J Mol Evol. 2024 Feb 28. doi: 10.1007/s00239-024-10156-1. Online ahead of print.

ABSTRACT

Ecological and evolutionary transitions offer an excellent opportunity to examine the molecular basis of adaptation. Fishes of the order Beloniformes include needlefishes, flyingfishes, halfbeaks, and allies, and comprise over 200 species occupying a wide array of habitats-from the marine epipelagic zone to tropical rainforest rivers. These fishes also exhibit a diversity of diets, including piscivory, herbivory, and zooplanktivory. We investigated how diet and habitat affected the molecular evolution of cone opsins, which play a key role in bright light and colour vision and are tightly linked to ecology and life history. We analyzed a targeted-capture dataset to reconstruct the evolutionary history of beloniforms and assemble cone opsin sequences. We implemented codon-based clade models of evolution to examine how molecular evolution was affected by habitat and diet. We found high levels of positive selection in medium- and long-wavelength beloniform opsins, with piscivores showing increased positive selection in medium-wavelength opsins and zooplanktivores showing increased positive selection in long-wavelength opsins. In contrast, short-wavelength opsins showed purifying selection. While marine/freshwater habitat transitions have an effect on opsin molecular evolution, we found that diet plays a more important role. Our study suggests that evolutionary transitions along ecological axes produce complex adaptive interactions that affect patterns of selection on genes that underlie vision.

PMID:38416218 | DOI:10.1007/s00239-024-10156-1

J Exp Bot. 2024 Feb 28;75(5):1205-1209. doi: 10.1093/jxb/erae008.

NO ABSTRACT

PMID:38416206 | DOI:10.1093/jxb/erae008

Elife. 2024 Feb 28;13:e91717. doi: 10.7554/eLife.91717.

ABSTRACT

Experiments involving periodic stimuli shed light on the interplay between hyper-osmotic stress and glucose starvation in yeast cells.

PMID:38416131 | DOI:10.7554/eLife.91717

New Phytol. 2024 Feb 28. doi: 10.1111/nph.19634. Online ahead of print.

NO ABSTRACT

PMID:38415795 | DOI:10.1111/nph.19634

Circ Arrhythm Electrophysiol. 2024 Feb 28:e012022. doi: 10.1161/CIRCEP.123.012022. Online ahead of print.

ABSTRACT

BACKGROUND: Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants.

METHODS: HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression.

RESULTS: ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs.

CONCLUSIONS: CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

PMID:38415356 | DOI:10.1161/CIRCEP.123.012022

Biochem Soc Trans. 2024 Feb 28:BST20230632C. doi: 10.1042/BST20230632C. Online ahead of print.

ABSTRACT

The formation of fluid- or gas-filled lumina surrounded by epithelial cells pervades development and disease. We review the balance between lumen pressure and mechanical forces from the surrounding cells that governs lumen formation. We illustrate the mechanical side of this balance in several examples of increasing complexity, and discuss how recent work is beginning to elucidate how nonlinear and active mechanics and anisotropic biomechanical structures must conspire to overcome the isotropy of pressure to form complex, non-spherical lumina.

PMID:38415294 | DOI:10.1042/BST20230632C

IEEE Open J Eng Med Biol. 2024 Feb 21;5:1-13. doi: 10.1109/OJEMB.2024.3351717. eCollection 2024.

ABSTRACT

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

PMID:38415197 | PMC:PMC10896418 | DOI:10.1109/OJEMB.2024.3351717

Front Immunol. 2024 Feb 13;14:1282859. doi: 10.3389/fimmu.2023.1282859. eCollection 2023.

ABSTRACT

INTRODUCTION: The COVID-19 Disease Map project is a large-scale community effort uniting 277 scientists from 130 Institutions around the globe. We use high-quality, mechanistic content describing SARS-CoV-2-host interactions and develop interoperable bioinformatic pipelines for novel target identification and drug repurposing.

