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This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced with consistent purity and potency in less than 24 hours. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstratedin vitroagainst SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressedin vivo. The proposed production process is efficient and can be readily scaled up and deployed anywhere in the world where a viral pathogen might emerge. The current emergence of viral variants has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

Wild-type P53-induced phosphatase 1 (WIP1), also known as

Quantitative high-throughput screening (qHTS) pharmacologically evaluates chemical libraries for therapeutic uses, toxicological risk and, increasingly, for academic probe discovery. Phenotypic high-throughput screening assays interrogate molecular pathways, often relying on cell culture systems, historically less focused on multicellular organisms. Caenorhabditis elegans has served as a eukaryotic model organism for human biology by virtue of genetic conservation and experimental tractability. Here, a paradigm enabling C. elegans qHTS using 384-well microtiter plate laser-scanning cytometry is described, in which GFP-expressing organisms revealing phenotype-modifying structure–activity relationships guide subsequent life-stage and proteomic analyses, and Escherichia coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over two life cycles, mitigating bacterial overgrowth complications. We demonstrate the method with libraries of anti-infective agents, or substances of toxicological concern. Each was tested in seven-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations with a physiological impact. We anticipate that this qHTS approach will enable analysis of C. elegans orthologous phenotypes of human pathologies to facilitate drug library profiling for a range of therapeutic indications.

XBP1 variant 1 (Xv1) is the most abundant XBP1 variant and is highly enriched across cancer types but nearly none in normal tissues. Its expression is associated with poor patients’ survival and is specifically required for survival of malignant cells, but the underlying mechanism is not known. Here we report that Xv1 upregulates the polyglutamylase tubulin tyrosine ligase-like 6 (TTLL6) and promotes mitosis of cancer cells. Like the canonical XBP1, Xv1 mRNA undergoes unconventional splicing by IRE1α under endoplasmic reticulum stress, but it is also constitutively spliced by IRE1β. The spliced Xv1 mRNA encodes the active form of Xv1 protein (Xv1s). RNA sequencing in HeLa cells revealed that Xv1s overexpression regulates expression of genes that are not involved in the canonical unfolded protein response, including TTLL6 as a highly upregulated gene. Gel shift assay and chromatin immunoprecipitation revealed that Xv1s bind to the TTLL6 promoter region. Knockdown of TTLL6 caused death of cancer cells but not benign and normal cells, similar to the effects of knocking down Xv1. Moreover, overexpression of TTLL6 partially rescued BT474 cells from apoptosis induced by either TTLL6 or Xv1 knockdown, supporting TTLL6 as an essential downstream effector of Xv1 in regulating cancer cell survival. TTLL6 is localized in the mitotic spindle of cancer cells. Xv1 or TTLL6 knockdown resulted in decreased spindle polyglutamylation and interpolar spindle, as well as congression failure, mitotic arrest and cell death. These findings suggest that Xv1 is essential for cancer cell mitosis, which is mediated, at least in part, by increasing TTLL6 expression.

RNF5 has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections. This study developed a small molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.

Quantitative high-throughput screening (qHTS) evaluates the pharmacology of drug and investigational agent libraries for potential therapeutic uses, toxicological risk assessment, and increasingly for academic chemical tool discovery. Phenotypic HTS assays aim to interrogate molecular pathways and networks, often relying on cell culture systems, historically with less emphasis on multicellular organisms. C. elegans has served as a powerful eukaryotic model organism for human biology and disease by virtue of genetic conservation and experimental tractability. Here we describe a paradigm to enable C. elegans in qHTS using 384-well microtiter plate laser scanning cytometry. GFP-expressing organisms are used to reveal phenotype-modifying structure-activity relationships to guide subsequent life stages and proteomic analysis. E. coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over 7-days spanning two life cycles to mitigate complications from bacterial overgrowth. We demonstrate the method with a library composed of anti-infective agents, or molecules of general toxicological concern. Each was tested in 7-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations having a physiological impact. We anticipate a broader application of this qHTS-coupled proteomics approach will enable the analysis of C. elegans orthologous transgenic phenotypes of human pathologies to facilitate drug and probe profiling from high-impact chemical libraries for a range of therapeutic indications and study of potential toxicological signatures.Graphic Abstract

