Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays

Atitudes do enfermeiro face às infecções associadas aos cuidados de saúde

ENQUADRAMENTO: As infecções adquiridas em consequência da prestação de cuidados constitui um problema grave, com morbilidade e mortalidade significativas. Estas infecções são evitáveis em cerca de um terço dos casos, pelo que a capacidade de as prevenir pode constituir um dos indicados de qualidade dos cuidados. É um problema escondido, transversal que nenhuma instituição ou país ainda conseguiu resolver. Em cada ano, centenas de milhares de doentes em todo o mundo são afectados pelas IACS, sendo considerado o evento adverso mais frequente durante a prestação de cuidados, em todo o mundo. OBJECTIVOS: Caracterizar as Atitudes adoptadas pelos Enfermeiros face às IACS; Analisar em que medida as variáveis sócio-profissionais e a formação acerca das IACS influenciam as atitudes do Enfermeiro MÉTODOS: O estudo transversal de natureza observacional assenta numa lógica de análise descritivo – correlaciona e foi realizado numa amostra definida pelo método não probabilístico, de forma acidental, durante um período de tempo, constituída por 142 Enfermeiros prestadores de cuidados na ULS da Guarda. O perfil médio revela enfermeiros do sexo feminino (76,80%), com idade média de 36 anos, a exercer a profissão entre os 10-19 anos (40,80%) com uma média 13,04 anos, licenciados (69,50%), com a categoria profissional de Enfermeiro graduado (50,00%), com horário na instituição de 35h/semana (70,40%), com vínculo à Função pública (61,30%) e sem duplo emprego (77,47%). De forma a caracterizar as atitudes do enfermeiro, utilizou-se um instrumento de colheita de dados constituído por uma caracterização sócio-profissional e formação acerca das IACS, a Escala de Atitudes face às IACS (Barroca, Cruz, Gaspar e Valdemar, 2006) e uma resposta aberta acerca das condições no serviço que favorecem a infecção nosocomial. RESULTADOS: Constatou-se que os enfermeiros adoptam atitudes consideradas adequadas, com uma média de 103 respostas adequadas. Nas variáveis pessoais (sexo e idade) infere-se que não há relação entre o sexo e as atitudes do enfermeiro face às IACS. Das variáveis profissionais dos enfermeiros, apenas o duplo emprego influência as atitudes do enfermeiro face às IACS. Por outro lado inferimos que existe uma relação de dependência entre os enfermeiros com formação acerca das IACS e as atitudes do enfermeiro face às IACS. A idade, o tempo de exercício profissional e o número de horas de formação não se revelam preditoras das atitudes do enfermeiro face às IACS. CONCLUSÕES: As evidências encontradas neste estudo referem que o duplo emprego e a formação dos profissionais acerca das IACS influência as atitudes do enfermeiro face a esta problemática. As soluções para os problemas das IACS baseiam-se em medidas de prevenção e controlo de infecção, que requerem a responsabilização e a mudança de comportamentos por parte dos profissionais de saúde.ABSTRACT FRAMEWORK: Acquired infections as a result of prevision of care remain a serious problem of significant morbidity and mortality. These infections are avoidable by almost a third of the cases thus the preventing capacity could be a quality indication of cares. This is a hidden transversal problem which still has been solved by neither institution nor country. Each year, many hundreds of thousands of patients worldwide are affected by the IACS, it may be viewed as the most frequent adverse event during the prevision of care elsewhere in the world. OBJECTIVES: To characterize the attitude adopted by nurses in the face of IACS; to analyse what extent the socio-professional variables and the formation regarding the IACS influence the nurse´s attitudes. METHODS: The transversal study of the observational nature is based on descriptive analysis defined accidentally not by the provavilistic method– It was made during a certain period of time with a random sample composed of 142 nurses care providers at the ULS in Guarda. The average profile shows nurses of the female sex (76,80%), with average of 36 years of age, practising the profession between 10 to 19 years (40,80%) an average of 13,04 years, graduates (69,50%), with the professional category of graduated nurse (50,00%), with institution's schedule of 35-hour workweek (70,40%), connected with the public service (61,30%) and without double employment (77,47%). In order to characterise the nurse´s attitudes, collection of data was made with an instrument constituted by a socio-professional characterization and formation , the scale of attitudes regarding the IACS (Barroca, Cruz, Gaspar e Valdemar, 2006) as well as an open answer regarding the conditions of the service which can lead to nosocomial infection. RESULTS: It was found the nurses adopt attitudes considered adequate, with an average of 103 adequate answers. In relation to the personal variables (gender and age) we can conclude that there is no relation between the gender of the nurse and his attitude towards the IACS. Relative to the nurse professional variables, only double employment influences his attitude towards the IACS. In other hand, we can conclude that there is a dependent relation between nurses with preparation on IACS and is attitude towards the IACS. Age, professional experience and number of formation hours cannot predict the conduct from nurses towards IACS. CONCLUSIONS: We found evidences in this study that double employment and formation from the professional about the IACS, influences his conduct towards this problem. Solutions to the IACS problem reside in prevention and control of infection which requires responsibility and change in the health professional’s behaviour

