Biology subjects

Albani, S.

Publications and source records attributed to Albani, S..

14 recordsLinked to original sources

bioRxiv2026

Multi-omics and functional analysis of a bioengineered vascularized pancreatic cancer model reveal an immunosuppressive and therapy-resistant niche

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by therapy resistance and an immunosuppressive tumor microenvironment. To comprehensively characterize the complex stromal-immune cell interactions that drive PDAC aggressiveness, we applied an integrated multi-modal approach combining single-cell RNA sequencing, spatial transcriptomics, proteomics, immunofluorescence, and microfluidic-based functional assays to bioengineered spheroid models with increasing cellular complexity (up to four cell types) integrating human pancreatic cancer cells, pancreatic stellate cells, endothelial cells, and monocyte-derived macrophages. By incorporating vascularization within the OrganiX microfluidic platform, we enable studies of immune cell trafficking in vascularized tumors. Multi-omics phenotyping revealed coordinated molecular programs in our four-cell organotypic spheroid models, including enhanced hypoxic and glycolytic pathways, NF-{kappa}B activation, and ECM remodeling. Stromal and immune cells acquired tumor-associated phenotypes mirroring patient heterogeneity, including IL-1{beta}+ macrophages, inflammatory cancer-associated fibroblasts (iCAFs), and antigen-presenting CAFs (apCAFs). The four-cell model exhibited superior clinical relevance, with gene expression signatures that correlated more closely with poor-prognosis patient cohorts and cancer hallmarks that were functionally validated through microfluidic-based assays demonstrating enhanced tumor invasion, angiogenesis, and therapeutic resistance. Finally, live imaging combined with transcriptomic readouts captures dynamic interactions between neutrophils and cancer cells in the vascularized microtumor, enabling direct observation of intravascular events relevant to metastatic dissemination. This integrated analysis demonstrated the recreation of a human-relevant aggressive PDAC niche, establishing a framework that bridges in vitro cellular crosstalk studies with patient-relevant therapeutic responses, offering a powerful translational tool for therapy development in PDAC.

cancer biology↗

bioRxiv2025

Computational screening and automatic filtering for the discovery of novel inhibitors of TMPRSS2, a type II transmembrane serine protease

Transmembrane Serine Protease 2 (TMPRSS2) is a membrane protein of the type II serine protease family of enzymes implied in epithelial homeostasis. It is involved in several diseases, notably prostate cancer and SARS-CoV-2 infections. Over the years, only a few tested TMPRSS2 inhibitors showed consistent results. This prompted us to select it as target of structure-based virtual screening, to search for novel inhibitors among a library of 475,770 small molecules. Two sets of TMPRSS2 structures were selected, one taken from molecular dynamics simulations, the other from recently solved X-ray crystallographic structures. We designed a workflow to filter docking results in a reproducible way, allowing for a faster and more reliable selection. The program uses four metrics: the pose consistency of the ligand, docking score, number of interactions with key protein residues, and cluster analysis. This led to the selection and visual inspection of two sets of 500 compounds, which yielded 10 reasonable hit candidates.

bioinformatics↗

bioRxiv2025

Functional analysis of ligand-gated chloride channels in a cnidarian sheds light on the evolution of inhibitory signalling

{psi}-aminobutyric acid (GABA) is the predominant inhibitory transmitter in the vertebrate nervous system. Fast inhibitory signalling is mediated by type A GABA receptors (GABAARs), which form pentameric ligand-gated ion channels. While GABA is also present in plants and prokaryotes, it is unknown when it was first used for fast neuronal transmission. Cnidaria represent a sister group to all Bilateria and possess a variety of putative GABAARs, none of which has been functionally characterized. In this study, we surveyed putative inhibitory ion channel receptors from the model cnidarians Nematostella and Hydra. Phylogenetic analysis revealed a surprising complexity of these receptors. The majority formed a cnidarian-specific radiation with some receptors forming a basal clade. We functionally analyzed seven putative Nematostella GABAARs of this radiation and found that none was activated by GABA or glycine, whereas three were activated by glutamate. Using site-directed mutagenesis, we identified a lysine residue in the canonical ligand-binding pocket that is important for activation by glutamate. Our results identified a group of inhibitory ion channel receptors in Cnidaria that uses glutamate as a ligand. Moreover, they suggest that inhibitory ion channel receptors in Cnidaria massively diversified, which may have been instrumental in the evolution of complex behaviors and sensory processing by the cnidarian nervous system. This work lays the foundation for understanding the diversity and evolution of inhibitory receptors in Cnidaria and the evolution of inhibitory signalling in animal nervous systems.

