Flaviviruses, such as Dengue, Zika, and West Nile virus, cause a significant global health burden and have earned attention as high-pandemic risk pathogens. Flaviviruses interact closely with cell membranes at every stage of their life cycle, and mounting evidence demonstrates that flaviviruses rely on specific lipids and lipid-remodeling proteins, presenting potential therapeutic opportunities for targeting the host's lipid metabolism. Our understanding of lipid function in infection has expanded considerably in recent years, partly thanks to advances in lipidomics, cryo-electron tomography, lipid-based chemical tools, and biophysical characterization techniques. In this review, we highlight recent breakthroughs that have clarified flavivirus lipid requirements and functions, as well as ongoing technological advances in the virus-lipid interaction field, which are poised to enable the next wave of discoveries.

Mycobacterium tuberculosis (Mtb) establishes infection by entering host phagocytes through phagocytosis. While host lipids are known to influence this process, the specific contribution of the signaling lipid diacylglycerol (DAG) remains poorly defined. Here, we identify DAG as a critical regulator of phagocytosis. Disruption of DAG production, through inhibition or genetic deletion of adipose triglyceride lipase (ATGL) and phospholipase C gamma 2 (PLCγ2), two major pathways that generate cellular DAG pools, markedly reduced uptake of both Mtb and zymosan-coated beads. Notably, loss of ATGL or PLCγ2 did not impair receptor trafficking to the cell surface or cargo binding, indicating that DAG is not required for phagocytic recognition or initiation, but instead for a later step in phagosome formation. Mechanistically, cells lacking ATGL or PLCγ2 displayed constitutive phosphoinositide 3-kinase (PI3K) phosphorylation, suggesting that dysregulated intracellular signaling prevents completion of phagocytosis. These findings uncover a previously unappreciated role for DAG biosynthesis in coordinating intracellular signaling required for phagocytosis and provide new insight into host pathways that govern Mtb entry.

Cellular lipids shape health and disease through specific protein interactions, yet lipid–protein networks remain poorly defined. Despite rapid advances in functional lipid probes, the field still lacks a practical, dedicated protocol for conducting lipid–protein interaction studies. We describe detailed methods for determining lipid interactomes within cells using multifunctionalized lipid derivatives. We provide a protocol that details how to (i) treat cells with lipid derivatives and perform photochemistry to obtain lipid–protein conjugates, (ii) extract cellular lysates for downstream analysis, (iii) perform click chemistry on lysates with a fluorophore and observe lipid–protein conjugates by in-gel fluorescence and (iv) perform click chemistry on lysates with azide beads and prepare lipid–protein conjugates for proteomic analysis. We provide context on important parameters for each step and include guidelines for controls, as well as suggestions for troubleshooting based on common problems encountered during the preparation of this protocol. This protocol enables identification of proteins that bind to specific lipids across diverse biological systems and cellular states. The entire workflow from cell treatment to complete proteomic sample preparation requires ~15 h over 4 d, depending on the type of experimental readout (in-gel fluorescence or proteomics) and the usage of pause points. Practitioners are expected to be familiar with standard biochemical techniques, such as sterile sample handling and tissue culture and gel electrophoresis. Additional skills are needed for mass spectrometric analysis, and collaboration with a proteomics core facility is recommended. The described procedures uniquely enable the identification of the protein interactors (the interactome) of select lipid species, providing for a major advance in the characterization of the biological roles of lipids in cellular systems. Cellular lipids are known to have specific protein interactions. This protocol describes how to study the lipid–protein interactome using multifunctional lipid probes capable of photo-cross-linking and click chemistry by in-gel fluorescence or mass spectrometry.