Major advances have been made to improve the sensitivity of mass analyzers, spectral quality, and speed of data processing enabling more comprehensive proteome discovery and quantitation. While focus has recently begun shifting toward robust proteomics sample preparation efforts, a high-throughput proteomics sample preparation is still lacking. We report the development of a highly automated universal 384-well plate sample preparation platform with high reproducibility and adaptability for extraction of proteins from cells within a culture plate. Digestion efficiency was excellent in comparison to a commercial digest peptide standard with minimal sample loss while improving sample preparation throughput by 20- to 40-fold (the entire process from plated cells to clean peptides is complete in ~300 min). Analysis of six human cell types, including two primary cell samples, identified and quantified ~4000 proteins for each sample in a single high-performance liquid chromatography (HPLC)–tandem mass spectrometry injection with only 100 – 10,000 cells, thus demonstrating universality of the platform. The selected protein was further quantified using a developed HPLC-multiple reaction monitoring method for HeLa digests with a heavy labeled internal standard peptide spiked in. Excellent linearity was achieved across different cell numbers indicating a potential for target protein quantitation in clinical research.

Clinical translation of human pluripotent stem cells (hPSCs) requires advanced strategies that ensure safe and robust long-term growth and functional differentiation. Pluripotent cells are capable of extensive self-renewal, yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. Here, we deployed innovative high-throughput screening strategies to identify a small molecule cocktail that dramatically improves viability of hPSCs and their differentiated progeny. The combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique polypharmacology strategy for comprehensive cytoprotection, providing a new rationale for efficient and safe utilization of hPSCs. Conferring cell fitness by multi-target drug combinations may become a common approach in cryobiology, drug development, and regenerative medicine.

Classic galactosemia is a rare disease caused by inherited deficiency of galactose-1 phosphate uridylyltransferase (GALT). Accumulation of galactose-1 phosphate (gal-1P) is thought to be the major cause of the chronic complications associated with this disease, which currently has no treatment. Inhibiting galactokinase (GALK1), the enzyme that generates galactose-1 phosphate, has been proposed as a novel strategy for treating classic galactosemia. Our previous work identified a highly selective unique dihydropyrimidine inhibitor against GALK1. With the determination of a co-crystal structure of this inhibitor with human GALK1, we initiated a structure-based structure–activity relationship (SAR) optimization campaign that yielded novel analogs with potent biochemical inhibition (IC(50) < 100 nM). Lead compounds were also able to prevent gal-1P accumulation in patient-derived cells at low micromolar concentrations and have pharmacokinetic properties suitable for evaluation in rodent models of galactosemia.

Wildtype P53-induced phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, inorganic phosphate. We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

Creatine transporter deficiency (CTD) is caused by a defect in the X-linked creatine transporter SLC6A8 gene leading to severe neurologic and physiologic conditions. Cyclocreatine and phosphocyclocreatine supplementation is seen as a potential treatment, but the presence of these compounds within commercially available dietary supplements presents the risk of self-medication. High-performance liquid chromatography-mass spectrometry (HPLC-MS) is an excellent technique to assess composition of complex amino acid mixtures. Herein, we have developed a facile HPLC-MS method using a cyano column in hydrophilic interaction liquid chromatography (HILIC) mode with isocratic elution over 4 min to identify the main components of two commercially available dietary supplements. The relative standard deviation (RSD) for retention time and extracted ion integrated area are

