[Development of clinically relevant vital signs of goat lambs of different breeds between birth and 28th day of life post natum]. / Entwicklung klinisch relevanter Vitalwerte von Ziegenlämmern verschiedener Rassen zwischen Geburt und 28. Lebenstag post natum.

Monitoring caprine neonates is of great importance to minimize losses of valuable offspring potential. In addition to experience and ethological knowledge, it requires clinically relevant basic values for vitality assessment. Therefore, the aim of the study was to register appropriate basic parameters in healthy caprine lambs. MATERIAL AND METHODS: Included in the study were 46 healthy, vital born lambs from 28 dams of the breeds White German Edel goat (WGE, n=15), Boer goat (n=7); Toggenburg goat (n=6). The three groups of subjects included 26 WGE, 9 Boer goat and 11 Toggenburg goat lambs. From birth, they were under intensive veterinary control. Vital signs respiratory rate, heart rate, and rectal temperature were measured immediately p.n.; 3rd, 12th, 24th h p.n.; 2nd, 3rd, 5th, 7th, 9th, 14th, 21st, and 28th d p.n. Furthermore, crown-rump length (CRL), birth weight, and weight development until 28th d p.n. Statistical analysis of the data was performed using BMDP/Dynamic Release 7.0 and two-factorial analysis of variance. RESULTS: Vital signs showed a typical course in the first 4 weeks p.n. with a significant decrease in values from the 5th d. p.n. Time as well as breed had significant influence on the development of the respective profiles (covariance analysis; p<0.001). There was a linear correlation for SSL and birth weight (y=0.0037 ×28969; r value 0.7805). Daily weight gain was independent of race and diet type. In contrast, there was a significant dependence between birth type (singleton or multiple birth) and weight gain with p<0.01 and between birth weight and sex (p<0.05). CONCLUSION: The presented typical, breed-dependent postnatal changes of vital parameters in goat lambs in the period 0-28 days p.n. allow a more specific differentiation between clinically healthy and abnormal individuals in herd management using the physiological value tables. The age-correlated weight gain serves as a further evaluation parameter.

Evidence for direct interaction between the oncogenic proteins E6 and E7 of high-risk human papillomavirus (HPV).

Human papillomaviruses (HPVs) are DNA tumor viruses that infect mucosal and cutaneous epithelial cells of more than 20 vertebrates. High-risk HPV causes about 5% of human cancers worldwide, and the viral proteins E6 and E7 promote carcinogenesis by interacting with tumor suppressors and interfering with many cellular pathways. As a consequence, they immortalize cells more efficiently in concert than individually. So far, the networks of E6 and E7 with their respective cellular targets have been studied extensively but independently. However, we hypothesized that E6 and E7 might also interact directly with each other in a novel interaction affecting HPV-related carcinogenesis. Here, we report a direct interaction between E6 and E7 proteins from carcinogenic HPV types 16 and 31. We demonstrated this interaction via cellular assays using two orthogonal methods: coimmunoprecipitation and flow cytometry-based FRET assays. Analytical ultracentrifugation of the recombinant proteins revealed that the stoichiometry of the E6/E7 complex involves two E7 molecules and two E6 molecules. In addition, fluorescence polarization showed that (I) E6 binds to E7 with a similar affinity for HPV16 and HPV31 (in the same micromolar range) and (II) that the binding interface involves the unstructured N-terminal region of E7. The direct interaction of these highly conserved papillomaviral oncoproteins may provide a new perspective for studying HPV-associated carcinogenesis and the overall viral life cycle.

Master mitotic kinases regulate viral genome delivery during papillomavirus cell entry.

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2's chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.

