Methods in molecular biology (Clifton, N.J.)Jason Ngo, Emily Lee, Marta Olah, Falak Sher
Human induced pluripotent stem cell (iPSC)-derived microglia (iMG) provide an in vitro experimental system for studying human microglial biology, neuroinflammation, and genetic risk mechanisms associated with neurological disease. This chapter describes a standardized, scalable, and reproducible protocol for the differentiation of human iPSCs into functional microglia-like cells, with particular emphasis on applications in transcriptional and epigenomic network analysis. The protocol supports high-viability floating iMG production, compatibility with pooled CRISPR perturbation approaches, and downstream multiomic profiling, including single-cell RNA sequencing, chromatin accessibility assays, and proteomics. Detailed procedures are provided for iPSC maintenance, hematopoietic progenitor cell generation, microglial maturation, functional genomics integration, and quality control.
Methods in molecular biology (Clifton, N.J.)Ece Cakiroglu, Serif Senturk
Clustered regularly interspaced short palindromic repeats (CRISPR) and associated (Cas) systems have revolutionized the field of genome engineering by providing versatile, efficient, and programmable tools for precise genetic manipulation. Originally identified as an adaptive immune mechanism in prokaryotes, CRISPR/Cas systems have been extensively repurposed for a wide range of applications across molecular biology, biotechnology, and medicine. This chapter provides a comprehensive overview of the molecular mechanisms underlying CRISPR/Cas immunity. Furthermore, the classification of CRISPR/Cas systems into distinct types and subtypes is discussed, highlighting their structural and functional diversity. Advances in genome editing technologies, including CRISPR-mediated knockout, base editing, and prime editing, are explored with an emphasis on their mechanisms and applications. The chapter also examines emerging CRISPR-based platforms for transcriptional regulation, epigenome editing, and RNA targeting, which enable precise and reversible modulation of gene expression without altering genomic DNA. In addition, the transformative impact of CRISPR technologies on functional genomics is addressed, particularly through high-throughput screening approaches that facilitate the identification of gene function and genetic vulnerabilities. CRISPR-based diagnostic tools and therapeutic strategies are also reviewed, underscoring their potential in disease detection and treatment. Despite significant progress, challenges such as off-target effects, delivery limitations, and safety concerns remain critical considerations. Overall, this chapter highlights the expanding capabilities of CRISPR/Cas systems and their growing importance in both fundamental research and clinical applications.
Methods in molecular biology (Clifton, N.J.)Gijs Wilbrink, Roman Pleskot, Peter Grones
The plasma membrane (PM) is the primary interface between plant cells and their environment, and its resident proteins mediate key processes such as extracellular signal perception and downstream cellular reprogramming. Yet, PM proteins are typically underrepresented in total protein extracts, and existing enrichment strategies are often laborious and require extensive optimization. Here, a simple and robust workflow is described for enriching PM proteins from Arabidopsis thaliana seedlings using total microsomal membranes obtained by differential centrifugation as starting material. Sequential low- and high-speed spins are used to isolate total microsomal membranes and progressively deplete contaminating organelles, thereby increasing the relative abundance of PM proteins. Coupled with the rich genetic toolkit available in Arabidopsis, this protocol provides an accessible platform for systematic characterization of the PM proteome.
Methods in molecular biology (Clifton, N.J.)Zhenlan Yao, Melody Li
Affinity purification-mass spectrometry (AP-MS) is a powerful proteomic approach for dissecting the interaction network between virus and host. Traditional AP-MS employs overexpression of viral proteins as baits to enrich host interactors. However, overexpressed viral proteins may mislocalize to inappropriate cellular compartments and trigger endoplasmic reticulum stress by overwhelming the protein-folding machinery, which leads to false identification of host factors. To overcome these limitations, we introduce an AP-MS strategy based on direct infection with an epitope-tagged chikungunya virus (CHIKV/myc-E2), which we used to successfully uncover two new antiviral factors in CHIKV cellular reservoirs-macrophages. In this protocol, we will describe this technique step by step: (1) design and construction of myc-tagged virus by advanced multi-fragment assembly, (2) in vitro transcription and preparation of infectious myc-tagged virus stocks, and (3) immunoprecipitation of myc-tagged viral protein and its interactome for mass spectrometry analysis. This strategy enables accurate identification of viral interactors in a physiologically relevant context, providing a framework for future proteomic studies using tagged viruses.
Methods in molecular biology (Clifton, N.J.)Xiaoqian Shi-Kunne, Jan A L van Kan
The availability of a high-quality genome assembly facilitates the analysis of fungal genomes. This chapter outlines the tools and steps involved in genome sequence assembly and annotation of a plant pathogen, Botrytis cinerea. We describe the use of Illumina short-read and Oxford Nanopore long-read sequencing data to assemble the B. cinerea genome. The steps include the pre-processing of sequencing data, genome assembly using Flye, scaffolding with NtLink, and polishing with Racon, Medaka, and NextPolish. The quality of the final assembly is evaluated using BUSCO, which serves as a benchmark for the completeness of a genome. We also provide details on the identification and masking of repetitive elements using the EarlGrey pipeline, as well as the gene prediction and annotation process with Funannotate. The methodologies and insights described can be applied to genome research in other fungal species.
Methods in molecular biology (Clifton, N.J.)Fanju W Meng, Duy K Nguyen, Patrick J Murphy
Alterations in chromatin state, mediated through histone modifications and the incorporation of histone variants, are fundamental to establishing transcriptional networks and cell identity. Recent advances in low-input epigenome profiling methods, such as CUT&Tag and CUT&RUN, have enabled the study of chromatin states from very limited starting materials. In this chapter, we describe procedures for generating CUT&Tag libraries to profile histone modifications and histone variants in early-developing zebrafish embryos.
Global genomic surveillance has emerged as a foundational pillar of public health in the twenty-first century, enabling real-time tracking of pathogen evolution and informing outbreak response. This chapter examines the strategic architecture of global genomic surveillance, focusing on its application to arboviruses such as chikungunya virus (CHIKV). It explores the integration of genomic data with epidemiological, clinical, and environmental information within a One Health framework, while addressing critical challenges in governance, equity, and interoperability. The discussion covers the entire genomic surveillance workflow, from sample collection and sequencing to bioinformatic analysis and phylogenetic inference, and highlights the transformative role of artificial intelligence (AI) in predictive surveillance. By analyzing global initiatives, operational barriers, and emerging technologies, this chapter underscores the necessity of sustainable, equitable, and interoperable genomic systems to proactively address current and future infectious disease threats.
