Navigating colorectal cancer prognosis: A Treg-related signature discovered through single-cell and bulk transcriptomic approaches.

BACKGROUND: The significance of regulatory T cells (Tregs) in colorectal cancer is unclear. METHODS: The single-cell sequencing data for colorectal cancer, specifically GSE132465 and GSE188711, were retrieved from the GEO database. Simultaneously, bulk transcriptome data were obtained from the UCSC Xena website. To delve into the heterogeneity of Treg cells and identify key genes at the single-cell sequencing level, we employed dimensionality reduction techniques alongside clustering and conducted differential expression gene analysis. For the bulk transcriptome data, we utilized weighted co-expression network analysis to investigate critical gene modules. Additionally, we employed COX regression and Lasso regression methodologies to construct prognostic models, thereby assessing patient outcomes. To facilitate outcome evaluation, nomograms were constructed. The integration of these diverse approaches aims to comprehensively study colorectal cancer, encompassing single-cell heterogeneity, key gene identification, and prognosis modeling using both single-cell and bulk transcriptome data. Polymerase chain reaction (PCR) experiments are used to verify mRNA expression levels of key genes. The analysis software was R software (version 4.3.2). RESULTS: Through single-cell sequencing analysis and bulk transcriptome analysis, we constructed a prognostic model composed with Treg-associated signatures. The high-risk group demonstrated significantly worse prognosis compared with the low-risk group, highlighting the clinical relevance of our models. PCR confirmed that the key gene DEAH-box helicase 15 (DHX15) was significantly overexpressed in colorectal cancer. CONCLUSIONS: The prognostic models developed in this study offer a potential tool for risk assessment, guiding treatment decisions for colorectal cancer patients.

Development of a Novel Circulating Autoantibody Biomarker Panel for the Identification of Patients with 'Actionable' Pulmonary Nodules.

Due to poor compliance and uptake of LDCT screening among high-risk populations, lung cancer is often diagnosed in advanced stages where treatment is rarely curative. Based upon the American College of Radiology's Lung Imaging and Reporting Data System (Lung-RADS) 80-90% of patients screened will have clinically "non-actionable" nodules (Lung-RADS 1 or 2), and those harboring larger, clinically "actionable" nodules (Lung-RADS 3 or 4) have a significantly greater risk of lung cancer. The development of a companion diagnostic method capable of identifying patients likely to have a clinically actionable nodule identified during LDCT is anticipated to improve accessibility and uptake of the paradigm and improve early detection rates. Using protein microarrays, we identified 501 circulating targets with differential immunoreactivities against cohorts characterized as possessing either actionable (n = 42) or non-actionable (n = 20) solid pulmonary nodules, per Lung-RADS guidelines. Quantitative assays were assembled on the Luminex platform for the 26 most promising targets. These assays were used to measure serum autoantibody levels in 841 patients, consisting of benign (BN; n = 101), early-stage non-small cell lung cancer (NSCLC; n = 245), other early-stage malignancies within the lung (n = 29), and individuals meeting United States Preventative Screening Task Force (USPSTF) screening inclusion criteria with both actionable (n = 87) and non-actionable radiologic findings (n = 379). These 841 patients were randomly split into three cohorts: Training, Validation 1, and Validation 2. Of the 26 candidate biomarkers tested, 17 differentiated patients with actionable nodules from those with non-actionable nodules. A random forest model consisting of six autoantibody (Annexin 2, DCD, MID1IP1, PNMA1, TAF10, ZNF696) biomarkers was developed to optimize our classification performance; it possessed a positive predictive value (PPV) of 61.4%/61.0% and negative predictive value (NPV) of 95.7%/83.9% against Validation cohorts 1 and 2, respectively. This panel may improve patient selection methods for lung cancer screening, serving to greatly reduce the futile screening rate while also improving accessibility to the paradigm for underserved populations.

Argonaute, Vault, and Ribosomal Proteins Targeted by Autoantibodies in Systemic Lupus Erythematosus.

