CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal in vivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providing insights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair.

Muscle denervation promotes functional interactions between glial and mesenchymal cells through NGFR and NGF.

We performed scRNA-seq/snATAC-seq of skeletal muscles post sciatic nerve transection to delineate cell type-specific patterns of gene expression/chromatin accessibility at different time points post-denervation. Unlike myotrauma, denervation selectively activates glial cells and Thy1/CD90-expressing mesenchymal cells. Glial cells expressed Ngf receptor (Ngfr) and were located near neuromuscular junctions (NMJs), close to Thy1/CD90-expressing cells, which provided the main cellular source of NGF post-denervation. Functional communication between these cells was mediated by NGF/NGFR, as either recombinant NGF or co-culture with Thy1/CD90-expressing cells could increase glial cell number ex vivo. Pseudo-time analysis in glial cells revealed an initial bifurcation into processes related to either cellular de-differentiation/commitment to specialized cell types (e.g., Schwann cells), or failure to promote nerve regeneration, leading to extracellular matrix remodeling toward fibrosis. Thus, interactions between denervation-activated Thy1/CD90-expressing and glial cells represent an early abortive process toward NMJs repair, ensued by the conversion of denervated muscles into an environment hostile for NMJ repair.

SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of peripheral heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. Yet, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. A genome-wide analysis of gene expression in cultures of primary HGPS fibroblasts identified SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1), as central gene that propels a cell-autonomous pathogenic signaling from the altered nuclear lamina. Indeed, siRNA-mediated downregulation and pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by the correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector and target for therapeutic interventions in HGPS pathogenesis.

LncRNA EPR-induced METTL7A1 modulates target gene translation.

EPR is a long non-coding RNA (lncRNA) that controls cell proliferation in mammary gland cells by regulating gene transcription. Here, we report on Mettl7a1 as a direct target of EPR. We show that EPR induces Mettl7a1 transcription by rewiring three-dimensional chromatin interactions at the Mettl7a1 locus. Our data indicate that METTL7A1 contributes to EPR-dependent inhibition of TGF-ß signaling. METTL7A1 is absent in tumorigenic murine mammary gland cells and its human ortholog (METTL7A) is downregulated in breast cancers. Importantly, re-expression of METTL7A1 in 4T1 tumorigenic cells attenuates their transformation potential, with the putative methyltransferase activity of METTL7A1 being dispensable for its biological functions. We found that METTL7A1 localizes in the cytoplasm whereby it interacts with factors implicated in the early steps of mRNA translation, associates with ribosomes, and affects the levels of target proteins without altering mRNA abundance. Overall, our data indicates that METTL7A1-a transcriptional target of EPR-modulates translation of select transcripts.

Methods for the Differential Analysis of Hi-C Data.

The 3D organization of chromatin within the nucleus enables dynamic regulation and cell type-specific transcription of the genome. This is true at multiple levels of resolution: on a large scale, with chromosomes occupying distinct volumes (chromosome territories); at the level of individual chromatin fibers, which are organized into compartmentalized domains (e.g., Topologically Associating Domains-TADs), and at the level of short-range chromatin interactions between functional elements of the genome (e.g., enhancer-promoter loops).The widespread availability of Chromosome Conformation Capture (3C)-based high-throughput techniques has been instrumental in advancing our knowledge of chromatin nuclear organization. In particular, Hi-C has the potential to achieve the most comprehensive characterization of chromatin 3D interactions, as it is theoretically able to detect any pair of restriction fragments connected as a result of ligation by proximity.This chapter will illustrate how to compare the chromatin interactome in different experimental conditions, starting from pre-computed Hi-C contact matrices, how to visualize the results, and how to correlate the observed variations in chromatin interaction strength with changes in gene expression.

MyoD induces ARTD1 and nucleoplasmic poly-ADP-ribosylation during fibroblast to myoblast transdifferentiation.

While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation.

Transcription Factor-Directed Re-wiring of Chromatin Architecture for Somatic Cell Nuclear Reprogramming toward trans-Differentiation.

MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers, and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs led to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation.

Sarcopenia as a potential cause of chronic hyponatremia in the elderly.

