A network-based approach reveals long non-coding RNAs associated with disease activity in lupus nephritis: key pathways for flare and potential biomarkers to be used as liquid biopsies.

Objective: A blood-based biomarker is needed to assess lupus nephritis (LN) disease activity, minimizing the need for invasive kidney biopsies. Long non-coding RNAs (lncRNAs) are known to regulate gene expression, appear to be stable in human plasma, and can serve as non-invasive biomarkers. Methods: Transcriptomic data of whole blood samples from 74 LN patients and 20 healthy subjects (HC) were analyzed to identify differentially expressed (DE) lncRNAs associated with quiescent disease and flares. Weighted gene co-expression network analysis (WGCNA) was performed to uncover lncRNAs with a central role (hub lncRNAs) in regulating key biological processes that drive LN disease activity. The association of hub lncRNAs with disease activity was validated using RT-qPCR on an independent cohort of 15 LN patients and 9 HC. cis- and trans-targets of validated lncRNAs were explored in silico to examine potential mechanisms of their action. Results: There were 444 DE lncRNAs associated with quiescent disease and 6 DE lncRNAs associated with flares (FDR <0.05). WGCNA highlighted IFN signaling and B-cell activity/adaptive immunity as the most significant processes contributing to nephritis activity. Four disease-activity-associated lncRNAs, namely, NRIR, KLHDC7B-DT, MIR600HG, and FAM30A, were detected as hub genes and validated in an independent cohort. NRIR and KLHDC7B-DT emerged as potential key regulators of IFN-mediated processes. Network analysis suggests that FAM30A and MIR600HG are likely to play a central role in the regulation of B-cells in LN through cis-regulation effects and a competing endogenous RNA mechanism affecting immunoglobulin gene expression and the IFN-λ pathway. Conclusions: The expression of lncRNAs NRIR, KLHDC7B-DT, FAM30A, and MIR600HG were associated with disease activity and could be further explored as blood-based biomarkers and potential liquid biopsy on LN.

Combined - whole blood and skin fibroblasts- transcriptomic analysis in Psoriatic Arthritis reveals molecular signatures of activity, resistance and early response to treatment.

Background: An interplay between immune cells and resident skin and joint stromal cells is implicated in psoriatic arthritis (PsA), yet the mechanisms remain elusive with a paucity of molecular biomarkers for activity and response. Combined transcriptomic and immunophenotypic analysis of whole blood and skin fibroblasts could provide further insights. Methods: Whole blood RNA-seq was performed longitudinally in 30 subjects with PsA at the beginning, one and six months after treatment, with response defined at six months. As control groups, 10 healthy individuals and 10 subjects with rheumatoid arthritis (RA) were recruited combined with public datasets from patients with psoriasis (PsO) and systemic lupus erythematous (SLE). Differential expression analysis and weighted gene co-expression network analysis were performed to identify gene expression signatures, while deconvolution and flow cytometry to characterize the peripheral blood immune cell profile. In a subset of affected and healthy individuals, RNA-seq of skin fibroblasts was performed and subjected to CellChat analysis to identify the blood-skin fibroblast interaction network. Results: PsA demonstrated a distinct "activity" gene signature in the peripheral blood dominated by TNF- and IFN-driven inflammation, deregulated cholesterol and fatty acid metabolism and expansion of pro-inflammatory non-classical monocytes. Comparison with the blood transcriptome of RA, PsO, and SLE revealed a "PsA-specific signature" enriched in extracellular matrix remodeling. This was further supported by the skin fibroblast gene expression profile, displaying an activated, proliferating phenotype, and by skin-blood interactome analysis revealing interactions with circulating immune cells through WNT, PDGF and immune-related semaphorins. Of note, resistance to treatment was associated with upregulation of genes involved in TGFß signaling and angiogenesis and persistent increase of non-classical monocytes. Differentially expressed genes related to platelet activation and hippo signaling discriminated responders and non-responders as early as one month after treatment initiation. Conclusion: Transcriptome analysis of peripheral blood and skin fibroblasts in PsA reveals a distinct disease activity signature and supports the involvement of skin fibroblasts through their activation and interaction with circulating immune cells. Aberrant TGFß signaling and persistently increased non-classical monocytes characterize treatment-resistant PsA, with pro-inflammatory pathways related to platelet activation and Hippo signaling predicting early response to treatment.

Mitophagy Balance in Various Cell Subsets in Patients with ANCA-Associated Vasculitis and Correlation with the Presence of Anti-Neutrophil Cytoplasmic Antibodies.

