Background: Cerebrospinal fluid (CSF) is a valuable source of biomarkers for neurological diseases, but detection of low-abundance proteins is often masked by highly abundant proteins. Enrichment strategies can enhance proteomic coverage and improve biomarker discovery, yet comparative evaluations of such methods in equine CSF are limited. Objectives: This study compared the ProteoMiner Small-Capacity Enrichment Kit and the PreOmics Enrich-iST Kit for their ability to deplete high-abundance proteins and enhance detection of low-abundance proteins relevant to neuropathology. Methods: Equine CSF samples were processed with either a native in-solution trypsin digestion without further enrichment, ProteoMiner Small-Capacity Enrichment Kit, or PreOmics enrichment. Samples were analysed by label-free liquid chromatography-tandem mass spectrometry. Proteins were identified and quantified using emPAI scores, and gene ontology pathway analyses were performed to evaluate enrichment efficiency and biological relevance. Results: The PreOmics Enrich-iST Kit identified the highest number of proteins overall, including neurobiology-relevant low-abundance proteins not detected by other methods, and achieved superior depletion of high-abundance proteins. Gene ontology pathway analysis revealed broader enrichment of neuropathology-relevant pathways. Conclusions: The PreOmics Enrich-iST Kit outperformed the ProteoMiner Small-Capacity Enrichment Kit and native digestion in equine CSF proteomics, providing greater depletion of high-abundance proteins and enhanced detection of neurobiology-relevant low-abundance proteins. This method offers a robust tool for comprehensive proteomic profiling and may facilitate the discovery of novel biomarkers for equine neurological disorders.

BACKGROUND:Ageing and dental disease in horses lead to structural and functional deterioration of dental tissues, yet their molecular signatures remain poorly characterised. Understanding how these processes alter the protein composition of enamel, dentin, cementum and pulp is essential for improving equine oral health and identifying biomarkers of degeneration. OBJECTIVES:To characterise the proteomic profiles of equine dental tissues and determine age- and disease-associated changes, with the aim of identifying proteins consistently altered across conditions. STUDY DESIGN:Ex vivo proteomic study. METHODS:Fifteen equine cheek teeth were allocated to three groups (n = 5/group): mature adult, old and diseased. Label-free quantitative mass spectrometry was used to profile proteins across enamel, dentin, cementum and pulp. Differential abundance analysis and clustering assessed age- and disease-related effects. A Shiny-based interactive tool was developed for data exploration. RESULTS:A total of 1950 proteins were identified, with 58% unique to dental pulp. Ageing affected 329 proteins across all tissues, while disease impacted 379. Ageing consistently disrupted extracellular matrix (ECM) homeostasis in hard tissues. Cementum demonstrated increased catabolic markers the metalloproteinases -2 and -13. In dentin, the mineralisation protein dentin sialophosphoprotein was significantly reduced with age. Altered proteins in diseased pulp demonstrated predicted inhibition of ECM organisation (z-score = -3.05, p = 0.032) and elastic fibre formation (z-score = -2.82, p = 0.0033), while retinoic acid receptor activation was increased (z-score = 2.11, p = 0.006). Similarity analysis revealed overlapping molecular responses between ageing and disease. MAIN LIMITATIONS:Small sample size and inclusion of only cheek teeth limit generalisability. Proteomic findings require functional validation. CONCLUSIONS:Ageing and disease induce distinct yet overlapping proteomic alterations in equine dental tissues, primarily affecting ECM homeostasis. A subset of consistently altered proteins may serve as targets for regenerative strategies, with implications for improving equine dental health and welfare.

Objective:Temporomandibular joint osteoarthritis (TMJ-OA) is painful and causes masticatory dysfunction, but current treatment is limited to symptom relief due to an incomplete appreciation of aetiology. Herein, we develop morphological and histological methods for quantitative evaluation of TMJ-OA severity and examine whether STR/Ort mice, which are genetically predisposed to spontaneous knee OA, exhibit protection against TMJ-OA upon genetic gain-of-function modification of an aggrecanase-selective mutant of tissue inhibitor of metalloproteinase (TIMP)-3. Design:We established morphological changes in mandibular condylar head adapted from human TMJ-OA criteria, and developed and verified the utility of TMJ-OA histological damage scoring adapted from the OARSI system. Mutant TIMP3 containing an extra alanine at the N-Terminus ([-1A] TIMP-3 was overexpressed in STR/Ort and CBA mice. Morphological changes in mandibular condyle and TMJ cartilage degradation were evaluated and quantified using micro-CT and histology in mice aged 10, 20 and 40 weeks. Results:Whilst no evidence of TMJ-OA was observed in STR/Ort mice aged 10 weeks, bone erosion and osteophyte formation appeared in the mandibular condyle by 20 weeks, with remarkable deformity and bone resorption at 40 weeks in STR/Ort, but not the parental CBA strain. TMJ-OA was less severe in 40 week-old [-1A]TIMP-3 overexpressing STR/Ort and CBA compared to wild-type mice. Conclusions:Using our new mouse TMJ-OA scoring system we have found that OA affects joints other than the knee in the STR/Ort strain. Genetic gain-of-function modification of STR/Ort mice with an aggrecanase-selective mutant of tissue inhibitor of metalloproteinase (TIMP)-3 also affords in vivo chondroprotection against this TMJ-OA.