The antigen presenting molecule major histocompatibility complex class I-related protein 1 (MR1) binds small molecule metabolites derived from the microbial riboflavin biosynthetic pathway and presents them at the cell surface for surveillance by MR1-restricted mucosal-associated invariant T cells (MAIT cells). MR1 ligands can originate in the extracellular space or in endosomal compartments that contain microbial pathogens. Distinct, complementary antigen processing and presentation pathways enable MR1 to survey diverse intracellular locations and present both exogenous and intracellular antigens. Here, we generated a panel of BEAS-2B MR1 KO cells reconstituted with MR1 proteins mutated at amino acids 9 to 16. The overexpressed mutated MR1 molecules differentially translocated to the cell surface in response to 6-formylpterin and differed in their ability to present mycobacterial antigens to MAIT cell clones. While they barely presented Mycobacterium smegmatis supernatant and other exogenous MAIT cell antigens, their ability to present antigens derived from mycobacterial infection and a 5-amino-6-D-ribitylaminouracil prodrug that requires endosomal processing remained largely intact. Protein coimmunoprecipitation and mass spectrometry-based proteomic analysis showed that mutated MR1 differentially associated with calnexin and β2-microglobulin (B2M). Knock down of B2M in cells overexpressing wildtype MR1 phenocopied the loss of exogenous antigen presentation but did not impact presentation of intracellular antigens. Thus, the MR1-mediated presentation of exogenous antigen appears to be limited by binding to B2M whereas a lower sensitivity to B2M deficiency suggests that MAIT cell activation via the endosomal antigen presentation pathway may be limited by the availability of MR1 itself.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that can be sensed by T cells, which are essential for the control of infection. In comparison to viral infections, Mtb antigens are relatively limited and hence, challenging to sample. Specialized antigen presentation pathways enable the presentation of such scarce antigens to CD8+ T cells, which are, thus, uniquely poised to survey intracellular environments. A subset of CD8+ T cells prevalent in the airways, known as mucosal-associated invariant T (MAIT) cells, can be activated through the presentation of Mtb antigens via the major histocompatibility complex class I-related protein 1 (MR1) molecule. Prior work demonstrates that endosomal calcium signaling is critical for MR1-mediated presentation of Mtb-derived antigens. Here, we show that the calcium-sensing trafficking proteins Synaptotagmin (Syt) 1 and Syt7 specifically promote MAIT cell activation in response to Mtb-infected cells. In bronchial epithelial cells, Syt1 and Syt7 localize to late endo-lysosomes and MR1 vesicles. Loss of Syt1 and Syt7 results in enlarged MR1 vesicles and an increased number of MR1 vesicles in close proximity to Mtb-containing vacuoles during infection. This study identifies a specialized pathway in which Syt1 and Syt7 facilitate the translocation of MR1 from Mtb-containing vacuoles, potentially to the cell surface for antigen presentation.

SUMMARY:Lipid-protein interactions play essential roles in cellular signaling and membrane dynamics, yet their systematic characterization has long been hindered by the inherent biochemical properties of lipids. Recent advances in functionalized lipid probes-equipped with photoactivatable crosslinkers, affinity handles, and photocleavable protecting groups-have enabled proteomics-based identification of lipid interacting proteins with unprecedented specificity and resolution. Despite the growing number of published lipid interactomes, there remains no centralized effort to harmonize, compare, or integrate these datasets. The Lipid Interactome addresses this gap by providing a structured, interactive web portal that adheres to FAIR data principles-ensuring that lipid interactome studies are Findable, Accessible, Interoperable, and Reusable. Through standardized data formatting, interactive visualizations, and direct cross-study comparisons, this resource enables researchers to systematically explore the protein-binding partners of diverse bioactive lipids. By consolidating and curating lipid interactome proteomics data from multiple studies, the Lipid Interactome database serves as a critical tool for deciphering the biological functions of lipids in cellularsystems. AVAILABILITY AND IMPLEMENTATION:This site can be viewed at LipidInteractome.org. All data are available for download. No user information is collected or necessary for data navigation, interaction, or download.

