RIG-I Uses an ATPase-Powered Translocation-Throttling Mechanism for Kinetic Proofreading of RNAs and Oligomerization.

RIG-I has a remarkable ability to specifically select viral 5'ppp dsRNAs for activation from a pool of cytosolic self-RNAs. The ATPase activity of RIG-I plays a role in RNA discrimination and activation, but the underlying mechanism was unclear. Using transient-state kinetics, we elucidated the ATPase-driven "kinetic proofreading" mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T cell receptors. Even in the autoinhibited state of RIG-I, the C-terminal domain kinetically discriminates against self-RNAs by fast off rates. ATP binding facilitates dsRNA engagement but, interestingly, makes RIG-I promiscuous, explaining the constitutive signaling by Singleton-Merten syndrome-linked mutants that bind ATP without hydrolysis. ATP hydrolysis dissociates self-RNAs faster than 5'ppp dsRNA but, more importantly, drives RIG-I oligomerization through translocation, which we show to be regulated by helicase motif IVa. RIG-I translocates directionally from the dsRNA end into the stem region, and the 5'ppp end "throttles" translocation to provide a mechanism for threading and building a signaling-active oligomeric complex.

DDX58 variant triggers IFN-ß-induced autophagy in trabecular meshwork and influences intraocular pressure.

Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.

A loosened gating mechanism of RIG-I leads to autoimmune disorders.

DDX58 encodes RIG-I, a cytosolic RNA sensor that ensures immune surveillance of nonself RNAs. Individuals with RIG-IE510V and RIG-IQ517H mutations have increased susceptibility to Singleton-Merten syndrome (SMS) defects, resulting in tissue-specific (mild) and classic (severe) phenotypes. The coupling between RNA recognition and conformational changes is central to RIG-I RNA proofreading, but the molecular determinants leading to dissociated disease phenotypes remain unknown. Herein, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) and single molecule magnetic tweezers (MT) to precisely examine how subtle conformational changes in the helicase insertion domain (HEL2i) promote impaired ATPase and erroneous RNA proofreading activities. We showed that the mutations cause a loosened latch-gate engagement in apo RIG-I, which in turn gradually dampens its self RNA (Cap2 moiety:m7G cap and N1-2-2'-O-methylation RNA) proofreading ability, leading to increased immunopathy. These results reveal HEL2i as a unique checkpoint directing two specialized functions, i.e. stabilizing the CARD2-HEL2i interface and gating the helicase from incoming self RNAs; thus, these findings add new insights into the role of HEL2i in the control of antiviral innate immunity and autoimmunity diseases.

DDX58(RIG-I)-related disease is associated with tissue-specific interferon pathway activation.

BACKGROUND: Singleton-Merten syndrome (SGMRT) is a rare immunogenetic disorder that variably features juvenile open-angle glaucoma (JOAG), psoriasiform skin rash, aortic calcifications and skeletal and dental dysplasia. Few families have been described and the genotypic and phenotypic spectrum is poorly defined, with variants in DDX58 (DExD/H-box helicase 58) being one of two identified causes, classified as SGMRT2. METHODS: Families underwent deep systemic phenotyping and exome sequencing. Functional characterisation with in vitro luciferase assays and in vivo interferon signature using bulk and single cell RNA sequencing was performed. RESULTS: We have identified a novel DDX58 variant c.1529A>T p.(Glu510Val) that segregates with disease in two families with SGMRT2. Patients in these families have widely variable phenotypic features and different ethnic background, with some being severely affected by systemic features and others solely with glaucoma. JOAG was present in all individuals affected with the syndrome. Furthermore, detailed evaluation of skin rash in one patient revealed sparse inflammatory infiltrates in a unique distribution. Functional analysis showed that the DDX58 variant is a dominant gain-of-function activator of interferon pathways in the absence of exogenous RNA ligands. Single cell RNA sequencing of patient lesional skin revealed a cellular activation of interferon-stimulated gene expression in keratinocytes and fibroblasts but not in neighbouring healthy skin. CONCLUSIONS: These results expand the genotypic spectrum of DDX58-associated disease, provide the first detailed description of ocular and dermatological phenotypes, expand our understanding of the molecular pathogenesis of this condition and provide a platform for testing response to therapy.

