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Recurrent pneumonia is one of the most frequent reasons for referral to paediatric chest physicians. The diagnostic work-up is dependent on whether infection repeatedly occurs in the same lung lobe, or affects multiple lobes and/or different areas in different episodes. A 13-year-old girl was admitted with a second episode of right lower lobe pneumonia. The chest x-ray demonstrated an inhomogeneous opacity, without a clearly recognizable segmental distribution. A contrast-enhanced CT scan, was therefore performed that showed a polycyclic consolidation with blood supply from a systemic artery, originated from the thoracic aorta. A diagnosis of superinfection of an intralobar sequestration was made. The patient was treated with systemic antibiotics and, four weeks later, a segmental resection of the lesion was performed. The histological evaluation of the surgical specimen confirmed the diagnosis.

Well-being in a small group can be tied to how much its members interact. Small group tours are social occasions, and the discussion that ensues has been shown by ethnographers to be important for a more enriching experience. Increasing conversation can thus be seen as a way to improve social and psychological well-being.We present DramaTric, a mobile presentation system that delivers hour-long dramas to groups in museums. DramaTric gets sensor data from its environment and analyzes group behavior to deliver dynamically adapted dramatic scenes designed to stimulate conversation. Each scene contains slight differences in the story, leading visitors to understand their own drama only by talking with other group members. We describe an experiment with a full-scale drama to test if switching from presenting a drama with one technique to another results in more conversation. This shows that by using adaptive techniques we can modify social behavior, which can in turn promote well-being.

This study aimed to evaluate the diagnostic accuracy of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), midregional proatrial natriuretic peptide (MR-proANP) and midregional proadrenomedullin (MR-proADM) to distinguish bacterial from viral community-acquired pneumonia (CAP) and to identify severe cases in children hospitalized for radiologically confirmed CAP. Index test results were compared with those derived from routine diagnostic tests, i.e., white blood cell (WBC) counts, neutrophil percentages, and serum C-reactive protein (CRP) and procalcitonin (PCT) levels.This prospective, multicenter study was carried out in the most important children's hospitals (n = 11) in Italy and 433 otherwise healthy children hospitalized for radiologically confirmed CAP were enrolled. Among cases for whom etiology could be determined, CAP was ascribed to bacteria in 235 (54.3%) children and to one or more viruses in 111 (25.6%) children. A total of 312 (72.2%) children had severe disease.CRP and PCT had the best performances for both bacterial and viral CAP identification. The cut-off values with the highest combined sensitivity and specificity for the identification of bacterial and viral infections using CRP were ≥7.98 mg/L and ≤7.5 mg/L, respectively. When PCT was considered, the cut-off values with the highest combined sensitivity and specificity were ≥0.188 ng/mL for bacterial CAP and ≤0.07 ng/mL for viral CAP. For the identification of severe cases, the best results were obtained with evaluations of PCT and MR-proANP. However, in both cases, the biomarker cut-off with the highest combined sensitivity and specificity (≥0.093 ng/mL for PCT and ≥33.8 pmol/L for proANP) had a relatively good sensitivity (higher than 70%) but a limited specificity (of approximately 55%).This study indicates that in children with CAP, sTREM-1, MR-proANP, and MR-proADM blood levels have poor abilities to differentiate bacterial from viral diseases or to identify severe cases, highlighting that PCT maintains the main role at this regard.

Embryonic stem cells (ESCs) preserve the unique ability to differentiate into any somatic cell lineage while maintaining their self-renewal potential, relying on a complex interplay of extracellular signals regulating the expression/activity of pluripotency transcription factors and their targets. Leukemia inhibitory factor (LIF)-activated STAT3 drives ESCs' stemness by a number of mechanisms, including the transcriptional induction of pluripotency factors such as Klf4 and the maintenance of a stem-like epigenetic landscape. However, it is unknown if STAT3 directly controls stem-cell specific non-coding RNAs, crucial to balance pluripotency and differentiation. Applying a bioinformatic pipeline, here we identify Lncenc1 in mouse ESCs as an STAT3-dependent long non-coding RNA that supports pluripotency. Lncenc1 acts in the cytoplasm as a positive feedback regulator of the LIF-STAT3 axis by competing for the binding of microRNA-128 to the 3'UTR of the Klf4 core pluripotency factor mRNA, enhancing its expression. Our results unveil a novel non-coding RNA-based mechanism for LIF-STAT3-mediated pluripotency., (© The Author(s) 2023. Published by Oxford University Press.)

