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bioRxiv2021

Cholesterol dynamics are essential for the growth and development of Plasmodium falciparum within the erythrocyte

Cholesterol is the most abundant lipid in the erythrocyte. During its blood stage development, the malaria parasite establishes an active cholesterol gradient across the various membrane systems within the infected erythrocyte. Interestingly, some antimalarial compounds have recently been shown to disrupt cholesterol homeostasis in intraerythrocytic stages of Plasmodium falciparum. These studies point to the importance of cholesterol for parasite growth. Previously, reduction of cholesterol from the erythrocyte membrane by treatment with methyl-{beta}-cyclodextrin (M{beta}CD) was shown to inhibit parasite invasion and growth. In addition, M{beta}CD treatment of trophozoite stage P. falciparum was shown to result in parasite expulsion from the host cell. We have revisited these phenomena by using live video microscopy, ultrastructural analysis, and response to antimalarial compounds. By using time-lapse video microscopy of fluorescently tagged parasites, we show that M{beta}CD treatment for just 30 min causes dramatic expulsion of the trophozoite stage parasites. This forceful expulsion occurs within 10 sec. Remarkably, the plasma membrane of the host cell from which the parasite has been expelled does not appear to be compromised. The parasitophorous vacuolar membrane (PVM) continued to surround the extruded parasite, but the PVM appeared damaged. Treatment with antimalarial compounds targeting PfATP4 or PfNCR1 prevented M{beta}CD-mediated extrusion of the parasites, pointing to a potential role of cholesterol dynamics underlying the expulsion phenomena. We also confirmed the essential role of erythrocyte plasma membrane cholesterol for invasion and growth of P. falciparum. This defect can be partially complemented by cholesterol and desmosterol but not with epicholesterol, revealing stereospecificity underlying cholesterol function. Overall, our studies advance previous observations and reveal unusual cell biological features underlying cholesterol depletion of infected erythrocyte plasma membrane. ImportanceMalaria remains a major challenge in much of the world. Symptoms of malaria are caused by the growth of parasites belonging to Plasmodium spp. inside the red blood cells (RBC), leading to their destruction. The parasite depends upon its host for much of its nutritional need. Cholesterol is a major lipid in the RBC plasma membrane, which is the only source of this lipid for malarial parasites. We have previously shown that certain new antimalarial compounds disrupt cholesterol homeostasis in P. falciparum. Here we use live time-lapse video microscopy to show dramatic expulsion of the parasite from the host RBC when the cholesterol content of the RBC is reduced. Remarkably, this expulsion is inhibited by the antimalarials that disrupt lipid homeostasis. We also show stereospecificity of cholesterol in supporting parasite growth inside RBC. Overall, these results point to a critical role of cholesterol in physiology of malaria parasites.

microbiology↗

bioRxiv2022

Subcapsular sinus macrophage sensing of extracellular matrix rigidity alters membrane topography and immune complex mobility

Subcapsular sinus macrophages (SSMs) play a key role in immune defence by forming immunological barriers that control the transport of pathogens from lymph into lymph node follicles. SSMs participate in antibody responses by presenting antigens directly to naive B cells and by supplying antigens to follicular dendritic cells to propagate germinal centre reactions. Despite the prominent roles that SSMs play during immune responses, little is known about their cell biology because they are technically challenging to isolate and study in vitro. Here, we used multi-colour fluorescence microscopy to identify lymph nodederived SSMs in culture. We focused on the role of SSMs as antigen-presenting cells and found that their actin cytoskeleton regulates the spatial organisation and mobility of immune complexes displayed on the cell surface. Moreover, we determined that SSMs are mechanosensitive cells that respond to changes in extracellular matrix (ECM) rigidity by altering the architecture of the actin cytoskeleton, leading to changes in cell morphology, membrane topography, and immune complex mobility. Our results reveal a new mechanism regulating physical aspects of antigen presentation by antigen-presenting cells, which may have implications for B cell activation and antibody responses.

immunology↗

bioRxiv2017

Evolutionary Origin of the Mammalian Hematopoietic System Found in a Colonial Chordate

Hematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are hematopoietic stem cells (HSCs), which are multipotent, self-renewing and generate the entire repertoire of blood and immune cells throughout life. Here we studied the hematopoietic system of Botryllus schlosseri, a colonial tunicate that has vasculature, circulating blood cells, and interesting characteristics of stem cell biology and immunity. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. Using flow-cytometry, whole-transcriptome sequencing of defined cell populations, and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, the HSC niche, and demonstrated that self-recognition inhibits cytotoxic reaction. Our study implies that the HSC and myeloid lineages emerged in a common ancestor of tunicates and vertebrates and suggests that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from the same origin.

immunology↗

bioRxiv2018

Evaluation of S1PR1/pSTAT3 and S1PR2/FOXP1 Expression in Aggressive, Mature B Cell Lymphomas

Aggressive, mature B-cell lymphomas represent a heterogeneous group of diseases including Burkitt Lymphoma (BL), High Grade B Cell Lymphomas (HGBL) (eg, Double-Hit B cell lymphomas (HGBL-DH: HGBL with MYC and BCL2 and/or BCL6 translocations)), HGBL, Not Otherwise Specified (HGBL, NOS) and Diffuse Large B Cell Lymphoma. The overlapping morphologic and immunohistochemical features of these lymphomas may pose diagnostic challenges in some cases, and a better understanding of potential diagnostic biomarkers and possible therapeutic targets is needed. Sphingosine 1 Phosphate Receptors (S1PR1-5) represent a family of G-protein coupled receptors that bind the sphingolipid (S1P) and influence migration and survival pathways in a variety of cell types, including lymphocytes. S1PRs are emerging as biomarkers in B cell biology and interaction between S1PR pathways and STAT3 or FOXP1 has been reported, especially in DLBCL. Our aim was to extend the understanding of the S1PR1, STAT3 and S1PR2, FOXP1 expression beyond DLBCL, into additional aggressive, mature B cell lymphomas such as BL, HGBL-DH and HGBL,NOS.\n\nHerein, we report that S1PR1 and S1PR2 showed different patterns of expression in mantle zones and follicle centers in reactive lymphoid tissue and, among the lymphomas in this study, Burkitt lymphomas showed a unique pattern of expression compared to HGBL and DLBCL. Additionally, we found that S1PR1 and S1PR2 expression was typically mutually exclusive and were expressed in a low proportion of cases (predominantly HGBL involving extranodal sites). Lastly, FOXP1 was expressed in a high proportion of the various case types and pSTAT3 was detected in a significant proportion of HGBL and DLBCL cases. Taken together, these findings provide further evidence that S1PR1, pSTAT3, S1PR2 and FOXP1 play a role in a subset of aggressive mature B cell lymphomas.

pathology↗

bioRxiv2026

Long-range transport of biomolecular condensates by Marangoni effect

How mesoscale cargos such as biomolecular condensates are transported and spatially positioned within cells remains a fundamental question in cell biology. During Caenorhabditis elegans zygote polarization, a substantial fraction of P granules undergoes persistent anterior-to-posterior motion that cannot be fully explained by dissolution-recondensation. Here, combining microfluidic reconstitution, quantitative micropipette aspiration and live-cell imaging, we show that a MEG-3 concentration gradient generates interfacial-tension asymmetry and drives the directed transport of PGL-3-RNA co-condensates in vitro and P granules in vivo through the Marangoni effect. This framework enables parameter-free quantitative predictions of condensate transport. Orthogonal molecular perturbations abolish or reverse directional migration, directly linking interfacial properties to transport behavior and establishing the specificity of this mechanism. Together, our findings identify the Marangoni effect as a previously unrecognized mechanism for intracellular transport of biomolecular condensates and provide direct causal evidence that condensate emergent properties can contribute to physiological organization.

biophysics↗

bioRxiv2024

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

microbiology↗

bioRxiv2022

Pancreatic tumors activate arginine biosynthesis to adapt to myeloid-driven amino acid stress

