Turbinal bones are still one of the last frontiers of the tetrapod skull: hypotheses, challenges and perspectives.

Turbinals are bony or cartilaginous structures that are present in the nasal cavity of most tetrapods. They are involved in key functions such as olfaction, heat, and moisture conservation, as well as protection of the respiratory tract. Despite recent studies that challenged long-standing hypotheses about their physiological and genomic correlation, turbinals remain largely unexplored, particularly for non-mammalian species. Herein, we review and synthesise the current knowledge of turbinals using an integrative approach that includes comparative anatomy, physiology, histology and genomics. In addition, we provide synonyms and correspondences of tetrapod turbinals from about 80 publications. This work represents a first step towards drawing hypotheses of homology for the whole clade, and provides a strong basis to develop new research avenues.

Tumor-secreted extracellular vesicles counteract therapy response by triggering inflammatory mesenchymal stem cell development.

PURPOSE: Therapy resistance is a major clinical hurdle in bone cancer treatment and seems to be largely driven by poorly understood microenvironmental factors. Recent evidence suggests a critical role for a unique subpopulation of mesenchymal stem cells with inflammatory features (iMSCs), though their origin and function remained unexplored. We demonstrate that cancer-secreted extracellular vesicles (EVs) trigger the development of iMSCs, which hinder therapy response in vivo, and set out to identify strategies to counteract their function. EXPERIMENTAL DESIGN: The role of iMSCs in therapy resistance was evaluated in an orthotopic xenograft mouse model of osteosarcoma. EV-induced alterations of the MSC transcriptome were analyzed and compared with scRNA-seq data of osteosarcoma and multiple myeloma patient biopsies. Functional assays identified EV components driving iMSC development. We assessed the efficacy of clinical drugs in blocking iMSC-induced resistance in vivo. RESULTS: We found that iMSCs are induced by interaction with cancer EVs and completely abrogate the antimetastatic effect of TGFb signaling inhibition. Importantly, EV-induced iMSCs faithfully recapitulate the inflammatory single-cell RNA signature of stromal cells enriched in multiple myeloma and osteosarcoma patient biopsies. Mechanistically, cancer EVs act through two distinct mechanisms. EV-associated TGFb induces IL6 production, while the EV-RNA cargo enhances TLR3-mediated chemokine production. We reveal that simultaneous blockade of downstream EV-activated pathways with ladarixin and tocilizumab disrupts metastasis formation and overcomes iMSC-induced resistance. CONCLUSIONS: Our observations establish iMSCs as major contributors to drug resistance, reveal EVs as physiological triggers of iMSC development and highlight a promising combination strategy to improve therapy response in bone cancer patients.

Elective full pulpotomy in mature permanent teeth diagnosed with symptomatic irreversible pulpitis: a two years retrospective study.

AIM: To investigate the outcome of elective full pulpotomy, using calcium silicate-based cements (CSBC), after 2 years, in symptomatic mature permanent teeth with carious lesions, diagnosed as irreversible pulpitis, and analyse the capacity of Wolters et al. (2017) classification to predict the likelihood of treatment failure. METHODS: The treatment records of 56 patients with symptomatic mature teeth with carious lesions, diagnosed as irreversible pulpitis and treated by elective full pulpotomy, using CSBCs as pulp capping materials, were reviewed. Thirteen teeth were excluded. The remaining 43 teeth were evaluated retrospectively at 24 months. Fisher`s exact test with the Lancaster's mid-P adjustment was used to assess different outcomes amongst the diagnostic categories. RESULTS: Four of the cases failed before 24 months and required root canal treatment (RCT). Overall success rate at 2 years was 90.7% (39 of 43). An inverse, but non-significant, correlation was observed between the severity of pulpitis according to the Wolters classification and the treatment success rate (p > 0.05). The type of CSBC used was associated to the success rate (OR = 10.5; 95% C.I. = 0.5 - 207.4; p = 0.027), being 82% with Endosequence and 100% with Biodentine. Postoperative pain associated significantly to lower success rate (66.7%) (Odds ratio = 8.0; 95% C.I. = 0.7 - 95.9; p = 0.047). CONCLUSIONS: Elective full pulpotomy using a CSBC was a successful choice for the treatment of mature permanent teeth with symptoms indicative of irreversible pulpitis. There were no significant differences between the success rate of mild, moderate and severe pulpitis. Postoperative pain could be considered a risk marker for failure of full pulpotomy. The term "irreversible pulpitis" should be re-signified to indicate the need for access to the pulp chamber, rather than an indication for extraction or RCT.