Lipids are fundamental organizers of biological membranes, yet visualizing lipid species within subcellular organelles has remained beyond experimental reach. Lennartz et al. introduce Lipid-correlative light and electron microscopy (CLEM), a CLEM workflow that maps lipid species onto membrane ultrastructure with nanoscale precision, uncovering active sphingomyelin sorting within the early endosome.

Cellular lipids shape health and disease through specific protein interactions, yet lipid-protein networks remain poorly defined. Despite rapid advances in functional lipid probes, the field still lacks a practical, dedicated protocol for conducting lipid-protein interaction studies. We describe detailed methods for determining lipid interactomes from cells using multifunctionalized lipid derivatives. We provide protocols that detail 1) how to treat cells with lipid derivatives and perform photochemistry to obtain lipid-protein conjugates; 2) how to perform click chemistry with a fluorophore and observe lipid-protein conjugates by in-gel fluorescence; 3) how to perform click chemistry with azide beads and prepare lipid-protein conjugates for proteomic analysis. We provide context on important parameters for each step and include guidelines for controls, as well as suggestions for troubleshooting based on common problems encountered during the preparation of this protocol. This protocol enables mapping lipid interactomes across diverse biological systems. The entire workflow from cell treatment to complete proteomic sample preparation requires ∼15 hours over four days, depending on the type of experimental readout (in-gel fluorescence or proteomics), and the usage of pause points. Practitioners are expected to be familiar with standard biochemical techniques, such as sterile sample handling and tissue culture and gel electrophoresis. Additional skills are needed for mass spectrometric analysis, and collaboration with a proteomics core facility is recommended. The described procedures uniquely enable the identification of the protein interactors (the interactome) of select lipid species, providing for a major shift in the characterization of the biological roles of lipids in cellular systems.

MR1 is a non-polymorphic, ubiquitously expressed, MHC class I-like antigen-presenting molecule that presents small-molecule metabolites to T cells. Studies have shown that MR1 plays a role in microbial infection, inflammation, and tumor immunity. The antigens it presents include metabolites of microbial and self-origin as well as small-molecule drugs and form stable complexes with MR1 that are displayed on the cell surface to activate T cells. However, unlike classical MHC I and II molecules, the fundamental biology of MR1 remains poorly understood, particularly the mechanisms governing antigen loading and intracellular trafficking. This knowledge gap is largely due to the lack of molecular tools available to precisely manipulate MR1 function. In this study, we describe a high-affinity (1.6 nM KD) anti-MR1 nanobody, MR1Nb1. We characterize the binding of this nanobody including affinity by ELISA and kinetics by BLI. Crucially, we map the binding epitope of MR1Nb1 on MR1 by HDX-MS, providing key insights into the mechanism through which it blocks MR1T cell activation. In functional assays MR1Nb1 effectively and specifically blocks MR1T cell activation by cells infected with M. tuberculosis or treated with M. smegmatis supernatant or the synthetic ligand deazalumazine. MR1Nb1 further stains MR1-ligand complexes on the cell surface in a flow cytometry assay. This nanobody represents a unique and versatile tool for the field, as it can be produced inexpensively and expressed intracellularly within antigen presenting cells. Hence, our study provides a powerful new molecular probe for dissecting the mechanistic underpinnings of MR1 biology and uncover its broader roles in immunity.

The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the protein's structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential.

Cells continuously metabolize lipids, converting them into diverse molecules that influence cellular functions- in health and disease. Sphingolipids have been specifically linked to a wide range of - cellular processes and have been shown to play a crucial role in the onset and progression of various diseases. Here, we detail a method for measuring lipid flux through a combination of labeled lipids and thin-layer chromatography (TLC). This method describes the exogenous addition of radioactive and fluorescent lipid analogues and the subsequent tracing of their metabolism. This metabolic tracing allows for conclusions to be drawn about the fate of a lipid in a given system. Though the described method focuses on sphingolipids, many alternatives are discussed for adaptation of the method to measurement of any specific lipid of interest. Overall, this method offers a robust, easy-to-adapt approach for measuring lipid flux across different cell types and experimental systems.

