The use of insect cells to identify potent and selective inhibitors of the replication of the Dengue and Chikungunya viruses and unravel their molecular mechanism of action

Dengue and Chikungunya viruses cause the most important arthropod-borne viral infections for humans. These viruses are predominant in tropical and subtropical regions. In addition, these viruses are predominant in tropical and subtropical regions. Dengue mortality rate is around 1.2 to 3.5% and deaths due to chikungunya fever are around 1 in 1000; however, half of chikungunya-infected patients evolve into a chronic state that can persist for months up to years. There are no antiviral drugs available for DENV and CHIKV treatment and prevention. Moreover, vector control strategies have failed so far. Thus, the development of potent inhibitors for a broad spectrum of RNA viruses is urgently needed. We established and characterized a new embryonic insect cell line from Culex quinquefasciatus mosquito. Also we established the flaviviruses and alphavirus replication, both in C6/36 and Lulo insect cell lines, as well as in Vero cell line. In addition we carried out a reference compound library and reference panel of assays and data for DENV, which provides a benchmark for further studies. During this study, a panel of 9 antiviral molecules, with proven in vitro anti-dengue virus activity and that act at different stages of the DENV life cycle, was selected. Finally, Favipiravir or T-705, was identified as inhibitor in vitro and in vivo of alphaviruses and the mutation K291R in nsP4, which is responsible of the polymerase activity, was found as the mode of action in CHIKV. Interestingly, lysine in motif F1 is also highly conserved in positive-stranded RNA viruses and this might explain the broad spectrum of T-705 antiviral activity.

Estudi interlingüístic de les construccions que expressen estats patològics en català i en mandinga

La tesi de Crous (2009) descriu les característiques de les estructures gramaticals del català i del mandinga que expressen malalties, signes i símptomes, trets físics i psíquics, etc. (com ser diabètic, estar refredat, tenir febre, tenir un bony (a la cama), tenir mal de panxa o tenir mal a la panxa, venir un atac de tos, agafar febre o fer mal el cap). La tesi demostra que la conceptualització i la categorització dels estats patològics, la manera com els parlants perceben alguns aspectes dels estats (com la temporalitat, l'abast corporal, la causalitat o el grau d'afectació) i les relacions entre aquests estats i els altres dos participants de la situació d'afectació (una persona i les seves parts del cos), no només es posa de manifest en els mots d'una llengua sinó que també intervé en la construcció i en la distribució dels diferents participants dins d'una oració.

Discoespondilite Canina : Estudo Retrospectivo de 10 Casos Clínicos

A discoespondilite é uma doença infecciosa rara que afecta, de forma crónica, os discos intervertebrais e as extremidades adjacentes dos corpos vertebrais. Geralmente advém de uma infecção disseminada por via hematógena e os agentes mais frequentes são bacterianos e são principalmente Staphylococcus spp., Streptococcus spp., E. Coli e Brucella spp. Também pode ser devida a infecções fúngicas, parasitárias ou migração de corpos estranhos. É caracterizada pela degenerescência do disco intervertebral e lesões escleróticas e proliferativas das extremidades dos corpos vertebrais. O principal sinal clínico desta doença é a hiperestesia paravertebral e alterações da marcha ou relutância ao movimento. Febre e anorexia são menos frequentes do que seria de esperar e os sinais neurológicos são considerados raros. O diagnóstico desta doença é geralmente radiográfico e a determinação do agente pode ser conseguida por cultura de material discal, hemocultura ou urocultura. Podem ser usados meios de imagiologia avançada como TAC e RM para melhor avaliar a extensão das lesões e o envolvimento dos tecidos circunvizinhos. A realização de hemogramas raramente revela alterações significativas embora possa existir leucocitose. O tratamento médico é eficaz em aproximadamente 76% dos casos e deve ser feito com base em cultura e TSA mas, de forma empírica, as cefalosporinas de primeira geração são frequentemente utilizadas. Em alguns casos pode ser necessária a estabilização ou desbridamento cirúrgicos. O estudo retrospectivo realizado no âmbito deste trabalho, teve como objectivo avaliar os sinais clínicos, radiográficos e laboratoriais , assim como o maneio médico e cirúrgico de 10 casos de discoespondilite confirmada radiográfica e clinicamente, num período de 2 anos. Observou-se maior prevalência da doença em machos, em cães jovens e adultos, e raças de grande porte. A região mais afectada foi a junção lombossagrada, e o sinal mais observado foi a dor paraespinhal. No entanto os sinais neurológicos foram mais frequentes do que o descrito. Os agentes isolados em cultura de material discal não foram os mais comuns. O tratamento médico instituído pelos veterinários foi eficaz em 6 dos casos, Foi necessária intervenção cirúrgica em 3 e 1 animal não recuperou totalmente até à conclusão deste estudo.