METHODS: Extensive community work allowed an impressive step forward in building interfaces between Systems Biology tools and platforms. Our framework can link biomolecules from omics data analysis and computational modelling to dysregulated pathways in a cell-, tissue- or patient-specific manner. Drug repurposing using text mining and AI-assisted analysis identified potential drugs, chemicals and microRNAs that could target the identified key factors.

RESULTS: Results revealed drugs already tested for anti-COVID-19 efficacy, providing a mechanistic context for their mode of action, and drugs already in clinical trials for treating other diseases, never tested against COVID-19.

DISCUSSION: The key advance is that the proposed framework is versatile and expandable, offering a significant upgrade in the arsenal for virus-host interactions and other complex pathologies.

PMID:38414974 | PMC:PMC10897000 | DOI:10.3389/fimmu.2023.1282859

iScience. 2024 Feb 9;27(3):109164. doi: 10.1016/j.isci.2024.109164. eCollection 2024 Mar 15.

ABSTRACT

Myogenic differentiation is integral for the regeneration of skeletal muscle following tissue damage. Though high-energy post-mitotic muscle relies predominantly on mitochondrial respiration, the importance of mitochondrial remodeling in enabling muscle differentiation and the players involved are not fully known. Here we show that the mitochondrial fusion protein OPA1 is essential for muscle differentiation. Our study demonstrates that OPA1 loss or inhibition, through genetic and pharmacological means, abolishes in vivo muscle regeneration and in vitro myotube formation. We show that both the inhibition and genetic deletion of OPA1 prevent the early onset metabolic switch required to drive myoblast differentiation. In addition, we observe an OPA1-dependent upregulation of the supercomplex assembly factor, SCAF1, at the onset of differentiation. Importantly, preventing the upregulation of SCAF1, through OPA1 loss or siRNA-mediated SCAF1 knockdown, impairs metabolic reprogramming and muscle differentiation. These findings reveal the integral role of OPA1 and mitochondrial reprogramming at the onset of myogenic differentiation.

PMID:38414856 | PMC:PMC10897915 | DOI:10.1016/j.isci.2024.109164

Front Cell Neurosci. 2024 Feb 13;18:1347436. doi: 10.3389/fncel.2024.1347436. eCollection 2024.

ABSTRACT

The vertebrate retina is made up of six specialized neuronal cell types and one glia that are generated from a common retinal progenitor. The development of these distinct cell types is programmed by transcription factors that regulate the expression of specific genes essential for cell fate specification and differentiation. Because of the complex nature of transcriptional regulation, understanding transcription factor functions in development and disease is challenging. Research on the Cone-rod homeobox transcription factor CRX provides an excellent model to address these challenges. In this review, we reflect on 25 years of mammalian CRX research and discuss recent progress in elucidating the distinct pathogenic mechanisms of four CRX coding variant classes. We highlight how in vitro biochemical studies of CRX protein functions facilitate understanding CRX regulatory principles in animal models. We conclude with a brief discussion of the emerging systems biology approaches that could accelerate precision medicine for CRX-linked diseases and beyond.

PMID:38414750 | PMC:PMC10896975 | DOI:10.3389/fncel.2024.1347436

Anim Cells Syst (Seoul). 2023 Nov 14;27(1):321-328. doi: 10.1080/19768354.2023.2275613. eCollection 2023.

