Resistance to epidermal growth factor receptor (EGFR)-targeted therapies remains a major obstacle in the treatment of EGFR-mutant non-small cell lung cancer (NSCLC). Here, we report a metabolic adaptation in osimertinib-resistant (OsiR) cells characterized by elevated acetate levels and activation of an unconventional pyruvate-acetaldehyde-acetate (PAA) shunt. While our previous work explored the PAA pathway from a functional perspective, including mitochondrial remodeling and in vivo analyses in transgenic models, the present study focuses on detailed biochemical and metabolite-level adaptations occurring in osimertinib-resistant NSCLC cells. Integrated transcriptomic, exometabolomic, and targeted protein analyses were performed in parental (Par) and OsiR H1975 NSCLC cells. Extracellular metabolites were quantified using high-resolution chromatographic platforms including LC-HRMS, HPLC-DAD, and GC-MS, and pathway analysis was performed using MetaboAnalyst. Resistant cells displayed suppression of canonical NADPH-producing pathways, particularly the oxidative branch of the pentose phosphate pathway (PPP), concomitant with activation of an alternative metabolic route marked by elevated extracellular pyruvate, acetaldehyde, and acetate. Transcriptomic and protein analyses revealed upregulation of the aldehyde dehydrogenases ALDH2 and ALDH7A1, enzymes capable of catalyzing NADP+-dependent oxidation of acetaldehyde to acetate. These findings support engagement of a PAA metabolic shunt that contributes to adaptive NADPH turnover and supports redox balance despite severe NADPH depletion when canonical pathways are compromised. Resistant cells also exhibited enhanced glycolytic flux, increased acetate production, and elevated expression of enzymes involved in acetate utilization and acetyl-CoA metabolism. Together, these findings identify a resistance-associated metabolic adaptation involving activation of an unconventional PAA pathway that may serve as a de novo alternative source of reducing power. By focusing on specific metabolic intermediates and enzyme-level changes, this work provides mechanistic insights complementary to our previous functional study. The PAA pathway may represent a metabolic vulnerability in osimertinib-resistant EGFR-mutant NSCLC.

Paired mesoderm homeobox protein 2B (PHOX2B) is a transcription factor essential for autonomic nervous system development. Heterozygous mutations in the PHOX2B gene are associated with neurodevelopmental disorders, including congenital central hypoventilation syndrome and Hirschsprung's disease. Additionally, PHOX2B plays a role in the genetic landscape of neuroblastoma, with mutations detected in both familial and sporadic forms of this rare cancer. Notably, PHOX2B is highly expressed in most neuroblastoma cells. Despite its significance, little is known about the regulation of PHOX2B gene expression, and limited attention has been given to the genomic features of the antisense strand at the PHOX2B locus, although the presence of an antisense transcript is suggested by bioinformatics analyses. In this study, we characterize the recently annotated human antisense transcript PHOX2B-AS1 and the previously unidentified mouse antisense Phox2b transcript. Our findings reveal that PHOX2B positively regulates PHOX2B-AS1 expression and that inhibiting the antisense transcript reduces PHOX2B protein levels. Together, these results provide strong evidence for the existence of a gene antisense to PHOX2B and highlight a strict correlation and reciprocal regulation between PHOX2B and PHOX2B-AS1.

Tunicates, including ascidians, are recognized as the true ‘sister group’ of vertebrates and are emerging as models to study the development and degeneration of central nervous system (CNS). Ascidian larvae have the typical chordate body plan that includes a dorsal neural tube. During their metamorphosis, a deep tissue reorganization takes place, with some tissues that degenerate while others develop to become functional during the adult life. The larval CNS also degenerates and most neurons disappear, making room for the formation of adult CNS. The genome of the ascidian Ciona intestinalis has been sequenced and annotated, with several CNS specific genes that have been characterized, revealing specification mechanisms shared with humans. These features make ascidian metamorphosis a good model to study the mechanisms underlying physiological CNS degeneration and to compare them to the pathological conditions typical of neurodegenerative diseases. In order to shed light on the molecular determinants of C. intestinalis metamorphosis and neurodegeneration, we analyzed the proteome at three stages of development: swimming larva (SwL, Hotta stage 28), settled larva (SetL, Hotta stage 32) and metamorphosing larva (MetL, Hotta stage 34). A total of 405 modulated proteins were identified by mass spectrometry by comparing the three stages. Enrichment and network analysis showed the involvement of several processes/pathways, including autophagy and mTOR pathway, and actin cytoskeleton organization and remodeling among the most significant ones. This study elucidates molecular pathways underlying ascidian metamorphosis and highlights shared mechanisms between physiological neurodegeneration in ascidians and pathological neurodegeneration in humans. ### Competing Interest Statement The authors have declared no competing interest. 'Piano Sviluppo Unimi - Linea 3 - Bando SoE' Grant, University of Milan, RV\_PSR\_SOE\_2020\_RPENN PRIN2022 Grant, Italian Ministry of University and Research, 2022SF7HY9