Objective A fundamental understanding of redox homeostasis in Anopheles gambiae midgut cells under different oxidative conditions is missing. Such knowledge can aid in the development of new malaria transmission-blocking strategies aimed at disrupting natural homeostatic processes in the mosquito during Plasmodium parasite uptake (i.e. blood feeding). The aim of this study was to understand how the An. gambiae midgut regulates oxidative stress to reactive oxygen species (ROS), especially to a potent ROS-inducer such as tert-Butyl hydroperoxide (tBHP). Results Initial studies using quantitative immunoblot indicated that the expression of the classical antioxidant protein An. gambiae thioredoxin-1 (AgTrx-1) remained unchanged across challenges with different concentrations of tBHP suggesting that additional mechanisms to regulate ROS may be involved. We therefore conducted a global proteomic survey, which revealed that An. gambiae midguts under low (50 μM) and high (200 μM) tBHP concentrations were enriched in proteins indicative of ribosomal/nucleolar stress. Ribosomal stress is an inherent cellular response to an imbalance in ribosomal proteins (RPs) due to cellular stress such as oxidative stress. Our data suggest that ribosomal/nucleolar stress is the primary cellular response in An. gambiae midguts under tBHP challenge. Considering these results, we discuss harnessing the ribosomal stress response as a potential malaria transmission-blocking strategy. Electronic supplementary material The online version of this article (10.1186/s13104-019-4196-1) contains supplementary material, which is available to authorized users.

Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

Chemical-peptide conjugation is the molecular initiating event in skin sensitization. The OECD test guideline uses a high-performance liquid chromatography/ultraviolet (HPLC/UV) detection method to quantify chemical-peptide conjugation in a direct peptide reactivity assay (DPRA), which measures the depletion of two synthetic peptides containing lysine or cysteine residues. To improve assay throughput, sensitivity and accuracy, an automated 384-well plate-based RapidFire solid-phase extraction (SPE) system coupled with tandem mass spectrometry (MS/MS) DPRA was developed and validated in the presence of a newly designed internal standard. Compared to the HPLC/UV-based DPRA, the automated SPE-MS/MS-based DPRA improved throughput from 16 min to 10 s per sample, and substrate peptides usage was reduced from 100 mM to 5 μM. When implementing the SPE-MS/MS-based DPRA into a high-throughput platform, we found 10 compounds that depleted lysine peptide and 24 compounds that depleted cysteine peptide (including 7 unreported chemicals from 55 compounds we tested) in a concentration-response manner. The adduct formation between cysteine and cinnamic aldehyde and ethylene glycol dimethacrylate were further analyzed using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) to confirm the conjugation. Overall, the automated SPE-MS/MS-based platform is an efficient, economic, and accurate way to detect skin sensitizers.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug–target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.

Proteins are widely used as drug targets, enzyme substrates, and biomarkers for numerous diseases. The emerging demand for proteins quantitation has been increasing in multiple fields. Currently, there is still a big gap for high-throughput protein quantitation at intact protein level using label-free method. Here we choose ribonuclease B (RNB) as a model, which is the substrate for human endo-β-N-acetylglucosaminidase (hENGase), a promising drug target for the treatment of N-Glycanase deficiency. Intact proteinlevel multiple reaction monitoring (MRM) methods were initally developed and optimized to quantify RNB and deglycosylated RNB (RNB-deg), with the S/N ratio improved by nearly 20-fold compared to the traditional full MS scan methods. To further increase the throughput making it possible for hENGase inhibitors screen, the protein MRM methods were introduced to the RapidFire-MS/MS system, achieving at least 12-fold throughput improvement. This assay was further optimized into 384-well plate format for compound screening with S/B ratio >37-fold and Z’ factor >0.7 that is suitable for high-throughput screening of compound collections with a speed of 2 h per 384-well plate and an ability to screen over 3000 compounds per day at a single concentration dose. This 384-well plate based automated SPE-MS/MS assay is efficient and robust for compound screening and the assay format has a wide applicability to protein targets for other disease models.

Clinical translation of human pluripotent stem cells (hPSCs) requires advanced strategies that ensure safe and robust long-term growth and functional differentiation. Pluripotent cells are capable of extensive self-renewal, yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. Here, we deployed innovative high-throughput screening strategies to identify a small molecule cocktail that dramatically improves viability of hPSCs and their differentiated progeny. We discovered that the combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. In proof-of-principle experiments we then demonstrated the strong improvements that CEPT provided for several key applications in stem cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body and organoid formation, single-cell cloning, genome editing, and new iPSC line generation. Thus, CEPT represents a unique polypharmacology strategy for comprehensive cytoprotection, providing a new rationale for efficient and safe utilization of hPSCs. Conferring cell fitness by multi-target drug combinations may become a common approach in cryobiology, drug development, and regenerative medicine.