P4-ATPases:lipid flippases in cell membranes

Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates, including lysophospholipids and synthetic alkylphospholipids. At the same time, the cellular processes known to be directly or indirectly affected by this class of transporters have expanded to include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell

Biophysical study of resin acid effects on phospholipid membrane 1 structure and properties

Hydrophobic resin acids (RAs) are synthesized by conifer trees as part of their defense mechanisms. One of the functions of RAs in plant defense is suggested to be the perturbation of the cellular membrane. However, there is a vast diversity of chemical structures within this class of molecules, and there are no clear correlations to the molecular mechanisms behind the RA's toxicity. In this study we unravel the molecular interactions of the three closely related RAs dehydroabietic acid, neoabietic acid, and the synthetic analogue dichlorodehydroabietic acid with dipalmitoylphosphatidylcholine (DPPC) model membranes and the polar lipid extract of soybeans. The complementarity of the biophysical techniques used (NMR, DLS, NR, DSC, Cryo-TEM) allowed correlating changes at the vesicle level with changes at the molecular level and the co-localization of RAs within DPPC monolayer. Effects on DPPC membranes are correlated with the physical chemical properties of the RA and their toxicity

Aminoglycerophospholipid flipping and P4-ATPases in Toxoplasma gondii

Lipid flipping in the membrane bilayers is a widespread eukaryotic phenomenon that is catalyzed by assorted P4-ATPases. Its occurrence, mechanism, and importance in apicomplexan parasites have remained elusive, however. Here we show that Toxoplasma gondii, an obligate intracellular parasite with high clinical relevance, can salvage phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) but not phosphatidylcholine (PtdCho) probes from its milieu. Consistently, the drug analogs of PtdCho are broadly ineffective in the parasite culture. NBD-PtdSer imported to the parasite interior is decarboxylated to NBD-PtdEtn, while the latter is not methylated to yield PtdCho, which confirms the expression of PtdSer decarboxylase but a lack of PtdEtn methyltransferase activity and suggests a role of exogenous lipids in membrane biogenesis of T. gondii. Flow cytometric quantitation of NBD-probes endorsed the selectivity of phospholipid transport and revealed a dependence of the process on energy and protein. Accordingly, our further work identified five P4-ATPases (TgP4-ATPase1-5), all of which harbor the signature residues and motifs required for phospholipid flipping. Of the four proteins expressed during the lytic cycle, TgP4-ATPase1 is present in the apical plasmalemma; TgP4-ATPase3 resides in the Golgi network along with its noncatalytic partner Ligand Effector Module 3 (TgLem3), whereas TgP4-ATPase2 and TgP4-ATPase5 localize in the plasmalemma as well as endo/cytomembranes. Last but not least, auxin-induced degradation of TgP4-ATPase1-3 impaired the parasite growth in human host cells, disclosing their crucial roles during acute infection. In conclusion, we show selective translocation of PtdEtn and PtdSer at the parasite surface and provide the underlying mechanistic and physiological insights in a model eukaryotic pathogen.Peer Reviewe

Transbilayer dynamics of phospholipids in the plasma membrane of the <em>Leishmania</em> genus

Protozoans of the Leishmania genus are the etiological agents of a wide spectrum of diseases commonly known as leishmaniases. Lipid organization of the plasma membrane of the parasite may mimic the lipid organization of mammalian apoptotic cells and play a role in phagocytosis and parasite survival in the mammal host. Here, we analyzed the phospholipid dynamics in the plasma membrane of both the L. (Leishmania) and the L. (Viannia) subgenera. We found that the activity and substrate specificity of the inward translocation machinery varied between Leishmania species. The differences in activity of inward phospholipid transport correlated with the different sensitivities of the various species towards the alkyl-phospholipid analogue miltefosine. Furthermore, all species exhibited a phospholipid scramblase activity in their plasma membranes upon stimulation with calcium ionophores. However, binding of annexin V to the parasite surface was only detected for a subpopulation of parasites during the stationary growth phase and only marginally enhanced by scramblase activation

The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2:A phosphatidylserine flippase

Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In the present study, we succeeded in purifying recombinant aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in Arabidopsis thaliana, in complex with the ALA-interacting subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2-ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Small changes in the stereochemistry or the functional groups of the phosphatidylserine head group affected enzymatic activity, whereas alteration in the length and composition of the acyl chains only had minor effects. Likewise, the enzymatic activity of the ALA2-ALIS5 complex was stimulated by both mono- and di-acyl phosphatidylserines. Taken together, the results identify the lipid head group as the key structural element for substrate recognition by the P4 ATPase