Lateral flow assays (LFAs) are among the most successful technologies for point-of-care and at-home testing, but further advances are needed to reduce costs and accelerate development. Alpaca-derived nanobodies (Nbs), single-domain antibody fragments, are promising immunoassay reagents across diverse applications. Their small size and ease of recombinant production make them particularly well suited for diagnostics. Here, we present a paper-based LFA targeting the SARS-CoV-2 nucleocapsid (N) protein that exclusively uses Nbs for direct antigen detection. We also demonstrate in-house synthesis of Nb-coated gold nanoparticles, enabling instrument-free visual readout and detection of N protein down to 40 ng/mL. This design avoids components that require mammalian cell culture and can be produced entirely from in-house reagents, simplifying manufacturing and lowering component costs. Because the assay is read visually without an external reader, it is well suited for deployment in resource-limited settings. Together, these results highlight the speed and practicality of developing Nb-based LFAs and suggest a broadly applicable strategy for detecting other clinically important disease biomarkers.

Sphingomyelin (SM) is a major component of mammalian cell membranes and is particularly abundant in the myelin sheath that surrounds nerve fibers. Its production is catalyzed by SM synthases, SMS1 and SMS2, which interconvert phosphatidylcholine and ceramide into diacylglycerol and SM, respectively, in the Golgi and at the plasma membrane. As the lipids participating in this reaction fulfill both structural and signaling functions, SMS enzymes have considerable potential to influence diverse important cellular processes. The nematode Caenorhabditis elegans is an attractive model for studying both animal development and human disease. The organism contains five SMS homologues, but none of these have been characterized in any detail. Here, we carried out the first systematic analysis of SMS family members in C. elegans. Using heterologous expression systems, genetic ablation, metabolic labeling, and lipidome analyses, we show that C. elegans harbors at least three distinct SM synthases and one ceramide phosphoethanolamine (CPE) synthase. Moreover, C. elegans SMS family members have partially overlapping but also unique subcellular distributions, and together they occupy all principal compartments of the secretory pathway. Our findings shed light on crucial aspects of sphingolipid metabolism in a valuable animal model and open avenues for exploring the role of SM and its metabolic intermediates in organismal development.

BACKGROUND:As COVID-19 becomes endemic and vaccines are annually adapted, exposure intervals and immune imprinting become critical considerations for vaccination strategy. Imprinting by the ancestral spike protein affected bivalent Wuhan-Hu-1/BA.4-5 vaccine responses. We assess the persistence of imprinting in antibody responses to the more recent XBB.1.5 monovalent formulation. METHODS:We quantified live virus-neutralizing antibodies by focus reduction neutralization test and ancestral spike receptor-binding isotype titers by immunosorbent assay in individuals before and after XBB.1.5 vaccination. We compared responses between those who previously received three to four doses of Wuhan-Hu-1 vaccine and one dose of bivalent Wuhan-Hu-1/BA.4-5 (bivalent recipients) and those who received three to four doses of Wuhan-Hu-1 (bivalent non-recipients). RESULTS:We report that before XBB.1.5 vaccination, bivalent non-recipients have decreased breadth and potency of neutralization. At post-vaccination, non-recipients exhibit greater boosting of neutralizing antibodies against XBB.1.5 (18.4X versus 6.2X), EG.5.1 (30.9X versus 7.0X), and JN.1 (9.2X versus 3.7X) variants with comparable breadth and trends toward greater potency. Greater boosting in non-recipients is similarly observed for spike-binding IgA and total IgG/A/M but not IgG nor IgM. Bivalent non-recipients had longer intervals between vaccination, which may enhance antibody responses; however, bivalent receipt and interval are tightly linked, preventing isolation of individual contributions to boosting. Nonetheless, back-boosting of ancestral SARS-CoV-2 titers in both participant groups provides interval-independent evidence that imprinting persists. CONCLUSIONS:Our findings indicate that immune imprinting continues to affect humoral immunity elicited by the XBB.1.5 vaccine. Both imprinting and exposure intervals are important phenomena underlying immunogenicity of future variant-adapted COVID-19 vaccines.

