bims-pideca 2024-12-15 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2024–12–15
28 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

Profiling PIK3CA variants in disorders of somatic mosaicism.
Molecular landscape and classification of vascular anomalies.
AnVILWorkflow: A runnable workflow package for Cloud-implemented bioinformatics analysis pipelines.
Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.
How to build the virtual cell with artificial intelligence: Priorities and opportunities.
Improving genetic diagnostic yield in a large cohort of children with rare vascular anomalies or PIK3CA-related overgrowth spectrum.
Genetic and pharmacological targeting of mTORC1 in mouse models of arteriovenous malformation expose non-cell autonomous signalling in HHT.
iFlpMosaics enable the multispectral barcoding and high-throughput comparative analysis of mutant and wild-type cells.
Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34+ hematopoietic progenitor cells and humanized mice.
qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.
Pleiotropic tumor suppressive functions of PTEN missense mutations during gliomagenesis.
Central control of dynamic gene circuits governs T cell rest and activation.
Optimized reporters for multiplexed detection of transcription factor activity.
State of the interactomes: an evaluation of molecular networks for generating biological insights.
A statistical approach for systematic identification of transition cells from scRNA-seq data.
Single-cell RNA sequencing algorithms underestimate changes in transcriptional noise compared to single-molecule RNA imaging.
Identification of cell type-specific cell-penetrating peptides through in vivo phage display leveraged by next generation sequencing.
Structure and organization of full-length Epidermal Growth Factor Receptor in extracellular vesicles by cryo-electron tomography.
Circadian Control of Protein Synthesis.
scTrends: A living review of commercial single-cell and spatial 'omic technologies.
A framework for neural organoids, assembloids and transplantation studies.
Decoding Angiocrine Signaling: Endothelial Cells as Drivers of Organ Regeneration and Homeostasis.
Neuropilin-1 controls vascular permeability through juxtacrine regulation of endothelial adherens junctions.
Alk1/Endoglin signaling restricts vein cell size increases in response to hemodynamic cues.
Intracellular endothelial cell metabolism in vascular function and dysfunction.
Comparative analysis of PI3K-AKT and MEK-ERK1/2 signaling-driven molecular changes in granulosa cells.
Bad bar charts are pervasive across biology.
Emergence of power-law distributions in protein-protein interaction networks through study bias.

Genet Med Open. 2023 ;1(1): 100815

Profiling PIK3CA variants in disorders of somatic mosaicism.

Bahareh A Mojarad, Patricia V Hernandez, Michael J Evenson, Meagan M Corliss, Sarah L Stein, Amy Theos, Carrie C Coughlin, Bryan Sisk, Maithilee Menezes, Molly C Schroeder, Jonathan W Heusel, Julie A Neidich, Yang Cao.

   Purpose: Variants in PIK3CA (encoding p110α; the catalytic subunit of PI3K) characterize some disorders of somatic mosaicism (DoSM) conditions with clinical features, including sporadic overgrowth and vascular malformations. Here, we profile PIK3CA variants in DoSM.
Methods: We applied a next-generation, sequencing-based, laboratory-developed test, using an average coverage of approximately 2000× for up to 37 genes associated with DoSM, on a cohort of 1197 patients with DoSM referred for clinical genomics services between 2013 and 2022.
Results: We identified clinically reportable variants in 747 (62.4%) individuals in this cohort. Notably, 371 clinically reportable variants in PIK3CA were identified in 368 patients, constituting approximately 49.2% of all patients with reportable findings. Variants in the C2 domain of p110α are enriched in DoSM (this cohort) compared with those of cancer (Catalogue of Somatic Mutations in Cancer [COSMIC] database), highlighting the role of the C2 domain in driving uncontrolled cell proliferation in DoSM. Furthermore, we report 17 novel variants in PIK3CA that are not previously reported in DoSM and describe clinical presentation correlation for 4 novel variants.
Conclusion: Our findings from the largest single-center cohort of patients with DoSM expand the spectrum of variants in PIK3CA and shed light on the less-studied role of the C2 domain in the pathogenesis of DoSM.

Keywords:  NGS; PIK3CA; PIK3CA-related overgrowth spectrum; PROS; Somatic mosaicism

DOI:  https://doi.org/10.1016/j.gimo.2023.100815
Hematology Am Soc Hematol Educ Program. 2024 12 06. 2024(1): 700-708

Molecular landscape and classification of vascular anomalies.

Emmanuel Seront, Angela Queisser, Laurence M Boon, Miikka Vikkula.

Vascular malformations, which result from anomalies in angiogenesis, include capillary, lymphatic, venous, arteriovenous, and mixed malformations and affect specific vessel types. Historically, treatments such as sclerotherapy and surgery have shown limited efficacy in complicated cases. Most vascular malformations occur sporadically, but some can be inherited. They result from mutations similar to oncogenic alterations, activating pathways such as PI3K-AKT-mTOR or Ras-MAPK-ERK. Recognizing these parallels, we highlight the potential of targeted molecular inhibitors, repurposing anticancer drugs for the treatment of vascular malformations. This case-based review explores recent developments in precision medicine for slow-flow and fast-flow vascular malformation.

DOI: https://doi.org/10.1182/hematology.2024000598
F1000Res. 2024 ;13 1257

AnVILWorkflow: A runnable workflow package for Cloud-implemented bioinformatics analysis pipelines.

