Extracellular Vesicles Regulate Biofilm Formation and Yeast-to-Hypha Differentiation in Candida albicans.

In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation in vitro. By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. IMPORTANCE The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.

Loss of lamin-B1 and defective nuclear morphology are hallmarks of astrocyte senescence in vitro and in the aging human hippocampus.

The increase in senescent cells in tissues, including the brain, is a general feature of normal aging and age-related pathologies. Senescent cells exhibit a specific phenotype, which includes an altered nuclear morphology and transcriptomic changes. Astrocytes undergo senescence in vitro and in age-associated neurodegenerative diseases, but little is known about whether this process also occurs in physiological aging, as well as its functional implication. Here, we investigated astrocyte senescence in vitro, in old mouse brains, and in post-mortem human brain tissue of elderly. We identified a significant loss of lamin-B1, a major component of the nuclear lamina, as a hallmark of senescent astrocytes. We showed a severe reduction of lamin-B1 in the dentate gyrus of aged mice, including in hippocampal astrocytes, and in the granular cell layer of the hippocampus of post-mortem human tissue from non-demented elderly. The lamin-B1 reduction was associated with nuclear deformations, represented by an increased incidence of invaginated nuclei and loss of nuclear circularity in senescent astrocytes in vitro and in the aging human hippocampus. We also found differences in lamin-B1 levels and astrocyte nuclear morphology between the granular cell layer and polymorphic layer in the elderly human hippocampus, suggesting an intra-regional-dependent aging response of human astrocytes. Moreover, we described senescence-associated impaired neuritogenic and synaptogenic capacity of mouse astrocytes. Our findings show that reduction of lamin-B1 is a conserved feature of hippocampal cells aging, including astrocytes, and shed light on significant defects in nuclear lamina structure which may contribute to astrocyte dysfunctions during aging.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and glial cells: Insights and perspectives.

In December 2019, a pneumonia outbreak was reported in Wuhan, Hubei province, China. Since then, the World Health Organization declared a public health emergency of international concern due to a growing number of deaths around the globe, as well as unparalleled economic and sociodemographic consequences. The disease called coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel form of human coronavirus. Although coronavirus infections have been associated with neurological manifestations such as febrile seizures, convulsions, change in mental status, and encephalitis, less is known about the impact of SARS-CoV-2 in the brain. Recently, emerging evidence suggests that SARS-CoV-2 is associated with neurological alterations in COVID-19 patients with severe clinical manifestations. The molecular and cellular mechanisms involved in this process, as well as the neurotropic and neuroinvasive properties of SARS-CoV-2, are still poorly understood. Glial cells, such as astrocytes and microglia, play pivotal roles in the brain response to neuroinflammatory insults and neurodegenerative diseases. Further, accumulating evidence has shown that those cells are targets of several neurotropic viruses that severely impact their function. Glial cell dysfunctions have been associated with several neuroinflammatory diseases, suggesting that SARS-CoV-2 likely has a primary effect on these cells in addition to a secondary effect from neuronal damage. Here, we provide an overview of these data and discuss the possible implications of glial cells as targets of SARS-CoV-2. Considering the roles of microglia and astrocytes in brain inflammatory responses, we shed light on glial cells as possible drivers and potential targets of therapeutic strategies against neurological manifestations in patients with COVID-19. The main goal of this review is to highlight the need to consider glial involvement in the progression of COVID-19 and potentially include astrocytes and microglia as mediators of SARS-CoV-2-induced neurological damage.

Protective effect of fungal extracellular vesicles against murine candidiasis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFß, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.

Uptake and persistence of bacterial magnetite magnetosomes in a mammalian cell line: Implications for medical and biotechnological applications.

Magnetotactic bacteria biomineralize intracellular magnetic nanocrystals surrounded by a lipid bilayer called magnetosomes. Due to their unique characteristics, magnetite magnetosomes are promising tools in Biomedicine. However, the uptake, persistence, and accumulation of magnetosomes within mammalian cells have not been well studied. Here, the endocytic pathway of magnetite magnetosomes and their effects on human cervix epithelial (HeLa) cells were studied by electron microscopy and high spatial resolution nano-analysis techniques. Transmission electron microscopy of HeLa cells after incubation with purified magnetosomes showed the presence of magnetic nanoparticles inside or outside endosomes within the cell, which suggests different modes of internalization, and that these structures persisted beyond 120 h after internalization. High-resolution transmission electron microscopy and electron energy loss spectra of internalized magnetosome crystals showed no structural or chemical changes in these structures. Although crystal morphology was preserved, iron oxide crystalline particles of approximately 5 nm near internalized magnetosomes suggests that minor degradation of the original mineral structures might occur. Cytotoxicity and microscopy analysis showed that magnetosomes did not result in any apparent effect on HeLa cells viability or morphology. Based on our results, magnetosomes have significant biocompatibility with mammalian cells and thus have great potential in medical, biotechnological applications.

