Mapping the cancer surface proteome in search of target antigens for immunotherapy.

Immune-based therapeutic interventions recognizing proteins localized on the cell surface of cancer cells are emerging as a promising cancer treatment. Antibody-based therapies and engineered T cells are now approved by the Food and Drug Administration to treat some malignancies. These therapies utilize a few cell surface proteins highly expressed on cancer cells to release the negative regulation of immune activation that limits antitumor responses (e.g., PD-1, PD-L1, CTLA4) or to redirect the T cell specificity toward blood cancer cells (e.g., CD19 and B cell maturation antigen). One limitation preventing broader application of these novel therapeutic strategies to all cancer types is the lack of suitable target antigens for all indications owing in part to the challenges in identifying such targets. Ideal target antigens are cell surface proteins highly expressed on malignant cells and absent in healthy tissues. Technological advances in mass spectrometry, enrichment protocols, and computational tools for cell surface protein isolation and annotation have recently enabled comprehensive analyses of the cancer cell surface proteome, from which novel immunotherapeutic target antigens may emerge. Here, we review the most recent progress in this field.

Mass spectrometry-based methods for investigating the dynamics and organization of the surfaceome: exploring potential clinical implications.

INTRODUCTION: Cell-surface proteins are extremely important for many cellular events, such as regulating cell-cell communication and cell-matrix interactions. Aberrant alterations in surface protein expression, modification (especially glycosylation), and interactions are directly related to human diseases. Systematic investigation of surface proteins advances our understanding of protein functions, cellular activities, and disease mechanisms, which will lead to identifying surface proteins as disease biomarkers and drug targets. AREAS COVERED: In this review, we summarize mass spectrometry (MS)-based proteomics methods for global analysis of cell-surface proteins. Then, investigations of the dynamics of surface proteins are discussed. Furthermore, we summarize the studies for the surfaceome interaction networks. Additionally, biological applications of MS-based surfaceome analysis are included, particularly highlighting the significance in biomarker identification, drug development, and immunotherapies. EXPERT OPINION: Modern MS-based proteomics provides an opportunity to systematically characterize proteins. However, due to the complexity of cell-surface proteins, the labor-intensive workflow, and the limit of clinical samples, comprehensive characterization of the surfaceome remains extraordinarily challenging, especially in clinical studies. Developing and optimizing surfaceome enrichment methods and utilizing automated sample preparation workflow can expand the applications of surfaceome analysis and deepen our understanding of the functions of cell-surface proteins.

The cell surface contains many important proteins such as receptors and transporters. These proteins are responsible for cells to communicate with each other, take nutrients from outside, and interact with their surroundings. Aberrant changes in surface protein expression, modifications, and interactions with other molecules directly result in various diseases, including infections, immune disorders, and cancer. Currently, mass spectrometry (MS)-based proteomics is very powerful to study proteins on a large scale, and there has been a strong interest in employing MS to investigate cell-surface proteins. In this review, we discuss different methods combining mass spectrometry with other approaches to systematically characterize protein abundance, dynamics, modification, and interaction on the cell surface. These methods help uncover protein functions and specific cell-surface proteins related to human diseases. A better understanding of the functions and properties of cell-surface proteins can facilitate the discovery of surface proteins as effective biomarkers for disease early detection and the identification of drug targets for disease treatment.

Touchpad-based immunochromatographic strip for detecting the skin surface proteins.

This study demonstrates, for the first time, the proof-of-concept of a novel immunosensor, a touchpad-based immunochromatographic strip, that non-invasively extracts and detects skin surface proteins. The strip was composed of a nitrocellulose membrane at the center, where a spot of anti-human IgG capture antibody was physically adsorbed. The capture antibody spot was covered with a glass fiber membrane impregnated with phosphate-buffered saline (PBS) to extract skin surface proteins, avoiding direct contact of the human skin with the capture antibodies. Skin surface IgG was detected in two steps: (1) touching the capture antibody via a glass fiber membrane containing PBS, and (2) dipping the strip into the Au-nanoparticle-labeled secondary antibody to visualize the existence of the captured skin surface IgG on the strip. We qualitatively demonstrated that using a very small amount of PBS while maintaining contact with the skin, skin surface proteins can be concentrated and detected, even with a relatively low-sensitivity immunochromatographic chip. This sensor is expected to be a potential biosensor for the non-invasive diagnosis of the integrity of human skin.

