The function of the NHS gene that is responsible for the Nance-Horan Syndrome has remained elusive at the cellular level. Using CRISPR/Cas9, we inactivated the NHS gene in MCF10A cells and characterized the isoforms expressed in these cells. NHS KO cells displayed reduced migration persistence, a phenotype that was fully rescued by the long isoform 1 (i1) that contains a N-terminal WAVE Homology Domain (WHD), but only partially rescued by the short isoform 2 (i2), which does not. Patient mutations resulting in NHS proteins truncated at their C-terminus also reduced the ability of NHS i1 to rescue migration persistence. Using Tandem Affinity Purification (TAP) of NHS i1 and mass spectrometry, we identified as major NHS partners, all subunits of the WAVE Regulatory Complex (WRC) except WAVE subunits themselves, indicating that the WHD of NHS assembles a WAVE Shell Complex (WSC). The Arp2/3 complex and the Nucleation Promoting Factor (NPF) WAVE that activates it are critical for migration persistence. To investigate the role of the NHS-containing WSC, we performed TAP of the ABI1 subunit in parental and NHS KO cells and identified differential partners associated with ABI1 only in parental cells, but not in NHS KO cells. The most abundant of these were the WIPF2/N-WASP complex, which together with the kinase ABL2, was also critical for migration persistence. These results suggest that NHS controls cell migration by remodeling NPF complexes and their higher order assembly.

The detection and identification of unknown organic pollutants in complex environmental and forensic samples remain major analytical challenges. While numerous finely tuned liquid chromatography-mass spectrometry (LC-MS) methods exist for specific compound classes, no transversal strategy has been proposed to date. Here we propose an "all-in-one" LC-MS strategy that is robust enough to handle the unknown and broad enough to encompass multiple chemical families within a single analytical framework. Our approach combines a structured, literature-derived analysis of LC-based methods with experimental validation on model mixtures and real forensic samples. From a systematic review of hundreds of reported conditions, we established a structured database of key LC parameters for selected pesticides, household products, and dyes. This enabled the design of a cross-family LC-MS method validated on representative compounds and successfully applied to authentic casework, including suspicious delivery packages and household cleaning agents in forensic investigations. Rather than relying on novel instrumentation, this work provides the first literature-driven, experimentally validated workflow for broad-spectrum, nontargeted LC-MS detection. This strategy, widely applicable across environmental and forensic applications, is a valuable resource for harmonization, spectral library growth, and automated annotation. Moreover, the compiled reference database of chromatographic conditions serves as a unique tool for method development in the community.

Two of the most used hormones in sport, included in the WADA list of banned molecules, are human chorionic gonadotropin (hCG) and insulin-like growth factor (IGF-I). Male athletes may use pharmaceutical preparations of hCG to stimulate the production of testosterone before competition and IGF-I to stimulate muscle protein synthesis, promote glycogen storage, and improve lipolysis. Dry matrix microsampling and dried blood spots (DBS) are very promising strategies to improve current doping control activities, as they can lead to a significant improvement in compound stability, the simplification of sample collection and pre-analytic phases, the facilitation of their automation and a reduction of the risks of sample contamination and tampering. In this study, a protein extraction protocol from DBS, specifically hCG and IGF-I, was developed. Through the testing of various extraction solvents and subsequent analysis by Qubit fluorometry and nanoLC-MS/MS, we established an effective protein extraction procedure from DBS, specifically targeting hCG and IGF-I. The nanoLC-MS/MS bottom-up proteomic study reported a good protein extraction protocol from DBS, demonstrating good reproducibility and abundance related to the peptide sequences of hCG and IGF-I detected.

