Inflammatory responses of bovine endometrial epithelial cells are increased under in vitro heat stress conditions.

Cattle exposed to heat stress have reduced fertility, reduced milk production and increased incidence of postpartum uterine infection. Heat stress is suggested to alter immune function of cattle; however, the mechanisms underlying heat stress mediated uterine infection are unknown. We hypothesized that exposure of endometrial cells to heat stress would further increase expression of inflammatory mediators in response to bacterial components due to altered heat-shock protein expression. Bovine endometrial epithelial cells (BEND) were exposed to Escherichia coli lipopolysaccharide (LPS) or a synthetic triacylated lipopeptide (Pam3CSK4) under heat stress (41.0 °C) or thermoneutral (38.5 °C) conditions for 24 h. Exposure of BEND cells to LPS or Pam3CSK4 increased the expression of the proinflammatory mediators IL1B, IL6, and CXCL8 compared to control medium. However, exposure of BEND cells to heat stress increased LPS and Pam3CSK4 induced expression of IL1B compared to cells exposed to thermoneutral conditions, and expression of LPS induced IL6 was also increased when BEND cells were exposed to heat stress. To determine if heat shock proteins increased BEND cell expression of inflammatory mediators, HSP1A1 and HSF1 were targeted by siRNA knock down. Expression of HSP1A1 and HSF1 were reduced following siRNA knockdown; however, knockdown of HSP1A1 or HSF1 further increased heat stress mediated increased expression of inflammatory mediators. These data suggest that heat stress increased BEND cell inflammatory responses to bacterial components, while heat shock proteins HSP1A1 and HSF1 help to restrain inflammatory responses. These mechanisms may contribute to the increased incidence of uterine infection observed in cows under heat stress conditions.

Pre-treatment with IL-6 potentiates ß-cell death induced by pro-inflammatory cytokines.

BACKGROUND: Type I Diabetes mellitus (T1D) is characterized by a specific destruction of ß-cells by the immune system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for ß-cells demise. Cytokine-induced iNOS activation, via NF-κB, is implicated in induction of ß-cells death, which includes ER stress activation. Physical exercise has been used as an adjunct for better glycemic control in patients with T1D, since it is able to increase glucose uptake independent of insulin. Recently, it was observed that the release of IL-6 by skeletal muscle, during physical exercise, could prevent ß-cells death induced by pro-inflammatory cytokines. However, the molecular mechanisms involved in this beneficial effect on ß-cells are not yet completely elucidated. Our aim was to evaluate the effect of IL-6 on ß-cells exposed to pro-inflammatory cytokines. RESULTS: Pre-treatment with IL-6 sensitized INS-1E cells to cytokine-induced cell death, increasing cytokine-induced iNOS and Caspase-3 expression. Under these conditions, however, there was a decrease in cytokines-induced p-eIF2-α but not p-IRE1expression, proteins related to ER stress. To address if this prevention of adequate UPR response is involved in the increase in ß-cells death markers induced by IL-6 pre-treatment, we used a chemical chaperone (TUDCA), which improves ER folding capacity. Use of TUDCA increased cytokines-induced Caspase-3 expression and Bax/Bcl-2 ratio in the presence of IL-6 pre-treatment. However, there is no modulation of p-eIF2-α expression by TUDCA in this condition, with increase of CHOP expression. CONCLUSION: Treatment with IL-6 alone is not beneficial for ß-cells, leading to increased cell death markers and impaired UPR activation. In addition, TUDCA has not been able to restore ER homeostasis or improve ß-cells viability under this condition, suggesting that other mechanisms may be involved.

Prepartum heat stress in dairy cows increases postpartum inflammatory responses in blood of lactating dairy cows.