Integrins protect sensory neurons in models of paclitaxel-induced peripheral sensory neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect from cancer treatment with no known method for prevention or cure in clinics. CIPN often affects unmyelinated nociceptive sensory terminals. Despite the high prevalence, molecular and cellular mechanisms that lead to CIPN are still poorly understood. Here, we used a genetically tractable Drosophila model and primary sensory neurons isolated from adult mouse to examine the mechanisms underlying CIPN and identify protective pathways. We found that chronic treatment of Drosophila larvae with paclitaxel caused degeneration and altered the branching pattern of nociceptive neurons, and reduced thermal nociceptive responses. We further found that nociceptive neuron-specific overexpression of integrins, which are known to support neuronal maintenance in several systems, conferred protection from paclitaxel-induced cellular and behavioral phenotypes. Live imaging and superresolution approaches provide evidence that paclitaxel treatment causes cellular changes that are consistent with alterations in endosome-mediated trafficking of integrins. Paclitaxel-induced changes in recycling endosomes precede morphological degeneration of nociceptive neuron arbors, which could be prevented by integrin overexpression. We used primary dorsal root ganglia (DRG) neuron cultures to test conservation of integrin-mediated protection. We show that transduction of a human integrin ß-subunit 1 also prevented degeneration following paclitaxel treatment. Furthermore, endogenous levels of surface integrins were decreased in paclitaxel-treated mouse DRG neurons, suggesting that paclitaxel disrupts recycling in vertebrate sensory neurons. Altogether, our study supports conserved mechanisms of paclitaxel-induced perturbation of integrin trafficking and a therapeutic potential of restoring neuronal interactions with the extracellular environment to antagonize paclitaxel-induced toxicity in sensory neurons.

Periscope Proteins are variable-length regulators of bacterial cell surface interactions.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

Surface Proteins of Bifidobacterium Bifidum DNG6 Growing in 2'-FL Alleviating LPS-Induced Intestinal Barrier Injury in vitro.

Human milk oligosaccharides (HMOs) promote the growth and adhesion of bifidobacteria, thus exerting multiple biological functions on intestinal epithelial cells. Bacterial surface proteins play an important role in bacterial-host intestinal epithelial interactions. In this study, we aim to investigate the effects of surface proteins extracted from Bifidobacterium bifidum DNG6 (B. bifidum DNG6) consuming 2'-fucosyllactose (2'-FL) on Caco-2 cells monolayer barrier injury induced by lipopolysaccharide, compared with lactose (Lac) and galacto-oligosaccharides (GOS). Our results indicated that 2'-FL may promote the surface proteins of B. bifidum DNG6 to improve intestinal barrier injury by positively regulating the NF-κB signaling pathway, reducing inflammation(TNF-α reduced to 50.34%, IL-6 reduced to 22.83%, IL-1ß reduced to 37.91%, and IL-10 increased to 63.47%)and strengthening tight junction (ZO-1 2.39 times, Claudin-1 2.79 times, and Occludin 4.70 times). The findings of this study indicate that 2'-FL can further regulate intestinal barrier damage by promoting the alteration of B. bifidum DNG6 surface protein. The findings of this research will also provide theoretical support for the development of synbiotic formulations.

Targeted Degradation of Cell-Surface Proteins via Chaperone-Mediated Autophagy by Using Peptide-Conjugated Antibodies.

Cell-surface proteins are important drug targets but historically have posed big challenges for the complete elimination of their functions. Herein, we report antibody-peptide conjugates (Ab-CMAs) in which a peptide targeting chaperone-mediated autophagy (CMA) was conjugated with commercially available monoclonal antibodies for specific cell-surface protein degradation by taking advantage of lysosomal degradation pathways. Unique features of Ab-CMAs, including cell-surface receptor- and E3 ligase-independent degradation, feasibility towards different cell-surface proteins (e.g., epidermal growth factor receptor (EGFR), programmed cell death ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2)) by a simple change of the antibody, and successful tumor inhibition in vivo, make them attractive protein degraders for biomedical research and therapeutic applications. As the first example employing CMA to degrade proteins from the outside in, our findings may also shed new light on CMA, a degradation pathway typically targeting cytosolic proteins.