ABSTRACT

The internal ribosome entry site (IRES) is a unique structure found in the 5' untranslated region (5'-UTR) of specific messenger RNAs (mRNAs) that allows ribosomes to bind and initiate translation without the need for a cap structure. In this study, we investigated the presence and functional properties of the IRES activity of nitric oxide synthase 2 (NOS2) mRNA, which encodes an enzyme that produces nitric oxide in response to various stimuli such as inflammation. Nitric oxide is a signaling molecule that plays a crucial role in various physiological processes, including immune responses and neuronal signaling. Our results showed the existence of IRES activity in the 5'-UTR of Nos2 mRNA in various cell types. IRES-mediated translation of NOS2 mRNA was higher in neuronal cells and its activity increased in response to lipopolysaccharide (LPS). Despite inhibition of cap-dependent translation, nitrite production was partially maintained. These results demonstrate the presence of IRES activity in the 5'-UTR of NOS2 mRNA and suggest that IRES-mediated translation plays a key role in controlling nitric oxide production in response to LPS, an inflammatory stimulus.

PMID:38414531 | PMC:PMC10898816 | DOI:10.1080/19768354.2023.2275613

Mol Ther. 2024 Feb 26:S1525-0016(24)00094-7. doi: 10.1016/j.ymthe.2024.02.025. Online ahead of print.

ABSTRACT

Exosomes are extracellular vesicles (EVs) (∼50-150 nm) have emerged as promising vehicles for therapeutic applications and drug delivery. These membrane-bound particles, released by all actively dividing cells have the ability to transfer effector molecules, including proteins, RNA, and even DNA, from donor cells to recipient cells, thereby modulating cellular responses. RNA-based therapeutics, including miRNAs, mRNAs, lncRNAs, and circRNAs, hold great potential in controlling gene expression and treating a spectrum of medical conditions. RNAs encapsulated in EVs are protected from extracellular degradation, making them attractive for therapeutic applications. Understanding the intricate biology of cargo loading and transfer within EVs is pivotal in unlocking their therapeutic potential. This review discusses the biogenesis and classification of EVs, methods for loading RNA into EVs, their advantages as drug carriers over synthetic lipid-based systems, and the potential applications in treating neurodegenerative diseases, cancer and viral infections. Notably, EVs show promise in delivering RNA cargo across the blood-brain barrier and targeting tumour cells, offering a safe and effective approach to RNA-based therapy in these contexts.

PMID:38414242 | DOI:10.1016/j.ymthe.2024.02.025

Plant Dis. 2024 Feb 27. doi: 10.1094/PDIS-10-23-2195-PDN. Online ahead of print.