Effects of Diathermy on Pain in Women with Fibromyalgia: A Randomized Controlled Trial.

(1) Background: The main characteristic of fibromyalgia (FM) is generalized musculoskeletal pain. This may be accompanied by muscle and joint stiffness, sleep and mood disorders, anxiety and depression, cognitive dysfunction, and chronic fatigue. It is endemic in developed countries, with a higher prevalence among women than men, and its etiology is still unknown. Diagnosis is made based on chronic generalized pain and through the presence of tender points. The objective of this study was to analyze the efficacy of diathermy on pain in patients with fibromyalgia. (2) Methods: A single, blind, randomized experimental study was developed with a sample of 31 participants. Measurements were taken and recorded at three different intervals using the following measurement tools: the pressure pain threshold (PPT) at the tender points (TP) of the right and left trochanteric prominence with an algometer, the pain measurement scale, the Fibromyalgia Impact Questionnaire, the sleep quality index (PSQI, Pittsburgh), the Multidimensional Fatigue Inventory (MFI-S), and the scale for anxiety and depression (Hospital Anxiety and Depression Scale). Sociodemographic data were collected through Google Forms (age, height, weight, Body Mass Index). The intervention took place twice weekly across four weeks of sessions. (3) Results: Statistically significant results were obtained in the right and left trochanter PPT, as well as for anxiety and fatigue in the experimental group. The results obtained show that this treatment has managed to improve the quality of sleep, the impact of disease, chronic fatigue, and anxiety in patients with FM. (4) Conclusions: Diathermy is a tool that can help reduce pain. It can also improve the baseline levels of chronic fatigue, anxiety, the impact of the disease, and sleep quality in patients with fibromyalgia.

Regression Models for In Vivo Discrimination of the Iberian Pig Feeding Regime after Near Infrared Spectroscopy Analysis of Faeces.

The Iberian pig is a native breed of the Iberian Peninsula, which holds an international reputation due to the superior quality and the added value of its products. Different rearing practices and feeding regimes are regulated, resulting in different labelling schemes. However, there is no official analytical methodology that is standardised for certification purposes in the sector. Near Infrared Spectroscopy (NIRS) is a technology that provides information about the physicochemical composition of a sample, with several advantages that have enabled its implementation in different fields. Although it has already been successfully used for the analysis of Iberian pig's final products, samples evaluated with NIRS technology are characterised by a postmortem collection. The goal of this study was to evaluate the potential of NIRS analysis of faeces for in vivo discrimination of the Iberian pig feeding regime, using the spectral information per se for the development of modified partial least squares regressions. Faecal samples were used due to their easy collection, especially in extensive systems where pig handling is difficult. A total of 166 individual samples were collected from 12 farms, where the three different feeding regimes available in the sector were ensured. Although slight differences were detected depending on the chemometric approach, the best models obtained a classification success and a prediction accuracy of over 94% for feeding regime discrimination. The results are considered very satisfactory and suggest NIRS analysis of faeces as a promising approach for the in vivo discrimination of the Iberian pigs' diet, and its implementation during field inspections, a significative achievement for the sector.

Assessing Predictive Value of SARS-CoV-2 Epitope-Specific CD8+ T-Cell Response in Patients with Severe Symptoms.