Methods in molecular biology (Clifton, N.J.)Mark Roosjen, Dolf Weijers
Mass spectrometry-based proteomics allows the unbiased identification and quantification of proteins and phosphopeptides in biological materials. The nature of walled plant cells requires specific protocols for effective and efficient protein isolation, and, in general, the plant sciences can benefit from more accessible, optimized proteomics workflows. Advances in MS instrumentation now allow the measurement of large numbers of samples, shifting constraints in proteomics toward the accurate, high-throughput preparation of samples. Here, we describe a high-throughput (phospho)proteomics protocol that enables processing of samples using different filter types in a 96-well format.
Aberrant three-dimensional genome organization is a hallmark of cancer, often driving oncogene activation through mechanisms such as enhancer hijacking. High-throughput chromosome conformation capture (Hi-C) maps these interactions on a genome-wide scale. Unlike earlier dilution-based methods, in situ Hi-C performs proximity ligation within intact nuclei, minimizing random ligation noise and enabling fine-scale structure detection. This chapter describes an optimized in situ Hi-C protocol tailored for cancer cell lines using MboI digestion and biotin-mediated pull-down to generate high-complexity libraries. We further outline a computational workflow that extends beyond standard topological mapping of compartments and topologically associating domains to identify cancer-specific aberrations. Specifically, we focus on detecting chromosomal rearrangements (structural variants) and characterizing the distinct circular topology of extrachromosomal DNA. This integrated experimental and analytical framework provides the necessary tools to dissect the spatial dysregulation underlying tumor evolution.
Methods in molecular biology (Clifton, N.J.)Ana Ruiz-Padilla, Julio L Rodríguez-Romero, Marco Chiapello, María A Ayllón
High-throughput sequencing of total RNA has permitted the detection of novel mycoviruses in fungi with different types of genomes, including mycoviruses with double-stranded RNA, single-stranded positive- or negative-stranded RNA, or single-stranded DNA genomes. However, in silico detection of mycoviruses is not always sufficient to guarantee their presence in sequenced samples, especially in the case of the discovery of unique mycoviruses, and additional analyses are required to validate in vivo the data obtained by bioinformatics analysis. This chapter provides comprehensive protocols for the extraction of total RNA from the plant pathogenic fungus Botrytis cinerea for next-generation sequencing (NGS), outlines the bioinformatics pipeline designed and followed to detect mycoviruses in the sequenced samples, and details the detection in vivo and the complete molecular characterization of the mycoviruses identified in silico.
Single-cell transcriptomics has revolutionized our understanding of cellular heterogeneity by enabling high-resolution gene expression profiling at the individual cell level. However, traditional single-cell RNA sequencing (scRNA-seq) lacks direct protein quantification, limiting comprehensive immunophenotyping. Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) overcomes this limitation by integrating antibody-derived tag (ADT) quantification with scRNA-seq, allowing simultaneous measurement of surface protein and gene expression from the same cell. This multimodal approach enhances immune cell characterization, revealing new functional states and rare subpopulations in complex biological systems. Here, we provide a detailed protocol for performing CITE-seq, from sample preparation to sequencing and data analysis. We highlight key experimental considerations, discuss challenges related to antibody selection and batch effects, and provide troubleshooting strategies to ensure robust and reproducible results. The integration of transcriptomic and proteomic data through CITE-seq provides unparalleled insights into cellular function, with broad applications in immunology, oncology, and systems biology.
Studying the transcriptome and the proteome of cells is essential for gaining a detailed understanding of cellular behavior, development, drug action, and disease progression. Spatial biology emerges to advance our ability to study the expression of molecules within tissues while preserving their natural spatial context. These cutting-edge technologies enable the mapping of thousands of individual cells in their original environment by detecting the location and biological quantity of cellular contents, such as RNAs and proteins. Here, we present a protocol to combine spatial transcriptomics (Xenium) and spatial proteomics (PhenoCycler-Fusion) within 8 days on the same tissue section to successfully study the expression of hundreds of RNAs and tens of proteins simultaneously. The combination of these two technologies and consequent integration of the two data layers together with high-resolution H&E images allows for the extraction of a maximum of information from a single tissue section. Application of this protocol and the resulting integrated data will help researchers to understand complex biological processes and disease mechanisms, supporting more nuanced research in molecular biology and pathology.
Proteomics, the large-scale study of proteins, enables the identification, quantification, and functional characterization of proteins, revealing post-translational modifications and protein interactions that are not apparent from transcriptomic data. Human organoids, which recapitulate the structural and functional complexity of native epithelial tissues, provide powerful tools to study disease mechanisms and personalize therapies. However, their culture poses challenges for efficient protein extraction and reproducible analysis. Here, we present a proteomics workflow optimized to maximize protein recovery from Matrigel-encased organoids. Samples were processed using S-Trap microcolumns to minimize losses, followed by liquid chromatography-mass spectrometry (LC-MS) in data-independent acquisition (DIA/SWATH-MS) mode for comprehensive, untargeted quantification. Library-free computational analysis using DIA-NN, combined with differential expression analysis, enabled sensitive detection of key proteins in intestinal organoids.
Methods in molecular biology (Clifton, N.J.)Talita Émile Ribeiro Adelino, Joilson Xavier, Vagner Fonseca, Marta Giovanetti
Nanopore sequencing is transforming viral genomics through real-time, portable, long-read analysis of RNA and DNA. Unlike traditional short-read platforms, it detects nucleotide sequences by measuring ionic current changes as nucleic acids pass through nanoscale pores, enabling direct single-molecule sequencing and base modification detection. Its simplicity, flexibility, and capacity for ultra-long reads make it ideal for resolving complex genomic regions, structural variants, and full viral genomes. These advantages have accelerated its use in pathogen surveillance and outbreak response, especially in resource-limited settings. For chikungunya virus (CHIKV), nanopore sequencing allows rapid, culture-independent recovery of complete genomes from clinical and vector samples, enabling real-time tracking of viral diversity, evolution, and spread. Experiences from Ebola, Zika, and COVID-19 have demonstrated the power of portable sequencing, now applied to CHIKV monitoring. Advances in tools such as Guppy, Dorado, Minimap2, and Medaka enhance read quality, consensus accuracy, and downstream analyses. Despite challenges in basecalling and error correction, robust quality control pipelines ensure reliable results. Ongoing improvements in chemistry, flow cell design, and machine learning will further enhance fidelity and throughput, establishing nanopore sequencing as a cornerstone of CHIKV genomic surveillance and epidemic preparedness.