OBJECTIVE: To expand, in an unbiased manner, our knowledge of autoantigens and autoantibodies in patients with systemic lupus erythematosus (SLE) and evaluate their associations with serological and clinical variables. METHODS: Human proteome arrays (> 21,000 proteins) were screened with serum from patients with SLE (n = 12) and healthy controls (n = 6) for IgG and IgA binding. Top hits were validated with 2 cohorts of patients with SLE (cohort 1, n = 49; cohort 2, n = 46) and other rheumatic diseases by ELISA. Clinical associations of the autoantibodies were tested. RESULTS: Ro60 was the top hit in the screen, and the 10 following proteins included 2 additional known SLE autoantigens plus 8 novel autoantigens involved in microRNA processing (Argonaute protein 1 [AGO1], AGO2, and AGO3), ribosomes (ribosomal protein lateral stalk subunit P2 and ovarian tumor deubiquitinase 5 [OTUD5]), RNA transport by the vault (major vault protein), and the immune proteasome (proteasome activator complex subunit 3). Patient serum contained IgG reactive with these proteins and IgA against the AGO proteins. Using the 95th percentile of healthy donor reactivity, 5-43% were positive for the novel antigens, with OTUD5 and AGO1 showing the highest percentages of positivity. Autoantibodies against AGO1 proteins were more prevalent in patients with oral ulcers in a statistically significant manner. IgG autoantibodies against AGO proteins were also seen in other rheumatic diseases. CONCLUSION: We discovered new autoantigens existing in cytosolic macromolecular protein assemblies containing RNA (except the proteasome) in cells. A more comprehensive list of autoantigens will allow for a better analysis of how proteins are targeted by the autoimmune response. Future research will also reveal whether specific autoantibodies have utility in the diagnosis or management of SLE.

Neoadjuvant enoblituzumab in localized prostate cancer: a single-arm, phase 2 trial.

B7 homolog 3 (B7-H3; CD276), a tumor-associated antigen and possible immune checkpoint, is highly expressed in prostate cancer (PCa) and is associated with early recurrence and metastasis. Enoblituzumab is a humanized, Fc-engineered, B7-H3-targeting antibody that mediates antibody-dependent cellular cytotoxicity. In this phase 2, biomarker-rich neoadjuvant trial, 32 biological males with operable intermediate to high-risk localized PCa were enrolled to evaluate the safety, anti-tumor activity and immunogenicity of enoblituzumab when given before prostatectomy. The coprimary outcomes were safety and undetectable prostate-specific antigen (PSA) level (PSA0) 1 year postprostatectomy, and the aim was to obtain an estimate of PSA0 with reasonable precision. The primary safety endpoint was met with no notable unexpected surgical or medical complications, or surgical delay. Overall, 12% of patients experienced grade 3 adverse events and no grade 4 events occurred. The coprimary endpoint of the PSA0 rate 1 year postprostatectomy was 66% (95% confidence interval 47-81%). The use of B7-H3-targeted immunotherapy in PCa is feasible and generally safe and preliminary data suggest potential clinical activity. The present study validates B7-H3 as a rational target for therapy development in PCa with larger studies planned. The ClinicalTrials.gov identifier is NCT02923180.

Influence of Taijiquan Exercise on Mentality and Emotion Regulation by Intelligent Medical Big Data Analysis.

