Multimode chromatography-based techniques for high purity isolation of extracellular vesicles from human blood plasma.

Extracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation-based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar biophysical characteristics of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography-based strategies. On average, we identified 716 ± 68 and 1054 ± 35 protein groups in EV isolates from 100 µL of plasma using multimode chromatography- and ultracentrifugation-based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications.

Circulating extracellular vesicles in human cardiorenal syndrome promote renal injury in a kidney-on-chip system.

BACKGROUNDCardiorenal syndrome (CRS) - renal injury during heart failure (HF) - is linked to high morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear.METHODSWe investigated the role of circulating EVs from patients with CRS on renal epithelial/endothelial cells using a microfluidic kidney-on-chip (KOC) model. The small RNA cargo of circulating EVs was regressed against serum creatinine to prioritize subsets of functionally relevant EV-miRNAs and their mRNA targets investigated using in silico pathway analysis, human genetics, and interrogation of expression in the KOC model and in renal tissue. The functional effects of EV-RNAs on kidney epithelial cells were experimentally validated.RESULTSRenal epithelial and endothelial cells in the KOC model exhibited uptake of EVs from patients with HF. HF-CRS EVs led to higher expression of renal injury markers (IL18, LCN2, HAVCR1) relative to non-CRS EVs. A total of 15 EV-miRNAs were associated with creatinine, targeting 1,143 gene targets specifying pathways relevant to renal injury, including TGF-ß and AMPK signaling. We observed directionally consistent changes in the expression of TGF-ß pathway members (BMP6, FST, TIMP3) in the KOC model exposed to CRS EVs, which were validated in epithelial cells treated with corresponding inhibitors and mimics of miRNAs. A similar trend was observed in renal tissue with kidney injury. Mendelian randomization suggested a role for FST in renal function.CONCLUSIONPlasma EVs in patients with CRS elicit adverse transcriptional and phenotypic responses in a KOC model by regulating biologically relevant pathways, suggesting a role for EVs in CRS.TRIAL REGISTRATIONClinicalTrials.gov NCT03345446.FUNDINGAmerican Heart Association (AHA) (SFRN16SFRN31280008); National Heart, Lung, and Blood Institute (1R35HL150807-01); National Center for Advancing Translational Sciences (UH3 TR002878); and AHA (23CDA1045944).

Comparison of Four Purification Methods on Serum Extracellular Vesicle Recovery, Size Distribution, and Proteomics.

In recent decades, the role played by extracellular vesicles in physiological and pathological processes has attracted attention. Extracellular vesicles are released by different types of cells and carry molecules that could become biomarkers for the diagnosis of diseases. Extracellular vesicles are also moldable tools for the controlled release of bioactive substances in clinical and therapeutic applications. However, one of the significant challenges when studying these exciting and versatile vesicles is the purification process, which presents significant difficulties in terms of lack of purity, yield, and reproducibility, reflected in unreliable data. Therefore, our objective in the present study was to compare the proteomic profile of serum-derived EVs purified using ExoQuick™ (Systems Biosciences), Total Isolation Kit (Life Technologies), Ultracentrifugation, and Ultrafiltration. Each technique utilized for purification has shown different concentrations and populations of purified particles. The results showed marked differences in distribution, size, and protein content, demonstrating the need to develop reproducible and reliable protocols to isolate extracellular vesicles for their clinical application.

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Evaluation of alveolar process resorption after tooth extraction using the socket shield technique without immediate installation of implants: a randomised controlled clinical trial.

