Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited therapeutic options and high recurrence rates. Given the high immunogenicity of TNBC, immune checkpoint inhibitors have been recently incorporated into its treatment. However, immunotherapy extends survival in only a subset of patients, denoting an urgent need to better understand the regulatory mechanisms that shape TNBC-immune microenvironment interactions. In this study, we identified the promyelocytic leukemia PML protein as a new modulator of TNBC immunogenicity. By measuring transcriptional responses to PML perturbation in TNBC cells we found that PML supports chronic activation of inflammatory pathways, resulting in constitutive upregulation of immunosuppressive genes, including PD-L1. Accordingly, PML silencing in a TNBC mouse model led to a substantial reorganization of the tumor microenvironment, with accumulation of various T cell populations linked to immune activation and tumor rejection. Importantly, analysis of human TNBC datasets linked high PML levels to chronic expression of inflammatory gene sets and impaired immune activation in the tumor microenvironment. In parallel, mechanistic studies, including PML interactome mapping and genome-wide DNA methylation profiling, showed that PML cooperates with DNMT1 to repress HLA gene expression via CpG methylation, thereby impairing tumor recognition and T cell-mediated killing. In sum, this study identifies PML as a key mediator of immune evasion in TNBC by concomitantly fostering an immunosuppressive tumor microenvironment and suppressing antigen presentation and tumor recognition by T cells. As PML-targeting therapies are clinically available for leukemia patients, our findings position PML as a promising actionable target to enhance immune responses and improve outcomes for patients with TNBC.

CONTEXT:Non-obstructive azoospermia (NOA) is the most severe form of male infertility, affecting 1% of all men, with a clinical picture characterized by no sperm production, hyalinization of the basal membrane of the seminiferous tubules, primary hypogonadism, and earlier onset of age-related comorbidities compared with fertile men. NOA is also characterized by etiologic heterogeneity and the non-genetic form has higher incidence of testicular germ cell cancer (TGCC) compared to the forms with genetic abnormalities. OBJECTIVE:We aimed to establish molecular pathways in the testicular somatic cells that are either shared or specific for non-genetic and genetic forms of NOA, such as complete androgen insensitivity syndrome (CAIS) and Klinefelter syndrome (KS). METHODS:We performed single-cell RNA sequencing of the testicular somatic cells of an individual with CAIS, and data integration with published scRNA-seq datasets of testis with normal spermatogenesis, NOA, KS, and germinal testicular cancer. Detailed clinical data of the CAIS patient, testosterone and estradiol levels in age-matched men (120 fertile, 155 infertile, 116 NOA, 18 KS, and 343 with TGCC) were analyzed. RESULTS:In all conditions, Leydig cells are immature and senescent, but those of NOA associated with primary hypogonadism depict the highest expression of transcripts associated with the seminoma microenvironment, including estrogen-responsive genes. An oncological transcriptional signature in the Leydig cells has been confirmed at the systemic levels by showing a prognostic role of the decreasing testosterone/estradiol ratio for TGCC in men with non-genetic NOA. CONCLUSION:This study offers molecular insights into the prediction of TGCC in persons with NOA and eligibility for the use of aromatase inhibitors.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the 3'-UTR of the DMPK gene that lead to nuclear foci accumulation and splicing defects. Circular RNAs (circRNAs) are emerging regulators of muscular disorders, but their role in DM1 remains largely unknown. By analyzing available RNA-sequencing datasets from DM1 patients, followed by validation in patients and matching control muscle biopsies, we identified seven circRNAs that were significantly increased in DM1 muscles and displayed high circular-to-linear isoform ratios. Among them, circARHGAP10 correlated positively with CTG repeat length and inversely with muscle strength, indicating its potential as a biomarker. Silencing of circARHGAP10 in DM1 myogenic cells reduced DMPK expression, decreased nuclear foci, and partially rescued normal splicing. Bioinformatics prediction and pull-down of circARHGAP10 indicated that circARHGAP10 binds miR-409-3p. circARHGAP10 and miR-409-3p were both found to be upregulated in DM1 muscle biopsies and silencing of circARHGAP10 led to the downregulation of miR-409-3p, indicating their co-regulation. Interestingly, miR-409-3p overexpression blocked the beneficial effects of circARHGAP10 silencing on DMPK levels, foci, and splicing. Thus, circARHGAP10-dependent regulation of DM1-associated mechanisms is mediated, at least in part, via interaction with miR-409-3p. In conclusion, circARHGAP10 exhibits promising potential as a biomarker and therapeutic target for DM1.

