Phosphatidylinositol-3-kinase-Akt pathway in negative-stranded RNA virus infection: a minireview.

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.

Entry of Newcastle Disease Virus into the host cell: role of acidic pH and endocytosis.

Most paramyxoviruses enter the cell by direct fusion of the viral envelope with the plasma membrane. Our previous studies have shown the colocalization of Newcastle Disease Virus (NDV) with the early endosome marker EEA1 and the inhibition of NDV fusion by the caveolin-phosphorylating drug phorbol 12-myristate 13-acetate (PMA) prompted us to propose that NDV enters the cells via endocytosis. Here we show that the virus-cell fusion and cell-cell fusion promoted by NDV-F are increased by about 30% after brief exposure to low pH in HeLa and ELL-0 cells but not in NDV receptor- deficient cell lines such as GM95 or Lec1. After a brief low-pH exposure, the percentage of NDV fusion at 29 °C was similar to that at 37 °C without acid-pH stimulation, meaning that acid pH would decrease the energetic barrier to enhance fusion. Furthermore, preincubation of cells with the protein kinase C inhibitor bisindolylmaleimide led to the inhibition of about 30% of NDV infectivity, suggesting that a population of virus enters cells through receptor-mediated endocytosis. Moreover, the involvement of the GTPase dynamin in NDV entry is shown as its specific inhibitor, dynasore, also impaired NDV fusion and infectivity. Optimal infection of the host cells was significantly affected by drugs that inhibit endosomal acidification such as concanamycin A, monensin and chloroquine. These results support our hypothesis that entry of NDV into ELL-0 and HeLa cells occurs through the plasma membrane as well as by dynamin- low pH- and receptor- dependent endocytosis.

[A randomised clinical trial to evaluate the effectiveness of an educational intervention developed for adult asthmatics in a primary care centre]. / Ensayo clínico aleatorio para evaluar la eficacia de una intervención educativa desarrollada en atención primaria sobre asmáticos adultos.

OBJECTIVE: To assess the effect of an educational intervention on asthma control and quality of life. DESIGN: A randomised clinical trial of patients with asthma, with an intervention group (IG) and a control (GC). Asthma control and quality of life was measured in both groups at baseline and every three months for one a year. Blinding was only possible in the collection and analysis of data. LOCATION: Two urban Primary Care Health Centres PARTICIPANTS: A total of 163 patients aged 18 to 55 years were included: 84 were assigned to the IG and 79 to the CG. The follow-up was completed by 104 patients (GI: 55 and GC 49). INTERVENTION GI: Three educational sessions in small interactive groups. The first session was at the beginning of spring, the second 15 days later, and the third 6 months later, to recall the knowledge. MAIN MEASUREMENTS: Asthma control level and quality of life using ACT(Asthma Control Test) and the AQLQ (Asthma Quality of life Questionnaire). RESULTS: In the third month, statistically significant differences were detected in the percentage of patients with good control [(P=.0002), 75% in the GI, and 48.5% in the GC, Relative Risk (RR)=1.6 [1.2 to 2.1], Number Needed to Treat (NNT)=3.8 [2.4 to 9.4], and an improvement in levels of quality of life from baseline (P=.005), RR=2.3 [1.3 to 4.1], NNT: 4.3 [2.6 to 12.4]. No differences were detected in the remaining sessions. CONCLUSIONS: These interventions are effective in improving the control and quality of life in short-term, which can guide us in choosing the best time to do it.

A Synergistic Approach Therapy for Colorectal Cancer Based on Exosomes and Exploitation of Metabolic Pathways.