Single-incision drilling technique to achieve hemiepiphysiodesis of the distal metacarpus - complications and outcome in 207 foals with metacarpophalangeal varus deformities.

OBJECTIVE: To report the outcome of foals treated for metacarpophalangeal varus deformity with a single-incision drilling technique for hemiepiphysiodesis of the distal lateral metacarpal physis. STUDY DESIGN: Retrospective case-control cohort study. ANIMALS: Thoroughbred foals (n = 207), 171 age- and sex-matched maternal siblings. METHODS: Medical records (2017-2020) were reviewed for signalment, limb(s) treated, location of the surgery, and any reported complications. Follow-up radiographs obtained for the yearling sale were assessed for abnormalities. Horses were matched to maternal siblings using an online database. Sales and racing performance data were compared between cohorts. RESULTS: The average age at the time of surgery was 97 days. The treated limb was the left front in 52, right front in 31, both fronts in 119, unknown in 5. Three horses developed calcinosis circumscripta lesions adjacent to the physis, which were removed successfully. No radiographic abnormalities associated with the surgery site were detected on yearling prepurchase radiographs. There were no differences in sales and racing performance data between treated horses and maternal controls. CONCLUSION: Hemiepiphysiodesis is a safe and effective treatment for metacarpophalangeal varus deformities in foals. No negative effect on sales or racing performance was identified. CLINICAL SIGNIFICANCE: This technique avoids risks, costs, and the need for second surgery associated with an orthopedic implant. The surgeon should be aware of the potential for development of a calcinosis circumscripta lesion with this technique.

Psoriasis-like skin disorder in transgenic mice expressing a RIG-I Singleton-Merten syndrome variant.

Mutations in DDX58 (DExD/H-box helicase 58), which encodes the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I), were recently identified in the rare autoimmune disease Singleton-Merten syndrome (SMS). We report the spontaneous development of psoriasis-like skin lesions as an SMS-like symptom in transgenic mice harboring one of the RIG-I SMS variants, E373A. Histological analysis revealed typical characteristics of psoriasis, including the abnormal proliferation and differentiation of keratinocytes leading to epidermal hyperplasia, and infiltrates consisting of neutrophils, dendritic cells and T cells. Levels of the IL-23/IL-17 immune axis cytokines were high in the skin lesions. Rag2-/- transgenic mice showed partial amelioration of the phenotype, with down-regulation of inflammatory cytokines, including IL-17A, suggesting the importance of lymphocytes for the pathogenesis similar to that of human psoriasis. Of note, IL-17A deficiency abolished the skin phenotype, and treatment using the JAK inhibitor tofacitinib not only prevented onset, but also improved the skin manifestations even after onset. Our study provides further evidence for the involvement of RIG-I activation in the onset and progression of psoriasis via type I interferon signaling and the IL-23/IL-17 axis.

A novel pathogenic variant p.Asp797Val in IFIH1 in a Japanese boy with overlapping Singleton-Merten syndrome and Aicardi-Goutières syndrome.

Pathogenic-activating variants of interferon induced with Helicase C domain 1 (IFIH1) cause Singleton-Merten (S-M) syndrome, which accompanies acro-osteolysis, loss of permanent teeth, and aortic calcification, as well as causing Aicardi-Goutières (A-G) syndrome, which shows progressive encephalopathy, spastic paraplegia, and calcification of basal ganglia. Recently, patients with overlapping syndromes presenting with features of S-M syndrome and A-G syndrome were reported. However, progression of clinical features of this condition has not been fully understood. We report a Japanese boy with a novel pathogenic IFIH1 variant who presented with clinical features of S-M syndrome and A-G syndrome.

Hereditary Disorders of Cardiovascular Calcification.