Uterine diseases and heat stress (HS) are major challenges for the dairy cow. Heat stress alters host immune resilience, making cows more susceptible to the development of uterine disease. Although HS increases the incidence of uterine disease, the mechanisms by which this occurs are unclear. We hypothesize that evaporative cooling (CL) to alleviate HS in prepartum cows has carry-over effects on postpartum innate immunity. Nulliparous pregnant Holstein heifers were assigned to receive either forced CL that resulted in cool conditions (shade with water soakers and fans; n = 14) or to remain under HS conditions (barn shade only; n = 16) for 60 d prepartum. Postpartum, all cows were housed in a freestall barn equipped with shade, water soakers, and fans. Respiratory rate and rectal temperature during the prepartum period were greater in HS heifers compared with CL heifers, indicative of HS. Although milk production was decreased in HS cows compared with CL cows, the incidence of uterine disease and content of total or pathogenic bacteria in vaginal mucus on d 7 or d 21 postpartum was not affected by treatment. Whole blood was collected on d 21 and subjected to in vitro stimulation with lipopolysaccharide. Lipopolysaccharide-induced accumulation of IL-1ß, IL-10, and MIP-1α was greater in blood collected from HS cows compared with CL cows. Our results imply that prepartum HS during late pregnancy has carry-over effects on postpartum innate immunity, which may contribute to the increased incidence of uterine disease observed in cows exposed to prepartum HS.

Effect of calving season on metritis incidence and bacterial content of the vagina in dairy cows.

Heat stress and uterine diseases, including metritis and endometritis, both reduce milk yields and reduce reproductive performance. Bacterial growth is promoted by elevated temperature while heat stress reduces host immune cell function, but it is not known whether increased environmental temperature promotes uterine disease by altering host immunity or bacterial growth. We hypothesize that seasonal variations in environmental temperature influence metritis incidence in the dairy cow independent of bacterial prevalence in the reproductive tract. To investigate how environmental temperature may impact metritis incidence, records of 3507 calvings in Florida over a 5-year period were evaluated. The incidence of metritis increased from 21.1% in the cool season (October through March) to 24.2% during the warm season (April through September, P < 0.05). To elucidate a link between environmental temperature and uterine disease, 102 cows were enrolled during the warm season (September 2017; n = 51) and cool season (February-March 2018; n = 51). Cows were maintained on pasture during the dry period and moved to free stall barns with fans and water soakers immediately prior to calving and remained in that environment after calving. Vaginal mucus was collected and scored on days 7 (to evaluate metritis) and 21 (to evaluate endometritis) postpartum to evaluate the incidence of uterine disease and quantify bacterial content and species using qPCR. Daily milk yield for the first 60 DIM was reduced during the warm season compared with the cool season (32.6 ± 1.62 vs 37.23 ± 1.60 kg, P < 0.05) consistent with effects of prepartum heat stress. Interestingly, more cows had persistent uterine disease on both d 7 and d 21 in the warm season compared with the cool season (58.0 vs 29.4%, P < 0.05). Regardless of calving season the total bacterial content in the vagina was greater on d 7 compared to d 21. While metritis incidence was increased in the warm season, the vaginal content of total bacteria, Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum and Prevotella melaninogenica were similar during the cool season and the warm season. Our data suggests that prepartum heat stress related to season of calving increased the incidence of metritis and persistence of uterine disease in the dairy cow independent of vaginal bacteria content. The possibility that prepartum heat stress perturbs host immune function and increases the risk of metritis when cows are exposed to an equivalent number of pathogenic bacteria requires further investigation.

Bovine endometrial cells do not mount an inflammatory response to Leptospira.