Hyponatremia is the most frequent electrolyte disorder found in clinical practice, particularly in hospitalized elderly patients, where it is associated with fractures, falls, hospital readmission, prolonged hospital stay and increased mortality. Pathophysiologically, hyponatremia can be induced by the reduction in sodium or potassium body content, and/or the increase in water body content. Sarcopenia is an ageing-associated progressive and generalized loss of musculoskeletal mass and strength which leads to low physical performance, particularly in the frail elderly. Since muscle mass is the main potassium body store, this condition usually represents a reduced body potassium content. In the present article it is hypothesized that sarcopenia, as a cause of low potassium body content, could induce or co-induce hyponatremia, particularly in elderly individuals suffering from frailty phenotype.

The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration.

Metabolic reprogramming is an active regulator of stem cell fate choices, and successful stem cell differentiation in different compartments requires the induction of oxidative phosphorylation. However, the mechanisms that promote mitochondrial respiration during stem cell differentiation are poorly understood. Here we demonstrate that Stat3 promotes muscle stem cell myogenic lineage progression by stimulating mitochondrial respiration in mice. We identify Fam3a, a cytokine-like protein, as a major Stat3 downstream effector in muscle stem cells. We demonstrate that Fam3a is required for muscle stem cell commitment and skeletal muscle development. We show that myogenic cells secrete Fam3a, and exposure of Stat3-ablated muscle stem cells to recombinant Fam3a in vitro and in vivo rescues their defects in mitochondrial respiration and myogenic commitment. Together, these findings indicate that Fam3a is a Stat3-regulated secreted factor that promotes muscle stem cell oxidative metabolism and differentiation, and suggests that Fam3a is a potential tool to modulate cell fate choices.

Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy.

Fibro-adipogenic progenitors (FAPs) are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, and ability to adopt multiple lineages in vitro. Gene expression analysis at single-cell level reveals that FAPs undergo dynamic transitions through a spectrum of cell states that can be identified by differential expression levels of Tie2 and Vcam1. Different patterns of Vcam1-negative Tie2high or Tie2low and Tie2low/Vcam1-expressing FAPs are detected during neonatal myogenesis, response to acute injury and Duchenne Muscular Dystrophy (DMD). RNA sequencing analysis identified cell state-specific transcriptional profiles that predict functional interactions with satellite and inflammatory cells. In particular, Vcam1-expressing FAPs, which exhibit a pro-fibrotic expression profile, are transiently activated by acute injury in concomitance with the inflammatory response. Aberrant persistence of Vcam1-expressing FAPs is detected in DMD muscles or upon macrophage depletion, and is associated with muscle fibrosis, thereby revealing how disruption of inflammation-regulated FAPs dynamics leads to a pathogenic outcome.

Denervation-activated STAT3-IL-6 signalling in fibro-adipogenic progenitors promotes myofibres atrophy and fibrosis.

Fibro-adipogenic progenitors (FAPs) are typically activated in response to muscle injury, and establish functional interactions with inflammatory and muscle stem cells (MuSCs) to promote muscle repair. We found that denervation causes progressive accumulation of FAPs, without concomitant infiltration of macrophages and MuSC-mediated regeneration. Denervation-activated FAPs exhibited persistent STAT3 activation and secreted elevated levels of IL-6, which promoted muscle atrophy and fibrosis. FAPs with aberrant activation of STAT3-IL-6 signalling were also found in mouse models of spinal cord injury, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS) and in muscles of ALS patients. Inactivation of STAT3-IL-6 signalling in FAPs effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and ALS (SODG93A mice). Activation of pathogenic FAPs following loss of integrity of neuromuscular junctions further illustrates the functional versatility of FAPs in response to homeostatic perturbations and suggests their potential contribution to the pathogenesis of neuromuscular diseases.

Computational methods for analyzing genome-wide chromosome conformation capture data.

In all organisms, chromatin is packed to fulfil structural constraints and functional requirements. The hierarchical model of chromatin organization in the 3D nuclear space encompasses different topologies at diverse scale lengths, with chromosomes occupying distinct volumes, further organized in compartments, inside which the chromatin fibers fold into large domains and short-range loops. In the recent years, the combination of chromosome conformation capture (3C) techniques and high-throughput sequencing allowed probing chromatin spatial organization at the whole genome-scale. 3C-based methods produce enormous amounts of genomic data that are analyzed using ad-hoc computational procedures. Here, we review the common pipelines and methods for the analysis of genome-wide chromosome conformation capture data, highlighting recent developments in key steps for the identification of chromatin structures.