The rapid evolution of SARS-CoV-2 has led to the emergence of numerous variants with enhanced transmissibility and immune evasion. Despite widespread vaccination, infections persist, and the mechanisms by which SARS-CoV-2 reprograms host metabolism remain incompletely understood. Here, we investigated whether virus-induced lipid remodeling is conserved across variants and whether changes in lipid abundance correlate with alterations in lipid biosynthetic enzymes. Using global untargeted lipidomics and quantitative proteomics, we analyzed A549-ACE2 cells infected with the Delta (B.1.617.2) or Omicron (B.1.1.529) variants and compared them to cells infected with the ancestral WA1 strain. In parallel, we conducted quantitative proteomics to assess virus-induced changes in the host proteome. Our results reveal that SARS-CoV-2 drives a remarkably consistent pattern of metabolic rewiring at both the lipidomic and proteomic levels across all three variants. We mapped changes in the expression of host metabolic enzymes and compared these to corresponding shifts in lipid abundance. This integrative analysis identified key host proteins involved in virus-mediated lipid remodeling, including fatty acid synthase (FASN), lysosomal acid lipase (LIPA), and ORM1-like protein 2 (ORMDL2). Together, these findings highlight conserved metabolic dependencies of SARS-CoV-2 variants and underscore host lipid metabolism as a potential target for broad-spectrum antiviral strategies.

As COVID-19 transitions into endemicity and vaccines are annually updated to circulating SARS-CoV-2 lineages such as JN.1, exposure intervals and immune imprinting become critical considerations for vaccination strategy. Imprinting by the ancestral spike protein has been observed with the bivalent Wuhan-Hu-1/BA.4-5 vaccine and its persistence can be further evaluated in the context of the more recent XBB.1.5 monovalent vaccine. We assessed antibody responses in individuals who received three to four doses of Wuhan-Hu-1, one dose of bivalent Wuhan-Hu-1/BA.4-5, and one dose of XBB.1.5 vaccine (bivalent recipients). We compared these to individuals who received three to four doses of Wuhan-Hu-1 and one dose of XBB.1.5 vaccine without prior bivalent vaccination (bivalent non-recipients). Before XBB.1.5 vaccination, bivalent non-recipients demonstrated decreased breadth and potency of neutralizing antibodies compared to recipients, but at post-vaccination exhibited greater boosting of neutralizing antibodies against XBB.1.5 (18.4X versus 6.2X), EG.5.1 (30.9X versus 7.0X), and JN.1 (9.2X versus 3.7X) variants with trends toward higher neutralizing titers and comparable variant cross-neutralization. Increased boosting in non-recipients were similarly observed for IgA and total IgG/A/M isotypes binding the spike receptor-binding domain but not IgG nor IgM. Bivalent non-recipients had longer intervals between exposures, which has been reported to enhance antibody boosting; however, bivalent receipt and interval were tightly linked variables, preventing the isolation of individual contributions to boosting. Nonetheless, significant "back-boosting" of ancestral SARS-CoV-2 titers upon XBB.1.5 vaccination in both participant groups indicate that immune imprinting continues to affect contemporary vaccines. Altogether, our findings highlight imprinting and exposure intervals as important phenomena underlying variant-adapted COVID-19 vaccine immunogenicity.

Cells derived from diseased tissue and their related cell lines exhibit numerous metabolic changes, including variations in lipid composition and metabolism. Indeed, lipids are important biomarkers of various diseases and exhibit crucial signaling roles during disease states. However, lipids, especially low-abundant and transient lipids like phosphoinositides, are difficult to study due to a lack of sophisticated tools. Here, we describe a unique targeted lipidomics method that allows us to define and compare the phosphoinositide composition of diseased and healthy tissues as well as related cell lines.