Calidad de vida en pacientes con enfermedades tiroideas

Introducción: Las enfermedades del tiroides son relativamente frecuentes, constituyen un importante grupo dentro de las enfermedades crónicas no transmisibles. Objetivo: Determinar la calidad de vida en pacientes con enfermedades tiroideas. Métodos: Se realizó un estudio observacional, descriptivo y transversal. El universo estuvo constituido por 210 pacientes con diagnóstico de enfermedades tiroideas, que cumplieron con los criterios de inclusión y exclusión previstos en el estudio, previo consentimiento informado y la muestra no probabilística de 198 pacientes. Se utilizaron las variables: edad, sexo, enfermedad tiroidea diagnosticada (hipertiroidismo, hipotiroidismo, bocio difuso eutiroideo) y calidad de vida (facetas según dimensiones, según la clasificación de expertos y enfermedades tiroideas y la calidad de vida integral según enfermedades tiroideas). Se utilizó el cuestionario World Health Organization Quality of Life. Resultados: Predominó el hipotiroidismo como afección tiroidea más frecuente en las féminas entre 50 a 59 años. Las manifestaciones clínicas que puntuaron las medias más bajas fueron el dolor y malestar, seguido de indicaciones médicas y sentimientos negativos; las dimensiones físicas y psicológicas puntuaron con medias bajas, al igual que el ambiente, valores considerados como deficientes. Conclusiones: Predominó una calidad de vida integral media en el mayor por ciento de los pacientes, estas enfermedades deben ser identificadas a tiempo, para evitar otras complicaciones en diferentes sistemas del organismo que pudieran comprometer la vida del paciente.

Introduction: Thyroid diseases are relatively frequent; they constitute an important group within chronic non-communicable diseases. Objective: To determine the quality of life in patients with thyroid diseases. Methods: An observational, descriptive and cross-sectional study was carried out. The universe (210 patients) with a diagnosis of thyroid diseases, who met the inclusion and exclusion criteria provided for in the study, prior informed consent, and the non-probabilistic sample (198 patients). The variables used were: age, sex, diagnosed thyroid disease (hyperthyroidism, hypothyroidism, diffuse euthyroid goiter) and quality of life (facets according to dimensions, according to expert classification and thyroid diseases and comprehensive quality of life according to diseases thyroid). The World Health Organization Quality of Life questionnaire was used. Results: Hypothyroidism predominated as the most frequent thyroid condition in women between 50 and 59 years of age. The clinical manifestations that scored the lowest average were pain and discomfort, followed by medical indications and negative feelings; the physical and psychological dimensions scored with low averages, as well as the environment, values ​​considered deficient. Conclusions: An average comprehensive quality of life prevailed in the highest percentage of patients; these diseases must be identified in time, to avoid other complications in different body systems that could compromise the patient's life.

Cottontail rabbit papillomavirus E6 proteins: Interaction with MAML1 and modulation of the Notch signaling pathway.

Animal models are necessary to study how cutaneous human papillomaviruses (HPVs) are associated with carcinogenesis. The cottontail rabbit papillomavirus (CRPV) induces papilloma in the -cutaneous skin of rabbits and serves as an established animal model for HPVlinked carcinogenesis where viral E6 proteins play crucial roles. Several studies have reported the dysregulation of the Notch signaling pathway by cutaneous beta HPV, bovine PV and mouse PV E6 via their association with Mastermind-like 1 protein (MAML1), thus interfering with cell proliferation and differentiation. However, the CRPV E6 gene encodes an elongated E6 protein (long E6, LE6) and an N-terminally truncated product (short E6, SE6) making it unique from other E6 proteins. Here, we describe the interaction between both CRPV E6 proteins and MAML1 and their ability to downregulate the Notch signaling pathway which could be a way CRPV infection induces carcinogenesis similar to beta HPV.

[Management and hygiene measures during an outbreak of herpes, influenza, strangles or infections with multidrug resistant bacteria]. / Vorgehensweise und Hygienemaßnahmen beim Ausbruch von Herpes, Influenza, Druse oder Infektionen mit multiresistenten Keimen.

The aim of this review is to describe general guidelines of hygiene measures in the horse stable as well as to provide current recommendations for an outbreak of a common infectious disease. General cleanliness, hand hygiene, avoidance of stress, regular deworming, and vaccinations belong to the basic hygiene measures in a horse herd. All new or returning equids should be submitted to a quarantine period as an important prevention measure. Repeated washing and disinfection of hands may prevent spreading of infectious agents to people and horses.The conception of a hygiene plan, including general biosecurity procedures and standard operating procedures in a case of an outbreak of an infectious disease, zoonosis, or colonization with multi-resistant bacteria is strongly recommended. As soon as the disease is suspected, extended hygiene measures including protective clothing, cleaning, disinfection, and isolation of potentially infected animals should be implemented. Prompt confirmation of the causative agent by examination of appropriate samples is crucial. It is important to adjust all safety measures based on the contagious nature of the respective pathogen and its major transmission routes. Apart from a lock-down of the stable, clinic or show grounds, the segregation of horses plays an important role. Implementation of the "traffic light system" is recommended. In this, the red group ("infected") include animals with clinical signs of the disease or that have been tested positive. All horses with possible pathogen contact should be allocated to a yellow group ("suspected") and regularly controlled for the signs of infection and fever. Clinically normal horses without contact to the infected animals belong to the green group ("healthy"). A change of protective clothing and an extensive disinfection should be performed when moving between the groups.The extended hygiene measures are to be maintained until all animals have been tested negative or fail to exhibit clinical signs of the disease for a certain time period.