neuroscience↗

bioRxiv2024

CyNET- a network analysis framework for high dimensionality, system level analyses of the functional Immunome

The immune system is a complex "Network of Networks", in which various immune cell subsets interact and influence each others functions, ultimately determining immune competence and the control or onset of disease. The coordinated interactions between these cell subsets determine whether the outcome is a normal physiological state or a pathological condition. Established statistical procedures largely ignore the interactions between subsets and rely on statistically significant changes in the frequency of cell subsets. We developed CyNET (Cytometry Network)-- an analysis platform based on a network science approach to understand the immune system holistically. CyNET enables us to quantify the systems level and subsets level properties. We show that changes in the centrality of the nodes (immune subsets) reflect better biological functions than changes in frequency. We used CyNET to analyze the immune development along the chronological age gradient. Peripheral blood cells from healthy newborns (cord blood), adults (20 to 55 years), and elderly (70 years and above) human subjects were further used for validation using single-cell transcriptomics. We found that network edge density, degree centralization, and assortativity score reflect the maturation and development of the immune system along the age axis, thus enabling the characterisation of the functional architecture of the Immunome and the identification of key functional hubs within the immune networks.

immunology↗

bioRxiv2024

Single-cell and spatial atlas of steatotic liver-related hepatocellular carcinoma

Steatotic liver disease-related hepatocellular carcinoma (SLD-HCC) poses significant challenges in liver cancer management. Our current study investigates the tumor microenvironment (TME) of SLD-HCC using single-cell transcriptomic, proteomic and spatial transcriptomic analyses. We identified altered immune-related and lipid metabolism pathways, particularly in regulatory T cells (Tregs) and cancer-associated fibroblasts (CAFs) within the SLD-HCC microenvironment, suggesting distinct cellular adaptations to a high-fat TME and general immunosuppression. Cytometry by time-of-flight revealed a cold and immunosuppressive TME depleted with CD8+ T cells and enriched with Tregs, while spatial transcriptomics uncovered a unique spatial architecture with Treg/CAF clusters specifically located at the tumor margins in SLD-HCC. Crucially, we identified Treg-CAF interactions as a key mediator associated with lack of response to immunotherapy in SLD-HCC. Our findings highlight the intricate immune dynamics of SLD-HCC, indicating potential therapeutic targets to counteract immune evasion and restore anti-tumor immunity in SLD-HCC. HighlightsO_LIThe TME of SLD-HCC exhibits altered immune and lipid pathways C_LIO_LIImmunosuppressive SLD-HCC TME is depleted with CD8+ T cells and enriched with Tregs C_LIO_LISLD-HCC has a unique spatial architecture with Treg-CAF clusters at tumor margins C_LIO_LITreg-CAF interaction via TNFSF14-TNFRSF14 axis mediates immunotherapy resistance C_LI

immunology↗

bioRxiv2024

Cholesterol modifies Nav1.7 - an in-silico and in-vitro analysis

Cholesterol is a major component of plasma membranes and unsurprisingly plays a significant role in actively regulating the functioning of several membrane proteins in humans. Notably, recent studies have shown that cholesterol depletion can also impact transmission of potentially painful signals in the context of peripheral inflammation, via hyperexcitability of the voltage-gated sodium channel (Nav) subtype 1.9, but the structural mechanisms underlying this regulation remain to be elucidated. In this study, we focus on the role of cholesterol depletion on Nav1.7, which is primarily expressed in the peripheral sensory neurons and linked to various chronic inherited pain syndromes. Coarse-grained molecular dynamics simulations shed light on the dynamic changes of the geometry of Nav1.7 upon membrane cholesterol depletion: A loss of rigidity at key structural motifs linked to activation and fast-inactivation is observed, as well as changes in the geometry of drug-binding regions in the channel. Loss of rigidity in cholesterol depleted conditions should allow the channel to transition between different gating states more easily. In-vitro whole-cell patch clamp experiments on HEK293t cells expressing Nav1.7 validated these predictions made in silico at the functional level. Hyperpolarizing shifts in the voltage-dependence of activation and fast-inactivation were observed along with an acceleration of the time to peak and onset kinetics of fast inactivation. These results underline the critical role of membrane composition, and of cholesterol in particular, in influencing Nav1.7 gating characteristics. Furthermore, our results hint to a key role of the membrane environment in affecting drug effects and in pathophysiological dysregulation, sharpening our approaches for analgesics design. Supplementary datahttps://doi.org/10.5281/zenodo.10829175

physiology↗

bioRxiv2023

Comparative analysis of single-cell RNA sequencing methods with and without sample multiplexing