Auranofin (AF), a gold(I)-thiolate complex, exhibits diverse therapeutic potential, but its precise molecular targets within cells remain incompletely understood. This study aimed to develop and evaluate direct and indirect metallomics-based approaches for identifying AF protein targets. We first explored the stability of gold-protein adducts under standard digestion conditions and mass spectrometry analysis. We evaluated a Precursor Ion Scan-like strategy utilizing High-Collision Energy (HCE) fragmentation to monitor the diagnostic elemental gold ion at m/z 196.96. This direct approach, coupled to the classical data dependent acquisition, successfully identified in vitro AF target in complex protein samples. Direct LC-MS/MS analysis of AF-treated cells presented challenges due to low intracellular gold concentrations. Therefore, we implemented an indirect enrichment strategy, combining biotin labeling of gold-coordinated thiols with SILAC, to enhance detection sensitivity and reduce false positives. This indirect approach detected, in cellulo, known AF target while also revealed endoplasmic reticulum (ER) redox folding proteins as potential targets, consistent with AF-induced ER stress showed in our previous study. The study highlights the complexities of direct gold speciation in cells and contributes to a more comprehensive understanding of AF's multi-target mechanisms of action.

The development of ribosomal profiling (Riboseq) revealed the immense coding capacity of human and viral genomes. Here, we used Riboseq to delineate the translatome of HIV-1 in infected CD4+ T cells. In addition to canonical viral protein coding sequences (CDSs), we identify 98 alternative open reading frames (ARFs), corresponding to small Open Reading Frames (sORFs) that are distributed across the HIV genome including the UTR regions. Using a database of HIV genomes, we observe that most ARF amino-acid sequences are likely conserved among clade B and C of HIV-1, with 8 ARF-encoded amino-acid sequences being more conserved than the overlapping CDSs. Using T cell-based assays and mass spectrometry-based immunopeptidomics, we demonstrate that ARFs encode viral polypeptides. In the blood of people living with HIV, ARF-derived peptides elicit potent poly-functional T cell responses mediated by both CD4+ and CD8+ T cells. Our discovery expands the list of conserved viral polypeptides that are targets for vaccination strategies and might reveal the existence of viral microproteins or pseudogenes.

AbstractThe molecular mechanisms underlying cell migration remain incompletely understood. Here, we show that knock-out cells for NHSL3, the most recently identified member of the Nance-Horan Syndrome family, are more persistent than parental cells in single cell migration, but that, in wound healing, follower cells are impaired in their ability to follow leader cells. The NHSL3 locus encodes several isoforms. We identify the partner repertoire of each isoform using proteomics and predict direct partners and their binding sites using an AlphaFold2-based pipeline. Rescue with specific isoforms, and lack of rescue when relevant binding sites are mutated, establish that the interaction of a long isoform with MENA/VASP proteins is critical at cell-cell junctions for collective migration, while the interaction of a short one with 14-3-3θ in lamellipodia is critical for single cell migration. Taken together, these results demonstrate that NHSL3 regulates single and collective cell migration through distinct mechanisms.

Proteomics aims to achieve the large-scale characterization and quantification of proteins within complex biological systems. In mass spectrometry (MS)-based proteomics, the bottom-up approach, where proteins are proteolyzed into peptides, remains predominant due to the enhanced detectability of peptides. Conventional proteolysis relies on enzymatic or chemical reactions, whereas electrochemical proteolysis represents a promising, low-cost, eco-friendly, and rapid alternative. However, its implementation remains limited due to extensive protein adsorption on electrode surfaces, which is primarily governed by hydrophobic interactions that hinder electrochemical activity. In this work, we developed an electrochemical proteolysis platform employing carbon screen-printed electrodes (SPEs) directly coupled to MS detection. The main challenges addressed were (i) reducing protein adsorption on carbon surfaces to enhance proteolytic efficiency and (ii) enabling direct coupling of the electrolyzed sample to the MS without purification. To decrease surface hydrophobicity, the electrodes underwent electrochemical and plasma treatments, characterized by contact angle measurements (CAM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Electrochemically treated SPEs exhibited superior stability (30 days), an enhanced heterogeneous rate constant (k0 = (2.01 ± 0.15) × 10-4 cm2/s), and a larger electroactivity area (Ae = 0.47 ± 0.01 cm2). Reduced BSA adsorption confirmed the improved surface properties. Using this platform, insulin was successfully digested in a one-pot electrochemical process (-1.2 V, 30 min; +1.1 V, 20 min) and directly analyzed by MALDI-TOF MS. These findings demonstrate for the first time the feasibility of carbon SPE-based electrochemical proteolysis, enabling miniaturized, inexpensive, and chemical-free protein digestion compatible with MS-based bottom-up proteomics.