Contribuição para o estudo das transfusões sanguíneas no cão e no gato : situação em Portugal e estudo de 61 transfusões

Os avanços na área da Medicina transfucional, nomeadamente a descoberta de nova informação sobre os grupos sanguíneos de várias espécies, a introdução rotineira da tipificação sanguínea e das provas de compatibilidade eritrocitária, o estudo das reacções transfusionais adversas, o despiste de doenças infeciosas no dador e a aplicação da terapia por componentes, têm contribuído para aumentar a segurança da transfusão sanguínea em Medicina Veterinária e, por consequência, a sua utilização é cada vez mais frequente. O presente trabalho é constituído por três objectivos: perspectivar a medicina transfusional em Portugal através da análise dos resultados de um inquérito, dirigido aos CAMV, nomeadamente sobre o uso da terapia por componentes, as principais indicações de transfusão e a ocorrência de reacções transfusionais; caracterizar a população de gatos e cães receptores de transfusões sanguíneas, com enfoque na prevalência dos diferentes grupos sanguíneos, na indicação para a realização da transfusão e tipo de produto administrado; determinar a ocorrência de reacções transfusionais através da monotorização do doente antes, durante e após a administração das transfusões. No presente estudo, 86% dos Centros de Atendimento Médico Veterinário (CAMV) inquiridos recorrem à transfusão sanguínea como terapia complementar. Destes, 54.7% utiliza sangue total e produtos do sangue, 41.3% apenas sangue total e 4% apenas produtos do sangue. Os produtos do sangue mais utilizados são o concentrado de glóbulos vermelhos e o plasma fresco congelado (34.2% e 31.6% respectivamente). A anemia constitui o principal motivo para a realização de transfusões sanguíneas e a hipertermia a reacção transfusional mais frequente. Relativamente à caracterização da população de cães e gatos que receberam transfusão sanguínea conclui-se que 77.8% dos cães pertenciam ao grupo DEA 1.1 negativo e 22.2% ao grupo DEA 1.1 positivo. Todos os gatos incluídos neste estudo pertenciam ao grupo sanguíneo A. A anemia por hemorragia foi a indicação predominante para a administração de sangue nos cães (54.3%). Nos gatos a anemia por não produção de eritrócitos prevalece (60%). No que respeita às reacções transfusionais, das 61 transfusões realizadas apenas se registou uma dispneia num gato.

Estudo restrospectivo de pancreatite canina em 41 casos

A pancreatite, apesar de relativamente comum na medicina canina, continua a constituir um desafio devido à sua elevada complexidade patogénica, sinais clínicos não patognomónicos, diagnóstico por vezes difícil e tratamento inespecífico. De forma a realizar um estudo retrospectivo foram avaliados registos médicos de 41 canídeos diagnosticados com pancreatite, apresentados à consulta entre 2007 e 2013, de forma a caracterizar o doente pancreático. As características do animal, história clinica, sinais clínicos apresentados, métodos de diagnóstico e tratamento instituído foram analisados. Como critérios de inclusão neste estudo encontram-se a medição de cPLI com resultados acima do valor de referência e a realização de ecografia abdominal. A ecografia demonstrou evidências de pancreatite em 81% dos casos, sendo este valor superior aos 68% encontrados na literatura. Os sinais clínicos apresentados mais comuns foram vómito (39), prostração (38), dor abdominal (36), perda de peso e anorexia (26), desidratação (25), diarreia (21) e febre (5). Todos os animais foram submetidos a tratamento médico, tendo sido apenas 4 deles sujeitos também a cirurgia. A taxa de sobrevivência nos animais com tratamento médico foi de 95% e de 50 % nos animais com tratamento médico e cirúrgico.