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful technique for investigating biological heterogeneity at the single-cell level in human systems and model organisms. Recent advances in scRNA-seq have enabled the pooling of cells from multiple samples into single libraries, thereby increasing sample throughput while reducing technical batch effects, library preparation time, and the overall cost. However, a comparative analysis of scRNA-seq methods with and without sample multiplexing is lacking. In this study, we benchmarked methods from two representative platforms: Parse Biosciences (Parse; with sample multiplexing) and 10X Genomics (10x; without sample multiplexing). By using peripheral blood mononuclear cells (PBMCs) obtained from two healthy individuals, we demonstrate that demultiplexed scRNA-seq data obtained from Parse showed similar cell type frequencies compared to 10X data where samples are not multiplexed. Despite a relatively lower library and cell capture efficiencies, Parse can detect rare cell types (e.g. plasmablasts and dendritic cells) which is likely due to its relatively higher sensitivity in gene detection. Moreover, comparative analysis of transcript quantification between the two platforms revealed platform-specific distributions of gene length and GC content. These results offer guidance for researchers in designing high-throughput scRNA-seq studies.

genomics↗

bioRxiv2022

Compartmentalization and persistence of dominant (regulatory) T cell clones indicates antigen skewing in juvenile idiopathic arthritis

Autoimmune inflammation is characterized by tissue infiltration and expansion of antigen-specific T cells. Although this inflammation is often limited to specific target tissues, it remains yet to be explored whether distinct affected sites are infiltrated with the same, persistent T cell clones. Here we performed CyTOF analysis and T cell receptor (TCR) sequencing to study immune cell composition and (hyper-)expansion of circulating and joint-derived Tregs and non-Tregs in Juvenile Idiopathic Arthritis (JIA). We studied different joints affected at the same time, as well as over the course of relapsing-remitting disease. We found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient, and observed a strong overlap between dominant T cell clones, especially Treg, of which some could also be detected in circulation and persisted over the course of relapsing remitting disease. Moreover, these T cell clones were characterized by a high degree of sequence similarity, indicating the presence of TCR clusters responding to the same antigens. These data suggest that in localized autoimmune disease there is auto-antigen driven expansion of both Teffector and Treg clones, that are highly persistent and are (re)circulating. These dominant clones might represent interesting therapeutic targets.

immunology↗

bioRxiv2020

Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 M and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 [A], showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/422677v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@17ca2aeorg.highwire.dtl.DTLVardef@19c5159org.highwire.dtl.DTLVardef@1a0adf6org.highwire.dtl.DTLVardef@1fd05cd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure.C_FLOATNO Workflow for identification and profiling of inhibitors of SARS-CoV-2 3CL-Pro using a large scale repurposing and bioactive compound collection (rhs). Primary assay principle based on quenched FRET peptide substrate of SARS-CoV-2 3CL-Pro (lhs). Inhibiting compounds reduce fluorescence signal relative to DMSO controls. Hit profiling using X-ray. C_FIG

molecular biology↗

bioRxiv2020

A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics

The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unfortunately, unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ~30,000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ~200 virtual screenings of compound libraries on selected protein structures, we redefine the proteins druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.

biophysics↗

bioRxiv2019

Fibroblast-specific IL11 signaling is required for lung fibrosis and inflammation

Tissue injury leads to activation of resident stromal, parenchymal and immune cells to initiate reparative processes that, if unresolved, can lead to fibrosis and organ damage. The directionality of effect between fibrosis and inflammation in the lung has been a point of debate for many years. Here, we tested the hypothesis that Interleukin 11 (IL11) signaling in fibroblasts is of primary importance for pulmonary fibrosis and that this event is upstream of lung inflammation. We generated mice with loxP-flanked Il11ra1 alleles and crossed them to a Col1a2-CreERT strain to enable Il11ra1 deletion in adult fibroblasts (Il11ra1-CKO mice). Lung fibroblasts from Il11ra1-CKO mice were selectively deleted for Il11ra1 and refractory to TGF{beta}1 stimulation. In the mouse model of bleomycin-induced lung fibrosis, Il11ra1-CKO mice had markedly reduced pulmonary fibrosis and lesser lung damage, which was accompanied by diminished ERK activation in the stromal compartment. Bleomycin lung injury in Il11ra1-CKO mice was also associated with diminished STAT3 activation in inflammatory cells, fewer pulmonary immune cell infiltrates and almost complete inhibition of NF-kB activation. These data reveal an essential role for IL11 signaling in fibroblasts for lung fibrosis and show that inflammation in the lung can be secondary to stromal activation.