Specific T cell responses against SARS-CoV-2 provided an overview of acquired immunity during the pandemic. Anti-SARS-CoV-2 immunity determines the severity of acute illness, but also might be related to the possible persistence of symptoms (long COVID). We retrospectively analyzed ex vivo longitudinal CD8+ T cell responses in 26 COVID-19 patients diagnosed with severe disease, initially (1 month) and long-term (10 months), and in a cohort of 32 vaccinated healthcare workers without previous SARS-CoV-2 infection. We used peptide-human leukocyte antigen (pHLA) dextramers recognizing 26 SARS-CoV-2-derived epitopes of viral and other non-structural proteins. Most patients responded to at least one of the peptides studied, mainly derived from non-structural ORF1ab proteins. After 10 months follow-up, CD8+ T cell responses were maintained at long term and reaction against certain epitopes (A*01:01-ORF1ab1637) was still detected and functional, showing a memory-like phenotype (CD127+ PD-1+). The total number of SARS-CoV-2-specific CD8+ T cells was significantly associated with protection against long COVID in these patients. Compared with vaccination, infected patients showed a less effective immune response to spike protein-derived peptides restricted by HLA. So, the A*01:01-S865 and A*24:02-S1208 dextramers were only recognized in vaccinated individuals. We conclude that initial SARS-CoV-2-specific CD8+ T cell response could be used as a marker to understand the evolution of severe disease and post-acute sequelae after SARS-CoV-2 infection.

Association of MC4R rs17782313 Genotype With Energy Intake and Appetite: A Systematic Review and Meta-analysis.

CONTEXT: The melanocortin-4 receptor gene (MC4R) is associated with a higher risk of obesity by the presence of the C allele in rs17782313, but the mechanisms are not clear. OBJECTIVE: The present systematic review and meta-analysis aimed to explore the association between the different genotypes of MC4R rs17782313 and energy intake and appetite. DATA SOURCES: A literature search was conducted up to June 2023 in PubMed, Scopus, Web of Science, and Cochrane Collaboration databases, following PRISMA guidelines. DATA EXTRACTION: Inclusion criteria were studies in humans measuring energy intake, appetite, or satiety in all ages and physiological conditions. Studies dealing solely with body mass index were excluded. Twenty-one articles representing 48 560 participants were included in the meta-analysis. DATA ANALYSIS: According to the NHLBI (National Heart, Lung, and Blood Institute) quality-assessment criteria, all case-control studies and 6 out of 17 cohort and cross-sectional studies were classified as "good," while the rest scored as "fair." Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in a (CT+CC) vs TT dominant model, and both random-effects and fixed-effects models were used. A statistically significant association between the presence of the C allele and increased appetite was found (OR = 1.25; 95% CI: 1.01-1.49; P = .038) using the fixed-effects model, but the random-effects model proved nonsignificant. However, no association with energy intake was found. None of the variables considered (sample size, year of publication, sex, age group, type of population, origin, and quality) were identified as effect modifiers, and no publication biases were found after subgroup and meta-regression analyses. CONCLUSION: To our knowledge, this is the first systematic review and meta-analysis that has analyzed the association between rs17782313 of MC4R and energy intake and appetite. Identifying people genetically predisposed to increased appetite may be of great interest, not only to prevent obesity in younger populations but also to avoid malnutrition in elderly persons. This paper is part of the Nutrition Reviews Special Collection on Precision Nutrition. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration no. CRD42023417916.

Fluorescence lifetime nanothermometer based on the equilibrium formation of anthracene AIE-excimers in living cells.

The effective measurement of temperature in living systems at the nano and microscopic scales continues to be a challenge to this day. Here, we study the use of 2-(anthracen-2-yl)-1,3-diisopropylguanidine, 1, as a nanothermometer based on fluorescence lifetime measurements and its bioimaging applications. In aqueous solution, 1 is shown in aggregated form and the equilibrium between the two main aggregate types (T-shaped and π-π) is highly sensitive to the temperature. The heating of the medium shifts the equilibrium toward the formation of highly emissive T-shaped aggregates. This species shows a high fluorescence emission and a long lifetime in comparison with the π-π aggregates and the freé monomer. A linear relationship between the fluorescence lifetime and the temperature both in aqueous solution and in a synthetic intracellular buffer was found. Fluorescence lifetime imaging microscopy (FLIM) also showed a linear relationship between lifetime and temperature with an excellent sensitivity in MCF7 breast cancer cells, which opens the door for its potential use as FLIM nanothermometer in the biomedical field.