Circular RNAs (circRNAs) are involved in the pathogenesis of several cardiovascular diseases, including heart failure. In this study, we report that circular PVT1 (circPVT1) was upregulated in the left ventricle of 31 ischemic heart failure patients compared to 11 non-ischemic controls. RNA sequencing analysis following circPVT1 knockdown in immortalized human cardiomyocytes identified differentially expressed genes, mainly involved in fibrosis. Notably, in human cardiac fibroblasts, circPVT1 expression significantly increased after TGF-β1 treatment and circPVT1 silencing attenuated the levels of pro-fibrotic markers induced by TGF-β1. RNA pull-down assays validated the interaction between circPVT1 and two fibrosis-related miRNAs, miR-30a-5p and miR-125b-5p. The levels of these miRNAs were not altered upon circPVT1 knockdown. However, the expression of their mRNA targets was deregulated upon circPVT1 silencing, suggesting that circPVT1 modulates miRNA cellular bioavailability. Accordingly, inhibition of either miR-30a-5p or miR-125b-5p restored the expression of TGF-β1-induced pro-fibrotic markers following circPVT1 silencing, indicating that both miR-30a-5p and miR-125b-5p act as downstream effectors of circPVT1 in cardiac fibroblast activation. In conclusion, these findings highlight a pro-fibrotic role for circPVT1, which can regulate cardiac fibroblast activation interacting with the anti-fibrotic miR-30a-5p and miR-125b-5p. The modulation of circPVT1 expression may represent a potential strategy to reduce cardiac fibrosis and remodeling.

Circular RNAs (circRNAs) are involved in the pathogenesis of several cardiovascular diseases, including heart failure. In this study, we report that circular PVT1 (circPVT1) was upregulated in the left ventricle of 31 ischemic heart failure patients compared to 11 non-ischemic controls. RNA-Sequencing analysis following circPVT1 knockdown in immortalized human cardiomyocytes identified differentially expressed genes mainly involved in fibrosis. Notably, in human cardiac fibroblasts, circPVT1 expression significantly increased after TGF-β1 treatment and circPVT1 silencing attenuated the levels of pro-fibrotic markers induced by TGF-β1. RNA pull-down assays validated the interaction between circPVT1 and two fibrosis-related miRNAs, miR-30a-5p and miR-125b-5p. The levels of these miRNAs were not altered upon circPVT1 knockdown. However, the expression of their mRNA targets was deregulated upon circPVT1 silencing, suggesting that circPVT1 modulates miRNA cellular bioavailability. Accordingly, inhibition of either miR-30a-5p or miR-125b-5p restored the expression of TGF-β1-induced pro-fibrotic markers following circPVT1 silencing, indicating that both miR-30a-5p and miR-125b-5p act as downstream effectors of circPVT1 in cardiac fibroblast activation. In conclusion, these findings highlight a pro-fibrotic role for circPVT1, which can regulate cardiac fibroblast activation via sponging the anti-fibrotic miR-30a-5p and miR-125b-5p. The modulation of circPVT1 expression may represent a potential strategy to reduce cardiac fibrosis and remodeling.