Overproduction of reactive oxygen species and antioxidant superoxide dismutases (SOD1, SOD2) dysregulation contribute to chronic inflammation such as generated in inflammatory bowel diseases (IBD). A kinetic redox shotgun proteomic strategy (OcSILAC for Oxidized cysteine Stable Isotope Labelling by Amino acids in Cell culture) was used to explore the lipopolysaccharide (LPS) effects including LPS-induced oxidation and inflammation cascades on a dedicated intestinal epithelial cell line (HT29-MD2) together with the potential mitigating role of a Mn-based SOD-mimic Mn1. While LPS induced transient oxidative damages at early times (15 min), cells incubated with Mn1 showed, in this time frame, a significantly reduced cysteine oxidation, highlighting Mn1 antioxidant properties. Over time, cysteine oxidation of LPS-treated cells was counteracted by an overexpression of antioxidant proteins (SOD1, NQO1) and a late (6 h) preponderant increase in SOD2 level. Mn1, when co-incubated with LPS, attenuated the level of most LPS-modified proteins, that is, proteins involved in the inflammatory response. Our results highlight Mn1 as a potentially effective antioxidant and anti-inflammatory agent to consider in the treatment of IBD, as well as a useful tool for exploring the interconnection between oxidative stress and inflammation.

Advances in metagenomics have led to the identification of new intestinal temperate bacteriophages. However, their experimental characterization remains challenging due to a limited understanding of their lysogenic-lytic cycle and the common lack of plaque formation in vitro. In this study, we investigated the hankyphage, a widespread transposable phage of prominent Bacteroides symbionts. Hankyphages spontaneously produced virions in laboratory conditions even in the absence of inducer, but virions did not show any evidence of infectivity. To increase virion production and raise the chances of observing infection events, we identified a master repressor of the hankyphage lytic cycle, RepCHP, whose silencing amplified hankyphage gene expression, and enhanced replicative transposition and virion production. However, attempts to infect or lysogenize new host cells with different capsular types remained unsuccessful. Transmission electron microscopy and capsid DNA sequencing revealed an abnormal virion morphology and incomplete DNA packaging of the hankyphage, suggesting that it cannot complete its assembly in laboratory conditions for reasons that are yet to be identified. Still, metavirome and phylogenetic analyses were suggestive of hankyphage horizontal transmission. We could also detect the activity of diversity-generating retroelements (DGRs) that mutagenize the hankyphage tail fiber, and likely contribute to its broad host range. This study sheds light on the life cycle of this abundant intestinal bacteriophage and highlights important gaps in our understanding of the factors required for the completion of its life cycle. Elucidating this puzzle will be critical to gain a better understanding of the hankyphage biology and ecological role.

The detection and identification of unknown organic pollutants in complex environmental and household samples remains a significant challenge. This review focuses on the LC-MS analysis of three major classes of compounds: pesticides, household products, and colorants, which are often poorly analyzed by Gas Chromatography (GC) and benefit significantly from Liquid Chromatography (LC) analysis. This review aims to provide valuable insights into the development of robust and efficient LC methods for the untargeted analysis of complex environmental and household samples. Initiating LC method development for the analysis of unknown compounds can be challenging, as LC parameters must be adapted to a wide range of compounds while ensuring good resolution of similar pollutants. By analyzing a vast amount of literature, we have identified and classified key LC parameters, including stationary phase selection, mobile phase composition, gradient elution profiles, and detection methods, that are crucial for robust wide-range LC analysis. To illustrate the practical application of this bibliographic classification, we have extracted a consensus LC-MS method designed to emphasize broad applicability for the compounds of interest. As a proof of concept, this method has been successfully applied to a diverse set of model compounds, demonstrating its versatility and effectiveness even within complex and "dirty" samples.