Hepatitis B Virus Small Envelope Protein Promotes Hepatocellular Carcinoma Angiogenesis via Endoplasmic Reticulum Stress Signaling To Upregulate the Expression of Vascular Endothelial Growth Factor A.

Hepatocellular carcinoma (HCC) is a hypervascular tumor, and accumulating evidence has indicated that stimulation of angiogenesis by hepatitis B virus (HBV) may contribute to HCC malignancy. The small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV protein and has a close clinical association with HCC; however, whether SHBs contributes to HCC angiogenesis remains unknown. This study reports that the forced expression of SHBs in HCC cells promoted xenograft tumor growth and increased the microvessel density (MVD) within the tumors. Consistently, HBsAg was also positively correlated with MVD counts in HCC patients' specimens. The conditioned media from the SHBs-transfected HCC cells increased the capillary tube formation and migration of human umbilical vein endothelial cells (HUVECs). Intriguingly, the overexpression of SHBs increased vascular endothelial growth factor A (VEGFA) expression at both the mRNA and protein levels. Higher VEGFA expression levels were also observed in xenograft tumors transplanted with SHBs-expressing HCC cells and in HBsAg-positive HCC tumor tissues than in their negative controls. As expected, in the culture supernatants, the secretion of VEGFA was also significantly enhanced from HCC cells expressing SHBs, which promoted HUVEC migration and vessel formation. Furthermore, all three unfolded protein response (UPR) sensors, inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK), and activating transcription factor 6 (ATF6), associated with ER stress were found to be activated in SHBs-expressing cells and correlated with VEGFA protein expression and secretion. Taken together, these results suggest an important role of SHBs in HCC angiogenesis and may highlight a potential target for preventive and therapeutic intervention for HBV-related HCC and its malignant progression. IMPORTANCE Chronic hepatitis B virus infection is one of the important risk factors for the development and progression of hepatocellular carcinoma (HCC). HCC is characteristic of hypervascularization even at early phases of the disease due to the overexpression of angiogenic factors like vascular endothelial growth factor A (VEGFA). However, a detailed mechanism of HBV-induced angiogenesis remains to be established. In this study, we demonstrate for the first time that the most abundant HBV protein, i.e., small surface antigen (SHBs), can enhance the angiogenic capacity of HCC cells by the upregulation of VEGFA expression both in vitro and in vivo. Mechanistically, SHBs induced endoplasmic reticulum (ER) stress, which consequently activated unfolded protein response (UPR) signaling to increase VEGFA expression and secretion. This study suggests that SHBs plays an important proangiogenic role in HBV-associated HCC and may represent a potential target for antiangiogenic therapy in HCC.

COVID-19db linkage maps of cell surface proteins and transcription factors in immune cells.

The highly contagious SARS-CoV-2 and its associated disease (COVID-19) are a threat to global public health and economies. To develop effective treatments for COVID-19, we must understand the host cell types, cell states and regulators associated with infection and pathogenesis such as dysregulated transcription factors (TFs) and surface proteins, including signaling receptors. To link cell surface proteins with TFs, we recently developed SPaRTAN (Single-cell Proteomic and RNA-based Transcription factor Activity Network) by integrating parallel single-cell proteomic and transcriptomic data based on Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and gene cis-regulatory information. We apply SPaRTAN to CITE-seq data sets from patients with varying degrees of COVID-19 severity and healthy controls to identify the associations between surface proteins and TFs in host immune cells. Here, we present COVID-19db of Immune Cell States (https://covid19db.streamlit.app/), a web server containing cell surface protein expression, SPaRTAN-inferred TF activities, and their associations with major host immune cell types. The data include four high-quality COVID-19 CITE-seq data sets with a toolset for user-friendly data analysis and visualization. We provide interactive surface protein and TF visualizations across major immune cell types for each data set, allowing comparison between various patient severity groups for the discovery of potential therapeutic targets and diagnostic biomarkers.

Recent advances in nitrogen-containing heterocyclic compounds as receptor tyrosine kinase inhibitors for the treatment of cancer: Biological activity and structural activity relationship.