BackgroundEquine odontoclastic tooth resorption and hypercementosis (EOTRH) is a painful disorder primarily affecting the incisor teeth of horses over 15 years of age. Clinical signs of the disease include prehension problems, halitosis and in severe cases weight loss. The disease predominately affects the reserve crown and presents as a loss of dental tissue and excessive build-up of cementum.ObjectivesTo determine the radiographic scores of horses with EOTRH and age-matched controls and to increase understanding of EOTRH using microCT to compare teeth from horses with EOTRH and age-matched controls.Study designIn vivo and ex vivo studies.MethodsThis study used radiography (in vivo) and microcomputed tomography (microCT) (ex vivo) to help understand and help characterise the imaging changes that occur in EOTRH. For radiography, 87 patients were assessed using a radiographic scoring system for EOTRH. The microCT study was undertaken on 20 extracted cadaver incisor teeth which were scanned and segmented to measure the different dental tissues. These were assessed using a descriptive analysis (surface roughening, tooth resorption, root blunting and pulp cavity).ResultsRadiographic scoring demonstrated that 03s were more severely affected than 01s in EOTRH. Total radiographic score and age had a weak positive correlation. Following microCT, we identified that EOTRH teeth had a lower pulp and enamel volume and therefore significantly higher pulp and enamel ratios relative to the whole tooth volume, compared with control teeth. Cementum and dentine volumes were more variable in EOTRH teeth. Thus, their ratios relative to the whole tooth volume were not different to control teeth.Main limitationsThe number of horses was relatively small.ConclusionsResults suggest differing degrees of tooth resorption and hypercementosis, in different affected teeth potentially indicating multiple phenotypes of the disease. We provide further evidence of the presence of subclinical EOTRH imaging changes in some teeth.

Background Equine dental diseases significantly impact a horse's overall health, performance and quality of life. They can result in secondary infections and digestive disturbances, potentially leading to colic. A recently described disease affecting the incisors of horses is equine odontoclastic tooth resorption and hypercementosis (EOTRH). Understanding EOTRH is crucial for early diagnosis, effective management and prevention of its severe consequences. Objectives To determine proteomic differences in incisor cementum in horses with and without clinical EOTRH. Study design Comparative and observational clinical study. Methods Teeth were extracted (N = 5) and cementum was isolated using a diamond wire. Proteins were extracted using an optimised sequential workflow, and trypsin was digested for mass spectrometry. Protein identification and label-free quantification were undertaken. Results In total 1149 unique proteins were detected in cementum across all samples. We identified four proteins exclusively in EOTRH-affected cementum. EOTRH samples showed a higher heterogeneity than healthy samples. In total, 54 proteins were increased in EOTRH, and 64 proteins were reduced (adjusted p-value <0.05). Inflammatory proteins, such as cathepsin G (p = 0.004), neutrophil elastase (p = 0.003), bactericidal permeability-increasing protein (p = 0.002), azurocidin (p = 0.003) and lactotransferrin (p = 0.002) were all increased in EOTRH. Pathway analysis revealed that antimicrobial peptides (Z score 2.65, p = 1.93E-09) and neutrophil degranulation (Z-score 1.89, p = 1.7E-04) were commonly up-regulated canonical pathways. Main limitations The sample size was limited. Lack of age-matched healthy controls. Conclusion EOTRH leads to biochemical changes within the cementum proteome, which are important in explaining the physiological changes occurring in disease. Differentially abundant proteins may represent promising biomarkers for earlier disease detection and the establishment of a cell-based model could provide further insight into the role these proteins play in hypercementosis and resorption.