Exploring kidney allograft rejection: A proof-of-concept study using spatial transcriptomics.

In this proof-of-concept study, spatial transcriptomics combined with public single-cell ribonucleic acid-sequencing data were used to explore the potential of this technology to study kidney allograft rejection. We aimed to map gene expression patterns within diverse pathologic states by examining biopsies classified across nonrejection, T cell-mediated acute rejection, interstitial fibrosis, and tubular atrophy. Our results revealed distinct immune cell signatures, including those of T and B lymphocytes, monocytes, mast cells, and plasma cells, and their spatial organization within the renal interstitium. We also mapped chemokine receptors and ligands to study immune cell migration and recruitment. Finally, our analysis demonstrated differential spatial enrichment of transcription signatures associated with kidney allograft rejection across various biopsy regions. Interstitium regions displayed higher enrichment scores for rejection-associated gene expression patterns than tubular areas, which had negative scores. This implies that these signatures are primarily driven by processes unfolding in the renal interstitium. Overall, this study highlights the value of spatial transcriptomics for revealing cellular heterogeneity and immune signatures in renal transplant biopsies and demonstrates its potential for studying the molecular and cellular mechanisms associated with rejection. However, certain limitations must be borne in mind regarding the development and future applications of this technology.

Challenges of diagnosing homologous recombination deficiencies in metastatic prostate cancer: a six-year experience from a single institution.

PURPOSE: We evaluated the prevalence of homologous recombination deficiencies (HRD) to determine the efficacy of different techniques and clinical characteristics of patients. METHODS: This retrospective study included patients with metastatic prostate cancer who underwent molecular testing at our hospital between 2016 and 2022. We used tumor tissue, ctDNA, and lymphocytes for somatic or germline testing. We analyzed the clinical characteristics and survival outcomes. RESULTS: 144 patients were tested (113 somatic, 21 germline, and 10 both). Technical issues prevented the analysis of 23 prostatic samples (18.7%). 12 (8.3%) patients had HRD. BRCA2 was the most frequent mutation (66.7%). Patients with HRD were younger (57.5 years). Patients with BRCA mutations had poorer survival (31.9 vs 56.3 months, p = 0.048). CONCLUSION: In our institution, 8.3% of the patients had HRD. Tumor tissue analysis failed in 18.7% of tests. ctDNA analysis is an alternative detection method. BRCA mutations are correlated with poor prognosis.

Propionate and butyrate counteract renal damage and progression to chronic kidney disease.

BACKGROUND: Short-chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, are produced by gut microbiota through fermentation of complex carbohydrates that cannot be digested by the human host. They affect gut health and can contribute at the distal level to the pathophysiology of several diseases, including renal pathologies. METHODS: SCFA levels were measured in chronic kidney disease (CKD) patients (n = 54) at different stages of the disease and associations with renal function and inflammation parameters were examined. The impact of propionate and butyrate in pathways triggered in tubular cells under inflammatory conditions was analysed using genome-wide expression assays. Finally, a pre-clinical mouse model of folic acid-induced transition from acute kidney injury to CKD was used to analyse the preventive and therapeutic potential of these microbial metabolites in the development of CKD. RESULTS: Faecal levels of propionate and butyrate in CKD patients gradually reduce as the disease progresses, and do so in close association with established clinical parameters for serum creatinine, blood urea nitrogen and the estimated glomerular filtration rate. Propionate and butyrate jointly downregulated the expression of 103 genes related to inflammatory processes and immune system activation triggered by TNF-α in tubular cells. In vivo, the administration of propionate and butyrate, either before or soon after injury, respectively prevented and slowed the progression of damage. This was indicated by a decrease in renal injury markers, the expression of pro-inflammatory and pro-fibrotic markers, and recovery of renal function over the long term. CONCLUSIONS: Propionate and butyrate levels are associated with a progressive loss of renal function in CKD patients. Early administration of these SCFAs prevents disease advancement in a pre-clinical model of acute renal damage, demonstrating their therapeutic potential independently of the gut microbiota.

