A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit

Abstract Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3′-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.This study was funded by grants from the National Natural Science Foundation of China (U0931003/L01) and the National High-Tech R&D Program of China (2011AA10A208) to EMZ, the National Natural Science Foundation of China (31302103) to WCB, the European Community’s Seventh Frame-work Programme (PoRRSCon, FP7-KBBE-2009-3-245141) and the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16075) to FA and LE.Peer Reviewe

Comparação de pH e matéria seca em três processos de silagem com forragem de gergelim (Sesamum indicum) nos trópicos secos.

El objetivo de este estudio fue comparar el pH y la materia seca (MS) en tres procesos de ensilaje con planta de ajonjolí en climas secos. Los experimentos se realizaron en la Facultad de Medicina Veterinaria y Zootecnia No. 1 de la Universidad Autónoma de Guerrero. En el experimento 1, se ensiló forraje de maíz combinado con forraje de ajonjolí (T1: 100:00, T2: 75:25, T3: 50:50, T4: 25:75 y T5: 00:100% respectivamente). En el experimento 2 se ensiló forraje de ajonjolí mezclado con melaza (T1:0, T2:4, T3:8 y T4:12% respectivamente) En el experimento 3 se ensiló planta de ajonjolí sola (T1), con melaza 8% (T2), planta presecada sola (T3) y planta presecada con melaza 8% (T4). Se realizaron síes microsilos por tratamiento (6 réplicas). Los diseños experimentales fueron completamente al azar. En el experimento 1, el pH más bajo fue para el T1 con un valor de 3.83, en el experimento 2 fue para el T4 con un valor de 3.94 y en el experimento 3 fue para el T2 con un valor de 4.00. Para la MS, el contenido fue mayor para el T1 con 28.55%, en el experimento 2 fue para el T4 con 27.63% y en el experimento 3 fue para el T3 con 36.00%. Todos los experimentos mostraron diferencia estadística (p< 0.0001) entre tratamientos. Sin embargo, los valores para pH y MS son aceptables para prácticamente todos los tratamientos, en acuerdo con otras investigaciones. Por lo tanto, se concluye que al ensilar forraje de ajonjolí mezclado con forraje de maíz, melaza o planta presercada se logra mejorar la fermentación ácida y se incrementa el contenido de materia seca. Sin embargo, es necesario realizar evaluaciones posteriores de las propiedades físico-químicas y determinar su valor nutritivo.The objective of this study was to compare the pH and dry matter (DM) in three silage processes with sesame plants in dry climates. The experiments were carried out at the Faculty of Veterinary Medicine and Zootechnics No. 1 of the Autonomous University of Guerrero. In experiment 1, corn forage combined with sesame forage was ensiled (T1: 100:00, T2: 75:25, T3: 50:50, T4: 25:75 and T5: 00:100% respectively). In experiment 2 sesame forage mixed with molasses (T1:0, T2:4, T3:8 and T4:12% respectively) was ensiled. In experiment 3 sesame plant alone (T1) was ensiled with 8% molasses ( T2), pre-dried plant alone (T3) and pre-dried plant with 8% molasses (T4). Six microsilos per treatment (6 replicates) were made. The experimental designs were completely randomized. In experiment 1, the lowest pH was for T1 with a value of 3.83, in experiment 2 it was for T4 with a value of 3.94 and in experiment 3 it was for T2 with a value of 4.00. For DM, the content was higher for T1 with 28.55%, in experiment 2 it was for T4 with 27.63% and in experiment 3 it was for T3 with 36.00%. All the experiments showed statistical difference (p< 0.0001) between treatments. However, the values for pH and DM are acceptable for practically all treatments, in agreement with other investigations. Therefore, it is concluded that ensiling sesame forage mixed with corn forage, molasses or presercated plant improves acid fermentation and increases the dry matter content. However, it is necessary to carry out further evaluations of the physical-chemical properties and determine its nutritive value.O objetivo deste estudo foi comparar o pH e a matéria seca (MS) em três processos de silagem com plantas de gergelim em climas secos. Os experimentos foram realizados na Faculdade de Medicina Veterinária e Zootecnia nº 1 da Universidade Autônoma de Guerrero. No experimento 1, a forragem de milho combinada com a forragem de gergelim foi ensilada (T1: 100:00, T2: 75:25, T3: 50:50, T4: 25:75 e T5: 00:100% respectivamente). No experimento 2 a forragem de gergelim foi ensilada misturada com melaço (T1:0, T2:4, T3:8 e T4:12% respectivamente) - planta seca sozinha (T3) e planta pré-seca com 8% de melaço (T4). Seis microsilos por tratamento (6 réplicas) foram feitos. Os delineamentos experimentais foram inteiramente casualizados. No experimento 1, o menor pH foi para T1 com valor de 3,83, no experimento 2 foi para T4 com valor de 3,94 e no experimento 3 foi para T2 com valor de 4,00. Para MS o teor foi maior para T1 com 28,55%, no experimento 2 foi para T4 com 27,63% e no experimento 3 foi para T3 com 36,00%. Todos os experimentos apresentaram diferença estatística (p< 0,0001) entre os tratamentos. No entanto, os valores de pH e MS são aceitáveis ​​para praticamente todos os tratamentos, concordando com outras investigações. Portanto, conclui-se que a ensilagem de forragem de gergelim misturada com forragem de milho, melaço ou planta preservada melhora a fermentação ácida e aumenta o teor de matéria seca. No entanto, é necessário realizar mais avaliações das propriedades físico-químicas e determinar seu valor nutritivo