Nutrient stress in the tumor microenvironment requires cancer cells to adopt adaptive metabolic programs to maintain survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Previously, we performed quantitative metabolomics of the interstitial fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the microenvironment of these tumors (Sullivan et al., 2019a). Here, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling study of PDAC metabolism under physiological nutrition. We show that PDAC cells cultured in TIFM, compared to standard laboratory models, adopt a cellular state more similar to PDAC cells in tumors. Further, using the TIFM model we identified arginine biosynthesis as a metabolic adaptation PDAC cells engage to cope with microenvironmental arginine starvation driven by myeloid cells in PDAC tumors. Altogether, these data show that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity and enable the discovery of novel cancer metabolic phenotypes.

cancer biology↗

bioRxiv2015

Feedback Control in Planarian Stem Cell Systems

Background In planarian flatworms, the mechanisms underlying the activity of collectively pluripotent adult stem cells (neoblasts) and their descendants can now be studied from the level of the individual gene to the entire animal. Flatworms maintain startling developmental plasticity and regenerative capacity in response to variable nutrient conditions or injury. We develop a model for cell dynamics in such animals, using methods of nonlinear dynamics, assuming that fully differentiated cells exert feedback control on neoblast activity. Results Our model predicts a number of whole organism level and general cell biological and behaviours, some of which have been empirically observed or inferred in planarians and others that have not. As previously observed empirically we find: 1) a curvilinear relationship between external food and planarian steady state size; 2) the fraction of neoblasts in the steady state is constant regardless of planarian size; 3) a burst of controlled apoptosis during regeneration after amputation as the number of differentiated cells are adjusted towards their correct homeostatic/steady state level. In addition our model describes the following properties that can inform and be tested by future experiments: 4) the strength of feedback control from differentiated cells to neoblasts (i.e. the activity of the signalling system) and from neoblasts on themselves in relation to absolute number depends upon the level of food in the environment; 5) planarians adjust size when food level reduces initially through increased apoptosis and then through a reduction in neoblast self-renewal activity; 6) following wounding or excision of differentiated cells, different time scales characterize both recovery of size and the two feedback functions; 7) the temporal pattern of feedback controls differs noticeably during recovery from a removal or neoblasts or a removal of differentiated cells; and 8) the signaling strength for apoptosis of differentiated cells depends upon both the absolute and relative deviations of the number of differentiated cells from their homeostatic level.\n\nConclusions We offer the first analytical framework for organizing experiments on planarian flatworm stem cell dynamics in a form that allows models to be compared with quantitative cell data based on underlying molecular mechanisms and thus facilitates the interplay between empirical studies and modeling. This framework is the foundation for studying cell migration during wound repair, the determination of homeostatic levels of differentiated cells by natural selection, and stochastic effects.

Systems Biology↗

bioRxiv2025

Lactate cannot replace glucose for maintaining the viability of mouse and human glioma cells

ObjectivesAerobic lactic acid fermentation (the "Warburg effect") is associated with OxPhos insufficiency and altered energy metabolism in most cancers. Whether lactate is a major fuel in cancer cells remains debated. This study investigated whether lactate could serve as a metabolic fuel in glioma cells and replace glucose to support viability. MethodsA bioluminescence ATP assay and calcein-AM/EthD-III double-staining were used to measure ATP content and viability in mouse (VM-M3, CT-2A) and human (U-87MG) glioma cells differing in cell biology and genetic background. Viability was assessed in thioglycollate-elicited peritoneal macrophages (TPMs) from VM/Dk and C57BL/6J mice, used as syngeneic non-neoplastic controls for VM-M3 and CT-2A gliomas, respectively. Oxygen consumption rate (OCR) was determined using the Resipher system. ResultsLactate alone failed to sustain ATP content and viability in all glioma cell lines. ATP content and viability were lower in cancer cells cultured in glutamine and lactate than in glucose and glutamine. In contrast, lactate alone sustained over 45% viability in both TPM models. Moreover, viability was similar between TPMs cultured in glutamine and lactate and in glucose and glutamine. In human U-87MG, lactate addition under severe glucose restriction increased OCR and viability. A low dose of the glycolysis inhibitor 2-deoxy-D-glucose abolished both increases. ConclusionsOur results suggest non-neoplastic mouse TPMs utilize lactate more effectively than mouse glioma cells. In U-87MG, lactate utilization appears glycolysis-dependent, given its sensitivity to 2-deoxy-D-glucose. In conclusion, our data does not support lactate as a major oxidative fuel for viability in mouse and human glioma cells.