Osteoporosis is characterized by loss of bone mineral density and increased fracture risk. Reduction of hip fracture incidence is of major clinical importance. Hip protectors aim to attenuate the impact force transmitted to the femur upon falling, however different conclusions on their efficacy have been reported; some authors suggest this may be due to differences in compliance. The aim of this study was to apply an In Silico trial methodology to predict the effectiveness of hip protectors and its dependence on compliance. A cohort of 1044 virtual patients (Finite Element models of proximal femur) were generated. A Markov chain process was implemented to predict fracture incidence with and without hip protectors, by simulating different levels of compliance. At each simulated follow-up year, a Poisson distribution was randomly sampled to determine the number of falls sustained by each patient. Impact direction and force were stochastically sampled from a range of possible scenarios. The effect of wearing a hip protector was simulated by applying attenuation coefficients to the impact force (12.9 %, 19 % and 33.8 %, as reported for available devices). A patient was considered fractured when impact force exceeded the femur strength. Without hip protector, virtual patients experienced 66 ± 5 fractures in 10 years. Wearing the three devices, fracture incidence was reduced to 43 ± 4, 35 ± 4 and 17 ± 2 respectively, at full compliance. As expected, effectiveness was dependent on compliance. This In Silico trial technology can be applied in the future to test multiple interventions, optimise intervention strategies, improve clinical trial design and drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The risk of aseptic loosening in cementless hip stems can be reduced by improving osseointegration with osteoinductive coatings favoring long-term implant stability. Osseointegration is usually evaluated in vivo studies, which, however, do not reproduce the mechanically driven adaptation process. This study aims to develop an in silico model to predict implant osseointegration and the effect of induced micromotion on long-term stability, including a calibration of the material osteoinductivity with conventional in vivo studies. A Finite Element model of the tibia implanted with pins was generated, exploiting bone-to-implant contact measures of cylindrical titanium alloys implanted in rabbits' tibiae. The evolution of the contact status between bone and implant was modeled using a finite state machine, which updated the contact state at each iteration based on relative micromotion, shear and tensile stresses, and bone-to-implant distance. The model was calibrated with in vivo data by identifying the maximum bridgeable gap. Afterward, a push-out test was simulated to predict the axial load that caused the macroscopic mobilization of the pin. The bone-implant bridgeable gap ranged between 50 μm and 80 μm. Predicted push-out strength ranged from 19 N to 21 N (5.4 MPa-3.4 MPa) depending on final bone-to-implant contact. Push-out strength agrees with experimental measurements from a previous animal study (4 ± 1 MPa), carried out using the same implant material, coated, or uncoated. This method can partially replace in vivo studies and predict the long-term stability of cementless hip stems., (© 2024. The Author(s).)