Arterial calcification is a common phenomenon in the elderly, in patients with atherosclerosis or renal failure and in diabetes. However, when present in very young individuals, it is likely to be associated with an underlying hereditary disorder of arterial calcification. Here, we present an overview of the few monogenic disorders presenting with early-onset cardiovascular calcification. These disorders can be classified according to the function of the respective disease gene into (1) disorders caused by an altered purine and phosphate/pyrophosphate metabolism, (2) interferonopathies, and (3) Gaucher disease. The finding of arterial calcification in early life should alert the clinician and prompt further genetic work-up to define the underlying genetic defect, to establish the correct diagnosis, and to enable appropriate therapy.

Stance Phase Detection by Inertial Measurement Unit Placed on the Metacarpus of Horses Trotting on Hard and Soft Straight Lines and Circles.

The development of on-board technologies has enabled the development of quantification systems to monitor equine locomotion parameters. Their relevance among others relies on their ability to determine specific locomotor events such as foot-on and heel-off events. The objective of this study was to compare the accuracy of different methods for an automatic gait events detection from inertial measurement units (IMUs). IMUs were positioned on the cannon bone, hooves, and withers of seven horses trotting on hard and soft straight lines and circles. Longitudinal acceleration and angular velocity around the latero-medial axis of the cannon bone, and withers dorso-ventral displacement data were identified to tag the foot-on and a heel-off events. The results were compared with a reference method based on hoof-mounted-IMU data. The developed method showed bias less than 1.79%, 1.46%, 3.45% and -1.94% of stride duration, respectively, for forelimb foot-on and heel-off, and for hindlimb foot-on and heel-off detection, compared to our reference method. The results of this study showed that the developed gait-events detection method had a similar accuracy to other methods developed for straight line analysis and extended this validation to other types of exercise (circles) and ground surface (soft surface).

Efficacy of a commercial dry sleeve cryotherapy system for cooling the equine metacarpus.

OBJECTIVE: To determine the ability of a commercial cryotherapy system (Game Ready Equine) to cool the metacarpal subcutaneous tissue and the superficial digital flexor tendon (SDFT) in horses. STUDY DESIGN: Experimental study. ANIMALS OR SAMPLE POPULATION: Six healthy adult horses. METHODS: Thermocouples were implanted into the metacarpal subcutaneous tissues and the SDFT of six horses. Two treatments (cryotherapy or cryotherapy with 5-50 mmHg intermittent compression) were randomly assigned to forelimbs and performed for 20 minutes. Temperatures were compared to the target range of 10-19°C and between groups. RESULTS: Only one limb in the cryotherapy/compression group reached the target range after cryotherapy. Temperatures did not differ between treatment groups at time 0. Lowest temperatures achieved in the subcutaneous tissue (p = .0043) and SDFT (p = .005) were 4.9 and 7.6°C lower when intermittent compression was applied. Similarly, applying compression induced a maximum change in temperature of approximately 7.0°C in the subcutaneous tissue (p = .014) and 10.2°C in the SDFT (p = .0001). CONCLUSION: The cryotherapy system did not cool equine subcutaneous tissue or SDFT to the target temperature range, except in one limb. Combining cryotherapy with intermittent compression did result in lower temperatures and a greater change in temperature of the subcutaneous tissue and SDFT. CLINICAL SIGNIFICANCE: When using this cryotherapy system, the addition of intermittent compression should be considered to achieve lower temperatures and potentially greater reduction in inflammation. Further studies are warranted to determine the effect of longer treatment times, higher compression settings, and the optimal temperature for benefits in normal and diseased equine tissues.

Arthroscopic removal of osteochondral fragments located within the condylar fossa of the third metacarpus/metatarsus in Thoroughbred yearlings.