Background/Objectives: Comparative sociogenomics combines multiple scientific fields to investigate the genetic basis of social behavior across species. Our aim was to uncover the genetic roots of human sociability with possible implications for autism, a neurodevelopmental disorder characterized by social and communication deficits. Methods: We conducted molecular network analysis on 659 sociability-related genes from different animal species, including humans. Results: We identified a network of 240 genes strongly associated with autism (p < 10(-15)), with 194 inferred. These genes were grouped into 23 functional communities related to cell-cell junctions and communication, inflammatory and synaptic signaling, neurotransmitter receptors and semaphorin signaling among the more enriched meta-pathways. Some network genes were clustered in nine chromosomal bands (FDR < 0.25), indicating genes' functional cooperation, shared evolutionary history, and coordinated regulation, and few genes are physically in linkage with ASD genes (within 0.5 cM) or controlled by human-accelerated regions. Conclusions: The most compelling inferred autism risk genes are MED12, FZD9, and DMD since they are differentially expressed in autistic brains, physically linked to key autism genes, controlled by human-accelerated regions, or mapped to chromosomal regions enriched in network genes. If validated, they could represent novel biomarkers, advancing the understanding of autism's genetic makeup.

Psychiatric disorders often arise from the interaction between genetic predisposition and chronic psychosocial stress, yet the molecular programs determining resilience versus susceptibility remain incompletely understood. Building on evidence that the transcriptional corepressor LSD1 links environmental stress to neuronal gene regulation, we investigated whether isoform-specific regulation of LSD1 splicing contributes to stress adaptation. Using a mouse model of chronic social defeat stress, we analyzed LSD1 microexon E8a splicing in the hippocampus of resilient and susceptible animals. RNA-seq was performed after the last stress session to capture genome-wide transcriptional responses during the window of LSD1 splicing regulation. Comparative analyses with published LSD1 knockdown, LSD1 ChIP-seq and chronic stress datasets were conducted. Hippocampal samples from suicide victims were analyzed to assess translational relevance. Analysis of LSD1 splicing dynamics revealed that resilient mice, but not susceptible animals, retained the ability to reiterate acute stress-induced exon E8a skipping after repeated stress exposure, preserving the capacity to upregulate the enzymatically active ubLSD1 isoform in the hippocampus. In susceptible mice this inducible splicing response was absent. Mechanistically, splicing regulation involved the long non-coding RNA MALAT1, which controls the neurospecific splicing factor nSR100, a regulator of LSD1 exon E8a inclusion. Reduced MALAT1 expression in susceptible mice coincided with marked overactivation of stress-responsive genes revealed by RNA-seq. Approximately 15% (86 of 595) of genes deregulated in susceptible versus resilient hippocampi overlapped with transcripts modulated by LSD1 knockdown in an independent neuronal system. Of these, 25 were direct LSD1 ChIP-seq targets. ESR1 emerged as a regionally divergent upstream regulator associated with susceptibility. The MALAT1-nSR100-LSD1 axis represents a regulatory pathway modulating stress adaptation. Downregulation of ubLSD1 and MALAT1 in the hippocampus of suicide victims recapitulates the molecular phenotype observed in stress-susceptible mice, linking disruption of this pathway to pathological behavioral outcomes.

Clonally established tumor cell lines often do not recapitulate the behavior of cells in tumors. The sequencing of a whole tumor tissue may not uncover transcriptome profiles induced by the interactions of all different cell types within a tumor. Interferons for instance have a vast number of binding sites in their target genes. Access to the DNA binding sites is determined by the epigenomic state of each different cell type within a tumor mass. To understand how genes such as interferons appear to have both tumor-promoting and tumor-inhibiting functions, single-cell transcript analysis was performed in the breast cancer tissue of HER2+ (epidermal growth factor receptor 2) patients. We identified that potential antagonistic oncogenic activities of cells can be due to diverse expression patterns of genes with pleiotropic functions. Molecular pathways both known and novel were identified and were similar with those previously identified for patients with rheumatoid arthritis. Our study demonstrates the efficacy in using single-cell transcript analysis to gain insight into genes with apparent contradictory or paradoxical roles in oncogenesis.