Leptospirosis causes abortion, premature birth, and stillbirth in cattle, but the mechanisms remain unclear. Infected cattle shed Leptospira intermittently and present a range of clinical symptoms, making diagnosis difficult. The primary route of Leptospira transmission in any animal is the colonization of the renal tubule and excretion by urine; however, Leptospira can also colonize the female reproductive tract of cows and can be transmitted by semen. Vaccination against Leptospira in the US is routine in cattle, but immunity is not guaranteed. The cell wall of Leptospira contains toll-like receptor agonists including peptidoglycan and lipopolysaccharide. The capacity of Leptospira to initiate an innate inflammatory response from uterine endometrial cells is unknown but may be a cause of reproductive failure. Using cell culture, we tested the capacity of bovine endometrial epithelial cells or human monocytes to elicit an inflammatory response to Leptospira borgpetersenii serovar Hardjo strain TC273. Cells were exposed to either heat-killed Leptospira, Leptospira outer membrane, Escherichia coli lipopolysaccharide, Pam3CSK4 or medium alone for 2 to 24 h. Exposure of bovine endometrial epithelial cells or human monocytes to heat-killed Leptospira or Leptospira outer membrane did not induce the expression of IL1A, IL1B, IL6, or CXCL8, while exposure to E. coli lipopolysaccharide or Pam3CSK4 increased the expression of IL1A, IL1B, IL6, and CXCL8 compared to control cells. This data suggest that Leptospira does not trigger a classical inflammatory response in endometrial cells. Understanding the interaction between Leptospira and the female reproductive tract is important in determining the mechanisms of Leptospirosis associated with reproductive failure. LAY SUMMARY: Cows infected with the Leptospira have abortion and stillbirth. It is not known how Leptospira causes pregnancy failure in the cow. We tested if Leptospira causes inflammation in cells of the uterus which triggers pregnancy failure. We collected cells from the uterus of healthy cows at the abattoir and placed them into culture with Leptospira and measured the expression of genes associated with inflammation. To our surprise, cells of the uterus did not respond to Leptospira; however, the same cells did respond to other disease-causing bacteria found in the uterus. This suggests that cells of the uterus can recognize bacteria and produce an inflammatory reaction but not in response to Leptospira. This finding suggests the immune system of the uterus cannot detect Leptospira which may go on to cause reproductive failure in cows. Understanding how Leptospira interact with cells of the uterus will help reduce pregnancy failure of cows with leptospirosis.

Kinematic Comparison of the Roundhouse Kick Between Taekwondo, Karate, and Muaythai.

ABSTRACT: Diniz, R, Del Vecchio, FB, Schaun, GZ, Oliveira, HB, Portella, EG, da Silva, ES, Formalioni, A, Campelo, PCC, Peyré-Tartaruga, LA, and Pinto, SS. Kinematic comparison of the roundhouse kick between taekwondo, karate, and muaythai. J Strength Cond Res 35(1): 198-204, 2021-The roundhouse kick (RHK) is frequently executed in taekwondo, karate, and muaythai because of its high technical effectiveness during combat. The purpose of this study was to compare kinematic characteristics during RHK performance between taekwondo, karate, and muaythai athletes. Forty-seven male athletes (25.5 ± 4.7 years, 1.75 ± 0.1 m, and 75.8 ± 11.5 kg) volunteered to participate (taekwondo: 17; karate: 15; and muaythai: 15). Self-selected distance from target, mean and peak fifth metatarsus linear velocity (LV5mean; LV5peak), mean and peak hip (HAVmean; HAVpeak) and knee (KAVmean; KAVpeak) angular velocities, as well as target linear acceleration (TLA) were analyzed with a 3D video motion analysis system. Comparisons between modalities were performed with 1-way analysis of variances and Bonferroni's post hoc test (α = 0.05). Self-selected distance was lower in muaythai compared with taekwondo and karate (p < 0.001). Also, karate had greater LV5mean compared with muaythai (p = 0.001), and muaythai showed higher HAVmean than karate (p = 0.011). In addition, HAVpeak was greater in muaythai than in taekwondo and karate (p < 0.001). No differences were found for KAVmean, KAVpeak, and TLA. Although it is similarly described between modalities, RHK showed distinct kinematic characteristics between taekwondo, karate, and muaythai. Based on these results, coaches and athletes can improve their RHK technique according to the specificities of each combat sport. Specifically, it is suggested that combat strategies should aim to increase the distance from the opponent during combat for muaythai athletes, whereas taekwondo and karate athletes should focus on decreasing it.

Preventing postpartum uterine disease in dairy cattle depends on avoiding, tolerating and resisting pathogenic bacteria.

Up to forty percent of dairy cows develop metritis or endometritis when pathogenic bacteria infect the uterus after parturition. However, resilient cows remain healthy even when exposed to the same pathogens. Here, we provide a perspective on the mechanisms that dairy cows use to prevent postpartum uterine disease. We suggest that resilient cows prevent the development of uterine disease using the three complementary defensive strategies of avoiding, tolerating and resisting infection with pathogenic bacteria. Avoidance maintains health by limiting the exposure to pathogens. Avoidance mechanisms include intrinsic behaviors to reduce the risk of infection by avoiding pathogens or infected animals, perhaps signaled by the fetid odor of uterine disease. Tolerance improves health by limiting the tissue damage caused by the pathogens. Tolerance mechanisms include neutralizing bacterial toxins, protecting cells against damage, enhancing tissue repair, and reprogramming metabolism. Resistance improves health by limiting the pathogen burden. Resistance mechanisms include inflammation driven by innate immunity and adaptive immunity, with the aim of killing and eliminating pathogenic bacteria. Farmers can also help cows prevent the development of postpartum uterine disease by avoiding trauma to the genital tract, reducing stress, and feeding animals appropriately during the transition period. Understanding the mechanisms of avoidance, tolerance and resistance to pathogens will inform strategies to generate resilient animals and prevent uterine disease.

