BACKGROUND:A prospective, randomized, placebo-controlled trial showed reduced retears after arthroscopic rotator cuff (RC) repair in patients without osteoporosis by a systemic single-dose of zoledronic acid. Distinct micro-ribonucleic acids (miRNAs) related to inflammation, fibrosis, and tendon-to-bone healing are associated with RC injuries. PURPOSE:To investigate the longitudinal effects of a single-dose of zoledronic acid in patients without osteoporosis undergoing arthroscopic RC repair on circulating miRNAs in order to explore the molecular mechanism of this treatment. STUDY DESIGN:Controlled laboratory study. METHODS:Data were collected in the course of a single-center, prospective, randomized, placebo-controlled, triple-blinded (investigator, surgeon, patient) phase II trial. A total of 80 patients underwent arthroscopic RC repair and were intraoperatively randomized to the zoledronic acid group (n = 40) or the control group (n = 40). Circulating plasma miRNAs were assessed preoperatively, 2 days postoperatively, and 3 months postoperatively using small RNA sequencing and reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS:Six miRNAs were selected for validation by RT-qPCR based on small RNA-sequencing analysis. No statistical differences in miRNA plasma levels were observed preoperatively between the 2 study groups. Two days after surgery, plasma levels were significantly lower for miR-140-3p (P = .047) in the zoledronic acid group. In the control group, plasma levels were significantly lower for miR-29a-3p (P < .001), miR-21-5p (P = .036), miR-192-5p (P = .034), and miR-146a-5p (P < .001) 2 days after surgery. Three months after surgery, miRNA plasma levels of the 2 study groups equalized without any significant differences. Gene target analysis identified collagen type I alpha 1 chain, collagen type III alpha 1 chain, vascular endothelial growth factor, insulin-like growth factor 1, and transforming growth factor-β1 as targets of miRNAs impacted by zoledronic acid. CONCLUSION:Longitudinal plasma miRNA expressions suggest a molecular response to zoledronic acid, potentially indicating a reduction of inflammation and fibrosis at the enthesis as well as increased collagen synthesis and vascularization, possibly explaining improved tendon-to-bone healing. CLINICAL RELEVANCE:Zoledronic acid is a safe and easy-to-apply augmentation technique for patients undergoing arthroscopic RC repair. REGISTRATION:NCT05677152 (ClinicalTrials.gov identifier).

MicroRNAs (miRNAs) are epigenetic regulators of gene activity. Analysis of circulating miRNAs enables minimal-invasive studies of disease mechanisms and identification of novel disease biomarkers. The aim of this case-control validation study was to investigate previously identified circulating miRNAs in Swedish adolescents with long-duration (8.0-16.5 years) type 1 diabetes (T1D), and healthy matched controls to confirm their utility as biomarker candidates to diagnose and monitor the progression of T1D. Quantitative PCR analysis of 23 previously reported miRNAs was performed in 24 T1D and 24 control individuals. Body composition was assessed by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Prospectively collected clinical data were retrieved from the Swedish diabetes quality registry. The selected miRNAs showed higher variability in both male and female T1D groups compared to controls. Statistical analysis confirmed differences for 12 miRNAs in comparison with controls, including miR-223-3p and miR-135a-5p, which previously were reported to be associated with T1D. MiR-34a-5p and miR-210-3p were positively associated with T1D duration and HbA1c (average from the last year), respectively. In conclusion, 12 previously reported miRNAs showed consistent differential expression between individuals with T1D and controls. Among these were miR-223-3p and miR-135a-5p, which are associated with cardiovascular/inflammatory disease and cancer, respectively. These findings suggest potential clinical utility of circulating miRNAs for T1D diagnosis and disease monitoring, although extended validation of the identified miRNA biomarkers in larger, independent cohorts is required to establish the necessary scientific evidence for clinical translation.