ABSTRACT

Luteoviruses (family Tombusviridae) and poleroviruses (family Solemoviridae) are economically important pathogens of cereals such as wheat (Triticum aestivum), barley (Hordeum vulgare) and oat (Avena sativa). In Australia, the luteoviruses barley yellow dwarf virus PAV (BYDV PAV) and barley yellow dwarf virus MAV (BYDV MAV), along with the poleroviruses cereal yellow dwarf virus RPV (CYDV RPV) and maize yellow dwarf virus RMV (MYDV RMV), were distinguished from each other and reported in the 1980s (Sward and Lister 1988; Waterhouse and Helms 1985). The poleroviruses barley virus G (BVG) and cereal yellow dwarf virus RPS (CYDV RPS) were reported in Australia more recently (Nancarrow et al. 2019; Nancarrow et al. 2023), while the luteovirus barley yellow dwarf virus PAS (BYDV PAS) has not previously been reported in Australia. During 2010, an oat plant exhibiting yellow/ red leaf discoloration and stunted growth was collected from a roadside in Horsham, Victoria, Australia. The plant tested positive for BYDV PAV and negative for BYDV MAV, CYDV RPV and MYDV RMV by tissue blot immunoassay (TBIA) as described by Trębicki et al (2017). The virus isolate has since been continuously maintained in a glasshouse in live wheat plants using aphids (Rhopalosiphum padi). In 2021, total RNA extracted from a wheat plant infected with this isolate (Nancarrow et al. 2023) tested positive for BYDV PAV by RT-PCR using the primers BYDV-1/BYDV-2 (Rastgou et al. 2005), but negative for BYDV PAV, CYDV RPV and MYDV RMV using other published primers (Deb and Anderson 2008). A high-throughput sequencing (HTS) library was prepared from the total RNA with the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) without ribosomal RNA depletion and sequenced on a NovaSeq 6000 (Illumina). Raw reads were trimmed and filtered using fastp v0.20.0 (Chen et al. 2018) while de novo assembly of all of the resulting 5,049,052 reads was done using SPAdes v3.15.3 (Nurk et al. 2017). BLASTn analysis of the resulting 4,067 contigs (128- 12,457 bp in length) revealed only one large virus-like contig (5,649 bp) which was most similar to BYDV PAS isolates on NCBI GenBank, sharing 87% nucleotide (nt) identity with BYDV PAS isolate OH2 (MN128939), 86% nt identity with the BYDV PAS reference sequence (NC_002160) and 82% nt identity with the BYDV PAV reference sequence (NC_004750). Additionally, 4,008 HTS reads were mapped to the assembled genome sequence with Bowtie2 v2.4.5. (Langmead and Salzberg 2012) with 100% genome coverage and an average coverage depth of 101X. Primers were designed to the assembled genome sequence to generate overlapping amplicons across the genome, and the resulting amplicons were Sanger sequenced. This confirmed the genome sequence of BYDV PAS isolate PT from Australia (5649 bp, GC content 47.9%), which was deposited in GenBank (LC782749). Ten additional plant samples collected from western Victoria, Australia, all tested positive for BYDV PAS by RT-PCR using the primers PASF and PASR (Laney et al. 2018). The additional samples consisted of one oat sample collected in 2005, one barley sample collected in 2007, three wheat samples collected in 2016 and one barley, one brome grass (Bromus sp.) and three wheat samples collected in 2020. BYDV PAS is also efficiently transmitted by R. padi but is often more prevalent and severe than BYDV PAV; it can also overcome some sources of virus resistance that are effective against BYDV PAV (Chay et al. 1996, Robertson and French 2007). To our knowledge, this is the first report of BYDV PAS in Australia. Further work is needed to determine the extent of its distribution, incidence, impacts and epidemiology in Australia, along with its relationship to other BYDV PAS isolates.

PMID:38414196 | DOI:10.1094/PDIS-10-23-2195-PDN

Biol Lett. 2024 Feb;20(2):20230480. doi: 10.1098/rsbl.2023.0480. Epub 2024 Feb 28.

ABSTRACT

Active electroreception-the ability to detect objects and communicate with conspecifics via the detection and generation of electric organ discharges (EODs)-has evolved convergently in several fish lineages. South American electric fishes (Gymnotiformes) are a highly species-rich group, possibly in part due to evolution of an electric organ (EO) that can produce diverse EODs. Neofunctionalization of a voltage-gated sodium channel gene accompanied the evolution of electrogenic tissue from muscle and resulted in a novel gene (scn4aa) uniquely expressed in the EO. Here, we investigate the link between variation in scn4aa and differences in EOD waveform. We combine gymnotiform scn4aa sequences encoding the C-terminus of the Nav1.4a protein, with biogeographic data and EOD recordings to test whether physiological transitions among EOD types accompany differential selection pressures on scn4aa. We found positive selection on scn4aa coincided with shifts in EOD types. Species that evolved in the absence of predators, which likely selected for reduced EOD complexity, exhibited increased scn4aa evolutionary rates. We model mutations in the protein that may underlie changes in protein function and discuss our findings in the context of gymnotiform signalling ecology. Together, this work sheds light on the selective forces underpinning major evolutionary transitions in electric signal production.

PMID:38412964 | DOI:10.1098/rsbl.2023.0480

Biochem Pharmacol. 2024 Feb 25:116091. doi: 10.1016/j.bcp.2024.116091. Online ahead of print.

