Post-exercise management of exertional hyperthermia in dogs participating in dog sport (canicross) events in the UK.

Exercise is a common trigger of heat-related illness (HRI) events in dogs, accounting for 74% of canine HRI cases treated under primary veterinary care in the United Kingdom. However, few empirical studies have evaluated the effectiveness of differing cooling methods for dogs with exertional hyperthermia or HRI. This study aimed to prospectively evaluate effects of ambient conditions and post-exercise management practices (cooling methods and vehicular confinement) on the post-exercise temperature change of dogs participating in UK canicross events. Canine temperature was recorded at three intervals post-exercise: as close as possible to 0- (immediately post-exercise), 5-, and 15-min post-exercise. Ambient conditions and post-exercise management were recorded for 115 cooling profiles from 52 dogs. In 28/115 (24.4%) profiles, the dog's temperature increased during the first 5-min post-exercise. Overall, 68/115 (59.1%) profiles included passive cooling (stood or walked outside), 35 (30.4%) active cooling (cold-water immersion or application of a cooling coat), and 12 (10.4%) involved no cooling and were immediately housed in vehicles. No dogs developed hypothermia during the study and no adverse effects were observed from any cooling method. In hyperthermic dogs, overall post-exercise body temperature change was significantly negatively associated (i.e. the dogs cooled more) with 0-min post-exercise body temperature (ß = -0.93, p < 0.001), and not being housed in a vehicle (ß = -0.43, p = 0.013). This study provides evidence cold-water immersion (in water at 0.1-15.0 °C) can be used to effectively and safely cool dogs with exertional hyperthermia. Progressive temperature increases in many dogs - even after exercise has terminated - supports the message to "cool first, transport second" when managing dogs with HRI. When transporting dogs post-exercise or with HRI even after active cooling, care should be taken to cool the vehicle before entry and promote air movement around the dog during transport to facilitate ongoing cooling and prevent worsening of hyperthermia during travel.

Caudal-dependent cell positioning directs morphogenesis of the C. elegans ventral epidermis.

Strikingly, epithelial morphogenesis remains incomplete at the end of C. elegans embryonic development; newly hatched larvae undergo extensive remodelling of their ventral epidermis during the first larval stage (L1), when newly-born epidermal cells move ventrally to complete the epidermal syncytium. Prior to this remodelling, undivided lateral seam cells produce anterior adherens junction processes that are inherited by the anterior daughter cells following an asymmetric division during L1. These adherens junction processes provide the ventral migratory route for these anterior daughters. Here, we show that these processes are perturbed in pal-1/caudal mutant animals, resulting in their inheritance by posterior, seam-fated daughters. This causes aberrant migration of seam daughter cells, disrupting the ventral epidermis. Using 4D-lineaging, we demonstrate that this larval epidermal morphogenesis defect in pal-1 mutants can be traced directly back to an initial cell positioning defect in the embryo. pal-1 expression, driven by a single intronic enhancer, is required to correctly position the seam cells in embryos such that the appropriate cell junctions support the correct migratory paths of seam daughters later in development, irrespective of their fate. Thus, during ventral epithelial remodelling in C. elegans, we show that the position of migrating cells, specified by pal-1/caudal, appears to be more important than their fate in driving morphogenesis.

Regulation of microRNA-221, -222, -21 and -27 in articular cartilage subjected to abnormal compressive forces.

