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Understanding the biomolecular interactions between graphene and human immune cells is a prerequisite for its utilization as a diagnostic or therapeutic tool. To characterize the complex interactions between graphene and immune cells, we propose an integrative analytical pipeline encompassing the evaluation of molecular and cellular parameters. Herein, we use single-cell mass cytometry to dissect the effects of graphene oxide (GO) and GO functionalized with amino groups (GONH 2 ) on 15 immune cell populations, interrogating 30 markers at the single-cell level. Next, the integration of single-cell mass cytometry with genome-wide transcriptome analysis shows that the amine groups reduce the perturbations caused by GO on cell metabolism and increase biocompatibility. Moreover, GONH 2 polarizes T-cell and monocyte activation toward a T helper-1/M1 immune response. This study describes an innovative approach for the analysis of the effects of nanomaterials on distinct immune cells, laying the foundation for the incorporation of single-cell mass cytometry on the experimental pipeline. Understanding the interaction of nanomaterials and immune cells at the biomolecular level is of great significance in therapeutic applications. Here, the authors investigated the interaction of graphene oxide nanomaterials and several immune cell subpopulations using single-cell mass cytometry and genome-wide transcriptome analysis.

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene. mTOR signalling both inhibits autophagy and promotes cell proliferation. Cecconi and colleagues report that AMBRA1 links these two processes by facilitating dephosphorylation and degradation of the proto-oncogene c-Myc.

The identification of the large protein Ambra-1, which regulates autophagy and plays a crucial role in embryogenesis is described. The absence of Ambra-1 function during development results in severe neural tube defects associated with autophagy impairment, accumulation of ubiquitinated proteins, unbalanced cell proliferation and excessive apoptotic cell death. Autophagy is a self-degradative process involved both in basal turnover of cellular components and in response to nutrient starvation or organelle damage in a wide range of eukaryotes 1 , 2 , 3 . During autophagy, portions of the cytoplasm are sequestered by double-membraned vesicles called autophagosomes, and are degraded after fusion with lysosomes for subsequent recycling 4 . In vertebrates, this process acts as a pro-survival or pro-death mechanism in different physiological and pathological conditions, such as neurodegeneration and cancer 2 , 5 , 6 , 7 ; however, the roles of autophagy during embryonic development are still largely uncharacterized 3 . Beclin1 (Becn1; coiled-coil, myosin-like BCL2-interacting protein) is a principal regulator in autophagosome formation, and its deficiency results in early embryonic lethality 8 , 9 . Here we show that Ambra1 (activating molecule in Beclin1-regulated autophagy), a large, previously unknown protein bearing a WD40 domain at its amino terminus, regulates autophagy and has a crucial role in embryogenesis. We found that Ambra1 is a positive regulator of the Becn1-dependent programme of autophagy, as revealed by its overexpression and by RNA interference experiments in vitro . Notably, Ambra1 functional deficiency in mouse embryos leads to severe neural tube defects associated with autophagy impairment, accumulation of ubiquitinated proteins, unbalanced cell proliferation and excessive apoptotic cell death. In addition to identifying a new and essential element regulating the autophagy programme, our results provide in vivo evidence supporting the existence of a complex interplay between autophagy, cell growth and cell death required for neural development in mammals.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by a mutation in the X-linked Dytrophin gene preventing the expression of the functional protein. Exon skipping therapy using antisense oligonucleotides (AONs) is a promising therapeutic strategy for DMD. While benefits of AON therapy have been demonstrated, some challenges remain before this strategy can be applied more comprehensively to DMD patients. These include instability of AONs due to low nuclease resistance and poor tissue uptake. Delivery systems have been examined to improve the availability and stability of oligonucleotide drugs, including polymeric carriers. Previously, we showed the potential of a hydrogel-based polymeric carrier in the form of injectable PEG-fibrinogen (PF) microspheres for delivery of chemically modified 2′-O-methyl phosphorothioate (2OMePs) AONs. The PF microspheres proved to be cytocompatible and provided sustained release of the AONs for several weeks, causing increased cellular uptake in mdx dystrophic mouse cells. Here, we further investigated this delivery strategy by examining in vivo efficacy of this approach. The 2OMePS/PEI polyplexes loaded in PF microspheres were delivered by intramuscular (IM) or intra-femoral (IF) injections. We examined the carrier biodegradation profiles, AON uptake efficiency, dystrophin restoration, and muscle histopathology. Both administration routes enhanced dystrophin restoration and improved the histopathology of the mdx mice muscles. The IF administration of the microspheres improved the efficacy of the 2OMePS AONs over the IM administration. This was demonstrated by a higher exon skipping percentage and a smaller percentage of centered nucleus fibers (CNF) found in H&E-stained muscles. The restoration of dystrophin expression found for both IM and IF treatments revealed a reduced dystrophic phenotype of the treated muscles. The study concludes that injectable PF microspheres can be used as a carrier system to improve the overall therapeutic outcomes of exon skipping-based therapy for treating DMD.