Full-length tRNAs lacking a functional CCA tail are selectively sorted into the lumen of extracellular vesicles.

Small extracellular vesicles (sEVs) are heterogenous lipid membrane particles typically less than 200 nm in size and secreted by most cell types either constitutively or upon activation signals. sEVs isolated from biofluids contain RNAs, including small non-coding RNAs (ncRNAs), that can be either encapsulated within the EV lumen or bound to the EV surface. EV-associated microRNAs (miRNAs) are, despite a relatively low abundance, extensively investigated for their selective incorporation and their role in cell-cell communication. In contrast, the sorting of highly-structured ncRNA species is understudied, mainly due to technical limitations of traditional small RNA sequencing protocols. Here, we adapted ALL-tRNAseq to profile the relative abundance of highly structured and potentially methylated small ncRNA species, including transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and Y RNAs in bulk EV preparations. We determined that full-length tRNAs, typically 75 to 90 nucleotides in length, were the dominant small ncRNA species (>60% of all reads in the 18-120 nucleotides size-range) in all cell culture-derived EVs, as well as in human plasma-derived EV samples, vastly outnumbering 21 nucleotides-long miRNAs. Nearly all EV-associated tRNAs were protected from external RNAse treatment, indicating a location within the EV lumen. Strikingly, the vast majority of luminal-sorted, full-length, nucleobase modification-containing EV-tRNA sequences, harbored a dysfunctional 3' CCA tail, 1 to 3 nucleotides truncated, rendering them incompetent for amino acid loading. In contrast, in non-EV associated extracellular particle fractions (NVEPs), tRNAs appeared almost exclusively fragmented or 'nicked' into tRNA-derived small RNAs (tsRNAs) with lengths between 18 to 35 nucleotides. We propose that in mammalian cells, tRNAs that lack a functional 3' CCA tail are selectively sorted into EVs and shuttled out of the producing cell, offering a new perspective into the physiological role of secreted EVs and luminal cargo-selection.

Unlocking the potential of Opuntia species mucilage in chemistry.

Herein, we describe a detailed protocol to extract the mucilage from different species of the genus Opuntia spp. (i.e., Opuntia Ficus (OFi), Opuntia Dillenii (ODi) and Opuntia Robusta (ORo)). The extracted mucilage was characterized by NMR, FTIR-ATR, HPLC, and TGA. OFi was found to have the highest phenolic content, 7.84 ± 1.93 mg catechol/g mucilage. The mucilage from the three species were characterized by having a high content of monosaccharides, being mannose and glucose the most abundant components (ca. 48-73 % and 23-35 %, respectively). In the context of biomass revalorization, the mucilage was proven to serve as a reducing and stabilizing agent in the synthesis of gold nanoparticles (AuNP/mucilage). The synthesis was optimized with a mucilage concentration of 2 mg/mL using 12.5 µL of KAuCl4 and was carried out at 80 °C for 90 min. This protocol afforded spherical nanoparticles with an average size of 9.7 ± 4.0 nm that were stable for at least 14 days, as demonstrated by TEM. Synthesized AuNP/mucilage was evaluated as a plasmonic catalyst for the reduction of 4-nitrophenol as model reaction, showing a considerable enhancement in its kapp of 97 % under white light and a decrease of 24.8 % in its activation energy.

Defining predictors of responsiveness to advanced therapies in Crohn's disease and ulcerative colitis: protocol for the IBD-RESPONSE and nested CD-metaRESPONSE prospective, multicentre, observational cohort study in precision medicine.