Methods in molecular biology (Clifton, N.J.)Almudena Escobar-Niño, Olivier Coste, Maria Victoria Calcis-Marzán, Francisco Javier Fernandez-Acero
Proteomics has been revealed as a key set of technologies that provide a detailed description of the molecular processes involved in the development of a specific phenotype. "Omics" technologies can collect an incredible amount of information. Among them, proteomics is an invaluable tool for defining specific biological information by studying the complete set of proteins under specific conditions, the proteome; or specific subsets of proteins, the subproteome. It is a crucial instrument for describing protein post-translational modifications, the functional annotation of the genome, and the detection of orphan genes. Protein extraction procedures are necessary to obtain B. cinerea protein extracts of sufficient quality to be analyzed by LC-MS/MS, avoiding contaminants that interfere with the identification process. After experimental design, collect the samples and replicates as defined in each experimental approach; we will describe protocols and procedures for the next steps of proteome and subproteome extraction and LC-MS analysis.
The Immuno-Oncology Biological Research (IOBR) package is an R-based analysis tool for exploring the tumor microenvironment (TME) and its influence on anti-tumor immunity. Built for high-throughput data-spanning both transcriptomic and genomic profiles-IOBR integrates six analytical modules, including transcriptomic data preprocessing, TME profiling, TME pattern identification, ligand-receptor interaction analysis, genome-TME interaction assessment, and visualization. In this chapter, we walk through a multi-omics workflow using example datasets, illustrating data preparation, distribution analyses, result interpretation, and graphical output. IOBR is open source and is available at https://github.com/IOBR/IOBR and a detailed GitBook ( https://iobr.github.io/book/ ) offers a complete manual and analysis guide for each function.
Methods in molecular biology (Clifton, N.J.)Benjamin L Kidder
Teratoma formation is the gold standard assay for evaluating the developmental pluripotency of human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Following subcutaneous injection into immunodeficient mice, pluripotent stem cells spontaneously differentiate into derivatives representing all three embryonic germ layers-ectoderm, mesoderm, and endoderm. Beyond serving as a functional assay for pluripotency, teratomas provide a unique three-dimensional model system for studying early human development and lineage specification in vivo. This chapter describes comprehensive protocols for teratoma formation in immunodeficient mice, tissue processing for multiple downstream genomic applications, and multi-omics profiling approaches. We detail methods for embryonic stem cell culture, teratoma generation via subcutaneous injection, tissue dissection and processing for chromatin immunoprecipitation followed by sequencing (ChIP-Seq), RNA sequencing (RNA-Seq), single-cell multiome profiling combining chromatin accessibility (ATAC-Seq) and gene expression (scRNA-Seq), and histological analysis using hematoxylin and eosin (H&E) staining. Additionally, we provide bioinformatics workflows for analyzing the resulting genomic datasets to characterize the epigenetic and transcriptional landscapes of teratoma-derived tissues. These methods enable comprehensive molecular characterization of developmental processes and provide valuable resources for stem cell biologists studying pluripotency, differentiation, and early embryonic development.
The plant genomeSamuel A Adewale, Md Ali Babar, Diego Jarquin, Naeem Khan, Stephen Harrison, Noah DeWitt, Rick Boyles, Shuyu Liu, Ellen Melson, Daniel Hathcoat, Jason D Fiedle…
Genetic gains of oat (Avena sativa L.) grain yield have been historically low compared to other major cereal crops. The use of machine learning models to capture complex interactions and leveraging data types other than genomic information in prediction models has great potential for improving complex traits in oat breeding programs. This study assessed the performance of deep learning model for genomic prediction compared to other statistical models, examined the optimal training set size for grain yield prediction, and investigated the potential of incorporating environmental covariates for enhancing oat grain yield prediction. A total of 463 oat lines were evaluated in five environments in Southern United States, and genotyping of the lines gave 12,657 single-nucleotide polymorphism markers. Our results showed that training set sizes 200-350 could be the optimal size for our panel, indicating the possibility of reducing phenotyping costs by reducing the size of the oat panel tested. The deep learning model was less superior to genomic best linear unbiased prediction and other models for grain yield, test weight, and heading days in the different environments. Incorporating interaction effects (G × E or G × W) into the multikernel prediction models across environments improved predictive abilities for grain yield by up to 0.21 compared to the baseline models. This reveals the potential of incorporating weather data to enhance predictive abilities in genomic prediction models. Our findings provide important information for improving genetic gains in oat breeding programs by integrating genomics and environmental information.
Pakistan journal of pharmaceutical sciencesOrcun Can
BACKGROUND: The biology of the estrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2-) breast cancers is heterogeneous even when they are categorized by their risk via genomics. Transcriptomic PGR expression reflects endocrine pathway activity and may provide complementary biological information within established GENE70-derived genomic-risk categories. Whether this molecular marker improves the biological interpretation of genomic-risk stratification beyond conventional clinicopathological assessment remains uncertain. OBJECTIVES: The aim of this study was to determine whether transcriptomic PGR expression provides complementary biological and prognostic information within reconstructed GENE70-derived genomic-risk categories and refines the characterization of endocrine-related tumour biology in ER-positive/HER2-negative breast cancer. METHODS: This study analysed publicly available transcriptomic and clinical data from three cohorts: METABRIC (discovery cohort), GSE96058/SCAN-B cohort (validation cohort) and TCGA-BRCA cohort (molecular validation cohort). The GENE70-derived genomic-risk score was reconstructed for each cohort using matched genes. Cox regression, Kaplan-Meier analysis and subgroup comparisons were used to assess relationships between PGR expression, clinicopathologic variables, molecular features and survival outcomes. RESULTS: Across the three independent cohorts, low transcriptomic PGR expression was consistently associated with higher GENE70-derived genomic risk, increased MKI67 expression, reduced ESR1 expression and enrichment of the Luminal B subtype. Survival findings differed between cohorts. In the discovery METABRIC cohort, transcriptomic PGR expression showed heterogeneous associations with survival, particularly within GENE70-derived high-risk subgroups, whereas the external GSE96058/SCAN-B validation cohort demonstrated consistent associations between low PGR expression and poorer overall survival in both the overall ER-positive/HER2-negative population and GENE70-derived high-risk subgroups. CONCLUSION: These findings suggest that transcriptomic PGR provides complementary biological and prognostic information within GENE70-derived genomic-risk categories. However, because treatment response was not evaluated in the present study, the findings should not be interpreted as evidence of predictive or pharmacogenomic utility and prospective studies incorporating treatment-response analyses are required before such applications can be established.