Effect of seminal plasma or transforming growth factor on bovine endometrial cells.

Semen induces post-coital inflammation of the endometrium in several species. Post-coital inflammation is proposed to alter the endometrial environment of early pregnancy, mediate embryonic development and modulate the maternal immune response to pregnancy. In cattle, it is common for pregnancies to occur in the absence of whole semen due to the high utilization of artificial insemination. Here, we have utilized a cell culture system to characterize semen-induced expression of inflammatory mediators in bovine endometrial cells and test the efficacy of transforming growth factor beta as the active agent in mediating any such change. We hypothesize that seminal plasma-derived transforming growth factor beta increases the expression of inflammatory mediators in bovine endometrial cells. Initially, we describe a heat-labile cytotoxic effect of seminal plasma on BEND cells, and a moderate increase in IL1B and IL6 expression. In addition, we show that transforming growth factor beta is present in bovine semen and can increase the expression of endometrial IL6, whereas blocking transforming growth factor beta in semen ameliorates this effect. However, intra-uterine infusion of seminal plasma, sperm or transforming growth factor beta did not alter the endometrial expression of inflammatory mediators. We conclude that bovine semen can modulate endometrial gene expression in vitro, which is partially due to the presence of transforming growth factor beta. It is likely that additional, unidentified, bioactive molecules in semen can alter the endometrial environment. Characterizing bioactive molecules in bovine semen may lead to the development of additives to improve artificial insemination in domestic species.

A model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes.

Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows.

Intracellular reactive oxygen species production and phagocytosis of Staphylococcus aureus by milk neutrophils as tool to diagnose mastitis and identify susceptible dairy cows / Produção intracelular de espécies reativas de oxigênio e fagocitose de Staphylococcus aureus por neutrófilos lácteos como ferramentas para o diagnóstico da mastite e identificação de vacas leiteiras susceptíveis à mastite

The immune response capacity of the mammary gland plays a major role to determine if mastitis will or not be established. Thus, we hypothesize that a better understanding of polymorphonuclear neutrophil leukocyte (PMN) function will elucidate mechanisms that will improve our knowledge of how we could avoid an inflammatory process by increasing the immune capacity of the cow, and even further, to search for a tool to diagnose mastitis or a possible way to select and identify non-susceptible animals. The present study utilized 112 quarters from 28 Holstein dairy cows that were divided into quarters milk samples with somatic cell count (SCC) <2×105 cells mL-1 (n=72) and SCC >2×105 cells mL-1 (n=40). The percentages of milk PMNs and the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus by milk neutrophils were evaluated by flow cytometry. Our results showed a higher percentage of neutrophils in quarter milk samples with high SCC (P=0.0003), and this group also had a significantly higher percentage of neutrophils that produced ROS (P=0.008). On the other hand, the phagocytosis intensity of S. aureus by milk neutrophils was higher in quarters with low SCC (P=0.003), suggesting a better mammary gland immunity against invading pathogens. Analyzing the results of the predictive values of the measured PMN functions, they cannot be used isolated as a good diagnosis test since none of them had a satisfactory sensitivity and specificity values, which was also confirmed by the Youden index values being far from one. In conclusion, the assessment of milk bovine neutrophil functions could improve our understanding of the cellular basis of mastitis. Although, the intracellular ROS production and S. aureus phagocytosis by milk neutrophil did not have high predictive values to detect intramammary infections, our results strengthen the idea that that poor bovine mammary gland neutrophil phagocytic ability may be associated with high SCC, and might be considered to identify susceptible dairy cows to mastitis.(AU)