RNA interference (RNAi) mediated by the small interfering RNA (siRNA) pathway is a major antiviral mechanism in insects. This pathway is triggered when double-stranded RNA (dsRNA) produced during virus replication is recognized by Dicer-2, leading to the formation of virus-derived siRNA duplexes. These siRNAs are loaded onto the programmable nuclease Argonaute-2 (AGO2), with one strand serving as a guide to target and cleave fully complementary sequences of viral RNAs. While siRNAs are generated from viral dsRNA, the specific viral RNA species targeted for silencing during RNA virus replication remains unclear. In this study, we characterized the primary viral RNA targets of the Drosophila siRNA pathway during infections caused by negative and positive RNA viruses, namely Vesicular stomatitis virus (VSV) and Sindbis virus (SINV). Our findings reveal that polyadenylated transcripts of VSV and SINV are the major targets of silencing by the siRNA pathway during infection, likely when they are poised for translation. Consistent with earlier findings, we show that AGO2 is associated with ribosomes in control and virus infected cells. Therefore, we propose that the inhibition of the replication of RNA viruses in Drosophila results from the silencing of incoming viral transcripts, facilitated by the association of AGO2 with ribosomes.

The small interfering RNA pathway is the primary antiviral defense mechanism in invertebrates and plants. This systemic mechanism relies on the recognition, transport, and internalization of double-stranded RNA (dsRNA). Our aim was to identify cell surface proteins that bind extracellular dsRNA and mediate its internalization in Drosophila cells. We used coimmunoprecipitation coupled with proteomics analysis and found that silencing heat shock cognate protein 70-4 (Hsc70-4), a constitutively expressed heat shock protein, impairs dsRNA internalization. Unexpectedly, despite lacking a predicted transmembrane domain, Hsc70-4 localizes to the cell membrane via lipid interactions. Antibody blocking experiments revealed an extracellular domain on Hsc70-4 that is essential for dsRNA internalization. Intriguingly, this dsRNA-specific binding capacity of Hsc70-4 functions independently of its chaperone activity. These findings not only highlight Hsc70-4 as a previously uncharacterized and essential component in the dsRNA internalization process but also offer promising insights for advancing RNA interference-based technologies to combat pests and vector-borne diseases.

NEuroBioStand is a European consortium funded within the EPM Health call 2022, aimed at developing a metrological research framework for standardizing biomarkers of neurodegenerative diseases (NDDs), ensuring equivalent results among different measurement procedures, and facilitating the establishment of common reference intervals and cut-off values. In clinical chemistry, the highest level of standardization for end-user measurement procedures is achieved through the development of reference materials (RMs) and/or reference measurement procedures (RMPs) to establish a calibration hierarchy to the International System of Units (SI). This work is accomplished through tight collaboration among NMIs/Dis, academics, clinicians, and industry, under the umbrella of the IFCC WG-BND. NEuroBioStand is structured into three technical work packages (WPs): WP1) P-tau measurements in plasma: Recent advances in ultrasensitive assays have enabled the measurement of phosphorylated tau (P-tau) in plasma, which has become a promising biomarker for Alzheimer's disease (AD). The first step in standardising this biomarker is defining the measurand, focusing on specific P-tau isoforms, including P-tau181, P-tau217, and P-tau231. The consortium is developing an isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference measurement procedure (RMP). Traceability to SI units is achieved through the production of fully characterized protein and/or peptide phosphorylated primary calibrators. In particular, the full characterisation of the phosphorylation status of the protein material has been carried out to determine the site occupancy of the most relevant phosphorylation sites by using the SI-traceable phosphorylated peptide materials. The candidate RMP will certify P-tau concentration in pilot matrix-based reference materials. Discussions about potential formats of these materials include plasma pools, pools spiked with recombinant protein, and pools spiked with cerebrospinal fluid (CSF), to evaluate for commutability. WP2) Standardization of neurofilament light chain (NfL): Neurofilament light chain (NfL) has been established as a robust biomarker for neuroaxonal damage in various neurological conditions. The consortium has defined the target analyte in the matrix, and a reference measurement procedure by isotope dilution mass spectrometry (IDMS) is under development for the quantification of neurofilament with a target uncertainty below 15%. A clinical LC-MS method using immunoprecipitation is being optimised for sensitivity, using highly specific antibodies. Once optimised, serum samples from patients will be analysed, comparing different cohorts and immunoassays. WP3) Emerging biomarkers and novel approaches for protein structural conformation determination: The consortium has selected GFAP as an emerging biomarker candidate to promote its implementation in clinical practice by developing an LC-MS method to evaluate the feasibility for SI-traceable quantification in biological fluids. NEuroBioStand is also exploring the use of chemical cross-linking and native mass spectrometry to understand protein conformational states relevant to neurodegenerative diseases. Studies on tau and phosphorylated tau samples using native and intact LC-MS analysis are under development. Crosslinking-MS revealed significant structural differences between Tau and phosphorylated Tau, with a new method developed for pair-wise network and cluster analysis of crosslinks. The long-term goal of this project is to have robust and accurate blood tests capable of providing reliable values for the diagnosis, prognosis, and care of patients affected by NDDs.