Sehyun Oh, Kai Gravel-Pucillo, Marcel Ramos, Michael C Schatz, Sean Davis, Vincent Carey, Martin Morgan, Levi Waldron.

  Advancements in sequencing technologies and the development of new data collection methods produce large volumes of biological data. The Genomic Data Science Analysis, Visualization, and Informatics Lab-space (AnVIL) provides a cloud-based platform for democratizing access to large-scale genomics data and analysis tools. However, utilizing the full capabilities of AnVIL can be challenging for researchers without extensive bioinformatics expertise, especially for executing complex workflows. We present the AnVILWorkflow R package, which enables the convenient execution of bioinformatics workflows hosted on AnVIL directly from an R environment. AnVILWorkflow simplifies the setup of the cloud computing environment, input data formatting, workflow submission, and retrieval of results through intuitive functions. We demonstrate the utility of AnVILWorkflow for three use cases: bulk RNA-seq analysis with Salmon, metagenomics analysis with bioBakery, and digital pathology image processing with PathML. The key features of AnVILWorkflow include user-friendly browsing of available data and workflows, seamless integration of R and non-R tools within a reproducible analysis pipeline, and accessibility to scalable computing resources without direct management overhead. AnVILWorkflow lowers the barrier to utilizing AnVIL's resources, especially for exploratory analyses or bulk processing with established workflows. This empowers a broader community of researchers to leverage the latest genomics tools and datasets using familiar R syntax. This package is distributed through the Bioconductor project ( https://bioconductor.org/packages/AnVILWorkflow), and the source code is available through GitHub ( https://github.com/shbrief/AnVILWorkflow).

Keywords:  AnVIL; Cloud computing; Genomics; R/Bioconductor; Workflows

DOI:  https://doi.org/10.12688/f1000research.155449.1
Stem Cell Reports. 2024 Dec 05. pii: S2213-6711(24)00316-3. [Epub ahead of print] 102372

Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.

Antje K Rottner, Anders Lundin, Songyuan Li, Mike Firth, Marcello Maresca, Grzegorz Sienski.

  Gene editing strategies to safely and robustly modify the Alzheimer's disease-associated APOE4 isoform are still lacking. Prime editing (PE) enables the precise introduction of genetic variants with minimal unintended editing and without donor templates. However, it requires optimization for each target site and has not yet been applied to APOE4 gene editing. Here, we screened PE guide RNA (pegRNA) parameters and PE systems for introducing the APOE4 variant and applied the optimized PE strategy to generate disease-relevant human induced pluripotent stem cell models. We show that introducing a single-nucleotide difference required for APOE4 correction inhibits PE activity. To advance efficient and robust genome engineering of precise genetic variants, we further present a reliable PE enrichment strategy based on diphtheria toxin co-selection. Our work provides an optimized and reproducible genome engineering pipeline to generate APOE4 disease models and outlines novel strategies to accelerate genome editing in cellular disease model generation.

Keywords:  APOE4; Alzheimer’s disease; cellular disease models; co-selection; gene editing; genome engineering; human induced pluripotent stem cells; pegRNA optimization; prime editing

DOI:  https://doi.org/10.1016/j.stemcr.2024.11.002
Cell. 2024 Dec 12. pii: S0092-8674(24)01332-1. [Epub ahead of print]187(25): 7045-7063

How to build the virtual cell with artificial intelligence: Priorities and opportunities.

Charlotte Bunne, Yusuf Roohani, Yanay Rosen, Ankit Gupta, Xikun Zhang, Marcel Roed, Theo Alexandrov, Mohammed AlQuraishi, Patricia Brennan, Daniel B Burkhardt, Andrea Califano, Jonah Cool, Abby F Dernburg, Kirsty Ewing, Emily B Fox, Matthias Haury, Amy E Herr, Eric Horvitz, Patrick D Hsu, Viren Jain, Gregory R Johnson, Thomas Kalil, David R Kelley, Shana O Kelley, Anna Kreshuk, Tim Mitchison, Stephani Otte, Jay Shendure, Nicholas J Sofroniew, Fabian Theis, Christina V Theodoris, Srigokul Upadhyayula, Marc Valer, Bo Wang, Eric Xing, Serena Yeung-Levy, Marinka Zitnik, Theofanis Karaletsos, Aviv Regev, Emma Lundberg, Jure Leskovec, Stephen R Quake.

  Cells are essential to understanding health and disease, yet traditional models fall short of modeling and simulating their function and behavior. Advances in AI and omics offer groundbreaking opportunities to create an AI virtual cell (AIVC), a multi-scale, multi-modal large-neural-network-based model that can represent and simulate the behavior of molecules, cells, and tissues across diverse states. This Perspective provides a vision on their design and how collaborative efforts to build AIVCs will transform biological research by allowing high-fidelity simulations, accelerating discoveries, and guiding experimental studies, offering new opportunities for understanding cellular functions and fostering interdisciplinary collaborations in open science.

Keywords:  AI; ML; cell biology; virtual cell

DOI:  https://doi.org/10.1016/j.cell.2024.11.015
Genet Med Open. 2024 ;2 100837

Improving genetic diagnostic yield in a large cohort of children with rare vascular anomalies or PIK3CA-related overgrowth spectrum.

Timothy E Green, Denisse Garza, Natasha J Brown, Michelle G de Silva, Mark F Bennett, Caitlin Tubb, Roderic J Phillips, Duncan MacGregor, Susan J Robertson, Phillip Bekhor, Jodie Simpson, Anthony J Penington, Michael S Hildebrand.