OBJECTIVE: To describe an arthroscopic technique for the removal of osteochondral fragments located within the condylar fossa of the third metacarpal/tarsal bone. STUDY DESIGN: Retrospective study. ANIMALS: Thoroughbred yearlings (n = 11). METHODS: Osteochondral fragments located within the condylar fossa of the third metacarpal/tarsal bone identified during presale radiographic examination were removed via arthroscopy, assisted with ultrasonography in select cases. Complete fragment removal was confirmed by intraoperative radiography. RESULTS: Fragments were successfully removed using rongeurs following dissection of soft tissue attachments using a bipolar radiofrequency probe. No postoperative complications occurred. CONCLUSIONS: An ipsilateral arthroscopic and instrument portal coupled with ultrasound assistance and a radiofrequency probe allowed for successful removal of fragments located within the condylar fossa of the third metacarpal/tarsal bone. The technique allowed for the removal of the unstable osteochondral fragment and associated physical debris. CLINICAL SIGNIFICANCE: The described surgical technique enables the removal of osteochondral fragments located within the condylar fossa of the third metacarpal/tarsal bone with minimal disruption to the surrounding soft tissues.

Relation between palm and finger cortical representations in primary somatosensory cortex: A 7T fMRI study.

Many studies focused on the cortical representations of fingers, while the palm is relatively neglected despite its importance for hand function. Here, we investigated palm representation (PR) and its relationship with finger representations (FRs) in primary somatosensory cortex (S1). Few studies in humans suggested that PR is located medially with respect to FRs in S1, yet to date, no study directly quantified the somatotopic organization of PR and the five FRs. Importantly, the link between the somatotopic organization of PR and FRs and their activation properties remains largely unexplored. Using 7T fMRI, we mapped PR and the five FRs at the single subject level. First, we analyzed the cortical distance between PR and FRs to determine their somatotopic organization. Results show that PR was located medially with respect to D5. Second, we tested whether the observed cortical distances would predict the relationship between PR and FRs activations. Using three complementary measures (cross-activations, pattern similarity and resting-state connectivity), we show that the relationship between PR and FRs activations were not determined by their somatotopic organization, that is, there was no gradient moving from D5 to D1, except for resting-state connectivity, which was predicted by the somatotopy. Instead, we show that the representational geometry of PR and FRs activations reflected the physical structure of the hand. Collectively, our findings suggest that the spatial proximity between topographically organized neuronal populations do not necessarily predicts their functional properties, rather the structure of the sensory space (e.g., the hand shape) better describes the observed results.

Singleton-Merten syndrome: A rare cause of femoral head necrosis.

Singleton-Merten syndrome (SMS) is a type I interferonopathy. In this report, we disclose the first-to the best of our knowledge-direct association of SMS with femoral head necrosis (FHN). The following case report presents the condition of a 38-year-old male suffering from SMS with FHN, characterized by acute symptoms and rapid disease progression. As per the recommendations of the Association Research Circulation Osseous (ARCO) and the S3-guidelines, we successfully treated the FHN with core decompression. Our histological results correlate with the changes described in medical literature in patients with SMS and MDA5-knockout in vivo experiments such as osteopenia, widened medullary cavity, and thin cortical bone. Moreover, the conducted immunohistochemistry shows strong CD56 positivity of the osteoblasts and osteocytes, as well as significant CD68 and CD163 positivity of the middle-sized osteoclasts. Collectively, these findings suggest an underlying syndrome in the FHN. A six-month post-operative follow-up revealed complete recovery with the absence of the initial symptoms and ability to resume normal daily activities. Taken together, our findings suggest that SMS is an additional cause of FHN in young adults. Early detection and adequate treatment using well-established joint-preserving techniques demonstrate a favorable improvement of the patient's clinical condition.

Singleton-Merten Syndrome-like Skeletal Abnormalities in Mice with Constitutively Activated MDA5.

