Optimization of ultracentrifugation-based method to enhance the purity and proteomic profiling depth of plasma-derived extracellular vesicles and particles.

Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as "PS"), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery.

Ragas: integration and enhanced visualization for single cell subcluster analysis.

SUMMARY: Subcluster analysis is a powerful means to improve clustering and characterization of single cell RNA-Seq data. However, there are no existing tools to systematically integrate results from multiple subclusters, which creates hurdles for accurate data quantification, visualization, and interpretation in downstream analysis. To address this issue, we developed Ragas, an R package that integrates multi-level subclustering objects for streamlined analysis and visualization. A new data structure was implemented to seamlessly connect and assemble miscellaneous single cell analyses from different levels of subclustering, along with several new or enhanced visualization functions. Moreover, a re-projection algorithm was developed to integrate nearest-neighbor graphs from multiple subclusters in order to maximize their separability on the combined cell embeddings, which significantly improved the presentation of rare and homogeneous subpopulations. AVAILABILITY AND IMPLEMENTATION: The Ragas package and its documentation can be accessed through https://github.com/jig4003/Ragas and its source code is also available at https://zenodo.org/records/11244921.

Non-criteria antiphospholipid antibodies and calprotectin as potential biomarkers in pediatric antiphospholipid syndrome.

Our study aimed to evaluate the presence, clinical associations, and potential mechanistic roles of non-criteria antiphospholipid antibodies (aPL) and circulating calprotectin, a highly stable marker of neutrophil extracellular trap release (NETosis), in pediatric APS patients. We found that 79% of pediatric APS patients had at least one non-criteria aPL at moderate-to-high titer. Univariate logistic regression demonstrated that positive anti-beta-2 glycoprotein I domain 1 (anti-D1) IgG (p = 0.008), anti-phosphatidylserine/prothrombin (aPS/PT) IgG (p < 0.001), and aPS/PT IgM (p < 0.001) were significantly associated with venous thrombosis. Positive anti-D1 IgG (p < 0.001), aPS/PT IgG (p < 0.001), and aPS/PT IgM (p = 0.001) were also associated with non-thrombotic manifestations of APS, such as thrombocytopenia. Increased levels of calprotectin were detected in children with APS. Calprotectin correlated positively with absolute neutrophil count (r = 0.63, p = 0.008) and negatively with platelet count (r = -0.59, p = 0.015). Mechanistically, plasma from pediatric APS patients with high calprotectin levels impaired platelet viability in a dose-dependent manner.

Distinct baseline immune characteristics associated with responses to conjugated and unconjugated pneumococcal polysaccharide vaccines in older adults.

Pneumococcal infections cause serious illness and death among older adults. The capsular polysaccharide vaccine PPSV23 and conjugated alternative PCV13 can prevent these infections; yet, underlying immunological responses and baseline predictors remain unknown. We vaccinated 39 older adults (>60 years) with PPSV23 or PCV13 and observed comparable antibody responses (day 28) and plasmablast transcriptional responses (day 10); however, the baseline predictors were distinct. Analyses of baseline flow cytometry and bulk and single-cell RNA-sequencing data revealed a baseline phenotype specifically associated with weaker PCV13 responses, which was characterized by increased expression of cytotoxicity-associated genes, increased frequencies of CD16+ natural killer cells and interleukin-17-producing helper T cells and a decreased frequency of type 1 helper T cells. Men displayed this phenotype more robustly and mounted weaker PCV13 responses than women. Baseline expression levels of a distinct gene set predicted PPSV23 responses. This pneumococcal precision vaccinology study in older adults uncovered distinct baseline predictors that might transform vaccination strategies and initiate novel interventions.

Up to date: Genética de la enfermedad celíaca / No disponible

La enfermedad celíaca (EC) es una enfermedad sistémica caracterizada por enteropatía crónica y mediada por el sistema inmune, que se desencadena en individuos genéticamente susceptible tras la ingesta de gluten[1]. En Europa, y probablemente también a nivel mundial, la prevalencia en la población general es aproximadamente del 1 %, pero aumenta en ciertos grupos de riesgo, en que se incluyen los familiares de primer grado de los pacientes. En estos individuos el riesgo puede elevarse hasta un 38 %, lo que da idea de la fuerte carga genética asociada a esta patología. De hecho, la concordancia entre gemelos monocigóticos ronda el 80 %, y disminuye hasta el 20 % en gemelos dicigóticos[2-3]. Los diversos estudios que se han desarrollado para desengranar los factores que determinan la heredabilidad de esta enfermedad, describen la principal asociación con genes localizados en la región del HLA (Human Leukocytes Antigen), así como un gran número de variantes génicas distribuidas por todo el genoma que aparecen con mayor frecuencia en la población enferma (AU)

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