Identification of microRNA biomarkers simultaneously expressed in circulating extracellular vesicles and atherosclerotic plaques.

Background: Atherosclerosis is a widespread disorder of the cardiovascular system. The early detection of plaques by circulating biomarkers is highly clinically relevant to prevent the occurrence of major complications such as stroke or heart attacks. It is known that extracellular vesicles (EVs) are important in intercellular communication in atherosclerotic disorders and carry many components of their cells of origin, including microRNAs (miRNAs). In this study, we test the assumption that miRNAs present in material acquired from plaques in patients undergoing surgery for atherosclerotic carotid artery stenosis are also expressed in circulating EVs obtained from the identical patients. This would allow the adoption of a liquid biopsy approach for the detection of plaques. Methods: We studied 22 surgical patients with atherosclerotic carotid arterial stenosis and 28 healthy controls. EVs were isolated from serum by precipitation. miRNA expression profiles of serum-derived EVs were obtained by small RNA sequencing and in plaque material simultaneously acquired from patients. A comparative analysis was performed to identify circulating atherosclerosis-associated miRNAs that are also detectable in plaques. Results: Seven miRNAs were found to be differentially regulated in patient serum compared with the serum of healthy controls. Of these, miR-193b-5p, miR-193a-5p, and miR-125a-3p were significantly upregulated in patients compared with that in healthy controls and present in both, circulating EVs and plaque material. An overrepresentation analysis of experimentally validated mRNA targets revealed an increased regulation of inflammation and vascular growth factors, key players in atherosclerosis and plaque formation. Conclusion: Our findings suggest that circulating EVs reflect plaque development in patients with symptomatic carotid artery stenosis, which can serve as biomarker candidates for detecting the presence of atherosclerotic plaques.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Decrypting the molecular basis of cellular drug phenotypes by dose-resolved expression proteomics.

Proteomics is making important contributions to drug discovery, from target deconvolution to mechanism of action (MoA) elucidation and the identification of biomarkers of drug response. Here we introduce decryptE, a proteome-wide approach that measures the full dose-response characteristics of drug-induced protein expression changes that informs cellular drug MoA. Assaying 144 clinical drugs and research compounds against 8,000 proteins resulted in more than 1 million dose-response curves that can be interactively explored online in ProteomicsDB and a custom-built Shiny App. Analysis of the collective data provided molecular explanations for known phenotypic drug effects and uncovered new aspects of the MoA of human medicines. We found that histone deacetylase inhibitors potently and strongly down-regulated the T cell receptor complex resulting in impaired human T cell activation in vitro and ex vivo. This offers a rational explanation for the efficacy of histone deacetylase inhibitors in certain lymphomas and autoimmune diseases and explains their poor performance in treating solid tumors.

The proteome of bacterial membrane vesicles in Escherichia coli-a time course comparison study in two different media.

Introduction: Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. Methods: To gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1ß and IL-8 in the human monocyte cell line THP-1 upon bMV treatment. Results: Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1ß and IL-8 expressions. Conclusion: Our study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

A pipeline for the development and analysis of extracellular vesicle-based transcriptomic biomarkers in molecular diagnostics.

Extracellular vesicles are shed by every cell type and can be found in any biofluid. They contain different molecules that can be utilized as biomarkers, including several RNA species which they protect from degradation. Here, we present a pipeline for the development and analysis of extracellular vesicle-associated transcriptomic biomarkers that our group has successfully applied multiple times. We highlight the key steps of the pipeline and give particular emphasis to the necessary quality control checkpoints, which are linked to numerous available guidelines that should be considered along the workflow. Our pipeline starts with patient recruitment and continues with blood sampling and processing. The purification and characterization of extracellular vesicles is explained in detail, as well as the isolation and quality control of extracellular vesicle-associated RNA. We point out the possible pitfalls during library preparation and RNA sequencing and present multiple bioinformatic tools to pinpoint biomarker signature candidates from the sequencing data. Finally, considerations and pitfalls during the validation of the biomarker signature using RT-qPCR will be elaborated.

Prostaglandins as local regulators of ovarian physiology in ruminants.