Erratic cell proliferation is the initial symptom of cancer, which can eventually metastasize to other organs. Before cancer becomes metastatic, its spread is triggered by pro-angiogenic factors including vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), Platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR) and Platelet Factor (PF4), all of which are part of receptor tyrosine kinase (RTK) family. Receptor tyrosine kinases (RTKs) are cell-surface proteins and aresignaling enzymes that transfer ATP-phosphate to tyrosine residue substrates. Important biological processes like proliferation, differentiation, motility, and cell-cycle regulation are all possessedby these proteins. Unusual RTK expression is typically associated with cell growth abnormalities, which is linked to tumor acquisition, angiogenesis, and cancer progression. In addition to the already available medications, numerous other heterocyclic are being studied for their potential action against a variety of cancers. In the fight against cancer, in particular, these heterocycles have been used for their dynamic core scaffold and their inherent adaptability. In this review article, we have compiled last five years research work including nitrogen containing heterocycles that have targeted RTK. Herein, the SAR and activity of various compounds containing diverse heterocyclic (pyrimidine, indole, pyridine, pyrazole, benzimidazole, and pyrrole) scaffolds are discussed, and they may prove useful in the future for designing new leads against RTKs. Our focus in this manuscript is to comprehensively review the latest research on the biological activity and structural activity relationship of nitrogen compounds as RTK inhibitors. We believe that this may be an important contribution to the field, as it can help guide future research efforts and facilitate the development of more effective cancer therapies.

Comprehensive Strategy for Identifying Extracellular Vesicle Surface Proteins as Biomarkers for Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a liver disorder that has become a global health concern due to its increasing prevalence. There is a need for reliable biomarkers to aid in the diagnosis and prognosis of NAFLD. Extracellular vesicles (EVs) are promising candidates in biomarker discovery, as they carry proteins that reflect the pathophysiological state of the liver. In this review, we developed a list of EV proteins that could be used as diagnostic biomarkers for NAFLD. We employed a multi-step strategy that involved reviewing and comparing various sources of information. Firstly, we reviewed papers that have studied EVs proteins as biomarkers in NAFLD and papers that have studied circulating proteins as biomarkers in NAFLD. To further identify potential candidates, we utilized the EV database Vesiclepedia.org to qualify each protein. Finally, we consulted the Human Protein Atlas to search for candidates' localization, focusing on membrane proteins. By integrating these sources of information, we developed a comprehensive list of potential EVs membrane protein biomarkers that could aid in diagnosing and monitoring NAFLD. In conclusion, our multi-step strategy for identifying EV-based protein biomarkers for NAFLD provides a comprehensive approach that can also be applied to other diseases. The protein candidates identified through this approach could have significant implications for the development of non-invasive diagnostic tests for NAFLD and improve the management and treatment of this prevalent liver disorder.

Staphylococcus lugdunensis: a Skin Commensal with Invasive Pathogenic Potential.

Staphylococcus lugdunensis is a species of coagulase-negative staphylococcus (CoNS) that causes serious infections in humans akin to those of S. aureus It was often misidentified as S. aureus, but this has been rectified by recent routine use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in diagnostic laboratories. It encodes a diverse array of virulence factors for adhesion, cytotoxicity, and innate immune evasion, but these are less diverse than those encoded by S. aureus It expresses an iron-regulated surface determinant (Isd) system combined with a novel energy-coupling factor (ECF) mechanism for extracting heme from hemoproteins. Small cytolytic S. lugdunensis synergistic hemolysins (SLUSH), peptides related to phenol-soluble modulins of S. aureus, act synergistically with ß-toxin to lyse erythrocytes. S. lugdunensis expresses a novel peptide antibiotic, lugdunin, that can influence the nasal and skin microbiota. Endovascular infections are initiated by bacterial adherence to fibrinogen promoted by a homologue of Staphylococcus aureus clumping factor A and to von Willebrand factor on damaged endothelium by an uncharacterized mechanism. S. lugdunensis survives within mature phagolysosomes of macrophages without growing and is released only following apoptosis. This differs fundamentally from S. aureus, which actively grows and expresses bicomponent leukotoxins that cause membrane damage and could contribute to survival in the infected host. S. lugdunensis is being investigated as a probiotic to eradicate S. aureus from the nares of carriers. However, this is contraindicated by its innate virulence. Studies to obtain a deeper understanding of S. lugdunensis colonization, virulence, and microbiome interactions are therefore warranted.