Genetic epidemiologyKristen J Sutton, Julie E Gervis, Moomal Jatoi, Liang-Dar Hwang, Audrey E Hendricks, Debashis Ghosh, Kenneth E Westerman, Joanne B Cole
Most Mendelian randomization (MR) of dietary intake use the full set of genome-wide significant (GWS) variants in the instrumental variable (IV), likely biasing causal estimates due to pleiotropy. To characterize the common methods to handle pleiotropy in dietary intake MR, we conducted a scoping review of the literature on dietary intake MR studies. We extracted information on IV construction, assessment of pleiotropy, and sensitivity analyzes revealing that only 20% of studies used an IV with functional plausibility. In the absence of functionally-informed IVs, we tested if two-sample MR using GWS variants filtered for pleiotropic associations through phenome-wide association studies (PheWAS) could identify diet-health relationships supported by existing nutrition science, focusing on oily fish and alcohol intake, the latter of which has a functionally-informed IV for comparison (rs1229984 in the ADH1B gene). To further explore this question, we performed multivariable MR and employed MR-CAUSE. The numerous models consistently supported that oily fish reduced triglycerides. In contrast, GWS and PheWAS-filtered IVs suggested that alcohol decreased alanine aminotransferase levels, whereas the functional IV (rs1229984) found the opposite expected relationship. Isolating the direct effect of dietary intake from GWS IV remains challenging. Future work should focus on identifying functional variants impacting dietary behavior.
Journal of inherited metabolic diseasePatryk Lipiński, Jaak Jaeken
Congenital disorders of glycosylation (CDG) are a rapidly expanding group of inherited metabolic diseases affecting glycoconjugate glycan biosynthesis and attachment. This review provides a structured overview of major advances in human CDG reported from 2023 to 2026, including newly recognized CDG, emerging biochemical and pathophysiological insights, advances in diagnostic strategies and biomarkers, and progress in treatment. Recent developments include the recognition of RPN1-CDG, UGGT1-CDG, and DHRSX-CDG, refinement of phenotypes in established disorders, including PMM2-CDG, ALG13-CDG, ALG8-CDG, GMPPA-CDG, TRAPPC11-CDG, and PGAP3-CDG, and increasing use of glycomics, glycoproteomics, cellular models, and multi-omics approaches. Therapeutic progress remains uneven: no FDA/EMA-approved disease-modifying therapy is currently available for any CDG, and most interventions remain off-label, experimental, or supported by limited observational evidence. Despite substantial progress, most CDG still lack disease-modifying treatment, validated biomarkers, and prospective natural history data.
Journal of inherited metabolic diseaseNandaki Keshavan, Julia Neugebauer, Marcello Bellusci, Enrico Bertini, Garry Brown, Niklas Darin, Suzanne DeBrosse, Lucy Drexler, Stefan Drexler, Gregory M Enn…
Primary pyruvate dehydrogenase complex deficiency (PDCD) comprises a group of monogenic disorders caused by pathogenic variants in genes encoding subunits of, or regulatory components affecting, the pyruvate dehydrogenase complex. The clinical phenotype spans a broad continuum, ranging from early onset congenital lactic acidosis to infantile or childhood onset global developmental delay with epilepsy, through to more attenuated adult-onset neurological presentations. Reports from patients and advocacy groups indicate substantial variability in clinical management across both emergency and outpatient settings and between centres internationally. This variability underscores the need for systematic evaluation of the evidence base and the development of harmonised, consensus-driven clinical guidelines to standardise care and improve outcomes. An international consortium of experts from Europe and North America, including metabolic physicians, neurologists, dietitians, geneticists and patient representatives, was convened. The group undertook a structured review of the literature and developed guideline statements addressing disease classification, recognition, diagnostic evaluation, dietary and non-dietary management, surveillance for complications, genetic counselling and transition to adult services. Each recommendation was assigned a GRADE rating reflecting strength and quality of evidence. Consensus was achieved using a Delphi methodology. In total, 199 recommendations reached consensus and constitute the core of these guidelines. These recommendations provide a framework for consistent, high-quality, multidisciplinary care. The process also identified key evidence gaps, highlighting priorities for future research and the ongoing need to develop effective disease-modifying therapies.
Basic & clinical pharmacology & toxicologyAbraham García-López, Andrea Guzmán-de Antonio, Tania S Rubio-Lepe, Elena Diago-Sempere, Juan Raffo-Nogueira, Pedro Arias, Belén Ferrer-Martínez, Sonia Rodrigu…
VKORC1 genetic variants are major determinants of interindividual variability in vitamin K antagonist (VKA) dose requirements and are incorporated into several pharmacogenetic dosing guidelines. The promoter variant rs9923231 is considered the clinically relevant marker for genotype-guided VKA dosing. However, the intronic variant rs9934438 is sometimes used as a surrogate marker because it is assumed to be in strong linkage disequilibrium with rs9923231. We aimed to assess whether rs9934438 can reliably substitute rs9923231 in clinical practice. We conducted a retrospective, single-centre study including 939 patients who underwent pharmacogenetic testing. Both variants were genotyped using a TaqMan OpenArray custom panel. Concordance between rs9934438 and rs9923231 was evaluated, and the diagnostic performance of rs9934438 for predicting clinically relevant rs9923231 diplotypes was assessed. Although concordance was high overall, rs9934438 misclassified two patients who, according to rs9923231, required a reduced VKA dose, resulting in potential false-negative classifications. These findings indicate that, while rs9934438 provides informative results in most cases, it may fail to identify some patients requiring dose adjustment. Our results highlight the clinical implications of relying on surrogate markers in pharmacogenetic-guided VKA therapy and support the preferential use of rs9923231 to ensure accurate individualized dosing and optimize treatment safety.
GliaRobert W Lewis, Breana C Dogan, Amy L Stanek, Elliot B Evans, Madelyn G Coble, Hayli E Spence-Osorio, Karen L G Farizatto, Angie L Mordant, C Allie Mills, Laur…
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy characterized by early-onset macrocephaly, white matter edema, seizures, and motor and cognitive decline. Approximately 25% of MLC patients carry HEPACAM pathogenic variants, many of which are dominant missense variants causing remitting MLC Type 2b. HEPACAM encodes hepatic and glial cell adhesion molecule (hepaCAM), also known as GlialCAM, an astrocyte-enriched transmembrane protein with important roles in astrocyte territory establishment, gap junction coupling, branching organization, synaptic function, and development of the gliovascular unit. The molecular mechanisms through which pathogenic variants in HEPACAM alter hepaCAM protein function in vivo and facilitate MLC pathogenesis during brain development remain largely unknown. Here, we used new viral tools and proximity-based proteomics to examine how three different dominant pathogenic variants alter hepaCAM subcellular localization and protein interactome in astrocytes of the developing mouse cortex. We found dramatic changes in hepaCAM distribution throughout the astrocyte, which were common to all mutants tested. We also observed significant changes in protein interactome between wild type and mutant hepaCAM, including decreased association with previously described hepaCAM-interacting proteins Connexin 43 and CLC-2. Moreover, we identified the epilepsy-associate potassium channel KCNQ2 as a novel hepaCAM interaction partner and found reduced association between KCNQ2 and pathogenic variants. Collectively, our data provide new insights into hepaCAM protein function in astrocytes during brain development, reveal altered protein dynamics of pathogenic variants, and provide a new resource to explore the molecular underpinnings of MLC pathogenesis.