Background: Therapeutic applications require large amounts of extracellular vesicles (EVs) that cannot be obtained by standard laboratory protocols. Since culturing parameters and isolation methods can significantly affect the molecular composition and therapeutic efficacy of EVs, the development of a new scale-up protocol should be followed by the molecular fingerprinting and validation of therapeutic potential in vivo.Methods: We developed a new scale-up protocol based on microcarrier culture (3D) of immortalized human dental pulp stem cells in a spinning bioreactor and subsequent isolation of EVs by 2-step tangential flow filtration (TFF) and size exclusion chromatography (SEC).Results: A new scale-up protocol increased EV yields by 463-fold. When compared with ultracentrifugation (UC), isolation using TFF/SEC substantially reduced the complexity of proteomic cargo, whereas culture conditions (2D vs. 3D) affected miRNA, but not mRNA and proteomic content of the EVs. We next compared the therapeutic efficacy of both EV products in 6-hydroxydopamine rat model of Parkinson's disease (PD). The same amounts of EVs derived from standard 2D cultures by UC and a new large-scale protocol were intranasally administered to PD rats, where they similarly improved gait and cognitive functions, preserved nigrostriatal tyrosine hydroxylase density and suppressed neuroinflammation. Notably, both EV preparations were enriched in proteins and miRNAs associated with anti-oxidative and anti-inflammatory responses.Conclusion: Our protocol allows large-scale production of EVs that are therapeutically effective in the pre-clinical model of PD.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have emerged as promising and safe therapeutic agents, however, donor heterogeneities, limited replicative life span and changes in the cellular phenotype throughout in vitro cultivation remain major hurdles for scalable EV production. For these reasons, this study aims to investigate the use of hTERT immortalized ('telomerized') MSCs as a potential source for efficient, standardized, reliable MSC-EVs production by comparing parental primary to their telomerized MSC counterparts. We observed that hTERT expression does not affect cell morphology or cellular doubling time, while ensuring unlimited, stable in vitro propagation. In addition, telomerized WJ-MSCs maintained the canonical expression profile of surface markers and the tri-lineage differentiation potential of their primary counterparts. In terms of EV characteristics, the immortalization by hTERT expression did not affect size, number, cargo composition or biological activity regarding anti-inflammatory, anti-fibrotic and wound healing properties in vitro. In summary, the use of hTERT to immortalize MSCs leads to the creation of cell lines that continuously produce MSC-EVs without altering any key functionalities of the cells or resulting EVs. This suggests that telomerization of human cells from single donors is a promising strategy for generating cell factories that can produce EVs in standardized conditions at scale and with standardization.

The coordinated activity of macrophages is essential for bone repair, with pro-inflammatory M1 macrophages driving early responses and anti-inflammatory M2 macrophages supporting later tissue remodeling. While both phenotypes are required, prolonged persistence of either subtype can impair healing, underscoring the correct transition between the two states. Macrophage polarization is closely linked to cellular metabolism, and human macrophages display distinct metabolic profiles. Macrophage-derived extracellular vesicles (EVs) carry bioactive cargo and reflect parental polarization, influencing recipient cell function. This raises critical questions about how metabolic regulation influences human macrophage function, their EVs and their effect on angiogenesis and osteogenesis. This study investigates EVs derived from polarized primary human macrophages and from macrophages exposed to DASA-58, a small molecule which activates the metabolic enzyme pyruvate kinase M2 (PKM2). Alterations in macrophage metabolism modify the molecular cargo of their EVs, including microRNAs (miRNAs), to modulate regenerative activity. These findings demonstrate that human macrophage-derived EVs exert metabolically dependent effects on angiogenesis and osteogenesis, and that metabolic modulation enables the generation of EVs with hybrid pro-regenerative properties intermediate between M1 and M2. This establishes metabolic reprogramming within human macrophages using small molecules as a strategy to engineer novel phenotypes and EVs for bone repair.