Prostaglandins are synthesized from arachidonic acid through the catalytic activities of cyclooxygenase, while the production of different prostaglandin types, prostaglandin F2 alpha (PGF) and prostaglandin E2 (PGE), are regulated by specific prostaglandin synthases (PGFS and PGES). Prostaglandin ligands (PGF and PGE) bind to specific high-affinity receptors and initiate biologically distinct signalling pathways. In the ovaries, prostaglandins are known to be important endocrine regulators of female reproduction, in addition to maintaining local function through autocrine and/or paracrine effect. Many research groups in different animal species have already identified a variety of factors and molecular mechanisms that are responsible for the regulation of prostaglandin functions. In addition, prostaglandins stimulate their intrafollicular and intraluteal production via the pathway of prostaglandin self-regulation in the ovary. Therefore, the objective of the review article is to discuss recent findings about local regulation patterns of prostaglandin ligands PGF and PGE during different physiological stages of ovarian function in domestic ruminants, especially in bovine. In conclusion, the discussed local regulation mechanisms of prostaglandins in the ovary may stimulate further research activities in different methodological approaches, especially during final follicle maturation and ovulation, as well as corpus luteum formation and function.

Local Expression Dynamics of Various Adipokines during Induced Luteal Regression (Luteolysis) in the Bovine Corpus Luteum.

The study aimed to evaluate the mRNA expression levels of various local novel adipokines, including vaspin, adiponectin, visfatin, and resistin, along with their associated receptors, heat shock 70 protein 5, adiponectin receptor 1, and adiponectin receptor 2, in the corpus luteum (CL) during luteal regression, also known as luteolysis, in dairy cows. We selected Fleckvieh cows in the mid-luteal phase (days 8-12, control group) and administered cloprostenol (PGF analog) to experimentally induce luteolysis. We collected CL samples at different time points following PGF application: before treatment (days 8-12, control group) and at 0.5, 2, 4, 12, 24, 48, and 64 h post-treatment (n = 5) per group. The mRNA expression was measured via real-time reverse transcription polymerase chain reaction (RT-qPCR). Vaspin was characterized by high mRNA levels at the beginning of the regression stage, followed by a significant decrease 48 h and 64 h after PGF treatment. Adiponectin mRNA levels were elevated 48 h after PGF. Resistin showed upregulation 4 h post PGF application. In summary, the alterations observed in the adipokine family within experimentally induced regressing CL tissue potentially play an integral role in the local regulatory processes governing the sequence of events culminating in functional luteolysis and subsequent structural changes in the bovine ovary.

Expression of Locally Produced Adipokines and Their Receptors during Different Physiological and Reproductive Stages in the Bovine Corpus Luteum.

This study aimed to determine the gene expression of different local novel adipokines, such as vaspin, adiponectin, visfatin, and resistin, and their known receptors, namely, heat shock 70 protein 5, adiponectin receptor 1, and adiponectin receptor 2, in the bovine corpus luteum (CL) during different phases of the estrous cycle (on days 1-2, 3-4, 5-7, 8-12, 13-18, >18) and pregnancy (at months 1-2, 3-4, 5-7, >7). The mRNA expression was measured by reverse transcription polymerase chain reaction (RT-qPCR). The mRNA expression levels were normalized to the geometric mean of all three constantly expressed reference genes (cyclophilin A, ubiquitin, ubiquitin C). Our findings suggest that adipokines are expressed and present in all investigated groups, and are specifically up- or downregulated during the estrus cycle and during pregnancy. Vaspin and adiponectin levels were upregulated in the middle and late cycle stages. Resistin was abundant during the CL regression stage and in the first months of pregnancy. The specific expression of adipokine receptors indicates their involvement in the local mechanisms that regulate CL function. Further investigations are required to elucidate the regulative mechanisms underlying the different local effects of adipokines on the ovarian physiology of cows.

Extracellular Vesicles and Endocannabinoid Signaling in Patients with COVID-19.

