Osteogenic differentiation is crucial in normal bone formation and pathological calcification, such as calcific aortic valve disease (CAVD). Understanding the proteomic and transcriptomic landscapes underlying this differentiation can unveil potential therapeutic targets for CAVD. In this study, we employed RNA sequencing transcriptomics and proteomics on a timsTOF Pro platform to explore the multiomics profiles of valve interstitial cells (VICs) and osteoblasts during osteogenic differentiation. For proteomics, we utilized 3 data acquisition/analysis techniques: data-dependent acquisition (DDA)–parallel accumulation serial fragmentation (PASEF) and data-independent acquisition (DIA)–PASEF with a classic library-based (DIA) and machine learning–based library-free search (DIA-ML). Using RNA sequencing data as a biological reference, we compared these 3 analytical techniques in the context of actual biological experiments. We use this comprehensive dataset to reveal distinct proteomic and transcriptomic profiles between VICs and osteoblasts, highlighting specific biological processes in their osteogenic differentiation pathways. The study identified potential therapeutic targets specific for VICs osteogenic differentiation in CAVD, including the MAOA and ERK1/2 pathway. From a technical perspective, we found that DIA-based methods demonstrate even higher superiority against DDA for more sophisticated human primary cell cultures than it was shown before on HeLa samples. While the classic library-based DIA approach has proved to be a gold standard for shotgun proteomics research, the DIA-ML offers significant advantages with a relatively minor compromise in data reliability, making it the method of choice for routine proteomics.

Many plants and animals have symbiotic relationships with microorganisms, including bacteria. The interactions between bacteria and their hosts result in different outcomes for the host organism. The outcome can be neutral, harmful or have beneficial effects for participants. Remarkably, these relationships are not static, as they change throughout an organism’s lifetime and on an evolutionary scale. One of the structures responsible for relationships in bacteria is O-antigen. Depending on the characteristics of its components, the bacteria can avoid the host’s immune response or establish a mutualistic relationship with it. O-antigen is a key component in Gram-negative bacteria’s outer membrane. This component facilitates interaction between the bacteria and host immune system or phages. The variability of the physical structure is caused by the genomic variability of genes encoding O-antigen synthesis components. The genes and pathways of O-polysaccharide (OPS) synthesis were intensively investigated mostly for Enterobacteriaceae species. Considering high genetic and molecular diversity of this structure even between strains, these findings may not have caught the entire variety possibly presented in non-model species. The current study presents a comparative analysis of genes associated with O-antigen synthesis in bacteria of the Oxalobacteraceae family. In contrast to existing studies based on PCR methods, we use a bioinformatics approach and compare O- anti gens at the level of clusters rather than individual genes. We found that the O-antigen genes of these bacteria are represented by several clusters located at a distance from each other. The greatest similarity of the clusters is observed within individual bacterial genera, which is explained by the high variability of O-antigens. The study describes similarities of OPS genes inherent to the family as a whole and also considers individual unique cases of O-antigen genetic variability inherent to individual bacteria.

Providencia is a genus of Gram-negative bacteria belonging to the Morganellaceae family. This genus includes nine species (P. stuartii, P. sneebia, P. rettgeri, P. rustigianii, P. heimbachae, P. burhodogranariea, P. alcalifaciens, P. huaxiensis , and P. vermicola ) with varying degrees of virulence, capable of infecting humans and insects [1, 2]. For Gram-negative bacteria, the somatic antigen (O-antigen) has become one of the key virulence factors. It is the highly immunogenic part of lipopolysaccharides due to the distal location. O-antigens are characterized by structural heterogeneity, providing varying degrees of inter- and intraspecific virulence. At the genetic level, somatic antigens have an operon structure. Operon genes responsible for the synthesis and transformation of O-polysaccharide are transcribed together. Analysis of O-antigen operon organization determines genes specific for each O-serogroup. It is beneficial for molecular typing of strains and for studies of bacterial evolution. This study focuses on identifying and comparing candidates for O-antigen operons in Providencia species with different levels of virulence. The hypothesis is the presence of an association between the O-antigen operon composition and the bacteria lifestyle. Data processing and analysis are carried out by a pipeline developed by the authors. Pipeline combines five steps of the genome analysis: genome quality evaluation, assembly annotation, operon identification with verification of operon boundaries, and visualization of O-antigen operons. The results reveal previously undescribed O-antigen genes and the changes in the O-antigen operons structure. Among the changes are a transposon insertion leading to tetracycline resistance and the presence of IS elements.

Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling and immune dysregulation. Myeloid angiogenic cells (MACs) are blood-derived mononuclear cells that acquire endothelial-like features at sites of vascular injury, but their transcriptomic phenotype in the pulmonary vascular compartment is poorly characterized. We isolated adherent cells from blood aspirated from the wedged pulmonary artery of four patients with severe PAH during right heart catheterization. The cells were vWF-positive and ACTA2-negative by immunocytochemistry and co-expressed canonical endothelial (CDH5, PECAM1, ERG, FLT1) and myeloid (PTPRC/CD45, CD14, SPI1, IRF8) transcripts, consistent with MAC-like adherent cultures rather than a conventional endothelial population. Bulk RNA sequencing of these cultures was compared with human pulmonary artery endothelial cells (HPAECs) and HUVECs used as endothelial reference cultures. Relative to both references, PAH MACs showed higher transcript levels of cytokines (IL1A, TNF, IL15, IL10) and matrix metalloproteinases (MMP8/9/12); relative to HPAECs, they showed lower levels of Notch-associated genes (DLL4, HEYL). Protein–protein interaction analysis identified modules centered on complement components, myeloid immune receptors and CC chemokines. Because MACs from non-PAH subjects were not available, these differences describe the phenotype of PAH-derived MACs relative to endothelial reference cells rather than a PAH-specific effect. Adherent cultures obtained from wedged pulmonary arterial blood are therefore dominated by a mononuclear phagocyte transcriptional program, which has direct implications for the design of patient-derived pulmonary vascular cell models.