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Background: Late-onset or sporadic Alzheimer’s disease (sAD) is a neurodegenerative disease leading to cognitive impairment and memory loss. The underlying pathological changes take place several years prior to the appearance of the first clinical symptoms, however, the early diagnosis of sAD remains obscure. Objective: To identify changes in circulating microRNA (miR) expression in an effort to detect early biomarkers of underlying sAD pathology. Methods: A set of candidate miRs, earlier detected in biofluids from subjects at early stage of sAD, was linked to the proposed tau-driven adverse outcome pathway for memory loss. The relative expression of the selected miRs in serum of 12 cases (mild cognitive impairment, MCI) and 27 cognitively normal subjects, recruited within the ongoing Aiginition Longitudinal Biomarker Investigation Of Neurodegeneration (ALBION) study, was measured by RT-qPCR. Data on the protein levels of amyloid-β (Aβ42) and total/phosphorylated tau (t-tau/p-tau), in cerebrospinal fluid (CSF), and the cognitive z-scores of the participants were also retrieved. Results: Each doubling in relative expression of 13 miRs in serum changed the odds of either having MCI (versus control), or having pathological Aβ42 or pathological Aβ42 and tau (versus normal) proteins in their CSF, or was associated with the global composite z-score. Conclusion: These candidate human circulating miRs may be of great importance in early diagnosis of sAD. There is an urgent need for confirming these proposed early predictive biomarkers for sAD, contributing not only to societal but also to economic benefits.

Context MicroRNAs (miRNAs)—short, single-stranded, noncoding RNAs—regulate several biological processes, including bone metabolism. Objective We investigated circulating miRNAs as promising biomarkers for treatment monitoring in women with postmenopausal osteoporosis on denosumab (DMAB) therapy. Methods In this prospective, observational, single-center study, 21 postmenopausal women treated with DMAB were included for a longitudinal follow-up of 2 years. Next-generation sequencing (NGS) was performed to screen for serological miRNAs at baseline, month 6, and month 24. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to confirm NGS findings in the entire cohort. Bone turnover markers (BTM) P1NP and CTX, and bone mineral density (BMD) by dual x-ray absorptiometry were assessed and correlated to miRNAs. Results BMD at the hip (5.5%, P = 0.0006) and lumbar spine significantly increased (11.4%, P = 0.017), and CTX (64.1%, P < 0.0001) and P1NP (69.3%, P < 0.0001) significantly decreased during treatment. NGS analysis revealed significant changes in miRNAs after 2 years of DMAB treatment but not after 6 months. Seven miRNAs were confirmed by RT-qPCR to be significantly changed during a 2-year course of DMAB treatment compared to baseline. Four of these were mainly transcribed in blood cells, including monocytes. Correlation analysis identified significant correlation between change in miRNA and change in BTMs as well as BMD. Based on effect size and correlation strength, miR-454-3p, miR-26b-5p, and miR-584-5p were defined as top biomarker candidates, with the strongest association to the sustained effect of denosumab on bone in osteoporotic patients. Conclusion Two years of DMAB treatment resulted in upregulation of 7 miRNAs, 4 of which are mainly transcribed in monocytes, indicating a potential impact of DMAB on circulating osteoclast precursor cells. These changes were associated to BMD gain and BTM suppression and could therefore be useful for monitoring DMAB treatment response.

Background aims: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow–derived MSCs, this study focused on improving the MSC-EV production for clinical application. Methods: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. Results: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. Conclusions: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Patients discovered recumbent, helpless and incapacitated, awake or unresponsive are referred to as "long lie trauma" (LLT) in the German medical jargon. Yet, a characterization of this cohort is missing.We retrospectively analyzed all LLT patients admitted to the emergency department of the University Hospital Cologne from July 2018 to December 2020.A total of 50 LLT patients (median age 76 years, median time on the ground 13.5 h) were identified. The FD was most often attributed to primary cerebral causes in 40% of the cases (20% ischemic stroke, 16% intracranial hemorrhage, 4% epilepsy), intoxication/overdose (12%), and trauma (10%). It was often associated with infection (52%), injury (22%), hypovolemia (66%), acute kidney injury (20%), and severe rhabdomyolysis (creatine kinase ≥ 5000 U/l, 21%) as well as severe hypothermia 32 °C (20%). Overall, 69% of the patients were admitted to an intensive care unit and in-hospital mortality was 50%.The term "long lie trauma" describes a complex clinical situation, in which various conditions lead to an incapacitated state with acute onset, which then causes further adverse health effects. Trauma or tissue damage were no obligatory requirement in this syndrome. Considering the high morbidity and in-hospital mortality, patients should initially be treated in the emergency room by an interdisciplinary team.HINTERGRUND: Bisher fehlen Versorgungsdaten für Patienten mit Liegetrauma (LT).Deskriptive retrospektive Analyse aller rettungsdienstlich mit einem LT der Notaufnahme des Universitätsklinikums Köln von 07.2018 bis 12.2020 zugeführten Patienten.Insgesamt konnten 50 Patienten mit LT (Altersmedian 76 Jahre, Liegedauer im Median 13,5 h) im Untersuchungszeitraum identifiziert werden. Die zugrunde liegende Ursache für das LT war in 40 % primär neurologisch (ischämischer Schlaganfall: 20 %, intrakranielle Blutung: 16 %, Epilepsie: 4 %), in 12 % eine Intoxikation und in 10 % ein häusliches Trauma. Häufige assoziierte Diagnosen waren Infektionen (52 %), Traumafolgen (22 %), Exsikkose (66 %), akute Nierenfunktionsstörung (20 %), schwere Rhabdomyolyse (Kreatininkinase ≥ 5000 U/l, 21 %) und schwere Hypothermie 32 °C (20 %). Insgesamt wurden 69 % der Patienten auf einer Intensivstation aufgenommen und die Krankenhausletalität betrug 50 %.Das LT beschreibt einen Patientenzustand, bei dem infolge vielfältiger Ursachen plötzlich die eigenständige Mobilisierung und ein selbstständiges Hilfeholen verhindert werden und dadurch weitere Gesundheitsschäden entstehen. Bei diesem Syndrom sind Gewebsschäden als Folge des Liegens keine notwendige Voraussetzung für das Vorliegen eines LT. Aufgrund der hohen Morbidität und Letalität sollten diese Patienten in einem nichttraumatologischen Schockraum aufgenommen werden.