MargheRita_library.xlsx: Spectral library of reference standards: metabolite MS information and metadata. MargheRita_library_MSP.txt: Spectral library of reference standards: metabolite MS/MS spectra in msp format. Standards_RP_NEG.txt: standard dataset used to assess the performance of metabolite identification in margheRita containing a panel of 33 standard metabolites acquired in IDA mode, negative polarity upon chromatographic separation on C18 column. Standards_RP_POS.txt: standard dataset used to assess the performance of metabolite identification in margheRita containing a panel of 33 standard metabolites acquired in IDA mode, positive polarity upon chromatographic separation on C18 column. Urine_RP_NEG_norm.txt: urine dataset used to assess the performance of metabolite identification in margheRita containing 72 urine samples acquired in SWATH mode, negative polarity upon chromatographic separation on C18 column. Urine_RP_POS_norm.txt: urine dataset used to assess the performance of metabolite identification in margheRita containing 72 urine samples acquired in SWATH mode, positive polarity upon chromatographic separation on C18 column.

ABSTRACT Alterations in glycoconjugate profiles are thought to promote changes in cell‐to‐cell and cell‐to‐intracellular and extracellular scaffold interactions in human disease. The nearly unlimited number of “glycoforms” that may exist in nature are difficult to study due to glycosylation and glycoconjugate modifications being associated with non‐genome coded posttranscription and post‐translation processes. Specific products generated by glycosylation are dependent on concentration and sub‐cellular locations of glycan synthesis and processing enzymes. An indirect “high‐throughput” approach to study glycosylation is to characterize glycan processing enzymes (hydrolases and transferases) by single cell sequencing of all cell types in tissue of human diseases. We previously identified TMEM230 as an endoplasmic reticulum (ER) associated protein that regulates NOTCH glycoprotein receptor and ligand signaling in zebrafish blood vessel formation and destructive remodeling capacities of diverse cell types including fibroblast, phagocytic and immune system cells in patients with cancer or granulomatous systemic vasculitis autoimmune disorder. NOTCH signaling represents a paradigm in glycan mediated signal transduction and supports the role of TMEM230 in glycan modifications. The ER initiates the earliest steps of glycoconjugate synthesis, sorting, and trafficking. As blood vessel and tissue remodeling, and Notch signaling are hallmarks of autoimmune disorders, we investigated whether aberrant TMEM230 expression was also associated with changes in expression of glycan processing enzymes in patients with rheumatoid arthritis (RA). In this current study, single cell sequencing analysis supported that TMEM230 expression was downregulated in all cell types associated with synovial tissue of RA patients while glycan processing enzymes were predominantly upregulated. In contrast, TMEM230 was upregulated in patients with high‐grade compared to low‐grade gliomas as it was N‐linked glycosylation (GlcNAc), and glycoprotein and glycosaminoglycan expression. Our collective results support that TMEM230 regulates glycan/glycoconjugate processing enzymes in RA and the expression of protein glycoconjugate in aggressive gliomas. TMEM230 may therefore be a therapeutic target and marker for clinical treatment for glycosylation induced human autoimmunity disorders or cancer.

Disease phenotypes can be described as the consequence of interactions among molecular processes that are altered beyond resilience. Here, we address the challenge of assessing the possible alteration of intra- and inter-cellular molecular interactions among processes or cells. We present an approach, designated as "Ulisse", which complements the existing methods in the domains of enrichment analysis, pathway crosstalk analysis and cell-cell communication analysis. It applies to gene lists that contain quantitative information about gene-related alterations, typically derived in the context of omics or multi-omics studies. Ulisse highlights the presence of alterations in those components that control the interactions between processes or cells. Considering the complexity of statistical assessment of network-based analyses, crosstalk quantification is supported by two distinct null models, which systematically sample alternative configurations of gene-related changes and gene-gene interactions. Further, the approach provides an additional way of identifying the genes associated with the phenotype. As a proof-of-concept, we applied Ulisse to study the alteration of pathway crosstalks and cell-cell communications in triple negative breast cancer samples, based on single-cell RNA sequencing. In conclusion, our work supports the usefulness of crosstalk analysis as an additional instrument in the "toolkit" of biomedical research for translating complex biological data into actionable insights.