Applications of Magnetotactic Bacteria, Magnetosomes and Magnetosome Crystals in Biotechnology and Nanotechnology: Mini-Review.

Magnetotactic bacteria (MTB) biomineralize magnetosomes, which are defined as intracellular nanocrystals of the magnetic minerals magnetite (Fe3O4) or greigite (Fe3S4) enveloped by a phospholipid bilayer membrane. The synthesis of magnetosomes is controlled by a specific set of genes that encode proteins, some of which are exclusively found in the magnetosome membrane in the cell. Over the past several decades, interest in nanoscale technology (nanotechnology) and biotechnology has increased significantly due to the development and establishment of new commercial, medical and scientific processes and applications that utilize nanomaterials, some of which are biologically derived. One excellent example of a biological nanomaterial that is showing great promise for use in a large number of commercial and medical applications are bacterial magnetite magnetosomes. Unlike chemically-synthesized magnetite nanoparticles, magnetosome magnetite crystals are stable single-magnetic domains and are thus permanently magnetic at ambient temperature, are of high chemical purity, and display a narrow size range and consistent crystal morphology. These physical/chemical features are important in their use in biotechnological and other applications. Applications utilizing magnetite-producing MTB, magnetite magnetosomes and/or magnetosome magnetite crystals include and/or involve bioremediation, cell separation, DNA/antigen recovery or detection, drug delivery, enzyme immobilization, magnetic hyperthermia and contrast enhancement of magnetic resonance imaging. Metric analysis using Scopus and Web of Science databases from 2003 to 2018 showed that applied research involving magnetite from MTB in some form has been focused mainly in biomedical applications, particularly in magnetic hyperthermia and drug delivery.

Culture-independent characterization of a novel magnetotactic member affiliated to the Beta class of the Proteobacteria phylum from an acidic lagoon.

Magnetotactic bacteria (MTB) comprise a group of motile microorganisms common in most mesothermal aquatic habitats with pH values around neutrality. However, during the last two decades, a number of MTB from extreme environments have been characterized including: cultured alkaliphilic strains belonging to the Deltaproteobacteria class of the Proteobacteria phylum; uncultured moderately thermophilic strains belonging to the Nitrospirae phylum; cultured and uncultured moderately halophilic or strongly halotolerant bacteria affiliated with the Deltaproteobacteria and Gammaproteobacteria classes and an uncultured psychrophilic species belonging to the Alphaproteobacteria class. Here, we used culture-independent techniques to characterize MTB from an acidic freshwater lagoon in Brazil (pH ∼ 4.4). MTB morphotypes found in this acidic lagoon included cocci, rods, spirilla and vibrioid cells. Magnetite (Fe3 O4 ) was the only mineral identified in magnetosomes of these MTB while magnetite magnetosome crystal morphologies within the different MTB cells included cuboctahedral (present in spirilla), elongated prismatic (present in cocci and vibrios) and bullet-shaped (present in rod-shaped cells). Intracellular pH measurements using fluorescent dyes showed that the cytoplasmic pH was close to neutral in most MTB cells and acidic in some intracellular granules. Based on 16S rRNA gene phylogenetic analyses, some of the retrieved gene sequences belonged to the genus Herbaspirillum within the Betaproteobacteria class of the Proteobacteria phylum. Fluorescent in situ hybridization using a Herbaspirillum-specific probe hybridized with vibrioid MTB in magnetically-enriched samples. Transmission electron microscopy of the Herbaspirillum-like MTB revealed the presence of many intracellular granules and a single chain of elongated prismatic magnetite magnetosomes. Diverse populations of MTB have not seemed to have been described in detail in an acid environment. In addition, this is the first report of an MTB phylogenetically affiliated with Betaproteobacteria class.

