Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc.

Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic ß-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.

Malignant fibrous histiocytoma of the head and neck: a case series.

PURPOSE: The study objective is to evaluate the clinical features and outcomes of patients treated for head and neck malignant fibrous histiocytoma at a tertiary care medical facility. MATERIALS AND METHODS: This is a retrospective case series of 17 adult subjects with malignant fibrous histiocytoma of the head and neck who were treated between January 1, 1965, and December 31, 2010. This study was conducted using patient charts at a tertiary medical center. Subject selection was conducted using Current Procedural Terminology numbers; International Classification of Diseases, Ninth Revision, codes; and a search of the tumor registry. RESULTS: Chart review of the 17 identified subjects revealed an overwhelming male predominance (88%) with an overall mean age of 69 years(52-87 years). Thirteen patients (78%) underwent some form of surgical resection, 6 patients (35%) received radiation therapy, and 6 (35%) were given chemotherapy over the course of treatment. Nine tumors (53%) had a cutaneous origin, whereas 8 lesions (47.1%) were found in the soft tissue of the head and neck region. The local recurrence rate following a single resection was 46%. Overall median survival following diagnosis was found to be 65 months, with a 5-year survival rate of 52%. Median disease-free survival was 20 months, with a 5-year disease-free survival rate of 37%. Overall median and 5-year survival rates were found to increase with clear surgical margins, as was 5-year survival. CONCLUSIONS: Aggressive surgical management to achieve clear margins is central to the effective treatment of malignant fibrous histiocytoma of the head and neck. Metastatic disease portends a dismal prognosis.

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos.

DNA accessibility of cis-regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many genes regulating embryonic development have been identified, the underlying CRE dynamics controlling their expression remain largely uncharacterized. To address this, we produced a multimodal resource and genomic regulatory map for the zebrafish community, which integrates single-cell combinatorial indexing assay for transposase-accessible chromatin with high-throughput sequencing (sci-ATAC-seq) with bulk histone PTMs and Hi-C data to achieve a genome-wide classification of the regulatory architecture determining transcriptional activity in the 24-h post-fertilization (hpf) embryo. We characterized the genome-wide chromatin architecture at bulk and single-cell resolution, applying sci-ATAC-seq on whole 24-hpf stage zebrafish embryos, generating accessibility profiles for ∼23,000 single nuclei. We developed a genome segmentation method, ScregSeg (single-cell regulatory landscape segmentation), for defining regulatory programs, and candidate CREs, specific to one or more cell types. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization and identified new regulatory principles between chromatin modalities prevalent during zebrafish development. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos identified novel cellular roles for this hematovascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work defines regulatory architecture and principles in the zebrafish embryo and establishes a resource of cell-type-specific genome-wide regulatory annotations and candidate CREs, providing a valuable open resource for genomics, developmental, molecular, and computational biology.

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

Space-time variations of human capital assets across U.S. metropolitan areas, 1980 to 2000.

This article examines the changing structure of human capital in U.S. metropolitan regions from 1980 to 2000. Data are drawn from the Dictionary of Occupational Titles and from the Integrated Public Use Microdata Series. Intensive empirical investigation leads to three main conclusions. First, forms of human capital in the United States are becoming more oriented to labor tasks that call for cognitive-cultural skills. Second, cognitive-cultural skills are accumulating most intensively in large metropolitan areas. Third, physical or practical forms of human capital are increasingly being relegated to smaller metropolitan areas. That said, important residues of human capital, focused on physical or practical tasks, remain a durable element of the economies of large metropolitan areas. I offer a brief theoretical explanation of these results.

Micro-Clotting of Platelet-Rich Plasma Upon Loading in Hydrogel Microspheres Leads to Prolonged Protein Release and Slower Microsphere Degradation.