In order to reduce the side effects of traditional chemotherapy in the treatment of colorectal cancer (CRC), a new drug delivery system has been developed in this work, based on exosomes that can host two drugs that act synergistically: farnesol (that stops the cell cycle) and paclitaxel (prevents microtubule system depolymerization). Firstly, exosomes were isolated from different cell cultures (from colorectal cancer and from fibroblast as example of normal cell line) by different methods and characterized by western blot, TEM and DLS, and results showed that they express classical protein markers such as CD9 and HSP-70 and they showed spherical morphology with sizes from 93 nm to 129 nm depending on the source. These exosomes were loaded with both drugs and its effect was studied in vitro. The efficacy was studied by comparing the viability of cell cultures with a colorectal cancer cell line (HCT-116) and a normal cell line (fibroblast HS-5). Results showed that exosomes present a specific effect with more reduction in cell viability in tumour cultures than healthy ones. In summary, exosomes are presented in this work as a promising strategy for colorectal cancer treatment.

Cholesterol dependence of Newcastle Disease Virus entry.

Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-ß-cyclodextrin (MßCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MßCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.

Measuring loneliness: Psychometric properties of the three-item loneliness scale among community-dwelling adults.

Loneliness is a prevalent set of negative feelings associated with unsatisfactory and reduced social interactions, inadequate social support, poor satisfaction with life and health, negative emotions, and economic burden. Thus, its measurement is of foremost importance. Therefore, this study aimed (i) to devise the Portuguese version of the three-Item Loneliness Scale (T-ILS), which is ideal for epidemiological studies, and (ii) to evaluate its psychometric properties. Three hundred forty-five community-dwelling Portuguese adults with a mean age of 54.6 ± 19.5 years, 61.7% women, recruited door-to-door, were assessed with the Portuguese versions of T-ILS, Satisfaction With Life Scale-SWLS, Lubben Social Network Scale 6-items-LSNS-6, a question regarding Happiness/Unhappiness, and a sociodemographic questionnaire. The T-ILS showed good psychometric properties and correlated moderately with SWLS and LSNS-6, and happiness, and weakly with the number of people in the household. The Portuguese version of the T-ILS proved to be a valid and reliable instrument, easy and quick to administer. It proved to be a valuable tool in screening loneliness in Portugal, being potentially useful to the identification of lonelier people in need of intervention.

α2-3- and α2-6- N-linked sialic acids allow efficient interaction of Newcastle Disease Virus with target cells.

Receptor recognition and binding is the first step in the viral cycle. It has been established that Newcastle Disease Virus (NDV) interacts with sialylated molecules such as gangliosides and glycoproteins at the cell surface. Nevertheless, the specific receptor(s) that mediate virus entry are not well known. We have analysed the role of the sialic acid linkage in the early steps of the viral infection cycle. Pretreatment of ELL-0 cells with both α2,3 and α2,6 specific sialidases led to the inhibition of NDV binding, fusion and infectivity, which were restored after α2,3(N)- and α2,6(N)-sialyltransferase incubation. Moreover, α2,6(N)-sialyltransferases also restored NDV activities in α2-6-linked sialic acid deficient cells. Competition with α2-6 sialic acid-binding lectins led to a reduction in the three NDV activities (binding, fusion and infectivity) suggesting a role for α2-6- linked sialic acid in NDV entry. We conclude that both α2-3- and α2-6- linked sialic acid containing glycoconjugates may be used for NDV infection. NDV was able to efficiently bind, fuse and infect the ganglioside-deficient cell line GM95 to a similar extent to that of its parental MEB4, suggesting that gangliosides are not essential for NDV binding, fusion and infectivity. Nevertheless, the fact that the interaction of NDV with cells deficient in N-glycoprotein expression such as Lec1 was less efficient prompted us to conclude that NDV requires N-linked glycoproteins for efficient attachment and entry into the host cell.

Mutations in the ectodomain of newcastle disease virus fusion protein confer a hemagglutinin-neuraminidase-independent phenotype.

The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable postfusion form is critical for the promotion of fusion, although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle disease virus, located at critical regions of the protein, such as the C terminus of the N-terminal heptad repeat (HRA) and the N terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild type. The N211A, L461A, I463A, and I463F mutants showed a hyperfusogenic phenotype both in syncytium and in dye transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type did, meaning they probably had lower energy requirements for activation. Moreover, the N211A, I463A, and I463F mutants exhibited hemagglutinin-neuraminidase (HN)-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells.