Our aim was to evaluate resorption of the alveolar ridge using the socket shield technique (SST) without immediate placement of dental implants. This randomised controlled clinical trial included 27 patients: 14 maxillary non-molar teeth were partially extracted using the SST (test group) and 13 were extracted using a minimally traumatic extraction approach (control group). Alterations in height and thickness of the alveolar ridge were evaluated by cone beam computed tomograms taken immediately after, and 100 days after, surgery. Minor resorption was observed in the height of the buccal and palatal plates, without intergroup difference (p ≥ 0.10). The test group showed significantly better preservation of the buccal-to-palatal crest dimension (p ≤ 0.05). In the control group, preservation of buccal plate thickness was significantly greater (p ≤ 0.05), but intragroup vertical resorption of the buccal plate and reduction in the buccal-to-palatal crest distance were greater (p ≤ 0.05). The SST without the immediate placement of implants showed greater preservation of the buccal-to-palatal crest dimension and lower preservation of buccal wall thickness compared with minimally traumatic extraction. In addition, it provided superior maintenance of the baseline buccal wall height. The modified SST is a promising approach, but factors that interfere with the results should be investigated.

Effects of endurance racing on horse plasma extracellular particle miRNA.

BACKGROUND: Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non-coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long-duration/low intensity exercise in plasma EP miRNAs. OBJECTIVES: To evaluate the effects of 160 km endurance racing on horse plasma extracellular particles and their miRNA population. STUDY DESIGN: Cohort study. METHODS: We collected plasma from five Arabian horses during five time-points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano) and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. RESULTS: Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2 hours after the race, which returned to baseline 15 hours after the race. MicroRNA differential expression analysis revealed increasing levels of eca-miR-486-5p during and after the race, and decreasing levels of eca-miR-9083 after the end. CONCLUSIONS: This study adds new data about the variation in plasma EP concentrations after long-distance exercise and brings new insights about the roles of exercise-derived EP miRNAs during low-intensity endurance exercise.

Transcriptional Remodeling Patterns in Murine Dendritic Cells Infected with Paracoccidioides brasiliensis: More Is Not Necessarily Better.

Most people infected with the fungus Paracoccidioides spp. do not get sick, but approximately 5% develop paracoccidioidomycosis. Understanding how host immunity determinants influence disease development could lead to novel preventative or therapeutic strategies; hence, we used two mouse strains that are resistant (A/J) or susceptible (B10.A) to P. brasiliensis to study how dendritic cells (DCs) respond to the infection. RNA sequencing analysis showed that the susceptible strain DCs remodeled their transcriptomes much more intensely than those from the resistant strain, agreeing with a previous model of more intense innate immunity response in the susceptible strain. Contrastingly, these cells also repress genes/processes involved in antigen processing and presentation, such as lysosomal activity and autophagy. After the interaction with P. brasiliensis, both DCs and macrophages from the susceptible mouse reduced the autophagy marker LC3-II recruitment to the fungal phagosome compared to the resistant strain cells, confirming this pathway's repression. These results suggest that impairment in antigen processing and presentation processes might be partially responsible for the inefficient activation of the adaptive immune response in this model.

Laccase Affects the Rate of Cryptococcus neoformans Nonlytic Exocytosis from Macrophages.

Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans, we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis.IMPORTANCECryptococcus neoformans is a yeast that causes severe disease, primarily in immunosuppressed people. It has many attributes that allow it to survive and cause disease, such as a polysaccharide capsule and the dark pigment melanin produced by the laccase enzyme. Upon infection, the yeast is ingested by cells called macrophages, whose function is to kill them. Instead, these fungal cells can exit from macrophages in a process called nonlytic exocytosis. We know that this process is controlled by both host and fungal factors, only some of which are known. As part of an ongoing study, we observed that C. neoformans isolates that produce melanin faster are more-frequent targets of nonlytic exocytosis. Further experiments showed that this is probably due to higher production of laccase, because fungi lacking this enzyme are nonlytically exocytosed less often. This shows that laccase is an important signal/regulator of nonlytic exocytosis of C. neoformans from macrophages.

Detection of Extracellular Vesicle RNA Using Molecular Beacons.

Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets.

Effects of Acute Aerobic Exercise on Rats Serum Extracellular Vesicles Diameter, Concentration and Small RNAs Content.