Rapid communications in mass spectrometry : RCMTing-Yu Wei, Joao A Paulo
RATIONALE: Multiplexed quantitative proteomics enables simultaneous analysis of multiple biological samples, increasing throughput while reducing instrument time and sample requirements. However, integrating sample multiplexing with data-independent acquisition (DIA) remains challenging. We present a TMTpro plexDIA strategy leveraging MS1-level mass differences between nonisobaric TMTpro reagent variants to enable multiplexed quantification without compromising DIA sensitivity. METHODS: Three Saccharomyces cerevisiae deletion strains (Δmet6, Δpfk2, and Δura2) were labeled with TMTproZero (light), TMTpro16 (standard), and super-heavy TMTpro (heavy), mixed in three permutations, and analyzed by narrow-window DIA (2 m/z isolation, 300 scan events) on an Orbitrap Astral mass spectrometer. Database searching was performed using FragPipe/MSFragger with plex-DIA quantification enabled. RESULTS: Over 2000 protein groups and over 20 000 precursors were identified per channel per mixture, with closely matched identification rates across all three channels. All nine expected deletion patterns were correctly identified, with channel-specific depletion reproduced consistently across precursor charge states (2+, 3+, and 4+). CONCLUSIONS: TMTpro plex-DIA enables accurate, multiplexed quantitative proteomics through MS1-level mass separation of the nonisobaric TMTpro isotopologs. The characteristic deletion patterns observed for each knockout strain serve as intrinsic molecular barcodes, validating sample identity and demonstrating the broad utility of plex-DIA for high-throughput, multiplexed proteomics applications.
Chembiochem : a European journal of chemical biologyStefania De Chiara, Angela Di Somma, Valentina Mazziotti, Serena Coppola, Franca Oglio, Lidia Tammaro, Maria Pia Riccio, Carmela Bravaccio, Antonio Molinaro, R…
The etiology of pediatric autism spectrum disorder (ASD) has been increasingly linked to alterations in the gut-brain axis, highlighting the intricate bidirectional communication between gut microbiota and central nervous system. The molecular mechanisms of this communication are poorly understood. Here we investigated whether ASD-associated gut microbiota could exhibit altered inflammatory molecular outputs by integrating chemistry-driven profiling of fecal lipopolysaccharides (LPS), functional bacterial proteomics, and neuroimmune cellular assays. Structural analyses revealed that LPS from non-autistic healthy donors (NASD) displayed highly conserved carbohydrate and lipid A signatures dominated by hypo-acylated mono-phosphorylated species typically associated with immunomodulatory Bacteroides-derived LPS. In contrast, LPS from ASD children exhibited increased structural heterogeneity. These molecular alterations were functionally reflected in human HMC3 microglial cells, where ASD-derived fecal LPS induced stronger IL-6 and IL-8 release compared with NASD-derived LPS, indicating enhanced neuroinflammatory potential. Functional proteomic profiling disclosed broadly comparable microbial compositions but marked metabolic divergence. These findings suggest that gut microbiota in ASD children are associated with a functionally heterogeneous microbial ecosystem characterized by altered immunostimulatory molecular outputs despite the absence of massive taxonomic shifts, with potential relevance for neuroimmune dysregulation.
Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseasesC Z Zhang, S S Lyu, X D Han, H Wang, Y Sun, X D Liu
Objective: To investigate the molecular epidemiological characteristics, antimicrobial resistance mechanisms, and clinical risk factors for infection with carbapenem-resistant Klebsiella pneumoniae (CRKP), and to examine the relationship between antimicrobial resistance and virulence. Methods: A total of 528 Klebsiella pneumoniae (KP) isolates and corresponding clinical data were collected from hospitalized patients at Qingdao Municipal Hospital between November 2016 and October 2023. Based on antimicrobial susceptibility testing results, 80 patients with CRKP infection were assigned to the CRKP group. Patients with carbapenem-susceptible K. pneumoniae (CSKP) isolates recovered within 7 days before or after the CRKP isolation date were selected as matched controls for each patient in the CRKP group. If insufficient eligible controls were available, the matching window was expanded to 30 days before or after the isolation date. Ultimately, 160 matched patients were included in the CSKP group. Whole-genome sequencing was performed on all included isolates to systematically characterize their molecular epidemiological features and resistance and virulence gene profiles. A multivariable logistic regression model was used to identify independent risk factors for CRKP infection. Spearman rank correlation analysis was performed to assess the correlation between resistance and virulence gene scores. Results: Multivariable logistic regression analysis showed that a history of antimicrobial agent use in the 30 days before infection (OR=4.411, 95%CI: 1.749-11.128; Wald χ²=9.882; P<0.01) and invasive abdominal procedures in 30 days before infection (OR=6.846, 95%CI: 3.141-14.922; Wald χ²=23.411; P<0.01) were independent risk factors for CRKP infection. ST11 was the predominant sequence type among CRKP isolates (80.0%), and the blaKPC-2 gene was the predominant resistance determinant (88.8%). The predominant CRKP clone, ST11, underwent a serotype transition from ST11-K25/O5 to ST11-K64/O2a. At the overall population level, the antimicrobial resistance gene score was negatively correlated with the virulence gene score (Spearman ρ=-0.321, P<0.001). However, subgroup analysis showed no significant correlation between the two scores within the same ST11 clone (Spearman ρ=0.020, P=0.872). These findings suggest that the overall negative correlation was a population-level artifact caused by inherent phenotypic differences among distinct clonal groups, rather than an evolutionary trade-off in which the acquisition of antimicrobial resistance within the same clone resulted in reduced virulence. The intensive care unit (ICU) was the central hub for nosocomial clonal transmission of CRKP. The predominant clone may have disseminated from the ICU to general wards through patient transfers, healthcare workers, or medical equipment, thereby forming interdepartmental transmission chains. Conclusions: A history of antimicrobial agent use and invasive abdominal procedures in the 30 days before CRKP infection were independent risk factors for CRKP infection. CRKP isolates frequently carried plasmids coharboring multiple antimicrobial resistance determinants and exhibited extensive antimicrobial resistance.