High-grade gliomas (HGGs) are the most aggressive adult brain tumors, with a dismal median survival of approximately 15 months, highlighting the need for novel therapeutic strategies. In a prior immunotherapy trial using dendritic cells against glioblastoma, miR-216b emerged as a potential predictive biomarker. Thus, we hypothesize that miR-216b impacts glioma aggressiveness and thereby therapeutic success. Here, we demonstrate that miR-216b is significantly downregulated in the majority of Isocitrate dehydrogenase 1/2 (IDH) wild-type HGG tissue samples (n = 42) and cell models (n = 18). Functional assays revealed that miR-216b overexpression impairs glioma cell proliferation, migration, and stemness characteristics. Transcriptomic and target prediction analyses identified CDK4, a key cell cycle regulator, as a direct target of miR-216b, confirmed via luciferase reporter assays. Correspondingly, upregulating miR-216b (mimic) via transfection decreased CDK4 mRNA and protein levels accompanied by a p21-dependent increase of cells in G0/G1 phase. In addition, miR-216b expression correlated with increased sensitivity to the CDK4/6 inhibitor Abemaciclib. Notably, miR-216b levels were significantly higher in less aggressive IDH-mutant gliomas (n = 21), linking its downregulation to malignancy grade. Collectively, our findings discovered miR-216b as a tumor suppressor in HGGs, modulating CDK4 expression and affecting the responsiveness to CDK4/6 inhibitors. The observed results support the potential of miR-216b as both a prognostic and predictive indicator in HGGs.

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.

Background Pathological conditions often arise from dysregulation of complex gene networks. MicroRNAs (miRNAs) are central modulators of these networks, with upregulation of disease-promoting miRNAs suppressing beneficial pathways, and downregulation of protective/therapeutic miRNAs normally restraining pathological programs. Because individual miRNAs coordinately regulate multiple genes, they represent powerful therapeutic targets. We hypothesized that pathology-associated gene expression imbalances could be corrected by placing downregulated protective/therapeutic miRNAs under the control of promoters driving overexpression of disease-promoting miRNAs, thereby simultaneously disabling pathogenic programs and inducing therapeutic ones. We termed this concept castling, after the chess move. Methods Candidate miRNA pairs for castling were identified as inversely regulated in CAR T cells at early and late stages of the chronic antigen stimulation eventually leading to their dysfunction. Proof-of-concept experiments with castling of selected miRNA clusters (knock in of miR-17~92 into a miR15/16 locus) was then implemented in both primary T cells and in CAR T cells using a newly developed genome-editing procedure TRIPLE (Targeted Replacement Induced by Persistent Locus Editing) that enhances homology-directed repair via sequential cleavage. This was followed by differential expression of miRNA and mRNAs resulting from castling compared to similarly treated non-castled corresponding control cells. In addition, castled CAR T cells were evaluated functionally in the chronic antigen stimulation assay. Results In primary T-cells, swapped expression patterns of the castled miR17~92 and miR15-16 clusters elicited expected bidirectional changes of expression of their predicted target mRNA subsets. In CAR T cells under chronic antigen stimulation, castling of these miRNA clusters delayed dysfunction, enhanced cytokine production, and reshaped transcriptional programs consistent with restored T cell fitness. This was accompanied by up- and downregulation of multiple supporting genes. Conclusions Castling, implemented via TRIPLE or any other suitable gene editing technology, offers a broadly applicable strategy to reprogram disease-driven gene regulatory networks by converting pathological regulatory loops into self-correcting circuits. As a conceptual therapeutic approach, castling may be broadly applicable for improvement of multiple types of cell therapies in a variety of indications as well as for manipulation of not only miRNA but also of protein coding mRNAs. ### Competing Interest Statement H.K., S.A., S.A.N., E.S., D.Z. are employed at Lepton Pharmaceuticals. M.H., M.P. and A.D. are employed at TAmiRNA GmbH. E.F. and C.M. are advisors to Lepton Pharmaceuticals. T.C. is an advisor to AaviGen, AstraZeneca, Cimeio Therapeutics, Excision BioTherapeutics, GenCC, and Novo Nordisk. All other authors declare no conflicts of interest. Lepton Pharmaceuticals German Federal Ministry of Research, Technology and Space (BMFTR) CRACK IT Challenge