Introduction: Endocannabinoids in COVID-19 have immunomodulatory and anti-inflammatory properties but the functional role and the regulation of endocannabinoid signaling in this pandemic disorder is controversial. To exercise their biologic function, endocannabinoids need to travel across the intercellular space and within the blood stream to reach their target cells. How the lipophilic endocannabinoids are transported in the vascular system and how these hydrophobic compounds cross cell membranes is still unclear. Extracellular vesicles (EVs) are released and incorporated by many cell types including immune cells. EVs are small lipid-membrane covered particles and contain RNA, lipids and proteins. They play an important role in intercellular communication by transporting these signaling molecules from their cells of origin to specific target cells. EVs may represent ideal transport vehicles for lipophilic signaling molecules like endocannabinoids and this effect could also be evident in COVID-19. Materials and Methods: We measured the endocannabinoids anandamide, 2-AG, SEA, PEA and OEA in patients with COVID-19 in EVs and plasma. RNA sequencing of microRNAs (miRNAs) derived from EVs (EV-miRNAs) and mRNA transcripts from blood cells was used for the construction of signaling networks reflecting endocannabinoid and miRNA communication by EVs to target immune cells. Results: With the exception of anandamide, endocannabinoid concentrations were significantly enriched in EVs in comparison to plasma and increased with disease severity. No enrichment in EVs was seen for the more hydrophilic steroid hormones cortisol and testosterone. High EV-endocannabinoid concentrations were associated with downregulation of CNR2 (CB2) by upregulated EV-miRNA miR-146a-5p and upregulation of MGLL by downregulated EV-miR-199a-5p and EV-miR-370-5p suggesting counterregulatory effects. In contrast, low EV-levels of anandamide were associated with upregulation of CNR1 by downregulation of EV-miR-30c-5p and miR-26a-5p along with inhibition of FAAH. Immunologically active molecules in immune cells regulated by endocannabinoid signaling included VEGFA, GNAI2, IGF1, BDNF, IGF1R and CREB1 and CCND1 among others. Discussion and Conclusions: EVs carry immunologically functional endocannabinoids in COVID-19 along with miRNAs which may regulate the expression of mRNA transcripts involved in the regulation of endocannabinoid signaling and metabolism. This mechanism could fine-tune and adapt endocannabinoid effects in recipient cells in relationship to the present biological context.

Replicative Study in Performance-Related Genes of Brazilian Elite Soccer Players Highlights Genetic Differences from African Ancestry and Similarities between Professional and U20 Youth Athletes.

Classically, genetic association studies have attempted to assess genetic polymorphisms related to human physiology and physical performance. However, the heterogeneity of some findings drives the research to replicate, validate, and confirmation as essential aspects for ensuring their applicability in sports sciences. Genetic distance matrix and molecular variance analyses may offer an alternative approach to comparing athletes' genomes with those from public databases. Thus, we performed a complete sequencing of 44 genomes from male Brazilian first-division soccer players under 20 years of age (U20_BFDSC). The performance-related SNP genotypes were obtained from players and from the "1000 Genomes" database (European, African, American, East Asian, and South Asian). Surprisingly, U20_BFDSC performance-related genotypes had significantly larger FST levels (p < 0.00001) than African populations, although studies using ancestry markers have shown an important similarity between Brazilian and African populations (12-24%). U20_BFDSC were genetically similar to professional athletes, showing the intense genetic selection pressure likely to occur before this maturation stage. Our study highlighted that performance-related genes might undergo selective pressure due to physical performance and environmental, cognitive, and sociocultural factors. This replicative study suggests that molecular variance and Wright's statistics can yield novel conclusions in exercise science.

Uptake and effects of polystyrene nanoplastics in comparison to non-plastic silica nanoparticles on small intestine cells (IPEC-J2).

Nanoplastics smaller than 1 µm accumulate as anthropogenic material in the food chain, but only little is known about their uptake and possible effects on potentially strongly exposed cells of the small intestine. The aim of the study was to observe the uptake of 100 nm polystyrene nanoplastics into a non-tumorigenic small intestine cell culture model (IPEC-J2 cells) and to monitor the effects on cell growth and gene regulation, compared to a 100 nm non-plastic silica nanoparticle reference. The intracellular uptake of both types of nanoparticles was proven via (confocal) fluorescence microscopy and complemented with transmission electron microscopy. Fluorescence microscopy showed a growth phase-dependent uptake of nanoparticles into the cells, hence further experiments included different time points related to epithelial closure, determined via electric cell substrate impedance sensing. No retardations in epithelial closure of cells after treatment with polystyrene nanoparticles could be found. In contrast, epithelial cell closure was partly negatively influenced by silica nanoparticles. An increased production of organic nanoparticles, like extracellular vesicles, was not measurable via nanoparticle tracking analysis. An assessment of messenger RNA by next generation sequencing and subsequent pathway analysis revealed that the TP53 pathway was influenced significantly by the polystyrene nanoparticle treatment. In both treatments, dysregulated mRNAs were highly enriched in the NOTCH signaling pathway compared to the non-particle control.

The potential role of serum extracellular vesicle derived small RNAs in AML research as non-invasive biomarker.