Physical exercise stimulates organs, mainly the skeletal muscle, to release a broad range of molecules, recently dubbed exerkines. Among them, RNAs, such as miRNAs, piRNAs, and tRNAs loaded in extracellular vesicles (EVs) have the potential to play a significant role in the way muscle and other organs communicate to translate exercise into health. Low, moderate and high intensity treadmill protocols were applied to rat groups, aiming to investigate the impact of exercise on serum EVs and their associated small RNA molecules. Transmission electron microscopy, resistive pulse sensing, and western blotting were used to investigate EVs morphology, size distribution, concentration and EVs marker proteins. Small RNA libraries from EVs RNA were sequenced. Exercise did not change EVs size, while increased EVs concentration. Twelve miRNAs were found differentially expressed after exercise: rno-miR-128-3p, 103-3p, 330-5p, 148a-3p, 191a-5p, 10b-5p, 93-5p, 25-3p, 142-5p, 3068-3p, 142-3p, and 410-3p. No piRNA was found differentially expressed, and one tRNA, trna8336, was found down-regulated after exercise. The differentially expressed miRNAs were predicted to target genes involved in the MAPK pathway. A single bout of exercise impacts EVs and their small RNA load, reinforcing the need for a more detailed investigation into EVs and their load as mediators of health-promoting exercise.

The Effects of Acute and Chronic Exercise on Skeletal Muscle Proteome.

Skeletal muscle plasticity and its adaptation to exercise is a topic that is widely discussed and investigated due to its primary role in the field of exercise performance and health promotion. Repetitive muscle contraction through exercise stimuli leads to improved cardiovascular output and the regulation of endothelial dysfunction and metabolic disorders such as insulin resistance and obesity. Considerable improvements in proteomic tools and data analysis have broth some new perspectives in the study of the molecular mechanisms underlying skeletal muscle adaptation in response to physical activity. In this sense, this review updates the main relevant studies concerning muscle proteome adaptation to acute and chronic exercise, from aerobic to resistance training, as well as the proteomic profile of natural inbred high running capacity animal models. Also, some promising prospects in the muscle secretome field are presented, in order to better understand the role of physical activity in the release of extracellular microvesicles and myokines activity. Thus, the present review aims to update the fast-growing exercise-proteomic scenario, leading to some new perspectives about the molecular events under skeletal muscle plasticity in response to physical activity. J. Cell. Physiol. 232: 257-269, 2017. © 2016 Wiley Periodicals, Inc.

Circulating miR-1, miR-133a, and miR-206 levels are increased after a half-marathon run.

CONTEXT: Circulating miRNAs are potential biomarkers that can be important molecules driving cell-to-cell communication. OBJECTIVE: To investigate circulating muscle-specific miRNAs in recreational athletes. MATERIALS AND METHODS: Three miRNAs from whole plasma before and after a half-marathon were analyzed by qPCR. RESULTS: MiR-1, -133a, and -206 significantly increased after the race. DISCUSSION: Increased levels of miRNAs after exercise point to potential biomarkers and to the possibility of being functional players following endurance training. CONCLUSION: These miRNAs are potential biomarkers of muscle damage or adaptation to exercise.

Cirurgias periodontais com finalidade protética / Periodontal surgery with prosthetic purpose

O sucesso de um tratamento restaurador é verificado mediante a integração das restaurações ou próteses com o sistema estomatognático. Sob o ponto de vista periodontal, nenhum tratamento reabilitador protético deve ser realizado sem antes estabelecer a saúde periodontal. Diversas são as técnicas cirúrgicas utilizadas com esta finalidade. Sob o ponto de vista protético, as cirurgias periodontais podem contribuir para uma maior retenção, longevidade e estética da prótese

Nova técnica para remoção de retentores intra-radiculares / New technique to intraradicular posts removal

A remoção de retentores intraradiculares constitui um procedimento difícil, arriscado e demorado, sendo, muitas vezes, um desafio para o cirurgião-dentista. A literatura descreve várias técnicas e aparelhos para a solução deste problema, como o em prego de ultra-som, o desgaste por meio de instrumentos rotatórios e a tração por alicates ou fórceps especiais. Este trabalho relata uma nova técnica para a remoção de retentores intra-radiculares metálicos fundidos, cimentados com fosfato de zinco.