Multiple myeloma (MM) is linked to chronic NF-κB activity in myeloma cells, but this activity is generally considered a cell-autonomous property of the cancer cells. The precise extent of NF-κB activation and the contributions of the physical microenvironment and of cell-to-cell communications remain largely unknown. By quantitative immunofluorescence, we found that NF-κB is mildly and heterogeneously activated in a fraction of MM cells in human BMs, while only a minority of MM cells shows a strong activation. To gain quantitative insights on NF-κB activation in living MM cells, we combined advanced live imaging of endogenous p65 Venus-knocked-in in MM.1S and HS-5 cell lines to model MM and mesenchymal stromal cells (MSCs), cell co-cultures, microfluidics and custom microbioreactors to mimic the 3D-interactions within the bone marrow (BM) microenvironment. We found that i) reciprocal MM-MSC paracrine crosstalk and cell-to-scaffold interactions shape the inflammatory response in the BM; ii) the pro-inflammatory cytokine IL-1β, abundant in MM patients' plasma, activates MSCs, whose paracrine signals are responsible for strong NF-κB activation in a minority of MM cells; iii) IL-1β, but not TNF-α, activates NF-κB in vivo in BM-engrafted MM cells, while its receptor inhibitor Anakinra reduces the global NF-κB activation. We propose that NF-κB activation in the BM of MM patients is mild, restricted to a minority of cells and modulated by the interplay of restraining physical microenvironmental cues and activating IL-1β-dependent stroma-to-MM crosstalk.

Noncoding RNAs (ncRNAs), which include circular RNAs (circRNAs) and microRNAs (miRNAs), regulate the development of cardiovascular diseases (CVD). Notably, circRNAs can interact with miRNAs, influencing their specific mRNA targets’ levels and shaping a competing endogenous RNAs (ceRNA) network. However, these interactions and their respective functions remain largely unexplored in ischemic heart failure (IHF). This study is aimed at identifying circRNA-centered ceRNA networks in non-end-stage IHF. Approximately 662 circRNA-miRNA-mRNA interactions were identified in the heart by combining state-of-the-art bioinformatics tools with experimental data. Importantly, KEGG terms of the enriched mRNA indicated CVD-related signaling pathways. A specific network centered on circBPTF was validated experimentally. The levels of let-7a-5p, miR-18a-3p, miR-146b-5p, and miR-196b-5p were enriched in circBPTF pull-down experiments, and circBPTF silencing inhibited the expression of HDAC9 and LRRC17, which are targets of miR-196b-5p. Furthermore, as suggested by the enriched pathway terms of the circBPTF ceRNA network, circBPTF inhibition elicited endothelial cell cycle arrest. circBPTF expression increased in endothelial cells exposed to hypoxia, and its upregulation was confirmed in cardiac samples of 36 end-stage IHF patients compared to healthy controls. In conclusion, circRNAs act as miRNA sponges, regulating the functions of multiple mRNA targets, thus providing a novel vision of HF pathogenesis and laying the theoretical foundation for further experimental studies.

Mutations of Cystic Fibrosis Transmembrane conductance Regulator ( CFTR ) lead to Cystic Fibrosis (CF), but the substantial phenotypic variations are determined by non- CFTR allelic diversity. To map novel disease phenotypes in a CF mouse model, we used Collaborative Cross (CC) mice, a highly genetically diverse mouse resource population. ΔF508-Cftr homozygosity produced a fully penetrant lethal phenotype by eight weeks in two CC lines. The lethality of CC006 ΔF508/ΔF508 was fully prenatal while CC037 ΔF508/ΔF508 showed either prenatal or postnatal lethality. Novel phenotypes of CC037 ΔF508/ΔF508 were revealed early in life including respiratory and systemic inflammatory profiles, and blood, bone marrow, pancreas, heart, and reproductive tract pathologies. Severe intestinal blockage was observed as common in other CF mouse models. These results suggest that the exploration of CF disease phenotypes in a mouse population with diverse genetic profiles is needed to map the genetic origin of currently unidentified disease traits and their potential translation to humans.

Translocations producing rearranged versions of the transcription factor double homeobox 4 (DUX4-r) are one of the most frequent causes of B cell acute lymphoblastic leukemia (B-ALL). DUX4-r retains the DNA binding domain of wild-type DUX4 but is truncated on the C-terminal transcription activation domain. The precise mechanism through which DUX4-r causes leukemia is unknown, and no targeted therapy is currently available. We found that the rearrangement leads to both a loss and a gain of function in DUX4-r. Loss of CBP/EP300 transcriptional coactivator interaction leads to an inability to bind and activate repressed chromatin. Concurrently, a gain of interaction with the general transcription factor 2 I (GTF2I) redirects DUX4-r toward leukemogenic targets. This neomorphic activity exposes an Achilles' heel whereby DUX4-r-positive leukemia cells are exquisitely sensitive to GTF2I targeting, which inhibits DUX4-r leukemogenic activity. Our work elucidates the molecular mechanism through which DUX4-r causes leukemia and suggests a possible therapeutic avenue tailored to this B-ALL subtype.