A resposta imune da glândula mamária desempenha um papel importante ao determinar o estabelecimento da infecção. Desta forma, a melhor compreensão da função dos neutrófilos irá nos subsidiar conhecimentos, pelo qual podemos evitar o processo inflamatório pela otimização da resposta imune de bovinos leiteiros, e fornece ferramentas para diagnosticar a mastite ou um possível instrumento para identificar e selecionar animais resistentes à infecção intramamária, aumentando a produtividade do rebanho. O presente estudo utilizou 112 amostras provenientes de quartos mamários de 28 vacas Holandesas que foram divididos em amostras de leite com baixa (n=72; <2×105 células mL-1) ou alta (n=40; 2×105 células mL-1) contagem de células somáticas (CCS). A porcentagem de neutrófilos no leite, a produção intracelular de espécies reativas de oxigênio (ERO) e a fagocitose de Staphylococcus aureus pelos neutrófilos do leite foram avaliadas por citometria de fluxo. Os resultados do presente estudo demonstraram maior percentagem de neutrófilos (CH138+; P=0,0003) e percentagem de neutrófilos que produziram ERO (P=0,008) em amostras de leite com alta CCS. Por outro lado, a intensidade de fagocitose de S. aureus por neutrófilos em amostras de leite com baixa CCS (P=0,003), que demonstra maior atividade funcional destas células neste grupo. As funções neutrofílicas para o diagnóstico da mastite não apresentaram valores de sensibilidade e especificidade altos, que foram confirmados pelo índice Youden. Desta forma, conclui-se que a produção intracelular de ERO e fagocitose de S. aureus pelos neutrófilos do leite não apresentaram valores preditivos altos para detecção de mastite. Além disto, os resultados do presente estudo reforçam a ideia de neutrófilos do leite com menor capacidade fagocítica podem ser associados à alta CCS, e pode ser considerado como uma ferramenta para identificar animais mais susceptíveis à infecções intramamárias.(AU)

The liver transcriptome of suckermouth armoured catfish (Pterygoplichthys anisitsi, Loricariidae): Identification of expansions in defensome gene families.

Pterygoplichthys is a genus of related suckermouth armoured catfishes native to South America, which have invaded tropical and subtropical regions worldwide. Physiological features, including an augmented resistance to organic xenobiotics, may have aided their settlement in foreign habitats. The liver transcriptome of Pterygoplichthys anisitsi was sequenced and used to characterize the diversity of mRNAs potentially involved in the responses to natural and anthropogenic chemicals. In total, 66,642 transcripts were assembled. Among the identified defensome genes, cytochromes P450 (CYP) were the most abundant, followed by sulfotransferases (SULT), nuclear receptors (NR) and ATP binding cassette transporters (ABC). A novel expansion in the CYP2Y subfamily was identified, as well as an independent expansion of the CYP2AAs. Two expansions were also observed among SULT1. Thirty-two transcripts were classified into twelve subfamilies of NR, while 21 encoded ABC transporters. The diversity of defensome transcripts sequenced herein could contribute to this species' resistance to organic xenobiotics.

An RNA-based approach to sequence the mitogenome of Hypoptopoma incognitum (Siluriformes: Loricariidae).

Hypoptopoma incognitum is a fish of the fifth most species-rich family of vertebrates and abundant in rivers from the Brazilian Amazon. Only two species of Loricariidae fish have their complete mitogenomes sequence deposited in the Genbank. An innovative RNA-based approach was used to assemble the complete mitogenome of H. incognitum with an average coverage depth of 5292×. The typical vertebrate mitochondrial features were found; 22 tRNA genes, two rRNA genes, 13 protein-coding genes, and a non-coding control region. Moreover, the use of this approach allowed the measurement of mtRNA expression levels, the punctuation pattern of editing, and the detection of heteroplasmies.

The complete mitochondrial genome of Corydoras nattereri (Callichthyidae: Corydoradinae)

The complete mitogenome of Corydoras nattereri , a species of mailed catfishes from southeastern Brazil, was reconstructed using next-generation sequencing techniques. The mitogenome was assembled using mitochondrial transcripts from the liver transcriptomes of three individuals, and produced a circular DNA sequence of 16,557 nucleotides encoding 22 tRNA genes, two rRNA genes, 13 protein-coding genes and two noncoding control regions (D-loop, OrigL). Phylogeographic analysis of closely related sequences of Cytochrome Oxydase C subunit I (COI) demonstrates high diversity among morphologically similar populations of C. nattereri . Corydoras nattereri is nested within a complex of populations currently assigned to C. paleatus and C. ehrhardti . Analysis of mitogenome structure demonstrated that an insertion of 21 nucleotides between the ATPase subunit-6 and COIII genes may represent a phylogenetically informative character associated with the evolution of the Corydoradinae.