Comparison of computational methods for Hi-C data analysis.

Hi-C is a genome-wide sequencing technique used to investigate 3D chromatin conformation inside the nucleus. Computational methods are required to analyze Hi-C data and identify chromatin interactions and topologically associating domains (TADs) from genome-wide contact probability maps. We quantitatively compared the performance of 13 algorithms in their analyses of Hi-C data from six landmark studies and simulations. This comparison revealed differences in the performance of methods for chromatin interaction identification, but more comparable results for TAD detection between algorithms.

Post-orchidectomy retroperitoneal seminoma: A case report.

Between 2 and 5% of malignant germ cell tumors in males arise at extragonadal sites. The origin of extragonadal retroperitoneal germ cell tumors remains controversial. Whether these develop primarily in the retroperitoneum or are metastases of a primary testicular tumor has long been debated. We report a 38-year-old male who presented with abdominal pain and was diagnosed with retroperitoneal seminoma. The patient gave a history of having undergone a right orchidectomy for an undescended testis via the inguinal route 10 years previously with a reported histology of benign inflammatory mass.

Arterial erectile dysfunction: different severities of endothelial apoptosis between diabetic patients "responders" and "non responders" to sildenafil.

BACKGROUND: The low pharmacological response to phosphodiesterase type 5 inhibitors represents an expression of higher endothelial damage in certain categories of patients with erectile dysfunction and high cardiovascular risk. The present study evaluated this objective in type 2 diabetic patients with erectile dysfunction, classified as "non responders" to Sildenafil. METHODS: Eighteen "responder" and twelve "non responder" type 2 diabetic patients were evaluated, relatively to different levels of endothelial damage, through the diagnostic use of a new immunophenotype of circulating endothelial progenitor cells (CD45neg/CD34pos/CD144pos) and endothelial microparticles (CD45neg/CD144pos/Annexin Vpos), recently developed and published by our group. RESULTS: "Non responder" patients showed a significant higher severity [8.0±3.0 (International Index of Erectile Function-abbreviated version with 5 questions) vs 14.0±3.0] and duration (10.0±2.0 vs 7.0±2.0 years) of erectile dysfunction, higher level of penile arterial insufficiency (peak systolic velocity=13.0±16.0 vs 28.0±26.0cm/s; acceleration time=153±148 vs 125±128 mm/s) and finally a significant higher level of endothelial apoptosis [0.15±0.13 vs 0.05±.0.03% (serum concentrations of endothelial microparticles)] associated with higher serum concentrations of circulating late immunophenotype of endothelial progenitor cells (0.40±0.35 vs 0.12±.0.10%). CONCLUSIONS: The results of this study corroborate the clinical value of the low clinical response to phosphodiesterase type 5 inhibitors in the treatment of erectile dysfunction in the patients with high cardiovascular risk profile, such as diabetics. In addition, the markers used in this study confirm their potential application in clinical practice as useful indicators of endothelial alteration. However, in the future we will have to assess a larger number of patients and for a longer period of observation in order to better understand the causal and temporal relations.

Bone demineralization in postmenopausal women: role of anamnestic risk factors.

This study evaluated the effects of LT4 administration on the bone mineral density (BMD) in physiological postmenopausal women after two years of continuative treatment. 110 postmenopausal women with nodular goiter aged between 50 and 55 years were examined before and after 2 years of therapy with a fixed dose of LT4 (1.6 mcg/kg/die) for the treatment of nodular thyroid disease. The results showed that the patients on treatment with LT4 have a slight, but significant reduction of the BMD after 2 years of treatment, associated with increased serum levels of alkaline phosphatase and urinary excretion of hydroxyproline, confirming our data conducted on the same group after one year of therapy. Comparison between patients receiving LT4 (group A) or not (group B) showed that group A patients had significantly lower BMD. We demonstrated the statistically significant influence of the following risk factors on BMD: (1) body mass index <19 kg/m(2); (2) the onset of menarche after the age of 15 years; (3) positive history for period of amenorrhoea; (4) nulliparity.