Combined treatment with PTH(1-34) and mechanical loading confers increased structural benefits to bone than monotherapies. However, it remains unclear how this longitudinal adaptation affects the bone mechanics. This study quantified the individual and combined longitudinal effects of PTH(1-34) and mechanical loading on the bone stiffness and strength evaluated in vivo with validated micro-finite element (microFE) models. C57BL/6 mice were ovariectomised at 14-week-old and treated either with injections of PTH(1-34), compressive tibia loading or both interventions concurrently. Right tibiae were in vivo microCT-scanned every 2 weeks from 14 until 24-week-old. MicroCT images were rigidly registered to reference tibia and the cortical organ level (whole bone) and tissue level (midshaft) morphometric properties and bone mineral content were quantified. MicroCT images were converted into voxel-based homogeneous, linear elastic microFE models to estimate the bone stiffness and strength. This approach allowed us for the first time to quantify the longitudinal changes in mechanical properties induced by combined treatments in a model of accelerated bone resorption. Both changes of stiffness and strength were higher with co-treatment than with individual therapies, consistent with increased benefits with the tibia bone mineral content and cortical area, properties strongly associated with the tibia mechanics. The longitudinal data shows that the two bone anabolics, both individually and combined, had persistent benefit on estimated mechanical properties, and that benefits (increased stiffness and strength) remained after treatment was withdrawn., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Patients with breast cancer show altered expression of genes within the pectoralis major skeletal muscle cells of the breast. Through analyses of The Cancer Genome Atlas (TCGA)-breast cancer (BRCA), we identified three previously uncharacterized putative novel tumor suppressor genes expressed in normal muscle cells, whose expression was downregulated in breast tumors. We found that NEDD4 binding protein 2-like 1 ( N4BP2L1 ), pleckstrin homology domain-containing family A member 4 ( PLEKHA4 ), and brain-enriched guanylate kinase-associated protein ( BEGAIN ) that are normally highly expressed in breast myoepithelial cells and smooth muscle cells were significantly downregulated in breast tumor tissues of a cohort of 50 patients with this cancer. Our data revealed that the low expression of PLEKHA4 in patients with menopause below 50 years correlated with a higher risk of breast cancer. Moreover, we identified N4BP2L1 and BEGAIN as potential biomarkers of HER2-positive breast cancer. Furthermore, low BEGAIN expression in breast cancer patients with blood fat, heart problems, and diabetes correlated with a higher risk of this cancer. In addition, protein and RNA expression analysis of TCGA-BRCA revealed N4BP2L1 as a promising diagnostic protein biomarker in breast cancer. In addition, the in silico data of scRNA-seq showed high expression of these genes in several cell types of normal breast tissue, including breast myoepithelial cells and smooth muscle cells. Thus, our results suggest their possible tumor-suppressive function in breast cancer and muscle development., 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 © 2024 Dastsooz, Anselmi, Lauria, Cicconetti, Proserpio, Mohammadisoleimani, Firoozi, Mansoori, Haghi-Aminjan, Caizzi and Oliviero.)

Gut microbiota is responsible for essential functions in human health. Several communication axes between gut microbiota and other organs via neural, endocrine, and immune pathways have been described, and perturbation of gut microbiota composition has been implicated in the onset and progression of an emerging number of diseases. Here, we analyzed peripheral nerves, dorsal root ganglia (DRG), and skeletal muscles of neonatal and young adult mice with the following gut microbiota status: a) germ-free (GF), b) gnotobiotic, selectively colonized with 12 specific gut bacterial strains (Oligo-Mouse-Microbiota, OMM12), or c) natural complex gut microbiota (CGM). Stereological and morphometric analyses revealed that the absence of gut microbiota impairs the development of somatic median nerves, resulting in smaller diameter and hypermyelinated axons, as well as in smaller unmyelinated fibers. Accordingly, DRG and sciatic nerve transcriptomic analyses highlighted a panel of differentially expressed developmental and myelination genes. Interestingly, the type III isoform of Neuregulin1 (NRG1), known to be a neuronal signal essential for Schwann cell myelination, was overexpressed in young adult GF mice, with consequent overexpression of the transcription factor Early Growth Response 2 ( Egr2 ), a fundamental gene expressed by Schwann cells at the onset of myelination. Finally, GF status resulted in histologically atrophic skeletal muscles, impaired formation of neuromuscular junctions, and deregulated expression of related genes. In conclusion, we demonstrate for the first time a gut microbiota regulatory impact on proper development of the somatic peripheral nervous system and its functional connection to skeletal muscles, thus suggesting the existence of a novel 'Gut Microbiota-Peripheral Nervous System-axis.'

