A methodological primer of extracellular vesicles isolation and characterization via different techniques.

We present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at -80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the "best method" for their work under their individual conditions.

Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Annexin A5 Inhibits Endothelial Inflammation Induced by Lipopolysaccharide-Activated Platelets and Microvesicles via Phosphatidylserine Binding.

Sepsis is caused by a dysregulated immune response to infection and is a leading cause of mortality globally. To date, no specific therapeutics are available to treat the underlying septic response. We and others have shown that recombinant human annexin A5 (Anx5) treatment inhibits pro-inflammatory cytokine production and improves survival in rodent sepsis models. During sepsis, activated platelets release microvesicles (MVs) with externalization of phosphatidylserine to which Anx5 binds with high affinity. We hypothesized that recombinant human Anx5 blocks the pro-inflammatory response induced by activated platelets and MVs in vascular endothelial cells under septic conditions via phosphatidylserine binding. Our data show that treatment with wildtype Anx5 reduced the expression of inflammatory cytokines and adhesion molecules induced by lipopolysaccharide (LPS)-activated platelets or MVs in endothelial cells (p < 0.01), which was not observed with Anx5 mutant deficient in phosphatidylserine binding. In addition, wildtype Anx5 treatment, but not Anx5 mutant, improved trans-endothelial electrical resistance (p < 0.05) and reduced monocyte (p < 0.001) and platelet (p < 0.001) adhesion to vascular endothelial cells in septic conditions. In conclusion, recombinant human Anx5 inhibits endothelial inflammation induced by activated platelets and MVs in septic conditions via phosphatidylserine binding, which may contribute to its anti-inflammatory effects in the treatment of sepsis.

Hemodialysis-Related Acute Brain Injury Demonstrated by Application of Intradialytic Magnetic Resonance Imaging and Spectroscopy.

SIGNIFICANCE STATEMENT: Hemodialysis (HD) results in reduced brain blood flow, and HD-related circulatory stress and regional ischemia are associated with brain injury over time. However, studies to date have not provided definitive direct evidence of acute brain injury during a HD treatment session. Using intradialytic magnetic resonance imaging (MRI) and spectroscopy to examine HD-associated changes in brain structure and neurochemistry, the authors found that multiple white (WM) tracts had diffusion imaging changes characteristic of cytotoxic edema, a consequence of ischemic insult and a precursor to fixed structural WM injury. Spectroscopy showed decreases in prefrontal N -acetyl aspartate (NAA) and choline concentrations consistent with energy deficit and perfusion anomaly. This suggests that one HD session can cause brain injury and that studies of interventions that mitigate this treatment's effects on the brain are warranted. BACKGROUND: Hemodialysis (HD) treatment-related hemodynamic stress results in recurrent ischemic injury to organs such as the heart and brain. Short-term reduction in brain blood flow and long-term white matter changes have been reported, but the basis of HD-induced brain injury is neither well-recognized nor understood, although progressive cognitive impairment is common. METHODS: We used neurocognitive assessments, intradialytic anatomical magnetic resonance imaging, diffusion tensor imaging, and proton magnetic resonance spectroscopy to examine the nature of acute HD-associated brain injury and associated changes in brain structure and neurochemistry relevant to ischemia. Data acquired before HD and during the last 60 minutes of HD (during maximal circulatory stress) were analyzed to assess the acute effects of HD on the brain. RESULTS: We studied 17 patients (mean age 63±13 years; 58.8% were male, 76.5% were White, 17.6% were Black, and 5.9% were of Indigenous ethnicity). We found intradialytic changes, including the development of multiple regions of white matter exhibiting increased fractional anisotropy with associated decreases in mean diffusivity and radial diffusivity-characteristic features of cytotoxic edema (with increase in global brain volumes). We also observed decreases in proton magnetic resonance spectroscopy-measured N -acetyl aspartate and choline concentrations during HD, indicative of regional ischemia. CONCLUSIONS: This study demonstrates for the first time that significant intradialytic changes in brain tissue volume, diffusion metrics, and brain metabolite concentrations consistent with ischemic injury occur in a single dialysis session. These findings raise the possibility that HD might have long-term neurological consequences. Further study is needed to establish an association between intradialytic magnetic resonance imaging findings of brain injury and cognitive impairment and to understand the chronic effects of HD-induced brain injury. CLINICAL TRIALS INFORMATION: NCT03342183 .

Cognitive Impairment Early After Initiating Maintenance Hemodialysis: A Cross Sectional Study.