ObjectivemiRNAs are small non-coding RNAs that control gene expression. Specific intra- and extracellular miRNA signatures have been identified in various diseases. Whether certain miRNA signatures are associated with psoriasis (PsO) and PsA is currently unknown. We aimed to search for circulating miRNA signatures associated with PsO and PsA patients.MethodsExpression of miRNAs was analysed by reverse transcription quantitative real-time PCR (RT-qPCR) in the serum of PsA, PsO patients and healthy controls. Demographic and disease-specific characteristics and imaging data from hand MRI were recorded. In the discovery phase, 192 miRNA assays were analysed in 48 samples (PsA, PsO, controls: each N = 16). For validation, 17 selected miRNAs were measured in the total population.ResultsA total of 141 patients and controls were analysed (51 PsA, 40 PsO, 50 controls). In the discovery phase 51 miRNAs in PsO and 64 miRNAs in PsA were down- or upregulated compared with controls, with 33 miRNAs being changed in both (adj. P ConclusionmiRNA signatures in PsA and PsO patients differ from controls. Nine miRNAs were differentially regulated in PsA and PsO patients, five of them previously reported to be involved in bone and cartilage metabolism, indicating an intimate association of psoriatic inflammation and bone/cartilage changes.

Despite significant advances in the diagnosis and treatment of cardiovascular diseases, recent calls have emphasized the unmet need to improve precision-based approaches in cardiovascular disease. Although some studies provide preliminary evidence of the diagnostic and prognostic potential of circulating coding and non-coding RNAs, the complex RNA biology and lack of standardization have hampered the translation of these markers into clinical practice. In this position paper of the CardioRNA COST action CA17129, we provide recommendations to standardize the RNA development process in order to catalyse efforts to investigate novel RNAs for clinical use. We list the unmet clinical needs in cardiovascular disease, such as the identification of high-risk patients with ischaemic heart disease or heart failure who require more intensive therapies. The advantages and pitfalls of the different sample types, including RNAs from plasma, extracellular vesicles, and whole blood, are discussed in the sample matrix, together with their respective analytical methods. The effect of patient demographics and highly prevalent comorbidities, such as metabolic disorders, on the expression of the candidate RNA is presented and should be reported in biomarker studies. We discuss the statistical and regulatory aspects to translate a candidate RNA from a research use only assay to an in-vitro diagnostic test for clinical use. Optimal planning of this development track is required, with input from the researcher, statistician, industry, and regulatory partners., peer-reviewed

Platelets are the main effectors of primary hemostasis but also cause thrombosis in pathological conditions. Antiplatelet drugs are the cornerstone for the prevention of adverse cardiovascular events. Monitoring the extent of platelet inhibition is essential. Currently available platelet function tests come with constraints, limiting use in antiplatelet drug development as well as in clinical routine. With this study, we aim to investigate whether plasma miRNAs might be suitable biomarkers for monitoring antiplatelet treatment. Platelet-poor plasma was obtained from a trial including 87 healthy male volunteers that either received ticagrelor (n = 44) or clopidogrel (n = 43). Blood was collected before drug intake and after 2 h, 6 h, and 24 h. We measured a panel of 11 platelet-enriched miRNAs (thrombomiRs) by RT-qPCR and selected four biomarker candidates (i.e., miR-223-3p, miR-150-5p, miR-126-3p, miR-24-3p). To further characterize those miRNAs, we performed correlation analyses with the number of extracellular vesicles and clotting time dependent on procoagulant vesicles (PPL assay). We show that platelet-enriched miRNAs in the circulation are significantly reduced upon P2Y12-mediated platelet inhibition. This effect occurred fast, reaching its peak after 2 h. Additionally, we demonstrate that higher baseline levels of thrombomiRs are linked to a stronger reduction upon antiplatelet therapy. Finally, we show that miRNAs from our panel might be the cargo of platelet-derived and procoagulant vesicles. In conclusion, we provide evidence that thrombomiR levels change within 2 h after pharmacological platelet inhibition and circulate the body within platelet-derived and procoagulant extracellular vesicles, rendering them potential biomarker candidates for the assessment of in vivo platelet function.

Osteoarthritis of the equine distal interphalangeal joint is a common cause of lameness. MicroRNAs from biofluids are promising biomarkers and therapeutic candidates. Synovial fluid samples from horses with mild and severe equine distal interphalangeal joint osteoarthritis were submitted for small RNA sequencing. The results demonstrated that miR-92a was downregulated in equine synovial fluid from horses with severe osteoarthritis and there was a significant increase in COMP, COL1A2, RUNX2 and SOX9 following miR-92a mimic treatment of equine chondrocytes in monolayer culture. This is the first equine study to evaluate the role of miR-92a in osteoarthritic chondrocytes in vitro.

MicroRNAs (miRNAs) are short (18–24 nucleotides) non-coding RNA sequences that regulate gene expression via binding of messenger RNA. It is estimated that miRNAs co-regulate the expression of more than 70% of all human genes, many of which fulfil important roles in bone metabolism and muscle function. In-vitro and in-vivo experiments have shown that the targeted loss of miRNAs in distinct bone cell types (osteoblasts and osteoclasts) results in altered bone mass and bone architecture. These results emphasize the biological relevance of miRNAs for bone health. MiRNAs are not only considered as novel bone biomarkers because of their biological importance to bone metabolism, but also on the basis of other favorable properties: 1) Secretion of miRNAs from cells enables “minimally invasive” detection in biological fluids such as serum. 2) High stability of miRNAs in serum enables the retrospective analysis of frozen blood specimens. 3) Quantification of miRNAs in the serum is based on the RT-PCR - a robust method that is considered as the gold standard for the analysis of nucleic acids in clinical diagnostics. With regard to osteoporosis, it has been shown that many of the known risk factors are characterized by distinct miRNA profiles in the affected tissues: i) age-related loss of bone mass, ii) sarcopenia, iii) changes in estrogen metabolism and related changes Loss of bone mass, and iv) diabetes. Therefore, numerous studies in recent years have dealt with the characterization of miRNAs in the serum of osteoporosis patients and healthy controls, and were able to identify recurring miRNA patterns that are characteristic of osteoporosis. These novel biomarkers have great potential for the diagnosis and prognosis of osteoporosis and its clinical outcomes. The aim of this article is to give a summary of the current state of knowledge on the research and application of miRNA biomarkers in osteoporosis. MikroRNAs (miRNAs) sind kurze (18–24 Nukleotide) RNA Molekule, welche uber die Bindung an Botenstoff-RNA (messengerRNA) die Genexpression regulieren. Derzeit sind circa 2500 humane miRNAs annotiert. Circa 70% aller humanen Gene werden durch diese miRNAs co-reguliert, darunter viele Gene mit wesentlichen Funktionen fur den Knochenstoffwechsel und die Muskelfunktion. Experimente in der Petrischale und im Tiermodell konnten zeigen, dass der zielgerichtete Verlust von miRNAs in Zelltypen des Knochens (Osteoblasten und Osteoklasten) zu starken Veranderungen der Knochenstruktur fuhren, woraus sich eine grose Relevanz von miRNAs fur die Knochengesundheit ableiten lasst. Als Knochenbiomarker zeichnen sich miRNAs aber nicht nur wegen ihrer biologischen Funktionen im Knochenstoffwechsel aus, sondern auch auf Basis weiterer gunstiger Eigenschaften: 1) Die Sekretion von miRNAs aus Zellen ermoglicht eine „minimal-invasive“ Detektion in Bioflussigkeiten wie zum Beispiel Serum. 2) Die hohe Stabilitat von miRNAs im Serum vereinfacht retrospektive Analysen von gefrorenen Blutproben. 3) Die Quantifizierung von miRNAs im Serum erfolgt auf Basis der RT-PCR – eine robuste Methode die als Goldstandard fur die Analytik von Nukleinsauren in der klinischen Diagnostik gilt. In Bezug auf die Osteoporose konnte gezeigt werden, dass viele der bekannten Risikofaktoren zu Veranderungen von miRNA Profilen in den betroffenen Geweben fuhren: i) Altersbedingter Verlust an Knochenmasse, ii) Sarkopenie, iii) Veranderungen im Ostrogenstoffwechsel und daraus bedingter Verlust an Knochenmasse, und iv) Diabetes. In weiterer Folge haben sich in den vergangenen Jahren zahlreiche Studien mit der Charakterisierung von miRNAs im Serum von Osteoporosepatient*innen und gesunden Kontrollen auseinandergesetzt, und konnten miRNA Muster identifizieren, welche mit niedriger Knochendichte und erhohtem Frakturrisiko assoziiert sind. Diese neuartigen Biomarker haben groses Potenzial fur die Diagnose und Prognose der Osteoporose, insbesondere der klinischen Konsequenz in Form von Fragilitatsfrakturen. Dieser Artikel fasst den derzeitigen Wissenstand in Bezug auf die Erforschung und Anwendung von miRNAs als Biomarker fur Osteoporose zusammen.