Skeletal muscle atrophy is a hallmark of cachexia, a wasting condition typical of chronic pathologies, that still represents an unmet medical need. Bone morphogenetic protein (BMP)-Smad1/5/8 signaling alterations are emerging drivers of muscle catabolism, hence, characterizing these perturbations is pivotal to develop therapeutic approaches. We identified two promoters of "BMP resistance" in cancer cachexia, specifically the BMP scavenger erythroferrone (ERFE) and the intracellular inhibitor FKBP12. ERFE is upregulated in cachectic cancer patients' muscle biopsies and in murine cachexia models, where its expression is driven by STAT3. Moreover, the knock down of Erfe or Fkbp12 reduces muscle wasting in cachectic mice. To bypass the BMP resistance mediated by ERFE and release the brake on the signaling, we targeted FKBP12 with low-dose FK506. FK506 restores BMP-Smad1/5/8 signaling, rescuing myotube atrophy by inducing protein synthesis. In cachectic tumor-bearing mice, FK506 prevents muscle and body weight loss and protects from neuromuscular junction alteration, suggesting therapeutic potential for targeting the ERFE-FKBP12 axis., Competing Interests: Declaration of interests E.M., V.R., L.S., and P.E.P. declare to be inventors of the patent PCTIB2022050175 (WO2022/149113)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The influence of aging on intestinal stem cells and their niche can explain underlying causes for perturbation in their function observed during aging. Molecular mechanisms for such a decrease in the functionality of intestinal stem cells during aging remain largely undetermined. Using transcriptome-wide approaches, our study demonstrates that aging intestinal stem cells strongly upregulate antigen presenting pathway genes and over-express secretory lineage marker genes resulting in lineage skewed differentiation into the secretory lineage and strong upregulation of MHC class II antigens in the aged intestinal epithelium. Mechanistically, we identified an increase in proinflammatory cells in the lamina propria as the main source of elevated interferon gamma (IFNγ) in the aged intestine, that leads to the induction of Stat1 activity in intestinal stem cells thus priming the aberrant differentiation and elevated antigen presentation in epithelial cells. Of note, systemic inhibition of IFNγ-signaling completely reverses these aging phenotypes and reinstalls regenerative capacity of the aged intestinal epithelium., (© 2023. Springer Nature Limited.)

The nuclear receptor NR2F1 acts as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, mutations in the NR2F1 gene cause Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including vision impairment, intellectual disability and autistic traits. In this study, we identified, by genome-wide and in silico analyses, a set of nuclear-encoded mitochondrial genes as potential genomic targets under direct NR2F1 transcriptional control in neurons. By combining mouse genetic, neuroanatomical and imaging approaches, we demonstrated that conditional NR2F1 loss of function within the adult mouse hippocampal neurogenic niche results in a reduced mitochondrial mass associated with mitochondrial fragmentation and downregulation of key mitochondrial proteins in newborn neurons, the genesis, survival and functional integration of which are impaired. Importantly, we also found dysregulation of several nuclear-encoded mitochondrial genes and downregulation of key mitochondrial proteins in the brain of Nr2f1-heterozygous mice, a validated BBSOAS model. Our data point to an active role for NR2F1 in the mitochondrial gene expression regulatory network in neurons and support the involvement of mitochondrial dysfunction in BBSOAS pathogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Long non-coding RNAs (lncRNAs) regulate gene expression through different molecular mechanisms, including DNA binding via the formation of RNA:DNA:DNA triple helices (TPXs). Despite the increasing amount of experimental evidence, TPXs investigation remains challenging. Here we present 3plex , a software able to predict TPX interactions in silico . Given an RNA sequence and a set of DNA sequences, 3plex integrates 1) Hoogsteen pairing rules that describe the biochemical interactions between RNA and DNA nucleotides, 2) RNA secondary structure prediction and 3) determination of the TPX thermal stability derived from a collection of TPX experimental evidences. We systematically collected and uniformly re-analysed published experimental lncRNA binding sites on human and mouse genomes. We used these data to evaluate 3plex performance and showed that its specific features allow a reliable identification of TPX interactions. We compared 3plex with the other available software and obtained comparable or even better accuracy at a fraction of the computation time. Interestingly, by inspecting collected data with 3plex we found that TPXs tend to be shorter and more degenerated than previously expected and that the majority of analysed lncRNAs can directly bind to the genome by TPX formation. Those results suggest that an important fraction of lncRNAs can exert its biological function through this mechanism. The software is available at https://github.com/molinerisLab/3plex., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of β-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of β-Catenin depends on its ability to localize in and stabilize the β-Catenin destruction complex, promoting enhanced β-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the β-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response., (© 2023. The Author(s).)