Flow cytometry based-FRET: basics, novel developments and future perspectives.

Förster resonance energy transfer (FRET) is a widespread technology used to analyze and quantify protein interactions in multiple settings. While FRET is traditionally measured by microscopy, flow cytometry based-FRET is becoming popular within the last decade and more commonly used. Flow cytometry based-FRET offers the possibility to assess FRET in a short time-frame in a high number of cells thereby allowing stringent and statistically robust quantification of FRET in multiple samples. Furthermore, established, simple and easy to implement gating strategies facilitate the adaptation of flow cytometry based-FRET measurements to most common flow cytometers. We here summarize the basics of flow cytometry based-FRET, highlight recent novel developments in this field and emphasize on exciting future perspectives.

An enhanced triple fluorescence flow-cytometry-based assay shows differential activation of the Notch signaling pathway by human papillomavirus E6 proteins.

Human papillomaviruses are DNA tumor viruses. A persistent infection with high-risk HPV types is the necessary risk factor for the development of anogenital carcinoma. The E6 protein is a viral oncoprotein that directly interacts with different cellular regulatory proteins mainly affecting the cell cycle, cellular differentiation and polarization of epithelial cells. In dependency of the phylogenetic classification of HPV different interaction partners of E6 have been described. The Notch pathway seems to be one common target of HPV, which can be up or down regulated by different E6 proteins. Our novel triple fluorescence flow-cytometry-based assay allows a semi-quantitative comparison of the E6 proteins´ effect on the Notch pathway using a Notch-responsive reporter plasmid. As a result, all E6 proteins of beta-HPV repressed the Notch reporter expression, of which HPV38 E6 showed the greatest repression potential. In contrast, alpha-HPV E6 of HPV16, activates the reporter expression most significantly, whereas E6 of HPV31 and low-risk HPV6b showed significant activation only in a p53-null cell line. Interestingly, HPV18 E6, with the second highest carcinogenic risk, shows no effect. This high divergence within different genus of HPV is important for targeting the Notch pathway regarding a potential HPV therapy.

Native Isolation of 3×HA-Tagged Protein Complexes to Characterize Protein-Protein Interactions.

Co-immunoprecipitation (Co-IP) is a straightforward method that is widely used in studying direct protein-protein interactions in physiological environments. This technique is based on the antigen-antibody interaction: the protein of interest (bait) is captured by a specific antibody, followed by antibody-bait precipitation. The proteins interacting with the bait protein (prey) co-precipitate with the antibody-bait complex from a cell lysate as an antibody-bait/prey complex. Nowadays, a variety of surface-functionalized materials with antibodies immobilized on agarose or magnetic beads are available, replacing the precipitation of antibodies and simplifying the application. However, unspecific binding of cellular proteins to matrix surfaces and/or antibodies has become a common issue. Unspecific binding that leads to false-positive signals and a high background can hamper further analysis. Our protocol describes a strategy to tremendously reduce unspecific background when isolating native proteins and protein complexes. Instead of eluting our samples under denaturing conditions, we elute triple hemagglutinin (3×HA)-tagged bait/prey complexes in their native form with a competitive peptide simulating the 3×HA tag of the bait protein. Matrix-unspecific interacting proteins and Co-IP antibodies remain on the matrix instead of being eluted under conventionally applied denaturing conditions. We optimized the elution by altering incubation time, eluent concentration, and temperature. These improvements result in more pure proteins. This strategy not only reduces background in SDS-PAGE and western blot but also allows complex characterization in vitro. © 2021 Wiley Periodicals LLC.

Structure of High-Risk Papillomavirus 31 E6 Oncogenic Protein and Characterization of E6/E6AP/p53 Complex Formation.

The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (∼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.

