Development of a novel streamlined workflow (AACRE) and database (inCREDBle) for genomic analysis of carbapenem-resistant Enterobacterales.

In response to the threat of increasing antimicrobial resistance, we must increase the amount of available high-quality genomic data gathered on antibiotic-resistant bacteria. To this end, we developed an integrated pipeline for high-throughput long-read sequencing, assembly, annotation and analysis of bacterial isolates and used it to generate a large genomic data set of carbapenemase-producing Enterobacterales (CPE) isolates collected in Spain. The set of 461 isolates were sequenced with a combination of both Illumina and Oxford Nanopore Technologies (ONT) DNA sequencing technologies in order to provide genomic context for chromosomal loci and, most importantly, structural resolution of plasmids, important determinants for transmission of antimicrobial resistance. We developed an informatics pipeline called Assembly and Annotation of Carbapenem-Resistant Enterobacteriaceae (AACRE) for the full assembly and annotation of the bacterial genomes and their complement of plasmids. To explore the resulting genomic data set, we developed a new database called inCREDBle that not only stores the genomic data, but provides unique ways to filter and compare data, enabling comparative genomic analyses at the level of chromosomes, plasmids and individual genes. We identified a new sequence type, ST5000, and discovered a genomic locus unique to ST15 that may be linked to its increased spread in the population. In addition to our major objective of generating a large regional data set, we took the opportunity to compare the effects of sample quality and sequencing methods, including R9 versus R10 nanopore chemistry, on genome assembly and annotation quality. We conclude that converting short-read and hybrid microbial sequencing and assembly workflows to the latest nanopore chemistry will further reduce processing time and cost, truly enabling the routine monitoring of resistance transmission patterns at the resolution of complete chromosomes and plasmids.

An Integrated Approach for the Early Detection of Endometrial and Ovarian Cancers (Screenwide Study): Rationale, Study Design and Pilot Study.

Screenwide is a case-control study (2017−2021) including women with incident endometrial and ovarian cancers (EC and OC), BRCA1/2 and MMR pathogenic variant carriers, and age-matched controls from three centers in Spain. Participants completed a personal interview on their sociodemographic factors, occupational exposure, medication, lifestyle, and medical history. We collected biological specimens, including blood samples, self-collected vaginal specimens, cervical pap-brush samples, uterine specimens, and, when available, tumor samples. The planned analyses included evaluation of the potential risk factors for EC/OC; evaluation of molecular biomarkers in minimally invasive samples; evaluation of the cost-effectiveness of molecular tests; and the generation of predictive scores to integrate different epidemiologic, clinical, and molecular factors. Overall, 182 EC, 69 OC, 98 BRCA pathogenic variant carriers, 104 MMR pathogenic variant carriers, and 385 controls were enrolled. The overall participation rate was 85.7%. The pilot study using 61 samples from nine EC cases and four controls showed that genetic variants at the variant allele fraction > 5% found in tumors (n = 61 variants across the nine tumors) were detected in paired endometrial aspirates, clinician-collected cervical samples, and vaginal self-samples with detection rates of 90% (55/61), 79% (48/61), and 72% (44/61) by duplex sequencing, respectively. Among the controls, only one somatic mutation was detected in a cervical sample. We enrolled more than 800 women to evaluate new early detection strategies. The preliminary data suggest that our methodological approach could be useful for the early detection of gynecological cancers.

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia.

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets.

RNA release via membrane vesicles in Pseudomonas aeruginosa PAO1 is associated with the growth phase.

Pseudomonas aeruginosa PAO1 membrane vesicles (MVs) are known to play a role in cell-to-cell communication. Several studies have shown that the MV composition and physicochemical properties vary according to the bacterial growth stage, but the impact this might have on the externalization of RNA via MVs has not been addressed. Therefore, a study to characterize the RNA content from MVs retrieved at different growth phases was conducted. First, the transcriptome analyses revealed a higher abundance of around 300 RNA species in MVs when compared with the cells. The vesiculation rate along the growth curve was determined, showing that the release of MVs increased during the transition to the stationary phase, whereas it decreased in the late stationary phase. RNA-seq of MVs retrieved along the transition to the stationary phase demonstrated that the RNA cargo of vesicles did not vary. However, the amount of smaller RNAs (<200 nt) inside MVs retrieved in the late exponential phase was higher than in the stationary phase MVs. These results indicate that the externalization of RNA via MVs occurs during late exponential phase and implies the secretion of different types of MVs during growth.

A Mouse Brain-based Multi-omics Integrative Approach Reveals Potential Blood Biomarkers for Ischemic Stroke.