KEY POINTS: microRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression. miRs are involved in regulating cellular activities in response to mechanical loading in all physiological systems, although it is largely unknown whether this response differs with increasing magnitudes of load. miR-221, miR-222, miR-21-5p and miR-27a-5p were significantly increased in ex vivo cartilage explants subjected to increasing load magnitude and in in vivo joint cartilage exposed to abnormal loading. TIMP3 and CPEB3 are putative miR targets in chondrocytes Identification of mechanically regulated miRs that have potential to impact on tissue homeostasis provides a mechanism by which load-induced tissue behaviour is regulated, in both health and pathology, in all physiological systems. ABSTRACT: MicroRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression and are involved in mechano-regulation of cellular activities in all physiological systems. It is unknown whether such epigenetic mechanisms are regulated in response to increasing magnitudes of load. The present study investigated mechano-regulation of miRs in articular cartilage subjected to 'physiological' and 'non-physiological' compressive loads in vitro as a model system and validated findings in an in vivo model of abnormal joint loading. Bovine full-depth articular cartilage explants were loaded to 2.5 MPa (physiological) or 7 MPa (non-physiological) (1 Hz, 15 min) and mechanically-regulated miRs identified using next generation sequencing and verified using a quantitative PCR. Downstream targets were verified using miR-specific mimics or inhibitors in conjunction with 3'-UTR luciferase activity assays. A subset of miRs were mechanically-regulated in ex vivo cartilage explants and in vivo joint cartilage. miR-221, miR-222, miR-21-5p and miR-27a-5p were increased and miR-483 levels decreased with increasing load magnitude. Tissue inhibitor of metalloproteinase 3 (TIMP3) and cytoplasmic polyadenylation element binding protein 3 (CPEB3) were identified as putative downstream targets. Our data confirm miR-221 and -222 mechano-regulation and demonstrates novel mechano-regulation of miR-21-5p and miR-27a-5p in ex vivo and in vivo cartilage loading models. TIMP3 and CPEB3 are putative miR targets in chondrocytes. Identification of specific miRs that are regulated by increasing load magnitude, as well as their potential to impact on tissue homeostasis, has direct relevance to other mechano-sensitive physiological systems and provides a mechanism by which load-induced tissue behaviour is regulated, in both health and pathology.

Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage.

The composition and organisation of the extracellular matrix (ECM), particularly the pericellular matrix (PCM), in articular cartilage is critical to its biomechanical functionality; the presence of proteoglycans such as aggrecan, entrapped within a type II collagen fibrillar network, confers mechanical resilience underweight-bearing. Furthermore, components of the PCM including type VI collagen, perlecan, small leucine-rich proteoglycans-decorin and biglycan-and fibronectin facilitate the transduction of both biomechanical and biochemical signals to the residing chondrocytes, thereby regulating the process of mechanotransduction in cartilage. In this review, we summarise the literature reporting on the bidirectional reciprocity of the ECM in chondrocyte mechano-signalling and articular cartilage homeostasis. Specifically, we discuss studies that have characterised the response of articular cartilage to mechanical perturbations in the local tissue environment and how the magnitude or type of loading applied elicits cellular behaviours to effect change. In vivo, including transgenic approaches, and in vitro studies have illustrated how physiological loading maintains a homeostatic balance of anabolic and catabolic activities, involving the direct engagement of many PCM molecules in orchestrating this slow but consistent turnover of the cartilage matrix. Furthermore, we document studies characterising how abnormal, non-physiological loading including excessive loading or joint trauma negatively impacts matrix molecule biosynthesis and/or organisation, affecting PCM mechanical properties and reducing the tissue's ability to withstand load. We present compelling evidence showing that reciprocal engagement of the cells with this altered ECM environment can thus impact tissue homeostasis and, if sustained, can result in cartilage degradation and onset of osteoarthritis pathology. Enhanced dysregulation of PCM/ECM turnover is partially driven by mechanically mediated proteolytic degradation of cartilage ECM components. This generates bioactive breakdown fragments such as fibronectin, biglycan and lumican fragments, which can subsequently activate or inhibit additional signalling pathways including those involved in inflammation. Finally, we discuss how bidirectionality within the ECM is critically important in enabling the chondrocytes to synthesise and release PCM/ECM molecules, growth factors, pro-inflammatory cytokines and proteolytic enzymes, under a specified load, to influence PCM/ECM composition and mechanical properties in cartilage health and disease.

Subcutaneous administration of ciclosporin in 11 allergic cats - a pilot open-label uncontrolled clinical trial.