   Purpose: Drugs that attenuate hyperactivation of the phosphatidylinositol 3-kinase-Akt and Ras-mitogen-activated protein kinase signaling pathways are emerging treatments for children with rare, intractable vascular anomalies or PIK3CA-related overgrowth spectrum (PROS) with an eligible genetic diagnosis. However, access to genetic testing remains a barrier to genetic diagnosis. Here, we implement a targeted molecular diagnostic strategy for vascular anomalies or PROS.
Methods: We applied a novel genetic testing strategy to children with vascular anomalies or PROS using a tiered approach of (1) droplet digital PCR, (2) Sanger sequencing, (3) high-depth exome sequencing, and (4) reanalysis of existing clinical exome data.
Results: We applied this strategy to 60 individuals detecting pathogenic somatic variants in 33 of 60 (55%). This included 26 individuals with slow-flow lesions with variants in PIK3CA, TEK, GNAQ, GNA11, BRAF, or PIK3R1, 4 individuals with fast-flow lesions with variants in KRAS or MAP2K1, 1 individual with a PIK3CA variant and a mixed phenotype, and 2 individuals with PIK3CA variants and PROS without vascular anomalies.
Conclusion: We demonstrate an effective genetic diagnostic strategy for children with vascular anomalies or PROS identifying somatic variants in 55% of individuals. Increasing genetic diagnostic yield extends the clinicogenetic spectrum and may provide access for those with intractable disease to therapeutic drug trials.

Keywords:  Droplet digital PCR; Genetic diagnosis; Mosaicism; PROS; Vascular anomalies

DOI:  https://doi.org/10.1016/j.gimo.2023.100837
Angiogenesis. 2024 Dec 11. 28(1): 6

Genetic and pharmacological targeting of mTORC1 in mouse models of arteriovenous malformation expose non-cell autonomous signalling in HHT.

Antonio Queiro-Palou, Yi Jin, Lars Jakobsson.

  Arteriovenous malformations (AVMs) are abnormal high flow shunts between arteries and veins with major negative impact on the cardiovascular system. Inherited loss-of-function (LOF) mutations in endoglin, encoding an endothelial cell (EC) expressed co-receptor for BMP9/10, causes the disease HHT1/Osler-Weber-Rendu, characterized by bleeding and AVMs. Here we observe increased activity of the downstream signalling complex mTORC1 within the retinal vasculature of HHT mouse models. To investigate its importance in AVM biology, concerning subvascular action, cell specificity, signalling strength and kinetics we combine timed genetic and antibody-based models of HHT with genetic mTORC1 inhibition or activation through EC specific deletion of Rptor or Tsc1. Results demonstrate that EC mTORC1 activation is secondary to endoglin LOF and mainly a consequence of systemic effects following AVM. While genetic EC inhibition of mTORC1 only showed tendencies towards reduced AVM severity, EC overactivation counterintuitively reduced it, implying that mTORC1 must be within a certain range to facilitate AVM. Complete inhibition of mTORC1 signalling by rapamycin provided the strongest therapeutic effect, pointing to potential involvement of RAPTOR-independent pathways or AVM-promoting effects of non-ECs in this pathology.

Keywords:  Arteriovenous malformation; Endoglin; Endothelial; HHT; Raptor; Tsc1; mTORC1

DOI:  https://doi.org/10.1007/s10456-024-09961-5
Nat Methods. 2024 Dec 13.

iFlpMosaics enable the multispectral barcoding and high-throughput comparative analysis of mutant and wild-type cells.

Irene Garcia-Gonzalez, Stefano Gambera, Susana F Rocha, Alvaro Regano, Lourdes Garcia-Ortega, Mariya Lytvyn, Luis Diago-Domingo, Maria S Sanchez-Muñoz, Aroa Garcia-Cabero, Ivana Zagorac, Wen Luo, Macarena De Andrés-Laguillo, Macarena Fernández-Chacón, Verónica Casquero-Garcia, Federica Francesca Lunella, Carlos Torroja, Fátima Sánchez-Cabo, Rui Benedito.

To understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and, therefore, do not experience the same epigenetic changes and tissue microenvironment. The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here we present the iFlpMosaics toolkit comprising a large set of new genetic tools and mouse lines that enable recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wild-type and Cre-mutant cells within the same time window and tissue microenvironment. The labeled cells can be profiled by multispectral imaging or by fluorescence-activated flow cytometry and single-cell RNA sequencing. iFlpMosaics facilitate the induction and analysis of genetic mosaics in any quiescent or progenitor cell, and for any given single or combination of floxed genes, thus enabling a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis and disease.

DOI: https://doi.org/10.1038/s41592-024-02534-w
PLoS Pathog. 2024 Dec 11. 20(12): e1012285

Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34+ hematopoietic progenitor cells and humanized mice.

Nicole L Diggins, Andrew H Pham, Jennifer Mitchell, Christopher J Parkins, Luke Slind, Rebekah Turner, Byeong-Jae Lee, Andrew D Yurochko, Patrizia Caposio, Jay A Nelson, Meaghan H Hancock.