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

Intestinal colonization due to Escherichia coli ST131: risk factors and prevalence.

Background: Escherichia coli sequence type 131 (ST131) is a successful clonal group that has dramatically spread during the last decades and is considered an important driver for the rapid increase of quinolone resistance in E. coli. Methods: Risk factors for rectal colonization by ST131 Escherichia coli (irrespective of ESBL production) were investigated in 64 household members (18 were colonized) and 54 hospital contacts (HC; 10 colonized) of 34 and 30 index patients with community and nosocomial infection due to these organisms, respectively, using multilevel analysis with a p limit of < 0.1. Result: Colonization among household members was associated with the use of proton-pump inhibitors (PPI) by the household member (OR = 3.08; 95% CI: 0.88-10.8) and higher age of index patients (OR = 1.05; 95% CI; 1.01-1.10), and among HC, with being bed-ridden (OR = 21.1; 95% CI: 3.61-160.0) and having a urinary catheter (OR = 8.4; 95% CI: 0.87-76.9). Conclusion: Use of PPI and variables associated with higher need of person-to-person contact are associated with increased risk of rectal colonization by ST131. These results should be considered for infection control purposes.

The functioning of the Cuban home hospitalization programme: a descriptive analysis.

BACKGROUND: Over the last decades hospital at home (HaH) programmes have been set up in many, mainly European, countries. The Cuban HaH programme is not hospital driven, but the responsibility of the first line health services, and family doctors play a pivotal role. METHODS: We analyse the structure and functioning of the Cuban programme. In this descriptive study, information was prospectively collected on HaH patients admitted between July 1st 2001 and June 30th 2002. RESULTS: Admission rates varied between areas from 0.014 to 0.035 per person per year (ppy). The < 1 y and 1-4 y age groups had the highest admission rates. In one area the follow-up of pregnancy problems led to high 15-24 y and 25-49 y female admission rates (0,070 and 0,058 respectively). Respiratory affections were the most frequent reason for admission (32,6%), followed by early hospital discharge (16,0%) and gynaeco-obstetrical problems (10.8%). The median length of stay varied from 5 to 7 days between regions and from 5 days (early discharge) to 7 days (gynaeco-obstetrical problems) in function of the reason for admission. On average an HaH episode entailed 1.4 and 1.6 contacts per patient-day with the family doctor and nurse respectively. CONCLUSION: Difference in admission criteria in function of geography, distance to the hospital, transport facilities, and staff factors, as well as differences in hospital policy on early discharge explain the observed variability. The programme plays an important role in the integrated approach to quality care in the Cuban health system, but could benefit from more uniform admission criteria.

Comportamentos aditivos com e sem substância em adolescentes: relação com a idade e o sexo / Additive behaviors with and without substance in adolescents: relation with age and sex

Resumo Contexto: O fenómeno da adição, com e sem substância, constitui um problema na adolescência pelo impacto negativo em diferentes domínios, nomeadamente na saúde mental. Assim, é fundamental que as equipas de saúde escolar conheçam a prevalência e características dos consumos aditivos dos adolescentes. Objetivo(s): Caracterizar o consumo aditivo com substância (tabaco, álcool e drogas) e sem substância (internet, jogos e redes sociais) em adolescentes, e relacionar os consumos entre si e com as variáveis idade e sexo. Métodos: Estudo transversal, descritivo-correlacional, com a aplicação de um questionário de autopreenchimento em contexto escolar, com uma amostra não probabilística constituída por 571 adolescentes. Resultados: O tabaco, o álcool, a internet e as redes sociais foram as adições mais prevalentes nos adolescentes. Foi encontrada associação entre o consumo de álcool e drogas ilícitas e as variáveis idade e sexo; e entre o consumo de álcool e o grupo etário. Existe correlação positiva e significativa entre o consumo de tabaco e o consumo de drogas; e correlação entre o consumo de álcool com os consumos de tabaco e drogas. Conclusões: Os adolescentes com consumo aditivo a uma substância apresentavam também outras adições, com ou sem substância. O presente estudo realça a necessidade de reforçar os programas de educação para a saúde na comunidade educativa com enfoque na prevenção de comportamentos aditivos com e sem substância nos adolescentes.