Stroke remains a leading cause of death and disability worldwide. Despite continuous advances, the identification of key molecular signatures in the hyper-acute phase of ischemic stroke is still a primary interest for translational research on stroke diagnosis, prognosis, and treatment. Data integration from high-throughput -omics techniques has become crucial to unraveling key interactions among different molecular elements in complex biological contexts, such as ischemic stroke. Thus, we used advanced data integration methods for a multi-level joint analysis of transcriptomics and proteomics data sets obtained from mouse brains at 2 h after cerebral ischemia. By modeling net-like correlation structures, we identified an integrated network of genes and proteins that are differentially expressed at a very early stage after stroke. We validated 10 of these deregulated elements in acute stroke, and changes in their expression pattern over time after cerebral ischemia were described. Of these, CLDN20, GADD45G, RGS2, BAG5, and CTNND2 were next evaluated as blood biomarkers of cerebral ischemia in mice and human blood samples, which were obtained from stroke patients and patients presenting stroke-mimicking conditions. Our findings indicate that CTNND2 levels in blood might potentially be useful for distinguishing ischemic strokes from stroke-mimicking conditions in the hyper-acute phase of the disease. Furthermore, circulating GADD45G content within the first 6 h after stroke could also play a key role in predicting poor outcomes in stroke patients. For the first time, we have used an integrative biostatistical approach to elucidate key molecules in the initial stages of stroke pathophysiology and highlight new notable molecules that might be further considered as blood biomarkers of ischemic stroke.

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage.

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke.

Phosphatidylserine-Liposomes Promote Tolerogenic Features on Dendritic Cells in Human Type 1 Diabetes by Apoptotic Mimicry.

Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing ß-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow ß-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic ß-cells arrested autoimmunity to ß-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)-the main signal of the apoptotic cell membrane-and ß-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity.

Cinacalcet inhibits neuroblastoma tumor growth and upregulates cancer-testis antigens.

The calcium-sensing receptor is a G protein-coupled receptor that exerts cell-type specific functions in numerous tissues and some cancers. We have previously reported that this receptor exhibits tumor suppressor properties in neuroblastoma. We have now assessed cinacalcet, an allosteric activator of the CaSR approved for clinical use, as targeted therapy for this developmental tumor using neuroblastoma cell lines and patient-derived xenografts (PDX) with different MYCN and TP53 status. In vitro, acute exposure to cinacalcet induced endoplasmic reticulum stress coupled to apoptosis via ATF4-CHOP-TRB3 in CaSR-positive, MYCN-amplified cells. Both phenotypes were partially abrogated by phospholipase C inhibitor U73122. Prolonged in vitro treatment also promoted dose- and time-dependent apoptosis in CaSR-positive, MYCN-amplified cells and, irrespective of MYCN status, differentiation in surviving cells. Cinacalcet significantly inhibited tumor growth in MYCN-amplified xenografts and reduced that of MYCN-non amplified PDX. Morphology assessment showed fibrosis in MYCN-amplified xenografts exposed to the drug. Microarrays analyses revealed up-regulation of cancer-testis antigens (CTAs) in cinacalcet-treated MYCN-amplified tumors. These were predominantly CTAs encoded by genes mapping on chromosome X, which are the most immunogenic. Other modulated genes upon prolonged exposure to cinacalcet were involved in differentiation, cell cycle exit, microenvironment remodeling and calcium signaling pathways. CTAs were up-regulated in PDX and in vitro models as well. Moreover, progressive increase of CaSR expression upon cinacalcet treatment was seen both in vitro and in vivo. In summary, cinacalcet reduces neuroblastoma tumor growth and up-regulates CTAs. This effect represents a therapeutic opportunity and provides surrogate circulating markers of neuroblastoma response to this treatment.

Cross-pharmacology analysis of G protein-coupled receptors.

The degree of applicability of chemogenomic approaches to protein families depends on the accuracy and completeness of pharmacological data and the corresponding level of pharmacological similarity observed among their protein members. The recent public domain availability of pharmacological data for thousands of small molecules on 204 G protein-coupled receptors (GPCRs) provides a firm basis for an in-depth cross-pharmacology analysis of this superfamily. The number of protein targets included in the cross-pharmacology profile of the different GPCRs changes significantly upon varying the ligand similarity and binding affinity criteria. However, with the exception of muscarinic receptors, aminergic GPCRs distinguish themselves from the rest of the members in the family by their remarkably high levels of pharmacological similarity among them. Clusters of non-GPCR targets related by cross-pharmacology with particular GPCRs are identified and the implications for unwanted side-effects, as well as for repurposing opportunities, discussed.