BACKGROUND: Oral ciclosporin has been reported to be efficacious for feline inflammatory skin diseases; however, cats are often difficult to medicate orally. HYPOTHESIS/OBJECTIVE: To evaluate the efficacy and tolerability of subcutaneous ciclosporin administered to cats with allergic skin disease. ANIMALS: Eleven client-owned cats with nonseasonal clinical signs. METHODS: Prospective open label trial. Ciclosporin 50 mg/mL solution for injection (Sandimune®, Novartis; NJ, USA) was administered subcutaneously for 60 days with initial doses ranging from 2.5 mg/kg once daily (one cat) to every other day (five cats) and 5 mg/kg once daily (four cats) to every other day (one cat). Dosages were adjusted monthly if needed based on clinical response. Clinical response was assessed using a modified FeDESI (feline Dermatitis Extent and Severity Index) and PVAS (pruritus Visual Analog Scale) between days (D) 0, 30 and 60. RESULTS: Six cats completed the study and four of five cats withdrawn from the study were included in an intention-to-treat analysis. There was significant decrease in FeDESI and PVAS scores between D0 and D30, D0 and D60 and D30 and D60 (P < 0.05) in all ten cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Ciclosporin administered subcutaneously at initial doses of 2.5-5 mg/kg, once daily to alternate days, appears to be an efficacious therapy for feline allergic dermatitis and may be an alternative therapy for cats that cannot be treated orally. Randomized and controlled long term studies which include a larger number of cats are needed to confirm these findings.

AMPA/kainate glutamate receptors contribute to inflammation, degeneration and pain related behaviour in inflammatory stages of arthritis.

OBJECTIVES: Synovial fluid glutamate concentrations increase in arthritis. Activation of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) increase interleukin-6 (IL-6) release and cause arthritic pain, respectively. We hypothesised that AMPA and KA GluRs are expressed in human arthritis, and that intra-articular NBQX (AMPA/KA GluR antagonist) prevents pain and pathology in antigen-induced arthritis (AIA). METHODS: GluR immunohistochemistry was related to synovial inflammation and degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). A single intra-articular NBQX injection was given at induction, and knee swelling and gait of AIA and AIA+NBQX rats compared over 21 days, before imaging, RT-qPCR, histology and immunohistochemistry of joints. Effects of NBQX on human primary osteoblast (HOB) activity were determined. RESULTS: AMPAR2 and KA1 immunolocalised to remodelling bone, cartilage and synovial cells in human OA and RA, and rat AIA. All arthritic tissues showed degradation and synovial inflammation. NBQX reduced GluR abundance, knee swelling (p<0.001, days 1-21), gait abnormalities (days 1-2), end-stage joint destruction (p<0.001), synovial inflammation (p<0.001), and messenger RNA expression of meniscal IL-6 (p<0.05) and whole joint cathepsin K (p<0.01). X-ray and MRI revealed fewer cartilage and bone erosions, and less inflammation after NBQX treatment. NBQX reduced HOB number and prevented mineralisation. CONCLUSIONS: AMPA/KA GluRs are expressed in human OA and RA, and in AIA, where a single intra-articular injection of NBQX reduced swelling by 33%, and inflammation and degeneration scores by 34% and 27%, respectively, exceeding the efficacy of approved drugs in the same model. AMPA/KA GluR antagonists represent a potential treatment for arthritis.

Probing the substrate specificity of Trypanosoma brucei GlcNAc-PI de-N-acetylase with synthetic substrate analogues.

A series of synthetic analogues of 1-D-(2-amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate), consisting of 7 variants of either the D-myo-inositol, D-GlcpN or the phospholipid components, were prepared and tested as substrates and inhibitors of GlcNAc-PI de-N-acetylase, a genetically validated drug target enzyme responsible for the second step in the glycosylphosphatidylinositol (GPI) biosynthetic pathway of Trypanosoma brucei. The D-myo-inositol in the physiological substrate was successfully replaced by cyclohexanediol and is still a substrate for T. brucei GlcNAc-PI de-N-acetylase. However, this compound became sensitive to the stereochemistry of the glycoside linkage (the ß-anomer was neither substrate or inhibitor) and the structure of the lipid moiety (the hexadecyl derivatives were inhibitors). Chemistry was successfully developed to replace the phosphate with a sulphonamide, but the compound was neither a substrate or an inhibitor, confirming the importance of the phosphate for molecular recognition. We also replaced the glucosamine by an acyclic analogue, but this also was inactive, both as a substrate and inhibitor. These findings add significantly to our understanding of substrate and inhibitor binding to the GlcNAc-PI de-N-acetylase enzyme and will have a bearing on the design of future inhibitors.