The in-house ELISA SARS-CoV-2 serological assay, developed by the Armauer Hansen Research Institute (AHRI) in Ethiopia, measures anti-SARS-CoV-2 receptor binding domain (RBD) antibodies. This study aimed to compare the performance of our cost-effective in-house ELISA with two established commercially available SARS-CoV-2 antibody detection assays during the pre-Omicron COVID-19 pandemic. In April 2021, serum samples were collected from 1441 students across 60 schools in Oromia, from 15 hotspot districts and towns. Socio-demographic data were gathered using CSentryCSProDataEntry7.2.1. Performance agreements between AHRI's in-house ELISA and the two commercial assays were analyzed in these serum samples. Statistical analyses, including Cohen's kappa (κ), overall percentage agreement, positive percent agreement (PPA), and negative percent agreement (NPA), were performed using STATA software. Diagnostic parameters were presented with 95% confidence intervals (CI), calculated using the Clopper-Pearson method. The performance comparison of the in-house ELISA showed substantial agreement with the two commercial assays. The overall concordance rate between in-house ELISA and Elecsys CLIA was 80.8% (95% CI 75.0-86.5), while the agreement between in-house ELISA and the Rapid LFA test (IgG + IgM) was 75.8% (95% CI 70.1-81.5). The kappa coefficients were: in-house ELISA vs. Elecsys CLIA (κ = 0.61, 95% CI 0.55-0.67), in-house ELISA vs. Rapid LFA test (IgG + IgM) (κ = 0.52, 95% CI 0.46-0.58), and Elecsys CLIA vs. Rapid LFA test (IgG + IgM) (κ = 0.73, 95% CI 0.67-0.78). The in-house ELISA demonstrated strong agreement with the Elecsys CLIA, showing a PPA of 81.7% and an NPA of 80.1%. Compared to the Rapid LFA test (IgG + IgM), which had a PPA of 83% and an NPA of 70.4%, the in-house ELISA exhibited better overall agreement with Elecsys CLIA. This study's findings indicate substantial agreement between the in-house ELISA and Elecsys. However, only modest agreement was observed between the in-house ELISA and the rapid test (IgG + IgM). Together, these results suggest the utility of the in-house ELISA as a cost-effective tool for sero surveillance studies and monitoring the effect of interventions in resource-poor settings.

The rapid evolution of SARS-CoV-2 has produced myriad viral strains with increasing transmissibility and capacity for immune evasion. While effective vaccination campaigns have reduced the fatalities associated with SARS-CoV-2, infections continue, and a detailed understanding of how this virus manipulates host biochemical pathways remains elusive. We asked both whether the patterns of host lipid rewiring remained consistent across variants and whether the changes in the abundance of lipid classes are related to changes in the expression of the enzymes involved in their biosynthesis. We compared global nontargeted lipidomics on A549-ACE2 cells infected with the delta variant (B.1.617.2), or the omicron (B.1.1.529) variant to our previous results of global nontargeted lipidomics on A549-ACE2 cells infected with the original WA1 strain and further performed quantitative proteomics to assess changes in the host proteome. We found that metabolic rewiring, both on the lipid and the enzymatic level, is remarkably consistent across all three variants. We further mapped changes in the expression of host metabolic enzymes, linking enzyme expression to alterations in the abundance of specific lipids during infection. This analysis identified key proteins related to virus-mediated changes in lipid abundance, including fatty acid synthase (FASN), lysosomal acid lipase (LIPA), and ORMDL, a regulator of sphingolipid biosynthesis. These integrated lipidomic and proteomic experiments shed light on the importance of the complex network of host metabolism networks that support SARS-CoV-2 infection and suggest that lipid metabolism may be a promising avenue for uncovering conserved therapeutic targets.