Taijiquan training emphasizes the relaxation of the mind and the body, pay attention to maintain the peace of mind, and minimize the impact of external interference on the body so as to make the mind more comfortable. This study mainly explores the influence of Taijiquan practice on emotion regulation based on intelligent medical health big data analysis. The significance of Taijiquan in developing and improving the positive emotions of middle-aged people and maintaining physical and mental health is expounded. There are two methods of data collection: full collection and incremental collection. In this study, when the psychological testing equipment was launched, a full amount of historical data was collected; after the psychological testing equipment was online, the collection method was generally carried out in the way of incremental collection. The subjects exercised Taijiquan three times a week, one hour each time, and the exercise content was the 24-style Taijiquan designated by the workstation. At the same time, the subjects were asked not to engage in other regular physical exercise projects in their spare time. By longitudinal tracking and comparison of the Taijiquan intervention group after participating in the Taijiquan exercise intervention, the differences in the state of mind and emotion regulation strategies, and 12 subjects were selected voluntarily to participate in the emotional Stroop (the color words used in the classic Stroop paradigm were replaced with emotional and nonemotional words written in different colors, and the subjects were still tasked with responding to colors) experimental paradigm. In this paper, the moderate-intensity Taijiquan project is selected, which is in line with the effective value threshold theory of exercise load. It studies the effects of exercise on the body shape, cardiopulmonary function, flexibility, and balance ability of the body according to the metabolism theory and the movement balance theory of the human body adapting to the environment. Before the experiment, there was no significant difference between the Taijiquan training group and the control group, but after the experiment, there was a significant difference between the Taijiquan training group and the control group (P<0.05). Taijiquan has a significant effect on improving students' body shape, cardiopulmonary function, flexibility, balance, and mood.

Integration of IgA and IgG Autoantigens Improves Performance of Biomarker Panels for Early Diagnosis of Lung Cancer.

Lung cancer (LC) remains the leading cause of mortality from malignant tumors worldwide. In our previous study, we surveyed both IgG and IgM-bound serological biomarkers and validated a panel of IgG-bound autoantigens for early LC diagnosis with 50% sensitivity at 90% specificity. To further improve the performance of these serological biomarkers, we surveyed HuProt arrays, comprised of 20,240 human proteins, for IgA-bound autoantigens because IgAs are a major immunoglobulin isotype in the lung. Integrating with IgG-bound autoantigens, we discovered and validated a combined biomarker panel using ELISA-format tests. Specifically, in Phase I, we obtained IgA-based autoimmune profiles of 69 early stage LC patients, 30 healthy subjects and 25 patients with lung benign lesions (LBL) on HuProt arrays and identified 28 proteins as candidate autoantigens that were significantly associated with early stage LC. In Phase II, we re-purified the autoantigens and converted them into an ELISA-format testing to profile an additional large cohort, comprised of 136 early stage LC patients, 58 healthy individuals, and 29 LBL patients. Integration of IgG autoimmune profiles allowed us to identify and validate a biomarker panel of three IgA autoantigens (i.e. BCL7A, and TRIM33 and MTERF4) and three IgG autoantigens (i.e. CTAG1A, DDX4 and MAGEC2) for diagnosis of early stage LC with 73.5% sensitivity at >85% specificity. In Phase III, the performance of this biomarker panel was confirmed with an independent cohort, comprised of 88 early stage LC patients, 18 LBL patients, and 36 healthy subjects. Finally, a blind test on 178 serum samples was conducted to confirm the performance of the biomarker panel. In summary, this study demonstrates for the first time that an integrated panel of IgA/IgG autoantigens can serve as valuable biomarkers to further improve the performance of early diagnosis of LC.

Correction to: Protective Effects of Calcitonin Gene-Related Peptide-Mediated p38 Mitogen-Activated Protein Kinase Pathway on Severe Acute Pancreatitis in Rats.

The original version of the article unfortunately contained errors in Materials and Methods section, Figure 3 and Figure 4.

Protective Effects of Calcitonin Gene-Related Peptide-Mediated p38 Mitogen-Activated Protein Kinase Pathway on Severe Acute Pancreatitis in Rats.