Investigating mechanical and inflammatory pathological mechanisms in osteoarthritis using MSC-derived osteocyte-like cells in 3D.

Introduction: Changes to bone physiology play a central role in the development of osteoarthritis with the mechanosensing osteocyte releasing factors that drive disease progression. This study developed a humanised in vitro model to detect osteocyte responses to either interleukin-6, a driver of degeneration and bone remodelling in animal and human joint injury, or mechanical loading, to mimic osteoarthritis stimuli in joints. Methods: Human MSC cells (Y201) were differentiated in 3-dimensional type I collagen gels in osteogenic media and osteocyte phenotype assessed by RTqPCR and immunostaining. Gels were subjected to a single pathophysiological load or stimulated with interleukin-6 with unloaded or unstimulated cells as controls. RNA was extracted 1-hour post-load and assessed by RNAseq. Markers of pain, bone remodelling, and inflammation were quantified by RT-qPCR and ELISA. Results: Y201 cells embedded within 3D collagen gels assumed dendritic morphology and expressed mature osteocytes markers. Mechanical loading of the osteocyte model regulated 7564 genes (Padj p<0.05, 3026 down, 4538 up). 93% of the osteocyte transcriptome signature was expressed in the model with 38% of these genes mechanically regulated. Mechanically loaded osteocytes regulated 26% of gene ontology pathways linked to OA pain, 40% reflecting bone remodelling and 27% representing inflammation. Load regulated genes associated with osteopetrosis, osteoporosis and osteoarthritis. 42% of effector genes in a genome-wide association study meta-analysis were mechanically regulated by osteocytes with 10 genes representing potential druggable targets. Interleukin-6 stimulation of osteocytes at concentrations reported in human synovial fluids from patients with OA or following knee injury, regulated similar readouts to mechanical loading including markers of pain, bone remodelling, and inflammation. Discussion: We have developed a reproducible model of human osteocyte like cells that express >90% of the genes in the osteocyte transcriptome signature. Mechanical loading and inflammatory stimulation regulated genes and proteins implicated in osteoarthritis symptoms of pain as well as inflammation and degeneration underlying disease progression. Nearly half of the genes classified as 'effectors' in GWAS were mechanically regulated in this model. This model will be useful in identifying new mechanisms underlying bone and joint pathologies and testing drugs targeting those mechanisms.

Epidemiology of heat-related illness in dogs under UK emergency veterinary care in 2022.

BACKGROUND: Dogs are exposed to increasing environmental risk for developing heat-related illness (HRI), with 2022 recorded as the hottest year to date in the UK and most of Europe. METHODS: This study used VetCompass data to report the incidence risk, event fatality rate and canine risk factors for HRI in dogs presenting to Vets Now emergency care practices in the UK during 2022. RESULTS: From the clinical records of 167,751 dogs under care at Vets Now emergency clinics in 2022, 384 HRI events were identified. The 2022 incidence risk of HRI within the Vets Now caseload was 0.23% (95% confidence interval [CI]: 0.21%‒0.25%), with an event fatality rate of 26.56% (95% CI: 21.66%-32.25%). Multivariable analysis identified breed, age and sex/neuter status as risk factors for HRI. Brachycephalic dogs had 4.21 times the odds of HRI compared to mesocephalic dogs (95% CI: 3.22‒5.49, p < 0.001). LIMITATIONS: The clinical data used in this study were not primarily recorded for research and had some substantial levels of missing data (especially patient bodyweight). CONCLUSION: In order to protect canine welfare, improved long-term mitigation strategies are urgently needed to minimise HRI risk and associated fatality in UK dogs.

Comparative transcriptomic analysis of articular cartilage of post-traumatic osteoarthritis mouse models.