Abstract Background BACE1-antisense RNA (BACE1-AS) is a lncRNA antisense to the Beta-Secretase-1 (BACE1) gene encoding a key enzyme in the production of the β-amyloid peptide, associated to Alzheimer's disease (AD). In a previous study1, we showed that the BACE1-AS/BACE1 axis is activated in heart failure (HF) patients, leading to β-amyloid accumulation in failing hearts. Accordingly, BACE1-AS expression in different cultured cardiac cell types induced the expression of BACE1 and β-amyloid that, in turn, triggered apoptosis. The mechanisms underlying BACE1-AS action are not yet fully elucidated. Indeed, current models based on miRNA “sponging” or “masking” inducing BACE1 post-transcriptional stabilization may not recapitulate all BACE1-AS functions. Purpose Our aim is to investigate the molecular mechanisms regulated by BACE1-AS in heart failure disease. Methods BACE1-AS-pull-down, followed by RNA-Seq, was used to identify RNAs interacting with BACE1-AS in AC16 cardiomyocytes. Moreover, accessible chromatin sites induced by BACE1-AS overexpression were assayed by ATAC-Seq. Results BACE1-AS pull-down identified 698 BACE1-AS interacting RNAs. Among these enriched transcripts, 69 were mapping to genomic enhancer regions that have been found to be hypo-methylated in AD brains2. After qPCR validation of the interactions identified by RNA-seq, the modulation of SEMA4D, ABCG1, GFRA2 and RIMBP2, which were under the control of a subset of these enhancers, was assayed upon BACE1-AS overexpression in cardiomyocytes. It was found that their expression was induced, supporting the functional interaction between BACE1-AS and the identified enhancer loci. To gain further insight into BACE1-AS function in cardiomyocytes, accessible chromatin sites induced by BACE1-AS overexpression were assayed by ATAC-Seq. Interestingly, 17 regions identified by this approach overlapped with enhancers that are hypo-methylated in AD brains. One of the accessible chromatin sites was the locus encompassing RNF214/BACE1/BACE1-AS that maps to active enhancer marks, indicating that BACE1-AS may regulate the expression of BACE1 transcriptionally. Conclusion Collectively, these data suggest BACE1-AS as a node of shared disease-mechanisms between heart failure and AD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health

Mechanisms underlying severe male infertility are still largely elusive. However, recently, a single-cell transcription study by our group identified several differentially expressed coding genes in all the somatic cell types in testes of patients with idiopathic germ cell aplasia (iGCA). Here, we leverage this work by extending the analysis also to the non-coding portion of the genome. As a result, we found that 43 LncRNAs were differentially expressed in the somatic cells of these patients. Interestingly, a significant portion of the overexpressed LncRNAs was found to be a target of TAF9B, a transcription factor known to be involved in germ cell survival. Moreover, several overexpressed LncRNAs were also found to be activated in a mouse model of Sertoli cells treated with bisphenol A, a widespread environmental contaminant, long suspected to impair male fertility. Finally, a literature search for MEG3, a maternally imprinted LncRNA overexpressed as well in our patients, found it to be involved, among other things, in obesity and inflammation, known comorbidities of iGCA, ultimately suggesting that our findings deepen the understanding of the molecular insights coupled not only to the pathogenesis, but also to the clinical course of this class of patients.