Zusammenfassung Hintergrund Die aktuelle COVID-19-Pandemie stellt, trotz der Verfügbarkeit von Schnelltests und des Starts der Impfkampagne, die Notaufnahme weiterhin vor große Herausforderungen. Die strukturierte Erfassung von demographischen, klinischen sowie therapiebezogenen Daten stellt die Grundlage für die Erstellung evidenzbasierter Prozesse und Behandlungskonzepte dar. Ziel der Arbeit Darstellung der systematischen Erfassung klinischer Parameter bei Patienten mit COVID-19-Verdacht im Notaufnahmeregister ReCovER (Registry for COVID-19 in the Emergency Room) sowie deskriptive Darstellung der ersten 1000 Patienten. Material und Methoden In zentralen Notaufnahmen (ZNA) von 6 Universitätskliniken werden kontinuierlich Daten von Patienten mit COVID-19-Verdacht, unabhängig vom Nachweis einer SARS-CoV-2-Infektion, in ein webbasiertes, anonymisiertes Register eingegeben. Ergebnisse Zwischen dem 19.05.2020 und dem 13.01.2021 wurden 1000 Patienten in das Register eingegeben, hiervon waren 594 Patienten (59,4 %) in der SARS-CoV-2-positiven (PG) und 406 Patienten (40,6 %) in der negativen Gruppe (NG). Patienten der PG hatten signifikant weniger Vorerkrankungen und eine deutlich längere Latenz zwischen Symptombeginn und Vorstellung in der ZNA (Median 5 vs. 3 Tage), litten häufiger unter Husten, Myalgien, Fatigue sowie Geruchs/Geschmacksverlust und hatten einen signifikant höheren Sauerstoffbedarf als die NG-Patienten. Die Rate von schweren Krankheitsverläufen war in der PG signifikant erhöht und es bestanden nach Entlassung häufiger persistierende Symptome (11,1 vs. 4,6 %). Schlussfolgerungen Durch die multizentrische Erfassung von umfangreichen klinischen Daten zu COVID-19-Verdachtsfällen in der Notaufnahme können insbesondere auch für die Situation in Deutschland spezifische Aspekte analysiert werden. Dies ist essenziell für eine gezielte Überprüfung und Adaption international publizierter Strategien.

Parasites use different strategies of communication with their hosts. One communication channel that has been studied in recent years is the use of vesicle microRNAs to influence the host immune system by trematodes. sma-microRNA-10, secreted from

MicroRNAs (miRNAs) are short, single-stranded, non-coding RNAs which regulate gene expression. They originate from various tissues including bone and regulate different biological mechanisms including bone metabolism.The aim of this project was to investigate circulating miRNAs as promising biomarkers for treatment monitoring in women with postmenopausal osteoporosis on denosumab (DMAB) therapy.In this prospective, observational, single-centre study twenty-one postmenopausal women treated with DMAB were included for a longitudinal follow-up of two years.Next-generation sequencing (NGS) was performed to screen for serological miRNAs at defined time points (baseline, month 6 and month 24). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to confirm NGS findings in the entire cohort. Bone turnover markers (BTM) P1NP and CTX, and bone mineral density (BMD) by Dual X-Ray absorptiometry (DXA) were assessed and correlated to miRNAs.BMD at the hip (5,5%, p = 0.0006) and lumbar spine significantly increased (11,4%, p-value = 0.017) and CTX (64,1%, p 0.0001) and P1NP (69,3%, p 0.0001) significantly decreased during treatment. NGS analysis revealed significant changes in miRNAs after 2-years of DMAB treatment, but not after 6-months. Seven miRNAs were confirmed by RT-qPCR to be significantly changed during a 2-year course of DMAB treatment compared to baseline. Four of these were found to be mainly transcribed in blood cells including monocytes. Correlation analysis identified a significant correlation between change in miRNA and change in BTMs as well as BMD. Based on effect size and correlation strength, miR-454-3p, miR-26b-5p and miR-584-5p were defined as top biomarker candidates with the strongest association to the sustained effect of denosumab on bone in osteoporotic patients.Two years of DMAB-treatment resulted in the upregulation of 7 miRNAs, four of which are mainly transcribed in monocytes indicating a potential impact of DMAB on circulating osteoclast precursor cells. These changes were associated to BMD gain and BTM suppression and could therefore be useful for monitoring DMAB-treatment response.

ZusammenfassungBewusstseinsveränderungen sind eine typische Indikation für einen Notarzteinsatz. Dabei stellt das große Spektrum möglicher Differenzialdiagnosen für den Notarzt und die aufnehmende Klinik eine Herausforderung dar. Intoxikationen sind eine wichtige, jedoch insbesondere präklinisch oft schwierig zu identifizierende Ursache von Bewusstseinsveränderungen.