Animal models of post-traumatic osteoarthritis (PTOA) recapitulate the pathological changes observed in human PTOA. Here, skeletally mature C57Bl6 mice were subjected to either the rapid-onset, non-surgical, mechanical anterior cruciate ligament (ACL) rupture or surgical destabilisation of the medial meniscus (DMM) models. Transcriptome profiling of micro-dissected cartilage at day 7 and 42 post-ACL and DMM procedure respectively, showed that the two models were comparable and highly correlative (Spearman R =0.82, p<2.2E-16). Gene ontology enrichment analysis identified similarly enriched pathways, which were overrepresented by anabolic terms. To address the transcriptome changes more completely in the ACL model we also performed small RNA-seq, describing the first microRNA profile of this model. miR-199-5p was amongst the most abundant yet differentially expressed microRNAs and its inhibition in primary human chondrocytes led to a comparable transcriptome response to that observed in both human 'OA damaged vs intact cartilage' and murine DMM cartilage datasets. CELSR1, GIT1, ECE1 and SOS2 were all experimentally verified as novel miR-199-5p targets. Together, these data support the use of the ACL rupture model as a non-invasive companion to DMM.

A mixed-methods approach exploring acceptability and feasibility of trials designed to test drugs targeting prevention of post-traumatic osteoarthritis after knee injury.

Aims: To explore key stakeholder views around feasibility and acceptability of trials seeking to prevent post-traumatic osteoarthritis (PTOA) following knee injury, and provide guidance for next steps in PTOA trial design. Methods: Healthcare professionals, clinicians, and/or researchers (HCP/Rs) were surveyed, and the data were presented at a congress workshop. A second and related survey was then developed for people with joint damage caused by knee injury and/or osteoarthritis (PJDs), who were approached by a UK Charity newsletter or Oxford involvement registry. Anonymized data were collected and analyzed in Qualtrics. Results: Survey responses (n = 19 HCP/Rs, 39 PJDs) supported studies testing pharmacological agents preventing PTOA. All HCP/Rs and 30/31 (97%) PJDs supported the development of new treatments that improved or delayed knee symptoms and damage to knee structure. PJDs thought that improving structural knee damage was more important than knee symptoms. Both groups found studies more acceptable as expected future benefit and risk of PTOA increased. All drug delivery routes were acceptable. Workshop participants (around n = 60) reflected survey views. Discussions suggested that stratifying using molecular testing for likely drug response appeared to be more acceptable than using characteristics such as sex, age, and BMI. Conclusion: Our findings supported PTOA drug intervention studies, including situations where there is low risk of disease, no expected benefit of treatment, and frequent treatment administration. PJDs appeared less risk-averse than HCP/Rs. This work reinforces the benefits of consensus and involvement work in the co-creation of PTOA drug trial design. Involvement of key stakeholders, such as PJDs with different risks of OA and regulatory representatives, are critical for trial design success.

Applying clinical audit for quality improvement in canine dystocia cases seen at a UK primary-care emergency practice.

BACKGROUND: The paucity of published veterinary clinical audits suggests that clinical audit is an under-used tool for quality improvement (QI) in the veterinary profession. Therefore, a continuous QI process was designed and implemented at a UK multisite small animal emergency practice, focusing on audit of clinical management of canine dystocia. METHODS: Data collection phases were undertaken in 2014, 2019 and 2021, with intervening knowledge dissemination activities. Nine variables relating to clinical management of canine dystocia were selected as audit criteria in the initial dataset, and 21 variables were measured in each subsequent phase. RESULTS: Between 2014 and 2021, statistically significant increases (p < 0.05) were demonstrated in recording of bodyweight, use of diagnostic imaging, use of ultrasonography, recording of fetal heart rates, use of calcium gluconate, and use during caesarean section of intravenous fluid therapy, multimodal analgesia, full agonist opioids, paracetamol and local anaesthesia. Statistically significant decreases were demonstrated in median first quantity and median first dose of oxytocin, and in the use of NSAIDs during caesarean section. A clinical audit planning template was created for future audits. LIMITATIONS: Typical case presentation and management of canine dystocia cases may vary between dedicated emergency and non-emergency primary-care settings. CONCLUSION: This study demonstrates the feasibility of large-scale veterinary clinical audit and suggests that the application of the clinical audit process promotes learning within the veterinary team and improved clinical outcomes.

Cooling Methods Used to Manage Heat-Related Illness in Dogs Presented to Primary Care Veterinary Practices during 2016-2018 in the UK.