O mitogenoma completo de Corydoras nattereri , uma espécie de bagres encouraçados do sudeste do Brasil, foi reconstruído através de técnicas de sequencimento de DNA de próxima geração. O mitogenoma foi produzido a partir de produtos de transcrição mitocondrial dos transcriptomas hepáticos de três indivíduos, resultando numa sequência de DNA circular de 16.557 nucleotídeos abrangendo 22 genes de tRNA, dois genes de rRNA, 13 genes codificadores de proteínas e duas regiões de controle não codificadoras (D-loop, OrigL). A análise filogenética de sequências proximamente relacionadas da subunidade I do gene Citocrome Oxidase C (COI) demonstrou a existência de elevada diversidade entre populações morfologicamente similares de C. nattereri . Corydoras nattereri está inserida num complexo de populações atualmente identificadas como C. paleatus e C. ehrhardti . A análise da estrutura do mitogenoma demonstra que a inserção de uma sequência de 21 nucleotídeos entre os genes da subunidade 6 da ATPase e do COIII representa um caráter filogeneticamente informativo associado à evolução de Corydoradinae.

A representação social do enfermeiro para as crianças, em idade escolar / The social representation of the nurse for school-age children

Os desafios que se colocam hoje à profissão de enfermagem são muitos e não podem ser ganhos sem uma nova atitude profissional e institucional perante a profissão. Neste sentido, a necessidade de se refletir sobre a profissão de Enfermagem, levou a enveredar pelo "campo" das representações sociais, considerando que a representação social é uma forma específica de conhecimento, o conhecimento prático, que cada um de nós constrói tendo em vista a adequação do comportamento individual/social. Com o intuito de conhecer a representação social do enfermeiro, para as crianças em idade escolar, optou-se por um estudo de natureza analítica, indutivo e construtivista, adotando a técnica de análise de dados da Grounded Theory. Esta técnica pareceu ser a mais indicada pois permite, partindo da perspetiva dos participantes, a produção de conhecimento através de um processo indutivo. Como instrumentos de colheita de dados, serão utilizados a entrevista semi-estruturada, auxiliada pelo desenho. De facto, existe um sentimento de que, apesar da vertente técnica ser fundamental e valorizada, a vertente relacional é a mais valorizada pelas crianças, uma vez que é através dela que os enfermeiros estabelecem relações com os utentes, visando o seu cuidado como ser único. Esta questão assume particular importância no que se refere à interação enfermeirocriança.

The challenges facing the nursing profession today are many and can not be won without a new professional and institutional attitude towards the profession. In that sense, the need to reflect on the profession of Nursing, led the plunge into the "field" of social representations, whereas the social representation is a specific form of knowledge, practical knowledge, that each of us constructs a view the adequacy of the individual / social behavior. Aiming to meet the social representation of nurses for children of school age, it was decided by a study of analytical, inductive and constructive nature, adopting the technique of data analysis of Grounded Theory. This technique appeared to be the most suitable because it allows, starting from the perspective of the participants, the production of knowledge through an inductive process. As instruments of data collection, the semi-structured interview, aided by the design will be used. In fact, there is a feeling that, despite the technical aspect is fundamental and valued the relational aspect is valued by the children, since it is through it that nurses establish relationships with clients, targeting their care as being unique. This question is of particular importance with regard to nurse-child interaction

Application of pH-zone-refining countercurrent chromatography for the separation of indole alkaloids from Aspidosperma rigidum Rusby.