Common Problems in Surgical Patients

Presentation covering common acute conditions in surgical patients such as shortness of breath, chest pain and fever. Presentation offers diagnostic suggestions and procedures.

Predation in Ground-Nesting Birds: an Experimental Study Using Natural Egg-Color Variation

We tested the hypothesis that cryptically colored eggs would suffer less predation than conspicuous eggs in the ground-nesting red-legged partridge, Alectoris rufa. We used A. rufa as a model species because it has a wide range of natural egg colors, the eggs are widely available from breeding farms, and nests are easily mimicked because they are scrapes containing no vegetation. The study was conducted in the spring of 2001 in forest and fallow fields of central Spain in Castilla La Mancha, Ciudad Real. We used 384 clutches of natural eggs that were white, white spotted, brown, or brown spotted. Within clutches, eggs were consistent in color and size; among clutches, color differences were distributed across habitats. Clutches were checked once after 2 wk of exposure. Cryptic coloration had a survival advantage that was dependent on the local suite of predators. Rodent predation was nonselective with respect to clutch color; however, avian predation was significantly higher for conspicuous clutches. In addition, there was an interaction of landscape and egg color for avian predation. In forest landscapes, the clutches with highest survival were brown spotted, whereas in fallow landscapes, brown and brown spotted clutches had higher survival than white and white potted clutches. Thus, both the predator suite and the landscape had significant effects on the value of cryptic egg coloration. Our study is relevant for conservationists and managers in charge of restocking programs in hunting areas. The release of other partridge species or their hybrids could result in hybridization with wild partridges, potentially leading to nonoptimal clutch pigmentation and reduced survival of the native species. We therefore recommend that local authorities, managers, and conservationists be cautious with the use of alien species and hybrids and release only autochthonous species of partridges within their natural ranges.

Evaluation of PCR to detect Theileria parva in field-collected tick and bovine samples in Tanzania

The ability of PCR to detect infections of Theileria parva, the cause of East Coast Fever, in field-collected tick and bovine samples from Tanzania was evaluated. PCR-detected infection prevalence was high (15/20, 75%) in unfed adult Rhipicephalus appendiculatus ticks that fed as nymphs on an acutely-infected calf, but low (22/836, 2.6%) in unfed adult R. appendiculatus collected from field sites in Tanzania. Tick infection prevalence was comparable to that in previous studies that used salivary gland staining to detect T parva infection in field-collected host-seeking ticks. Of 282 naturally-exposed zebu calves, seven had PCR-positive buffy coat samples prior to detection of Theileria spp. parasites in stained huffy coat cells or lymph node biopsies. Evidence of Theileria spp. infections was detected in stained smears of lymph node biopsies from 109 calves (38.6%) and huffy coat samples from 81 (28.7%), while huffy coat samples from 66 (23.4%) were PCR-positive for T parva. Implications of these findings for the sensitivity and specificity of the PCR are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy

Here, we analyze the complete coding sequences of all recognized tick-borne flavivirus species, including Gadgets Gully, Royal Farm and Karshi virus, seabird-associated flaviviruses, Kadam virus and previously uncharacterized isolates of Kyasanur Forest disease virus and Omsk hemorrhagic fever virus. Significant taxonomic improvements are proposed, e.g. the identification of three major groups (mammalian, seabird and Kadam tick-borne flavivirus groups), the creation of a new species (Karshi virus) and the assignment of Tick-borne encephalitis and Louping ill viruses to a unique species (Tick-borne encephalitis virus) including four viral types (i.e. Western Tick-borne encephalitis virus, Eastern Tick-borne encephalitis virus, Turkish sheep Tick-borne encephalitis virus and Louping ill Tick-borne encephalitis virus). The analyses also suggest a complex relationship between viruses infecting birds and those infecting mammals. Ticks that feed on both categories of vertebrates may constitute the evolutionary bridge between the three distinct identified lineages.

