asoció con granjas en las cuales se veían aves en el interior de las naves (OR = 2,1; IC 95%: 1,3-3,2) y también con granjas que compartían trabajadores (OR = 2,5; IC 95%: 1,4-4,7). Se observó que la seropositividad se incrementaba cuando los trabajadores usaban botas exclusivas en la explotación (OR = 3,1; IC 95%: 1,3-7,3) o al alimentar a los cerdos principalmente con alimento granulado (OR = 1,7; IC 95%: 1,1-2,6). La seroprevalencia también fue superior durante los meses de otoño e invierno. La existencia en las granjas de vestuarios y duchas para el personal se asoció negativamente con la seroprevalencia (OR = 0,5; IC 95%: 0,3-0,8). La mayoría de los factores de riesgo podrían controlarse a través de estrictas medidas de higiene y bioseguridad, pero el gran número de rebaños que presentaron alta seroprevalencia (> 40%) puede poner a prueba la capacidad de intervención de las autoridades sanitarias oficiales

Serological characterization of Salmonella spp. infection in finishing pigs from NE Spain

The seroprevalence of salmonella spp. In finishing pigs in Aragón (NE of Spain) and the potential factors associated with it were assessed. Serum samples were collected directly from the Regional Diagnostic Laboratory (RDL). Only farms submitting a minimum of 30 serum samples to the RDL were included, i.e. exporting and farrow-to-finish farms, and those in the last stages of the Aujezsky\u27s disease eradication program

Companion Losses for Deep Neural Networks

Modern Deep Neuronal Network backends allow a great flexibility to define network architectures. This allows for multiple outputs with their specific losses which can make them more suitable for particular goals. In this work we shall explore this possibility for classification networks which will combine the categorical cross-entropy loss, typical of softmax probabilistic outputs, the categorical hinge loss, which extends the hinge loss standard on SVMs, and a novel Fisher loss which seeks to concentrate class members near their centroids while keeping these apartThe authors acknowledge financial support from the European Regional Development Fund and the Spanish State Research Agency of the Ministry of Economy, Industry, and Competitiveness under the project PID2019-106827GB-I00. They also thank the UAM–ADIC Chair for Data Science and Machine Learning and gratefully acknowledge the use of the facilities of Centro de Computaci´on Cient´ıfica (CCC) at UA

The addition of galacto-oligosaccharides on the feed for the control of salmonellosis in fattening pigs

Prebiotics that block intestinal harmful bacteria and stimulate both the activity of beneficial bacteria and the animal immune system may help in controlling pig salmonellosis. We added a galacto-oligosaccharide (Salmosan®) on the diet of pigs during the whole period of fattening to assess its potential effect on the prevalence of Salmonella spp. In a first trial 56 pigs from a small fattening unit (fewer than 200 animals) were fed with a diet where Salmosan® (0.5 kg/Ton of feed) was added, while the rest of the animals were fed with the same feed without the galacto-oligosaccharide

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

This work was funded by the US National Institutes of Health (NIH) grant P01GM099117, and by the National Science Foundation Graduate Research Fellowship under the grant DGE1144152 (J.R.H. and K.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.United States Department of Health & Human Services National Institutes of Health (NIH) - USA P01GM099117National Science Foundation (NSF) DGE114415