Influenza poses a persistent health burden worldwide. To design equitable vaccines effective across all demographics, it is essential to better understand how host factors such as genetic background and aging affect the single -cell immune landscape of influenza infection. Cytometry by time -of -flight (CyTOF) represents a promising technique in this pursuit, but interpreting its large, high -dimensional data remains difficult. We have developed a new analytical approach, in silico gating annotating training elucidating (iGATE), based on probabilistic support vector machine classification. By rapidly and accurately "gating" tens of millions of cells in silico into user -defined types, iGATE enabled us to track 25 canonical immune cell types in mouse lung over the course of influenza infection. Applying iGATE to study effects of host genetic background, we show that the lower survival of C57BL/6 mice compared with BALB/c was associated with a more rapid accumulation of inflammatory cell types and decreased IL -10 expression. Furthermore, we demonstrate that the most prominent effect of aging is a defective T cell response, reducing survival of aged mice. Finally, iGATE reveals that the 25 canonical immune cell types exhibited differential influenza infection susceptibility and replication permissiveness in vivo, but neither property varied with host genotype or aging. The software is available at https://github.com/ UmichWenLab/iGATE.

Current cancer vaccines using T cell epitopes activate antitumor T cell immunity through dendritic cell/macrophage-mediated antigen presentation, but they lack the ability to promote B/CD4 T cell crosstalk, limiting their anticancer efficacy. We developed antigen-clustered nanovaccine (ACNVax) to achieve long-term tumor remission by promoting B/CD4 T cell crosstalk. The topographic features of ACNVax were achieved using an iron nanoparticle core attached with an optimal number of gold nanoparticles, where the clusters of HER2 B/CD4 T cell epitopes were conjugated on the gold surface with an optimal intercluster distance of 5-10 nm. ACNVax effectively trafficked to lymph nodes and cross-linked with BCR, which are essential for stimulating B cell antigen presentation-mediated B/CD4 T cell crosstalk in vitro and in vivo. ACNVax, combined with anti-PD-1, achieved long-term tumor remission (>200 days) with 80% complete response in mice with HER2+ breast cancer. ACNVax not only remodeled the tumor immune microenvironment but also induced a long-term immune memory, as evidenced by complete rejection of tumor rechallenge and a high level of antigen-specific memory B, CD4, and CD8 cells in mice (>200 days). This study provides a cancer vaccine design strategy, using B/CD4 T cell epitopes in an antigen clustered topography, to achieve long-term durable anticancer efficacy through promoting B/CD4 T cell crosstalk.

BACKGROUND:Palmoplantar pustulosis (PPP) is an inflammatory disease characterized by relapsing eruptions of neutrophil-filled, sterile pustules on the palms and soles that can be clinically difficult to differentiate from non-pustular palmoplantar psoriasis (palmPP) and dyshidrotic palmoplantar eczema (DPE). OBJECTIVE:We sought to identify overlapping and unique PPP, palmPP, and DPE drivers to provide molecular insight into their pathogenesis. METHODS:We performed bulk RNA sequencing of lesional PPP (n = 33), palmPP (n = 5), and DPE (n = 28) samples, as well as 5 healthy nonacral and 10 healthy acral skin samples. RESULTS:Acral skin showed a unique immune environment, likely contributing to a unique niche for palmoplantar inflammatory diseases. Compared to healthy acral skin, PPP, palmPP, and DPE displayed a broad overlapping transcriptomic signature characterized by shared upregulation of proinflammatory cytokines (TNF, IL-36), chemokines, and T-cell-associated genes, along with unique disease features of each disease state, including enriched neutrophil processes in PPP and to a lesser extent in palmPP, and lipid antigen processing in DPE. Strikingly, unsupervised clustering and trajectory analyses demonstrated divergent inflammatory profiles within the 3 disease states. These identified putative key upstream immunologic switches, including eicosanoids, interferon responses, and neutrophil degranulation, contributing to disease heterogeneity. CONCLUSION:A molecular overlap exists between different inflammatory palmoplantar diseases that supersedes clinical and histologic assessment. This highlights the heterogeneity within each condition, suggesting limitations of current disease classification and the need to move toward a molecular classification of inflammatory acral diseases.