Platelet-rich plasma (PRP) is an autologous blood product that contains a variety of growth factors (GFs) that are released upon platelet activation. Despite some therapeutic potential of PRP in vitro, in vivo data are not convincing. Bolus injection of PRP is cleared rapidly from the body diminishing its therapeutic efficacy. This highlights a need for a delivery vehicle for a sustained release of PRP to improve its therapeutic effect. In this study, we used microfluidics to fabricate biodegradable PRP-loaded polyethylene glycol (PEG) microspheres. PRP was incorporated into the microspheres as a lyophilized PRP powder either as is (powder PRP) or first solubilized and pre-clotted to remove clots (liquid PRP). A high PRP loading of 10% w/v was achieved for both PRP preparations. We characterized the properties of the resulting PRP-loaded PEG microspheres including swelling, modulus, degradation, and protein release as a function of PRP loading and preparation. Overall, loading powder PRP into the PEG microspheres significantly affected the properties of microspheres, with the most pronounced effect noted in degradation. We further determined that microsphere degradation in the presence of powder PRP was affected by platelet aggregation and clotting. Platelet aggregation did not prevent but prolonged sustained PRP release from the microspheres. The delivery system developed and characterized herein could be useful for the loading and releasing of PRP to promote tissue regeneration and wound healing or to suppress tissue degeneration in osteoarthritis, and intervertebral disc degeneration.

Synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxamides with broad-spectrum human herpesvirus polymerase inhibition.

A versatile synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxylate esters has been developed which has lead to the identification of a new series of non-nucleoside inhibitors of human herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

Accelerated Solvent Extraction of Insecticides from Rice Hulls, Rice Bran, and Polished Rice Grains.

Analysis of pesticide residues in irrigated rice grains is important for food security. In this study, we analyzed accelerated solvent extraction (ASE) conditions for the extraction of thiamethoxam and chlorantraniliprole insecticides from rice hulls, rice bran, and polished rice grains. Several variables, including extraction solvent, extraction temperature, extraction pressure, cell size, static extraction time, and sample concentration, were investigated. The average recoveries of the three matrixes were between 89.7 and 109.7% at the fortification level of 0.75 mg/kg. The optimum ASE operating conditions were acetonitrile (100%) as extraction solvent, extraction temperature of 75°C for rice hulls and 100°C for rice bran and polished rice grains, extraction cell pressure of 10.3 MPa, 22 mL cell size, and two extraction cycles. The total extraction time was approximately 25 min. The extracted volume was evaporated to dryness and the residues were redissolved in 2 mL acetonitrile after 1 min of vortex-shaking. Thiamethoxam and chlorantraniliprole were analyzed by ultra-HPLC with tandem MS. In conclusion, ASE in rice hulls, rice bran, and polished rice grains offers the possibility of a fast and simple method for obtaining a quantitative extraction of the studied pesticides.

Residues of thiamethoxam and chlorantraniliprole in rice grain.

Thiamethoxam and chlorantraniliprole insecticides have been important tools for controlling pests in rice. However, food safety issues related to pesticide residues are important to consider with a food crop such as rice. Therefore, the objective of this study was to analyze thiamethoxam and chlorantraniliprole residues in rice hull, bran, and polished rice grains. The study was conducted during the 2012 cropping season at the Texas A&M Agrilife Research, David R. Wintermann Rice Research Station, near Eagle Lake, TX, USA. Rice was planted on May 5, 2012, using the cultivar 'Presidio'. Pesticide applications were performed at 5, 15, 25, and 35 days after flowering (DAF) using 1 and 2 times the recommended rate of 30 g active ingredient (ai) ha(-1) for thiamethoxam and 30 g ai ha(-1) for chlorantraniliprole. Sequentially, two treatments using the insecticides at recommended rate were applied at 5 and 25 DAF and at 5, 25, and 35 DAF. Insecticide residues were analyzed in different sample fractions: rice hull, bran, and polished rice grains. The samples were subjected to extraction using an accelerated solvent extraction (ASE) technique. Sample aliquots were analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), with a limit of quantification (LOQ) of 5 × 10(-5) mg kg(-1). Residues of thiamethoxam and chlorantraniliprole were detected in rice hull, bran, and polished rice grains, and the quantified values were greater in hull and in rice bran.

Adsorption and desorption of atrazine, desethylatrazine, deisopropylatrazine, and hydroxyatrazine in vegetated filter strip and cultivated soil.