BACKGROUND: Extracellular vesicles (EV) are cell-derived vesicles released by all cells in health and disease. Accordingly, EVs are also released by cells in acute myeloid leukemia (AML), a hematologic malignancy characterized by uncontrolled growth of immature myeloid cells, and these EVs likely carry markers and molecular cargo reflecting the malignant transformation occurring in diseased cells. Monitoring antileukemic or proleukemic processes during disease development and treatment is essential. Therefore, EVs and EV-derived microRNA (miRNA) from AML samples were explored as biomarkers to distinguish disease-related patterns ex vivo or in vivo. METHODOLOGY: EVs were purified from serum of healthy (H) volunteers and AML patients by immunoaffinity. EV surface protein profiles were analyzed by multiplex bead-based flow cytometry (MBFCM) and total RNA was isolated from EVs prior to miRNA profiling via small RNA sequencing. RESULTS: MBFCM revealed different surface protein patterns in H versus AML EVs. miRNA analysis showed individual as well as highly dysregulated patterns in H and AML samples. CONCLUSIONS: In this study, we provide a proof-of-concept for the discriminative potential of EV derived miRNA profiles as biomarkers in H versus AML samples.

Characterization of the Staphylococcus xylosus methylome reveals a new variant of type I restriction modification system in staphylococci.

Restriction modification (RM) systems are known to provide a strong barrier to the exchange of DNA between and within bacterial species. Likewise, DNA methylation is known to have an important function in bacterial epigenetics regulating essential pathways such as DNA replication and the phase variable expression of prokaryotic phenotypes. To date, research on staphylococcal DNA methylation focused mainly on the two species Staphylococcus aureus and S. epidermidis. Less is known about other members of the genus such as S. xylosus, a coagulase-negative commensal of mammalian skin. The species is commonly used as starter organism in food fermentations but is also increasingly considered to have an as yet elusive function in bovine mastitis infections. We analyzed the methylomes of 14 S. xylosus strains using single-molecular, real-time (SMRT) sequencing. Subsequent in silico sequence analysis allowed identification of the RM systems and assignment of the respective enzymes to the discovered modification patterns. Hereby the presence of type I, II, III and IV RM systems in varying numbers and combinations among the different strains was revealed, clearly distinguishing the species from what is known for other members of the genus so far. In addition, the study characterizes a newly discovered type I RM system, encoded by S. xylosus but also by a variety of other staphylococcal species, with a hitherto unknown gene arrangement that involves two specificity units instead of one (hsdRSMS). Expression of different versions of the operon in E. coli showed proper base modification only when genes encoding both hsdS subunits were present. This study provides new insights into the general understanding of the versatility and function of RM systems as well as the distribution and variations in the genus Staphylococcus.

Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Extensive blood transcriptome analysis reveals cellular signaling networks activated by circulating glycocalyx components reflecting vascular injury in COVID-19.

Background: Degradation of the endothelial protective glycocalyx layer during COVID-19 infection leads to shedding of major glycocalyx components. These circulating proteins and their degradation products may feedback on immune and endothelial cells and activate molecular signaling cascades in COVID-19 associated microvascular injury. To test this hypothesis, we measured plasma glycocalyx components in patients with SARS-CoV-2 infection of variable disease severity and identified molecular signaling networks activated by glycocalyx components in immune and endothelial cells. Methods: We studied patients with RT-PCR confirmed COVID-19 pneumonia, patients with COVID-19 Acute Respiratory Distress Syndrome (ARDS) and healthy controls (wildtype, n=20 in each group) and measured syndecan-1, heparan sulfate and hyaluronic acid. The in-silico construction of signaling networks was based on RNA sequencing (RNAseq) of mRNA transcripts derived from blood cells and of miRNAs isolated from extracellular vesicles from the identical cohort. Differentially regulated RNAs between groups were identified by gene expression analysis. Both RNAseq data sets were used for network construction of circulating glycosaminoglycans focusing on immune and endothelial cells. Results: Plasma concentrations of glycocalyx components were highest in COVID-19 ARDS. Hyaluronic acid plasma levels in patients admitted with COVID-19 pneumonia who later developed ARDS during hospital treatment (n=8) were significantly higher at hospital admission than in patients with an early recovery. RNAseq identified hyaluronic acid as an upregulator of TLR4 in pneumonia and ARDS. In COVID-19 ARDS, syndecan-1 increased IL-6, which was significantly higher than in pneumonia. In ARDS, hyaluronic acid activated NRP1, a co-receptor of activated VEGFA, which is associated with pulmonary vascular hyperpermeability and interacted with VCAN (upregulated), a proteoglycan important for chemokine communication. Conclusions: Circulating glycocalyx components in COVID-19 have distinct biologic feedback effects on immune and endothelial cells and result in upregulation of key regulatory transcripts leading to further immune activation and more severe systemic inflammation. These consequences are most pronounced during the early hospital phase of COVID-19 before pulmonary failure develops. Elevated levels of circulating glycocalyx components may early identify patients at risk for microvascular injury and ARDS. The timely inhibition of glycocalyx degradation could provide a novel therapeutic approach to prevent the development of ARDS in COVID-19.