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3-5% and early development of P. falciparum exoerythrocytic forms-a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.

Nearly one third of the encoded proteome is comprised of secretory proteins that enable communication between cells and organ systems, playing a ubiquitous role in human health and disease. High-throughput detection of secreted proteins would enhance efforts to identify therapies for secretion-related diseases. Using the Z mutant of alpha-1 antitrypsin as a human secretory model, we have developed 1536-well high-throughput screening assays that utilize acoustic droplet ejection to transfer nanoliter volumes of sample for protein quantification. Among them, the acoustic reverse phase protein array (acoustic RPPA) is a multiplexable, low-cost immunodetection technology for native, endogenously secreted proteins from physiologically relevant model systems like stem cells that is compatible with plate-based instrumentation. Parallel assay profiling with the LOPAC(1280) chemical library validated performance and orthogonality between a secreted bioluminescent reporter and acoustic RPPA method by consistently identifying secretory modulators with comparable concentration response relationships. Here, we introduce a robust, multiplexed drug discovery platform coupling extracellular protein quantification by acoustic RPPA with intracellular and cytotoxicity analyses from single wells, demonstrating proof-of-principle applications for human induced pluripotent stem cell-derived hepatocytes.

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy. Plasmodium falciparum gametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples.

The recent decline in global malaria burden has stimulated efforts toward Plasmodium falciparum elimination. Understanding the biology of malaria transmission stages may provide opportunities to reduce or prevent onward transmission to mosquitoes. Immature P. falciparum transmission stages, termed stages I to IV gametocytes, sequester in human bone marrow before release into the circulation as mature stage V gametocytes. This process likely involves interactions between host receptors and potentially immunogenic adhesins on the infected red blood cell (iRBC) surface. Here, we developed a flow cytometry assay to examine immune recognition of live gametocytes of different developmental stages by naturally exposed Malawians. We identified strong antibody recognition of the earliest immature gametocyte-iRBCs (giRBCs) but not mature stage V giRBCs. Candidate surface antigens (n = 30), most of them shared between asexual- and gametocyte-iRBCs, were identified by mass spectrometry and mouse immunizations, as well as correlations between responses by protein microarray and flow cytometry. Naturally acquired responses to a subset of candidate antigens were associated with reduced asexual and gametocyte density, and plasma samples from malaria-infected individuals were able to induce immune clearance of giRBCs in vitro. Infected RBC surface expression of select candidate antigens was validated using specific antibodies, and genetic analysis revealed a subset with minimal variation across strains. Our data demonstrate that humoral immune responses to immature giRBCs and shared iRBC antigens are naturally acquired after malaria exposure. These humoral immune responses may have consequences for malaria transmission potential by clearing developing gametocytes, which could be leveraged for malaria intervention.

Additional file 7. Proteomic data on enriched (overexpressed) proteins with annotated functions in cellular trafficking and lysosomal exocytosis.

Paraquat is a potent superoxide (O2&minus, )-inducing agent that is capable of inducing an oxidative imbalance in the mosquito midgut. This oxidative imbalance can super-stress the malaria parasite, leading to arrested development in the mosquito midgut and reduced transmission. While several studies have explored the effect of paraquat on malaria parasites, a fundamental understanding of the mosquito response to this compound remains unknown. Here, we quantified the mosquito midgut proteomic response to a paraquat-laced sugar meal, and found that An. gambiae midguts were enriched in proteins that are indicative of cells under endoplasmic reticulum (ER) stress. We also carried out qRT-PCR analyses for nine prominent thioredoxin (Trx) and glutathione (GSH)-dependent genes in mosquito midguts post P. falciparum blood meal ingestion to evaluate the concordance between transcripts and proteins under different oxidative stress conditions. Our data revealed an absence of significant upregulation in the Trx and GSH-dependent genes following infected blood meal ingestion. These data suggest that the intrinsic tolerance of the mosquito midgut to paraquat-mediated oxidative stress is through an ER stress response. These data indicate that mosquitoes have at least two divergent pathways of managing the oxidative stress that is induced by exogenous compounds, and outline the potential application of paraquat-like drugs to act selectively against malaria parasite development in mosquito midguts, thereby blocking mosquito-to-human transmission.