cancer biology↗

bioRxiv2024

Dicarbonyl stress debilitates mesothelial defense against metastasizing ovarian cancer

Chronic metabolic disorders and aging result in accumulation of active dicarbonyls that glycate biomolecules rendering them dysfunctional. Although metabolic aberrations are known to be epidemiologically associated with faster cancer progression, cell biological determinants of such associations remain elusive. The formation of micro-metastases in epithelial ovarian cancer involves its colonization of visceral peritonea through clearance of mesothelia that line the coelom. In this study, we observe that cocultures of immortalized human coelomic MeT-5A mesothelia with human ovarian cancer cells OVCAR-3 and SK-OV-3 show greater infiltration by the latter when exposed to increasing concentrations of the dicarbonyl methylglyoxal (MG). Treatment with increasing concentrations of MG caused death and senescence within human and murine serosal mesothelia. Cells showed higher levels of advanced glycation end products, dysregulated occludens junction protein ZO-1, and disrupted localization of cortical filamentous actin and its regulator ezrin, indicating poor inter-cell adhesion. Time lapse imaging also showed impaired migration for MG-treated single mesothelia and for their collective monolayers. Agent-based computer modeling of coculture dynamics predicted that a combined effect of confluence and migration allows inter-adherent mesothelia to contain the spread of colonizing cancer cells, which was confirmed through coculture time lapses of cancer colonization within higher and lower mesothelial densities. We found ovarian cancer cells showed higher levels of glyoxalase-1 (GLO-1) enzyme, which catabolizes MG, suggesting how they escaped its cytotoxic effects. Consistent with this, treatment of OVCAR-3 with MG concurrently with pharmacological inhibition of GLO-1 showed greater cell death. Our results suggest dicarbonyl stress helps colonizing cancer cells overcome the resistance of natural homoeostatic barriers and its inhibition may, in supplementation with chemotherapy, stem metastasis.

cancer biology↗

bioRxiv2024

BRACE: A novel Bayesian-based imputation approach for dimension reduction analysis of alternative splicing at single-cell resolution

Bayesian approach is a powerful tool to solve challenging questions in life sciences. One such area of life sciences in which Bayesian approach has seen an increased utility in the recent years is single-cell biology. Alternative splicing represents an additional layer of complexity underlying gene expression profile that has the potential to reveal insights into the biological mechanisms underpinning heath and disease states. Dimension reduction analysis is the cornerstone of RNA-sequencing analysis and has the ability to guide selection of candidate biomarkers based on segregation of sample groups. Nevertheless, dimension reduction analysis at single- cell resolution remains a significant challenge for alternative splicing datasets, and therefore hitherto preclude the assessment of candidate isoforms. Here, we introduce BRACE (a Bayesian-based imputation approach for dimension Reduction Analysis of alternative splicing at single-CEll resolution). We demonstrated our Bayesian approach represents an improvement over existing methods for imputing missing percent spliced-in values, and subsequently applied our approach for the dimension reduction analysis of alternative splicing events at single-cell resolution. We further demonstrated the application of our Bayesian approach over a range of single-cell datasets with increasing complexity, namely cell populations that are transcriptomically distinct, similar, and heterogenous. We anticipate our approach to enable assessment and selection of cell state- or disease-specific biomarkers for downstream experimental validation.

bioinformatics↗

bioRxiv2017

Show me your neighbours, and I’ll tell you what you are – cellular microenvironment matters

To answer major questions of cell biology, it is essential to understand cellular complexity. Modern automated microscopes produce vast amounts of images routinely, making manual analysis nearly impossible. Due to their efficiency, machine learning-based analysis software have become essential tools to perform single-cell-level phenotypic analysis of large imaging datasets. However, an important limitation of such methods is that they do not use the information gained from the cellular micro- and macroenvironment: the algorithmic decision is based solely on the local properties of the cell of interest. Here, we present how various microenvironmental features contribute to identifying a cell and how such additional information can improve single-cell-level phenotypic image analysis. The proposed methodology was tested for different sizes of Euclidean and nearest neighbour-based cellular environments both on tissue sections and cell cultures. Our experimental data verify that the microenvironment of a cell largely determines its entity. This effect was found to be especially strong for established tissues, while it was somewhat weaker in the case of cell cultures. Our analysis shows that combining local cellular features with the properties of the cell's microenvironment significantly improves the accuracy of machine learning-based phenotyping.