Human cytomegalovirus (HCMV) actively manipulates cellular signaling pathways to benefit viral replication. Phosphatidyl-inositol 3-kinase (PI3K)/Akt signaling is an important negative regulator of HCMV replication, and during lytic infection the virus utilizes pUL38 to limit Akt phosphorylation and activity. During latency, PI3K/Akt signaling also limits virus replication, but how this is overcome at the time of reactivation is unknown. Virally encoded microRNAs (miRNAs) are a key component of the virus arsenal used to alter signaling during latency and reactivation. In the present study we show that three HCMV miRNAs (miR-UL36, miR-UL112 and miR-UL148D) downregulate Akt expression and attenuate downstream signaling, resulting in the activation of FOXO3a and enhanced internal promoter-driven IE transcription. A virus lacking expression of all three miRNAs is unable to reactivate from latency both in CD34+ hematopoietic progenitor cells and in a humanized mouse model of HCMV infection, however downregulating Akt restores the ability of the mutant virus to replicate. These findings highlight the negative role Akt signaling plays in HCMV replication in lytic and latent infection and how the virus has evolved miRNA-mediated countermeasures to promote successful reactivation.

DOI: https://doi.org/10.1371/journal.ppat.1012285
EMBO J. 2024 Dec 12.

qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.

Reuben Philip, Amit Sharma, Laura Matellan, Anna C Erpf, Wen-Hsin Hsu, Johnny M Tkach, Haley D M Wyatt, Laurence Pelletier.

  Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.

Keywords:  CRISPR; Endogenous Tagging; Gene-editing

DOI:  https://doi.org/10.1038/s44318-024-00337-5
iScience. 2024 Dec 20. 27(12): 111278

Pleiotropic tumor suppressive functions of PTEN missense mutations during gliomagenesis.

Hyun Jung Jun, Joao A Paulo, Victoria A Appleman, Tomer M Yaron-Barir, Jared L Johnson, Alan T Yeo, Vaughn A Rogers, Shan Kuang, Hemant Varma, Steven P Gygi, Lloyd C Trotman, Al Charest.

  PTEN plays a crucial role in preventing the development of glioblastoma (GBM), a severe and untreatable brain cancer. In GBM, most PTEN deficiencies are missense mutations that have not been thoroughly examined. Here, we leveraged genetically modified mice and isogenic astrocyte cell cultures to investigate the role of clinically relevant mutations (G36E, L42R, C105F, and R173H) in the development of EGFR-driven GBM. We report that the loss of tumor suppression from these mutants is unrelated to their lipid phosphatase activity and rather relate to elevated localization at the cell membrane. Moreover, expression of these PTEN mutations heightened EGFR activity by sequestering EGFR within endomembranes longer and affected its signaling behavior. Through comprehensive studies on global protein phosphorylation and kinase library analyses in cells with the G36E and L42R PTEN mutations, we identified distinct cancer-promoting pathways activated by EGFR, offering targets for treating GBM with these PTEN alterations.

Keywords:  Cancer; Cell biology

DOI:  https://doi.org/10.1016/j.isci.2024.111278
Nature. 2024 Dec 11.

Central control of dynamic gene circuits governs T cell rest and activation.

Maya M Arce, Jennifer M Umhoefer, Nadia Arang, Sivakanthan Kasinathan, Jacob W Freimer, Zachary Steinhart, Haolin Shen, Minh T N Pham, Mineto Ota, Anika Wadhera, Rama Dajani, Dmytro Dorovskyi, Yan Yi Chen, Qi Liu, Yuan Zhou, Danielle L Swaney, Kirsten Obernier, Brian R Shy, Julia Carnevale, Ansuman T Satpathy, Nevan J Krogan, Jonathan K Pritchard, Alexander Marson.

The ability of cells to maintain distinct identities and respond to transient environmental signals requires tightly controlled regulation of gene networks1-3. These dynamic regulatory circuits that respond to extracellular cues in primary human cells remain poorly defined. The need for context-dependent regulation is prominent in T cells, where distinct lineages must respond to diverse signals to mount effective immune responses and maintain homeostasis4-8. Here we performed CRISPR screens in multiple primary human CD4+ T cell contexts to identify regulators that control expression of IL-2Rα, a canonical marker of T cell activation transiently expressed by pro-inflammatory effector T cells and constitutively expressed by anti-inflammatory regulatory T cells where it is required for fitness9-11. Approximately 90% of identified regulators of IL-2Rα had effects that varied across cell types and/or stimulation states, including a subset that even had opposite effects across conditions. Using single-cell transcriptomics after pooled perturbation of context-specific screen hits, we characterized specific factors as regulators of overall rest or activation and constructed state-specific regulatory networks. MED12 - a component of the Mediator complex - serves as a dynamic orchestrator of key regulators, controlling expression of distinct sets of regulators in different T cell contexts. Immunoprecipitation-mass spectrometry revealed that MED12 interacts with the histone methylating COMPASS complex. MED12 was required for histone methylation and expression of genes encoding key context-specific regulators, including the rest maintenance factor KLF2 and the versatile regulator MYC. CRISPR ablation of MED12 blunted the cell-state transitions between rest and activation and protected from activation-induced cell death. Overall, this work leverages CRISPR screens performed across conditions to define dynamic gene circuits required to establish resting and activated T cell states.

DOI: https://doi.org/10.1038/s41586-024-08314-y
Cell Syst. 2024 Dec 03. pii: S2405-4712(24)00312-0. [Epub ahead of print]

Optimized reporters for multiplexed detection of transcription factor activity.

Max Trauernicht, Teodora Filipovska, Chaitanya Rastogi, Bas van Steensel.