Combining different "omics" approaches, such as genomics and proteomics, is necessary to generate a detailed and complete insight into microbiome comprehension. Proper sample collection and processing and accurate analytical methods are crucial in generating reliable data. We previously developed the ChipFilter device for proteomic analysis of microbial samples. We have shown that this device coupled to LC-MS/MS can successfully be used to identify microbial proteins. In the present work, we have developed our workflow to analyze concomitantly proteins and nucleic acids from the same sample. We performed lysis and proteolysis in the device using cultures of E. coli, B. subtilis, and S. cerevisiae. After peptide recovery for LC-MS/MS analysis, DNA from the same samples was recovered and successfully amplified by PCR for the 3 species. This workflow was further extended to a complex microbial mixture of known compositions. Protein analysis was carried out, enabling the identification of more than 5000 proteins. The recovered DNA was sequenced, performing comparable to DNA extracted with a commercial kit without proteolysis. Our results show that the ChipFilter device is suited to prepare samples for parallel proteomic and genomic analyses, which is particularly relevant in the case of low-abundant samples and drastically reduces sampling bias.

Background: Multiple myeloma (MM) produces monoclonal immunoglobulins (Igs) and free light chains (LCs), which serve as biomarkers for treatment response and relapse. Effective therapies can drastically reduce circulating monoclonal immunoglobulin (Ig) levels, rendering them undetectable using conventional methods. This lack of sensitivity in assessing minimal residual disease (MRD) is critical, as MRD positivity correlates strongly with poorer progression-free survival. Currently, MRD relies on bone marrow biopsy, which is an invasive procedure. Although informative, these blind biopsies can miss focal disease areas, leading to discrepancies in the assessment of low-burden MM. There is a need for sensitive, specific, and noninvasive methods to accurately measure and monitor MRD. We addressed this gap by developing two complementary mass spectrometry (MS) approaches targeting patient-specific LCs and specific sample and data processing to minimize the limitations of tryptic clonotypic peptide analysis and the nucleotide sequencing step. Methods: Our pilot study involved 10 serum samples collected from patients with MM at diagnosis and various treatment points in Parisian hospitals (Pitié-Salpêtrière & Saint-Antoine). We used a two-step approach : Intact LC enrichment Specific nanobeads were used to enrich intact LCs, enabling accurate detection using a “middle-down” strategy.Clonotypic peptide identification: The enriched LCs were enzymatically digested and the resulting clonotypic peptides were identified using a “bottom-up” strategy. This approach enhances the detection limits, especially for low-abundance peptides within monoclonal proteins. De novo sequencing complemented protein database searches to identify patient-specific clonotypic peptides. The selection criteria were: Overlap at least partly with a hypervariable (CDR) region and conserved “constant” region in the IGBMT protein database.A BLAST search of the human protein library revealed no significant homology with other human proteins.Demonstrably decreased peptide signal intensity between diagnostic and post-treatment blood samples.The de novo software identification score exceeded 70%. Results: In both approaches, an intact specific monoclonal LC was observed when it was no longer detectable using conventional methods. The accuracy of mass measurements increases the specificity of detection. Our study showed that tryptic clonotypic peptide monitoring is not a universal strategy for accurate and sensitive MRD follow-up. However, an efficient sequencing process makes the characterization of peptides of interest even more robust and sensitive. One patient was monitored over time from the initial diagnosis through the treatment phase until considered negative and after relapse. In all monitored serum samples, intact LC was detected at 22,604 Da, which was consistent with the clonal nucleotide sequence obtained by RACERepSeq. However, among the 11 theoretical tryptic peptides generated from this 106 amino acid sequence, most are not suited for the bottom-up strategy (mass above 4000 Da), with at most two clonotypic candidates after total proteolysis. None could be used for CDR1, one peptide could cover CDR2 at 1557.8 Da, and one could cover only one-third of CDR3 at 878.5 Da. Although well detected in the initial diseased state and after relapse, neither of the two candidates could be identified at the end of the treatment and before relapse, resulting in an unambiguous false-negative result. Changing the proteolysis experimental conditions allowed us to cover CDR1, CDR2, and CDR3 more efficiently with at least four clonotypic peptide candidates. Consequently, at least 3 sequences covering CDR2 (3145.7 Da and 2982.6 Da) and CDR3 (2253.2 Da) were identified in all serum samples. Conclusions: Our optimized toolbox aimed to significantly improve the sensitivity and specificity of MRD detection in patients with MM. Utilizing blood samples allows for accurate, frequent, highly sensitive, and specific MRD monitoring. Personalized clonotypic profiling is crucial for a robust analysis. This approach can reduce the risks associated with undetected disease persistence and enable the earlier detection of relapse. Ongoing studies are comparing this method with bone marrow assessment. We anticipate that further technological advances will also increase the analytical throughput.