Abstract Introduction Circular RNAs (circRNAs) are an emerging class of non-coding RNAs originating from the splicing and circularization of pre-mRNAs and long non-coding RNAs. CircRNAs can regulate transcription and splicing, sequester microRNAs acting as “sponge” and inducing the respective targets, and bind to RNA binding proteins. CircRNAs have been found deregulated in several cardiovascular diseases, including heart failure (HF). However, incomplete and sometimes contradictory results have been reported on their regulation and function in HF, possibly for the heterogeneity of patients analyzed, indicating that our understanding of the regulation and role of cardiac circRNAs is still very limited. Purpose We aim to identify novel circRNA candidates deregulated in ischemic HF and to functionally characterize them in HF. Methods To define the circRNA expression pattern in ischemic HF, a high-depth RNA-seq was performed on the remote zone of left ventricle (LV) samples of 20 non end-stage ischemic HF patients with reduced LV ejection fraction and 20 matched controls. Differentially expressed circRNAs identified using CIRIquant tool were validated by qRT-PCR. Samples from the border zone of non end-stage HF patients and LV biopsies from end-stage HF patients were also used to measure circRNAs expression. Results By RNA-seq, we identified 17 circRNAs as differentially expressed with FDR<0.1. qRT-PCR results confirmed the differential expression of 4 circRNAs, cSLC6A6, cNAA16, cMLIP and cHDCA9, which were upregulated in HF samples (t-test, p-value <0.001). To evaluate circRNAs deregulated independently of their linear counterparts, we also estimated the ratio between circular and linear isoforms. cSLC6A6, cMLIP and cHDCA9 showed also a statistically significant (t-test, p-value <0.002) increase of the circular-to-linear ratio between HF and control patients. To complement the unbiased strategy of RNA-seq, we evaluated the modulation of 18 candidate circRNAs identified by a literature analysis as dysregulated in ischemic or not-ischemic cardiomyopathies or in biological mechanisms relevant for ischemic HF. cPVT1, cANKRD17, cBPTF, displaying a concordant deregulation in different stages of the disease (non end-stage and end-stage HF) and in different regions of LV myocardium (remote and border zone), were chosen for further analysis. Using siRNAs targeting the backsplice junction, cPVT1 was specifically knockdown in a human cardiomyocyte cell line. RNA-seq analysis of cPVT1 knockdown samples identified 3577 significantly differentially expressed genes (FDR<0.01). Gene ontology and pathway analysis showed that these genes were mainly involved in the formation of extracellular matrix, fibrosis and in cellular senescence, suggesting the involvement of cPVT1 in these HF-related disease mechanisms. Conclusion We identified new deregulated circRNAs in ischemic HF patients that might play a pathogenic role in HF. Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Italian Ministry of Health, Ricerca Corrente 2021-2023 “CircularRNAs in heart failure: regulation, function and interaction with the transcriptome”

Molecular mechanisms associated with human germ cell aplasia in infertile men remain undefined. Here we perform single-cell transcriptome profiling to highlight differentially expressed genes and pathways in each somatic cell type in testes of men with idiopathic germ cell aplasia. We identify immaturity of Leydig cells, chronic tissue inflammation, fibrosis, and senescence phenotype of the somatic cells, as well markers of chronic inflammation in the blood. We find that deregulated expression of parentally imprinted genes in myoid and immature Leydig cells, with relevant changes in the ratio of Lamin A/C transcripts and an active DNA damage response in Leydig and peritubular myoid cells are also indicative of senescence of the testicular niche. This study offers molecular insights into the pathogenesis of idiopathic germ cell aplasia.

Lipids are fundamental components of biological organisms and have important applications in the pharmaceutical, food, and cosmetics industries. Thus, it is important that young students and the general public properly understand the basic properties of lipids and how these relate to their biological and industrial roles. Here, we use molecular dynamics computer simulations and a simple, safe, and inexpensive popular hands-on activity, to communicate to participants why and how lipid molecules play a fundamental role in all living organisms and in our bodies. The activity is called "Fats' Love-Hate Relationships", to highlight how the different parts of amphiphilic lipids interact with water. This "love-hate relationship" is vital to the biological functions of lipids and drives the formation of lipid structures that can be visualized at molecular scale with the computer simulations. The participants were encouraged to investigate the interactions between milk lipids and soap surfactants, creating beautiful complex artwork that they could then take home. The hands-on activity was accompanied by a video of a molecular simulation that illustrates milk-soap interactions at a molecular scale and helps to explain how the amphiphilicity of lipids creates the beautiful artwork at a molecular level. The outreach activity has been performed in science festivals and in classrooms and has been well received by participants of all ages with multiple learner comprehension levels (primary and secondary school students and the general public). By combining molecular simulation, explanations of the amphiphilic structure of the lipids, and an engaging hands-on activity, we explained how lipids interact with water and surfactants and inspired discussions on the link between the structure of the lipids and their biological function, namely, their structural and protective roles as a key component of cell membranes.