  In any given cell type, dozens of transcription factors (TFs) act in concert to control the activity of the genome by binding to specific DNA sequences in regulatory elements. Despite their considerable importance, we currently lack simple tools to directly measure the activity of many TFs in parallel. Massively parallel reporter assays (MPRAs) allow the detection of TF activities in a multiplexed fashion; however, we lack basic understanding to rationally design sensitive reporters for many TFs. Here, we use an MPRA to systematically optimize transcriptional reporters for 86 TFs and evaluate the specificity of all reporters across a wide array of TF perturbation conditions. We thus identified critical TF reporter design features and obtained highly sensitive and specific reporters for 62 TFs, many of which outperform available reporters. The resulting collection of "prime" TF reporters can be used to uncover TF regulatory networks and to illuminate signaling pathways. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords:  MPRA; TF; TF reporter assay; TF reporter design; massively parallel reporter assay; multiplexed TF reporter assay; reporter; signaling pathways; specificity; transcription factor

DOI:  https://doi.org/10.1016/j.cels.2024.11.003
Mol Syst Biol. 2024 Dec 09.

State of the interactomes: an evaluation of molecular networks for generating biological insights.

Sarah N Wright, Scott Colton, Leah V Schaffer, Rudolf T Pillich, Christopher Churas, Dexter Pratt, Trey Ideker.

  Advancements in genomic and proteomic technologies have powered the creation of large gene and protein networks ("interactomes") for understanding biological systems. However, the proliferation of interactomes complicates the selection of networks for specific applications. Here, we present a comprehensive evaluation of 45 current human interactomes, encompassing protein-protein interactions as well as gene regulatory, signaling, colocalization, and genetic interaction networks. Our analysis shows that large composite networks such as HumanNet, STRING, and FunCoup are most effective for identifying disease genes, while smaller networks such as DIP, Reactome, and SIGNOR demonstrate stronger performance in interaction prediction. Our study provides a benchmark for interactomes across diverse biological applications and clarifies factors that influence network performance. Furthermore, our evaluation pipeline paves the way for continued assessment of emerging and updated interaction networks in the future.

Keywords:  Gene Prioritization; Interaction Prediction; Interactome; Network; Systems Biology

DOI:  https://doi.org/10.1038/s44320-024-00077-y
Cell Rep Methods. 2024 Nov 28. pii: S2667-2375(24)00303-5. [Epub ahead of print] 100913

A statistical approach for systematic identification of transition cells from scRNA-seq data.

Yuanxin Wang, Merve Dede, Vakul Mohanty, Jinzhuang Dou, Ziyi Li, Ken Chen.

  Decoding cellular state transitions is crucial for understanding complex biological processes in development and disease. While recent advancements in single-cell RNA sequencing (scRNA-seq) offer insights into cellular trajectories, existing tools primarily study expressional rather than regulatory state shifts. We present CellTran, a statistical approach utilizing paired-gene expression correlations to detect transition cells from scRNA-seq data without explicitly resolving gene regulatory networks. Applying our approach to various contexts, including tissue regeneration, embryonic development, preinvasive lesions, and humoral responses post-vaccination, reveals transition cells and their distinct gene expression profiles. Our study sheds light on the underlying molecular mechanisms driving cellular state transitions, enhancing our ability to identify therapeutic targets for disease interventions.

Keywords:  CP: developmental biology; CP: systems biology; carcinogenesis; cell development; cell differentiation; cell transitions; differential equations; dynamic systems; gene expression correlation; gene regulatory network; single-cell RNA sequencing; statistical analysis

DOI:  https://doi.org/10.1016/j.crmeth.2024.100913
Cell Rep Methods. 2024 Dec 05. pii: S2667-2375(24)00323-0. [Epub ahead of print] 100933

Single-cell RNA sequencing algorithms underestimate changes in transcriptional noise compared to single-molecule RNA imaging.

Neha Khetan, Binyamin Zuckerman, Giuliana P Calia, Xinyue Chen, Ximena Garcia Arceo, Leor S Weinberger.

  Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability. However, how best to quantify genome-wide noise remains unclear. Here, we utilize a small-molecule perturbation (5'-iodo-2'-deoxyuridine [IdU]) to amplify noise and assess noise quantification from numerous single-cell RNA sequencing (scRNA-seq) algorithms on human and mouse datasets and then compare it to noise quantification from single-molecule RNA fluorescence in situ hybridization (smFISH) for a panel of representative genes. We find that various scRNA-seq analyses report amplified noise-without altered mean expression levels-for ∼90% of genes and that smFISH analysis verifies noise amplification for the vast majority of tested genes. Collectively, the analyses suggest that most scRNA-seq algorithms (including a simple normalization approach) are appropriate for quantifying noise, although all algorithms appear to systematically underestimate noise changes compared to smFISH. For practical purposes, this analysis further argues that IdU noise enhancement is globally penetrant-i.e., homeostatically increasing noise without altering mean expression levels-and could enable investigations of the physiological impacts of transcriptional noise.

Keywords:  CP: imaging; CP: systems biology; noise-enhancer molecule; scRNA-seq; single-cell RNA sequencing; single-molecule RNA FISH; smFISH; transcriptional noise

DOI:  https://doi.org/10.1016/j.crmeth.2024.100933
Biomed Pharmacother. 2024 Dec 12. pii: S0753-3322(24)01626-3. [Epub ahead of print]182 117740

Identification of cell type-specific cell-penetrating peptides through in vivo phage display leveraged by next generation sequencing.