Adsorption and desorption of atrazine and its metabolites in vegetated filter strip soil (VFS) has not been evaluated, yet these data are needed to predict the transport of these compounds through the VFS. Adsorption and desorption parameters for atrazine, desethylatrazine (DEA), deisopropylatrazine (DIA), and hydroxyatrazine (HA) were compared between a cultivated Houston Black clay (CS) and an adjacent 12-year-old VFS established in a mixed stand of bermudagrass [Cynodon dactylon (L.) Pers.] and buffalograss [Buchloe dactyloides (Nutt. Engelm)]. Adsorption and desorption isotherms were determined by batch equilibrium. The evaluated chemical and physical properties of the VFS and CS were similar with the exception of a 1.7-fold increase in the organic carbon content of the VFS. Adsorption and desorption coefficients for atrazine were at least 59% higher in VFS than in CS. The adsorption coefficient for HA was 48% higher in VFS compared with CS, but desorption was not statistically different between soils. Adsorption and desorption coefficients for DEA and DIA were not statistically different between soils. The predicted order of mobility in CS is HA < atrazine = DIA = DEA. In VFS, the predicted order of mobility is HA < atrazine = DIA < DEA. These data indicate that the higher organic carbon in VFS will likely retard the transport of atrazine and HA to surface and ground waters; however, the transport rates of DEA and DIA will be similar between soils.

Aggregate culture of human embryonic stem cell-derived hepatocytes in suspension are an improved in vitro model for drug metabolism and toxicity testing.

Early phase drug development relies on primary human hepatocytes for studies of drug metabolism, cytotoxicity, and drug-drug interactions. However, primary human hepatocytes rapidly lose metabolic functions ex vivo and are refractory to expansion in culture and thus are limited in quantity. Hepatocytes derived from human pluripotent stem cells (either embryonic stem (ES) or induced pluripotent stem (iPS) cells), have the potential to overcome many of the limitations of primary human hepatocytes, but to date the use of human pluripotent stem cell-derived hepatocytes has been limited by poor enzyme inducibility and immature metabolic function. Here, we present a simple suspension culture of aggregates of ES cell-derived hepatocytes that compared to conventional monolayer adherent culture significantly increases induction of CYP 1A2 by omeprazole and 3A4 by rifampicin. Using liquid chromatography-tandem mass spectrometry, we further show that ES cell-derived hepatocytes in aggregate culture convert omeprazole and rifampicin to their human-specific metabolites. We also show that these cells convert acetaminophen (APAP) to its cytotoxic metabolite (N-acetyl-p-benzoquinone imine (NAPQI)), although they fail to perform APAP glucuronidation. In summary, we show that human pluripotent stem cell-derived hepatocytes in aggregate culture display improved enzymatic inducibility and metabolic function and is a promising step toward a simple, scalable system, but nonetheless will require further improvements to completely replace primary human hepatocytes in drug development.

2-Aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines as broad-spectrum inhibitors of human herpesvirus polymerases.

A novel series of 2-aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potent antivirals against human herpesviruses. These compounds demonstrate broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

Brazilian university technology transfer to rural areas / Transferência de tecnologia de universidades brasileiras na área rural

In agriculture, there is a difference between average yield obtained by farmers and crop potential. There is technology available to increase yields, but not all farmers have access to it and/or use this information. This clearly characterizes an extension and technology transference problem. There are several technology transfer systems, but there is no system to fit all conditions. Therefore, it is necessary to create extension solutions according to local conditions. Another rural extension challenge is efficiency, despite continuous funding reductions. One proposal that has resulted from extension reform worldwide has suggested integration between the public and private sectors. The public universities could play the role of training and updating technical assistance of human resources, which is the one of the main aspects that has limited technology transfer. The objective of this study was to identify approaches to promote technology transfer generated in Brazilian public universities to rural areas through literature review. An experimental approach of technology transfer is presented here where a Brazilian university extension Vice-chancellor incorporates professionals from consolidated research groups according to demand. In this way, public universities take part of their social functions, by integrating teaching, research, and extension.