Recent advancements in high-resolution and high-throughput sequencing technologies have significantly enhanced the study of cell-cell communication inference using single-cell and spatial transcriptomics data. Over the past 6 years, this growing interest has led to the development of more than 100 bioinformatics tools and nearly 50 resources, primarily in the form of ligand-receptor databases. These tools vary widely in their requirements, scoring approaches, ability to infer inter- and/or intra-cellular communication, assumptions, and limitations. Similarly, cell-cell communication resources differ in many aspects, mainly in the number of annotated interactions, species coverage, and their focus on inter-cellular signaling or both inter- and intra-cellular communication. This abundance and diversity create challenges in identifying compatible and suitable tools and resources to meet specific user needs. In this collaborative effort, we aim to provide a comprehensive report on the current state of cell-cell communication analysis derived from single-cell or spatial transcriptomics data. The report reviews existing methods and resources, addressing all relevant aspects from the user's perspective. It also explores current limitations, pitfalls, and unresolved issues in cell-cell communication inference, offering an aggregated analysis of the existing literature on the topic. Furthermore, we highlight potential future directions in the field and consolidate the collected knowledge into CCC-Catalog (https://sysbiobig.gitlab.io/ccc-catalog), a centralized web platform designed to serve as a hub for bioinformaticians and researchers interested in cell-cell communication inference.

Hepatic mitochondrial maladaptation features the transition from metabolic dysfunction-associated steatotic liver disease (MASLD) to Steatohepatitis (MASH) up to fibrosis/cirrhosis. However, it is still unexplored whether mitochondrial alterations also affect adipose tissue, muscle and heart during disease progression. C57Bl/6 mice were fed an AMLN diet to recapitulate the human MASLD spectrum. In the liver, TEM depicted a progressive morphologic dysfunction of mitochondria, which appeared swollen in MASH, with disorganized cristae/matrix loss in MASH-fibrosis. The mitophagy pathway was reduced in MASH-fibrosis, thus explaining the accumulation of damaged mitochondria, whereas mitochondrial complexes activities alongside OXPHOS protein levels and ATP production were dampened across the disease in liver, adipose, muscle, and cardiac tissues. Finally, the release of cell-free circulating mitochondrial DNA into the bloodstream reflected tissue mitochondrial impairment. In sum, we demonstrated that alterations in mitochondrial morphology, life cycle, and activity feature all disease stages in the liver but also in other tissues engaged in MASLD evolution.

Background & Aims:Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease worldwide, paralleling the rising prevalence of obesity. We previously reported that the rs17618244 variant in the Klotho-beta (KLB) gene, which encodes the hepatic obligate co-receptor of fibroblast growth factor receptor 4 (FGFR4), reduces hepatic and circulating KLB levels, leading to more severe MASLD in both children and adults. The present study aimed to evaluate the impact of another KLB variant, the intronic rs12152703 G>T polymorphism, on histological liver damage in patients with MASLD. Methods:The rs12152703 variant was genotyped in 1,311 patients with biopsy-proven MASLD, including 261 children, and its association with the disease spectrum was assessed. We also investigated the relationship between this variant and hepatic and circulating KLB expression. Finally, we evaluated in an in vitro model whether KLB overexpression in HepG2 cells affects lipid homeostasis and inflammation. Results:In multivariate analyses, the KLB rs12152703 variant was associated with lower serum aminotransferase levels and protection against steatosis, lobular inflammation, and steatohepatitis in the overall cohort (p <0.05). Hepatic and circulating KLB levels were increased in both adult and pediatric patients with MASLD carrying the variant (p <0.05). In vitro, KLB overexpression reduced intracellular lipid accumulation in free fatty acid-loaded HepG2 cells by modulating the expression of genes involved in lipid metabolism. Moreover, KLB induction counteracted lipopolysaccharide-induced activation of inflammatory genes and NF-κB (p65) phosphorylation. Conclusions:The KLB rs12152703 variant confers protection against lobular inflammation and is associated with increased hepatic and circulating KLB levels, in contrast to the at-risk rs17618244 variant. Consistently, KLB overexpression ameliorated steatosis and the pro-inflammatory state in lipid-loaded hepatocytes. These findings suggest that KLB may represent a novel druggable target for the treatment of severe MASLD. Impact and implications:This study highlights the protective effect of a genetic variant in the Klotho-beta (KLB) gene in attenuating hepatic inflammation and disease severity in both adults and children with metabolic dysfunction-associated steatotic liver disease. The favorable clinical associations appear to be mediated by increased circulating and hepatic KLB protein levels. Consistently, KLB overexpression alleviates lipid overload in hepatocytes by modulating the expression of genes involved in lipid metabolism. Together, these findings support the FGF19/KLB axis as a promising therapeutic target for the treatment of severe metabolic dysfunction-associated steatotic liver disease.