Singleton-Merten syndrome (SMS) is a type I interferonopathy characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, and psoriasis. A missense mutation in IFIH1 encoding a cytoplasmic viral RNA sensor MDA5 has recently been identified in the SMS patients as well as in patients with a monogenic form of lupus. We previously reported that Ifih1gs/+ mice express a constitutively active MDA5 and spontaneously develop lupus-like nephritis. In this study, we demonstrate that the Ifih1gs/+ mice also exhibit SMS-like bone abnormalities, including decreased bone mineral density and thin cortical bone. Histological analysis revealed a low number of osteoclasts, low bone formation rate, and abnormal development of growth plate cartilages in Ifih1gs/+ mice. These abnormalities were not observed in Ifih1gs/+ ・Mavs-/- and Ifih1gs/+ ・Ifnar1-/- mice, indicating the critical role of type I IFNs induced by MDA5/MAVS-dependent signaling in the bone pathogenesis of Ifih1gs/+ mice, affecting bone turnover. Taken together, our findings suggest the inhibition of type I IFN signaling as a possible effective therapeutic strategy for bone disorders in SMS patients.

Cell engraftment, vascularization, and inflammation after treatment of equine distal limb wounds with endothelial colony forming cells encapsulated within hydrogel microspheres.

BACKGROUND: Endothelial colony forming cells (ECFCs) may be useful therapeutically in conditions with poor blood supply, such as distal limb wounds in the horse. Encapsulation of ECFCs into injectable hydrogel microspheres may ensure cell survival and cell localization to improve neovascularization and healing. Autologous ECFCs were isolated from 6 horses, labeled with quantum nanodots (QD), and a subset were encapsulated in poly(ethylene) glycol fibrinogen microspheres (PEG-Fb MS). Full-thickness dermal wounds were created on each distal limb and injected with empty PEG-Fb MS, serum, ECFCs, or ECFCs encapsulated into PEG- Fb MS (ECFC/MS). Analysis included wound surface area (WSA), granulation tissue scoring (GS), thermography, collagen density staining, and immunohistochemical staining for endothelial and inflammatory cells. The purpose of this study was to track cell location and evaluate wound vascularization and inflammatory response after injection of ECFC/MS or naked ECFCs in equine distal limb wounds. RESULTS: ECFCs were found near and within newly formed blood vessels up to 3 weeks after injection. ECFC and ECFC/MS groups had the greatest blood vessel quantity at week 1 in the wound periphery. Wounds treated with ECFCs and ECFC/MS had the lowest density of neutrophils and macrophages at week 4. There were no significant effects of ECFC or ECFC/MS treatment on other measured parameters. CONCLUSIONS: Injection of microsphere encapsulated ECFCs was practical for clinical use and well-tolerated. The positive ECFC treatment effects on blood vessel density and wound inflammation warrant further investigation.

Single-incision percutaneous drilling technique to achieve hemiepiphysiodesis of the distal metacarpus in foals with metacarpophalangeal varus deformities.

OBJECTIVE: To describe a drilling technique for hemiepiphysiodesis of the distal lateral metacarpal physis and report the outcome of treated foals. STUDY DESIGN: Retrospective case series. SAMPLE POPULATION: Eleven thoroughbred foals. METHODS: While horses were under general anesthesia, the lateral aspect of the distal metacarpal physis was approached through a single small incision by using a power drill. The drill bit was placed at the level of the physis under radiographic guidance. A 4.5-mm drill bit was passed several times through the lateral growth plate to remove the cartilage in a fan-like pattern. Postoperative outcomes consisted of clinical assessment and farm manager/owner satisfaction. Cosmetics and radiographic appearance of the surgical site were assessed when the horses were yearlings. RESULTS: The procedure was performed in 16 limbs of 11 thoroughbred foals with a median age of 113.5 days (range, 90-129). Median age at postoperative follow-up was 422 days (range, 366 to 452). The procedure stopped the progression of a metacarpophalangeal varus deformity in all the limbs treated, determined by visual clinical evaluation and farm manager's satisfaction with subjectively excellent radiographic images and cosmetic outcomes at yearling age. CONCLUSION: Physis ablation was consistently achieved in these 11 foals with developing growth deformities of the distal metacarpus. CLINICAL SIGNIFICANCE: This drilling technique may offer a minimally invasive, safe, and simple technique to manage distal limb conformation in foals without placement of implants. Additional quantitative data are required to assess its effectiveness relative to other options.