bioinformatics↗

bioRxiv2022

SARS-CoV2 associated secretion of nanoLuciferase reports on virus and Virus-Like Particle production

SARS-CoV2 is a positive-strand RNA virus in the Coronaviridae family that has caused world-wide morbidity and mortality. While much progress has been made we still need expanded rapid anti-virals. The top advanced antiviral candidates all target stages of RNA replication, leaving virus assembly an unexplored avenue of antiviral research. To address this gap, and explore the biochemical and cell biological features of viral assembly, we have employed an improved virus-like particle (VLP) system. We exploited the small nanoLuciferase protein for enhanced signal and surprisingly found that the protein itself appears to be packaged into both SARS CoV2 VLPs and virions and secreted from cells. Interestingly, nLuc is not co-secreted with dengue or Zika infection, suggesting the large virion of Coronavirus can encaspidate and secrete a cellularly expressed reporter protein. Our findings open the way for powerful new approaches to measure viral particle production, egress and viral entry mechanisms.

molecular biology↗

bioRxiv2025

Multiplexed single-cell transcriptomics reveals diverse phenotypic outcomes for pathogenic SHP2 variants

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is an important regulator of Ras/MAPK signaling that acts downstream of receptor tyrosine kinases and other transmembrane receptors. Germline PTPN11 mutations cause developmental disorders such as Noonan Syndrome, whereas somatic mutations drive various cancers. While many pathogenic mutations enhance SHP2 catalytic activity, others are inactivating or affect protein interactions, confounding our understanding of SHP2-driven disease. Here, we combine single-cell transcriptional profiling of cells expressing clinically diverse SHP2 variants with protein biochemistry, structural analysis, and cell biology to explain how pathogenic mutations dysregulate signaling. Our analyses reveal that loss of catalytic activity does not phenocopy SHP2 knock-out at the gene expression level, that some mechanistically distinct mutations have convergent phenotypic effects, and that different mutations at the same hotspot residue can yield divergent cell states. These findings provide a framework for understanding the connection between SHP2 structural perturbations, cellular outcomes, and human diseases.

molecular biology↗

bioRxiv2020

Three-Dimensional Label-Free Imaging and Quantification of Migrating Cells during Wound Healing

The wound healing assay provides essential information about collective cell migration and cell-to-cell interactions. It is a simple, effective, and widely used tool for observing the effect of numerous chemical treatments on wound healing speed. To perform and analyze a wound healing assay, various imaging techniques have been utilized. However, image acquisition and analysis are often limited in two-dimensional space or require the use of exogenous labeling agents. Here, we present a method for imaging large-scale wound healing assays in a label-free and volumetric manner using optical diffraction tomography (ODT). We performed quantitative high-resolution three-dimensional (3D) analysis of cell migration over a long period without difficulties such as photobleaching or phototoxicity. ODT enables the reconstruction of the refractive index (RI) tomogram of unlabeled cells, which provides both structural and biochemical information about the individual cell at subcellular resolution. Stitching multiple RI tomograms enables long-term (24 h) and large field-of-view imaging (> 800 x 400 m2) with a lateral resolution of 110 nm. We demonstrated the thickness changes of leading cells and studied the effects of cytochalasin D. The 3D RI tomogram also revealed increased RI values in leading cells compared to lagging cells, suggesting the formation of a highly concentrated subcellular structure. STATEMENT OF SIGNIFICANCEThe wound healing assay is a simple but effective tool for studying collective cell migration (CCM) that is widely used in biophysical studies and high-throughput screening. However, conventional imaging and analysis methods only address two-dimensional properties in a wound healing assay, such as gap closure rate. This is unfortunate because biological cells are complex 3D structures, and their dynamics provide significant information about cell physiology. Here, we presented three-dimensional (3D) label-free imaging for wound healing assays and investigated the 3D dynamics of CCM. High-resolution subcellular structures as well as their collective dynamics were imaged and analyzed quantitatively. Our label-free quantitative 3D analysis method provides a unique opportunity to study the behavior of migrating cells during the wound healing process.