Optimized production strategy of the major capsid protein HPV 16L1 non-assembly variant in E. coli.

The capsid of human papillomavirus (HPV) consists of two capsid proteins - the major capsid protein L1 and the minor capsid protein L2. Assembled virus-like particles, which only consist of L1 proteins, are successfully applied as prophylactic vaccines against HPV infections. The capsid subunits are L1-pentamers, which are also reported to protect efficiently against HPV infections in animals. The recombinant production of L1 has been previously shown in E. coli, yeast, insect cells, plants and mammalian cell culture. Principally, in E. coli-based expression system L1 shows high expression yields but the protein is largely insoluble. In order to overcome this problem reported strategies address fusion proteins and overexpression of bacterial chaperones. However, an insufficient cleavage of the fusion proteins and removal of co-purified chaperones can hamper subsequent down streaming. We report a significant improvement in the production of soluble L1-pentamers by combining (I) a fusion of a N-terminal SUMO-tag to L1, (II) the heterologous co-expression of the chaperon system GroEL/ES and (III) low expression temperature. The fusion construct was purified in a 2-step protein purification including efficient removal of GroEL/ES and complete removal of the N-terminal SUMO-tag. The expression strategy was transferred to process-controlled high-cell-density fermentation with defined media according to the guidelines of good manufacturing practice. The produced L1 protein is highly pure (>95%), free of DNA (260:280 = 0.5) and pentameric. The production strategy yielded 5.73 mg of purified L1-pentamers per gram dry biomass. The optimized strategy is a suitable alternative for high yield L1-pentamer production and purification as a cheaper process for vaccine production.

The human α-defensin-derived peptide HD5(1-9) inhibits cellular attachment and entry of human cytomegalovirus.

Human cytomegalovirus (HCMV) infection causes severe illness in newborns and immunocompromised patients. Since treatment options are limited there is an unmet need for new therapeutic approaches. Defensins are cationic peptides, produced by various human tissues, which serve as antimicrobial effectors of the immune system. Furthermore, some defensins are proteolytically cleaved, resulting in the generation of smaller fragments with increased activity. Together, this led us to hypothesize that defensin-derived peptides are natural human inhibitors of virus infection with low toxicity. We screened several human defensin HNP4- and HD5-derived peptides and found HD5(1-9) to be antiviral without toxicity at high concentrations. HD5(1-9) inhibited HCMV cellular attachment and thereby entry and was active against primary as well as a multiresistant HCMV isolate. Moreover, cysteine and arginine residues were identified to mediate the antiviral activity of HD5(1-9). Altogether, defensin-derived peptides, in particular HD5(1-9), qualify as promising candidates for further development as a novel class of HCMV entry inhibitors.

Síndrome coronario agudo con elevación del segmento ST en el Centro de Diagnóstico Integral La Macandona / Acute coronary syndrome with ST elevation in La Macandona Comprehensive Diagnostic Center

RESUMEN Introducción: el síndrome coronario agudo con elevación del segmento ST, se debe a la oclusión completa de una de las arterias responsables del miocardio. Objetivo: determinar el comportamiento clínico epidemiológico del SCACEST en la sala de terapia intensiva del CDI La Macandona, del municipio Maracaibo, estado Zulia desde el año 2014 al 2017. Métodos: se realizó una investigación, observacional, longitudinal, descriptiva y retrospectiva, el universo de estudio fue los 82 portadores de la enfermedad. Se utilizaron las historias clínicas de cada paciente, las variables se agruparon en tablas según frecuencias absolutas y relativas. Resultados: predominó en el sexo masculino (73,17 %), en las edades de 60 a 69 años (55,84 %), como principal factor de riesgo la hipertensión arterial (80,48 %), la mayoría recibió tratamiento trombolítico con estreptoquinasa (88,23 %) y al final de la investigación el 96, 34 % fueron egresados vivos. Conclusiones: se deben diseñar protocolos de actuación en los centros de diagnóstico integral que garanticen una atención pronta y segura del SCACEST, para de e forma garantizar un mejor tratamiento y supervivencia en los pacientes portadores del mismo.