A 65-year-old woman was admitted to the emergency department following out-of-hospital cardiac arrest (OHCA) of unknown origin and successful resuscitation (CPR) on-scene. However, shortly after admission, the patient developed pericardial tamponade (PT) in the trauma bay despite an initially unremarkable echocardiography. Emergency pericardiocentesis was required to relieve the tamponade but led to hemorrhagic shock. Subsequent imaging and intraoperative findings revealed injury to the left circumflex artery (CX), likely caused by a left atrial appendage occluder (LAAO) that had penetrated the CX during resuscitation efforts. The tamponade was promptly identified through repeat extended focused assessment with sonography for trauma (eFAST) imaging following hemodynamic deterioration, and the underlying cause was quickly confirmed using standardized postresuscitation imaging protocols. The frequency of this complication remains unclear and warrants further investigation, particularly as the use of LAAOs continues to increase. These devices, while beneficial, may pose a risk for life-threatening, resuscitation-related injuries such as PT. This case report highlights the complexity of managing critically ill nontraumatic OHCA patients, who are often elderly, have multiple comorbidities, and may be on anticoagulation therapy-all factors that can complicate acute care. It also underscores the challenges of post-CPR management, as chest compressions themselves can cause significant trauma, even after brief durations of CPR. Common resuscitation-related injuries must therefore be anticipated and promptly addressed. Prehospital eFAST plays a critical role in the initial management of critically ill patients but should never delay transport. Repeating eFAST imaging after hospital admission is essential. In cases of hemodynamic deterioration, the ABCDE (airway, breathing, circulation, disability, and exposure) approach including eFAST sonography should be revisited, and differential diagnoses that were previously excluded must be reconsidered. To ensure high-quality care in these complex scenarios, the search for the underlying cause of OHCA should include standardized postresuscitation imaging as an integral part of trauma bay management.

Long-bone fractures occasionally develop excessive callus formation in the presence of traumatic brain injury, a clinically relevant but poorly understood injury response. Although this phenomenon is known for decades, the causal factors are still underexplored, and no systemic biomarkers are currently available to predict the exuberant bone-mass-formation at an early timepoint after injury. In this study, we used small-RNA-seq, bioinformatic analyses, and in vitro assays to identify a set of micro-RNAs in sera of hypertrophic callus patients that could serve as potential biomarkers. The identified miRNAs are highly expressed in the human brain, particularly in the pituitary gland, and have been shown to regulate mRNA targets implicated in osteogenic processes.

Introduction Bone healing is a well-orchestrated process involving various bone cells and signaling pathways, where disruptions can result in delayed or incomplete healing. MicroRNAs (miRNAs) are small non-coding RNAs capable of influencing various cellular processes, including bone remodeling. Due to their biological relevance and stable presence in biofluids, miRNAs may serve as candidates for diagnosis and prognosis of delayed bone healing. The aim of the study was to investigate changes in miRNAs circulating in the blood during the healing of rat calvaria defects as biomarkers of successful bone regeneration.Methods Standardized calvaria defects were created in 36 Wistar rats with a trephine drill and treated with collagen hydroxyapatite (CHA) scaffolds. The treatment groups included CHA scaffolds only, CHA scaffolds containing a plasmid coding for bone morphogenetic protein 2 (BMP2) and miR-590-5p, CHA scaffolds containing mesenchymal stromal cell-derived extracellular vesicles, and empty defects as a control group. After 1, 4 and 8 weeks of healing, the animals were evaluated by microcomputed tomography (microCT), as well as subjected to histological analyses. Blood was sampled from the tail vein prior to surgeries and after 1, 4, and 8 weeks of healing. miRNAs circulating in the plasma were determined using next-generation sequencing.Results Variability of bone regeneration within the four groups was unexpectedly high and did not result in significant differences between the groups, as indicated by the microCT and histological analyses of the newly formed bone tissue. However, irrespective of the treatment group and regenerative activity, we identified miRNAs with distinct expression patterns of up- and downregulation at different time points. Furthermore, rats with high and low regenerative activity were characterized by distinct circulating miRNA profiles. miR-133-3p was identified as the top upregulated miRNA and miR-375-3p was identified as the top downregulated miRNA in animals exhibiting strong regeneration over all time points evaluated.Conclusion Our study indicates that regardless of the treatment group, success or lack of bone regeneration is associated with a distinct expression pattern of circulating microRNAs. Further research is needed to determine whether their levels in the blood can be used as predictive factors of successful bone regeneration.