Em agricultura, há diferenças entre a produtividade média obtida pelos produtores e o potencial produtivo dos cultivos. Há informação tecnológica disponível para aumentar a produtividade, mas nem todos os produtores têm acesso e/ou usam a informação. Isso caracteriza claramente um problema de extensão e transferência de tecnologia. Há vários sistemas de transferência de tecnologia, mas, como não há sistema que se ajuste a todas as condições, é necessário criar alternativas adequadas às condições de cada local. Outro desafio da extensão rural é ser eficiente, apesar da contínua redução de recursos. Uma proposta advinda das constantes reformas na extensão verificada ao redor do mundo é o trabalho integrado entre a iniciativa privada e o poder público. A universidade pública contribuiria para o treinamento e a atualização dos recursos humanos envolvidos com assistência técnica, apontado como um dos aspectos limitantes na transferência de tecnologia. O objetivo deste estudo foi identificar, por meio de revisão bibliográfica, alternativas de promover a transferência de tecnologias geradas nas universidades públicas brasileiras para a área rural. Assim, é apresentada uma proposta de transferência de tecnologia a ser gerenciada pelas Pró-reitorias de extensão das universidades brasileiras, tendo como base os grupos consolidados de pesquisa, nos quais poderiam ser incorporados outros profissionais de acordo com a necessidade. Dessa forma, a universidade pública recuperaria parte da sua função social, integrando ensino, pesquisa e extensão.

Profundidade de localização do herbicida imazetapir + imazapique no solo sobre a fitotoxicidade em de plantas de arroz não resistente / Depth of placement of the herbicide imazethapyr + imazapic in soil profile on non-tolerant rice injury

Os herbicidas imazetapir e imazapique, usados em cultivares de arroz Clearfield®, possuem alta persistência e mobilidade no solo, ocasionando danos em genótipos de arroz não resistentes cultivados em rotação. Tais herbicidas podem lixiviar e atingir maiores profundidades ao longo do perfil. Esse posicionamento em profundidade pode ser um fator de seletividade e explicar parcialmente os diferentes resultados encontrados na literatura sobre o efeito residual do herbicida no solo. O objetivo do estudo foi avaliar o efeito da profundidade de localização no solo e da mistura formulada pelos herbicidas imazetapir e imazapique (75 e 25g e.a. L-1) na fitotoxicidade em genótipos de arroz não resistentes. Nesse sentido, foram conduzidos dois experimentos em solo com 15 por cento de argila e 1,2 por cento de matéria orgânica, em casa-de-vegetação, no campus da Universidade Federal de Pelotas (UFPel), em Pelotas, Rio Grande do Sul (RS). O experimento I consistiu de estudo preliminar visando a determinar a profundidade máxima de localização do herbicida no solo que causa danos ao arroz não resistente, e o herbicida foi alocado nas profundidades de 0, 5, 10, 20, 30, 50 e 70cm. O experimento II também consistiu na alocação do herbicida em profundidades ao longo do perfil do solo de 3, 6, 9, 12, 15 e 18cm. As variáveis analisadas foram fitotoxicidade, massa da matéria seca e estatura das plantas. O herbicida resultante da mistura formulada de imazetapir com imazapique localizado próximo à superfície do solo causa danos intensos em plantas de arroz não resistente, porém, quando alocado em profundidades maiores que 20cm da superfície do solo, não prejudica o desenvolvimento de genótipos de arroz não resistentes a essa mistura de herbicidas.

The herbicides imazethapyr and imazapic, used in Clearfield® rice, have high mobility and persistence in the soil, causing injury to non-resistant rice grown in rotation. These herbicides can leach and reach greater depths along the profile. This positioning can be a in-depth selectivity factor and partially explain the different results found in literature about carryover of imidazolines. To understand this effect, this study had the objective of to evaluate the effect of the positioning of the mixture of imazethapyr and imazapic (75g ai L-1 and 25g ai L-1) on the injury to non-resistant rice crop. Two experiments were carried out in soil with 15 percent clay and 1.2 percent organic matter in a greenhouse at the Universidade Federal de Pelotas, Pelotas, RS, Brazil. Experiment I consisted of a preliminary study to verify the location depth of the herbicide in the soil profile that causes injury to rice non-tolerant rice, and the herbicides has been allocated at depths of 0, 5, 10, 20, 30, 50 e 70cm. Experiment II also consisted in the allocation of herbicide at depths in the soil profile of 3, 6, 9, 12, 15 and 18cm. The variables were visual plant injury, plant shoot dry weight and plant height. The formulated mixture of imazethapyr + imazapic located near the soil surface cause injury to non resistant rice plants but when allocated at depths greater than 20cm of the soil surface does not affect the development of non-resistant rice.