Species of Aspidosperma (Apocynaceae) are characterized by the occurrence of indole alkaloids, but few recent reports on Aspidosperma rigidum Rusby chemical constituents were found. The present work shows the application of pH-zone refining countercurrent chromatography on the separation of alkaloids from the barks of A. rigidum. In this study, the dichloromethane extract was fractionated with the solvent system composed of methyl-tert-butyl ether and water with different concentrations of the retainer triethylamine in the organic stationary phase and formic or hydrochloric acids as eluters in the aqueous mobile phase, in order to evaluate the most suitable condition. In each experiment, from circa 200mg of the dichloromethane extract of A. rigidum, three major alkaloids were isolated and identified as 3α-aricine (circa 17mg), isoreserpiline (ca. 22mg) and 3ß-reserpiline (ca. 40mg), with relative purity of 79%, 89% and 82% respectively, in a one-step separation of 2h. Two of them - 3α-aricine and isoreserpiline - were isolated and identified for the first time in this species.

Estrutura do dossel, consumo e desempenho animal em pastos de capim-marandu sob lotação contínua / Sward structure, herbage intake and animal performance on marandu palisadegrass pastures subjected to continuous stocking

Avaliaram-se o valor nutritivo, a estrutura do dossel, a ingestão de forragem e a produção animal de novilhos em pastos de capim-marandu submetidos a três intensidades de pastejo. O delineamento experimental foi de blocos completos ao acaso, com duas repetições e três intensidades de pastejo, representadas pelas alturas do pasto de 15, 30 e 45cm. Mensalmente, os pastos foram amostrados para as estimativas das características estruturais do dossel e o valor nutritivo da forragem. A ingestão de matéria seca pelos animais foi estimada no verão e no outono de 2008. A oferta de forragem decresceu à medida que aumentou a intensidade de pastejo. Pastos manejados com 15cm de altura apresentaram maior valor nutritivo e estrutura do dossel mais favorável à apreensão de forragem pelos animais. A oferta limitou a ingestão de forragem no pasto com 15cm de altura, consequentemente se verificaram menor ganho de peso e maior taxa de lotação. Foram observados ganhos por área semelhantes nos pastos com 15 e 30cm, e ganhos mais elevados no pasto com 45cm. Durante o período das águas, o capim-marandu deve ser utilizado entre 15 e 30cm de altura sob lotação contínua.

The objectives were to evaluate the nutritive value, sward structure, herbage intake and animal performance on marandu palisadegrass pastures subjected to three grazing intensities under continuous stocking. A randomized block design was used, with two replicates and three grazing intensities represented by 15, 30 and 45cm sward heights. Monthly, the pastures were sampled to estimate the sward structure and nutritive value, and the animals were weighted. The herbage intake was estimated twice, one in the summer and another during autumn. The forage allowance decreased with the increase of the grazing intensity. Pasture with 15cm height presented greatest nutritive value and sward structure more favorable to herbage apprehension by animals. However, the herbage allowance limited the herbage intake by animals on the pasture with 15cm height, consequently the average daily gain was lower, but greater stocking rate was utilized. Therefore, the live weight gain per area was similar between the 15 and 30cm pastures, and higher than the 45cm high pasture. During the wet season, the marandu palisadegrass, subjected to continuous stocking, must be utilized between 15 and 30cm height.

Isolation of Trypanosoma caninum in domestic dogs in Rio de Janeiro, Brazil.

SUMMARY: The domestic dog's involvement with different members of the Trypanosomatidae family has been the focus of several studies due to this animal's close proximity to man. Recently this animal has been infected by a new Trypanosoma species (T. caninum), described in Rio de Janeiro and 19 similar isolates were later obtained. The objective of this study was to identify these isolates. All samples were isolated from intact skin cultures and analysed morphologically, by biochemical isoenzyme electrophoresis assays and by several molecular PCR assays. Additionally, anti-Leishmania sp. antibodies were assessed using the indirect Immunofluorescence Antibody Test (IFAT) in all animals. The methodologies employed to identify the isolates, including partial nucleotide sequences of 18S rRNA gene, indicated patterns identical to T. caninum and patterns different from the other species, including T. cruzi and T. rangeli samples. A phylogenetic tree constructed with the partial 18S ribosomal sequence shows that T. caninum is clustered with T. pestanai. Ten (52.6%) animals presented anti-Leishmania sp. antibodies with titres varying from 1:40 to 1:320. Thus, the hypothesis that this protozoan has disseminated among the dogs in Rio de Janeiro must be considered. The importance of a correct diagnosis in those animals and the possible consequences in the areas where visceral leishmaniasis is found are discussed here.