Parthenin and parthenolide are natural products that are closely related in structure to artemisinin, which is also a sesquiterpene lactone (SQL) and one of the most important antimalarial drugs available. Parthenin, like artemisinin, has an effect on Plasmodium blood stage development. We extended the evaluation of parthenin as a potential therapeutic for the transmissible stages of Plasmodium falciparum as it transitions between human and mosquito, with the aim of gaining potential mechanistic insight into the inhibitory activity of this compound. We posited that if parthenin targets different biological pathways in the parasite, this in turn could pave the way for the development of druggable compounds that could prevent the spread of artemisinin-resistant parasites. We examined parthenin's effect on male gamete activation and the ookinete-to-oocyst transition in the mosquito as well as on stage V gametocytes that are present in peripheral blood. Parthenin arrested parasite development for each of the stages tested. The broad inhibitory properties of parthenin on the evaluated parasite stages may suggest different mechanisms of action between parthenin and artemisinin. Parthenin's cytotoxicity notwithstanding, its demonstrated activity in this study suggests that structurally related SQLs with a better safety profile deserve further exploration. We used our battery of assays to test parthenolide, which has a more compelling safety profile. Parthenolide demonstrated activity nearly identical to that of parthenin against P. falciparum , highlighting its potential as a possible transmission-blocking drug scaffold. We discuss the context of the evidence with respect to the next steps toward expanding the current antimalarial arsenal.

The recent decline in global malaria burden has stimulated efforts toward Plasmodium falciparum elimination. Understanding the biology of malaria transmission stages may provide opportunities to reduce or prevent onward transmission to mosquitoes. Immature P. falciparum transmission stages, termed stages I to IV gametocytes, sequester in human bone marrow before release into the circulation as mature stage V gametocytes. This process likely involves interactions between host receptors and potentially immunogenic adhesins on the infected red blood cell (iRBC) surface. Here, we developed a flow cytometry assay to examine immune recognition of live gametocytes of different developmental stages by naturally exposed Malawians. We identified strong antibody recognition of the earliest immature gametocyte-iRBCs (giRBCs) but not mature stage V giRBCs. Candidate surface antigens (n = 30), most of them shared between asexual- and gametocyte-iRBCs, were identified by mass spectrometry and mouse immunizations, as well as correlations between responses by protein microarray and flow cytometry. Naturally acquired responses to a subset of candidate antigens were associated with reduced asexual and gametocyte density, and plasma samples from malaria-infected individuals were able to induce immune clearance of giRBCs in vitro. Infected RBC surface expression of select candidate antigens was validated using specific antibodies, and genetic analysis revealed a subset with minimal variation across strains. Our data demonstrate that humoral immune responses to immature giRBCs and shared iRBC antigens are naturally acquired after malaria exposure. These humoral immune responses may have consequences for malaria transmission potential by clearing developing gametocytes, which could be leveraged for malaria intervention.

Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however, AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission have remained elusive. Here we present the 2.65-Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profiles of three monoclonal antibodies (mAbs) to AnAPN1, including mAb 4H5B7, which effectively blocks transmission of natural strains of Plasmodium falciparum. Using the AnAPN1 structure, we map the conformation-dependent 4H5B7 neoepitope to a previously uncharacterized region on domain 1 and further demonstrate that nonhuman-primate neoepitope-specific IgG also blocks parasite transmission. We discuss the prospect of a new biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV.