Franziska Kohl, Oliver Laufkötter, Mike Firth, Luc Krimpenfort, Priyanka Mangla, Mohammadhassan Ansarizadeh, Gökçe Geylan, Lauri Eklund, Leonardo De Maria, Lars Jakobsson, John Wiseman.

  Vascular anomalies (VA) refer to abnormal blood or lymphatic vessel architecture, most often as a result of dysregulated growth. Venous malformations (VM), a subgroup of VAs, are triggered by activating mutations in the Angiopoietin/TIE2-PI3K/AKT/mTOR signaling pathway with TIE2 L914F (gene name TEK) being one of the most frequent mutations in patients with VMs. Although systemic targeting of the overactivated pathway is possible, it would be a therapeutic advantage to restrict treatment to only the affected lesions. To identify peptides with potential selective binding to TIE2 L914F lesions we applied in vivo phage display to TIE2 L914F-overexpressing endothelial cells (ECs) in a subcutaneous matrigel xenograft mouse model of VMs. By panning for lesion-targeting phages in combination with subcellular fractionation, a screen for cell-penetrating candidate phages was established. Employing Next Generation Sequencing (NGS) and a refined bioinformatic analysis we were able to identify many novel cell-penetrating peptides (CPPs). To pinpoint the most selective and viable CCP candidates a hierarchical clustering algorithm was utilized. This method aggregated CPPs with highly similar sequences into a small number of clusters from which consensus sequences could be derived. Selected candidate CPPs exhibited uptake in TIE2 L914F-expressing human umbilical vein endothelial cells (HUVEC) in culture and were able to deliver siRNA into these cells. In conclusion, our NGS bioinformatic-supported approach led to the identification of novel and selective CPPs capable of transporting a siRNA cargo into targeted cells.

Keywords:  Bioinformatic analysis; Cell-penetrating peptides; In vivo phage display; Next generation sequencing; Vascular anomalies

DOI:  https://doi.org/10.1016/j.biopha.2024.117740
bioRxiv. 2024 Nov 28. pii: 2024.11.25.625301. [Epub ahead of print]

Structure and organization of full-length Epidermal Growth Factor Receptor in extracellular vesicles by cryo-electron tomography.

Monica Gonzalez-Magaldi, Anuradha Gullapalli, Ophelia Papoulas, Chang Liu, Adelaide Y-H Leung, Luqiang Guo, Axel Brilot, Edward M Marcotte, Zunlong Ke, Daniel J Leahy.

We report here transport of the Epidermal Growth Factor Receptor (EGFR), Insulin Receptor, 7-pass transmembrane receptor Smoothened, and 13-pass Sodium-iodide symporter to extracellular vesicles (EVs) for structural and functional studies. Mass spectrometry confirmed the transported proteins as the most abundant in EV membranes, and the presence of many receptor-interacting proteins demonstrates the utility of EVs for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in the presence of EGF with a ∼3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular regions do not form regular arrays, suggesting that clustering is mediated by the intracellular region. Subtomogram averaging of the EGFR extracellular region (ECR) yielded a 15 Å map into which the crystal structure of the ligand-bound EGFR ECR dimer fits well. These findings refine our understanding of EGFR activation, clustering, and signaling, and they establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in a native-like environment.
Significance Statement: Atomic or near-atomic resolution structural studies of proteins embedded in cell membranes have proven challenging. We show that transporting integral membrane proteins to cell-derived extracellular vesicles enables structural and functional studies of human membrane proteins in a native membrane environment. We have used this approach to visualize an active form of full-length Epidermal Growth Factor Receptor (EGFR) and show that it forms clusters in the membrane and projects its cytoplasmic signaling domains ∼3 nm away from the membrane surface. EGFR is essential for normal development, but abnormal EGFR activity is associated with several human cancers and is the target of many anticancer therapies. Our studies refine current models of how ligand binding to EGFR transmits signals across cell membranes.

DOI: https://doi.org/10.1101/2024.11.25.625301
Bioessays. 2024 Dec 12. e202300158

Circadian Control of Protein Synthesis.

Nathan R James, John S O'Neill.

  Daily rhythms in the rate and specificity of protein synthesis occur in most mammalian cells through an interaction between cell-autonomous circadian regulation and daily cycles of systemic cues. However, the overall protein content of a typical cell changes little over 24 h. For most proteins, translation appears to be coordinated with protein degradation, producing phases of proteomic renewal that maximize energy efficiency while broadly maintaining proteostasis across the solar cycle. We propose that a major function of this temporal compartmentalization-and of circadian rhythmicity in general-is to optimize the energy efficiency of protein synthesis and associated processes such as complex assembly. We further propose that much of this temporal compartmentalization is achieved at the level of translational initiation, such that the translational machinery alternates between distinct translational mechanisms, each using a distinct toolkit of phosphoproteins to preferentially recognize and translate different classes of mRNA.