ABSTRACT Introduction: acute coronary syndrome with ST elevation is due to complete occlusion of one of the arteries responsible for the myocardium. Objective: to determine the clinical epidemiological behavior of acute coronary syndrome with ST elevation in the intensive care unit of La Macandona Comprehensive Diagnostic Center, Maracaibo municipality, Zulia state from 2014 to 2017. Methods: an observational, longitudinal, descriptive and retrospective study was conducted where the target group comprised 82 patients suffering from this pathology, the variables used: age, sex, smoking habit, hypercholesterolemia, diabetes mellitus, ischemic heart disease, whether they received thrombolytic treatment and hospital discharge status. To obtain the data the source used was the clinical histories of each patient; the variables were grouped in tables according to absolute and relative frequencies. Results: male sex 60 (73,17 %) predominated, 45 of them were from ages 60 to 69 (55,84 %), 66 suffered from hypertension (80,48 %) which was the main risk factor, most of them underwent thrombolytic treatment with streptokinase 75 (88,23 %) and 76 (96,34 %) were discharged alive from the hospital. Conclusions: protocols of actions must be designed in the Comprehensive Diagnostic Center that guarantee prompt and safe care, in order to guarantee better treatment and the survival of patients with acute coronary syndrome with ST elevation.

Refolding and in vitro characterization of human papillomavirus 16 minor capsid protein L2.

The minor capsid protein L2 of papillomaviruses exhibits multiple functions during viral entry including membrane interaction. Information on the protein is scarce, because of its high tendency of aggregation. We determined suitable conditions to produce a functional human papillomavirus (HPV) 16 L2 protein and thereby provide the opportunity for extensive in vitro analysis with respect to structural and biochemical information on L2 proteins and mechanistic details in viral entry. We produced the L2 protein of high-risk HPV 16 in Escherichia coli as inclusion bodies and purified the protein under denaturing conditions. A successive buffer screen resulted in suitable conditions for the biophysical characterization of 16L2. Analytical ultracentrifugation of the refolded protein showed a homogenous monomeric species. Furthermore, refolded 16L2 shows secondary structure elements. The N-terminal region including the proposed transmembrane region of 16L2 shows alpha-helical characteristics. However, overall 16L2 appears largely unstructured. Refolded 16L2 is capable of binding to DNA indicating that the putative DNA-binding regions are accessible in refolded 16L2. Further the refolded protein interacts with liposomal membranes presumably via the proposed transmembrane region at neutral pH without structural changes. This indicates that 16L2 can initially interact with membranes via pre-existing structural features.

Contact investigation after a fatal case of extensively drug-resistant tuberculosis (XDR-TB) in an aircraft, Germany, July 2013.

In July 2013, a passenger died of infectious extensively drug-resistant tuberculosis (XDR-TB) on board of an aircraft after a 3-hour flight from Turkey to Germany. Initial information indicated the patient had moved about the aircraft coughing blood. We thus aimed to contact and inform all persons exposed within the aircraft and to test them for newly acquired TB infection. Two-stage testing within 8 weeks from exposure and at least 8 weeks after exposure was suggested, using either interferon gamma release assays (IGRAs) or tuberculin skin test (TST). The TST cut-off was defined at a diameter > 10 mm; for differentiation between conversion and boosting, conversion was defined as increase of skin induration > 5 mm. Overall, 155 passengers and seven crew members were included in the investigation: the questionnaire response rate was 83%; 112 (69%) persons were tested at least once for TB infection. In one passenger, who sat next to the area where the patient died, a test conversion was registered. As of March 2017, no secondary active TB cases have been reported. We describe an unusual situation in which we applied contact tracing beyond existing European guidelines; we found one latent tuberculosis infection in a passenger, which we consider probably newly acquired.

Caracterización clínico-epidemiológica de las infecciones respiratorias agudas en el menor de 5 años / Clinical and Epidemiological Characterization of Acute Respiratory Infections in Children under Five Years of Age