Cellulosic biofuel represents a sustainable alternative to fossil fuels, yet high cellulase costs hinder its development. Thermostable cellulosomes, which function at elevated temperatures, increase reaction rates and reduce cooling costs by using cohesin-dockerin interactions to colocalize hyperthermostable cellulases. Due to the noncovalent nature of the cohesin-dockerin interaction, cellulosome stability has been limited to 75 degrees C. Our study leverages computational design and rapid screening to introduce two different intermolecular disulfide bridges between the same cohesin and dockerin, creating two disulfide cohesin-dockerin pairs. Both disulfide pairs withstood 100 degrees C and denaturing conditions. Furthermore, the two disulfide bridges retained their orthogonality, expanding the number of orthogonal cohesin-dockerin interactions. Finally, at the cellulase optimal temperature of 80 degrees C, disulfide assembly improved the activity of a bivalent cellulosome by 26% compared to that of its noncovalent counterpart. These disulfide cohesin-dockerin interactions can be used as building blocks to construct covalent protein complexes that can endure extreme temperatures.

CD4+ T cells play a vital role in the immune response, and their function requires T cell receptor (TCR) recognition of peptide epitopes presented in complex with MHC class II (MHCII) molecules. Consequently, rapidly identifying peptides that bind MHCII is critical to understanding and treating infectious disease, cancer, autoimmunity, allergy, and transplant rejection. Computational methods provide a fast, ultrahigh-throughput approach to predict MHCII-binding peptides but lack the accuracy of experimental methods. In contrast, experimental methods offer accurate, quantitative results at the expense of speed. To address the gap between these two approaches, we developed a high-throughput, semiquantitative experimental screening strategy termed microsphere-assisted peptide screening (MAPS). Here, we use the Zika virus envelope protein as an example to demonstrate the rapid identification of MHCII-binding peptides from a single pathogenic protein using MAPS. This process involves several key steps including peptide library design, peptide exchange into MHCII, peptide-MHCII loading onto microspheres, flow cytometry screening, and data analysis to identify peptides that bind to one or more MHCII alleles.

Immunomodulators that remodel the tumor immunosuppressive microenvironment have been combined with anti-programmed death 1 (α-PD1) or anti-programmed death ligand 1 (α-PDL1) immunotherapy but have shown limited success in clinical trials. However, therapeutic strategies to modulate the immunosuppressive microenvironment of lymph nodes have been largely overlooked. Here, we designed an albumin nanoparticle, Nano-PI, containing the immunomodulators PI3Kγ inhibitor (IPI-549) and paclitaxel (PTX). We treated two breast cancer mouse models with Nano-PI in combination with α-PD1, which remodeled the tumor microenvironment in both lymph nodes and tumors. This combination achieved long-term tumor remission in mouse models and eliminated lung metastases. PTX combined with IPI-549 enabled the formation of a stable nanoparticle and enhanced the repolarization of M2 to M1 macrophages. Nano-PI not only enhanced the delivery of both immunomodulators to lymph nodes and tumors but also improved the drug accumulation in the macrophages of these two tissues. Immune cell profiling revealed that the combination of Nano-PI with α-PD1 remodeled the immune microenvironment by polarizing M2 to M1 macrophages, increasing CD4+ and CD8+ T cells, B cells, and dendritic cells, decreasing regulatory T cells, and preventing T cell exhaustion. Our data suggest that Nano-PI in combination with α-PD1 modulates the immune microenvironment in both lymph nodes and tumors to achieve long-term remission in mice with metastatic breast cancer, and represents a promising candidate for future clinical trials.

