Metabolic control of BRISC-SHMT2 assembly regulates immune signalling.

Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.

Fibroblast expression of CD248 may contribute to exacerbation of microvascular damage during systemic sclerosis.

OBJECTIVES: CD248 is a glycoprotein, highly expressed on pericytes and fibroblasts (FBs), that is implicated in the fibrotic process. During angiogenesis, CD248 can promote vessel regression, binding multimerin-2 (MMRN-2). Thus, we investigated the expression of MMRN-2 in systemic sclerosis (SSc)-skin and of CD248 in isolated SSc-FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by co-culturing these cells with healthy control endothelial cells (HC-ECs). The apoptotic effect of CD248 on HC-ECs was evaluated. Finally, the ability of CD248 to prevent activation of VEGF receptor 2 (VEGFR2) was assessed. METHODS: By IF, MMRN-2 was investigated in SSc-skin and CD248 in SSc FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by HC-ECs/SSc-FBs co-cultures. Lentiviral-induced CD248 short-hairpin RNA delivery was employed for loss-of-function studies in SSc-FBs. HC-ECs were cultured in the presence of CD248 to assess apoptosis by IF and VEGFR2 phosphorylation by western blot. RESULTS: MMRN-2 expression was increased in skin SSc-ECs, whereas CD248 expression was increased in SSc-FBs. Functionally, CD248+-SSc-FBs suppressed angiogenesis in the organotypic model, as assessed by the reduction in total tube length of HC-ECs. This anti-angiogenetic behaviour was reversed by CD248 silencing. Furthermore, the presence of CD248 promoted the apoptosis of HC-ECs. Finally, CD248 prevented activation of VEGFR2 by reducing its phosphorylation after VEGF stimulation. CONCLUSION: CD248 was anti-angiogenic in vitro due to a reduction in tube formation and to induction of apoptosis of ECs. Increased expression of CD248 in SSc could contribute to the microvascular rarefaction observed at the tissue level in SSc. Our results suggest a pathogenic role for CD248-MMRN-2 in SSc.

Filling the data gaps: Transferring models from data-rich to data-poor deep-sea areas to support spatial management.

Spatial management of the deep sea is challenging due to limited available data on the distribution of species and habitats to support decision making. In the well-studied North Atlantic, predictive models of species distribution and habitat suitability have been used to fill data gaps and support sustainable management. In the South Atlantic and other poorly studied regions, this is not possible due to a massive lack of data. In this study, we investigated whether models constructed in data-rich areas can be used to inform data-poor regions (with otherwise similar environmental conditions). We used a novel model transfer approach to identify to what extent a habitat suitability model for Desmophyllum pertusum reef, built in a data-rich basin (North Atlantic), could be transferred usefully to a data-poor basin (South Atlantic). The transferred model was built using the Maximum Entropy algorithm and constructed with 227 presence and 3064 pseudo-absence points, and 200 m resolution environmental grids. Performance in the transferred region was validated using an independent dataset of D. pertusum presences and absences, with assessments made using both threshold-dependent and -independent metrics. We found that a model for D. pertusum reef fitted to North Atlantic data transferred reasonably well to the South Atlantic basin, with an area under the curve of 0.70. Suitable habitat for D. pertusum reef was predicted on 20 of the assessed 27 features including seamounts. Nationally managed Marine Protected Areas provide significant protection for D. pertusum reef habitat in the region, affording full protection from bottom trawling to 14 of the 20 suitable features. In areas beyond national jurisdiction (ABNJ), we found four seamounts that provided suitable habitat for D. pertusum reef to be at least partially protected from bottom trawling, whilst two did not fall within fisheries closures. There are factors to consider when developing models for transfer including data resolution and predictor type. Nevertheless, the promising results of this application demonstrate that model transfer approaches stand to provide significant contributions to spatial planning processes through provision of new, best available data. This is particularly true for ABNJ and areas that have previously undergone little scientific exploration such as the global south.

Targeting human plasmacytoid dendritic cells through BDCA2 prevents skin inflammation and fibrosis in a novel xenotransplant mouse model of scleroderma.

OBJECTIVES: Plasmacytoid dendritic cells (pDC) have been implicated in the pathogenesis of autoimmune diseases, such as scleroderma (SSc). However, this has been derived from indirect evidence using ex vivo human samples or mouse pDC in vivo. We have developed human-specific pDC models to directly identify their role in inflammation and fibrosis, as well as attenuation of pDC function with BDCA2-targeting to determine its therapeutic application. METHODS: RNAseq of human pDC with TLR9 agonist ODN2216 and humanised monoclonal BDCA2 antibody, CBS004. Organotypic skin rafts consisting of fibroblasts and keratinocytes were stimulated with supernatant from TLR9-stimulated pDC and with CBS004. Human pDC were xenotransplanted into Nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) with CBS004 or human IgG control. Skin punch biopsies were used to assess gene and protein expression. RESULTS: RNAseq shows TLR9-induced activation of human pDC goes beyond type I interferon (IFN) secretion, which is functionally inactivated by BDCA2-targeting. Consistent with these findings, we show that BDCA2-targeting of pDC can completely suppress in vitro skin IFN-induced response. Most importantly, xenotransplantation of human pDC significantly increased in vivo skin IFN-induced response to TLR agonist and strongly enhanced fibrotic and immune response to bleomycin compared with controls. In these contexts, BDCA2-targeting suppressed human pDC-specific pathological responses. CONCLUSIONS: Our data indicate that human pDC play a key role in inflammation and immune-driven skin fibrosis, which can be effectively blocked by BDCA2-targeting, providing direct evidence supporting the development of attenuation of pDC function as a therapeutic application for SSc.

The intracellular chloride channel 4 (CLIC4) activates systemic sclerosis fibroblasts.

OBJECTIVES: Tissue fibrosis in SSc is driven by active fibroblasts (myofibroblasts). Previous studies have shown the intracellular chloride channel 4 (CLIC4) mediates the activation of cancer-associated fibroblasts. In this study we investigated the role of CLIC4 in SSc fibroblast activation. METHODS: Fibroblasts were obtained from full thickness skin biopsies from SSc patients (early-diffuse). RNA and protein were collected from the fibroblasts and CLIC4 transcript and protein levels were assessed by qPCR and western blot. SSc patient fibroblasts were treated with the chloride channel inhibitors nitro-2-(3-phenylpropylamino)benzoic acid and indyanyloxyacetic acid 94. RESULTS: CLIC4 was expressed at significantly higher levels in SSc patients' fibroblasts compared with healthy controls, at both the transcript (3.7-fold) and protein (1.7-fold) levels. Inhibition of the TGF-ß receptor and its downstream transcription factor SMAD3 led to a reduction in CLIC4 expression, confirming this pathway as the main driver of CLIC4 expression. Importantly, treatment of SSc fibroblasts with known pharmacological inhibitors of CLIC4 led to reduced expression of the myofibroblast markers collagen type 1 and α-smooth muscle actin, inferring a direct role for CLIC4 in disease pathogenesis. CONCLUSIONS: We have identified a novel role for CLIC4 in SSc myofibroblast activation, which strengthens the similarities of SSc fibroblasts with cancer-associated fibroblasts and highlights this channel as a novel target for therapeutic intervention.

Muscle Damage in Systemic Sclerosis and CXCL10: The Potential Therapeutic Role of PDE5 Inhibition.

Skeletal muscle damage is a common clinical manifestation of systemic sclerosis (SSc). C-X-C chemokine ligand 10 (CXCL10) is involved in myopathy and cardiomyopathy development and is associated with a more severe SSc prognosis. Interestingly, the phosphodiesterase type 5 inhibitor (PDE5i) sildenafil reduces CXCL10 sera levels of patients with diabetic cardiomyopathy and in cardiomyocytes. Here, we analyzed the levels of CXCL10 in the sera of 116 SSc vs. 35 healthy subjects and explored differences in 17 SSc patients on stable treatment with sildenafil. CXCL10 sera levels were three-fold higher in SSc vs. healthy controls, independent of subset and antibody positivity. Sildenafil treatment was associated with lower CXCL10 sera levels. Serum CXCL10 strongly correlated with the clinical severity of muscle involvement and with creatine kinase (CK) serum concentration, suggesting a potential involvement in muscle damage in SSc. In vitro, sildenafil dose-dependently reduced CXCL10 release by activated myocytes and impaired cytokine-induced Signal transducer and activator of transcription 1 (STAT1), Nuclear factor-κB (NFκB) and c-Jun N-terminal kinase (JNK) phosphorylation. This was also seen in cardiomyocytes. Sildenafil-induced CXCL10 inhibition at the systemic and human muscle cell level supports the hypothesis that PDE5i could be a potential therapeutic therapy to prevent and treat muscle damage in SSc.

Seascape genomics reveals population isolation in the reef-building honeycomb worm, Sabellaria alveolata (L.).

BACKGROUND: Under the threat of climate change populations can disperse, acclimatise or evolve in order to avoid fitness loss. In light of this, it is important to understand neutral gene flow patterns as a measure of dispersal potential, but also adaptive genetic variation as a measure of evolutionary potential. In order to assess genetic variation and how this relates to environment in the honeycomb worm (Sabellaria alveolata (L.)), a reef-building polychaete that supports high biodiversity, we carried out RAD sequencing using individuals from along its complete latitudinal range. Patterns of neutral population genetic structure were compared to larval dispersal as predicted by ocean circulation modelling, and outlier analyses and genotype-environment association tests were used to attempt to identify loci under selection in relation to local temperature data. RESULTS: We genotyped 482 filtered SNPs, from 68 individuals across nine sites, 27 of which were identified as outliers using BAYESCAN and ARLEQUIN. All outlier loci were potentially under balancing selection, despite previous evidence of local adaptation in the system. Limited gene flow was observed among reef-sites (FST = 0.28 ± 0.10), in line with the low dispersal potential identified by the larval dispersal models. The North Atlantic reef emerged as a distinct population and this was linked to high local larval retention and the effect of the North Atlantic Current on dispersal. CONCLUSIONS: As an isolated population, with limited potential for natural genetic or demographic augmentation from other reefs, the North Atlantic site warrants conservation attention in order to preserve not only this species, but above all the crucial functional ecological roles that are associated with their bioconstructions. Our study highlights the utility of using seascape genomics to identify populations of conservation concern.

Long non-coding RNA HOTAIR drives EZH2-dependent myofibroblast activation in systemic sclerosis through miRNA 34a-dependent activation of NOTCH.

BACKGROUND: Systemic sclerosis (SSc) is characterised by autoimmune activation, tissue and vascular fibrosis in the skin and internal organs. Tissue fibrosis is driven by myofibroblasts, that are known to maintain their phenotype in vitro, which is associated with epigenetically driven trimethylation of lysine 27 of histone 3 (H3K27me3). METHODS: Full-thickness skin biopsies were surgically obtained from the forearms of 12 adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted. HOX transcript antisense RNA (HOTAIR) was expressed in healthy dermal fibroblasts by lentiviral induction employing a vector containing the specific sequence. Gamma secretase inhibitors were employed to block Notch signalling. Enhancer of zeste 2 (EZH2) was blocked with GSK126 inhibitor. RESULTS: SSc myofibroblasts in vitro and SSc skin biopsies in vivo display high levels of HOTAIR, a scaffold long non-coding RNA known to direct the histone methyltransferase EZH2 to induce H3K27me3 in specific target genes. Overexpression of HOTAIR in dermal fibroblasts induced EZH2-dependent increase in collagen and α-SMA expression in vitro, as well as repression of miRNA-34A expression and consequent NOTCH pathway activation. Consistent with these findings, we show that SSc dermal fibroblast display decreased levels of miRNA-34a in vitro. Further, EZH2 inhibition rescued miRNA-34a levels and mitigated the profibrotic phenotype of both SSc and HOTAIR overexpressing fibroblasts in vitro. CONCLUSIONS: Our data indicate that the EZH2-dependent epigenetic phenotype of myofibroblasts is driven by HOTAIR and is linked to miRNA-34a repression-dependent activation of NOTCH signalling.

Passive anti-C acquired in the setting of Rh immune globulin administration following Rh mismatched apheresis platelet transfusion: A case series.

Rh immune globulin (RhIG) may be administered to Rh(D)-negative recipients of Rh(D)-positive platelet (PLT) transfusions to mitigate anti-D alloantibody formation. We report a series of seven patients in which anti-C was detected as a result of RhIG administered as immunoprophylaxis following Rh-mismatched apheresis PLT transfusion, persisting for a range of 27 to 167 days post-RhIG. The passively transferred anti-C antibodies created complexities for subsequent transfusion support. Based on these challenges, in combination with emerging evidence supporting an extremely low anti-D alloimmunization risk following Rh-mismatched apheresis PLTs, we have changed our practice and now limit RhIG immunoprophylaxis in this setting to women of reproductive age. In summary, the blood bank and apheresis communities should be aware that passive transfer of non-D antibodies is possible following RhIG administration. This phenomenon represents a compelling reason to consider the risk/benefit ratio of RhIG and to limit its use to situations in which it is clinically necessary.

Blood Utilization and Transfusion Reactions in Pediatric Patients Transfused with Conventional or Pathogen Reduced Platelets.

OBJECTIVES: To assess the safety and efficacy of a Food and Drug Administration-approved pathogen-reduced platelet (PLT) product in children, as ongoing questions regarding their use in this population remain. STUDY DESIGN: We report findings from a quality assurance review of PLT utilization, associated red blood cell transfusion trends, and short-term safety of conventional vs pathogen-reduced PLTs over a 21-month period while transitioning from conventional to pathogen-reduced PLTs at a large, tertiary care hospital. We assessed utilization in neonatal intensive care unit (NICU) patients, infants 0-1 year not in the NICU, and children age 1-18 years (PED). RESULTS: In the 48 hours after an index conventional or pathogen-reduced platelet transfusion, respectively, NICU patients received 1.0 ± 1.4 (n = 91 transfusions) compared with 1.2 ± 1.3 (n = 145) additional platelet doses (P = .29); infants 0-1 year not in the NICU received 2.8 ± 3.0 (n = 125) vs 2.6 ± 2.6 (n = 254) additional platelet doses (P = .57); and PEDs received 0.9 ± 1.6 (n = 644) vs 1.4 ± 2.2 (n = 673) additional doses (P < .001). Time to subsequent transfusion and red cell utilization were similar in every group (P > .05). The number and type of transfusion reactions did not significantly vary based on PLT type and no rashes were reported in NICU patients receiving phototherapy and pathogen-reduced PLTs. CONCLUSIONS: Conventional and pathogen-reduced PLTs had similar utilization patterns in our pediatric populations. A small, but statistically significant, increase in transfusions was noted following pathogen-reduced PLT transfusion in PED patients, but not in other groups. Red cell utilization and transfusion reactions were similar for both products in all age groups.

Governance and the effectiveness of public health subsidies: Evidence from Ghana, Kenya and Uganda.

Distributing subsidized health products through existing health infrastructure could substantially and cost-effectively improve health in sub-Saharan Africa. There is, however, widespread concern that poor governance - in particular, limited health worker accountability - seriously undermines the effectiveness of subsidy programs. We audit targeted bednet distribution programs to quantify the extent of agency problems. We find that around 80% of the eligible receive the subsidy as intended, and up to 15% of subsidies are leaked to ineligible people. Supplementing the program with simple financial or monitoring incentives for health workers does not improve performance further and is thus not cost-effective in this context.

Low-Energy Ultrasound Treatment Improves Regional Tumor Vessel Infarction by Retargeted Tissue Factor.

OBJECTIVES: To enhance the regional antitumor activity of the vascular-targeting agent truncated tissue factor (tTF)-NGR by combining the therapy with low-energy ultrasound (US) treatment. METHODS: For the in vitro US exposure of human umbilical vein endothelial cells (HUVECs), cells were put in the focus of a US transducer. For analysis of the US-induced phosphatidylserine (PS) surface concentration on HUVECs, flow cytometry was used. To demonstrate the differences in the procoagulatory efficacy of TF-derivative tTF-NGR on binding to HUVECs with a low versus high surface concentration of PS, we performed factor X activation assays. For low-energy US pretreatment, HT1080 fibrosarcoma xenotransplant-bearing nude mice were treated by tumor-regional US-mediated stimulation (ie, destruction) of microbubbles. The therapy cohorts received the tumor vessel-infarcting tTF-NGR protein with or without US pretreatment (5 minutes after US stimulation via intraperitoneal injection on 3 consecutive days). RESULTS: Combination therapy experiments with xenotransplant-bearing nude mice significantly increased the antitumor activity of tTF-NGR by regional low-energy US destruction of vascular microbubbles in tumor vessels shortly before application of tTF-NGR (P < .05). Mechanistic studies proved the upregulation of anionic PS on the outer leaflet of the lipid bilayer of endothelial cell membranes by low-energy US and a consecutive higher potential of these preapoptotic endothelial cells to activate coagulation via tTF-NGR and coagulation factor X as being a basis for this synergistic activity. CONCLUSIONS: Combining retargeted tTF to tumor vessels with proapoptotic stimuli for the tumor vascular endothelium increases the antitumor effects of tumor vascular infarction. Ultrasound treatment may thus be useful in this respect for regional tumor therapy.

National UK Survey of Radiation Doses During Endovascular Aortic Interventions.

PURPOSE: Endovascular aortic repair (EAR) interventions, endovascular abdominal aortic repair (EVAR) and thoracic endovascular aortic repair (TEVAR), are associated with significant radiation exposures. We aimed to investigate the radiation doses from real-world practice and propose diagnostic reference level (DRL) for the UK. MATERIALS AND METHODS: Radiation data and essential demographics were retrospectively collected from 24 vascular and interventional radiology centres in the UK for all patients undergoing EAR-standard EVAR or complex, branched/fenestrated (BEVAR/FEVAR), and TEVAR-between 2018 and 2021. The data set was further categorised according to X-ray unit type, either fixed or mobile. The proposed national DRL is the 75th percentile of the collective medians for procedure KAP (kerma area product), cumulative air kerma (CAK), fluoroscopy KAP and CAK. RESULTS: Data from 3712 endovascular aortic procedures were collected, including 2062 cases were standard EVAR, 906 cases of BEVAR/FEVAR and 509 cases of TEVAR. The majority of endovascular procedures (3477/3712) were performed on fixed X-ray units. The proposed DRL for KAP was 162 Gy cm2, 175 Gy cm2 and 266 Gy cm2 for standard EVAR, TEVAR and BEVAR/FEVAR, respectively. CONCLUSION: The development of DRLs is pertinent to EAR procedures as the first step to optimise the radiation risks to patients and staff while maintaining the highest patient care and paving the way for steps to reduce radiation exposures.

Systemic Sclerosis dermal fibroblast exosomes trigger a Type 1 interferon response in keratinocytes through the TBK/JAK/STAT signalling axis.

BACKGROUND: Activation of Type I IFN response has been shown to correlates with disease activity in systemic sclerosis. It is currently unknown whether the tissue-specific Type I IFN activation is a consequence of the response observed in blood or rather its source. Exosomes from SSc fibroblasts were recently shown to activate macrophages in vitro. Here, we aimed to determine the source of Type I IFN signature in SSc skin biopsies and the potential role of exosomes from SSc dermal fibroblasts in the process. METHODS: Skin biopsies were obtained from healthy and SSc patients' forearms and processed for dermal fibroblasts and keratinocytes. Exosomes were isolated from healthy and SSc dermal fibroblast supernatants by ultracentrifugation and added to human skin keratinocytes. Keratinocyte transcriptome was analysed by RNA-seq analysis. TANK-binding kinase (TBK) and JAK were inhibited using a small molecule inhibitor (GSK8612) and Tofacitinib, respectively. RESULTS: SSc skin biopsies showed highest levels of Type I IFN response in the epidermal layer. RNA-seq analysis of keratinocytes transcriptome following exposure to dermal fibroblast exosomes showed strong upregulation of IFN signature genes induced by SSc exosomes compared to Healthy control. Inhibition of TBK or JAK activity suppressed the upregulation of the IFN signature induced by SSc exosomes. CONCLUSION: IFN activation of SSc keratinocytes is dependent on their crosstalk with dermal fibroblasts and inducible by extracellular exosomes. Our data indicates that SSc fibroblasts exosomes contributes to theType I IFN activation in SSc skin through activation of pattern recognition receptors upstream of TBK.

Serum CCL24 as a Biomarker of Fibrotic and Vascular Disease Severity in Systemic Sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a heterogeneous disease, characterized by variable tissue and vascular fibrosis in the context of autoimmune activation. CCL24 (or Eotaxin2) has been shown to promote microangiopathic, proinflammatory, and profibrotic processes in preclinical models of SSc. Here, we study serum CCL24 levels in a real-life cohort of patients with SSc, to determine its distribution across disease features and its value in predicting disease progression and related mortality. METHODS: Serum CCL24 was assessed in an observational cohort of consecutively enrolled patients with SSc. A high CCL24 cutoff was defined based on its distribution in a matched cohort of healthy controls. Disease progression and mortality were analyzed from the date of serum assessment. RESULTS: Two-hundred thirteen consecutively enrolled patients with SSc were included in this analysis. Median disease duration was six years (interquartile range 3-14), 28.6% of patients presented with interstitial lung disease (ILD), 46.9% had digital ulcers, and 25.3% showed high CCL24 serum concentration. High-CCL24 patients were more frequently male and positive for anti-scl-70, with a diagnosis of ILD and synovitis (P < 0.05 for all). Notably, high-CCL24 patients had lower diffusion of carbon monoxide and higher prevalence of digital ulcers, telangiectasias, and calcinosis (P < 0.05 for all). In a longitudinal setting, high CCL24 was associated with greater lung function decline and with higher disease-related mortality. CONCLUSION: Serum CCL24 is a biomarker of disease severity across fibrotic and vascular disease manifestations. These data support the development of therapies targeting CCL24 as a novel comprehensive therapeutic target in SSc.

Optimized J to T peak and T peak to T end measurements in nonclinical species administered moxifloxacin and amiodarone.

INTRODUCTION: Cardiovascular safety and the risk of developing the potentially fatal ventricular tachyarrhythmia, Torsades de Pointes (TdP), have long been major concerns of drug development. TdP is associated with a delayed ventricular repolarization represented by QT interval prolongation in the electrocardiogram (ECG), typically due to block of the potassium channel encoded by the human ether-a-go-go related gene (hERG). Importantly however, not all drugs that prolong the QT interval are torsadagenic and not all hERG blockers prolong the QT interval. Recent clinical reports suggest that partitioning the QT interval into early (J to T peak; JTp) and late repolarization (T peak to T end; TpTe) components may be valuable for distinguishing low-risk mixed ion channel blockers (hERG plus calcium and/or late sodium currents) from high-risk pure hERG channel blockers. This strategy, if true for nonclinical animal models, could be used to de-risk QT prolonging compounds earlier in the drug development process. METHODS: To explore this, we investigated JTp and TpTe in ECG data collected from telemetered dogs and/or monkeys administered moxifloxacin or amiodarone at doses targeting relevant clinical exposures. An optimized placement of the Tpeak fiducial mark was utilized, and all intervals were corrected for heart rate (QTc, JTpc, TpTec). RESULTS: Increases in QTc and JTpc intervals with administration of the pure hERG blocker moxifloxacin and an initial QTc and JTpc shortening followed by prolongation with the mixed ion channel blocker amiodarone were detected as expected, aligning with clinical data. However, anticipated increases in TpTec by both standard agents were not detected. DISCUSSION: The inability to detect changes in TpTec reduces the utility of these subintervals for prediction of arrhythmias using continuous single­lead ECGs collected from freely moving dogs and monkeys.

The ß-Secretase BACE1 Drives Fibroblast Activation in Systemic Sclerosis through the APP/ß-Catenin/Notch Signaling Axis.

BACE1 is well-known for its role in the development of Alzheimer's disease. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic diseases. The aim of this study was to investigate the role of BACE1 in the autoimmune disease systemic sclerosis (SSc). BACE1 protein levels were elevated in the skin of patients with SSc. Inhibition of BACE1 with small-molecule inhibitors or small interfering RNA blocked SSc and fibrotic stimuli-mediated fibroblast activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on ß-catenin and Notch signaling. The neurotropic factor brain-derived neurotrophic factor negatively regulates BACE1 expression and activity in dermal fibroblasts. Finally, sera from patients with SSc show higher ß-amyloid and lower brain-derived neurotrophic factor levels than healthy controls. The ability of BACE1 to regulate SSc fibroblast activation reveals a therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in clinical trials for Alzheimer's disease and could be repurposed to ameliorate fibrosis progression.

Characterization of genetic landscape and novel inflammatory biomarkers in patients with adult-onset Still's disease.

OBJECTIVES: Adult-onset Still's disease (AOSD) is systemic autoinflammatory disorder of unknown aetiology. Genetic studies have been limited. Here, we conducted detailed genetic and inflammatory biomarker analysis of a large AOSD cohort to investigate the underlying pathology and identify novel targets for potential treatment. METHODS: We investigated AOSD cases (n=60) for rare germline and somatic variants using whole exome sequencing with virtual gene panels. Transcriptome profiles were investigated by bulk RNA sequencing whole blood. Cytokine profiling was performed on an extended patient cohort (n=106), alongside measurements of NLRP3 inflammasome activation using a custom assay, and Type I Interferon (IFN) score using a novel method. RESULTS: We observed higher-than-expected frequencies of rare germline variants associated with monogenic autoinflammatory disorders in AOSD cases (AOSD 38.4% vs healthy controls 20.4%), and earlier onset of putative somatic variants associated with clonal haematopoiesis of indeterminate potential. Transcriptome profiling revealed positive correlation between Still's activity score (SAS) and gene expression associated with the innate immune system. ASC/NLRP3 specks levels and Type I IFN scores were significantly elevated in AOSD cases compared to healthy controls (p=0.0001 and 0.0015 respectively), in addition to several cytokines: IL-6 (p<0.0001), IL-10 (p<0.0075), IL-12p70 (p=0.0005), IL-18 (p<0.0001), IL-23 (p<0.0001), IFN-α2 (p=0.0009), and IFNγ (p=0.0002). CONCLUSIONS: Our study shows considerable genetic complexity within AOSD and demonstrates the potential utility of the ASC/NLRP3 specks assay for disease stratification and targeted treatment. The enriched genetic variants identified may not, by themselves, be sufficient to cause disease but may contribute to a polygenic model for AOSD.

Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation.

Deubiquitylases (DUBs) play a pivotal role in cell signalling and are often regulated by homo- or hetero-interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by selectively cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). BRCC36 is a Zn2+-dependent JAMM/MPN DUB, a challenging ubiquitin protease class for the design of selective inhibitors. We identified first-in-class DUB inhibitors that act as BRISC molecular glues (BLUEs). BLUEs inhibit DUB activity by stabilising a BRISC dimer consisting of 16 subunits. The BLUE-stabilised BRISC dimer is an autoinhibited conformation, whereby the active sites and interactions with the recruiting subunit SHMT2 are blocked. This unique mode of action leads to highly selective inhibitors for BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. Structure-guided inhibitor resistant mutants confirm BLUEs on-target activity in cells, and BLUE treatment results in reduced interferon-stimulated gene (ISG) expression in human peripheral blood mononuclear cells from Scleroderma patients, a disease linked with aberrant IFNAR1 activation. BLUEs represent a new class of molecules with potential utility in Type I interferon-mediated diseases and a template for designing selective inhibitors of large protein complexes by promoting protein-protein interactions instead of blocking them.