Standing low-field MRI of the equine proximal metacarpal/metatarsal region is considered useful for diagnosing primary bone pathology and makes a positive contribution to case management: A prospective survey study.

High-field MRI of the proximal metacarpal/metatarsal region has been associated with great diagnostic potential and clinical reports of standing low-field MRI of the forelimb suggest the same. To better understand diagnostic outcomes with standing low-field MRI of the proximal suspensory region, a prospective survey study was conducted and users of a widely available system questioned on their experience, operating procedures, and interpretation of standing low-field MRI findings. Response data included scores on a modified Likert scale from which weighted ratings were calculated for statistical analyses. Depending on the question, responses were obtained from 17 to 29 of the 38 invited facilities. Users indicated that standing low-field MRI was most frequently performed in the face of equivocal diagnostic findings; compared to Sports horses, general purpose riding horses were thought less likely to have detectable abnormalities and standing low-field MRI was rated most useful for the detection of primary bone pathology in the proximal metacarpal region. Standing low-field MRI signal change involving both the suspensory ligament and adjacent bone concurrently was rated most relevant and abnormalities solely affecting the muscle/adipose tissue bundles least relevant for diagnosing suspensory ligament injury. Transverse scans and in decreasing order T1-weighted gradient echo, short-tau inversion recovery FSE, T2*-weighted gradient echo, and T2-weighted FSE sequences were most frequently acquired and judged most useful by the majority of users experienced in imaging of the target area. This survey supports the relevant impact of standing low-field MRI on clinical case management, particularly in the context of imaging the proximal metacarpal region.

DDX58 and Classic Singleton-Merten Syndrome.

PURPOSE: Singleton-Merten syndrome manifests as dental dysplasia, glaucoma, psoriasis, aortic calcification, and skeletal abnormalities including tendon rupture and arthropathy. Pathogenic variants in IFIH1 have previously been associated with the classic Singleton-Merten syndrome, while variants in DDX58 has been described in association with a milder phenotype, which is suggested to have a better prognosis. We studied a family with severe, "classic" Singleton-Merten syndrome. METHODS: We undertook clinical phenotyping, next-generation sequencing, and functional studies of type I interferon production in patient whole blood and assessed the type I interferon promoter activity in HEK293 cells transfected with wild-type or mutant DDX58 stimulated with Poly I:C. RESULTS: We demonstrate a DDX58 autosomal dominant gain-of-function mutation, with constitutive upregulation of type I interferon. CONCLUSIONS: DDX58 mutations may be associated with the classic features of Singleton-Merten syndrome including dental dysplasia, tendon rupture, and severe cardiac sequela.

JAK-inhibitors. New players in the field of immune-mediated diseases, beyond rheumatoid arthritis.

Janus kinase (JAK)/signal transducers and activators of transcription (STATs) are a group of molecules associated with one of the major pathways through which many cytokines exert and integrate their function, and as such they are increasingly recognized as playing critical role in the pathogenesis subserving various immune-mediated diseases, including RA, PsA, SpAs, IBD, skin disorders (e.g. alopecia areata, atopic dermatitis), single-gene disorders like interferonopathies, and others. JAKs are the key initiating players of the JAK/STAT pathway. Upon binding of their respective effector molecules (cytokines, IFNs, growth factors and others) to type I and type II receptors, JAKs are activated, and through phosphorylation of themselves and of other molecules (including STATs), they mediate signal transduction to the nucleus. A class of drugs-called JAK inhibitors or JAKinibs-that block one or more JAKs has been developed in the last decade, and now numbers >20 members. Although, so far, JAK inhibitors have been marketed only for RA and PsA, these drugs have been tested in phase 2 and phase 3 clinical trials for other inflammatory conditions and beyond. In this review, we summarize the clinical data, including efficacy and safety, available for JAK inhibitors used in some immune-mediated conditions other than RA.