Kupffer cells (KCs) are the first line of defense in the liver against pathogens, yet several microbes successfully target the liver, bypass immune surveillance, and effectively develop in this tissue. Our current, albeit poor, understanding of KC-pathogen interactions has been largely achieved through the study of primary cells, requiring isolation from large numbers of animals. To facilitate the study of KC biology, an immortalized rat KC line 1, RKC1, was developed. We performed a comparative global proteomic analysis of RKC1 and primary rat KCs (PRKC) to characterize their respective responses to lipopolysaccharide-mediated immune stimulation. We identified patent differences in the proteomic response profile of RKC1 and PRKC to lipopolysaccharide. We observed that PRKC upregulated more immune function pathways and exhibited marked changes in cellular morphology following stimulation. We consequently analyzed the cytoskeletal signaling pathways of these cells in light of the fact that macrophages are known to induce cytoskeletal changes in response to pathogens. Our findings suggest that KCs respond differently to inflammatory stimulus than do monocyte-derived macrophages, and such data may provide insight into how pathogens, such as the malaria parasite, may have evolved mechanisms of liver entry through KCs without detection.

Background Malaria parasites are transmitted by Anopheles mosquitoes. Although several studies have identified mosquito midgut surface proteins that are putatively important for Plasmodium ookinete invasion, only a few have characterized these protein targets and demonstrated transmission-blocking activity. Molecular information about these proteins is essential for the development of transmission-blocking vaccines (TBV). The aim of the present study was to test three monoclonal antibodies (mAbs), A-140, A-78 and A-10, for their ability to recognize antigens and block oocyst infection of the midgut of Anopheles albimanus, a major malaria vector in Latin America. Method Western-blot of mAbs on antigens from midgut brush border membrane vesicles was used to select antibodies. Three mAbs were tested by membrane feeding assays to evaluate their potential transmission-blocking activity against Plasmodium berghei. The cognate antigens recognized by mAbs with oocyst-reducing activity were determined by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Results Only one mAb, A-140, significantly reduced oocyst infection intensity. Hence, its probable protein target in the Anopheles albimanus midgut was identified and characterized. It recognized three high-molecular mass proteins from a midgut brush border microvilli vesicle preparation. Chemical deglycosylation assays confirmed the peptide nature of the epitope recognized by mAb A-140. Immunoprecipitation followed by proteomic identification with tandem mass spectrometry revealed five proteins, presumably extracted together as a complex. Of these, AALB007909 had the highest mascot score and corresponds to a protein with a myosin head motor domain, indicating that the target of mAb A-140 is probably myosin located on the microvilli of the mosquito midgut. Conclusion These results provide support for the participation of myosin in mosquito midgut invasion by Plasmodium ookinetes. The potential inclusion of this protein in the design of new multivalent vaccine strategies for blocking Plasmodium transmission is discussed. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1548-8) contains supplementary material, which is available to authorized users.

Statistical analysis GLMM (data corresponding to Fig. 1d and Table 1). (DOCX 39 kb)

Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.

An integrated multidimensional nano-flow liquid chromatography platform with the combination of protein and peptide separation via online digestion by an immobilized enzymatic reactor was established, and successfully applied for proteome analysis. By this platform, proteins were first separated by a weak anion and weak cation mixed-bed microcolumn under a series of salt steps, online digested by a trypsin immobilized enzymatic reactor, digests trapped and desalted by a C18 precolumn, separated by nano-reversed phase liquid chromatography, and finally identified by electrospray ionization-MS/MS. To evaluate the performance of such a platform, Escherichia coli whole cell lysate proteins were analyzed. Compared with the results obtained by shotgun approach, the identified protein number was increased by 6%, with the consumed time decreased from 38 to 14 h. We also compared with integrate platform based on micro-HPLC, and the required sample amount was decreased to 8 mu g. These results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.

A novel kind of hydrophilic monolith based immobilized enzyme reactors (IMERs) was prepared both in UV-transparent capillaries and on glass microchips by the photopolymerization of N-acryloxysuccinimide and poly(ethylene glycol)diacrylate, followed by trypsin immobilization. The performance of capillary IMERs for protein digestion was evaluated by the digestion of myoglobin with the residential time from 12 s to 71 s. With μRPLC–ESI-MS/MS analysis, the obtained sequence coverages were all over 80%, comparable to that obtained by in-solution digestion for 12 h. The nonspecific absorption of BSA on monolithic support was evaluated, and no obvious protein residue was observed by a fluorescence assay. Moreover, no carry-over of the digests on the capillary IMER was found after the digestion of myoglobin (24 μg) and BSA (9 μg), which further demonstrated the good hydrophilicity of such matrix. In addition, an integrated microchip-based system involving on-line protein digestion by microchip-based IMER, peptides separation by nanoRPLC and identification by ESI-MS/MS was established, by which a mixture of standard proteins and one RPLC fraction of Escherichia coli extract were successfully identified, indicating that the hydrophilic monolith based IMER might provide a promising tool for high-throughput proteomic analysis.