BACKGROUND: Calcitonin gene-related peptide (CGRP) has antioxidant and anti-inflammatory activities on the pathological damage of acute pancreatitis. However, its molecular mechanism on severe acute pancreatitis (SAP) remains unknown. AIMS: To evaluate the influence of CGRP-mediated p38MAPK signaling pathway in rats with SAP. METHODS: SD rats were randomly divided into Sham group, SAP group, CGRP group (SAP rats injected with CGRP), SB203580 group (rats injected with p38MAPK pathway inhibitor SB203580), and CGRP8-37 group (SAP rats injected with CGRP8-37). Serum amylase and lipase activities were determined. Histopathological observations were evaluated, and the expression of inflammatory cytokines and oxidative stress-related indexes were measured. RESULTS: Compared with Sham group, SAP rats were increased in the activities of serum amylase and lipase, the pathologic assessment of pancreatic tissue, the levels of TNF-α, IL-1ß, IL-6, and IL-8, the content of MDA and MPO, and the expressions of CGRP, and p-p38MAPK protein, but they were decreased in SOD activity and GSH content. The above alterations were aggravated in the CGRP8-37 group when compared with SAP group. Besides, in comparison with SAP group, rats in the CGRP and SB203580 groups presented a reduction in the activities of serum amylase and lipase, the levels of inflammatory cytokines, the content of MDA and MPO, and the expressions of p-p38MAPK protein, while showed an elevation in SOD activity and GSH content. CONCLUSION: Pretreatment with CGRP alleviated oxidative stress and inflammatory response of SAP rats possibly by suppressing the activity of p38MAPK pathway, and thereby postponing the disease progression.

High-Throughput Chip Assay for Investigating Escherichia coli Interaction with the Blood-Brain Barrier Using Microbial and Human Proteome Microarrays (Dual-Microarray Technology).

Bacterial meningitis in neonates and infants is an acute lethal disease and occurs in response to microbial exploitation of the blood-brain barrier (BBB), resulting in the intracranial inflammation. Several pathogens, such as Escherichia coli ( E. coli), can cause this devastating disease; however, the underlying molecular mechanisms by which these pathogens exploit the BBB remain incompletely understood. To identify important players on both the pathogen and host sides that govern the E. coli-BBB cell interactions, we took advantage of the E. coli and human proteome microarrays (i.e., HuProt) as an unbiased, proteome-wide tool for identification of important players on both sides. Using the E. coli proteome microarrays, we developed a unique high throughput chip-based cell probing assay to probe with fluorescent live human brain microvascular endothelial cells (HBMEC, which constitute the BBB). We identified several transmembrane proteins, which effectively bound to live HBMEC. We focused on YojI protein for further study. By probing the HuProt arrays with YojI, interferon-alpha receptor (IFNAR2) was identified as one of its binding proteins. The importance of YojI and IFNAR2 involved in E. coli-HBMEC interactions was characterized using the YojI knockout bacteria and IFNAR2-knock down HBMEC and further confirmed by E. coli binding assay in HBMEC. This study represents a new paradigm (dual-microarray technology) that enables rapid, unbiased discovery of both pathogen and host players that are involved in pathogen-host interactions for human infectious diseases in a high throughput manner.

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.

Baseline antibody profiles predict toxicity in melanoma patients treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1, or the combination) enhance anti-tumor immune responses, yielding durable clinical benefit in several cancer types, including melanoma. However, a subset of patients experience immune-related adverse events (irAEs), which can be severe and result in treatment termination. To date, no biomarker exists that can predict development of irAEs. METHODS: We hypothesized that pre-treatment antibody profiles identify a subset of patients who possess a sub-clinical autoimmune phenotype that predisposes them to develop severe irAEs following immune system disinhibition. Using a HuProt human proteome array, we profiled baseline antibody levels in sera from melanoma patients treated with anti-CTLA-4, anti-PD-1, or the combination, and used support vector machine models to identify pre-treatment antibody signatures that predict irAE development. RESULTS: We identified distinct pre-treatment serum antibody profiles associated with severe irAEs for each therapy group. Support vector machine classifier models identified antibody signatures that could effectively discriminate between toxicity groups with > 90% accuracy, sensitivity, and specificity. Pathway analyses revealed significant enrichment of antibody targets associated with immunity/autoimmunity, including TNFα signaling, toll-like receptor signaling and microRNA biogenesis. CONCLUSIONS: Our results provide the first evidence supporting a predisposition to develop severe irAEs upon immune system disinhibition, which requires further independent validation in a clinical trial setting.

Identification of Serological Biomarkers for Early Diagnosis of Lung Cancer Using a Protein Array-Based Approach.