ABSTRACT

Despite the worldwide prevalence and huge burden of pain, pain is an undertreated phenomenon. Currently used analgesics have several limitations regarding their efficacy and safety. The discovery of analgesics possessing a novel mechanism of action has faced multiple challenges, including a limited understanding of biological processes underpinning pain and analgesia and poor animal-to-human translation. Computational pharmacology is currently employed to face these challenges. In this review, we discuss the theory, methods, and applications of computational pharmacology in pain research. Computational pharmacology encompasses a wide variety of theoretical concepts and practical methodological approaches, with the overall aim of gaining biological insight through data acquisition and analysis. Data are acquired from patients or animal models with pain or analgesic treatment, at different levels of biological organization (molecular, cellular, physiological, and behavioral). Distinct methodological algorithms can then be used to analyze and integrate data. This helps to facilitate the identification of biological molecules and processes associated with pain phenotype, build quantitative models of pain signaling, and extract translatable features between humans and animals. However, computational pharmacology has several limitations, and its predictions can provide false positive and negative findings. Therefore, computational predictions are required to be validated experimentally before drawing solid conclusions. In this review, we discuss several case study examples of combining and integrating computational tools with experimental pain research tools to meet drug discovery challenges.

PMID:38412924 | DOI:10.1016/j.bcp.2024.116091

Cell Rep Methods. 2024 Feb 26;4(2):100708. doi: 10.1016/j.crmeth.2024.100708.

ABSTRACT

Tumor deconvolution enables the identification of diverse cell types that comprise solid tumors. To date, however, both the algorithms developed to deconvolve tumor samples, and the gold-standard datasets used to assess the algorithms are geared toward the analysis of gene expression (e.g., RNA sequencing) rather than protein levels. Despite the popularity of gene expression datasets, protein levels often provide a more accurate view of rare cell types. To facilitate the use, development, and reproducibility of multiomic deconvolution algorithms, we introduce Decomprolute, a Common Workflow Language framework that leverages containerization to compare tumor deconvolution algorithms across multiomic datasets. Decomprolute incorporates the large-scale multiomic datasets produced by the Clinical Proteomic Tumor Analysis Consortium (CPTAC), which include matched mRNA expression and proteomic data from thousands of tumors across multiple cancer types to build a fully open-source, containerized proteogenomic tumor deconvolution benchmarking platform. http://pnnl-compbio.github.io/decomprolute.

PMID:38412834 | DOI:10.1016/j.crmeth.2024.100708

Cell Rep Methods. 2024 Feb 26;4(2):100715. doi: 10.1016/j.crmeth.2024.100715.

ABSTRACT

Imaging flow cytometry (IFC) allows rapid acquisition of numerous single-cell images per second, capturing information from multiple fluorescent channels. However, the traditional process of staining cells with fluorescently labeled conjugated antibodies for IFC analysis is time consuming, expensive, and potentially harmful to cell viability. To streamline experimental workflows and reduce costs, it is crucial to identify the most relevant channels for downstream analysis. In this study, we introduce PXPermute, a user-friendly and powerful method for assessing the significance of IFC channels, particularly for cell profiling. Our approach evaluates channel importance by permuting pixel values within each channel and analyzing the resulting impact on machine learning or deep learning models. Through rigorous evaluation of three multichannel IFC image datasets, we demonstrate PXPermute's potential in accurately identifying the most informative channels, aligning with established biological knowledge. PXPermute can assist biologists with systematic channel analysis, experimental design optimization, and biomarker identification.

PMID:38412831 | DOI:10.1016/j.crmeth.2024.100715

Comput Biol Med. 2024 Feb 15;171:108164. doi: 10.1016/j.compbiomed.2024.108164. Online ahead of print.