Princípios biomecânicos dos núcleos metálicos fundidos: revisão de literatura / Biomechanical properties of cast post-core: literature review

A revisão de literatura apresentada neste artigo mostra opiniões relacionadas a restauração de dentes tratados endodonticamente. Os conceitos básicos para confecção de núcleo com retenção intra-radicular na reconstrução da parte coronária de dentes totalmente destruídos já foram largamente estudados, sendo utilizados importantes parâmetros. Sendo assim, o objetivo desse artigo é fazer uma revisão de literatura sobre a biomecânica dos pinos metálicos fundidos.

Resistência à tração de retentores intra-radiculares fundidos em cobre-alumínio cimentados e submetidos a preparo. Estudo in vitro / Traction resistance os posts and cores cast in copper-aluminum cimented and subjected to preparation. In vitro study

Este estudo avaliou a resistência à tração de retentores intra-radiculares fundidos em cobre-alumínio cimentados com cimento fosfato de zinco, submetidos a preparo da porção coronária com ponta diamantada em alta rotação durante 4 minutos. O objeto foi verificar a influência do preparo com alta rotação sobre a resistência à tração dos retentores intra-radiculares em períodos variados após a cimentação. Foram usados 36 incisivos centrais superiores humanos extraídos com canais radiculares instrumentados e obturados. O espaço para o pino metálico foi preparado com 9 mm de profundidade e os padrões foram obtidos pela técnica direta. Os 36 corpos-de-prova foram aleatoriamente divididos em três grupos de 12. O grupo I constituiu-se dos retentores que não foram submetidos a preparo pós-cimentação; o grupo II dos retentores submetidos a preparo 7 dias após a cimentação, e o grupo III dos retentores submetidos a preparo 15 minutos após cimentação. Todos os corpos-de-prova foram submetidos a 200 ciclos térmicos nas temperaturas de 5°C, 37°C e 55°C. O tracionamento foi feito numa máquina de ensaios com velocidade de 0,5 mm/min. Os resultados permitiram concluir que o preparo com alta rotação não produziu efeito sobre a resistência à tração dos retentores intra-radiculares fundidos em cobre-alumínio e não houve diferenças estatisticamente significantes na resistência à tração nos grupos preparados 15 minutos e 7 dias após a cimentação

Prevalência de perda de dentes posteriores tratados endodonticamente e não-reabilitados / Loss prevalence of posterior teeth endodontically treated and that still remained with no final restoration

Para estudar a prevalência de perda de dentes posteriores endodonticamente tratados na FOUFBA e que ainda s encontravam sem restaurações dentes foram utilizados. Foram avaliados 75 dentes através de exame clínico radiográfico (grupo comprovado) e 60 dentes mediante informações fornecidas pelos pacientes por ligação telefônica (grupo referido). Os molares apresentaram uma maior prevalência de perda do que os pré-molares em ambos os grupos. A prevalência de perda foi maior naqueles dentes que permanecerem sem reabilitação por mais tempo. O grupo comprovado apresentou uma prevalência de perda que variou de 4,5 à 85,7 por cento, de acordo com o ano em que o tratamento endodôntico foi realizado e o grupo referido apresentou uma prevalência de perda de 7,7 à 40 por cento, também de acordo com o ano em que o tratamento endodôntico foi realizado

Apresentaçäo de um novo molde vaginal empregado em neovaginoplastias / Presentation of a new acrylic molar employed in vaginal reconstruction

É descrito um novo molde de acrílico para neovaginoplastia