Epithelial cells that participated in wound repair elicit a more efficient response to future injuries, which is believed to be locally restricted. Here we show that cell adaptation resulting from a localized tissue damage has a wide spatial impact at a scale not previously appreciated. We demonstrate that a specific stem cell population, distant from the original injury, originates long-lasting wound memory progenitors residing in their own niche. Notably, these distal memory cells have not taken part in the first healing but become intrinsically pre-activated through priming. This cell state, maintained at the chromatin and transcriptional level, leads to an enhanced wound repair that is partially recapitulated through epigenetic perturbation. Importantly wound memory has long-term harmful consequences, exacerbating tumourigenesis. Overall, we show that sub-organ-scale adaptation to injury relies on spatially organized memory-dedicated progenitors, characterized by an actionable cell state that establishes an epigenetic field cancerization and predisposes to tumour onset., (© 2023. The Author(s).)

The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation of key developmental regulatory regions at epiblast-like cells (EpiLCs) provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing we dissected the specific role of DNMT3B in cell fate. We identify DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect EpiLCs towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, downmodulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination., (© 2023. The Author(s).)

Dysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an "exon definition" mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS- from CD28-mediated costimulation during early T-cell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4 + T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes., 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 Gigliotti, Boggio, Favero, Incarnato, Santoro, Oliviero, Rojo, Zucchelli, Persichetti, Baldanzi, Dianzani and Corà.)

Epithelial-mesenchymal transition (EMT) is a complex and pivotal process involved in organogenesis and is related to several pathological processes, including cancer and fibrosis. During heart development, EMT mediates the conversion of epicardial cells into vascular smooth muscle cells and cardiac interstitial fibroblasts. Here, we show that the oncogenic transcription factor EB (TFEB) is a key regulator of EMT in epicardial cells and that its genetic overexpression in mouse epicardium is lethal due to heart defects linked to impaired EMT. TFEB specifically orchestrates the EMT-promoting function of transforming growth factor (TGF) β, and this effect results from activated transcription of thymine-guanine-interacting factor (TGIF)1, a TGFβ/Smad pathway repressor. The Tgif1 promoter is activated by TFEB, and in vitro and in vivo findings demonstrate its increased expression when Tfeb is overexpressed. Furthermore, Tfeb overexpression in vitro prevents TGFβ-induced EMT, and this effect is abolished by Tgif1 silencing. Tfeb loss of function, similar to that of Tgif1, sensitizes cells to TGFβ, inducing an EMT response to low doses of TGFβ. Together, our findings reveal an unexpected function of TFEB in regulating EMT, which might provide insights into injured heart repair and control of cancer progression., (© 2022. The Author(s).)

Background: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function., Results: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina., Conclusions: Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions., (© 2022. The Author(s).)