cell biology↗

bioRxiv2019

An immunome perturbation is present in juvenile idiopathic arthritis patients who are in remission and will relapse upon anti-TNFα withdrawal

ObjectivesBiologics treatment with anti-TNF is efficacious in juvenile idiopathic arthritic (JIA) patients. Despite displaying clinical inactivity during treatment, many patients will flare upon cessation of therapy. The inability to definitively discriminate patients who will relapse or continue to remain in remission after therapy withdrawal is currently a major unmet medical need. CD4 T cells have been implicated in active disease, yet how they contribute to disease persistence despite treatment is unknown.\n\nMethodsWe interrogated the circulatory reservoir of CD4+ immune subsets at the single cell resolution with mass cytometry (CyToF) of JIA patients (n=20) who displayed continuous clinical inactivity for at least 6 months with anti-TNF, and were subsequently withdrawn from therapy for 8 months, and scored as relapse or remission. These patients were examined prior to therapy withdrawal for putative subsets that could discriminate relapse from remission. We verified on a separate JIA cohort (n=16), the continued dysregulation of these circulatory subsets 8 months into therapy withdrawal. The immunological transcriptomic signature of CD4 memory in relapse/remission patients was examined with Nanostring.\n\nResultsAn inflammatory memory subset of CD3+CD4+CD45RA-TNF+ T cells deficient in immune checkpoints (PD1-CD152-) was present in relapse patients prior to therapy withdrawal. Transcriptomic profiling reveals divergence between relapse and remission patients in disease centric pathways involving (a) TCR activation, (b) apoptosis, (c) TNF, (d) NF-kB and (e) MAPK signalling.\n\nConclusionsA unique discriminatory immunomic and transcriptomic signature is associated with relapse patients and may explain how relapse occurs.

immunology↗

bioRxiv2019

Systemic sclerosis is a disease of a prematurely senescent, inflammatory and activated immunome

Systemic sclerosis (SSc) is an autoimmune disease characterised by excessive fibrosis of skin and internal organs, and vascular dysfunction. Association of T and B cell subsets have been reported in SSc, however there is lack of systematic studies of functional relations between immune cell subsets in this disease. This lack of mechanistic knowledge hampers targeted intervention. In the current study we sought to determine differential immune cell composition and their interactions in peripheral blood of SSc patients. Mononuclear cells from blood of SSc patients and healthy controls (HC) were analysed by mass cytometry using a 36 marker (cell-surface and intracellular) panel. Transcriptome analysis (m-RNA sequencing) was performed on sorted T and B cell subsets. Unsupervised clustering analysis revealed significant differences in the frequencies of T and B cell subsets in patients. Correlation network analysis highlighted an overall dysregulated immune architecture coupled with domination of inflammatory senescent T cell modules in SSc patients. Transcriptome analysis of sorted immune cells revealed an activated phenotype of CD4 and MAIT cells in patients, accompanied with increased expression of inhibitory molecules, reminiscent of phenotype exhibited by functionally adapted, exhausted T cells in response to chronic stimulation. Overall this study provides an in-depth analysis of the systemic immunome in SSc, highlighting the potential pathogenic role of inflammation and chronic stimulation mediated \"premature senescence\" of immune cells.\n\nONE SENTENCE SUMMARYThe immune architecture in Systemic sclerosis is altered and reminiscent of chronic stimulatory environment led premature senescence of immune cells.

immunology↗

bioRxiv2018

IL-11 neutralising therapies target hepatic stellate cell-induced liver inflammation and fibrosis in NASH

The transformation of hepatic stellate cells (HSCs) into myofibroblasts is the defining pathobiology in non-alcoholic steatohepatitis (NASH). Here we show that key NASH factors induce IL-11, which drives an autocrine and ERK-dependent activation loop to initiate and maintain HSC-to-myofibroblast transformation, causing liver fibrosis. IL-11 is upregulated in NASH and Il11ra1-deleted mice are strongly protected from liver fibrosis, inflammation and steatosis in murine NASH. Therapeutic inhibition of IL11RA or IL-11 with novel neutralizing antibodies robustly inhibits NASH pathology in preclinical models and reverses established liver fibrosis by promoting HSC senescence and favourable matrix remodelling. When given early in NASH, IL-11 inhibition prevents liver inflammation and steatosis, reverses severe hepatocyte damage and reduces hepatic immune cells and TGF{beta}1 levels. Our findings show an unappreciated and central role for IL-11 in HSCs and prioritise IL-11 signalling as a new therapeutic target in NASH while revealing an unexpected pro-inflammatory function for IL-11 in stromal immunity.

molecular biology↗