biophysics↗

bioRxiv2024

Synthesis and mechanical characterization of polyacrylamide (PAAm) hydrogels with different stiffnesses for large-batch cell culture applications

The impact of mechanical cues on cell behavior is increasingly being recognized, rendering hydrogel platforms that mimic the extracellular matrix indispensable in in vitro cell biology research. Here, we present a step-by-step protocol for synthesis and rheological characterization of polyacrylamide (PAAm) hydrogels with varying stiffnesses, produced as large circular unattached gels customizable in shape and size. We outline methods for their use in cell culture and downstream applications involving secretome or cell analysis, and protein visualization by fluorescence microscopy. This protocol is based on the recent work of Shi & Janmey who describe a novel and straightforward method for the production of large PAAm hydrogels for bulk cell culture and mechanobiology studies.1 Their procedure results in one large gel that is not attached to a supporting surface and therefore can be transferred and/or stamped to generate PAAm gels of custom shapes and sizes. The aim of this step-by-step procedure is therefore not to improve the reported protocol, but to create a clearly outlined and repeatable protocol that enables a smooth implementation in any lab for a diverse audience. In addition, our protocol describes besides harvesting of cells also the collection of secretome for downstream biochemical analyses, as well as immunofluorescence labeling using antibodies that can readily be multiplexed for optimization of labeling conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/613503v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@cefcc3org.highwire.dtl.DTLVardef@1348c93org.highwire.dtl.DTLVardef@f935acorg.highwire.dtl.DTLVardef@132dbd0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2018

The essential genome of the crenarchaeal model Sulfolobus islandicus

Sulfolobus islandicus is a model experimental system in the TACK superphylum of the Archaea, a key lineage in the evolutionary history of cell biology. Here we report a genome-wide identification of the repertoire of genes essential to S. islandicus growth in culture. We confirm previous targeted gene knockouts, uncover the non-essentiality of functions assumed to be essential to the Sulfolobus cell, including the proteinaceous S-layer, and highlight key essential genes whose functions are yet to be determined. Phyletic distributions illustrate the potential transitions that have occurred during the evolution of this contemporary archaeal cell and highlight the sets of genes that may have been associated with each transition. We use this comparative context as a lens to focus future research on archaea-specific uncharacterized essential genes for which future functional data would provide valuable insights into the evolutionary history of the contemporary cell.

microbiology↗

bioRxiv2025

A vesicle-based platform for high-efficiency, high-viability CRISPR/Cas9 knockout in primary human myeloid cells

Myeloid cells, including monocytes, macrophages, and dendritic cells, are central to host defence, inflammation, and antigen presentation. However, functional genetics studies in corresponding primary cells remain limited due to inefficient delivery methods. Here we report a vesicle-based CRISPR/Cas9 platform for effective genome editing in primary human myeloid cells using engineered virus-like particles (VLPs) and extracellular vesicles (EVs) to deliver Cas9-gRNA ribonucleoproteins (RNPs). Targeting primary CD14+ monocytes enables rapid generation of robust polyclonal knockouts while preserving cell viability and subsequent differentiation into macrophages and dendritic cells. Because our workflow relies on standard cell-culture handling rather than electroporation instrumentation, it is inexpensive, highly scalable and readily transferable across laboratories. Edited cells remain compatible with diverse downstream assays and functional readouts, enabling scalable loss-of-function studies and mechanistic dissection of primary human myeloid cell biology in health and disease. Vesicle-mediated RNP delivery thus provides a broadly applicable route to genetic perturbation in primary human myeloid lineages. SummaryFiori and colleagues describe a vesicle-based platform for efficient CRISPR/Cas9 knockout in primary human myeloid cells. This approach preserves cell viability, enables near-complete gene disruption, and supports functional studies of innate immunity, antigen presentation, and HIV-1 infection.

immunology↗