Lung cancer (LC) remains the leading cause of mortality from malignant tumors worldwide. Currently, a lack of serological biomarkers for early LC diagnosis is a major roadblock for early intervention and prevention of LC. To undertake this challenge, we employed a two-phase strategy to discover and validate a biomarker panel using a protein array-based approach. In Phase I, we obtained serological autoimmune profiles of 80 LC patients and 20 healthy subjects on HuProt arrays, and identified 170 candidate proteins significantly associated with LC. In Phase II, we constructed a LC focused array with the 170 proteins, and profiled a large cohort, comprised of 352 LC patients, 93 healthy individuals, and 101 patients with lung benign lesions (LBL). The comparison of autoimmune profiles between the early stage LC and the combined group of healthy and LBL allowed us to identify and validate a biomarker panel of p53, HRas, and ETHE1 for diagnosis of early stage LC with 50% sensitivity at >90% specificity. Finally, the performance of this biomarker panel was confirmed in ELISA tests. In summary, this study represents one of the most comprehensive proteome-wide surveys with one of the largest (i.e. 1,101 unique samples) and most diverse (i.e. nine disease groups) cohorts, resulting in a biomarker panel with good performance.

High-resolution physicochemical characterization of different intravenous immunoglobulin products.

Intravenous immunoglobulin (IVIg) is a complex mixture drug comprising diverse immunoglobulins and non-IgG proteins purified from the plasma of thousands of healthy donors. Approved IVIg products on the market differ regarding source of plasma, isolation process, and formulation. These products are used widely, and often interchangeably, for the treatment of immunodeficiency and autoimmune and inflammatory diseases, but their mechanisms of action in different indications are not well understood. A primary limitation to understanding the therapeutic relevance of specific components within IVIg has been the limited resolution of analytics historically implemented to characterize its complex mixture. In this study, high-resolution analytics were applied to better understand the composition of IVIg and product variations. We characterized three approved IVIg products: Gammagard®, Privigen®, and Octagam®. Differences in the distribution of molecular weight species, IgG sequence variants, isoforms, glycoforms, and the repertoire of previously reported antibody specificities were identified. We also compared the effect of aging on these products to identify changes in size distribution and posttranslational modifications. This type of characterization may provide insights into the specific factors and components of IVIg that may influence its activity and ultimately lead to optimization of IVIg products for use in autoimmune diseases.

Structural and genetic relationships of closely related O-antigens of Cronobacter spp. and Escherichia coli: C. sakazakii G2594 (serotype O4)/E. coli O103 and C. malonaticus G3864 (serotype O1)/E. coli O29.

O-Antigen (O-polysaccharide) variation is the basis for bacterial serotyping and is important in bacterial virulence and niche adaptation. In this work, we present structural and genetic evidences for close relationships between the O-antigens of the Cronobacter spp. and Escherichia coli. Cronobacter sakazakii G2594 (serotype O4) and Cronobacter malonaticus G3864 (serotype O1) are structurally related to those of E. coli O103 and O29, respectively, and some other members of the Enterobacteriaceae family differing in the patterns of lateral glucosylation (C. sakazakii G2594) or O-acetylation (C. malonaticus G3864). The O-antigen gene clusters of the corresponding Cronobacter and E. coli strains contain the same genes with high-level similarity, and the structural differences within both O-antigen pairs were suggested to be due to modification genes carried by prophages.

Related structures of the O-polysaccharides of Cronobacter dublinensis G3983 and G3977 containing 3-(N-acetyl-L-alanyl)amino-3,6-dideoxy-D-galactose.