Dysregulation of platelet function is causally connected to thrombus formation and cardiovascular diseases. Therefore, assessing platelet reactivity is crucial. However, current platelet function tests come with pitfalls, limiting clinical use. Plasma miRNA signatures have been suggested as novel biomarkers for predicting/diagnosing cardiovascular diseases and monitoring antiplatelet therapy. Here, we provide results from a comprehensive study on the feasibility of using circulatory platelet miRNAs as surrogate markers of platelet activation. We performed small RNA-Seq on different blood cell types to confirm known and identify novel platelet-enriched miRNAs and validated a panel of 16 miRNAs using RT-qPCR. To identify the main carrier of these blood-based platelet miRNAs, we enriched and analyzed distinct microvesicle populations. Platelets were stimulated with GPVI and P2Y12 agonists in vitro to monitor the release of the selected miRNAs following activation. Finally, the miRNA panel was also measured in plasma from mice undergoing the Folts intervention (recurrent thrombus formation in the carotid artery). Applying an unbiased bioinformatics-supported workflow to our NGS data, we were able to confirm a panel of previously established miRNA biomarker candidates and identify three new candidates (i.e., miR-199a-3p, miR-151a-5p, and miR-148b-3p). Basal levels of platelet-derived miRNAs in plasma were mainly complexed with proteins, not extracellular vesicles. We show that changes in miRNA levels due to platelet activation are detectable using RT-qPCR. In addition, we highlight limitations of studying the in vitro release of miRNAs from platelets. In vivo thrombosis resulted in significant elevations of platelet-derived miRNA levels in mice. In conclusion, we provide in-depth evidence that activated platelets release miRNAs, resulting in measurable changes in circulatory miRNA levels, rendering them promising biomarker candidates.

Aims Coronavirus disease 2019 (COVID-19) can lead to multiorgan damage. MicroRNAs (miRNAs) in blood reflect cell activation and tissue injury. We aimed to determine the association of circulating miRNAs with COVID-19 severity and 28 day intensive care unit (ICU) mortality. Methods and results We performed RNA-Seq in plasma of healthy controls (n = 11), non-severe (n = 18), and severe (n = 18) COVID-19 patients and selected 14 miRNAs according to cell- and tissue origin for measurement by reverse transcription quantitative polymerase chain reaction (RT–qPCR) in a separate cohort of mild (n = 6), moderate (n = 39), and severe (n = 16) patients. Candidates were then measured by RT–qPCR in longitudinal samples of ICU COVID-19 patients (n = 240 samples from n = 65 patients). A total of 60 miRNAs, including platelet-, endothelial-, hepatocyte-, and cardiomyocyte-derived miRNAs, were differentially expressed depending on severity, with increased miR-133a and reduced miR-122 also being associated with 28 day mortality. We leveraged mass spectrometry-based proteomics data for corresponding protein trajectories. Myocyte-derived (myomiR) miR-133a was inversely associated with neutrophil counts and positively with proteins related to neutrophil degranulation, such as myeloperoxidase. In contrast, levels of hepatocyte-derived miR-122 correlated to liver parameters and to liver-derived positive (inverse association) and negative acute phase proteins (positive association). Finally, we compared miRNAs to established markers of COVID-19 severity and outcome, i.e. SARS-CoV-2 RNAemia, age, BMI, D-dimer, and troponin. Whilst RNAemia, age and troponin were better predictors of mortality, miR-133a and miR-122 showed superior classification performance for severity. In binary and triplet combinations, miRNAs improved classification performance of established markers for severity and mortality. Conclusion Circulating miRNAs of different tissue origin, including several known cardiometabolic biomarkers, rise with COVID-19 severity. MyomiR miR-133a and liver-derived miR-122 also relate to 28 day mortality. MiR-133a reflects inflammation-induced myocyte damage, whilst miR-122 reflects the hepatic acute phase response., Graphical Abstract Graphical Abstract

In contrast to traditional methods like real-time polymerase chain reaction, next-generation sequencing (NGS), and especially small RNA-seq, enables the untargeted investigation of the whole small RNAome, including microRNAs (miRNAs) but also a multitude of other RNA species. With the promising application of small RNAs as biofluid-based biomarkers, small RNA-seq is the method of choice for an initial discovery study. However, the presentation of specific quality aspects of small RNA-seq data varies significantly between laboratories and is lacking a common (minimal) standard. The miRNA NGS Discovery pipeline (miND) aims to bridge the gap between wet lab scientist and bioinformatics with an easy to setup configuration sheet and an automatically generated comprehensive report that contains all essential qualitative and quantitative results that should be reported. Besides the standard steps like preprocessing, mapping, visualization, and quantification of reads, the pipeline also incorporates differential expression analysis when given the appropriate information regarding sample groups. Although miND has a focus on miRNAs, other RNA species like tRNAs, piRNA, snRNA, or snoRNA are included and mapping statistics are available for further analysis. miND has been developed and tested on a multitude of data sets with various RNA sources (tissue, plasma, extracellular vesicles, urine, etc.) and different species. miND is a Snakemake based pipeline and thus incorporates all advantages using a flexible workflow management system. Reference databases are downloaded, prepared and built with an included (but separate) workflow and thus can easily be updated to the most recent version but also stored for reproducibility. In conclusion, the miND pipeline aims to streamline the bioinformatics processing of small RNA-seq data by standardizing the processing from raw data to a final, comprehensive and reproducible report.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (−37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

MicroRNAs (miRNAs) have emerged as key regulators orchestrating a wide range of inflammatory and fibrotic diseases. However, the role of miRNAs in degenerative shoulder joint disorders is poorly understood. The aim of this explorative case‐control study was to identify pathology‐related, circulating miRNAs in patients with chronic rotator cuff tendinopathy and degenerative rotator cuff tears (RCT). In 2017, 15 patients were prospectively enrolled and assigned to three groups based on the diagnosed pathology: (i) no shoulder pathology, (ii) chronic rotator cuff tendinopathy, and (iii) degenerative RCTs. In total, 14 patients were included. Venous blood samples (“liquid biopsies”) were collected from each patient and serum levels of 187 miRNAs were determined. Subsequently, the change in expression of nine candidate miRNAs was verified in tendon biopsy samples, collected from patients who underwent arthroscopic shoulder surgery between 2015 and 2018. Overall, we identified several miRNAs to be progressively deregulated in sera from patients with either chronic rotator cuff tendinopathy or degenerative RCTs. Importantly, for the several of these miRNAs candidates repression was also evident in tendon biopsies harvested from patients who were treated for a supraspinatus tendon tear. As similar expression profiles were determined for tendon samples, the newly identified systemic miRNA signature has potential as novel diagnostic or prognostic biomarkers for degenerative rotator cuff pathologies. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. Inc. J Orthop Res 38:202–211, 2020