Over the past decade, the increasingly globalized society has continually redefined the qualities and skills of an ideal engineering graduate for industry and academic careers, and, more recently, in light of a global pandemic in 2020, the pedagogical environment has shifted toward a virtual classroom setting. Because the engineering and social challenges of the modern world are rapidly evolving, it is important to adapt teaching methods that reflect these changing times. An increasingly attractive teaching method in the engineering classroom is project-based learning (PBL), which is known to improve engaged-learning outcomes, such as creativity, risk taking, social responsibility, teamwork, self-confidence, and communication. However, it is still unclear how various PBL practices differentially impact these engaged-learning outcomes. Toward the goal of elucidating this, the impact of two different project formats, a virtual presentation versus an in-person presentation, was evaluated for a junior-level chemical engineering core course, Mass and Heat Transfer, over 2 years (248 students total). In surveys conducted after the projects were completed, students were asked to what degree the project improved each of the learning outcomes on a scale of 0 (no impact) to 10 (great impact). Data from these postproject surveys showed no statistically significant differences in impact on teamwork, self-confidence, and communication skills between the two groups. However, the virtual presentation had statistically significant greater positive impacts on student creativity [mean score: 8.9/10 (virtual) vs 7.7/10 (in-person); p < 0.001] and risk taking [mean score: 7.7/10 (virtual) vs 6.1/10 (in-person); p < 0.001], whereas the in-person presentation had a significantly more positive impact on social responsibility [mean score: 6.5/10 (in-person) vs 5.5/10 (virtual); p < 0.05]. Qualitative insights into these results were gathered from discussions with students in focus groups. The results of this study underscore the unique advantages associated with different presentation formats. From the perspective of the current transitions to online learning, the results suggest that changing project deliverables from an in-person to a virtual format may actually yield net gains in engaged-learning outcomes.

Despite promising developments in computational tools, peptide-class II MHC (MHCII) binding predictors continue to lag behind their peptide-class I MHC counterparts. Consequently, peptide-MHCII binding is often evaluated experimentally using competitive binding assays, which tend to sacrifice throughput for quantitative binding detail. Here, we developed a high-throughput semiquantitative peptide-MHCII screening strategy termed microsphere-assisted peptide screening (MAPS) that aims to balance the accuracy of competitive binding assays with the throughput of computational tools. Using MAPS, we screened a peptide library from Zika virus envelope (E) protein for binding to four common MHCII alleles (DR1, DR4, DR7, DR15). Interestingly, MAPS revealed a significant overlap between peptides that promiscuously bind multiple MHCII alleles and antibody neutralization sites. This overlap was also observed for rotavirus outer capsid glycoprotein VP7, suggesting a deeper relationship between B cell and CD4(+) T cell specificity which can facilitate the design of broadly protective vaccines to Zika and other viruses.

Cooperative enzyme catalysis in nature has long inspired the application of engineered multi-enzyme assemblies for industrial biocatalysis. Despite considerable interest, efforts to harness the activity of cell-surface displayed multi-enzyme assemblies have been based on trial and error rather than rational design due to a lack of quantitative tools. In this study, we have developed a quantitative approach to whole-cell biocatalyst characterization, enabling a comprehensive study of how yeast-surface displayed multi-enzyme assemblies form. Here we show that the multi-enzyme assembly efficiency is limited by molecular crowding on the yeast-cell surface, and that maximizing enzyme density is the most important parameter for enhancing cellulose hydrolytic performance. Interestingly, we also observed that proximity effects are only synergistic when the average inter-enzyme distance is greater than ~130 nm. The findings and the quantitative approach developed in this work should help to advance the field of biocatalyst engineering from trial and error to rational design. Efforts to harness the cellulolytic activity of enzyme assemblies have been mainly empirical due to the lack of quantification tools. Now this work reports experimental and theoretical approaches to quantify enzyme assemblies, revealing the parameters that are important for cellulolytic activity.

ADVERTISEMENT RETURN TO ISSUEPREVViewpointNEXTEngineering Spatially Organized Multienzyme Assemblies for Complex Chemical TransformationLuke F. BugadaLuke F. BugadaDepartment of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United StatesMore by Luke F. Bugada, Mason R. SmithMason R. SmithDepartment of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United StatesMore by Mason R. Smith, and Fei Wen*Fei WenDepartment of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States*E-mail: [email protected]More by Fei Wenhttp://orcid.org/0000-0001-7970-4796Cite this: ACS Catal. 2018, 8, 9, 7898–7906Publication Date (Web):July 17, 2018Publication History Received15 May 2018Published online17 July 2018Published inissue 7 September 2018https://pubs.acs.org/doi/10.1021/acscatal.8b01883https://doi.org/10.1021/acscatal.8b01883editorialACS PublicationsCopyright © 2018 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views3581Altmetric-Citations37LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (2 MB) Get e-AlertscloseSUBJECTS:Chemical reactions,Genetics,Metal organic frameworks,Peptides and proteins,Polymer scaffolds Get e-Alerts