A Recombinant Alpha-Like Protein Subunit Vaccine (GBS-NN) Provides Protection in Murine Models of Group B Streptococcus Infection.

BACKGROUND: Group B Streptococcus (GBS) transmission during pregnancy causes preterm labor, stillbirths, fetal injury, or neonatal infections. Rates of adult infections are also rising. The GBS-NN vaccine, engineered by fusing N-terminal domains of GBS Alpha C and Rib proteins, is safe in healthy, nonpregnant women, but further assessment is needed for use during pregnancy. Here, we tested GBS-NN vaccine efficacy using mouse models that recapitulate human GBS infection outcomes. METHODS: Following administration of GBS-NN vaccine or adjuvant, antibody profiles were compared by ELISA. Vaccine efficacy was examined by comparing infection outcomes in GBS-NN vaccinated versus adjuvant controls during systemic and pregnancy-associated infections, and during intranasal infection of neonatal mice following maternal vaccination. RESULTS: Vaccinated mice had higher GBS-NN-specific IgG titers versus controls. These antibodies bound alpha C and Rib on GBS clinical isolates. Fewer GBS were recovered from systemically challenged vaccinated mice versus controls. Although vaccination did not eliminate GBS during ascending infection in pregnancy, vaccinated dams experienced fewer in utero fetal deaths. Additionally, maternal vaccination prolonged neonatal survival following intranasal GBS challenge. CONCLUSIONS: These findings demonstrate GBS-NN vaccine efficacy in murine systemic and perinatal GBS infections and suggest that maternal vaccination facilitates the transfer of protective antibodies to neonates.

EXCESP: A Structure-Based Online Database for Extracellular Interactome of Cell Surface Proteins in Humans.

The interactions between ectodomains of cell surface proteins are vital players in many important cellular processes, such as regulating immune responses, coordinating cell differentiation, and shaping neural plasticity. However, while the construction of a large-scale protein interactome has been greatly facilitated by the development of high-throughput experimental techniques, little progress has been made to support the discovery of extracellular interactome for cell surface proteins. Harnessed by the recent advances in computational modeling of protein-protein interactions, here we present a structure-based online database for the extracellular interactome of cell surface proteins in humans, called EXCESP. The database contains both experimentally determined and computationally predicted interactions among all type-I transmembrane proteins in humans. All structural models for these interactions and their binding affinities were further computationally modeled. Moreover, information such as expression levels of each protein in different cell types and its relation to various signaling pathways from other online resources has also been integrated into the database. In summary, the database serves as a valuable addition to the existing online resources for the study of cell surface proteins. It can contribute to the understanding of the functions of cell surface proteins in the era of systems biology.

Genomic Analysis of MSM Rectal Chlamydia trachomatis Isolates Identifies Predicted Tissue-Tropic Lineages Generated by Intraspecies Lateral Gene Transfer-Mediated Evolution.

Chlamydia trachomatis is an obligate intracellular bacterium that causes serious diseases in humans. Rectal infection and disease caused by this pathogen are important yet understudied aspects of C. trachomatis natural history. The University of Washington Chlamydia Repository has a large collection of male-rectal-sourced strains (MSM rectal strains) isolated in Seattle, USA and Lima, Peru. Initial characterization of strains collected over 30 years in both Seattle and Lima led to an association of serovars G and J with male rectal infections. Serovar D, E, and F strains were also collected from MSM patients. Genome sequence analysis of a subset of MSM rectal strains identified a clade of serovar G and J strains that had high overall genomic identity. A genome-wide association study was then used to identify genomic loci that were correlated with tissue tropism in a collection of serovar-matched male rectal and female cervical strains. The polymorphic membrane protein PmpE had the strongest correlation, and amino acid sequence alignments identified a set of PmpE variable regions (VRs) that were correlated with host or tissue tropism. Examination of the positions of VRs by the protein structure-predicting Alphafold2 algorithm demonstrated that the VRs were often present in predicted surface-exposed loops in both PmpE and PmpH protein structure. Collectively, these studies identify possible tropism-predictive loci for MSM rectal C. trachomatis infections and identify predicted surface-exposed variable regions of Pmp proteins that may function in MSM rectal versus cervical tropism differences.