ABSTRACT

Inflammation plays a pivotal role in various pathological processes, ranging from routine injuries and infections to cancer. Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) are two major enzymes involved in the formation of lipid mediators of inflammation, such as prostaglandins and leukotrienes, through the arachidonic acid pathway. Despite the frequent use of nonsteroidal anti-inflammatory drugs for managing inflammatory disorders by inhibiting these enzymes, there is a wide spectrum of adverse effects linked to their usage. Jeevaneeya Rasayana (JR), a polyherbal formulation traditionally used in India, is renowned for its anti-inflammatory properties. The present study aimed to identify the potential phytocompounds in JR plants against COX-2 and 5-LOX, utilizing molecular docking and dynamic simulations. Among the 429 identified phytocompounds retrieved from publicly available data sources, Terrestribisamide and 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine have shown potential binding affinity and favorable interactions with COX-2 and 5-LOX arachidonic acid binding sites. The physicochemical properties and ADMET profiles of these compounds determined their drug-likeness and pharmacokinetics features. Additional validation using molecular dynamics simulations, SASA, Rg, and MM-PBSA binding energy calculations affirmed the stability of the complex formed between those compounds with target proteins. Together, the study identified the effectual binding potential of those bioactive compounds against COX-2 and 5-LOX, providing a viable approach for the development of effective anti-inflammatory medications.

PMID:38412690 | DOI:10.1016/j.compbiomed.2024.108164

ACS Synth Biol. 2024 Feb 27. doi: 10.1021/acssynbio.3c00676. Online ahead of print.

ABSTRACT

Glucosinolates are plant-specialized metabolites that can be hydrolyzed by glycosyl hydrolases, called myrosinases, creating a variety of hydrolysis products that benefit human health. While cruciferous vegetables are a rich source of glucosinolates, they are often cooked before consumption, limiting the conversion of glucosinolates to hydrolysis products due to the denaturation of myrosinases. Here we screen a panel of glycosyl hydrolases for high thermostability and engineer theBrassica crop, broccoli (Brassica oleracea L.), for the improved conversion of glucosinolates to chemopreventive hydrolysis products. Our transgenic broccoli lines enabled glucosinolate hydrolysis to occur at higher cooking temperatures, 20 °C higher than in wild-type broccoli. The process of cooking fundamentally transforms the bioavailability of many health-relevant bioactive compounds in our diet. Our findings demonstrate the promise of leveraging genetic engineering to tailor crops with novel traits that cannot be achieved through conventional breeding and improve the nutritional properties of the plants we consume.

PMID:38412618 | DOI:10.1021/acssynbio.3c00676

Nat Metab. 2024 Feb 27. doi: 10.1038/s42255-024-00994-0. Online ahead of print.

ABSTRACT

Our current understanding of mitochondrial organelle physiology has benefited from two broad approaches: classically, cuvette-based measurements with suspensions of isolated mitochondria, in which bioenergetic parameters are monitored acutely in response to respiratory chain substrates and inhibitors1-4, and more recently, highly scalable genetic screens for fitness phenotypes associated with coarse-grained properties of the mitochondrial state5-10. Here we introduce permeabilized-cell mitochondrial function sequencing (PMF-seq) to combine strengths of these two approaches to connect genes to detailed bioenergetic phenotypes. In PMF-seq, the plasma membranes within a pool of CRISPR mutagenized cells are gently permeabilized under conditions that preserve mitochondrial physiology, where detailed bioenergetics can be probed in the same way as with isolated organelles. Cells with desired bioenergetic parameters are selected optically using flow cytometry and subjected to next-generation sequencing. Using PMF-seq, we recover genes differentially required for mitochondrial respiratory chain branching and reversibility. We demonstrate that human D-lactate dehydrogenase specifically conveys electrons from D-lactate into cytochrome c to support mitochondrial membrane polarization. Finally, we screen for genetic modifiers of tBID, a pro-apoptotic protein that acts directly and acutely on mitochondria. We find the loss of the complex V assembly factor ATPAF2 acts as a genetic sensitizer of tBID's acute action. We anticipate that PMF-seq will be valuable for defining genes critical to the physiology of mitochondria and other organelles.

PMID:38413804 | DOI:10.1038/s42255-024-00994-0