Background: Abnormalities in cognitive function are almost universal in patients receiving hemodialysis (HD) and are associated with worse quality of life, impaired decision making, increased healthcare utilization and mortality. While cognitive impairment in the HD population is increasingly recognized, it is unclear how quickly it develops after starting HD. Methods: This was a cross-sectional study of a cohort of low dialysis vintage HD patients (<12 months). We used the validated Cambridge Brain Science (CBS) battery of web-based tests to evaluate cognition compared to age- and sex matched controls across three cognitive domains: verbal processing, reasoning and short-term memory. Results: Forty-nine HD patients were included in this study; 43 completed the full battery of tests. The average scores for HD patients were consistently below the age and sex-matched controls. Fifty-five percent of HD patients had cognitive impairment in verbal skills, 43% in reasoning and 18% in short-term memory. Conclusions: There is a high prevalence of CI evident early after starting HD, with the largest deficits seen in reasoning and verbal processing. These deficits may be attributable to the HD treatment itself. Further studies are needed to characterize the natural history of CI in this patient population and to test interventions aimed at preventing or slowing its progression.

Leishmanicidal effect of 1,3,4-thiadiazolium mesoionic salts on Leishmania amazonensis in vitro.

Leishmaniasis is a neglected broad clinical spectrum disease caused by protozoa of the genus Leishmania, which affect millions of people annually in the world and the treatment has severe side effects and resistant strains have been reported. Mesoionic salts are a subclass of the betaine group with extensive biological activity such as microbicide and anti-inflammatory In this work, we analyze the cytotoxic effects of mesoionic salts, 4-phenyl-5-(X-phenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (X = 4 Cl; 3,4 diCl and 3,4 diF), on Leishmania amazonensis in vitro. Initially, Mesoionic salts toxicity were evaluated by XTT assay on L. amazonensis promastigotes. Our results show that the mesoionic salts MI-3,4 diCl, MI-4 Cl and MI-3,4 diF were toxic to the promastigote parasite with IC50 values of 14.3, 40.1 and 61.8 µM, respectively. The amastigote survival was evaluated in treated infected-macrophages, and the results demonstrate that MI-4 Cl (IC50 = 33 µM) and MI-3,4 diCl (IC50 = 43 µM) have a toxic effect against these forms. None of the mesoionic compounds tested present host cell toxicity up to the tested concentration of 100 µM. The selectivity index for MI-3,4 diCl and MI-4 Cl were 3.94 and 6.97, respectively. Nitric oxide (NO) production assayed by Griess reagent, in LPS-activated macrophages or not, in the presence of the salts showed that only the MI-3,4 diCl compound reduced NO levels. Lipid profile analysis of treated-promastigotes showed no alteration of neutral lipids. Evaluation of mitochondrial membrane potential (∆Ψm) showed that the MI-4Cl compound was able to reduce (∆Ψm) by 50%. Therefore, our results suggest that the chlorinated compounds are promising biomolecules, which cause inhibition of L.amazonensis promastigotes, amastigotes, leading to mitochondrial damage.

Analytical Considerations in Nanoscale Flow Cytometry of Extracellular Vesicles to Achieve Data Linearity.

BACKGROUND: Platelet microparticles (PMPs) and their abundance in the blood are a prognostic biomarker in thrombotic disorders and cancer. Nanoscale flow cytometry (nFC) is ideal for high-throughput analysis of PMPs but these clinical assays have not been developed previously. OBJECTIVE: This article demonstrates that nFC is a suitable technology to enumerate PMPs present in plasma samples in a clinical setting. MATERIALS AND METHODS: nFC was performed using the Apogee A50-Micro instrument. Instrument settings and acquisition parameters were developed with the use of fluorescent beads and plasma samples. Sample preparation and handling was also optimized. RESULTS: nFC allows for linear detection of particles between approximately 200 and 1,000 nm based on calibration beads and was dependent on dilution factor and flow rate. Linearity in event analysis as samples became more diluted was lost when events approximately 100 nm were gated while linearity was maintained despite dilution of sample in events larger than 200 nm in diameter. Higher flow rates lead to an under-estimation of events analysed per microlitre of analyte and this was more pronounced when plasma samples were not diluted more than 1/20×. CONCLUSION: nFC offers multi-parametric analysis of PMPs when optimal calibration of acquisition and sample processing settings is performed. Analysis of plasmas from metastatic prostate cancer patients and leukaemia patients revealed that PMP levels were larger than 100 nm and were equally abundant in patients that responded to or failed androgen deprivation therapy or between patients representing different stages of leukaemia.