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo.

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic ß-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic ß-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.

Anesthetic­specific lncRNA and mRNA profile changes in blood during colorectal cancer resection: A prospective, matched­case pilot study.

Prometastatic and antitumor effects of different anesthetics have been previously analyzed in several studies with conflicting results. Thus, the underlying perioperative molecular mechanisms mediated by anesthetics potentially affecting tumor phenotype and metastasis remain unclear. It was hypothesized that anesthetic­specific long non­coding RNA (lncRNA) expression changes are induced in the blood circulation and play a crucial role in tumor outcome. In the present study, high­throughput sequencing and quantitative PCR were performed in order to identify lncRNA and mRNA expression changes affected by two therapeutic regimes, total intravenous anesthesia (TIVA) and volatile anesthetic gas (VAG) in patients undergoing colorectal cancer (CRC) resection. Total blood RNA was isolated prior to and following resection and characterized using RNA sequencing. mRNA­lncRNA interactions and their roles in cancer­related signaling of differentially expressed lncRNAs were identified using bioinformatics analyses. The comparison of these two time points revealed 35 differentially expressed lncRNAs in the TIVA­group, and 25 in the VAG­group, whereas eight were shared by both groups. Two lncRNAs in the TIVA­group, and 23 in the VAG­group of in silico identified target­mRNAs were confirmed as differentially regulated in the NGS dataset of the present study. Pathway analysis was performed and cancer relevant canonical pathways for TIVA were identified. Target­mRNA analysis of VAG revealed a markedly worsened immunological response against cancer. In this proof­of­concept study, anesthesic­specific expression changes in lncRNA and mRNA profiles in blood were successfully identified. Moreover, the data of the present study provide the first evidence that anesthesia­induced lncRNA pattern changes may contribute further in the observed differences in CRC outcome following tumor resection.

Isolation and analysis of tumor­derived extracellular vesicles from head and neck squamous cell carcinoma plasma by galectin­based glycan recognition particles.

Extracellular vesicles (EVs) have recently come into the spotlight as potential cancer biomarkers. Isolation of pure EVs is complex, so wider use requires reliable and time­efficient isolation methods. In the present study, galectin­based magnetic glycan recognition particles, EXÖBead® were investigated for their practicality as a novel EV isolation technique, exemplified here for squamous cell carcinoma of the head and neck. Analysis of the isolation method showed a high concentration of pure EVs with detection of specific EV markers such as CD9, CD63, CD81 and TSG101. No apolipoprotein A1 was shown in the isolates, indicating low contamination of this isolation technique compared with size exclusion chromatography. In addition, common leukocyte antigen (CD45), three HNSCC [epithelial cell adhesion molecule (EpCAM), pan­cytokeratin and programmed death­ligand 1 (PD­L1)] and PanEV markers (premixed CD9, CD63 and CD81 antibodies) were measured by bead­based flow cytometry (BFC). BFC revealed that CD45Neg PanEV+, EpCAM+ PanEV+ and PD­L1+ PanEV+ were significantly higher in tumor patients compared with healthy control plasma. CD45Neg PanEV+ and CD45+ PanEV+ carrying two or three HNSCC biomarkers were also significantly higher in tumor patients compared with healthy controls (BFC). Comparison of the functional immunosuppression effect of eluted tumor patient plasma EVs from EXÖBead® and commercial polyethylene glycol isolation showed a significant tumor­dependent increase in concentration of EVs. A peripheral blood mononuclear cell activation assay also showed that the T­cell functionality of tumor patient plasma EVs isolated with EXÖBead® was preserved in vitro. In conclusion, isolation using galectin­based magnetic glycan recognition particles is a novel method for isolating plasma EVs with low lipoprotein contamination. Bead­based flow cytometry provided an easy way to understand EV subpopulations. EXÖBead® therefore showed great potential as a new isolation tool with high throughput capacity that could potentially be used in a clinical setting.

The successful use of -omic technologies to achieve the 'One Health' concept in meat producing animals.

Human health and wellbeing are closely linked to healthy domestic animals, a vital wildlife, and an intact ecosystem. This holistic concept is referred to as 'One Health'. In this review, we provide an overview of the potential and the challenges for the use of modern -omics technologies, especially transcriptomics and proteomics, to implement the 'One Health' idea for food-producing animals. These high-throughput studies offer opportunities to find new potential molecular biomarkers to monitor animal health, detect pharmacological interventions and evaluate the wellbeing of farm animals in modern intensive livestock systems.