Abstract Background: The addiction behaviour is problematic due to its negative impact on adolescents' psychosocial well-being, including their mental health. Thus, it is essential that school health teams know the prevalence and the characteristics of adolescent addictive behaviours. Aim: To characterize addictive behaviour with substance (tobacco, alcohol, and drugs) and without substance (internet, games, and social media) in adolescents and to relate the additive behaviours with and without substance and with the variables age and sex. Methods: Cross-sectional, descriptive-correlational study with the application of a self-administered questionnaire, in a school context, to a non-probabilistic sample of 571 adolescents. Results: Tobacco, alcohol, the internet, and social media are the most prevalent adolescents' additions. Alcohol and illicit drugs consumption are associated with the variables age and sex, and alcohol consumption is associated with age. There is a positive and significant correlation between tobacco use and drug; and correlation between alcohol consumption and tobacco and drug consumption. Conclusions: Adolescents with addictive consumption on one substance have also other additions, with or without substance. The present study highlights the need to reinforce health education programs in the educational community with a focus on preventing addictive behaviors with and without substance in adolescents.

Bem-estar Psicológico e Utilização Problemática da Internet em Adolescentes / Psychological Well-Being and Problematic Use of the Internet in Adolescents

Resumo Contexto: A internet mudou os comportamentos dos adolescentes e trouxe novas formas de socialização e comunicação. Para além das suas vantagens, a utilização excessiva pode ter implicações no domínio pessoal, familiar, social e académico, com possíveis consequências nefastas para a saúde e bem-estar psicológico dos mais jovens. Objetivo(s): Caracterizar o bem-estar psicológico e a utilização da internet; relacionar o uso problemático da Internet com o bem-estar psicológico dos adolescentes; e relacionar o bem-estar psicológico dos adolescentes com as variáveis sociodemográficas, escolares e utilização da internet. Métodos: Estudo descritivo-correlacional e transversal com uma amostra de 478 adolescentes, com média de M=14,5 anos (DP=1,9), em contexto escolar. Aplicou-se um questionário que incluía a caracterização sociodemográfica e questões sobre a utilização da internet, o Questionário de Bem-estar Psicológico, versão reduzida (QBEP-R) e a Escala do Uso Generalizado e Problemático da Internet (GPIUS2). O tratamento estatístico foi efetuado com recurso à análise descritiva e inferencial. Resultados: Foram observados níveis satisfatórios e elevados de bem-estar psicológico nos adolescentes e utilização problemática da Internet em 13,4% da amostra. Verificou-se uma correlação negativa entre a utilização problemática da internet e o bem-estar psicológico. Constataram-se diferenças estatisticamente significativas no bem-estar psicológico em relação às variáveis: idade, sexo, nível de ensino, presença de hobby, utilização das redes sociais e jogos online. Conclusões: Os adolescentes com utilização problemática da internet apresentavam menor bem-estar psicológico, o que justifica a intervenção dos profissionais de saúde, na implementação de estratégias que promovam comportamentos saudáveis e consequentemente o bem-estar psicológico.