Life science allianceLaura Alunno, Ilaria Massignani, Meriem Hadjer Hamadou, Fabio Mazza, Daniele Peroni, Romina Belli, Erik Dassi, Alessandro Romanel, Alberto Inga
To prioritize germline genetic variants affecting mRNA fate at the translational level, we used sucrose gradient-based isolation of 80S monosomes and polysomes, followed by RNA sequencing in RPE-1 cells, under mock and nutlin treatment conditions. Differential gene expression analysis confirmed a canonical p53 response. Heterozygous SNPs and SNVs were identified from the RNA-sequencing data, and allelic fractions were calculated for total, monosomal, and polysomal mRNAs. Variants showing reproducible allelic fraction differences across fractions beyond experimental variability were defined as tranSNPs. Among over 7,000 heterozygous variants analyzable in polysomal RNA and over 5,000 in mRNAs associated with monosome-enriched fractions, 1,247 in 1,015 genes displayed a significant imbalance. Reporter assays performed in RPE-1 and HCT116 cells validated allelic or haplotype effects for 17 selected variants in UTRs and coding regions, confirming differences in 15 cases, with evidence of cell line-specific responses. Proteomic analysis supported allelic imbalance for a few selected missense variants. Monosome-enriched fractions improved sensitivity in measuring allelic imbalances without introducing a tranSNP positional bias, suggesting that 80S profiling enhances detection of allele-specific translational regulation in RPE-1 cells.
Life science allianceSarah L Pashley, Molly Hair, Chiamaka V Ukegbu, Mohammad Zeeshan, Akancha Mishra, Declan Brady, Robert Markus, Sue Vaughan, Carla Pasquarello, Anthony A Holder…
Plasmodium parasites rely on diverse protein kinases to regulate critical processes, yet many remain uncharacterised. Protein kinase 2 (PK2) is required for asexual blood-stage proliferation and merozoite invasion, but its role in sexual stages and transmission is unknown. Here, using live-cell and super-resolution imaging, ultrastructural analysis, functional phenotyping, and quantitative phosphoproteomics, we define the role of PK2 in Plasmodium berghei transmission. PK2 was expressed in merozoites, ookinetes, and sporozoites: the three invasive forms of the parasite. Conditional depletion of PK2 impaired ookinete motility, disrupted apical microneme positioning, abolished oocyst formation, and prevented mosquito-to-host transmission. Direct haemocoel injection of mutant ookinetes failed to restore sporozoite production, demonstrating an additional requirement for PK2 downstream of midgut traversal. Although global protein abundance was largely preserved, phosphoproteomic profiling identified altered phosphorylation of proteins associated with cytoskeletal organisation, host-cell interaction, midgut invasion, and gene regulation. These findings highlight PK2's critical role across multiple invasive stages, advancing understanding of malaria parasite transmission and identifying putative PK2-regulated pathways as potential targets for transmission-blocking interventions.
Proceedings of the National Academy of Sciences of the United States of AmericaDiane Martin-Moya, Jean-Christophe Grenier, Liam Thomas Lanigan, Justin Pelletier, Evan K Irving Pease, Fernando Racimo, Claude Bhérer, Hannes Schroeder, Julie…
Historical narratives of early colonization in Quebec frequently portray French-Indigenous relations as cooperative and characterized by admixture; which are often mobilized in modern debates on false-indigeneity. These narratives have been studied through genealogical, genetic, and bioarchaeological analyses, yet individuals of African descent remain mostly absent from them, while Indigenous peoples are framed largely in relation to colonists. Notre-Dame Cemetery (1683-1796) in Tiohtià:ke-Montréal offers a unique window onto one of the earliest urban colonial populations in Nouvelle France, but previous bioarchaeological studies have produced conflicting interpretations regarding the presence of non-European or admixed individuals. Here, we generated ancient DNA from 45 individuals and applied genotype imputation, runs-of-homozygosity, and identity-by-descent analyses to reevaluate genetic relatedness and cemetery organization. Our results show exclusively French European ancestry. We observe low levels of close-kin unions and identify several genetic lineages distributed across the cemetery's successive burial phases rather than family clustering or religious ordering. The spatial organization of the graves is consistent with rapid demographic growth, with successive burials overlapping one another, before being disrupted by urban development. Rooted in Indigenous decolonial frameworks, these findings challenge the 'admixed founding population' narrative, aligning with historical evidence of Indigenous and African-descended marginalization while highlighting the social inequalities shaping this colonial urban cemetery. Québec's bioarchaeological practices must move beyond blood ties as the sole measure of kinship and commit to legal frameworks that better reflect reciprocal obligations of care, in order to overcome the colonial structures that still define heritage.
Environmental microbiology reportsArefeen Haider, Tasnuva Jahan Esha, Sezanur Rahman, Jannat Akther, Naimul Islam Faysal, Tahsin Khan, Shakhinur Islam Mondal, Anowara Begum, Sudhangshu Kumar Bi…
Bacillus species are environmentally resilient bacteria associated with soil, food, industrial settings and opportunistic infections, yet their bacteriophages remain incompletely characterised, particularly from underexplored environmental sources. We isolated and characterised phage APU1, a dsDNA phage from agricultural soil in Bangladesh. Partial 16S rRNA gene sequencing identified the enrichment host conservatively as Bacillus sp. APU1 has a 153,847 bp genome that encodes 236 predicted proteins, including 84 shorter than 100 amino acids. CheckV classified the genome as high-quality, with an estimated completeness of 99.91% and no detectable host-gene contamination. Functional annotation identified genes associated with DNA replication, structural assembly, packaging and host lysis. PhaBOX classified APU1 as virulent, with PhaTYP and CHERRY prediction scores of 1.0. CARD/VFDB screening did not detect CARD-listed antimicrobial resistance genes or VFDB-listed virulence factor genes. Comparative genomic analyses using VICTOR, VIRIDIC and VIPTree placed APU1 within a group of related Bacillus phages. APU1 shared 89.7%-89.8% intergenomic similarity with its closest relatives, supporting its classification as a genomically distinct phage within this group. This study expands the genomic catalogue of Bacillus-associated phages from Bangladeshi soil and provides a foundation for future studies on phage diversity, host range, morphology and biological properties.
Aging cellShangshu Zhao, Chia-Ling Kuo, Eric J Lenze, Julie L Wetherell, Laura Haynes, Perla El-Ahmad, Richard Fortinsky, George Kuchel, Trevor Harris, Breno S Diniz
The accumulation of senescent cells is a recognized hallmark of biological aging and is associated with the onset of multiple chronic medical conditions. Senescent cells exhibit a distinct secretory profile, known as the senescence-associated secretory phenotype (SASP), which can propagate cellular senescence to neighboring and distant tissues. Measuring SASP factors in blood serves as a practical proxy for cellular senescence burden and may help track disease states and intervention outcomes. We developed and validated a composite SASP Score by integrating large-scale population proteomics data with a semi-supervised deep learning framework. The analytical workflow included: (1) selection of biologically curated SASP proteins; (2) development of a Guided autoencoder with Transformer (GAET) model using data from the UK Biobank Pharma Proteomics Project (UKB-PPP); (3) internal evaluation and association analyses within the UK Biobank; and (4) external validation and longitudinal assessment in an independent randomized clinical trial cohort. The deep learning-based SASP Score was a strong, independent predictor of mortality risk and incident serious, chronic medical conditions (e.g., dementia, COPD, myocardial infarction, stroke). In an independent cohort, multimodal exercise significantly changed the SASP Score trajectory over 18 months. Our findings support the potential of a deep learning-derived SASP Score as a biomarker for systemic cellular senescence burden. Our statistical approach can offer enhanced interpretability and cross-platform utility, providing a valuable tool for aging research and the evaluation of geroscience-guided interventions.