Posthepatectomy liver failure (PHLF) continues to be the most significant factor-determining outcome after hepatic resection, accounting for nearly half of postoperative mortality. In this study, we evaluated whether a newly developed commercially available test measuring circulating microRNAs (miRs) could predict PHLF and compared it with other established liver function tests. A total of 329 patients undergoing liver resection were included and postoperative outcome was assessed. Our previously described P-score, calculated on the basis of three circulating microRNAs (miR-122-5p, miR-192-5p, miR-151a-5p) using the hepatomiR® CE-IVD test, was evaluated and compared with other predictors of PHLF, namely indocyanine green (ICG)-clearance as well as the combined aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and albumin–bilirubin grade (ALBI) score. Compared with both other liver function tests, P-scores were superior in predicting PHLF and PHLF grades B and C (PHLF B + C) (PHLF B + C: hepatomiR® AUC = 0.835, APRI + ALBI AUC = 0.807; retention rate at 15 min (R15) AUC = 0.690; plasma disappearance rate (PDR) AUC = 0.691). We also documented a superior positive (77

BACKGROUND:Sepsis is a life-threatening response to an infection, often complicated by sepsis-associated acute kidney injury (SA-AKI). Early recognition of SA-AKI is critical but challenged by the limited sensitivity of existing diagnostic markers. MicroRNAs (miRNAs), which regulate key SA-AKI pathways, have shown diagnostic promise, yet their clinical utility in early SA-AKI recognition remains unexplored. Moreover, validation in relevant clinical settings and populations remains limited. Therefore, this study aims to explore the potential of miRNAs for early recognition of SA-AKI at emergency department (ED) presentation, and explore the generalizability of findings by including a cohort of intensive care urine (ICU) patients with more advanced disease. METHODS:We conducted a post-hoc analysis of prospectively collected data from patients admitted to the ED and ICU. We performed a thorough literature review to select twelve miRNAs, previously implicated in kidney injury and sepsis or SA-AKI. MiRNAs were extracted from plasma and quantified using qPCR with and normalization to the global mean. We measured plasma levels of selected miRNAs upon ED arrival in 193 acutely ill patients (no infection, n = 65; sepsis, n = 67; SA-AKI, n = 61), and 47 critically ill patients (sepsis, n = 18; SA-AKI, n = 29). Statistical analyses included logistic and Cox regression adjusted for clinical variables, with correlations assessed between miRNA levels and disease severity markers. Diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis. RESULTS:MiR-21-5p (OR 2.28, 95% CI [1.40-3.73]; p < 0.01) and miR-16-5p (OR 0.74, 95% CI [0.59-0.93]; p = 0.01) levels were associated with SA-AKI at ED presentation. Furthermore, miR-21-5p was independently associated with 30-day mortality after adjusting for age, illness severity, and comorbidities (adjusted OR 2.30, 95% CI [1.38-3.86]; p < 0.01). Similarly, in the ICU cohort with more advanced sepsis, miR-21-5p was associated with SA-AKI (OR 3.48, 95% CI [1.27-9.53]; P = 0.02), achieving an AUC of 0.74 (95% CI [0.58-0.89]), although it was not associated with 30-day mortality in this cohort. CONCLUSION:We selected twelve miRNAs through literature review associated with kidney injury, sepsis or SA-AKI. Of these, only miR-21-5p was associated with SA-AKI and predicted 30-day mortality upon ED admission. This analysis effectively serves as a negative validation for most literature-derived miRNAs, challenging their clinical applicability identification of SA-AKI both at early presentation and in more advanced stages. TRIAL REGISTRATION:This study is embedded in the Acutelines data-biobank (www.acutelines.nl), registered in Clinicaltrials.gov (NCT04615065, November 3rd 2020) and the The Biobank Intensive Care Groningen registered in Clinicaltrials.gov (NCT04502511, August 6th 2020).