Two dimensional high performance liquid chromatography-electrospray ionization tandem mass spectrometry (2D-HPLC-ESI-MS/MS) is one of the most powerful techniques for high resolution, efficiency, and throughput separation and identification of proteomes. For a bottom-up strategy-based proteome analysis, usually multistep salt elution was needed in the first dimension separation by SCX, to simplify the peptides for the further second dimensional separation by RPLC. Here, by using a 30 cm-long serially coupled long column (SCLC) in the second dimension, we reduced the salt steps of SCX from 13 to 5 to shorten the total analysis time. Compared to the commonly applied 2D-HPLC with over 10-step salt elution in SCX and microRPLC with a short column (SC), named as SC-2D, the peak capacity of 2D-HPLC with a SCLC column, named as SCLC-2D, was increased 3.3-folds while the analysis time was increased by only 1.17-folds. Therefore, the time-based protein identification efficiency was similar to 55 protein groups/h, nearly 2-fold of that for SC-2D (similar to 28 protein groups/h). With the further combination of assisted solubilization by ionic liquids and SCLC-2D, 608 integral membrane proteins (IMPs) (27.66% of the total 2198 proteins, FDR < 1%) were identified from rat brain, more than those obtaind by the traditional urea method (252 unique IMPs, occupying 17.03% of total 1480 proteins). All of these results demonstrate the promise of the developed technique for large-scale proteome analysis.

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.

The solubility and digestion efficiency are two crucial factors that might affect the identification of integral membrane proteins (IMPs). In this work, 1% (v/v) ionic liquid (IL), 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIM BF(4)), added in NH(4)HCO(3) buffer (pH 8.3), was applied as a sample preparation buffer for IMPs analysis. Compared to the commonly used sodium dodecyl sulfate and methanol methods, the number of identified IMPs from rat brain by microcolumn reversed phase liquid chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was improved by over three times, which might be due to the fact that BMIM BF(4) offered high solubilizing ability for IMPs and good compatibility for tryptic digestion. Furthermore, compared to Rapigest and urea methods, with BMIM BF(4) method, the number of identified IMPs from rat brain could be improved 25% and 80%, respectively, which might be contributed to the good solubilizing ability and high thermal stability of such IL. With the sample treated by BMIM BF(4) method, by 2D-nanoSCX-RPLC-ESI-MS/MS, 1,450 non-redundant proteins and 7,978 unique peptides were identified from rat brain, and 418 proteins contained at least one predicted transmembrane domain, with false discovery rates of less than 1% for peptide identification, and at least two identified unique peptides per protein. All these results demonstrate that the BMIM BF(4) method is of high potential for the large-scale identification of IMPs.

Screening of tumor markers by proteomic technology is the research focus and key of early diagnosis of stomach cancer study. Aiming at the complexity of the extracted proteins from biological tissue, reversed-phase high performance liquid chromatography (RP-HPLC) was employed as one of the most efficient chromatographic methods. Based on the difference of hydrophobicity, RP-HPLC separation was performed to reduce the complexity of stomach cancer tissue and normal tissue samples, separately. By comparing the chromatograms, different components were collected. The fractions with the retention times from 45 min to 47 min were digested and identified by liquid chromatography-multistage mass spectrometry (LC-MS/MS). Nine common proteins were found in both tumor tissue and normal tissue. Six specific proteins were screened in normal tissue and seventeen specific proteins were found in tumor tissue under the same conditions. Two proteins with higher abundance in tumor tissue were selected for further investigation. These proteins provide more information for future drug target and drug pathway research by the analysis of biological information.