Molecular ecology resourcesAmanda S Ackiss, Mark R Vinson, Ann J Ropp, Kristen M Gruenthal, Trevor J Krabbenhoft, Joseph V Siegel, Wendylee Stott, Daniel L Yule, Wesley A Larson
A comprehensive understanding of life history is vital to successful species conservation and management. When different life history stages are accompanied by considerable morphological or cryptic variation, such as the egg and larval phases exhibited by most fishes, genomic tools are essential for identifying species so that early-life ecology questions can be studied. Genotyping-in-thousands by sequencing (GT-seq) has recently emerged as a targeted and efficient approach for species identification. We leveraged existing genomic and transcriptomic data to develop a GT-seq panel capable of differentiating the members of the Coregonus artedi complex, a radiation of salmonids in the Laurentian Great Lakes whose members are indistinguishable with mitochondrial DNA barcoding loci and are the focus of bi-national conservation initiatives. Our panel of 494 loci was able to assign fishes in the C. artedi complex to species and lake. We examined cross-amplification in other coregonines with overlapping distributions and found that congeneric Lake Whitefish (C. clupeaformis) cross-amplified at 94% of loci and confamilial Round and Pygmy Whitefish (Prosopium spp.) cross-amplified at 42% and 38% of loci, respectively. We adapted bioinformatic probes to account for Prosopium-specific variants including 22 new SNPs and developed a whitelist of 428 SNPs capable of distinguishing these whitefishes. Finally, we demonstrated performance by identifying 3066 coregonine larvae and juveniles collected in spring 2019-2021 from Lake Superior. These results hold promise for future insights into the species-specific ecology of early life coregonines and demonstrate the flexibility of GT-seq panels, which may cross-amplify hundreds of informative genome-wide loci in related taxa.
The clinical teacherOrlando Carlos Conceição-Neto, Frederico Medeiros Rosas da Silva, Erika Verissimo Villela, Paula Borges de Andrade Lemes, João Pedro Gotti Ferreira, Chiara Gur…
BACKGROUND: Medical biochemistry and molecular genetics stand out as some of the most demanding courses for first-year medical students, who often require repeated exposure to complex pathways and molecular processes. To provide additional support in this early stage of training, we created PodBases, a peer-led educational podcast integrated into an academic mentoring program and made available through a widely accessible global audio-streaming platform. The goal was to offer brief, clinically oriented episodes that reinforced key concepts and complemented traditional teaching. APPROACH: The initiative followed a defined workflow involving close collaboration between student tutors and faculty. Tutors prepared scripts aligned with the weekly course content, which were subsequently reviewed for accuracy. Recordings were made using smartphones and edited with freely accessible audio-editing tools and published on a podcast-hosting service. We documented the entire process to create a practical tutorial that facilitates replication in similar educational settings. EVALUATION: Between May 2023 and January 2026, 30 episodes were published, reaching 13,050 streams across 16 countries. Student feedback highlighted the usefulness of the podcast during commutes and study sessions. Mentors reported skill development in communication, simplification of content and teamwork. IMPLICATIONS: PodBases demonstrates that student-generated podcasts can effectively complement traditional teaching by reinforcing core topics, supporting learner autonomy, enhancing comprehension and developing tutors' pedagogical skills. The experience suggests that peer-led digital resources can enhance comprehension while promoting engagement and professional growth in the foundational years of training. TRIAL REGISTRATION: Not applicable.
Life science allianceZsolt Szilagyi, Pauline Michon, Carlos Pardo-Hernández, Mãdãlina Sãcultanu, Javier Miralles Fusté, Andrew M Griffin, Paul S Charifson, Cindy Phan, Yonghong Shi…
Lon protease 1 (LONP1) is a conserved hexameric protease implicated in mitochondrial disorders and cancer progression. In this study, we present PZL-26, a potent and selective small-molecule inhibitor that targets LONP1 without affecting the proteasome, leading to selective accumulation of mitochondrial proteins. Using PZL-26 in a whole-genome CRISPR-Cas9 screen, we identified genes essential for cell survival under protease inhibition, supporting a role for LONP1 in key mitochondrial processes, including complex I biogenesis, mitochondrial transcription, and translation. Our CRISPR screen results are consistent with proteomics analysis, with both approaches converging on the same mitochondrial pathways and highlighting functional interactions between LONP1 and other mitochondrial proteases, including potential compensatory mechanisms. These findings establish PZL-26 as an effective tool for exploring LONP1 function and pave the way for future therapeutic strategies targeting LONP1 in mitochondrial diseases and cancer.
Animal geneticsMonica Nielsen, Hanne Gredal, Åsa Karlsson, Jennifer R S Meadows, Maja Arendt
Ivermectin-associated neurotoxicity is a potentially life-threatening condition caused by disruption of the ABCB1 encoded P-glycoprotein drug transporter in certain dog breeds. Skye terriers are considered at increased risk of ivermectin toxicity despite the absence of an identified molecular cause. We searched the Dog10K variant dataset for ABCB1 variants potentially underlying ivermectin sensitivity in Skye terriers and screened an independent cohort of 27 dogs to confirm sequence variants. A 1-bp frameshift deletion predicted to cause loss of function in ABCB1 was identified in two of three Skye terriers in the Dog10K dataset and was absent from all 1869 non-Skye-terrier genomes. The variant had an allele frequency of 16.7% in the independent Skye terrier cohort. This is the first reported ABCB1 variant in Skye terriers and represents a plausible candidate genetic basis for the anecdotally observed ivermectin sensitivity in the breed.