Analysis of Yeast Extracellular Vesicles.

Extracellular vesicles (EV) are important carriers of biologically active components in a number of organisms, including fungal cells. Experimental characterization of fungal EVs suggested that these membranous compartments are likely involved in the regulation of several biological events. In fungal pathogens, these events include mechanisms of disease progression and/or control, suggesting potential targets for therapeutic intervention or disease prophylaxis. In this manuscript we describe methods that have been used in the last 10 years for the characterization of EVs produced by yeast forms of several fungal species. Experimental approaches detailed in this chapter include ultracentrifugation methods for EV fractionation, chromatographic approaches for analysis of EV lipids, microscopy techniques for analysis of both intracellular and extracellular vesicular compartments, interaction of EVs with host cells, and physical chemical analysis of EVs by dynamic light scattering.

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans.

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-ß) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.

Intercellular transfer of tissue factor via the uptake of tumor-derived microvesicles.

Coagulation proteins play a critical role in numerous aspects of tumor biology. Cancer cells express tissue factor (TF), the protein that initiates blood clotting, which frequently correlates with processes related to cell aggressiveness, including primary tumor growth, invasion, and metastasis. It has been demonstrated that TF gets incorporated into tumor-derived microvesicles (MVs), a process that has been correlated with cancer-associated thrombosis. Here, we describe the exchange of TF-bearing MVs between breast cancer cell lines with different aggressiveness potential. The highly invasive and metastatic MDA-MB-231 cells displayed higher surface levels of functional TF compared with the less aggressive MCF-7 cells. MVs derived from MDA-MB-231 cells were enriched in TF and accelerated plasma coagulation, but MCF-7 cell-derived MVs expressed very low levels of TF. Incubating MCF-7 cells with MDA-MB-231 MVs significantly increased the TF activity. This phenomenon was not observed upon pretreatment of MVs with anti-TF or annexin-V, which blocks phosphatidylserine sites on the surface of MVs. Our data indicated that TF-bearing MVs can be transferred between different populations of cancer cells and may therefore contribute to the propagation of a TF-related aggressive phenotype among heterogeneous subsets of cells in a tumor.

O percurso de resiliência da mulher vítima de violência conjugal / The resilience route of women victims of conjugal violence

Resumo: Trata-se de pesquisa descritiva com abordagem qualitativa a luz do conceito de resiliência, realizada em uma Delegacia da Mulher localizada no município de Guarapuava, Paraná, de dezembro de 2011 a fevereiro de 2012, com 10 mulheres vítimas de violência conjugal, que procuraram o serviço policial para representar judicialmente contra o seu agressor. Teve como objetivo identificar o percurso de resiliência da mulher vítima de violência conjugal. A coleta dos discursos ocorreu mediante entrevista aberta, gravada, e da análise de conteúdo temática, emergiram os seguintes temas: Ameaça à vida e a integridade da família: o início do percurso da resiliência, e Superação da experiência vivida: a adaptação à nova realidade. O perfil das participantes evidenciou que sua idade variou entre 19 e 47 anos, todas eram separadas e tinham filhos, e o ensino fundamental incompleto foi a escolaridade mais predominante entre elas. Em relação à profissão ou ocupação, foi mencionada a de doméstica, comerciante, cabeleireira, cozinheira e do lar. O tipo de violência que sofreram foi a física, psicológica, patrimonial e ameaça contra a vida. A análise dos depoimentos revelou que as participantes da pesquisa decidiram enfrentar a situação e representar contra seu companheiro-agressor quando perceberam que a sua vida, bem como a integridade física de seus filhos e familiares estavam ameaçadas. Os fatores como o apoio encontrado na família e na Delegacia da Mulher tiveram um papel essencial para que as mulheres se sentissem fortalecidas, recuperassem a auto estima e buscassem um novo sentido para sua vida. Este se deu por meio do retorno ao estudo, ou o desejo da retornar à escola, pela busca por um emprego formal, e pela capacidade de projetar o futuro. O processo de enfrentamento da violência fez com que as mulheres avaliassem os danos que a violência acarreta para todos que nela estão envolvidos, se afastassem do convívio com o agressor e procurassem um novo cenário para a sua existência, e, dessa forma, continuaram o percurso de resiliência, mediante a superação do trauma vivido e adaptação a uma nova realidade. Ao finalizar esta pesquisa, foi possível vislumbrar que o conceito de resiliência pode ser explorado na prática cotidiana das enfermeiras, no sentido de transformá-las em tutoras de resiliência.