During skeletal myogenesis, the zinc-finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro-differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA-mediated gene silencing in C2C12 myoblasts and whole-transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Interventions for bone diseases (e.g. osteoporosis) require testing in animal models before clinical translation and the mouse tibia is among the most common tested anatomical sites. In vivo micro-Computed Tomography (microCT) based measurements of the geometrical and densitometric properties are non-invasive and therefore constitute an important tool in preclinical studies. Moreover, validated micro-Finite Element (microFE) models can be used for predicting the bone mechanical properties non-invasively. However, considering that the image processing pipeline requires operator-dependant steps, the reproducibility of these measurements has to be assessed. The aim of this study was to evaluate the intra- and inter-operator reproducibility of several bone parameters measured from microCT images. Ten in vivo microCT images of the right tibia of five mice (at 18 and 22 weeks of age) were processed. One experienced operator (intra-operator analysis) and three different operators (inter-operator) aligned each image to a reference through a rigid registration and selected a volume of interest below the growth plate. From each image the following parameters were measured: total bone mineral content (BMC) and density (BMD), BMC in 40 subregions (ten longitudinal sections, four quadrants), microFE-based stiffness and failure load. Intra-operator reproducibility was acceptable for all parameters (precision error, PE < 3.71%), with lowest reproducibility for stiffness (3.06% at week 18, 3.71% at week 22). The inter-operator reproducibility was slightly lower (PE < 4.25%), although still acceptable for assessing the properties of most interventions. The lowest reproducibility was found for BMC in the lateral sector at the midshaft (PE = 4.25%). Densitometric parameters were more reproducible than most standard morphometric parameters calculated in the proximal trabecular bone. In conclusion, microCT and microFE models provide reproducible measurements for non-invasive assessment of the mouse tibia properties., 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 Oliviero, Cheong, Roberts, Orozco Diaz, Griffiths, Bellantuono and Dall’Ara.)

The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy., (© 2022. The Author(s).)

In the human brain, long non-coding RNAs (lncRNAs) are widely expressed in an exquisitely temporally and spatially regulated manner, thus suggesting their contribution to normal brain development and their probable involvement in the molecular pathology of neurodevelopmental disorders (NDD). Bypassing the classic protein-centric conception of disease mechanisms, some studies have been conducted to identify and characterize the putative roles of non-coding sequences in the genetic pathogenesis and diagnosis of complex diseases. However, their involvement in NDD, and more specifically in intellectual disability (ID), is still poorly documented and only a few genomic alterations affecting the lncRNAs function and/or expression have been causally linked to the disease endophenotype. Considering that a significant fraction of patients still lacks a genetic or molecular explanation, we expect that a deeper investigation of the non-coding genome will unravel novel pathogenic mechanisms, opening new translational opportunities. Here, we present evidence of the possible involvement of many lncRNAs in the etiology of different forms of ID and NDD, grouping the candidate disease-genes in the most frequently affected cellular processes in which ID-risk genes were previously collected. We also illustrate new approaches for the identification and prioritization of NDD-risk lncRNAs, together with the current strategies to exploit them in diagnosis.

The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear (1,2). We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In the tumor microenvironment, Cancer Associated Fibroblasts (CAFs) become activated by cancer cells and increase their secretory activity to produce soluble factors that contribute to tumor cells proliferation, invasion and dissemination to distant organs. The pro-tumorigenic transcription factor STAT3 and its canonical inducer, the pro-inflammatory cytokine IL-6, act conjunctly in a positive feedback loop that maintains high levels of IL-6 secretion and STAT3 activation in both tumor and stromal cells. Here, we demonstrate that STAT3 is essential for the pro-tumorigenic functions of murine breast cancer CAFs both in vitro and in vivo, and identify a STAT3 signature significantly enriched for genes encoding for secreted proteins. Among these, ANGPTL4, MMP13 and STC-1 were functionally validated as STAT3-dependent mediators of CAF pro-tumorigenic functions by different approaches. Both in vitro and in vivo CAFs activities were moreover impaired by MMP13 inhibition, supporting the feasibility of a therapeutic approach based on inhibiting STAT3-induced CAF-secreted proteins. The clinical potential of such an approach is supported by the observation that an equivalent CAF-STAT3 signature in humans is expressed at high levels in breast cancer stromal cells and characterizes patients with a shorter disease specific survival, including those with basal-like disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

While many single-cell proteomics techniques have been rapidly developed over the past decade, flow cytometry still remains the pillar of single-cell protein analysis, as it allows to rapidly analyze and characterize protein expression in millions of cells.In this chapter, we will describe the main steps to prepare and acquire samples for flow cytometry, with particular focus on the setup of the right controls that are instrumental in analyzing and interpreting the results., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)