There is an urgent need for an easily assessable preoperative test to predict postoperative liver function recovery and thereby determine the optimal time point of liver resection, specifically as current markers are often expensive, time consuming, and invasive. Emerging evidence suggests that microRNA (miRNA) signatures represent potent diagnostic, prognostic, and treatment‐response biomarkers for several diseases. Using next‐generation sequencing as an unbiased systematic approach, 554 miRNAs were detected in preoperative plasma of 21 patients suffering from postoperative liver dysfunction (LD) after liver resection and 27 matched controls. Subsequently, we identified a miRNA signature—consisting of miRNAs 151a‐5p, 192‐5p, and 122‐5p—that highly correlated with patients developing postoperative LD after liver resection. The predictive potential for postoperative LD was subsequently confirmed using real‐time PCR in an independent validation cohort of 98 patients. Ultimately, a regression model of the two miRNA ratios 151a‐5p to 192‐5p and 122‐5p to 151a‐5p was found to reliably predict postoperative LD, severe morbidity, prolonged intensive care unit and hospital stays, and even mortality before an operation with a remarkable accuracy, thereby outperforming established markers of postoperative LD. Ultimately, we documented that miRNA ratios closely followed liver function recovery after partial hepatectomy. Conclusion: Our data demonstrate the clinical utility of an miRNA‐based biomarker to support the selection of patients undergoing partial hepatectomy. The dynamical changes during liver function recovery indicate a possible role in individualized patient treatment. Thereby, our data might help to tailor surgical strategies to the specific risk profile of patients.

Engineering of Chinese Hamster Ovary cells by manipulating microRNA (miRNA) expression levels has been shown to induce advantageous, desired phenotypes. Most of these studies so far were concerned with increasing productivity or reducing growth rate (with the implied intention of thus freeing cellular resources to also increase productivity). Here we evaluated the ability of growth correlating miRNAs to increase the growth rate of CHO-K1 cells by transient overexpression or knock down, respectively. Candidates were selected based on the correlation between growth rate and miRNA expression levels as observed in previous studies. These candidates were then up- or downregulated initially by transfection of mimics or inhibitors and subsequently by transfection of plasmids bearing the corresponding miRNAs or sponges. None of the 40 selected candidates was able to induce a better growth phenotype under these conditions. Overlap between miRNAs identified to correlate to growth in published miRNA expression studies and those identified to actively increase growth rate in a functional screen is minimal, indicating that the here selected approach of traditional overexpression/knock down engineering of miRNAs may not be a suitable strategy for the purpose of increasing growth rate.

Blood circulating microRNAs (c‐miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti‐aging therapies. Based on a cross‐sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c‐miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR‐19a‐3p and miR‐19b‐3p, were found increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT‐qPCR, but only with a high‐resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR‐19a/b‐3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age‐related increase of plasma miR‐19a/b‐3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c‐miRs and isomiRs as additional parameter to track the onset of aging and age‐related diseases using new potential biomarkers., Small RNA deep sequencing on plasma‐derived RNA from 12 donors (young and old subjects, healthy, and unhealthy centenarians) was performed: 79 differentially expressed c‐miRs were identified. MiR‐19a/b‐3p increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 subjects. The decrease was more relevant in healthy centenarians, also confirmed by the modified pattern of isomiRs. Three common targets of miR‐19a/b‐3p were identified converging into the FoxO signaling pathway.

Context: On March 11, the World Health Organization (WHO) announced the current corona virus disease 2019 (COVID-19) outbreak as a pandemic. The first laboratory-confirmed case of COVID-19 in Austria was announced on February 27, 2020. Since then, the incidence of infection followed an exponential increase until a complete lockdown in March 2020. Thereafter easing of restrictions was gradually introduced and until mid-August daily infections remained mostly below 5 per 100.000 population. Objectives: The aims of this study are to determine i) how many employees in Austrian trauma hospitals and rehabilitation facilities have virus specific IgG and IgM, and/or neutralizing antibodies against SARS-CoV-2, ii) how many are active virus carriers (symptomatic and asymptomatic) during the study, iii) the antibody decline in seropositive subjects over a period of around six months, and iv) the utility of rapid antibody tests for outpatient screening.Study Design: Open uncontrolled observational cross-sectional study.Setting/Participants: A total of 3301 employees in 11 Austrian trauma hospitals and rehabilitation facilities of the Austrian Social Insurance for Occupational Risks (AUVA) participated in the study.Study Interventions and Measures: Rapid antibody tests for SARS-CoV-2 specific IgG and IgM antibodies, and RT-PCR tests based on oropharyngeal swab samples, as well as laboratory-based antibody tests using ELISA/PRNT were performed. The tests were conducted twice, with an interval of 42.4±7.7 (Min=30, Max=64) days. Additionally, participants filled out a questionnaire including questions related to personal health, traveling activities, living situation, as well as inquiries of symptoms and comorbidities. Antibody positive tested participants were re-tested with ELISA/PRNT tests at a third time point on average 188.0±12.8 days after their initial test.Results: In our study cohort, only 27 out of 3301 participants (0.81%) had a positive antibody test at any time point during the study confirmed via neutralization test. Among participants who had positive test results in either of the antibody tests, 50.4% did not report any symptoms consistent with common manifestations of COVID-19 during the study period or within the preceding six weeks. In the group who tested positive during or prior to study inclusion the most common symptoms of an acute viral illness were rhinitis (21.9%), and loss of taste and olfactory sense (21.9%).The rapid antibody test was generally more sensitive based on serum (sensitivity=86.6%) as compared to whole blood (sensitivity=65.4). Concerning both ELISA tests overall the Roche test detected 24 (sensitivity=88.9%) and the Diasorin test 22 positive participants (sensitivity=81.5%).In participants with a positive PRNT, a significant decrease in PRNT concentration from 31.8±22.9 (Md=32.0) at T1 to 26.1±17.6 (Md=21.3) at T2 to 21.4±13.4 (Md=16.0) at T3 (c2=23.848, df=2, p2=23.848, df=2, pConclusions: During the study period (May 11th – December 21th) only 0.81% were tested positive for antibodies in our study cohort. The antibody concentration decreases significantly over time with 14.8% (4 out of 27) losing detectable antibodies.

MicroRNAs (miRNAs) are continued to be investigated as biomarkers for several translational and clinical applications, including cardiovascular disease. Biological importance, tissue specificity, sequence conservation, presence in biofluids, assay availability, and robustness are key advantages of miRNAs as biomarkers. However, the devil is in the detail, and especially in the context of blood-based biofluids, preanalytical, analytical, and biological variability can mask disease-related biological effects and impair biomarker development and implementation in clinical practice. Here, we summarize published as well as our own experiences with preanalytical, analytical, and biological biases that can interfere with extracellular RNA analysis and which have been inherent to several years of miRNA biomarker discovery and development.