The skin surface, our first barrier against the external environment, is covered by the sebum oil, a lipid film composed of sebaceous and epidermal lipids, which is important in the regulation of the hydration level of our skin. Here, we investigate the pathways leading to the transfer of epidermal lipids from the skin lipid bilayer to the sebum. We show that the sebum triglycerides, a major component of sebum, interact strongly with the epidermal lipids and extract them from the bilayer. Using microsecond time scale molecular dynamics simulations, we identify and quantify the free energy associated with the skin lipid extraction process.

The SC membrane was constructed using the open source software Packmol1 to place 288 SC lipid molecules in each of the two leaflets of a bilayer, symmetrically about the bilayer mid-plane. Packmol was used to place the lipid and water molecules inside rectangular regions with a random distributions. To better reproduce the bilayer structure, the top oxygen atoms of the OH group of the hydrophilic head groups and the terminal carbon atom of the longest acyl tail of each lipid molecule were constrained to lie above or under predefined planes (as shown in Fig. S1). A distance of 1.4 Å between molecules was adopted to avoid overlapping between them. The system was then equilibrated in water, following the procedure described in ref. 2: i.e. the system was run for 5 ns with the stratum corneum molecules fixed (to hydrate the bilayer properly), and then for 2 ns with only the terminal methyl group on all the lipid tails frozen, so that the rest of the molecules reorient themselves to accommodate the water environment. A further 50 ns NPT equilibration was run before joining the SC bilayer to the TG slabs. See the main paper for details on the simulation parameters.

We investigate the general dependence of the thermal transport across nanoparticle-fluid interfaces using molecular dynamics computations. We show that the thermal conductance depends strongly both on the wetting characteristics of the nanoparticle-fluid interface and on the nanoparticle size. Strong nanoparticle-fluid interactions, leading to full wetting states in the host fluid, result in high thermal conductances and efficient interfacial transport of heat. Weak interactions result in partial drying or full drying states, and low thermal conductances. The variation of the thermal conductance with particle size is found to depend on the fluid-nanoparticle interactions. Strong interactions coupled with large interfacial curvatures lead to optimum interfacial heat transport. This complex dependence can be modelled using an equation that includes the interfacial curvature as a parameter. In this way, we rationalise the existing experimental and computer simulation results and show that the thermal transport across nanoscale interfaces is determined by the correlations of both interfacial curvature and nanoparticle-fluid interactions.

In recent years, sebum oil has been found to play a key role in the regulation of the hydration of the outermost layer of the skin, the stratum corneum. Understanding how a major component of the sebum oil, the triglyceride tri-cis-6-hexadecenoin (TG), interacts with water is an important step in gaining insight into the water regulation function of the sebum oil. Here we use molecular dynamics simulations to investigate the structural and interfacial properties of TG in bulk and at the air and water interface. Our model performs very well in reproducing experimental results, such as density, surface tensions and surface pressure area isotherms. We show that triglyceride molecules in the liquid phase assemble together, through the glycerol group, forming a single percolating network. TG-air interfaces orient the lipids with the interface enriched with the hydrophobic tails and the glycerol groups buried inside. When in contact with water, the TG molecules at the interface orient the glycerol group towards the water phase and adopt a characteristic trident conformation. Water is shown to penetrate the TG layer thanks to the interaction with the oxygen atoms of the TG molecules, which acts as a pathway for water diffusion. The activation energy for the passage of water is found to be ≈9.5kBT at 310 K, showing that the layer is permeable to water diffusion.