Parasitological diagnosis of canine visceral leishmaniasis: is intact skin a good target?

The objective of this study was to evaluate intact skin of seroreactive dogs as a possible target for the parasitological confirmation of canine visceral leishmaniasis (CVL). For this purpose, 394 dogs identified in serological surveys carried out in the metropolitan region of Belo Horizonte were studied. Blood was collected from all animals for serology and a tissue sample was obtained from two sites for parasitological diagnosis. Skin obtained from the ear and scapular region was simultaneously analyzed in 247 animals and lesion samples and ear skin were analyzed in 147 dogs. Leishmania parasites were isolated from 310 (78.7%) animals, and all isolates were identified as Leishmania chagasi. Simultaneous isolation from two sites was possible in 240 of the 310 animals, including ear and scapular skin in 151/247 (61.1%) and ear skin and skin lesions in 89/147 (60.5%). Ours results suggest that intact skin is one of the main target sites for the parasitological confirmation of CVL in seroreactive dogs.

Trypanosoma caninum n. sp. (Protozoa: Kinetoplastida) isolated from intact skin of a domestic dog ( Canis familiaris) captured in Rio de Janeiro, Brazil.

An unknown Trypanosoma species was isolated from an axenic culture of intact skin from a domestic dog captured in Rio de Janeiro, Brazil, which was co-infected with Leishmania (Viannia) braziliensis. Giemsa-stained smears of cultures grown in different media revealed the presence of epimastigotes, trypomastigotes, spheromastigotes, transitional stages, and dividing forms (epimastigotes or spheromastigotes). The highest frequency of trypomastigotes was observed in RPMI (15.2%) and DMEM (9.2%) media containing 5% FCS, with a mean length of these forms of 43.0 and 36.0 mum, respectively. Molecular analysis by sequential application of PCR assays indicated that this trypanosome differs from Trypanosoma cruzi and T. rangeli when specific primers were applied. On the other hand, a PCR strategy targeted to the D7 domain of 24salpha rDNA, using primers D75/D76, amplified products of about 250 bp in that isolate (stock A-27), different from the amplification products obtained with T. cruzi and T. rangeli. This organism differs from T. cruzi mainly by the size of its trypomastigote forms and kinetoplasts and the absence of infectivity for macrophages and triatomine bugs. It is also morphologically distinct from salivarian trypanosomes reported in Brazil. Isoenzyme analysis at 8 loci demonstrated a very peculiar banding pattern clearly distinct from those of T. rangeli and T. cruzi. We conclude that this isolate is a new Trypanosoma species. The name T. caninum is suggested.

Noncore components of the fission yeast gamma-tubulin complex.

Relatively little is known about the in vivo function of individual components of the eukaryotic gamma-tubulin complex (gamma-TuC). We identified three genes, gfh1+, mod21+, and mod22+, in a screen for fission yeast mutants affecting microtubule organization. gfh1+ is a previously characterized gamma-TuC protein weakly similar to human gamma-TuC subunit GCP4, whereas mod21+ is novel and shows weak similarity to human gamma-TuC subunit GCP5. We show that mod21p is a bona fide gamma-TuC protein and that, like gfh1Delta mutants, mod21Delta mutants are viable. We find that gfh1Delta and mod21Delta mutants have qualitatively normal microtubule nucleation from all types of microtubule-organizing centers (MTOCs) in vivo but quantitatively reduced nucleation from interphase MTOCs, and this is exacerbated by mutations in mod22+. Simultaneous deletion of gfh1p, mod21p, and alp16p, a third nonessential gamma-TuC protein, does not lead to additive defects, suggesting that all three proteins contribute to a single function. Coimmunoprecipitation experiments suggest that gfh1p and alp16p are codependent for association with a small "core" gamma-TuC, whereas mod21p is more peripherally associated, and that gfh1p and mod21p may form a subcomplex independently of the small gamma-TuC. Interestingly, sucrose gradient analysis suggests that the major form of the gamma-TuC in fission yeast may be a small complex. We propose that gfh1p, mod21p, and alp16 act as facultative "noncore" components of the fission yeast gamma-TuC and enhance its microtubule-nucleating ability.