Fragility fractures are an important hallmark of aging and an increasingly recognized complication of Type 2 diabetes (T2D). T2D individuals have been found to exhibit an increased fracture risk despite elevated bone mineral density (BMD) by dual x-ray absorptiometry (DXA). However, BMD and FRAX-scores tend to underestimate fracture risk in T2D. New, reliable biomarkers are therefore needed. MicroRNAs (miRNAs) are secreted into the circulation from cells of various tissues proportional to local disease severity. Serum miRNA-classifiers were recently found to discriminate T2D women with and without prevalent fragility fractures with high specificity and sensitivity (AUC 0.90). However, the association of circulating miRNAs with incident fractures in T2D has not been examined yet. In 168 T2D postmenopausal women in the AGES-Reykjavik cohort, miRNAs were extracted from baseline serum and a panel of 10 circulating miRNAs known to be involved in diabetic bone disease and aging was quantified by qPCR and Ct-values extracted. Unadjusted and adjusted Cox proportional hazard models assessed the associations between serum miRNAs and incident fragility fracture. Additionally, Receiver operating curve (ROC) analyses were performed. Of the included 168 T2D postmenopausal women who were on average 77.2 ± 5.6 years old, 70 experienced at least one incident fragility fracture during the mean follow-up of 5.8 ± 2.7 years. We found that 3 serum miRNAs were significantly associated with incident diabetic fragility fracture: while low expression of miR-19b-1-5p was associated with significantly lower risk of incident fragility fracture (HR 0.84 (95% CI: 0.71-0.99, p = 0.0323)), low expression of miR-203a and miR-31-5p was each significantly associated with a higher risk of incident fragility fracture per unit increase in Ct-value (miR-203a: HR 1.29 (95% CI: 1.12-1.49), p = 0.0004, miR-31-5p HR 1.27 (95% CI: 1.06-1.52), p = 0.009). Hazard ratios of the latter two miRNAs remained significant after adjustments for age, body mass index (BMI), areal bone mineral density (aBMD), clinical FRAX or FRAXaBMD. Women with miR-203a and miR-31-5p serum levels in the lowest expression quartiles exhibited a 2.4-3.4-fold larger fracture risk than women with miR-31-5p and miR-203a serum expressions in the highest expression quartile (0.002 ≤ p ≤ 0.039). Women with both miR-203a and miR-31-5p serum levels below the median had a significantly increased fracture risk (Unadjusted HR 3.26 (95% CI: 1.57-6.78, p = 0.001) compared to those with both expression levels above the median, stable to adjustments. We next built a diabetic fragility signature consisting of the 3 miRNAs that showed the largest associations with incident fracture (miR-203a, miR-31-5p, miR-19b-1-5p). This 3-miRNA signature showed with an AUC of 0.722 comparable diagnostic accuracy in identifying incident fractures to any of the clinical parameters such as aBMD, Clinical FRAX or FRAXaBMD alone. When the 3 miRNAs were combined with aBMD, this combined 4-feature signature performed with an AUC of 0.756 (95% CI: 0.680, 0.823) significantly better than aBMD alone (AUC 0.666, 95% CI: 0.585, 0.741) (p = 0.009). Our data indicate that specific serum microRNAs including senescent miR-31-5p are associated with incident fragility fracture in older diabetic women and can significantly improve fracture risk prediction in diabetics when combined with aBMD measurements of the femoral neck.

A specific signature of 19 circulating miRNAs (osteomiRs) has been reported to be associated with fragility fractures due to postmenopausal osteoporosis. However, it is unknown whether osteoporotic fractures or low BMD phenotypes are independently contributing to changes in osteomiR serum levels. The first aim was to characterize the abundance, sensitivity to hemolysis, and correlation of osteomiR serum levels, the second objective to evaluate the diagnostic accuracy of osteomiRs for osteoporosis according to the WHO criteria and on basis of major osteoporotic fracture history. Fifty postmenopausal women with osteoporosis (with or without fragility fracture) and 50 non-osteoporotic women were included in this cross-sectional study. The diagnostic performance of osteomiRs for osteoporosis based on the WHO definition or fracture history was evaluated using multiple logistic regression and receiver-operator curve (AUC) analysis. The osteomiR® signature is composed of four clusters of miRNAs providing good performance for the diagnosis of osteoporosis in postmenopausal women defined by WHO criteria (AUC = 0.830) and based on history of major osteoporotic fractures (AUC = 0.834). The classification performance for the WHO criteria and for fracture risk is driven by miR-375 and miR-203a, respectively. OsteomiRs, a signature of 19 emerging miRNA bone biomarkers, are measurable in human serum samples. They constitute a panel of independent bone and muscle biomarkers, which in combination could serve as diagnostic biomarkers for osteoporosis in postmenopausal women. Supplementary Information The online version of this article (doi:10.1007/s00223-020-00802-3) contains supplementary material, which is available to authorized users.

Phenotype-specific omic expression patterns in people with frailty could provide invaluable insight into the underlying multi-systemic pathological processes and targets for intervention. Classical approaches to frailty have not considered the potential for different frailty phenotypes. We characterized associations between frailty (with/without disability) and sets of omic factors (genomic, proteomic, and metabolomic) plus markers measured in routine geriatric care. This study was a prevalent case control using stored biospecimens (urine, whole blood, cells, plasma, and serum) from 1522 individuals (identified as robust (R), pre-frail (P), or frail (F)] from the Toledo Study of Healthy Aging (R=178/P=184/F=109), 3 City Bordeaux (111/269/100), Aging Multidisciplinary Investigation (157/79/54) and InCHIANTI (106/98/77) cohorts. The analysis included over 35,000 omic and routine laboratory variables from robust and frail or pre-frail (with/without disability) individuals using a machine learning framework. We identified three protective biomarkers, vitamin D3 (OR: 0.81 [95% CI: 0.68-0.98]), lutein zeaxanthin (OR: 0.82 [95% CI: 0.70-0.97]), and miRNA125b-5p (OR: 0.73, [95% CI: 0.56-0.97]) and one risk biomarker, cardiac troponin T (OR: 1.25 [95% CI: 1.23-1.27]). Excluding individuals with a disability, one protective biomarker was identified, miR125b-5p (OR: 0.85, [95% CI: 0.81-0.88]). Three risks of frailty biomarkers were detected: pro-BNP (OR: 1.47 [95% CI: 1.27-1.7]), cardiac troponin T (OR: 1.29 [95% CI: 1.21-1.38]), and sRAGE (OR: 1.26 [95% CI: 1.01-1.57]). Three key frailty biomarkers demonstrated a statistical association with frailty (oxidative stress, vitamin D, and cardiovascular system) with relationship patterns differing depending on the presence or absence of a disability.