Chemotherapy-induced peripheral neuropathy (CIPN) is a concerning late effect of taxane treatment. This study aimed to explore and compare long-term symptoms and consequences of CIPN after docetaxel and paclitaxel treatment. Patients with breast cancer who had followed Danish recommended adjuvant docetaxel or paclitaxel treatment regimens completed an online questionnaire 2-3 years after treatment. The questionnaire comprised the Michigan Neuropathy Screen Instrument, the European Organization for Research and Treatment of Cancer (EORTC) QLQ-CIPN20, EORTC QLQ C30, and CIPN-specific symptoms. Painful CIPN was assessed using the Douleur Neuropathique 4 Questions. Questionnaires from 411 patients (docetaxel: 192, paclitaxel: 219) were analyzed. No significant difference in the prevalence of possible CIPN between the two groups was observed (docetaxel: 48.4 % [93/192] vs. paclitaxel: 45.2 % [99/219]; 95 % CI: 6.4 - 12.9, p = 0.51). However, the EORTC-QLQ-CIPN20 sum score was higher in the docetaxel group (difference: 3.0; 95 % CI: 0.0-6.1, p = 0.05). Among patients with reported CIPN symptoms, significantly more in the docetaxel group reported painful CIPN (docetaxel: 53.8 % [50/93] than in the paclitaxel group: 34.3 % [34/99]; p = 0.01). Quality of life scores from the EORCT-QLQ-C30 questionnaire were significantly lower in those with possible CIPN than in those without and lower in patients with painful possible CIPN than in those with painless CIPN. Docetaxel caused more severe and painful CIPN symptoms than paclitaxel. These findings are highly relevant, as docetaxel remains a crucial component of cancer treatments.

BackgroundTreatment response assessment can be challenging in bone-metastasizing breast cancer. Whole-body magnetic resonance imaging (WB-MRI) is superior to contrast-enhanced computed tomography (CE-CT) for detecting progressive disease (PD) in bone-only metastatic breast cancer; however, the ability to detect PD in mixed visceral- and bone-metastasizing breast cancer remains to be assessed in prospective studies.PurposeTo prospectively compare WB-MRI and CE-CT for detecting PD in mixed visceral- and bone-metastasizing breast cancer and determine if WB-MRI could detect PD earlier than CE-CT.Material and MethodsA total of 43 women with mixed visceral- and bone-metastasizing breast cancer were prospectively followed with WB-MRI and CE-CT from the initiation of new chemotherapy. Kaplan-Meier analysis of time to progression was performed for both WB-MRI and CE-CT, and the time difference between the two was compared using a one-sample t-test. The ability to detect PD earliest was compared using McNemar's test.ResultsOf the 43 patients enrolled, 21 completed the study. Treatment was changed due to PD in 18 patients and due to side effects in three patients. Median time until change of treatment was 8 months (range=4-21 months). WB-MRI correctly detected PD in 16/18 patients compared to 13/18 for CE-CT. A total of 73 pairs of examinations were analyzed, of which WB-MRI detected PD earliest in 7/18 patients compared to 1/18 for CE-CT (P = 0.035).. There were no statistically significant differences in time to progression between WB-MRI and CE-CT (P = 0.41).ConclusionWB-MRI can detect PD in women with mixed visceral- and bone-metastasizing breast cancer earlier than CE-CT.

Objective:To determine the protein composition of equine platelet-rich plasma (PRP) and PRP-derived extracellular vesicles (EVs) and evaluate their effects on tendon inflammation in vitro. As tendon injuries are common in horses and treatment with PRP derived from the horse's own blood shows promise, but outcomes vary due to inconsistent composition. PRP contains EVs that facilitate cell communication. Methods:From December 2022 through May 2023, equine plasma (n = 3, adult) was isolated via double centrifugation and PRP produced using a commercial kit. Extracellular vesicles were isolated using differential ultracentrifugation and characterized with a tetraspanin assay. Equine tenocyte fibroblasts (n = 6) were stimulated with IL-1β and tumor necrosis factor-α for 24 hours to induce an inflammatory state simulating injury before treatment with PRP or PRP-derived EVs. Proteomic analysis employed data-dependent LC-MS-MS. Results:Compared to plasma, PRP and PRP EVs were enriched in proteins related to cellular waste disposal and inhibition of lipid metabolism. Experimental conditions significantly influenced the levels of 18 proteins in tenocyte fibroblasts. Collagen type 1 α chain 1 abundance decreased with treatment of PRP and PRP EVs, with implications for collagen metabolism. An increase in sequestosome 1 was also observed, having the potential to enhance inflammation or resolve it through autophagy-mediated degradation. Conclusions:PRP EVs influence the proteome of inflammatory tenocyte fibroblasts and may contribute to PRP's therapeutic effects. Clinical Relevance:Understanding the protein composition of PRP and PRP-derived EVs may help optimize PRP-based treatments for tendon injuries. Therapies could become more consistent and effective, reducing reinjury rates and improving tendon healing.