The management of heat-related illness (HRI) in dogs has received limited attention in the veterinary literature, especially regarding effective cooling methods. Guidelines published in 2016 for prehospital management of dogs with HRI advised "cool first, transport second", and recommended using cold-water immersion and evaporative cooling (water application with air movement) as the optimal approaches to reduce the patient's temperature. The current retrospective cross-sectional observation study analysed electronic patient records from the VetCompass programme to describe the cooling methods used in dogs with HRI presented to primary care veterinary practices during 2016-2018. Of 623 HRI events identified, 341 (54.74%, 95% CI 50.81-58.60%) included information on cooling in their clinical record. Of these, 74/341 (21.70%, 95% CI 17.65-26.38%) were cooled prior to transport for veterinary care. Overall, 23.97% (95% CI 19.24-29.44%) were cooled using one of the two recommended cooling methods, whilst the most common cooling method recorded was the application of wet towels (51.31%, 95% CI 45.34-57.24%). Canine cooling guidance and messaging in both the public and veterinary sectors requires urgent review to ensure that the most effective cooling methods are promoted because delays to canine temperature reduction worsen patient outcomes.

The unequal burden of human-wildlife conflict.

Human-wildlife conflict is one of the most pressing sustainable development challenges globally. This is particularly the case where ecologically and economically important wildlife impact the livelihoods of humans. Large carnivores are one such group and their co-occurrence with low-income rural communities often results in real or perceived livestock losses that place increased costs on already impoverished households. Here we show the disparities associated with the vulnerability to conflict arising from large carnivores on cattle (Bos taurus) globally. Across the distribution of 18 large carnivores, we find that the economic vulnerability to predation losses (as measured by impacts to annual per capita income) is between two and eight times higher for households in transitioning and developing economies when compared to developed ones. This potential burden is exacerbated further in developing economies because cattle keepers in these areas produce on average 31% less cattle meat per animal than in developed economies. In the lowest-income areas, our estimates suggest that the loss of a single cow or bull equates to nearly a year and a half of lost calories consumed by a child. Finally, our results show that 82% of carnivore range falls outside protected areas, and five threatened carnivores have over one third of their range located in the most economically sensitive conflict areas. This unequal burden of human-carnivore conflict sheds light on the importance of grappling with multiple and conflicting sustainable development goals: protecting life on land and eliminating poverty and hunger.

Type IX collagen interacts with fibronectin providing an important molecular bridge in articular cartilage.

Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the α1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library. 73% of the interacting clones encoded fibronectin. The interaction was confirmed using in vitro immunoprecipitation and was further characterized by surface plasmon resonance. Using whole and pepsin-derived preparations of type IX collagen, the interaction was shown to be specific for the NC4 domain with no interaction with the triple helical collagenous domains. The interaction was shown to be of high affinity with nanomolar K(d) values. Analysis of the fibronectin-interacting clones indicates that the constant domain is the likely site of interaction. Type IX collagen and fibronectin were shown to co-localize in cartilage. This novel interaction between the NC4 domain of type IX collagen and fibronectin may represent an in vivo interaction in cartilage that could contribute to the matrix integrity of the tissue.

IKKε Inhibitor Amlexanox Promotes Olaparib Sensitivity through the C/EBP-ß-Mediated Transcription of Rad51 in Castrate-Resistant Prostate Cancer.

The progression of prostate cancer (PC) is often characterized by the development of castrate-resistant PC (CRPC). Patients with CRPC are treated with a variety of agents including new generation hormonal therapies or chemotherapy. However, as the cancer develops more resistance mechanisms, these drugs eventually become less effective and finding new therapeutic approaches is critical to improving patient outcomes. Previously, we have shown that IKKε depletion and IKKε inhibitors, BX795 and Amlexanox, decrease CRPC cell proliferation in vitro and in vivo and that IKKε inhibitors induce a senescence phenotype accompanied by increased DNA damage and genomic instability in CRPC cells. Here, we describe a new role for IKKε in DNA damage repair involving Rad51 and examine the therapeutic potential of Amlexanox combined with the PARP inhibitor Olaparib in CRPC cell lines. Combining Amlexanox with Olaparib decreased CRPC cell proliferation and enhanced DNA damage through the inhibition of Olaparib-induced Rad51 recruitment and expression in CRPC cells or IKKε-depleted PC-3 cells. We demonstrated that Rad51 promoter activity, measured by luciferase assay, was decreased with Amlexanox treatment or IKKε depletion and that Amlexanox treatment decreased the occupancy of transcription factor C/EBP-ß on the Rad51 promoter. Our mouse model also showed that Amlexanox combined with Olaparib inhibited tumor growth of CRPC xenografts. Our study highlights a new role for IKKε in DNA damage repair through the regulation of Rad51 transcription and provides a rationale for the combination of Amlexanox and Olaparib in the treatment of patients with CRPC.