MicroRNA‑21 (miR‑21) is a small non‑coding RNA that is differentially expressed during tooth development, particularly during amelogenesis. Although orthodontic tooth movement and the innate immune response are impaired, miR‑21 knockout mice demonstrate no obvious skeletal phenotype. However, the consequence of miR‑21 knockout on tooth phenotype and corresponding alveolar bone is unknown. The current study utilized landmark‑based geometric morphometrics to identify anatomical dissimilarities of the three lower and upper molars, and the corresponding alveolar bone, in miR‑21 knockout and wild‑type control mice. The anatomical structures were visualized by microcomputer tomography. A total of 36 and 38 landmarks were placed on mandibular and maxillary molars, respectively. For the alveolar bone, 16 landmarks were selected on both anatomical sites. General Procrustes analysis revealed significantly smaller molars and dimensions of the alveolar bone in the mandible of the miR‑21 knockout mice when compared with wild‑type controls (P=0.03 and P=0.04, respectively). The overall dimension of the mandible was reduced by the lack of miR‑21 (P=0.02). In the maxilla, the dimension of the alveolar bone was significant (P=0.02); however, this was not observed in the molars (P=0.36). Based on principal component analysis, no changes in shape for any of the anatomical sites were observed. Dental and skeletal jaw length were calculated and no prognathism was identified. However, the fluctuating asymmetry of the molars in the mandible and the maxilla was reduced in the miR‑21 knockout mice by 38 and 27%, respectively. Taken together, the results of the present study revealed that the molars in the mandible and the dimension of the respective alveolar bone were smaller in miR‑21 mice compared with wild‑type littermates, suggesting that miR‑21 influences tooth development.

Bone loss is one of the consequences of aging, leading to diseases such as osteoporosis and increased susceptibility to fragility fractures and therefore considerable morbidity and mortality in humans. Here we identify microRNA 146a as an essential epigenetic switch controlling bone loss with age. Mice deficient in miR-146a show regular development of their skeleton. However, while WT mice start to lose bone with age, animals deficient in miR-146a continue to accrue bone throughout their life span. Increased bone mass is due to increased generation and activity of osteoblasts in miR-146a deficient mice as a result of sustained activation of bone anabolic Wnt signaling during aging. Deregulation of the miR-146a target genes Wnt1 and Wnt5a parallel bone accrual and osteoblast generation, which is accompanied by reduced development of bone marrow adiposity. Furthermore, miR-146a deficient mice are protected from ovariectomy induced bone loss. In humans, levels of miR-146a are increased in patients suffering fragility fractures in comparison to those who do not.These data identify miR-146a as a crucial epigenetic temporal regulator which essentially controls bone homeostasis during aging by regulating bone anabolic Wnt signaling. Therefore, miR-146a might be a powerful therapeutic target to prevent age related bone dysfunction such as the development of bone marrow adiposity and osteoporosis.

Plastin 3 (PLS3), encoded by PLS3, is a newly recognized regulator of bone metabolism, and mutations in the encoding gene result in severe childhood-onset osteoporosis. Because it is an X chromosomal gene, PLS3 mutation-positive males are typically more severely affected whereas females portray normal to increased skeletal fragility. Despite the severe skeletal pathology, conventional metabolic bone markers tend to be normal and are thus insufficient for diagnosing or monitoring patients. Our study aimed to explore serum microRNA (miRNA) concentrations in subjects with defective PLS3 function to identify novel markers that could differentiate subjects according to mutation status and give insight into the molecular mechanisms by which PLS3 regulates skeletal health. We analyzed fasting serum samples for a custom-designed panel comprising 192 miRNAs in 15 mutation-positive (five males, age range 8-76 years, median 41 years) and 14 mutation-negative (six males, age range 8-69 years, median 40 years) subjects from four Finnish families with different PLS3 mutations. We identified a unique miRNA expression profile in the mutation-positive subjects with seven significantly upregulated or downregulated miRNAs (miR-93-3p, miR-532-3p, miR-133a-3p, miR-301b-3p, miR-181c-5p, miR-203a-3p, and miR-590-3p; p values, range .004-.044). Surprisingly, gender subgroup analysis revealed the difference to be even more distinct in female mutation-positive subjects (congruent p values, range .007-.086) than in males (p values, range .127-.843) in comparison to corresponding mutation-negative subjects. Although the seven identified miRNAs have all been linked to bone metabolism and two of them (miR-181c-5p and miR-203a-3p) have bioinformatically predicted targets in the PLS3 3' untranslated region (3'-UTR), none have previously been reported to associate with PLS3. Our results indicate that PLS3 mutations are reflected in altered serum miRNA levels and suggest there is crosstalk between PLS3 and these miRNAs in bone metabolism. These provide new understanding of the pathomechanisms by which mutations in PLS3 lead to skeletal disease and may provide novel avenues for exploring miRNAs as biomarkers in PLS3 osteoporosis or as target molecules in future therapeutic applications. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Glioblastoma is the most prevalent and aggressive brain cancer. With a median overall survival of ~15–20 months under standard therapy, novel treatment approaches are desperately needed. A recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cell (DC) vaccination, however, failed to prolong survival. Here, we investigated tumor tissue from trial patients to explore glioblastoma survival-related factors. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n = 36) with microRNA sequencing plus RT-qPCR (n = 38). Protein quantification identified, e.g., huntingtin interacting protein 1 (HIP1), retinol-binding protein 1 (RBP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as factor candidates correlated with a dismal prognosis. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with favorable tissue characteristics as they were enriched in patients with a comparably longer survival. To illustrate the utility of integrated miRNomics and proteomics findings, focal adhesion was studied further as one example for a pathway of potential general interest. Taken together, we here mapped possible drivers of glioblastoma outcome under immunotherapy in one of the largest DC vaccination tissue analysis cohorts so far—demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy—potentially building on insights generated here., Glioblastoma: screening for survival-related factors Glioblastoma is an aggressive form of brain cancer and effective immunotherapeutics are limited, with treatment currently based on chemotherapy and radiotherapy. A recent phase II clinical trial tested a personalized, targeted dendritic cell-based immunotherapy but there was no observed improvement in patient survival or progression-free survival compared to standard-of-care therapy. Here, Carmen Visus and colleagues have used tumor tissue samples from glioblastoma patients involved in this trial and receiving immunotherapy. Using a combination of mass spectrometry-based proteomics, microRNA sequencing and RT-qPCR they identified factors associated with survival or poor prognosis. Proteomics associated poor prognosis with various proteins including focal adhesion kinase 2 (FAK2), whilst microRNAs, miR-216b, miR-216a, miR-708 and let-7i, were associated with longer survival. Focussing on one pathway, FAK2, they integrated the proteomic and microRNA datasets and saw a negative association with overall survival across all patients. To test this, they added an FAK inhibitor to glioblastoma cell lines, including cells isolated from trial patients, and observed inhibition of gliomaspheres in treated cells, providing insights into potential immunotherapy targets.

In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale.