Las infecciones respiratorias agudas son un complejo sindromático de entidades clínicas, con diversidad epidemiológica y causal; causa más común de consulta médica pediátrica y de hospitalización. El objetivo fue caracterizar el comportamiento de las infecciones respiratorias agudas en pacientes pediátricos menores de 5 años ingresados en el Hospital Provincial Pediátrico Docente Pepe Portilla, Pinar del Río, durante el último semestre del año 2013. Se realizó una investigación observacional descriptiva y transversal. La muestra fue constituida por 88 pacientes pediátricos menores de 5 años. Se calcularon estadígrafos de tendencia central para expresar el comportamiento de variable cuantitativa. El sexo masculino reporta mayor frecuencia de infecciones respiratorias agudas. La lactancia materna no arrojó datos significativos, pero se observó una mayor incidencia de neumonías y bronconeumonía en pacientes que no la practicaban. La mayoría de los pacientes con enfermedades respiratorias agudas fueron niños entre 1 y 2 años. Los antecedentes patológicos personales y familiares de alergia y asma bronquial son importantes en la aparición de las enfermedades respiratorias agudas. Se hace necesario desarrollar acciones de promoción y prevención de salud para disminuir la incidencia de factores de riesgo y aparición de las infecciones respiratorias agudas en pacientes menores de 5 años, así como implementar algunas estrategias para reducir la morbilidad y mortalidad por estas enfermedades(AU)

Acute respiratory infections (ARIs) constitute a complex gamut of clinical features, with epidemiological and causal diversity, a most common cause for children medical consultation and for hospitalization. The objective was to characterize the behavior of ARIs in pediatric patients under 5 years of age admitted to Pepe Portilla Children Province Hospital of Pinar de Río Province, during the last semester of the year 2013. An observation, descriptive and cross-sectional research was carried out, with a sample constituted by 88 pediatric patients under five years of age. Statistic values of central tendency were calculated to express quantitative variable behavior.Results: the male sex reported a higher frequency of ARIs. Maternal breastfeeding did not show significant data, but a higher incidence was observed in pneumonia and bronchopneumonia. Most of the patients with ARIs were children aged between one and two years. Individual and family pathological history of allergy and bronchial asthma are important in the onset of AIRs. It is necessary to develop actions of health promotion and prevention to decrease the incidence of risk factor and the onset of ARIs in patient under five years of age, as well as to implement some strategies for reducing morbidity and mortality on this diseases(AU)

Biophysical characterization of polyomavirus minor capsid proteins.

The murine polyomavirus encodes three structural proteins, VP1, VP2 and VP3, which together form the viral capsid. The outer shell of this capsid is composed of the major capsid protein VP1, the inner shell consists of VP2 and its N-terminally truncated form VP3. These two minor capsid proteins interact with their identical C-terminal part in the central cavity of VP1 pentamers, building the capsid assembly unit. While the VP1 structure and functions are well studied, VP2 and VP3 are poorly understood. In order to get a detailed insight into the structure and function of the minor capsid proteins, they were produced recombinantly in Escherichia coli as inclusion bodies and refolded in vitro. The success of refolding was strictly dependent on the presence of detergent in the refolding buffer. VP2 and VP3 are monomeric and their structures exhibit a high α-helical content. The function of both proteins could be monitored by complex formation with VP1. Furthermore, we could demonstrate a hemolytic activity of VP2/VP3 in vitro, which fits well into a postulated membrane interaction of VP2 during viral infection.

Disulfide linkage and structure of highly stable yeast-derived virus-like particles of murine polyomavirus.

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys(115)-Cys(20), Cys(12)-Cys(20), Cys(16)-Cys(16), Cys(12)/ Cys(16)-Cys(115), and Cys(274)-Cys(274)), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.

Glicemia de jejum do primeiro trimestre e fatores de risco de gestantes com diagnóstico de diabetes melito gestacional / First trimester fasting glycemia and risk factors of pregnant women diagnosed with gestational diabetes mellitus