Abstract Background: The internet has changed the behavior of teenagers and brought new forms of socialization and communication. However, beyond its advantages there are also some risks. Excessive use can have consequences in the personal, family, social and academic domain, with possible harmful effects on the health and psychological well-being of youngsters. Aim: To characterize the psychological well-being and the use of the internet and to relate the problematic use of the Internet to the psychological well-being of adolescents. Methods: Descriptive-correlational, cross-sectional, and quantitative study, with a sample about 478 adolescents, with an average age of M=14,5 years old (SD =1,9), in a school context. A questionnaire was applied that included the sociodemographic characterization and use of the internet, the Psychological Well-being Questionnaire, short version (QBEP-R) and the Scale of Generalized and Problematic Use of the Internet (GPIUS2). The statistical treatment was performed using descriptive and inferential analysis. Results: Satisfactory and high levels of psychological well-being were observed in adolescents, and problematic use of the Internet was held by 13,4% of the sample. There was a negative correlation between problematic use of the Internet and psychological well-being. There were statistically significant differences in psychological well-being in relation to the variables: age, sex, level of education, presence of hobby, use of social networks and online games. Conclusions: Adolescents with problematic use of the internet have lower psychological well-being, which justifies the intervention of health professionals, namely nurses, in the implementation of strategies that promote healthy online behaviours and, consequently, psychological well-being.

Bacteraemia due to non-ESBL-producing Escherichia coli O25b:H4 sequence type 131: insights into risk factors, clinical features and outcomes.

The epidemiology and outcomes of bloodstream infections (BSIs) caused by Escherichia coli ST131 isolates not producing extended-spectrum ß-lactamases (ESBLs) are not well defined despite being more prevalent than ESBL-producers. In this study, risk factors and the impact on outcome of BSIs caused by non-ESBL-producing ST131 E. coli versus non-ST131 E. coli were investigated. A case-control study was performed in two tertiary centres to identify risk factors for ST131. Molecular methods were used to investigate all E. coli isolates from blood cultures for those belonging to O25b:H4-ST131 clonal group. fimH alleles were characterised in ST131 isolates. Multivariate analysis was performed by logistic regression or Cox regression as appropriate. A total of 33 ST131 E. coli cases and 56 controls were studied. ST131 isolates showed higher rates of resistance to ampicillin and ciprofloxacin; fimH alleles were H30 in 14 isolates (42.4%) and H22 in 12 isolates (36.4%). Only recent surgery (OR = 7.03, 95% CI 1.71-28.84; P = 0.007) and unknown source of bacteraemia (OR = 5.37, 95% CI 0.93-30.81; P = 0.05) were associated with ST131. ST131 isolates showed no association with 30-day mortality, therapeutic failure, presentation with severe sepsis/shock or length of stay. Bacteraemia due to non-ESBL-producing O25b:H4-ST131 E. coli showed few differences in terms of risk factors as well as similar outcome to non-ST131 E. coli. These data support the notion that ST131 strains are not less clinically virulent despite showing increased antimicrobial resistance, but also that they are not more virulent than other clonal groups causing BSI.

A utilização da acupuntura no controlo da dor crónica em usuários com síndrome vertebral com irradiação / The use of acupuncture to control chronic pain in users with irradiated vertebral syndrome

Objetivos: a acupuntura é, cada vez mais, uma técnica utilizada, em usuários portadores de várias patologias, e, em especial, aquelas que são acompanhadas de dor crónica. Esta pesquisa teve como objetivo geral analisar o efeito da terapêutica não farmacológica de acupuntura e eletroacupuntura para o controle da dor crónica, em usuários com diagnóstico médico de síndrome vertebral com irradiação. Métodos: foi realizada uma pesquisa com dois momentos de avaliação ­ antes e após sessões de terapêutica não farmacológica de acupuntura e eletroacupuntura. Utilizou-se um questionário com variáveis de caracterização sociodemográfica e clínica, o Brief Pain Inventory (BPI)e Short Form 6 Dimensions (SF-6D). Resultados: participaram no estudo 43 usuários, tendo sido encontradas diferenças estaticamente significativas, do momento 1 para o momento 2 de avaliação, quer no domínio da severidade da dor (p<0,01), quer no domínio interferência da dor no funcionamento diário (p<0,01), assim como nas diferentes dimensões do SF-6D foram encontradas diferenças estatisticamente significativas. Conclusões: salienta-se que a terapêutica não farmacológica de acupuntura e eletroacupuntura em usuários com diagnóstico de síndrome vertebral com irradiação e dor crónica contribuiu para a diminuição da severidade da dor, da interferência da dor no funcionamento diário e melhoria da qualidade de vida relacionada com a saúde.