The metabolic perturbation caused by calorie restriction enhances muscle repair by playing a critical role in regulating satellite cell availability and activity in the muscles of young and old mice. To clarify the underlying mechanisms we asked whether myoblast replication and differentiation are affected by metformin, a calorie restriction-mimicking drug. C2C12, a mouse myoblast cell line, readily differentiate in vitro and fuse to form myotubes. However, when incubated with metformin, C2C12 slow their replication and do not differentiate. Interestingly, lower doses of metformin promote myogenic differentiation. We observe that metformin treatment modulates the expression of cyclins and cyclin inhibitors thereby inducing a cell cycle perturbation that causes a delay in the G2/M transition. The effect of metformin treatment is reversible since after drug withdrawal, myoblasts can re-enter the cell cycle and/or differentiate, depending on culture conditions. Myoblasts cultured under metformin treatment fail to up-regulate MyoD and p21cip1, a key step in cell cycle exit and terminal differentiation. Although the details of the molecular mechanisms underlying the effect of the drug on myoblasts still need to be clarified, we propose that metformin negatively affects myogenic differentiation by inhibiting irreversible exit from the cell cycle through reduction of MyoD and p21cip1 levels.

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo . Of note, upon implantation, the biofabricated myo‐substitutes support the formation of new blood vessels and neuromuscular junctions—pivotal aspects for cell survival and muscle contractile functionalities—together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo‐substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios. Synopsis The regenerative capability of skeletal muscle tissue is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. By exploiting the potentials of our biofabrication approach, one can manufacture advanced cell‐laden myo‐substitutes that ultimately may restore the functionalities of severely damaged skeletal muscles in vivo . Biofabricated myo‐substitutes may represent a valid candidate for volumetric muscle loss treatment. Upon implantation, biofabricated myo‐substitutes support the formation of new blood vessels and neuromuscular junctions together with an advanced muscle mass and force recovery. The employed biofabrication approach is compatible with human primary stem cells ‐ namely pericytes. Graphical Abstract The regenerative capability of skeletal muscle tissue is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. By exploiting the potentials of our biofabrication approach, one can manufacture advanced cell‐laden myo‐substitutes that ultimately may restore the functionalities of severely damaged skeletal muscles in vivo .

System vaccinology identified an early innate signature associated with vaccine-mediated protection whose induction is likely to involve both immune and non-immune cells. To dissect muscle and stromal cell contribution, we simulated anti-COVID19 BNT162b2 mRNA vaccine intramuscular administration in human primary cell systems composed of 3D muscle-like tissue (3D-MT), fibroblasts, and peripheral blood mononuclear cells (PBMC). BNT162b2 vaccine was recognized by all cell types, although fibroblasts only translated the spike antigen. Factors from vaccine-injected 3D-MT stimulated monocyte and macrophage recruitment and promoted inflammatory macrophage activation, while stromal factors improved dendritic cell frequency and activation. Conditioned media from vaccine-exposed 3D-MT and fibroblasts elicited in PBMC the expression of an early innate immune module previously associated with protective responses in BNT162b2 vaccinees. Thus, models combining human PBMC, stromal and muscle cells could be employed for the validation of system vaccinology findings and non-animal vaccine pre-clinical testing.