Keywords:  biphasic model; circadian rhythms; protein synthesis; temporal compartmentalization; translational initiation

DOI:  https://doi.org/10.1002/bies.202300158
Cell Genom. 2024 Dec 11. pii: S2666-979X(24)00352-5. [Epub ahead of print]4(12): 100723

scTrends: A living review of commercial single-cell and spatial 'omic technologies.

scTrends Consortium

Understanding the rapidly evolving landscape of single-cell and spatial omic technologies is crucial for advancing biomedical research and drug development. We provide a living review of both mature and emerging commercial platforms, highlighting key methodologies and trends shaping the field. This review spans from foundational single-cell technologies such as microfluidics and plate-based methods to newer approaches like combinatorial indexing; on the spatial side, we consider next-generation sequencing and imaging-based spatial transcriptomics. Finally, we highlight emerging methodologies that may fundamentally expand the scope for data generation within pharmaceutical research, creating opportunities to discover and validate novel drug mechanisms. Overall, this review serves as a critical resource for navigating the commercialization and application of single-cell and spatial omic technologies in pharmaceutical and academic research.

DOI: https://doi.org/10.1016/j.xgen.2024.100723
Nature. 2024 Dec 09.

A framework for neural organoids, assembloids and transplantation studies.

Sergiu P Pașca, Paola Arlotta, Helen S Bateup, J Gray Camp, Silvia Cappello, Fred H Gage, Jürgen A Knoblich, Arnold R Kriegstein, Madeline A Lancaster, Guo-Li Ming, Gaia Novarino, Hideyuki Okano, Malin Parmar, In-Hyun Park, Orly Reiner, Hongjun Song, Lorenz Studer, Jun Takahashi, Sally Temple, Giuseppe Testa, Barbara Treutlein, Flora M Vaccarino, Pierre Vanderhaeghen, Tracy Young-Pearse.

As the field of neural organoids and assembloids rapidly expands, there is an emergent need for guidance and advice on designing, conducting and reporting experiments to increase the reproducibility and utility of these models. Here, our consortium- representing specialized laboratories from around the world- presents a framework for the experimental process that ranges from ensuring the quality and integrity of human pluripotent stem cells to characterizing and manipulating neural cells in vitro, and from transplantation techniques to considerations for modeling human development, evolution, and disease. As with all scientific endeavors, we advocate for rigorous experimental designs tailored to explicit scientific questions, and transparent methodologies and data sharing, to provide useful knowledge for both current research practices and for developing regulatory standards.

DOI: https://doi.org/10.1038/s41586-024-08487-6
Bioessays. 2024 Dec 09. e202400278

Decoding Angiocrine Signaling: Endothelial Cells as Drivers of Organ Regeneration and Homeostasis.

Riikka Kivelä.



Keywords:  angiocrine; blood flow; endothelial cell; regeneration

DOI:  https://doi.org/10.1002/bies.202400278
Angiogenesis. 2024 Dec 12. 28(1): 7

Neuropilin-1 controls vascular permeability through juxtacrine regulation of endothelial adherens junctions.

Sagnik Pal, Yangyang Su, Emmanuel Nwadozi, Lena Claesson-Welsh, Mark Richards.

  Neuropilin-1 (NRP1) regulates endothelial cell (EC) biology through modulation of vascular endothelial growth factor receptor 2 (VEGFR2) signalling by presenting VEGFA to VEGFR2. How NRP1 impacts VEGFA-mediated vascular hyperpermeability has however remained unresolved, described as exerting either a positive or a passive function. Using EC-specific Nrp1 knock-out mice, we discover that EC-expressed NRP1 exerts an organotypic role. In the ear skin, VEGFA/VEGFR2-mediated vascular leakage was increased following loss of EC NRP1, implicating NRP1 in negative regulation of VEGFR2 signalling. In contrast, in the back skin and trachea, loss of EC NRP1 decreased vascular leakage. In accordance, phosphorylation of vascular endothelial (VE)-cadherin was increased in the ear skin but suppressed in the back skin of Nrp1 iECKO mice. NRP1 expressed on perivascular cells has been shown to impact VEGF-mediated VEGFR2 signalling. Importantly, expression of NRP1 on perivascular cells was more abundant in the ear skin than in the back skin. Global loss of NRP1 resulted in suppressed VEGFA-induced vascular leakage in the ear skin, implicating perivascular NRP1 as a juxtacrine co-receptor of VEGFA in this compartment. Altogether, we demonstrate that perivascular NRP1 is an active participant in EC VEGFA/VEGFR2 signalling and acts as an organotypic modifier of EC biology.

Keywords:  Neuropilin-1; Signalling; VE-cadherin; VEGFA; Vascular permeability

DOI:  https://doi.org/10.1007/s10456-024-09963-3
Angiogenesis. 2024 Dec 10. 28(1): 5

Alk1/Endoglin signaling restricts vein cell size increases in response to hemodynamic cues.

Zeenat Diwan, Jia Kang, Emma Tsztoo, Arndt F Siekmann.

  Hemodynamic cues are thought to control blood vessel hierarchy through a shear stress set point, where flow increases lead to blood vessel diameter expansion, while decreases in blood flow cause blood vessel narrowing. Aberrations in blood vessel diameter control can cause congenital arteriovenous malformations (AVMs). We show in zebrafish embryos that while arteries behave according to the shear stress set point model, veins do not. This behavior is dependent on distinct arterial and venous endothelial cell (EC) shapes and sizes. We show that arterial ECs enlarge more strongly when experiencing higher flow, as compared to vein cells. Through the generation of chimeric embryos, we discover that this behavior of vein cells depends on the bone morphogenetic protein (BMP) pathway components Endoglin and Alk1. Endoglin (eng) or alk1 (acvrl1) mutant vein cells enlarge when in normal hemodynamic environments, while we do not observe a phenotype in either acvrl1 or eng mutant ECs in arteries. We further show that an increase in vein diameters initiates AVMs in eng mutants, secondarily leading to higher flow to arteries. These enlarge in response to higher flow through increasing arterial EC sizes, fueling the AVM. This study thus reveals a mechanism through which BMP signaling limits vein EC size increases in response to flow and provides a framework for our understanding of how a small number of mutant vein cells via flow-mediated secondary effects on wildtype arterial ECs can precipitate larger AVMs in disease conditions, such as hereditary hemorrhagic telangiectasia (HHT).