Cronobacter spp. are emerging opportunistic human pathogens linked with life-threatening infections predominantly in neonates. O-Antigen (O-polysaccharide) is highly variable and plays an important role in virulence and niche adaptation. In this work, short-chain O-polysaccharides consisting on the average of 2-3 repeating units were obtained by mild acid or mild alkaline degradation of the lipopolysaccharides of C. dublinensis G3983 and G3977 and studied by composition analysis, Smith degradation, and (1)H and (13)C NMR spectroscopy. The following structures of the O-polysaccharides were established: [Formula: see text] where R indicates H in strain G3983 or α-D-Glcp in strain G3977, d-Fuc3NAlaAc indicates 3-(N-acetyl-L-alanyl)amino-3,6-dideoxy-D-galactose. Both strains share the O-antigen gene cluster, which is identical to that of C. dublinensis O1 (Foodborne Pathog. Dis.2013, 10, 343-352). The assigned gene functions are in agreement with the O-antigen structure of C. dublinensis G3983, and the side-chain glucosylation of the O-antigen of C. dublinensis G3977 is evidently encoded elsewhere in the genome.

Serine racemase regulated by binding to stargazin and PSD-95: potential N-methyl-D-aspartate-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (NMDA-AMPA) glutamate neurotransmission cross-talk.

D-Serine, an endogenous co-agonist for the glycine site of the synaptic NMDA glutamate receptor, regulates synaptic plasticity and is implicated in schizophrenia. Serine racemase (SR) is the enzyme that converts L-serine to D-serine. In this study, we demonstrate that SR interacts with the synaptic proteins, postsynaptic density protein 95 (PSD-95) and stargazin, forming a ternary complex. SR binds to the PDZ3 domain of PSD-95 through the PDZ domain ligand at its C terminus. SR also binds to the C terminus of stargazin, which facilitates the cell membrane localization of SR and inhibits its activity. AMPA receptor activation internalizes SR and disrupts its interaction with stargazin, therefore derepressing SR activity, leading to more D-serine production and potentially facilitating NMDA receptor activation. These interactions regulate the enzymatic activity as well as the intracellular localization of SR, potentially coupling the activities of NMDA and AMPA receptors. This shuttling of a neurotransmitter synthesizing enzyme between two receptors appears to be a novel mode of synaptic regulation.

Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain.

DNA methylation has critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single base-resolution DNA methylome from adult mouse dentate neurons consists of both CpG (~75%) and CpH (~25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in regions of low CpG density, depleted at protein-DNA interaction sites and anticorrelated with gene expression. Functionally, both methylated CpGs (mCpGs) and mCpHs can repress transcription in vitro and are recognized by methyl-CpG binding protein 2 (MeCP2) in neurons in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNA methyltransferase 3A (DNMT3A) for active maintenance in postmitotic neurons. These characteristics of CpH methylation suggest that a substantially expanded proportion of the neuronal genome is under cytosine methylation regulation and provide a new foundation for understanding the role of this key epigenetic modification in the nervous system.

DNA methylation presents distinct binding sites for human transcription factors.

DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI:http://dx.doi.org/10.7554/eLife.00726.001.

VirD: a virion display array for profiling functional membrane proteins.

To facilitate high-throughput biochemical analyses of membrane proteins, we have developed a novel display technology in a microarray format. Both single-pass (cluster of differentiation 4, CD4) and multiple-pass (G protein-coupled receptor 77, GPR77) human transmembrane proteins were engineered to be displayed in the membrane envelop of herpes simplex virions. These viruses produce large spherical virions displaying multiple copies of envelop proteins. Our aim was to engineer this virus to express these human proteins during the virus productive cycle and incorporate the human proteins into the virion during the assembly process. Another strategy presented includes engineering a fusion of glycoprotein C (gC), a major constituent of herpes simplex virus type 1 (HSV-1) virions, by hijacking the cis-acting signals to direct incorporation of the chimeric protein into the virion. The expression of the human proteins in infected cells, at the cell surface and in purified virions, is in the correct transmembrane orientation, and the proteins are biochemically functional. Purified virions printed on glass slides form a high-density Virion Display (VirD) Array, and the displayed proteins were demonstrated to retain their native conformations and interactions on the VirD Array judging by similar assays, such as antibody staining, as well as lectin and ligand binding. This method can be readily scaled or tailored for different modalities including a high-content, high-throughput platform for screening ligands and drugs of human membrane proteins.