The tagged microarray marker (TAM) method for high-throughput detection of single nucleotide and indel polymorphisms

The tagged microarray marker (TAM) method allows high-throughput differentiation between predicted alternative PCR products. Typically, the method is used as a molecular marker approach to determining the allelic states of single nucleotide polymorphisms (SNPs) or insertion-deletion (indel) alleles at genomic loci in multiple individuals. Biotin-labeled PCR products are spotted, unpurified, onto a streptavidin-coated glass slide and the alternative products are differentiated by hybridization to fluorescent detector oligonucleotides that recognize corresponding allele-specific tags on the PCR primers. The main attractions of this method are its high throughput (thousands of PCRs are analyzed per slide), flexibility of scoring (any combination, from a single marker in thousands of samples to thousands of markers in a single sample, can be analyzed) and flexibility of scale (any experimental scale, from a small lab setting up to a large project). This protocol describes an experiment involving 3,072 PCRs scored on a slide. The whole process from the start of PCR setup to receiving the data spreadsheet takes 2 d.

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

Gene-chip technology and its applications

Gene Chips are finding extensive use in animal and plant science. Generally microarrays are of two kind, cDNA or oligonucleotide. cDNA microarrays were developed at Stanford University, whereas oligonucleotide were developed by Affymetrix. The construction of cDNA or oligonucleotide on a glass slide helps to compare the gene expression level of treated and control samples by labeling mRNA with green (Cy3) and red (Cy5) dyes. The hybridized gene chip emit fluorescence whose intensity and colour can be measured. RNA labeling can be done directly or indirectly. Indirect method involves amino allyle modified dUTP instead of pre-labelled nucleotide. Hybridization of gene chip generally occurs in a minimum volume possible and to ensure the hetroduplex formation, a ten fold more DNA is spotted on slide than in the solutions. A confocal or semi confocal laser technologies coupled with CCD camera are used for image acquisition. For standardization, house keeping genes are used or cDNA are spotted in gene chip that are not present in treated or control samples. Moreover, statistical analysis (image analysis) and cluster analysis softwares have been developed by Stanford University. The gene-chip technology has many applications like expression analysis, gene expression signatures (molecular phenotypes) and promoter regulatory element co-expression.

Development of peptide-conjugated morpholino oligomers as pan-arenavirus inhibitors

Members of the Arenaviridae are a threat to public health and can cause meningitis and hemorrhagic fever, yet treatment options remain limited by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis, translation, or both. However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary to sequence that is highly conserved across arenaviruses and located at the 5’-termini of both genomic segments were effective against Junín, Tacaribe, Pichinde and Lymphocytic Choriomeningitis arenavirus-infected cell cultures, and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5’-genomic-termini represent promising targets for pan-arenavirus antiviral therapeutic development.

Panzootics and the poor: devising a global livestock disease prioritisation framework for poverty alleviation

Panzootics such as highly pathogenic avian influenza and Rift Valley fever have originated from the South, largely among poor communities. On a global level, approximately two-thirds of those individuals living on less than US$2 per day keep livestock. Consequently, there is a need to better target animal health interventions for poverty reduction using an evidence-based approach. Therefore, the paper offers a three-step prioritisation framework using calculations derived from standard poverty measures: the poverty gap and the head count ratio. Data from 265 poor livestock-keeping households in Kenya informed the study. The results demonstrate that, across a spectrum of producers, the dependence upon particular species varies. Furthermore, the same livestock disease has differing impacts on the depth and severity of poverty. Consequently, animal health interventions need to

Ownership Advantages in Cross-border Real Estate Development: Some Evidence from European Markets

Drawing upon European industry and country case studies, this paper investigates the scope and drivers of cross-border real estate development. It is argued that the real estate development process encompasses a diverse range of activities and actors. It is inherently localised, the production process is complex and emphermal, and the outputs are heterogeneous. It analyses a transactions database of European real estate markets to provide insights into the extent of, and variations in, market penetration by non-domestic real estate developers. The data were consistent with the expectation that non-domestic real estate developers from mature markets would have a high level of market penetration in immature markets. Compared to western European markets, the CEE real estate office sales by developers were dominated by US, Israeli and other EU developers. This pattern is consistent with the argument that non-domestic developers have substantial Dunning-type ownership advantages when entering immature real estate markets. However, the data also suggested some unexpected patterns. Relative to their GDP, Austria, Belgium, Denmark, Sweden, Netherlands and Israel accounted for large proportions of sales by developers. All are EU countries (except Israel) with small, open, affluent, highly traded economies. Further, the data also indicate that there may be a threshold where locational disadvantages outweigh ownership advantages and deter cross-border real estate development.