Coxsackievirus B (CVB) infection of pancreatic β cells is associated with β cell autoimmunity and type 1 diabetes. We investigated how CVB affects human β cells and anti-CVB T cell responses. β cells were efficiently infected by CVB in vitro, down-regulated human leukocyte antigen (HLA) class I, and presented few, selected HLA-bound viral peptides. Circulating CD8 + T cells from CVB–seropositive individuals recognized a fraction of these peptides; only another subfraction was targeted by effector/memory T cells that expressed exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with β cell antigen GAD. Infected β cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Our in vitro and ex vivo data highlight limited CD8 + T cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8 + T cells recognizing structural and nonstructural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

The metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as the detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity of extracting proteins from heterogeneous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup can be successfully used for the identification of microbial proteins. Using cultures of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow the identification of a higher number of proteins and peptides than that by standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amounts of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, particularly in the case of a low volume or cell density. The mass spectrometry data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD039581.

The siRNA pathway is the primary antiviral defense mechanism in invertebrates and plants. The systemic nature of this defense mechanism is one of its more fascinating characteristics and the recognition and transport of double-stranded RNA (dsRNA) of viral origin is required for the systemic activity of the siRNA pathway. Indeed, cellular internalization of dsRNA from the environment is a widespread phenomenon among insects. Here we aimed to identify cell surface proteins that bind to extracellular dsRNA and mediate its internalization. To this end, we developed a novel co-immunoprecipitation protocol that we followed with proteomics analysis. Among the hits from our screens was Hsc70-4, a constitutively expressed member of the heat shock protein family that has been implicated in clathrin-mediated endocytosis. We found that silencing Hsc70-4 impaired dsRNA internalization. Surprisingly, despite lacking a predicted transmembrane domain, Hsc70-4 localizes to the cell membrane and this localization was preserved when Hsc70-4 was expressed in mammalian cells, suggesting a conserved role at the cell surface. Furthermore, Hsc70-4 shows a previously undescribed dsRNA-specific binding capacity. Our results show that Hsc70-4 is a key element of the dsRNA internalization process and its detailed study may facilitate the development of RNA interference (RNAi)-based technologies for pest and vector borne disease control. Importance To protect plants from pathogens or pests, the technology of “Host-induced gene silencing” has emerged as a powerful alternative to chemical treatments. This is an RNAi-based technology where small RNAs made in the plant silence the genes of the pests or pathogens that attack the plant. The small RNAs are generally derived from dsRNA expressed in transgenic plants. Alternatively, dsRNA can be sprayed onto the plant surface, where it can be taken up into the plant or ingested by pests. We have identified a cell surface protein that mediates the early steps of extracellular dsRNA internalization in insect cells. This could facilitate the development of new strategies for pest management.