In Silico and in Vitro-Guided Identification of Inhibitors of Alkylquinolone-Dependent Quorum Sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections, posing a serious burden to public health, due to its antibiotic resistance. The P. aeruginosa Pseudomonas Quinolone System (pqs) quorum sensing system, driven by the activation of the transcriptional regulator, PqsR (MvfR) by alkylquinolone (AQ) signal molecules, is a key player in the regulation of virulence and a potential target for the development of novel antibacterial agents. In this study, we performed in silico docking analysis, coupled with screening using a P. aeruginosa mCTX::PpqsA-lux chromosomal promoter fusion, to identify a series of new PqsR antagonists. The hit compounds inhibited pyocyanin and alkylquinolone signal molecule production in P. aeruginosa PAO1-L and PA14 strains. The inhibitor Ia, which showed the highest activity in PA14, reduced biofilm formation in PAO1-L and PA14, increasing their sensitivity to tobramycin. Furthermore, the hepatic and plasma stabilities for these compounds were determined in both rat and human in vitro microsomal assays, to gain a further understanding of their therapeutic potential. This work has uncovered a new class of P. aeruginosa PqsR antagonists with potential for hit to lead optimisation in the search for quorum sensing inhibitors for future anti-infective drug discovery programs.

Genetic evidence for a novel variant of the pilus island 1 backbone protein in group B Streptococcus.

PURPOSE: Pili contribute significantly to the pathogenesis of infection of group B Streptococcus (GBS) by facilitating adhesion and invasion of host cells. GBS pilin subunits (the backbone pilin protein, BP, and the ancillary pilin proteins, AP) as well as the specific enzymes required for pilus assembly are encoded by genes located in two separate genomic regions, known as pilus island 1 (PI-1) and PI-2. Our aim was to characterize the pilus profile of a collection of GBS isolates from metropolitan Toronto, Canada. METHODOLOGY: The pilus profile of 1332 invasive and colonizing GBS isolates was determined by PCR and, in selected cases, by whole genome sequencing. RESULTS: While investigating the pilus profile of a collection of GBS organisms, we discovered that 51 isolates possessed a novel variant of the PI-1 BP, which we named BP-1b. The predicted translated sequences of archetypical GBS BP-1 and novel BP-1b variants shared only 63 % amino acid sequence homology. The novel BP-1b variant was most common among strains of serotype Ib and VI, but was also found among strains of serotypes Ia, II, III and VIII. CONCLUSION: We describe a relatively frequent occurrence of a novel PI-1 BP that cannot be detected by a commonly used multiplex PCR scheme, which could lead to strains being mistyped as PI-1 negative. We present PCR primers that can easily be incorporated into the multiplex PCR assay to identify strains with novel BP-1b variant.

Stakeholder Engagement in Trial Design: Survey of Visitors to Critically Ill Patients Regarding Preferences for Outcomes and Treatment Options during Weaning from Mechanical Ventilation.

RATIONALE: Stakeholder engagement in research is expected to provide unique insights, make research investments more accountable and transparent, and ensure that future research is applicable to patients and family members. OBJECTIVES: To inform the design of a trial of strategies for weaning from mechanical ventilation, we sought to identify preferences of patient visitors regarding outcome and treatment measures. METHODS: We conducted an interviewer-administered questionnaire of visitors of critically ill patients in two family waiting rooms serving three intensive care units (ICUs) in Toronto, Canada. Respondents rated the importance of general and ventilation-related outcomes in two hypothetical scenarios (before a first spontaneous breathing trial, and after a failed spontaneous breathing trial) and selected a preferred technique for the breathing trials. With regard to the patient they were visiting, respondents identified the most important outcome to them at ICU admission, during the ICU stay, and at ICU discharge. MEASUREMENTS AND MAIN RESULTS: We analyzed 322 questionnaires (95.5% response rate). All outcomes were highly rated (average range: 7.82-9.74). Across scenarios, outcomes rated as most important were ICU and hospital survival (9.72, 9.70), avoiding complications (9.45), quality of life (9.394), patient comfort (9.393), and returning to previous living arrangements (9.31). Overall, the most important ventilation-related outcomes were being ventilator-free (8.95), avoiding reintubation (8.905), and passing a spontaneous breathing trial (8.903). Passing a spontaneous breathing trial assumed greater importance after an initial failed attempt. "Time to event" outcomes were less important to visitors. We did not identify a preferred spontaneous breathing trial technique. Although ICU survival was the most important outcome at ICU admission and during the ICU stay, visitors rated quality of life higher than hospital survival at ICU discharge. CONCLUSIONS: Visitors to critically ill patients prioritized two general outcomes (ICU and hospital survival) and three ventilation-related outcomes (being ventilator free, avoiding reintubation, passing a spontaneous breathing trial), and valued avoiding complications, maintaining quality of life, comfort, and returning to previous living arrangements. The outcomes preferences of the survey respondents evolved temporally during the ICU stay.