Aims: acupuncture is increasingly a technique used in users with various pathologies, and especially those who are accompanied by chronic pain. This research aimed to analyze the effect of non-pharmacological acupuncture and electroacupuncture therapy for the control of chronic pain in users with medical diagnosis of irradiation vertebral syndrome. Methods: a research was conducted with two evaluation moments: before and after non-pharmacological acupuncture and electroacupuncture therapy sessions. A questionnaire with variables of sociodemographic and clinical characterization, the Brief Pain Inventory and Short Form 6 Dimensions (SF-6D) was used. Results: forty-three users participated in the study, and statistically significant differences were found from moment 1 to moment 2 of evaluation, either in the pain severity domain (p<0.01) or in the pain interference domain in daily functioning (p <0.01), as well as in the different dimensions of SF-6D, statistically significant differences were found. Conclusions: non-pharmacological acupuncture and electroacupuncture therapy in users diagnosed with irradiation and chronic pain have contributed to decrease pain severity, pain interference in daily functioning and improvement of health-related quality of life.

RhoA activation promotes transformation and loss of thyroid cell differentiation interfering with thyroid transcription factor-1 activity.

Highly specialized cells, the thyrocytes, express a thyroid-specific set of genes for thyroglobulin (Tg), thyroperoxidase, and the transcription factors TTF-1, TTF-2, and Pax-8. The implication of the small GTPase RhoA in TSH-mediated proliferation of FRTL-5 rat thyroid cells has been previously demonstrated. To further analyze RhoA function in thyroid cell proliferation and differentiation patterns, we combined transient and stable transfection assays to express different mutant RhoA forms in FRTL-5 cells. Constitutively active RhoA (FRTL-5-RhoA QL cells) exhibited a fibroblast-like phenotype with organized actin fibers, whereas cells expressing the RhoA negative dominant phenotype (FRTL-5-RhoA N19 cells) present a rounded morphology and lose normal cytoskeletal architecture. In addition, expression of the constitutively active form of RhoA results in TSH-independent proliferation and anchorage-independent growth and induces tumors when inoculated in nude mice. Interestingly, FRTL-5-RhoA QL cells express less Tg and TTF-1 than wild-type FRTL-5 (FRTL-5- vector) or FRTL-5-RhoA N19, suggesting a loss at the differentiation stage. This effect is mediated, at least in part, by a decrease in TTF-1 activity, since transient or stable expression of RhoA QL results in a reduction in the activity of the wild-type Tg promoter as well as an artificial promoter the activation of which depends exclusively on TTF-1. The similarity between RhoA effects and thyroid transformation by Ras suggests that RhoA may act as a downstream effector of Ras; in fact, the dominant negative RhoA N19 abolished the down- regulatory effect of Ras V12 over the Tg promoter. Taken together, these results show for the first time that active RhoA is able to transform FRTL-5 cells and that this effect is coupled to a loss of thyroid differentiation due to impaired TTF-1 activity.

Gangliosides and N-glycoproteins function as Newcastle disease virus receptors.

The interaction of enveloped viruses with cell surface receptors is the first step in the viral cycle and an important determinant of viral host range. Although it is established that the paramyxovirus Newcastle Disease Virus binds to sialic acid-containing glycoconjugates the exact nature of the receptors has not yet been determined. Accordingly, here we attempted to characterize the cellular receptors for Newcastle disease virus. Treatment of cells with tunicamycin, an inhibitor of protein N-glycosylation, blocked fusion and infectivity, while the inhibitor of O-glycosylation benzyl-N-acetyl-alpha-D-galactosamide had no effect. Additionally, the inhibitor of glycolipid biosynthesis 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol blocked viral fusion and infectivity. These results suggest that N-linked glycoproteins and glycolipids would be involved in viral entry but not O-linked glycoproteins. The ganglioside content of COS-7 cells was analyzed showing that GD1a was the major ganglioside component; the presence of GM1, GM2 and GM3 was also established. In a thin-layer chromatographic binding assay, we analyzed the binding of the virus to different gangliosides, detecting the interaction with monosialogangliosides such as GM3, GM2 and GM1; disialogangliosides such as GD1a and GD1b, and trisialogangliosides such as GT1b. Unlike with other viruses, our results seem to point to the absence of a specific pattern of gangliosides that interact with Newcastle disease virus. In conclusion, our results suggest that Newcastle disease virus requires different sialic acid-containing compounds, gangliosides and glycoproteins for entry into the target cell. We propose that gangliosides would act as primary receptors while N-linked glycoproteins would function as the second receptor critical for viral entry.