INTRODUCTION: Characterised by chronic inflammation of the gastrointestinal tract, inflammatory bowel disease (IBD) symptoms including diarrhoea, abdominal pain and fatigue can significantly impact patient's quality of life. Therapeutic developments in the last 20 years have revolutionised treatment. However, clinical trials and real-world data show primary non-response rates up to 40%. A significant challenge is an inability to predict which treatment will benefit individual patients.Current understanding of IBD pathogenesis implicates complex interactions between host genetics and the gut microbiome. Most cohorts studying the gut microbiota to date have been underpowered, examined single treatments and produced heterogeneous results. Lack of cross-treatment comparisons and well-powered independent replication cohorts hampers the ability to infer real-world utility of predictive signatures.IBD-RESPONSE will use multi-omic data to create a predictive tool for treatment response. Future patient benefit may include development of biomarker-based treatment stratification or manipulation of intestinal microbial targets. IBD-RESPONSE and downstream studies have the potential to improve quality of life, reduce patient risk and reduce expenditure on ineffective treatments. METHODS AND ANALYSIS: This prospective, multicentre, observational study will identify and validate a predictive model for response to advanced IBD therapies, incorporating gut microbiome, metabolome, single-cell transcriptome, human genome, dietary and clinical data. 1325 participants commencing advanced therapies will be recruited from ~40 UK sites. Data will be collected at baseline, week 14 and week 54. The primary outcome is week 14 clinical response. Secondary outcomes include clinical remission, loss of response in week 14 responders, corticosteroid-free response/remission, time to treatment escalation and change in patient-reported outcome measures. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Wales Research Ethics Committee 5 (ref: 21/WA/0228). Recruitment is ongoing. Following study completion, results will be submitted for publication in peer-reviewed journals and presented at scientific meetings. Publications will be summarised at www.ibd-response.co.uk. TRIAL REGISTRATION NUMBER: ISRCTN96296121.

BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

Targeting epigenetic mechanisms has emerged as a potential therapeutic approach for the treatment of kidney diseases. Specifically, inhibiting the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1 has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, its clinical translation faces challenges due to issues with poor pharmacokinetics and side effects. Here, we developed engineered liposomes loaded with JQ1 with the aim of enhancing kidney drug delivery and reducing the required minimum effective dose by leveraging cargo protection. These liposomes efficiently encapsulated JQ1 in both the membrane and core, demonstrating superior therapeutic efficacy compared to freely delivered JQ1 in a mouse model of kidney ischemia-reperfusion injury. JQ1-loaded liposomes (JQ1-NPs) effectively targeted the kidneys and only one administration, one-hour after injury, was enough to decrease the immune cell (neutrophils and monocytes) infiltration to the kidney-an early and pivotal step to prevent damage progression. By inhibiting BRD4, JQ1-NPs suppress the transcription of pro-inflammatory genes, such as cytokines (il-6) and chemokines (ccl2, ccl5). This success not only improved early the kidney function, as evidenced by decreased serum levels of BUN and creatinine in JQ1-NPs-treated mice, along with reduced tissue expression of the damage marker, NGAL, but also halted the production of extracellular matrix proteins (Fsp-1, Fn-1, α-SMA and Col1a1) and the fibrosis development. In summary, this work presents a promising nanotherapeutic strategy for AKI treatment and its progression and provides new insights into renal drug delivery.

Electrospun Polyvinylpyrrolidone-Based Dressings Containing GO/ZnO Nanocomposites: A Novel Frontier in Antibacterial Wound Care.