Circulating cell-free microRNAs (miRNAs) are emerging as promising biomarkers with broad potential for clinical applications. However, pre-analytical variability significantly affects miRNA quantification and hampers reproducibility across studies. This work aims to define the Minimum Preanalytical Information required for the publication of studies on circulating cell-free miRNA-based biomarkers. We review key pre-analytical factors that influence circulating miRNA levels quantified using RT-qPCR. Critical variables include blood collection timing, sample type, centrifugation protocols, transport and storage conditions, hemolysis, lipemia, medication, physical activity and pathogen inactivation methods. We introduce a standardized checklist to promote methodological transparency and inter-study comparability. The final aim is to enhance the reliability of miRNA-based biomarker research and support its successful translation into clinical practice.

Lupus nephritis (LN) is characterized by a variable pattern on kidney biopsy. Histopathological assessment of LN yields six classes. Although it is generally assumed that the histological patterns in the tissue denote different molecular pathways, little is known about the molecular identity of these pathways. We aim to identify the molecular signatures of affected glomeruli in LN histological classes, with the overarching goal of defining molecular markers for diagnostic purposes and molecular targets for therapy. To explore this, we have studied two distinct patterns, namely the membranoproliferative glomerulonephritis (MPGN) pattern, found in class IV, and the membranous pattern, which identifies class V of LN. These patterns are clearly distinguishable and have a homogeneous distribution of lesions in glomeruli. We furthermore included control samples, i.e. biopsies from donor kidneys prior to transplantation. Cryosections of selected biopsies were used for Laser Microdissection (LMD) to capture glomeruli. Whole kidney and glomerular RNA were isolated and used for bulk RNA sequencing. Differential gene expression (DGE) analysis to compare MPGN or class V versus healthy control, and MPGN versus class V, was determined using the bioinformatic R-package EdgeR. Gene ontology term (GO-term) analysis was applied to identify the biological pathways involved. Marker genes of interest were compared to findings in the literature. Relevant marker genes were selected for validation of DGE by RT-qPCR. Normalized gene counts measured in glomerular samples of MPGN versus class V revealed class-specific molecular signatures that were masked when analyzing whole kidney samples. DGE analysis identified several inflammation-driven biological pathways being involved in the MPGN pattern of LN. In contrast, the membranous pattern of LN was characterized by biological pathways involved in nephron rearrangement and proliferation. Profound marker genes for the MPGN pattern with potential for diagnostic purposes were related to the interferon stimulating gene family, such as IFIT1 and MX2. RT-qPCR validation of DGE revealed a significant 6.5 fold higher expression of MX2 in MPGN compared to class V. Future immunohistochemical localization of marker genes will be pursued to identify if, and which glomerular cells produce these proteins. Laser microdissection, combined with RNA sequencing, is a powerful tool to explore glomerulus-specific molecular profiles that discriminate between MPGN, and membranous patterns of LN. Identification of activated biological pathways provides insight into disease status and delivers potential gene targets as indicators for disease. The results from our study could be a first step towards the usability of marker genes for diagnostics purposes in LN class characterization, and ultimately, towards identification of biological targets for therapy.