Fibro/Adipogenic Progenitors (FAPs) define a stem cell population playing a pro-regenerative role after muscle damage. When removed from their natural niche, FAPs readily differentiate into adipocytes or fibroblasts. This digressive differentiation potential, which is kept under tight control in the healthy muscle niche, contributes to fat and scar infiltrations in degenerative myopathies, such as in Duchenne Muscular Dystrophy (DMD). Controlling FAP differentiation by means of small molecules may contribute to delay the adverse consequences of the progressive pathological degeneration while offering, at the same time, a wider temporal window for gene therapy and cell-based strategies. In a high content phenotypic screening, we identified the immunosuppressant, azathioprine (AZA) as a negative modulator of FAP adipogenesis. We show here that AZA negatively affects the adipogenic propensity of FAPs purified from wild type and mdx mice by impairing the expression of the master adipogenic regulator, peroxisome proliferator-activated receptor γ (PPARγ). We show that this inhibition correlates with a decline in the activation of the AKT-mTOR axis, the main pathway that transduces the pro-adipogenic stimulus triggered by insulin. In addition, AZA exerts a cytostatic effect that has a negative impact on the mitotic clonal process that is required for the terminal differentiation of the preadipocyte-committed cells.

The term micro-heterogeneity refers to non-genetic cell to cell variability observed in a bell-shaped distribution of the expression of a trait within a population. The contribution of micro-heterogeneity to physiology and pathology remains largely uncharacterised. To address such an issue, we investigated the impact of heterogeneity in skeletal muscle fibro/adipogenic progenitors (FAPs) isolated from an animal model of Duchenne muscular dystrophy (DMD), the mdx mouse. FAPs play an essential role in muscle homoeostasis. However, in pathological conditions or ageing, they are the source of intramuscular infiltrations of fibrotic or adipose tissue. By applying a multiplex flow cytometry assay, we characterised and purified from mdx muscles two FAP cell states expressing different levels of SCA-1. The two cell states are morphologically identical and repopulate each other after several growth cycles. However, they differ in their in vitro behaviour. Cells expressing higher levels of SCA-1 (SCA1-High-FAPs) differentiate more readily into adipocytes while, when exposed to a fibrogenic stimulation, increase the expression of Col1a1 and Timp1 mRNA. A transcriptomic analysis confirmed the adipogenic propensity of SCA1-High-FAPs. In addition, SCA1-High-FAPs proliferate more extensively ex vivo and display more proliferating cells in dystrophic muscles in comparison to SCA1-Low-FAPs. Adipogenesis of both FAP cell states is inhibited in vitro by leucocytes from young dystrophic mice, while leucocytes isolated from aged dystrophic mice are less effective in limiting the adipogenesis of SCA1-High-FAPs suggesting a differential regulatory effect of the microenvironment on micro-heterogeneity. Our data suggest that FAP micro-heterogeneity is modulated in pathological conditions and that this heterogeneity in turn may impact on the behaviour of interstitial mesenchymal cells in genetic diseases.

In human dystrophies, progressive muscle wasting is exacerbated by ectopic deposition of fat and fibrous tissue originating from fibro/adipogenic progenitors (FAPs). In degenerating muscles, the ability of these cells to promote successful healing is attenuated, and FAPs aberrantly expand and differentiate into adipocytes and fibroblasts. Thus, arresting the fibro/adipogenic fate of FAPs, without affecting their physiological role, represents a valuable therapeutic strategy for patients affected by muscle diseases. Here, using a panel of adipose progenitor cells, including human-derived FAPs, coupled with pharmacological perturbations and proteome profiling, we report that LY2090314 interferes with a genuine adipogenic program acting as WNT surrogate for the stabilization of a competent β-catenin transcriptional complex. To predict the beneficial impact of LY2090314 in limiting ectopic deposition of fat in human muscles, we combined a poly-ethylene-glycol-fibrinogen biomimetic matrix with these progenitor cells to create a miniaturized 3D model of adipogenesis. Using this scalable system, we demonstrated that a two-digit nanomolar dose of this compound effectively represses adipogenesis at higher 3D scale, thus indicating the potential for LY2090314 to limit FAP-derived fat infiltrates in dystrophic muscles.