Journal of medical virologyNicole Lardini Freitas, Yago Côrtes Pinheiro Gomes, Fábio César Sousa Nogueira, Juliana Echevarria-Lima, João Victor Carvalho Deus, Carlos Otávio Brandão, Cris…
Pathophysiological mechanisms of neurological manifestations in COVID-19 are not fully known yet. In this case-control study, proteomic analysis was used to characterize cerebrospinal fluid (CSF) samples from COVID-19 patients with acute neurological manifestations primarily categorized as: isolated refractory headache (n = 12), encephalopathy (n = 24), and inflammatory neurological diseases (IND) (n = 13). Individuals with non-inflammatory, non-infectious neurological conditions (n = 8) were included as controls. Differentially expressed proteins (DEPs) in neuro-COVID-19 were associated with the complement system, function/activation of microglia/macrophages, inflammatory responses, and neuronal function/homeostasis. Functional enrichment analysis of DEPs and/or proteins specifically detected in neuro-COVID-19 groups indicated activation of complement and coagulation cascades, and synapse pruning in encephalopathy and IND. Upregulated complement activation was confirmed by increased CSF levels of C3a and soluble C5b-9. Biological processes of neuroinflammation, migration of immune cells into the central nervous system, activation of glia cells (microglia and astrocytes), and tissue remodeling were also present in IND. In turn, processes associated with disturbed organization/formation of neuron projections were related to encephalopathy and isolated headache. CSF from patients with isolated refractory headache was enriched for proteins related to neurons and to the cerebral cortex, cerebellum, and basal ganglia. Meanwhile, CSF from encephalopathy and IND patients was mostly enriched for proteins mapped to the cerebral cortex, choroid plexus, and thalamus. Moreover, CSF from IND patients had an increased proportion of microglia-related proteins. These findings support a model in which cases of IND and encephalopathy are associated with blood-brain barrier disruption potentially induced by complement activation and glia-promoted neuroinflammation, resulting in the involvement of distinct brain structures in COVID-19.
Journal of genetic counselingNicole Daley, Alyssa Griswold, Laura Moreno, Alyson Evans Floyd, Dat Duong, Benjamin D Solomon, Rebekah L Waikel
AI-driven chatbots have been utilized in healthcare to automate administrative tasks, improve patient education, and expand access to medical information; however, their role in genetic counseling remains underexplored. To investigate the adoption, perceptions, and potential utility of AI-based chatbots in genetic counseling practice, 217 genetic counselors and genetic counseling students from across North America were surveyed regarding chatbot usage, confidence in their application, and perceived benefits and limitations. While most participants (166/217; 76.5%) reported using general AI chatbots outside of clinical settings, far fewer (18/204; 8.8%) reported using or recommending clinical genetics chatbots in clinical practice. For those that used clinical genetics chatbots, the primary purpose was for communication with at-risk family members (11/18; 61.1%) and patient education (10/18; 55.6%). Confidence in chatbot technology varied, with highest confidence in gathering family history information (81/199; 40.7%) and lowest confidence in their ability to disclose variants of uncertain significance or positive genetic testing results (5/199; 2.5%). The greatest perceived benefits included reducing repetitive tasks (165/195, 84.6%) and allowing for time for other tasks (141/195; 72.3%), while major concerns revolved around patient comprehension (167/195; 85.6%) and having accurate, up-to-date information (145/195; 74.4%). Despite some concern about AI replacing human counselors, most participants reported they felt there was potential for chatbots to enhance workflow efficiency (128/195; 65.6%) if properly integrated and regulated. Limited AI training was identified as a barrier to adoption (16/195; 8.2% received training), highlighting a need for structured education on AI applications in genetic counseling. These findings suggest that AI chatbots hold promise as supplementary tools, but significant challenges must be addressed before widespread implementation in genetic counseling practice.
International journal of dermatologyW Austin Wyant, Dorsa Moslehi, Jennifer A Lo
Melanoma incidence has increased approximately 6-fold over the past 40 years, driven primarily by melanoma in situ and thin invasive melanomas, which are among the most subjective lesions to diagnose on histopathology. This review examines whether genomics clarifies the "gray zone" between benign and malignant pigmented lesions, or instead recasts existing histopathologic ambiguity in molecular terms, while also considering whether genomic classification shifts overdiagnosis upstream instead of resolving underlying diagnostic uncertainty. Across cytogenetic, mutation-based, and gene-expression platforms, current assays perform best in unequivocal melanoma-versus-nevus comparisons and lose accuracy in diagnostically ambiguous lesions where clarification is most needed. Gene expression profile (GEP) tests can be useful diagnostic aids in tissue-rich equivocal lesions, including atypical Spitz tumors, MELTUMPs, and cellular blue nevus/blue nevus-like melanocytoma differentials. One clinicopathologic-genomic model, the Merlin CP-GEP test, has been prospectively validated for predicting sentinel lymph node status and is now recognized as supporting sentinel lymph node biopsy decisions in T1b/T2a melanomas. Importantly, a low-risk prognostic GEP class assignment should not displace the standard sentinel lymph node biopsy discussion. GEP tests are least useful for small, thin, equivocal junctional proliferations, precisely the lesions that are most difficult to diagnose. Pre-biopsy non-invasive assays carry the additional risk of lowering, rather than raising, the biopsy threshold. Overall, current genomic tools should be viewed as an adjunct to, rather than a replacement for, histopathology and clinical context.
GliaAlba M Lucart-Sanchez, Edward Sellés-Climent, Jorge Navarro-Calvo, Guillem Pont-Espinós, Jose A Gomez-Sanchez, Raúl Estévez, Luis M Valor, Rocío Pérez-González
Extracellular vesicles (EVs) mediate intercellular communication in the central nervous system (CNS) and are emerging as biomarkers of brain health and disease. However, the molecular composition of cell type-specific brain EVs, particularly astrocyte-derived EVs (ADEVs), remains poorly defined. We performed comparative proteomic analysis of neuronal (NDEVs), microglial (MDEVs), and ADEVs from mouse brain using magnetic immunocapture and LC-MS/MS proteomic profiling. Each EV subtype displayed distinct molecular fingerprints. NDEVs were enriched in synaptic and neurogenesis-related proteins (e.g., APP, SNAP25, GPR158, and BDNF), whereas MDEVs contained immune and phagocytic markers (e.g., TMEM119, CX3CR1, CD11b). Strikingly, the ADEV proteome closely mirrored the recently characterized GlialCAM interactome from leukodystrophy research, encompassing GlialCAM/MLC1 and associated partners involved in ion and water homeostasis (EAAT1/2, AQP4, GJA1), together with GPCRs such as GPRC5B. This overlap suggests that ADEVs encapsulate a molecular scaffold characteristic of astrocytic endfeet, potentially extending their signaling functions to the extracellular space. In conclusion, our study provides a detailed comparative proteomic characterization of brain cell type-specific EVs, revealing that ADEVs contain the GlialCAM/MLC1 network and GPCRs. These findings identify candidate molecular signatures that support the future investigation of ADEVs for biomarker development and provide a proteomic framework for exploring their relationship with astrocytic endfoot biology, blood-brain barrier (BBB)-associated pathways, and neurodegenerative disorders.