In recent years, the rapid emergence of antibiotic-resistant bacteria has become a significant concern in the healthcare field, and although bactericidal dressings loaded with various classes of antibiotics have been used in clinics, in addition to other anti-infective strategies, this alarming issue necessitates the development of innovative strategies to combat bacterial infections and promote wound healing. Electrospinning technology has gained significant attention as a versatile method for fabricating advanced wound dressings with enhanced functionalities. This work is based on the generation of polyvinylpyrrolidone (PVP)-based dressings through electrospinning, using a DomoBIO4A bioprinter, and incorporating graphene oxide (GO)/zinc oxide (ZnO) nanocomposites as a potent antibacterial agent. GO and ZnO nanoparticles offer unique properties, including broad-spectrum antibacterial activity for improved wound healing capabilities. The synthesis process was performed in an inexpensive one-pot reaction, and the nanocomposites were thoroughly characterized using XRD, TEM, EDX, SEM, EDS, and TGA. The antibacterial activity of the dispersions was demonstrated against E. coli and B. subtilis, Gram-negative and Gram-positive bacteria, respectively, using the well diffusion method and the spread plate method. Bactericidal mats were synthesized in a rapid and cost-effective manner, and the fiber-based structure of the electrospun dressings was studied by SEM. Evaluations of their antibacterial efficacy against E. coli and B. subtilis were explored by the disk-diffusion method, revealing an outstanding antibacterial capacity, especially against the Gram-positive strain. Overall, the findings of this research contribute to the development of next-generation wound dressings that effectively combat bacterial infections and pave the way for advanced therapeutic interventions in the field of wound care.

Assessing the complementary information from an increased number of biologically relevant features in liquid biopsy-derived RNA-Seq data.

Liquid biopsy-derived RNA sequencing (lbRNA-seq) exhibits significant promise for clinic-oriented cancer diagnostics due to its non-invasiveness and ease of repeatability. Despite substantial advancements, obstacles like technical artefacts and process standardisation impede seamless clinical integration. Alongside addressing technical aspects such as normalising fluctuating low-input material and establishing a standardised clinical workflow, the lack of result validation using independent datasets remains a critical factor contributing to the often low reproducibility of liquid biopsy-detected biomarkers. Considering the outlined drawbacks, our objective was to establish a workflow/methodology characterised by: 1. Harness the rich diversity of biological features accessible through lbRNA-seq data, encompassing a holistic range of molecular and functional attributes. These components are seamlessly integrated via a Machine Learning-based Ensemble Classification framework, enabling a unified and comprehensive analysis of the intricate information encoded within the data. 2. Implementing and rigorously benchmarking intra-sample normalisation methods to heighten their relevance within clinical settings. 3. Thoroughly assessing its efficacy across independent test sets to ascertain its robustness and potential utility. Using ten datasets from several studies comprising three different sources of biological material, we first show that while the best-performing normalisation methods depend strongly on the dataset and coupled Machine Learning method, the rather simple Counts Per Million method is generally very robust, showing comparable performance to cross-sample methods. Subsequently, we demonstrate that the innovative biofeature types introduced in this study, such as the Fraction of Canonical Transcript, harbour complementary information. Consequently, their inclusion consistently enhances prediction power compared to models relying solely on gene expression-based biofeatures. Finally, we demonstrate that the workflow is robust on completely independent datasets, generally from different labs and/or different protocols. Taken together, the workflow presented here outperforms generally employed methods in prediction accuracy and may hold potential for clinical diagnostics application due to its specific design.

Coactivation of the Pelvic Floor and Gluteus Medius Muscles While Walking and Running in Female Runners.

(1) Background: Pelvic-floor-muscle (PFM) activation acts synergistically with multiple muscles while performing functional actions in humans. The purpose of this study was to characterize the activity of the PFMs and gluteus medius (GM) while walking and running in physically active nulliparous females. (2) Methods: The peak and average amplitude of maximal voluntary contractions (MVCs) during 60 s of walking (5 and 7 km/h) and running (9 and 11 km/h) were measured with electromyography of the GM and PFMs in 10 healthy female runners. (3) Results: The activation of both muscles increased (p < 0.001) while walking and running. The MVC of the GM was reached when walking and tripled when running, while the PFMs were activated at half their MVC when running. The global ratio of the GM (75.3%) was predominant over that of the PFMs (24.6%) while static and walking. The ratio reached 9/1 (GM/PFM) while running. (4) Conclusion: The GM and PFMs were active while walking and running. The GM's MVC tripled at high speeds, while the PFMs reached only half of their maximum contraction.