A limited-size ensemble of homogeneous CNN/LSTMs for high-performance word classification

The strength of long short-term memory neural networks (LSTMs) that have been applied is more located in handling sequences of variable length than in handling geometric variability of the image patterns. In this paper, an end-to-end convolutional LSTM neural network is used to handle both geometric variation and sequence variability. The best results for LSTMs are often based on large-scale training of an ensemble of network instances. We show that high performances can be reached on a common benchmark set by using proper data augmentation for just five such networks using a proper coding scheme and a proper voting scheme. The networks have similar architectures (convolutional neural network (CNN): five layers, bidirectional LSTM (BiLSTM): three layers followed by a connectionist temporal classification (CTC) processing step). The approach assumes differently scaled input images and different feature map sizes. Three datasets are used: the standard benchmark RIMES dataset (French); a historical handwritten dataset KdK (Dutch); the standard benchmark George Washington (GW) dataset (English). Final performance obtained for the word-recognition test of RIMES was 96.6%, a clear improvement over other state-of-the-art approaches which did not use a pre-trained network. On the KdK and GW datasets, our approach also shows good results. The proposed approach is deployed in the Monk search engine for historical-handwriting collections

Exploring the Midgut Transcriptome and Brush Border Membrane Vesicle Proteome of the Rice Stem Borer, Chilo suppressalis (Walker)

The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac

Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication

Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins

Female Institutional Directors on Boards and Firm Value

The aim of this research is to examine what impact female institutional directors on boards have on corporate performance. Previous research shows that institutional female directors cannot be considered as a homogeneous group since they represent investors who may or may not maintain business relations with the companies on whose corporate boards they sit. Thus, it is not only the effect of female institutional directors as a whole on firm value that has been analysed, but also the impact of pressure-resistant female directors, who represent institutional investors (investment, pension and mutual funds) that only invest in the company, and do not maintain a business relation with the firm. We hypothesize that there is a non-linear association, specifically quadratic, between institutional and pressure-resistant female directors on boards and corporate performance. Our results report that female institutional directors on boards enhance corporate performance, but when they reach a certain threshold on boards (11.72 %), firm value decreases. In line with female institutional directors, pressure-resistant female directors on boards also increase firm value, but only up to a certain figure (12.71 % on boards), above which they have a negative impact on firm performance. These findings are consistent with an inverted U-shaped relationship between female institutional directors and pressure-resistant female directors and firm performance

An Ontological Approach to Inform HMI Designs for Minimizing Driver Distractions with ADAS

ADAS (Advanced Driver Assistance Systems) are in-vehicle systems designed to enhance driving safety and efficiency as well as comfort for drivers in the driving process. Recent studies have noticed that when Human Machine Interface (HMI) is not designed properly, an ADAS can cause distraction which would affect its usage and even lead to safety issues. Current understanding of these issues is limited to the context-dependent nature of such systems. This paper reports the development of a holistic conceptualisation of how drivers interact with ADAS and how such interaction could lead to potential distraction. This is done taking an ontological approach to contextualise the potential distraction, driving tasks and user interactions centred on the use of ADAS. Example scenarios are also given to demonstrate how the developed ontology can be used to deduce rules for identifying distraction from ADAS and informing future designs

Ligand-dependent Hedgehog pathway activation in Rhabdomyosarcoma : the oncogenic role of the ligands

Altres ajuts: This work was supported by grants from Institut Català d'Oncologia (ICO), Instituto de Salud Carlos III (RTICC-RD12/0036/0016, /0020, /0035, /0057; and PI14/00647), Fundació A BOSCH, Fundació Amics Joan Petit, ajuts predoctorals del VHIR and RIS3CAT grants COMRDI15-1-0014 (ACCIÓ and FEDER).Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. The Hedgehog (HH) pathway is known to develop an oncogenic role in RMS. However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. The expression of HH ligands was studied by qPCR, western blot and immunohistochemistry. Functional and animal model studies were carried out with cells transduced with shRNAs against HH ligands or treated with HH-specific inhibitors (Vismodegib and MEDI-5304). Finally, the molecular characterisation of an off-target effect of Vismodegib was also made. The results showed a prominent expression of HH ligands supporting an autocrine ligand-dependent activation of the pathway. A comparison of pharmacologic Smoothened inhibition (Vismodegib) and HH ligand blocking (MEDI-5304) is also provided. Interestingly, a first description of pernicious off-target effect of Vismodegib is also reported. The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols. Moreover, the description of a pernicious off-target effect of Vismodegib, via unfolded protein response activation, may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers