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Deubiquitylases (DUBs) are crucial in cell signalling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). As a Zn 2+ -dependent JAMM/MPN DUB, BRCC36 is challenging to target with selective inhibitors. We discovered first-in-class inhibitors, termed B RISC molecular g lues (BLUEs), which stabilise a 16-subunit BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit SHMT2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild type BRISC, and this effect was absent when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential new strategy to mitigate Type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting, rather than blocking, protein-protein interactions.

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., (This article is protected by copyright. All rights reserved.)

Objective: Activation of type I interferon (IFN) response has been shown to correlate with disease activity in systemic sclerosis (SSc). 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 the forearms of healthy patients and of those with SSc 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 analyzed by RNA sequencing (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 the 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 up-regulation of IFN signature genes induced by SSc exosomes compared to healthy control. Inhibition of TBK or JAK activity suppressed the up-regulation 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 indicate that SSc fibroblast exosomes contribute to the type I IFN activation in SSc skin through activation of pattern recognition receptors upstream of TBK., (© 2024 The Author(s). Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

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 β-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 β-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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2024 The Authors. Arthritis Care & Research published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Cryptococcus neoformans is a fungal pathogen responsible for >200,000 yearly cases with a mortality as high as 81%. This burden results, in part, from an incomplete understanding of its pathogenesis and ineffective antifungal treatments; hence, there is a pressing need to understand the biology and host interactions of this yeast to develop improved treatments. Protein palmitoylation is important for cryptococcal virulence, and we previously identified the substrates of its main palmitoyl transferase. One of them was encoded by the uncharacterized gene CNAG&#95;02129. In the filamentous fungus Neurospora crassa , a homolog of this gene named hyphal anastomosis protein 13 plays a role in proper cellular communication and filament fusion. In Cryptococcus , cellular communication is essential during mating; therefore, we hypothesized that CNAG&#95;02129, which we named hyphal anastomosis protein 1 ( HAM1 ), may play a role in mating. We found that ham1 Δ mutants produce more fusion products during mating, filament more robustly, and exhibit competitive fitness defects under mating and non-mating conditions. Additionally, we found several differences with the major virulence factor, the polysaccharide capsule, that may affect virulence, consistent with prior studies linking virulence to mating. We observed that ham1 Δ mutants have decreased capsule attachment and transfer but exhibit higher amounts of exopolysaccharide shedding and biofilm production. Finally, HAM1 expression is significantly lower in mating media relative to non-mating conditions, consistent with it acting as a negative regulator of mating. Understanding the connection between mating and virulence in C. neoformans may open new avenues of investigation into ways to improve the treatment of this disease., Importance: Fungal mating is a vital part of the lifecycle of the pathogenic yeast Cryptococcus neoformans . More than just ensuring the propagation of the species, mating allows for sexual reproduction to occur and generates genetic diversity as well as infectious propagules that can invade mammalian hosts. Despite its importance in the biology of this pathogen, we still do not know all of the major players regulating the mating process and if they are involved or impact its pathogenesis. Here, we identified a novel negative regulator of mating that also affects certain cellular characteristics known to be important for virulence. This gene, which we call HAM1 , is widely conserved across the cryptococcal family as well as in many pathogenic fungal species. This study will open new avenues of exploration regarding the function of uncharacterized but conserved genes in a variety of pathogenic fungal species and specifically in serotype A of C. neoformans ., Competing Interests: The authors declare no conflict of interest.

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for >150,000 deaths every year with a mortality rate as high as 81%. This high medical burden is due, in part, to an incomplete understanding of its pathogenesis. In a previous study, we identified a cryptococcal atypical pleiotropic drug resistance (PDR) transporter, PDR6 , that regulated antifungal resistance and host interactions. Here, we follow-up on the role of PDR6 in cryptococcal virulence. In vivo , mice infected with the pdr6 Δ strain display altered symptomatology and disease progression. Specifically, we observed a significant increase in the innate immune cell populations in the pdr6 Δ-infected mice when compared to their WT-infected littermates. Furthermore, quantification of pulmonary cytokines/chemokines revealed a robust increase of pro-inflammatory cytokines in mice infected with the pdr6 Δ mutant strain. Whereas antifungal treatment of pdr6 Δ-infected animals did not affect survival, treatment with a corticosteroid significantly extended survival, highlighting the importance of a balanced/controlled host immune response. We determined that the hyper-inflammatory immune response occurs, in part, because the loss of the Pdr6 transporter indirectly alters the cryptococcal cell wall architecture and results in the increased exposure of chitin, β-glucan, and other cryptococcal-specific pathogen associated molecular patterns. Taken together, this study provides clinical insights regarding cryptococcal pathogenesis while also providing additional functions of PDR-type ATP-binding cassette (ABC) transporters in pathogenic fungi.

Although fungi have been important model organisms for solving genetic, molecular, and ecological problems, recently, they are also becoming an important source of infectious disease. Despite their high medical burden, fungal pathogens are understudied, and relative to other pathogenic microbes, less is known about how their gene functions contribute to disease. This is due, in part, to a lack of powerful genetic tools to study these organisms. In turn, this has resulted in inappropriate treatments and diagnostics and poor disease management. There are a variety of reasons genetic studies were challenging in pathogenic fungi, but in recent years, most of them have been overcome or advances have been made to circumvent these barriers. In this minireview, we highlight how recent advances in genetic studies in fungal pathogens have resulted in the discovery of important biology and potential new antifungals and have created the tools to comprehensively study these important pathogens., Competing Interests: The authors declare no conflict of interest.

The role of post-transcriptional modification in biological processes has been an ongoing field of study for several decades. Improvements in liquid chromatography platforms and mass spectrometry instrumentation have resulted in the enhanced identification, characterization, and quantification of modified nucleosides in biological systems. One consequence of the rapid technological improvements in the analytical acquisition of modified nucleosides has been a dearth of robust data processing workflows for analyzing more than a handful of samples at a time. To improve the utility of LC-MS/MS for batch analyses of modified nucleosides, a workflow for automated nucleoside identification has been developed. We adapted the Thermo Fisher Scientific metabolomics identification software package, Compound Discoverer, to accurately identify modified nucleosides from batch LC-MS/MS acquisitions. Three points of identification are used: accurate mass from a monoisotopic mass list, spectral matching from a spectral library, and neutral loss identification. This workflow was applied to a batch ( n = 24) of urinary nucleosides, resulting in the accurate identification and relative quantification of 16 known nucleosides in less than 1 h.

This article describes a rare case of a traumatic splenic arteriovenous fistula (AVF) causing portal hypertension in a patient presenting with abdominal pain, diarrhea, and melena. A 78-year-old was admitted to the hospital with abdominal pain. The patient's history was notable for prior laparotomy and left nephrectomy for a gunshot wound. Workup demonstrated portal hypertension with a dilated splenic vein with aneurysmal changes and a saccular celiac artery aneurysm. Celiac angiogram demonstrated a communication between celiac and portal circulation. The patient underwent laparotomy with ligation of the splenic artery and resection of the celiac artery aneurysm. In conclusion, splenic AVFs are relatively rare in clinical practice. Once the diagnosis is established, operative intervention is required to avoid complications of portal hypertension. Surgical ligation has been used in this case with a successful outcome., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that N 6 -methyladenosine (m 6 A) RNA methylation by Mettl3 is required for developmental pausing in mouse blastocysts and embryonic stem (ES) cells. Mettl3 enforces transcriptional dormancy through two interconnected mechanisms: (1) it promotes global mRNA destabilization and (2) it suppresses global nascent transcription by destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a crucial anti-pausing factor. Knockdown of N-Myc rescues pausing in Mettl3 -/- ES cells, and forced demethylation and stabilization of Mycn mRNA in paused wild-type ES cells largely recapitulates the transcriptional defects of Mettl3 -/- ES cells. These findings uncover Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during developmental pausing, with implications for dormancy in adult stem cells and cancer., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle 1-4 . Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAs Glu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNA Trp CCA from five to four base pairs (bp). The canonical 5-bp tRNA Trp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Decision aids for breast cancer screening are increasingly being used by physicians, but the association between physician practice decision-aid use and mammography rates remains uncertain. Using national data, this study examines the association between practice-level decision-aid use and mammography use among older women., Methods: Physician practice responses to the 2017/2018 National Survey of Healthcare Organizations and Systems (n=1,236) were linked to 2016 and 2017 Medicare fee-for-service beneficiary data from eligible beneficiaries (n=439,684) aged 65-74 years. In 2021, multivariable generalized linear models estimated the association of practice decision-aid use for breast cancer screening and advanced health information technology functions with mammography use, controlling for practice and beneficiary characteristics., Results: Overall, 60.1% of eligible beneficiaries had a screening mammogram, and 37.3% of physician practices routinely used decision aids for breast cancer screening. In adjusted analyses, advanced health information technology functions (OR=1.19, p=0.04) were associated with mammography use, but practice use of decision aids was not (OR=0.95, p=0.21). Beneficiary clinical and socioeconomic characteristics, including race, comorbidities, Medicare and Medicaid eligibility, and median household income were more strongly associated with mammography use than practice-level decision-aid use or advanced health information technology functions., Conclusions: Health information technology‒enabled automation of mammography reminders and other advanced health information technology functions may support mammography, whereas breast cancer decision aids may reduce patients' propensities to be screened through the alignment of their preferences and screening decision. More resources may be needed for decision aids to be routinely implemented to improve solicitation of patient preferences and targeting of mammography services., (Copyright © 2022 American Journal of Preventive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Activation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc)., Methods: The effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-β (TGF-β)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed., Results: Prophylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-β-induced ACTA2 (encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117., Conclusions: MT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress., (© 2022. The Author(s).)

Systemic sclerosis (SSc) is a terminal disease characterized by vasculopathy, tissue fibrosis, and autoimmunity. Although the exact etiology of SSc remains unknown, endothelial dysfunction, oxidative stress, and calcium handling dysregulation have been associated with a large number of SSc-related complications such as neointima formation, vasculogenesis, pulmonary arterial hypertension, impaired angiogenesis, and cardiac arrhythmias. Hemeoxygenase-1 (HO-1) is an antioxidant enzyme involved in multiple biological actions in the cardiovascular system including vascular tone, angiogenesis, cellular proliferation, apoptosis, and oxidative stress. The aim of this work was to investigate the physiological role of HO-1 and its relevance in the cardiovascular complications occurring in SSc. We found that, in early phases of SSc, the expression of HO-1 in dermal fibroblast is lower compared to those isolated from healthy control individuals. This is particularly relevant as reduction of the HO-1/CO signaling pathway is associated with endothelial dysfunction and vasculopathy. We show evidence of the role of HO-1/carbon monoxide (CO) signaling pathway in calcium handling. Using an in vitro model of pulmonary arterial hypertension (PAH) we investigated the role of HO-1 in Ca 2+ mobilization from intracellular stores. Our results indicate that HO-1 regulates calcium release from intracellular stores of human pulmonary arterial endothelial cells. We interrogated the activity of HO-1 in angiogenesis using an organotypic co-culture of fibroblast-endothelial cell. Inhibition of HO-1 significantly reduced the ability of endothelial cells to form tubules. We further investigated if this could be associated with cell motility or migration of endothelial cells into the extracellular matrix synthesized by fibroblasts. By mean of holographic imaging, we studied the morphological and functional features of endothelial cells in the presence of an HO-1 activator and selective inhibitors. Our results demonstrate that inhibition of HO-1 significantly reduces cell proliferation and cell motility (migration) of cultured endothelial cells, whilst activation of HO-1 does not modify either morphology, proliferation or motility. In addition, we investigated the actions of CO on the Kv7.1 (KCQN1) channel current, an important component of the cardiac action potential repolarization. Using electrophysiology (whole-cell patch-clamp in a recombinant system overexpressing the KCQN1 channel), we assessed the regulation of KCQN1 by CO. CORM-2, a CO donor, significantly reduced the Kv7.1 current, suggesting that HO-1/CO signaling may play a role in the modulation of the cardiac action potential via regulation of this ion channel. In summary, our results indicate a clear link between: 1) downregulation of HO-1/CO signaling; and 2) pathophysiological processes occurring in early phases of SSc, such as calcium homeostasis dysregulation, impaired angiogenesis and cardiac arrhythmias. A better understanding of the canonical actions (mainly due to the biological actions of CO), and non-canonical actions of HO-1, as well as the interaction of HO-1/CO signaling with other gasotransmitters in SSc will contribute to the development of novel therapeutic approaches., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ross, Mavria, Del Galdo and Elies.)

Objectives: To assess antibody and T cell responses to SARS-CoV-2 vaccination in patients with rheumatoid arthritis (RA) on disease-modifying antirheumatic drugs (DMARDs)., Methods: This prospective study recruited 100 patients with RA on a variety of DMARDs for antibody and T cell analysis, pre-vaccination and 4 weeks post-vaccination. Positive antibody response was defined as sera IgG binding to ≥1 antigen. Those that remained seronegative after first vaccination were retested 4 weeks after second vaccination; and if still seronegative after vaccination three. A T cell response was defined an ELISpot count of ≥7 interferon (IFN)γ-positive cells when exposed to spike antigens. Type I IFN activity was determined using the luminex multiplex assay IFN score., Results: After vaccine one, in patients without prior SARS-CoV-2 exposure, 37/83 (45%) developed vaccine-specific antibody responses, 44/83 (53%) vaccine-specific T cell responses and 64/83 (77%) developed either antibody or T cell responses. Reduced seroconversion was seen with abatacept, rituximab (RTX) and those on concomitant methotrexate (MTX) compared to 100% for healthy controls (p<0.001). Better seroconversion occurred with anti-tumour necrosis factor (TNF) versus RTX (p=0.012) and with age ≤50 (p=0.012). Pre-vaccine SARS-CoV-2 exposure was associated with higher quantitative seroconversion (≥3 antibodies) (p<0.001). In the subgroup of non-seroconverters, a second vaccination produced seroconversion in 54% (19/35), and after a third in 20% (2/10). IFN score analysis showed no change post-vaccine., Conclusion: Patients with RA on DMARDs have reduced vaccine responses, particularly on certain DMARDs, with improvement on subsequent vaccinations but with approximately 10% still seronegative after three doses., Competing Interests: Competing interests: BS: Speaker fees for Pfizer and Galapagos, L-AB: Honoraria for UCB, Abbvie and Galapagos, KM: Personal: Abbvie, Lilly, UCB.Grants: Lilly, Gilead, FD: received research support and consultancies, not related to the topic of this manuscript from ABBVIE, AstraZeneca, Boehringer-Ingelheim, Capella, Chemomab, Kymab, Mitsubishi-TanabePaul Emery: Grants : AbbVie, BMS, Lilly, Samsung. Consulting fees: BMS, AbbVie, MSD, Pfizer, Novartis, and Roche. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events:Abbvie, Gilead, Lilly, Novartis., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for 'normal' growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Secreted Frizzled Receptor Protein 4 (SFRP4) has been shown to be increased in Scleroderma (SSc). To determine its role in immune-driven fibrosis, we analysed SSc and sclerotic Chronic Graft Versus Host Disease (sclGVHD) biosamples; skin biopsies ( n = 24) from chronic GVHD patients (8 with and 5 without sclGVHD), 8 from SSc and 3 healthy controls (HC) were analysed by immunofluorescence (IF) and SSc patient sera ( n = 77) assessed by ELISA. Epithelial cell lines used for in vitro Epithelial-Mesenchymal-Transition (EMT) assays and analysed by Western Blot, RT-PCR and immunofluorescence. SclGVHD skin biopsies resembled pathologic features of SSc. IF of fibrotic skin biopsies indicated the major source of SFRP4 expression were dermal fibroblasts, melanocytes and vimentin positive/caveolin-1 negative cells in the basal layer of the epidermis. In vitro studies showed increased vimentin and SFRP4 expression accompanied with decreased caveolin-1 expression during TGFβ-induced EMT. Additionally, SFRP4 serum concentration correlated with severity of lung and skin fibrosis in SSc. In conclusion, SFRP4 expression is increased during skin fibrosis in two different immune-driven conditions, and during an in vitro EMT model. Its serum levels correlate with skin and lung fibrosis in SSc and may function as biomarker of EMT. Further studies are warranted to elucidate the role of SFRP4 in EMT within the pathogenesis of tissue fibrosis.

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-β 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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems. However, since a lack of tRNA genes in T. brucei mitochondria makes it essential to import a complete set from the cytoplasm, we surprisingly found that Q-modified tRNAs are preferentially imported over their unmodified counterparts. In turn, their absence from mitochondria has a pronounced effect on organellar translation and affects function. Although Q modification in T. brucei is globally important for codon selection, it is more so for mitochondrial protein synthesis. These results provide a unique example of the combined regulatory effect of codon usage and wobble modifications on protein synthesis; all driven by tRNA intracellular transport dynamics., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Competing interests: SH is an employee of Capella Bioscience, which holds a patent for CBS004 (GB1911188.9)., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N 6 -methyladenosine (m 6 A), has been shown to play a role in cancer, but its contribution to tumor progression and the underlying mechanisms remain unclear. Here, we report widespread FTO downregulation in epithelial cancers associated with increased invasion, metastasis and worse clinical outcome. Both in vitro and in vivo, FTO silencing promotes cancer growth, cell motility and invasion. In human-derived tumor xenografts (PDXs), FTO pharmacological inhibition favors tumorigenesis. Mechanistically, we demonstrate that FTO depletion elicits an epithelial-to-mesenchymal transition (EMT) program through increased m 6 A and altered 3'-end processing of key mRNAs along the Wnt signaling cascade. Accordingly, FTO knockdown acts via EMT to sensitize mouse xenografts to Wnt inhibition. We thus identify FTO as a key regulator, across epithelial cancers, of Wnt-triggered EMT and tumor progression and reveal a therapeutically exploitable vulnerability of FTO-low tumors., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Patients with complex health care needs may suffer adverse outcomes from fragmented and delayed care, reducing well-being and increasing health care costs. Health reform efforts, especially those in primary care, attempt to mitigate risk of adverse outcomes by better targeting resources to those most in need. However, predicting who is susceptible to adverse outcomes, such as unplanned hospitalizations, ED visits, or other potentially avoidable expenditures, can be difficult, and providing intensive levels of resources to all patients is neither wanted nor efficient. Our objective was to understand if primary care teams can predict patient risk better than standard risk scores., Methods: Six primary care practices risk stratified their entire patient population over a 2-year period, and worked to mitigate risk for those at high risk through care management and coordination. Individual patient risk scores created by the practices were collected and compared to a common risk score (Hierarchical Condition Categories) in their ability to predict future expenditures, ED visits, and hospitalizations. Accuracy of predictions, sensitivity, positive predictive values (PPV), and c-statistics were calculated for each risk scoring type. Analyses were stratified by whether the practice used intuition alone, an algorithm alone, or adjudicated an algorithmic risk score., Results: In all, 40,342 patients were risk stratified. Practice scores had 38.6% agreement with HCC scores on identification of high-risk patients. For the 3,381 patients with reliable outcomes data, accuracy was high (0.71-0.88) but sensitivity and PPV were low (0.16-0.40). Practice-created scores had 0.02-0.14 lower sensitivity, specificity and PPV compared to HCC in prediction of outcomes. Practices using adjudication had, on average, .16 higher sensitivity., Conclusions: Practices using simple risk stratification techniques had slightly worse accuracy in predicting common outcomes than HCC, but adjudication improved prediction.

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.

Objectives: Systemic sclerosis (SSc) is characterised by tissue fibrosis of the major organs of the body including the skin, lungs and heart. We have previously reported that the lncRNA HOTAIR plays a central role in the activation of SSc myofibroblasts, the key cellular elements of fibrosis. HOTAIR induces fibroblast activation through H3K27me3-mediated activation of the Notch signalling pathway. Here we aimed to identify the signalling events downstream of Notch that drive SSc myofibroblast activation., Methods: Patient fibroblasts were obtained from full-thickness forearm skin biopsies of 3 adult patients with SSc of recent onset. The lncRNA HOTAIR was expressed in healthy dermal fibroblasts by lentiviral transduction. Hedgehog signalling pathway was inhibited with GANT61 and GLI2 siRNA. Gamma secretase inhibitors RO4929097 and DAPT were used to block Notch signalling. GSK126 was used to inhibit Enhancer of Zeste 2 (EZH2)., Results: Overexpression of HOTAIR in dermal fibroblasts induced the expression of the Hedgehog pathway transcription factor GLI2. This is mediated by activation of Notch signalling following epigenetic downregulation of miRNA-34a expression. Inhibition of H3K27 methylation and Notch signalling reduced expression of GLI2 in HOTAIR-expressing fibroblasts as well as in SSc dermal fibroblasts. Importantly, the inhibition of GLI2 function using GANT61 or siRNA mitigates the pro-fibrotic phenotype induced by HOTAIR., Conclusions: Our data indicates that GLI2 expression is stably upregulated in SSc myofibroblasts through HOTAIR and that GLI2 mediates the expression of pro-fibrotic markers downstream of Notch.

Every type of nucleic acid in cells may undergo some kind of post-replicative or post-transcriptional chemical modification. Recent evidence has highlighted their importance in biology and their chemical complexity. In the following pages, we will describe new discoveries of modifications, with a focus on tRNA and mRNA. We will highlight current challenges and advances in modification detection and we will discuss how changes in nucleotide post-transcriptional modifications may affect cell homeostasis leading to malfunction. Although, RNA modifications prevail in all forms of life, the present review will focus on eukaryotic systems, where the great degree of intracellular compartmentalization provides barriers and filters for the level at which a given RNA is modified and will of course affect its fate and function. Additionally, although we will mention rRNA modification and modifications of the mRNA 5'-CAP structure, this will only be discussed in passing, as many substantive reviews have been written on these subjects. Here we will not spend much time describing all the possible modifications that have been observed; truly a daunting task. For reference, Bujnicki and coworkers have created MODOMICS, a useful repository for all types of modifications and their associated enzymes. Instead we will discuss a few examples, which illustrate our arguments on the connection of modifications, metabolism and ultimately translation. The fact remains, a full understanding of the long reach of nucleic acid modifications in cells requires both a global and targeted study of unprecedented scale, which at the moment may well be limited only by technology.

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Extensive knowledge of both the nature and position of tRNA modifications in all cellular tRNAs has been limited to two bacteria, Escherichia coli and Mycoplasma capricolum . Bacillus subtilis sp subtilis strain 168 is the model Gram-positive bacteria and the list of the genes involved in tRNA modifications in this organism is far from complete. Mass spectrometry analysis of bulk tRNA extracted from B. subtilis , combined with next generation sequencing technologies and comparative genomic analyses, led to the identification of 41 tRNA modification genes with associated confidence scores. Many differences were found in this model Gram-positive bacteria when compared to E. coli . In general, B. subtilis tRNAs are less modified than those in E. coli , even if some modifications, such as m 1 A22 or ms 2 t 6 A, are only found in the model Gram-positive bacteria. Many examples of non-orthologous displacements and of variations in the most complex pathways are described. Paralog issues make uncertain direct annotation transfer from E. coli to B. subtilis based on homology only without further experimental validation. This difficulty was shown with the identification of the B. subtilis enzyme that introduces ψ at positions 31/32 of the tRNAs. This work presents the most up to date list of tRNA modification genes in B. subtilis , identifies the gaps in knowledge, and lays the foundation for further work to decipher the physiological role of tRNA modifications in this important model organism and other bacteria., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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., Competing Interests: Competing interests: H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, and advisor to 10x Genomics, Arsenal Biosciences, and Spring Discovery., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Primary care risk stratification (RS) has been shown to help practices better understand their patient populations' needs and may improve health outcomes and reduce expenditures by targeting and tailoring care to high-need patients. This study aims to understand key considerations practices faced and practice experiences as they began to implement RS models., Methods: We conducted semistructured interviews about experiences in RS with 34 stakeholders from 15 primary care practices in Oregon and Colorado and qualitatively analyzed the data., Results: Three decisions were important in shaping practices' experiences with RS: choosing established versus self-created algorithms or heuristics, clinical intuition, or a combination; selecting mechanisms for assigning risk scores; determining how to integrate RS approaches into care delivery. Practices using clinical intuition found stratification time-consuming and difficult to incorporate into existing workflows, but trusted risk scores more than those using algorithms. Trust in risk scores was influenced by data extraction capabilities; practices often lacked sufficient data to calculate their perceived optimal risk score. Displaying the scores to the care team was a major issue. Finally, obtaining buy-in from care team members was challenging, requiring repeated cycles of improvement and workflow integration., Discussion: Practices used iterative approaches to RS implementation. As a result, procedural and algorithmic changes were introduced and were influenced by practices' health IT, staffing, and resource capacities. Practices were most successful when able to make iterative changes to their approaches, incorporated both automation and human process in RS, educated staff on the importance of RS, and had readily accessible risk scores., Competing Interests: Conflict of interest: none declared., (© Copyright 2019 by the American Board of Family Medicine.)

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.

Introduction: Like most patient-centered medical home (PCMH) models, Oregon's program, the Patient-Centered Primary Care Home (PCPCH), aims to improve care while reducing costs; however, previous work shows that PCMH models do not uniformly achieve desired outcomes. Our objective was to describe a process for refining PCMH models to identify high value elements (HVEs) that reduce cost and utilization., Methods: We performed a targeted literature review of each PCPCH core attribute. Value-related concepts and their metrics were abstracted, and studies were assessed for relevance and strength of evidence. Focus groups were held with stakeholders and patients, and themes related to each attribute were identified; calculation of HVE attainment versus PCPCH criteria were completed on eight primary care clinics. Analyses consisted of descriptive statistics and criterion validity with stakeholder input., Results: 2,126 abstracts were reviewed; 22 met inclusion criteria. From these articles and focus groups of stakeholders/experts (n = 49; 4 groups) and patients (n = 7; 1 group), 12 HVEs were identified that may reduce cost and utilization. At baseline, clinics achieved, on average, 31.3 percent HVE levels compared to an average of 87.9 percent of the 35 PCMH measures., Discussion: A subset of measures from the PCPCH model were identified as "high value" in reducing cost and utilization. HVE performance was significantly lower than standard measures, and may better calibrate clinic ability to reduce costs., Conclusion: Through literature review and stakeholder engagement, we created a novel set of high value elements for advanced primary care likely to be more related to cost and utilization than other models., Competing Interests: The authors have no competing interests to declare.

A small set of ribonucleoside modifications have been found in different regions of mRNA including the open reading frame. Accurate detection of these specific modifications is critical to understanding their modulatory roles in facilitating mRNA maturation, translation and degradation. While transcriptome-wide next-generation sequencing (NGS) techniques could provide exhaustive information about the sites of one specific or class of modifications at a time, recent investigations strongly indicate cautionary interpretation due to the appearance of false positives. Therefore, it is suggested that NGS-based modification data can only be treated as predicted sites and their existence need to be validated by orthogonal methods. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an analytical technique that can yield accurate and reproducible information about the qualitative and quantitative characteristics of ribonucleoside modifications. Here, we review the recent advancements in LC-MS/MS technology that could help in securing accurate, gold-standard quality information about the resident post-transcriptional modifications of mRNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications., (© 2018 Sarin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Galectins are carbohydrate binding proteins (lectins), which characteristically bind β-galactosides. Galectins play a role in tumour progression through involvement in proliferation, metastasis, angiogenesis, immune evasion and drug resistance. There is need for inhibitors (antagonists) that are specific for distinct galectins and that can interfere with galectin-carbohydrate interactions during cancer progression. Here, we propose that lactulose, a non-digestible galactose-fructose disaccharide, presents a novel inhibitor scaffold for design of inhibitors against galectins. Thermodynamic evaluation displays binding affinity of lactulose against the galectin-1 and galectin-3 carbohydrate recognition domain (CRD). Crystal structures of galectin-1 and galectin-3 in complex with lactulose reveal for the first time the molecular basis of the galectin-lactulose interactions. Molecular modelling was implemented to propose novel lactulose derivatives as potent anti-cancer agents., (© 2018 John Wiley & Sons A/S.)

Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.

The analytical identification of positional isomers (e.g., 3-, N 4 -, 5-methylcytidine) within the > 160 different post-transcriptional modifications found in RNA can be challenging. Conventional liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approaches rely on chromatographic separation for accurate identification because the collision-induced dissociation (CID) mass spectra of these isomers nearly exclusively yield identical nucleobase ions (BH 2 + ) from the same molecular ion (MH + ). Here, we have explored higher-energy collisional dissociation (HCD) as an alternative fragmentation technique to generate more informative product ions that can be used to differentiate positional isomers. LC-MS/MS of modified nucleosides characterized using HCD led to the creation of structure- and HCD energy-specific fragmentation patterns that generated unique fingerprints, which can be used to identify individual positional isomers even when they cannot be separated chromatographically. While particularly useful for identifying positional isomers, the fingerprinting capabilities enabled by HCD also offer the potential to generate HPLC-independent spectral libraries for the rapid analysis of modified ribonucleosides. Graphical Abstract ᅟ.

Objective: Aberrant activation of Wnt signaling has been observed in tissues from patients with systemic sclerosis (SSc). This study aimed to determine the role of transforming growth factor β (TGFβ) in driving the increased Wnt signaling, through modulation of axis inhibition protein 2 (Axin-2), a critical regulator of the Wnt canonical pathway., Methods: Canonical Wnt signaling activation was analyzed by TOPflash T cell factor/lymphoid enhancer factor promoter assays. Axin-2 was evaluated in vitro by analysis of Axin-2 primary/mature transcript expression and decay, TGFβ receptor type I (TGFβRI) blockade, small interfering RNA-mediated depletion of tristetraprolin 1, and XAV-939-mediated Axin-2 stabilization. In vivo, Axin-2 messenger RNA (mRNA) and protein expression was determined in skin and lung biopsy samples from mice that express a kinase-deficient TGFβRII specifically on fibroblasts (TβRIIΔk-fib-transgenic mice) and from littermate controls., Results: SSc fibroblasts displayed an increased response to canonical Wnt ligands despite basal levels of Wnt signaling that were comparable to those in healthy control fibroblasts in vitro. Notably, we showed that SSc fibroblasts had reduced basal expression of Axin-2, which was caused by an endogenous TGFβ-dependent increase in Axin-2 mRNA decay. Accordingly, we observed that TGFβ decreased Axin-2 expression both in vitro in healthy control fibroblasts and in vivo in TβRIIΔk-fib-transgenic mice. Additionally, using Axin-2 gain- and loss-of-function experiments, we demonstrated that the TGFβ-induced increased response to Wnt activation characteristic of SSc fibroblasts depended on reduced bioavailability of Axin-2., Conclusion: This study highlights the importance of reduced bioavailability of Axin-2 in mediating the increased canonical Wnt response observed in SSc fibroblasts. This novel mechanism extends our understanding of the processes involved in Wnt/β-catenin-driven pathology and supports the rationale for targeting the TGFβ pathway to regulate the aberrant Wnt signaling observed during fibrosis., (© 2018, American College of Rheumatology.)

The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.

Clinical quality measures (CQMs) aim to identify gaps in care and to promote evidence-based guidelines. Official CQM definitions consist of a measure's logic and grouped, standardized codes to define key concepts. In this study, we used the official CQM update process to understand how CQMs' meanings change over time. First, we identified differences between the narrative description, logic, and the vocabulary specifications offour standardized CQMs' definitions in subsequent versions (2015, 2016, and 2017). Next, we implemented the various versions in a quality measure calculation registry to understand how the differences affected calculated prevalence of risk and measure performance. Global performance rates changed up to 5.32%, and an increase of up to 28% new patients was observed for key conditions between versions. Updates to definitions that change a measure's logic and choices to include/exclude codes in value set vocabularies changes measurement of quality and likely introduces variation by implementation.

Risk stratification (RS) in primary care is frequently used by policy-makers, payers, and health systems; the process requires risk assessment for adverse health outcomes across a population to assign patients into risk tiers and allow care management (CM) resources to be targeted effectively. Our objective was to understand the approach to and perception of RS in primary care practices. An online survey was developed, tested, and administered to 148 representatives of 37 primary care practices engaged in RS varying in size, location and ownership. The survey assessed practices' approach to, perception of, and confidence in RS, and its effect on subsequent CM activities. We examined psychometric properties of the survey to determine validity and conducted chi-square analyses to determine the association between practice characteristics and confidence and agreement with risk scores. The survey yielded a 68% response rate (100 respondents). Overall, participants felt moderately confident in their risk scores (range 41-53.8%), and moderately to highly confident in their subsequent CM workflows (range 46-68%). Respondents from small and independent practices were more likely to have higher confidence and agreement with their RS approaches and scores ( p < 0.01). Confidence levels were highest, however, when practices incorporated human review into their RS processes ( p < 0.05). This trend was not affected by respondents' professional roles. Additional work from a broad mixed-methods effort will add to our understanding of RS implementation processes and outcomes., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.