Background Osteoporosis poses an immense burden to the society in terms of morbidity, mortality and financial cost. To reduce this burden, it is essential to accurately assess the individual patient's fracture risk and, where indicated, to initiate appropriate treatment that reduces fracture probability. Current screening and monitoring approaches include utilization of FRAX®, a web-based country-specific fracture risk assessment tool, and bone mineral density measurement by Dual Energy X-ray Absorptiometry (DXA). Recently, microRNAs have been recognized as important regulators of bone physiology and potential biomarkers for fracture risk assessment and monitoring. A fracture risk assessment tool based on microRNAs (osteomiR™ test) is currently being developed. The aim of this study was to estimate the cost-effectiveness of fracture risk screening, monitoring, and resulting treatment decisions for the Austrian female population using the osteomiR™ test compared with DXA, with FRAX®, or with no screening/monitoring. Methods A cost-utility-model was developed to simulate long-term consequences of Austrian women from age 50 over lifetime or death with respect to osteoporosis. Markov-modelling techniques were used to calculate health state transitions of fracture incidence according to risk groups (high, intermediate, low). High-risk patients receive medical treatment. Probabilities were derived via systematic-literature-review; direct costs (2015, €) from published sources from the payer's perspective. Results evaluate the incremental cost-effectiveness ratios (ICER) for osteomiR™ against the comparators, gains or losses of fractures, life years (LYs), quality-adjusted life years (QALYs), and direct costs. QALYs, life years (LYs) and costs were discounted (3% p.a). Results Fracture risk assessment and monitoring using the osteomiR™ test reduces fracture incidence compared with no monitoring, DXA alone, or FRAX® alone. In the per-patient analysis, the ICER/QALY of osteomiR™ vs. no-monitoring was 13,103 €, vs. FRAX® 37,813 €, and vs. DXA -19,605 €, indicating that costs can be saved while gaining QALYs. Considering the total cohort over lifetime, the osteomiR™ test can avoid 57,919 fractures compared with DXA, 31,285 fractures compared with FRAX® and 133,394 fractures compared with no monitoring. Sensitivity analysis confirmed the robustness of these findings. Conclusion Fracture risk assessment and monitoring using the osteomiR™ test dominates DXA-strategy and constitutes a cost-effective alternative to FRAX®, and no-monitoring, respectively.

Deoxynivalenol (DON), a frequent mycotoxin worldwide, impairs human and animal health. The response of microRNAs, small non-coding RNAs, to DON has been scarcely investigated, but holds remarkable potential for biomarker applications. Hence, we aimed to investigate DON-induced changes in the microRNA expression in porcine liver, jejunum and serum by combining targeted and untargeted analyses. Piglets received uncontaminated feed or feed containing 900 µg/kg and 2500 µg/kg DON for four weeks, followed by a wash-out period. In tissue, only slight changes in microRNA expression were detected, with ssc-miR-10b being downregulated in liver of DON-exposed piglets. In serum, several microRNAs were differentially expressed upon DON exposure, four of which were validated by qPCR (ssc-miR-16, ssc-miR-128, ssc-miR-451, ssc-miR-205). The serum microRNA response to DON increased over time and declined after removal of contaminated diets. Receiver operating curve analyses for individual microRNAs were significant, and a combination of the four microRNAs increased the predictive capacity for DON exposure. Predicted microRNA target genes showed enrichment of several pathways including PIK3-AKT, Wnt/β-catenin, and adherens junctions. This study gives, for the first time, a comprehensive view of the porcine microRNA response to DON, providing a basis for future research on microRNAs as biomarkers for mycotoxins.

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular microRNAs in concentrated conditioned media (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEV miRNA profiles – but not cCM miRNAs – were impacted by lipedema. Seven sEV miRNAs (miR–16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR–144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy, whereas only one cCM miRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

Zearalenone (ZEN)-degrading enzymes are a promising strategy to counteract the negative effects of this mycotoxin in livestock. The reaction products of such enzymes need to be thoroughly characterized before technological application as a feed additive can be envisaged. Here, we evaluated the estrogenic activity of the metabolites hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) formed by hydrolysis of ZEN by the zearalenone-lactonase Zhd101p. ZEN, HZEN, and DHZEN were tested in two in vitro models, the MCF-7 cell proliferation assay (0.01&ndash, 500 nM) and an estrogen-sensitive yeast bioassay (1&ndash, 10,000 nM). In addition, we compared the impact of dietary ZEN (4.58 mg/kg) and equimolar dietary concentrations of HZEN and DHZEN on reproductive tract morphology as well as uterine mRNA and microRNA expression in female piglets (n = 6, four weeks exposure). While ZEN increased cell proliferation and reporter gene transcription, neither HZEN nor DHZEN elicited an estrogenic response, suggesting that these metabolites are at least 50&ndash, 10,000 times less estrogenic than ZEN in vitro. In piglets, HZEN and DHZEN did not increase vulva size or uterus weight. Moreover, RNA transcripts altered upon ZEN treatment (EBAG9, miR-135a-5p, miR-187-3p and miR-204-5p) were unaffected by HZEN and DHZEN. Our study shows that both metabolites exhibit markedly reduced estrogenicity in vitro and in vivo, and thus provides an important basis for further evaluation of ZEN-degrading enzymes.

The mycotoxin zearalenone (ZEN) poses a risk to animal health because of its estrogenic effects. Diagnosis of ZEN-induced disorders remains challenging due to the lack of appropriate biomarkers. In this regard, circulating microRNAs (small non-coding RNAs) have remarkable potential, as they can serve as indicators for pathological processes in tissue. Thus, we combined untargeted and targeted transcriptomics approaches to investigate the effects of ZEN on the microRNA expression in porcine uterus, jejunum and serum, respectively. To this end, twenty-four piglets received uncontaminated feed (Control) or feed containing 0.17 mg/kg ZEN (ZEN low), 1.46 mg/kg ZEN (ZEN medium) and 4.58 mg/kg ZEN (ZEN high). After 28 days, the microRNA expression in the jejunum remained unaffected, while significant changes in the uterine microRNA profile were observed. Importantly, 14 microRNAs were commonly and dose-dependently affected in both the ZEN medium and ZEN high group, including microRNAs from the miR-503 cluster (i.e. ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-503 and ssc-miR-542-3p). Predicted target genes for those microRNAs are associated with regulation of gene expression and signal transduction (e.g. cell cycle). Although the effects in serum were less pronounced, receiver operating characteristic analysis revealed that several microRNA ratios were able to discriminate properly between non-exposed and ZEN-exposed pigs (e.g. ssc-miR-135a-5p/ssc-miR-432-5p, ssc-miR-542-3p/ssc-miR-493-3p). This work sheds new light on the molecular mechanisms of ZEN, and fosters biomarker discovery.