Population structure and antimicrobial resistance of invasive serotype IV group B Streptococcus, Toronto, Ontario, Canada.

We recently showed that 37/600 (6.2%) invasive infections with group B Streptococcus (GBS) in Toronto, Ontario, Canada, were caused by serotype IV strains. We report a relatively high level of genetic diversity in 37 invasive strains of this emerging GBS serotype. Multilocus sequence typing identified 6 sequence types (STs) that belonged to 3 clonal complexes. Most isolates were ST-459 (19/37, 51%) and ST-452 (11/37, 30%), but we also identified ST-291, ST-3, ST-196, and a novel ST-682. We detected further diversity by performing whole-genome single-nucleotide polymorphism analysis and found evidence of recombination events contributing to variation in some serotype IV GBS strains. We also evaluated antimicrobial drug resistance and found that ST-459 strains were resistant to clindamycin and erythromycin, whereas strains of other STs were, for the most part, susceptible to these antimicrobial drugs.

Uma experiência de educação ambiental aplicada a trabalhadores do Sistema Único de Saúde / An experience on environmental education applied to public Health System Workers

Apesar do crescente interesse sobre a educação ambiental e saúde na sociedade, a formação acadêmica ainda tem se mantido distante quanto à capacitação de profissionais de saúde com conhecimento e envolvimento sobre as questões ambientais. A Universalidade, como um princípio do Sistema Único de Saúde de assegurar a saúde como um direito de todos e como um direito humano à manutenção da vida individual e social, torna imprescindível a formação dos profissionais de saúde neste âmbito. Trata-se de um relato de experiência de uma atividade desenvolvida por alunas do Mestrado Profissional em Ensino na Saúde da Faculdade de Medicina da Universidade Federal do Rio Grande do Sul que visou construir e aplicar estratégias pedagógicas de sensibilização aos trabalhadores de saúde para a educação ambiental, embasadas nos fundamentos do Sentipensar e no conceito de universalidade de modo a formar agentes multiplicadores para a educação ambiental. A experiência desenvolvida proporcionou interação com a temática contextualizada aos ambientes de trabalho.(AU)

Despite the growing interest in environmental education and health in society, academic courses of the health area still neglect the training of health professionals with knowledge about environmental issues. Universality, as a principle of the Public Health System to ensure health as a common right and as a human right to the maintenance of individual and social life, can only be attained through the training of health professionals with this conviction. This is an experience report of an activity developed by students of the Master Degree Program in Health Teaching of the Medicine School ate the Federal University of Rio Grande do Sul. The article reports the aim to build and apply teaching strategies to raise awareness on health workers for environmental education. This study is based in the fundamentals of Sentipensar and the universality concept in order to form multipliers for environmental education. The experience developed some interactions with its theme in the work environments.(AU)

As Coordenadorias Regionais de Saúde no processo de implementação do Sistema Único de Saúde / The Regional Cordination of Health in the process of implementation of the National Health Insurance

Trata-se de uma pesquisa de intervenção qualitativa no campo da gestão e da educação em saúde, focando o trabalho das Coordenadorias Regionais de Saúde (CRS) em relação a seu papel na efetivação do Sistema Único de Saúde (SUS) no Rio Grande do Sul (RS), na percepção de seus trabalhadores. Pensa o lugar das CRS, o que pode subsidiar a reflexão e o delineamento das ações de nosso lugar e da relação possível, o que é uma criação, uma operação e uma aposta junto aos trabalhadores em saúde. Revisita a trajetória da reforma sanitária brasileira, resgatando a influência italiana. Apresenta as construções e aprendizados realizados com o SUS em termos de direito à saúde. Compreende o direito à saúde como luta cotidiana que tem implicações objetivas e subjetivas ao lidar potencialmente com as adversidades decorrentes de um contexto de desigualdades sociais. Trabalha a dimensão da descentralização e a necessidade de melhor precisar papéis e funções entre as esferas, especialmente no âmbito das CRS. Situa a organização e as áreas de abrangência das CRS e a da saúde no estado em macrorregiões. Indica a necessidade de instâncias regionalizadas para a efetivação do trabalho do SUS, conforme a lei orgânica da saúde. Atualmente, o papel das CRS envolve articuação, descentralização/ regionalização, foco na gestão e relação/diálogo/ cooperação. Deste modo, cabe às CRS planejar, acompanhar e gerenciar as ações e serviços de saúde, em cooperação técnica, financeira e operacional com os gestores municipais, trabalhadores e prestadores de serviços de saúde em consonância com a SES/RS e tem como fundamento os princípios e diretrizes do SUS...