OBJETIVO: Avaliar a incidência de repercussões materno-fetais e controle glicêmico em gestantes com diagnóstico de Diabetes Melito Gestacional (DMG) tendo como corte a glicemia de jejum de 85 mg/dL no primeiro trimestre e correlacionar com fatores de risco. MÉTODOS: Foram revisados os prontuários das gestantes acompanhadas no ambulatório de Pré-Natal de Alto Risco (PNAR) no período de janeiro de 2011 a março de 2012 e selecionadas aquelas com diagnóstico de DMG para contato e verificação do cartão de pré-natal. Foram colhidos dados de idade, paridade, glicemia de jejum do primeiro trimestre, valor do Teste Oral de Tolerância à Glicose (TOTG), Índice de Massa Corpórea (IMC), via de parto, forma de controle, repercussões fetais e fatores de risco para DMG. A análise estatística foi realizada no software PSPP 0.6.2 e consistiu de análise descritiva de frequências, teste do χ2 para variáveis categóricas, teste t de Student para amostras independentes e teste de Pearson para as correlações com nível de significância de 5%. RESULTADOS: Das 408 gestantes atendidas, 105 tinham diagnóstico de Diabetes Melito Gestacional (DMG) e 71 tinham prontuários completos ou responderam ao contato para fornecer as informações faltantes. No grupo DMG-jejum <85, (com glicemia de jejum <85 mg/dL na primeira consulta no primeiro trimestre) foram incluídas 29 gestantes (40,8%) e no grupo DMG-jejum>85 (glicemia de jejum >85 mg/dL na primeira consulta, no primeiro trimestre) 42 gestantes (59,1%). Observou-se que poucas pacientes (5 no grupo DMG-jejum <85 e 3 no grupo DMG-jejum>85) não apresentavam fatores de risco para DMG. Houve maior frequência da necessidade de controle com insulina nas pacientes do grupo DMG-jejum >85. Não houve diferença significativa quanto às repercussões fetais e via de parto entre os grupos. CONCLUSÃO: A glicemia de jejum do primeiro trimestre, tendo como ponto de corte o valor 85 mg/dL, isoladamente, ou associado a fatores de risco, não seria bom preditor isolado de repercussões materno-fetais do DMG.

PURPOSE: To evaluate the incidence of maternal and fetal repercussions and glycemic control in women with Gestational Diabetes Mellitus (GDM) using a fasting glucose of 85 mg/dL in the first trimester as a cut-off point and to correlate it with risk factors. METHODS: The medical records of pregnant women followed in the outpatient antenatal high-risk service (PNAR) of HRAN from January 2011 to March 2012 were reviewed and those women diagnosed with GDM were selected for contact and for prenatal card verification. We collected data of age, parity, fasting glucose during the first quarter, the value of the Oral Glucose Tolerance Test (OGTT), Body Mass Index (BMI), mode of delivery, form of control, effects and fetal risk factors for GDM. Statistical analysis was performed using the PSPP 0.6.2 software and consisted of descriptive analysis of frequencies, χ2 test for categorical variables, Student's t-test for independent samples, and Pearson test for correlations, with the level of significance set at 5%. RESULTS: From 408 pregnant women enrolled, 105 were diagnosed with GDM and 71 had complete records or answered to the contact in order to provide the missing information. The GDM-fasting <85 (fasting glucose <85 mg/dL at the first prenatal visit, in the first trimester) group consisted of 29 (40.8%) women and the GDM-fasting >85 (fasting glucose >85 mg/dL at the first prenatal visit, in the first trimester) consisted of 42 (59.1%) women. It was observed that few patients (five in the GDM-fasting <85 group and three in the GDM-fasting >85 group) had no risk factors for GDM. There was a major need for control with insulin in patients of the GDM-fasting >85 group. There was no significant difference related to fetal impact or mode of delivery between the groups. CONCLUSIONS: The first trimester fasting glycemia, with a cut-off value of 85 mg/dL alone or associated with risk factors, does not seem to be a good single predictor of the maternal-fetal effects of GDM.

Production of polyomavirus-like particles in a Klgal80 knockout strain of the yeast Kluyveromyces lactis.

VP1, the major coat protein of polyomavirus, assembles intracellularly to virus-like particles if expressed in eukaryotes. Here, the nonconventional yeast Kluyveromyces lactis was used for production of virus-like particles of murine polyomavirus. The heterologous gene of VP1 was integrated in the LAC4 locus of the GAL/LAC genes. Consequently the expression of VP1 is regulated by the interplay of the activator KlGal4p and inhibitor KlGal80p. This cloning strategy couples the production of VP1 to that of the enzyme ß -galactosidase, allowing a fast alternative for monitoring the course of recombinant protein production by measuring the ß -galactosidase activity. A Klgal80 knockout strain was generated for a constitutive expression of VP1 and a continuous VLP production. High-cell-density fermentation showed that (1) Kluyveromyces lactis is generally suitable for VLP production and (2) the Klgal80 knockout strain produces higher amounts of recombinant VP1. Furthermore, VLPs could be purified chromatographically to 87% (w/w) of total protein, and showed a homogeneous species of 45-nm particles and a high resistance against proteolysis compared to conventional in vitro assembled VLPs. This demonstrates the superior stability of virus-like particles produced in yeast.