NR1D1 regulation by Ran GTPase via miR4472 identifies an essential vulnerability linked to aneuploidy in ovarian cancer.

While aneuploidy is a main enabling characteristic of cancers, it also creates specific vulnerabilities. Here we demonstrate that Ran inhibition targets epithelial ovarian cancer (EOC) survival through its characteristic aneuploidy. We show that induction of aneuploidy in rare diploid EOC cell lines or normal cells renders them highly dependent on Ran. We also establish an inverse correlation between Ran and the tumor suppressor NR1D1 and reveal the critical role of Ran/NR1D1 axis in aneuploidy-associated endogenous DNA damage repair. Mechanistically, we show that Ran, through the maturation of miR4472, destabilizes the mRNA of NR1D1 impacting several DNA repair pathways. We showed that NR1D1 interacts with both PARP1 and BRCA1 leading to the inhibition of DNA repair. Concordantly, loss of Ran was associated with NR1D1 induction, accumulation of DNA damages, and lethality of aneuploid EOC cells. Our findings suggest a synthetic lethal strategy targeting aneuploid cells based on their dependency to Ran.

Targeting IKKε in Androgen-Independent Prostate Cancer Causes Phenotypic Senescence and Genomic Instability.

Advanced prostate cancer will often progress to a lethal, castration-resistant state. We previously demonstrated that IKKε expression correlated with the aggressiveness of prostate cancer disease. Here, we address the potential of IKKε as a therapeutic target in prostate cancer. We examined cell fate decisions (proliferation, cell death, and senescence) in IKKε-depleted PC-3 cells, which exhibited delayed cell proliferation and a senescent phenotype, but did not undergo cell death. Using IKKε/TBK1 inhibitors, BX795 and Amlexanox, we measured their effects on cell fate decisions in androgen-sensitive prostate cancer and androgen-independent prostate cancer cell lines. Cell-cycle analyses revealed a G2-M cell-cycle arrest and a higher proportion of cells with 8N DNA content in androgen-independent prostate cancer cells only. Androgen-independent prostate cancer cells also displayed increased senescence-associated (SA)-ß-galactosidase activity; increased γH2AX foci; genomic instability; and altered p15, p16, and p21 expression. In our mouse model, IKKε inhibitors also decreased tumor growth of androgen-independent prostate cancer xenografts but not 22Rv1 androgen-sensitive prostate cancer xenografts. Our study suggests that targeting IKKε with BX795 or Amlexanox in androgen-independent prostate cancer cells induces a senescence phenotype and demonstrates in vivo antitumor activity. These results strengthen the potential of exploiting IKKε as a therapeutic target.

Patient perspectives on an intervention after suicide attempt: The need for patient centred and individualized care.

BACKGROUND: Many types of intervention exist for suicide attempters, but they tend not to sufficiently consider patient's views. AIM: To identify useful components of a previously evaluated intervention after a suicide attempt from the patient's views and to better understand the process of recovery. METHOD: Forty-one interviews with suicide attempters were qualitatively analysed. Views of participants (i) on the components of the intervention (nurse case-management, joint crisis plan, meetings with relatives/network and follow-up calls) and (ii) their recovery were explored. The material was analysed by means of thematic analysis with a deductive-inductive approach. RESULTS: Participants valued the human and professional qualities of the nurse case-manager, and appreciated follow-up calls and meetings. However, their views diverged regarding for instance frequency of phone calls, or disclosing information or lack thereof. Interpersonal relationship, suicide attempters' own resources and life changes emerged as core recovery factors. DISCUSSION: The study highlights the figure of an engaged clinician, with both professional and human commitment, aware that some suicide attempters put more emphasis on their own resources than on delivered health care. CONCLUSIONS: Interventions should consider the clinician as the cornerstone of the tailored care valued by suicide attempters.