BACKGROUND. Myelodysplastic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis (MDS/MPN-RS-T) is a rare hematologic malignancy with an uncertain pathobiology. Previous studies have identified coexisting driver mutations in proliferative (JAK2/MPL) and dysplastic (SF3B1) genes, contributing to its mixed myelodysplastic/ myeloproliferative neoplasm phenotype (PMID: 38714876). Despite these findings, the effects of these simultaneous mutations on the biology of hematopoietic stem cells (HSCs) and their differentiation trajectories remain unclear. The increased risk of progression to acute myeloid leukemia (AML) in patients with both SF3B1 and JAK2/MPL mutations compared to those with only SF3B1 mutations, combined with the lack of consolidated therapies, makes this a subject of significant interest for identifying molecular mechanisms that could be targeted. The aim of this study is to investigate the effect of co-mutations on clonal dynamics, differentiation trajectories, and transcriptional processes at the single-cell level. METHODS: We used Target-seq (PMID: 30765193), a single-cell multiomics approach that combines genotyping and transcriptomics on bone marrow CD34+ cells from six MDS/MPN-RS-T patients, paired with 10X 3' scRNA-seq and scATAC-seq. RESULTS: Single-cell analysis revealed a significant increase in erythroid differentiation in cells carrying both driver mutations compared to wild-type (WT) cells and those with only the SF3B1 mutation (p<0.001). This phenotype was confirmed through the analysis of transcription factors (TFs) and the activity of their putative downstream target genes, known as regulons. At the level of hematopoietic stem and progenitor cells (HSPCs), we observed a decrease in the activity of classical stemness regulons such as JUN, FOS, and IRF1, accompanied by an increase in erythroid regulators like GATA1 and HES6 in cells with double mutations compared to WT cells (Wilcoxon test, p<0.01). Additionally, RUNX3 and ZBTB7A emerged as potential drivers of direct erythroid differentiation at the HSPC level. Furthermore, gene activity modeling from scATAC-seq confirmed an increase in promoter and enhancer accessibility of genes involved in erythroid differentiation. Analyses of single-cell states revealed an additional differentiation trajectory branch from megakaryocyte-erythroid progenitor cells (MEPs) to megakaryocytes (MKs). In double-mutated cells, we identified a commitment of progenitor cells to megakaryocyte differentiation through the activation of SRF and TAL1, which could explain the presence of thrombocytosis in patients. Cell differentiation regulation was enhanced by the activation of proliferative pathways (Myc, E2F targets) and a reduction of apoptosis along the differentiation trajectory in cells carrying both mutations compared to WT and SF3B1 mutated cells (FDR-adjusted p<0.01). Using a machine learning approach, we identified a gene signature capable of detecting double-mutated cells through these trajectories. This signature, when applied to matched 10X experiments, increased the number of genotyped cells and confirmed previous results, identifying several pathways involved in mitochondrial membrane rearrangement, potentially responsible for ring sideroblast development. CONCLUSION. By employing an innovative single-cell approach that integrates genomics, transcriptomics, and epigenomics, we have unraveled the intricate transcriptional regulation of mixed phenotype neoplasms such as MDS/MPN-RS-T. Our study underscores the synergistic impact of JAK2/MPL and SF3B1 mutations in disease progression, primarily through an early commitment to dysplastic erythroid differentiation. This progression is orchestrated by the activation of specific transcriptional and proliferative pathways, in conjunction with oxidative stress and mitochondrial biology. Importantly, our research led to the identification of a distinct gene signature that can detect the presence of these mutations using single-cell transcriptomic data, bypassing the need for expensive single-cell genotyping approaches. These findings reveal potential molecular targets, creating new opportunities for innovative diagnostic and therapeutic strategies for MDS/MPN-RS-T.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.