Abstract: This is a descriptive qualitative approach focusing the concept of resilience, performed in a Women's Police Station located in Guarapuava, Parana, from December 2011 to February 2012, with 10 women victims of conjugal violence, who have looked for the police service to represent in court against their abuser. It aimed to identify the resilience route in women victim of domestic violence. The speeches collection occurred through open interview, it has been recorded, and through the analysis, the following subjects have emerged: Threat to life and the family integrity, the beginning of the resilience journey, and the overcoming of the lived experience: adaptation to new reality. The participants profile revealed that their ages ranged between 19 and 47 years old, all of them were divorced and had children, and elementary school education was predominant among them. About the profession or occupation, was mentioned maid, trader, hairdresser and cook. The kind of violence suffered was physical, psychological, patrimonial and threat to life. The statements analysis revealed that the participants have decided to confront the situation and act against their fellow offending when they realized that their lives as the physical integrity of their children and families were threatened. The support that these women received from their families and women police station were essential to make them feeling empowered, regain self esteem and seek for a new meaning to their life. This has happened by their return to school, or desire of returning to study, the search for formal employment, and the ability to project the future. the process of facing and overcoming the violence has made this women to evaluate the damages that it brings to all who are involved, get distance from the abuser and seek for a new place for their existence, and this way, continued the resilience route by overcoming the trauma experienced and adapting to a new reality. At the end of this research, it was possible to understand that the concept of resilience can be explored in the nurses everyday practice in order to transform them into resilience tutors.

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans.

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.

Causas de internação hospitalar de idosos residentes em um município do Paraná, uma análise dos últimos 5 anos / Causes of hospitalization of elderly residents in a city of Parana, an analysis of the last 5 years

O objetivo da pesquisa foi descrever o perfil das internações hospitalares de idosos residentes em Guarapuava-PR, segundo sexo e idade nos últimos 5 anos. Estudo descritivo cujos dados foram coletados na base de dados do Sistema de Informações Hospitalares no período de setembro a dezembro de 2010. As quatro doenças, com maior prevalência de internações foram do aparelho circulatório, respiratório, digestório e neoplasias, com um total de 7.069, 6.123, 1.931 e 1.676 internações, respectivamente. As internações pelo sexo feminino foram maiores nas doenças do aparelho circulatório, respiratório, já nas doenças do sistema digestório e nas neoplasias, a prevalência foi maior no sexo masculino. Com estes dados observa-se a necessidade de novas políticas públicas, revisão na assistência hospitalar, para oferecer ao estrato idoso um atendimento efetivo e com qualidade.

The research objective was to describe the profile of hospital morbidity and mortality of elderly residents in Guarapuava/PR, by sex and age in the last five years. Descriptive study with data collected in the database of the Hospital Information System in the period September to December 2010. The four diseases with higher prevalence of hospitalizations were the circulatory, respiratory, digestive and neoplasms, with a total of 7,069, 6,123, 1,931 and 1,676 admissions, respectively. Hospitalizations were higher for females, diseases of the circulatory, respiratory, already in the digestive system diseases and cancer, the prevalence was higher in males. With these data there is a need for new policies, review in hospital care, to offer to this age population with an effective and quality service.

El objetivo de la pesquisa fue describir el perfil de las internaciones hospitalarias de ancianos residentes en Guarapuava/PR, por sexo y edad en los últimos cinco años. Estudio descriptivo con los datos recogidos en la base de datos del Sistema de Información Hospitalaria en el período de septiembre a diciembre de 2010. Las cuatro enfermedades con mayor prevalencia de hospitalizaciones fueron del sistema circulatorio, respiratorio, digestivo y neoplasias, con un total de 7.069, 6.123, 1.931 y 1.676 internaciones, respectivamente. Las hospitalizaciones por las mujeres fueron mayores en las enfermedades del aparato circulatorio y respiratorio, además en las enfermedades del sistema digestivo y en las neoplasias, la prevalencia fue mayor en los hombres. Con estos datos hay una necesidad de nuevas políticas, las revisiones en la atención hospitalaria, para ofrecer a esta población de edad con un servicio eficaz y de calidad.