Keywords:  Alk1; Artery; Endoglin; Endothelial Cell Size; Hereditary hemorrhagic telangiectasia; Shear Stress Set Point; Vein; Zebrafish

DOI:  https://doi.org/10.1007/s10456-024-09955-3
Trends Endocrinol Metab. 2024 Dec 12. pii: S1043-2760(24)00296-0. [Epub ahead of print]

Intracellular endothelial cell metabolism in vascular function and dysfunction.

Kathryn M Citrin, Balkrishna Chaube, Carlos Fernández-Hernando, Yajaira Suárez.

  Endothelial cells (ECs) form the inner lining of blood vessels that is crucial for vascular function and homeostasis. They regulate vascular tone, oxidative stress, and permeability. Dysfunction leads to increased permeability, leukocyte adhesion, and thrombosis. ECs undergo metabolic changes in conditions such as wound healing, cancer, atherosclerosis, and diabetes, and can influence disease progression. We discuss recent research that has revealed diverse intracellular metabolic pathways in ECs that are tailored to their functional needs, including lipid handling, glycolysis, and fatty acid oxidation (FAO). Understanding EC metabolic signatures in health and disease will be crucial not only for basic biology but can also be exploited when designing new therapies to target EC-related functions in different vascular diseases.

Keywords:  angiogenesis; atherosclerosis; endometabolism; endothelial cell; lipid metabolism

DOI:  https://doi.org/10.1016/j.tem.2024.11.004
Reproduction. 2024 Dec 01. pii: REP-24-0317. [Epub ahead of print]

Comparative analysis of PI3K-AKT and MEK-ERK1/2 signaling-driven molecular changes in granulosa cells.

Vijay Simha Baddela, Marten Michaelis, Xuelian Tao, Dirk Koczan, Julia Brenmoehl, Jens Vanselow.

The PI3K-AKT and MEK-ERK signaling pathways are integral to fundamental cellular processes such as proliferation, viability, and differentiation. In granulosa cells (GCs), these pathways are activated by FSH and IGF1 through respective receptors. We investigated the comparative transcriptome changes induced by AKT and ERK pathways using corresponding inhibitors in GCs. GCs isolated from antral follicles showed positive signals for phospho-AKT and -ERK proteins. Treatment of cultured GCs with FSH and IGF1 induced phospho-AKT and -ERK levels. Transcriptome analysis revealed 1436 genes regulated by AKT and 654 genes regulated by ERK pathway. Among these, 94 genes were commonly downregulated, and 11 genes were commonly upregulated in both data sets, while 110 genes were oppositely regulated. Bioinformatics analysis revealed that inhibition of PI3K-AKT and MEK-ERK pathways downregulates key reproductive processes and upstream molecules. Notably, AKT inhibition affected FSH, ESRRG, and HIF1 pathways, while ERK inhibition impacted CG, FOS, TGFβ, EGR1, and LH pathways. Transcriptome data showed that genes related to estradiol production were inhibited by ERK and induced by the AKT pathway. This was verified by radioimmunoassays, and mRNA and protein analysis of CYP19A1 and STAR genes. In addition, transcriptome data suggested downregulation of glucose metabolism in GCs. Using validation experiments, we confirm that both pathways are essential for glucose uptake, lactate production, and mitochondrial activity in GCs. These data provide a resource for informing future research for analyzing various novel candidate genes regulated by AKT and ERK pathways in GCs and other cell types.

DOI: https://doi.org/10.1530/REP-24-0317
Nature. 2024 Dec;636(8042): 512

Bad bar charts are pervasive across biology.

Amanda Heidt.



Keywords:  Publishing; Research data; Technology

DOI:  https://doi.org/10.1038/d41586-024-03996-w
Elife. 2024 Dec 11. pii: e99951. [Epub ahead of print]13

Emergence of power-law distributions in protein-protein interaction networks through study bias.

David B Blumenthal, Marta Lucchetta, Linda Kleist, Sándor P Fekete, Markus List, Martin H Schaefer.

  Degree distributions in protein-protein interaction (PPI) networks are believed to follow a power law (PL). However, technical and study bias affect the experimental procedures for detecting PPIs. For instance, cancer-associated proteins have received disproportional attention. Moreover, bait proteins in large-scale experiments tend to have many false-positive interaction partners. Studying the degree distributions of thousands of PPI networks of controlled provenance, we address the question if PL distributions in observed PPI networks could be explained by these biases alone. Our findings are supported by mathematical models and extensive simulations and indicate that study bias and technical bias suffice to produce the observed PL distribution. It is, hence, problematic to derive hypotheses about the topology of the true biological interactome from the PL distributions in observed PPI networks. Our study casts doubt on the use of the PL property of biological networks as a modeling assumption or quality criterion in network biology.

Keywords:  computational biology; human; systems biology

DOI:  https://doi.org/10.7554/eLife.99951