The long and the short of Periscope Proteins.

Bacteria sense, interact with, and modify their environmental niche by deploying a molecular ensemble at the cell surface. The changeability of this exposed interface, combined with extreme changes in the functional repertoire associated with lifestyle switches from planktonic to adherent and biofilm states necessitate dynamic variability. Dynamic surface changes include chemical modifications to the cell wall; export of diverse extracellular biofilm components; and modulation of expression of cell surface proteins for adhesion, co-aggregation and virulence. Local enrichment for highly repetitive proteins with high tandem repeat identity has been an enigmatic phenomenon observed in diverse bacterial species. Preliminary observations over decades of research suggested these repeat regions were hypervariable, as highly related strains appeared to express homologues with diverse molecular mass. Long-read sequencing data have been interrogated to reveal variation in repeat number; in combination with structural, biophysical and molecular dynamics approaches, the Periscope Protein class has been defined for cell surface attached proteins that dynamically expand and contract tandem repeat tracts at the population level. Here, I review the diverse high-stability protein folds and coherent interdomain linkages culminating in the formation of highly anisotropic linear repeat arrays, so-called rod-like protein 'stalks', supporting roles in bacterial adhesion, biofilm formation, cell surface spatial competition, and immune system modulation. An understanding of the functional impacts of dynamic changes in repeat arrays and broader characterisation of the unusual protein folds underpinning this variability will help with the design of immunisation strategies, and contribute to synthetic biology approaches including protein engineering and microbial consortia construction.

Assessment of Listeria monocytogenes Surface Proteins Identified from Proteomics Analysis for Use as Diagnostic Biomarkers.

The Gram-positive bacterium Listeria monocytogenes is an important pathogen that causes a foodborne illness with a high percentage of fatalities. Surface proteins, specifically expressed from a wide range of L. monocytogenes serotypes under selective enrichment culture conditions, can serve as targets for the detection and isolation of this pathogen using antibody-based methods. Among a number of surface proteins identified by mass spectrometry in a previous proteomic study, six candidates (annotated as LMOf2365_0148, LMOf2365_0312, LMOf2365_0546, LMOf2365_1883, LMOf2365_2111, and LMOf2365_2742) were selected here for investigating their expression in the bacterial cells cultured in vitro by raising rabbit polyclonal antibodies (PAbs) against the recombinant form of each candidate. These protein candidates contained regions conserved among various L. monocytogenes isolates but variable in other Listeria species. LMOf2365_0148, an uncharacterized protein with a LPXTG motif accountable for covalent linkage to the cell wall peptidoglycan, exhibited a strong reaction signal from anti-LMOf2365_0148 PAb binding to the cell surface, as detected by immunofluorescence microscopy. Further study, through the generation of a panel of mouse monoclonal antibodies (MAbs) to the recombinant LMOf2365_0148, showed that one of the MAbs, M3686, reacted to bacterial isolates belonging to all three lineages of L. monocytogenes under Health Canada's standard enrichment culture conditions (MFHPB-07 and MFHPB-30). These results demonstrated the potential of using LMOf2365_0148 as a surface biomarker, in conjunction with specific MAbs developed here, for the isolation and detection of L. monocytogenes from foods and food processing environments. IMPORTANCE Strains of Listeria monocytogenes are differentiated serologically into at least 13 serotypes and grouped phylogenetically into 4 distinct lineages (I, II, III, and IV). No single monoclonal antibody (MAb) reported to date is capable of binding to the surface of L. monocytogenes strains representing all the serotypes. This study assessed the expression of six surface proteins selected from a previous proteomic study and demonstrated that surface protein LMOf2365_0148 has the greatest potential as a surface biomarker. A panel of 24 MAbs to LMOf2365_0148 were assessed extensively, revealing that one of the MAbs, M3686, reacted to a wide range of L. monocytogenes isolates (lineage I, II, and III isolates) grown under standard enrichment culture conditions and thus led to the conclusion that LMOf2365_0148 is a useful novel surface biomarker for identifying, detecting, and isolating the pathogen from food and environmental samples.