[Methods for handling incomplete data in health research: a critical look]. / Datos incompletos: una mirada crítica para su manejo en estudios sanitarios.

OBJECTIVE: To illustrate methods for handling incomplete data in health research. METHODS: Two strategies for handling missing data are presented: complete-case analysis and imputations. The imputations used were mean imputations, regression imputations, and multiple imputations. These strategies are illustrated in the context of logistic regression through an example using data from the "Second Cuban national survey on risk factors and non communicable disease", carried out in 2001. RESULTS: The results obtained via mean and regression imputation were similar. The odds ratios were overestimated by 10%. The results of complete-case analysis showed the greatest difference from the reference odds ratios, with a variation of between 2 and 65%. The three methods distorted the relationship between age and hypertension. Multiple imputations produced estimates closest to those of the reference estimates with a variation of less than 16%. This was the only procedure preserving the relationship between age and hypertension. CONCLUSIONS: Selecting methods for handling missing data is difficult, since the same procedure can give precise estimations in certain circumstances and not in others. Complete-case analysis should be used with caution due to the substantial loss of information it produces. Mean and regression imputations produce unreliable estimates under missing at random (MAR) mechanisms.

Identification of cellular proteins that interact with Newcastle Disease Virus and human Respiratory Syncytial Virus by a two-dimensional virus overlay protein binding assay (VOPBA).

Although it is well documented that the initial attachment receptors for Newcastle Disease Virus (NDV) and Respiratory Syncytial Virus (RSV) are sialic acid-containing molecules and glycosaminoglycans respectively, the exact nature of the receptors for both viruses remains to be deciphered. Moreover, additional molecules at the host cell surface might be involved in the entry mechanism. With the aim of identifying the cellular proteins that interact with NDV and RSV at the cell surface, we performed a virus overlay protein binding assay (VOPBA). Cell membrane lysates were separated by two dimensional (2D) gel electrophoresis and electrotransferred to PVDF membranes, after which they were probed with high viral concentrations. NDV interacted with a Protein Disulfide Isomerase from chicken fibroblasts. In the case of RSV, we detected 15 reactive spots, which were identified as six different proteins, of which nucleolin was outstanding. We discuss the possible role of PDI and nucleolin in NDV and RSV entry, respectively.

Thermal stability of matrix protein from Newcastle disease virus.

The thermal stability of the matrix protein (M protein) of Newcastle disease virus (NDV) has been investigated using high-sensitivity differential scanning calorimetry (DSC) at pH 7.4. The thermal folding/unfolding of M protein at this pH value is a reversible process involving a highly cooperative transition between folded and unfolded monomers with a transition temperature (Tm) of 63 °C, an unfolding enthalpy, ΔH(Tm), of 340 kcal mol(-1), and the difference in heat capacity between the native and denatured states of the protein, ΔCp, of 5.1 kcal K(-1) mol(-1). The heat capacity of the native state of the protein is in good agreement with the values calculated using a structure-based parameterization, whereas the calculated values for the hypothetical fully-unfolded state of the protein is higher than those determined experimentally. This difference between the heat capacity of denatured M protein and the heat capacity expected for an unstructured polypeptide of the same sequence, together with the data derived from the heat-induced changes in the steady-state fluorescence of the protein, indicates that the polypeptide chain maintains a significant amount of residual structure after thermal denaturation.