ANCA-associated vasculitides (AAVs) are characterised by heterogeneous molecular and pathophysiological traits, causing ambiguous differential diagnosis and taxonomy. Response to therapy has proven far from successful, contributing to high mortality. Transcriptome analysis of different vasculitis subtypes adds new leads in elucidating mechanisms of disease and the role of specific cell subsets to them. Recent findings have shown that mitophagy is a procedure whose imbalance could lead to immune dysregulation with certain involvement to autoimmunity. Inflammatory response related mitophagy is yet to be described in AAVs. We here describe a research protocol to investigate mitophagy in monocytes, neutrophils, and T cells in AAV patients, and the relationship of disturbed mitophagy with ANCA seropositivity.

Targeting Long Non-Coding RNAs in Nervous System Cancers: New Insights in Prognosis, Diagnosis and Therapy.

Long non-coding RNAs (lncRNAs) constitute one of the most broad and diverse classes of cellular transcripts, playing key roles as regulatory molecules in many biological processes. Although the biology of lncRNAs is a new and emerging field of research, several studies have already shown that alterations in the expression of lncRNAs are associated with the development and progression of cancer in different organs and tissues, including central and peripheral nervous system. In this review, we summarize the oncogenic and tumor suppressive roles of lncRNAs in malignant tumors of the nervous system, such as glioma and neuroblastoma, focusing on their functional interactions with DNA, other RNA and protein molecules. We further discuss the potential use of lncRNAs as biomarkers for diagnosis, prognosis and tumor treatment. Gaining insight into the functional association between nervous system malignancies and lncRNAs could offer new perspectives to the development of promising therapeutic tools against cancer.

Dimerization is required for GARS-mediated neurotoxicity in dominant CMT disease.

Charcot-Marie-Tooth (CMT) disease is a genetically heterogeneous group of peripheral neuropathies. Mutations in several aminoacyl-tRNA synthetase (ARS) genes have been implicated in inherited CMT disease. There are 12 reported CMT-causing mutations dispersed throughout the primary sequence of the human glycyl-tRNA synthetase (GARS). While there is strong genetic evidence linking GARS mutations to CMT disease, the molecular pathology underlying the neuromuscular and sensory phenotypes is still not fully understood. In particular, it is unclear whether the mutations result in a toxic gain of function, a partial loss of activity related to translation, or a combination of these mechanisms. We identified a zebrafish allele of gars (gars(s266)). Homozygous mutant embryos carry a C->A transversion, that changes a threonine to a lysine, in a residue next to a CMT-associated human mutation. We show that the neuromuscular phenotype observed in animals homozygous for T209K Gars (T130K in GARS) is due to a loss of dimerization of the mutated protein. Furthermore, we show that the loss of function, dimer-deficient and human disease-associated G319R Gars (G240R in GARS) mutant protein is unable to rescue the above phenotype. Finally, we demonstrate that another human disease-associated mutant G605R Gars (G526 in GARS) dimerizes with the remaining wild-type protein in animals heterozygous for the T209K Gars and reduces the function enough to elicit a neuromuscular phenotype. Our data indicate that dimerization is required for the dominant neurotoxicity of disease-associated GARS mutations and provide a rapid, tractable model for studying newly identified GARS variants for a role in human disease.

Intracardiac flow dynamics regulate atrioventricular valve morphogenesis.

AIMS: Valvular heart disease is responsible for considerable morbidity and mortality. Cardiac valves develop as the heart contracts, and they function throughout the lifetime of the organism to prevent retrograde blood flow. Their precise morphogenesis is crucial for cardiac function. Zebrafish is an ideal model to investigate cardiac valve development as it allows these studies to be carried out in vivo through non-invasive imaging. Accumulating evidence suggests a role for contractility and intracardiac flow dynamics in cardiac valve development. However, these two factors have proved difficult to uncouple, especially since altering myocardial function affects the intracardiac flow pattern. METHODS AND RESULTS: Here, we describe novel zebrafish models of developmental valve defects. We identified two mutant alleles of myosin heavy chain 6 that can be raised to adulthood despite having only one functional chamber-the ventricle. The adult mutant ventricle undergoes remodelling, and the atrioventricular (AV) valves fail to form four cuspids. In parallel, we characterized a novel mutant allele of southpaw, a nodal-related gene involved in the establishment of left-right asymmetry, which exhibits randomized heart and endoderm positioning. We first observed that in southpaw mutants the relative position of the two cardiac chambers is altered, affecting the geometry of the heart, while myocardial function appears unaffected. Mutant hearts that loop properly or exhibit situs inversus develop normally, whereas midline, unlooped hearts exhibit defects in their transvalvular flow pattern during AV valve development as well as defects in valve morphogenesis. CONCLUSION: Our data indicate that intracardiac flow dynamics regulate valve morphogenesis independently of myocardial contractility.