Temporal dynamics of Plasmodium falciparum population in Metehara, east-central Ethiopia.

BACKGROUND: Plasmodium falciparum is the most serious, genetically most complex and fastest-evolving malaria parasite. Information on genetic diversity of this parasite would guide policy decision and malaria elimination endeavors. This study explored the temporal dynamics of P. falciparum population in two time points in Metehara, east-central Ethiopia. METHODS: The participants were quantitative real-time polymerase chain reaction-confirmed patients who were recruited for uncomplicated falciparum malaria therapeutic efficacy test in 2015 and 2019. Dry blood spot samples were analysed by the nested PCR to genotype P. falciparum merozoite surface protein (msp1, msp2) and glutamate-rich protein (glurp) genes. RESULTS: While msp1, msp2 and glurp genotypes were successfully detected in 26(89.7%), 24(82.8%) and 14(48.3%) of 2015 samples (n = 29); the respective figures for 2019 (n = 41) were 31(68.3%), 39(95.1%), 25(61.0%). In 2015, the frequencies of K1, MAD20 and RO33 allelic families of msp1, and FC27 and IC/3D7 of msp2 were 19(73.1%), 8(30.6%), 14(53.8%), 21(87.5%), 12(50.5%); and in 2019 it was 15(48.4%), 19(61.3%), 15(48.4%), 30(76.9%), 27(69.2%) respectively. MAD20 has shown dominance over both K1 and RO33 in 2019 compared to the proportion in 2015. Similarly, although FC27 remained dominant, there was shifting trend in the frequency of IC/3D7 from 50.5% in 2015 to 69.2% in 2019. The multiplicity of infection (MOI) and expected heterozygosity index (He) in 2015 and 2019 were respectively [1.43 ± 0.84] and [1.15 ± 0.91], 0.3 and 0.03 for msp1. However, there was no significant association between MOI and age or parasitaemia in both time points. CONCLUSION: The lower genetic diversity in P. falciparum population in the two time points and overall declining trend as demonstrated by the lower MOI and He may suggest better progress in malaria control in Metehara. But, the driving force and selective advantage of switching to MAD20 dominance over the other two msp1 allelic families, and the dynamics within msp2 alleles needs further investigation.

Identification of Toxoplasma gondii adhesins through a machine learning approach.

Toxoplasma gondii, as other apicomplexa, employs adhesins transmembrane proteins for binding and invasion to host cells. Search and characterization of adhesins is pivotal in understanding Apicomplexa invasion mechanisms and targeting new druggable candidates. This work developed a machine learning software called ApiPredictor UniQE V2.0, based on two approaches: support vector machines and multilayer perceptron, to predict adhesins proteins from amino acid sequences. By using ApiPredictor UniQE V2.0, five SAG-Related Sequences (SRSs) were identified within the Toxoplasma gondii proteome. One of those candidates, TgSRS12B, was cloned in plasmid pEXP5-CT/TOPO and expressed in E. coli BL21 DE3. The resulting recombinant protein was purified via affinity chromatography. Co-precipitation assays in CaCo and Muller cells showed interactions between TgSRS12B-His-tagged and the membrane fractions from both human cell lines. In conclusion, we demonstrated that ApiPredictor UniQE V2.0, a bioinformatic free software, was able to identify TgSRS12B as a new adhesin protein.

Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method.

Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of the cell surface subproteome of individual cells. Here we present a high-throughput technique which relies on the biotinylation of the accessible primary amino groups in the extracellular segments of the proteins, using HL60 as a model cell line. Several steps of the method have been thoroughly optimized to capture labeled surface proteins selectively and in larger quantities. These include the following: improving the efficiency of the cell surface biotinylation; reducing the endogen protease activity; applying an optimal amount of affinity column and elution steps for labeled peptide enrichment; and examining the effect of various solid-phase extraction methods, different HPLC gradients, and various tandem mass spectrometry settings. Using the optimized workflow, we identified at least 1700 surface-associated individual labeled peptides (~6000-7000 redundant peptides) from the model cell surface in a single nanoHPLC-MS/MS run. The presented method can provide a comprehensive and specific list of the cell surface available protein segments that could be potential targets in various bioinformatics and molecular biology research.