Extracellular vesicles (EVs) allow the exchange of protein, lipids and genetic material for communication between cells. We developed a method to track protein exchange between cells via EVs, using stable isotope labelling of amino acids in cell culture (SILAC). We compared EV isolation methods: ultracentrifugation and size exclusion chromatography (SEC) and undertook characterisation through nanoparticle tracking analysis (NTA), then optimised the requirement (0-10%) for foetal calf serum (FCS) and EV application concentration (6.5 million-200 EVs/cell seeded). We employed heavy amino acids L-Arg-HCl and L-Lys-2HCl to label donor cells and subsequent EV proteins. Unlabelled recipient cells were treated for 12 hours with EVs, at various concentrations. Mass spectrometry proteomics was used to assess uptake of heavy labelled EVs into recipient cells. A labelling efficiency of 62% was achieved. There was no relationship between the number of EVs applied to cells and the number of heavy proteins detected in the recipient cells. Pathway analysis indicated an inflammatory effect of applying EVs to cells, but the potential cause of this was unclear. We identified transferred protein cargo from EVs in recipient cells. Further optimisation of this method is still necessary. ### Competing Interest Statement The authors have declared no competing interest.

Improved survival after breast cancer treatment comes at a cost in the form of increased risk of late effects. A number of these are summarised in this review. The late effects can be divided in 1) late effects after locoregional treatment, e.g., lymphoedema, impaired shoulder movement, and pain; 2) consequences of systemic treatment, e.g. polyneuropathy, problems related to premature menopause, and increased risk of cardio-vascular disease; and 3) general late effects, commonly seen across all cancer types, including fatigue, insomnia, and cognitive impairment. There is a need for more knowledge about risk factors, prognoses, and the most effective treatments.

Immunocompromised patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can have a longer duration of viral shedding and persistence of symptoms. The optimal treatment strategy for these patients remains to be established. This case describes a male in his late sixties with follicular lymphoma and persistent symptoms of infection with SARS-CoV-2 variant BA.2 who was treated with remdesivir five times over a period of six months. The clinical effect of remdesivir treatment decreased over time, and further viral sequencing revealed the emergence of mutations across the SARS-CoV-2 genome. Due to the lack of other treatment options, the patient was treated with a combination of remdesivir and molnupiravir for 10 days, and epcoritamab was discontinued, which led to the cessation of symptoms. This case illustrates the risk of a diminished effect of remdesivir with prolonged use and the need for treatment guidelines for immunocompromised patients with persistent COVID-19.

Receptor-mediated endocytosis provides a mechanism for the selective uptake of specific molecules thereby controlling the composition of the extracellular environment and biological processes. The low-density lipoprotein receptor-related protein 1 (LRP1) is a widely expressed endocytic receptor that regulates cellular events by modulating the levels of numerous extracellular molecules via rapid endocytic removal. LRP1 also participates in signalling pathways through this modulation as well as in the interaction with membrane receptors and cytoplasmic adaptor proteins. LRP1 SNPs are associated with several diseases and conditions such as migraines, aortic aneurysms, cardiopulmonary dysfunction, corneal clouding, and bone dysmorphology and mineral density. Studies using Lrp1 KO mice revealed a critical, nonredundant and tissue-specific role of LRP1 in regulating various physiological events. However, exactly how LRP1 functions to regulate so many distinct and specific processes is still not fully clear. Our recent proteomics studies have identified more than 300 secreted proteins that either directly interact with LRP1 or are modulated by LRP1 in various tissues. This review will highlight the remarkable ability of this receptor to regulate secreted molecules in a tissue-specific manner and discuss potential mechanisms underpinning such specificity. Uncovering the depth of these "hidden" specific interactions modulated by LRP1 will provide novel insights into a dynamic and complex extracellular environment that is involved in diverse biological and pathological processes.

This review investigates that effective cancer treatment enhances the number of survivors in Denmark, but late complications like chemotherapy-induced peripheral neuropathy (CIPN) are a challenge. CIPN affects sensory nerves and can persist post-treatment. Diagnosis relies on symptoms and the medical history. Treatment options are limited and management faces geographic disparities and clinical capacity issues. Future research targets predictive markers and possible preventative interventions. CIPN, among other late complications, remains a significant concern in survivorship care.