Development of a biomarker signature using grating-coupled fluorescence plasmonic microarray for diagnosis of MIS-C.

Multisystem inflammatory syndrome in children (MIS-C) is a rare but serious condition that can develop 4-6 weeks after a school age child becomes infected by SARS-CoV-2. To date, in the United States more than 8,862 cases of MIS-C have been identified and 72 deaths have occurred. This syndrome typically affects children between the ages of 5-13; 57% are Hispanic/Latino/Black/non-Hispanic, 61% of patients are males and 100% have either tested positive for SARS-CoV-2 or had direct contact with someone with COVID-19. Unfortunately, diagnosis of MIS-C is difficult, and delayed diagnosis can lead to cardiogenic shock, intensive care admission, and prolonged hospitalization. There is no validated biomarker for the rapid diagnosis of MIS-C. In this study, we used Grating-coupled Fluorescence Plasmonic (GCFP) microarray technology to develop biomarker signatures in pediatric salvia and serum samples from patients with MIS-C in the United States and Colombia. GCFP measures antibody-antigen interactions at individual regions of interest (ROIs) on a gold-coated diffraction grating sensor chip in a sandwich immunoassay to generate a fluorescent signal based on analyte presence within a sample. Using a microarray printer, we designed a first-generation biosensor chip with the capability of capturing 33 different analytes from 80  µ L of sample (saliva or serum). Here, we show potential biomarker signatures in both saliva and serum samples in six patient cohorts. In saliva samples, we noted occasional analyte outliers on the chip within individual samples and were able to compare those samples to 16S RNA microbiome data. These comparisons indicate differences in relative abundance of oral pathogens within those patients. Microsphere Immunoassay (MIA) of immunoglobulin isotypes was also performed on serum samples and revealed MIS-C patients had several COVID antigen-specific immunoglobulins that were significantly higher than other cohorts, thus identifying potential new targets for the second-generation biosensor chip. MIA also identified additional biomarkers for our second-generation chip, verified biomarker signatures generated on the first-generation chip, and aided in second-generation chip optimization. Interestingly, MIS-C samples from the United States had a more diverse and robust signature than the Colombian samples, which was also illustrated in the MIA cytokine data. These observations identify new MIS-C biomarkers and biomarker signatures for each of the cohorts. Ultimately, these tools may represent a potential diagnostic tool for use in the rapid identification of MIS-C.

Draft Genome Sequence of Plesiomonas shigelloides MD22D9, Isolated from the Digestive Tract of Macrobdella decora.

Here, we present a draft genome sequence of Plesiomonas shigelloides MD22D9, isolated from the digestive tract of the North American medicinal leech Macrobdella decora. The gut microbiome of the medicinal leech is hypothesized to be critical for maintaining host fitness. This genome can provide insights into this uncharacterized microbe-host relationship.

Phylogenetic spread of sequence data affects fitness of consensus enzymes: Insights from triosephosphate isomerase.

The concept of consensus in multiple sequence alignments (MSAs) has been used to design and engineer proteins previously with some success. However, consensus design implicitly assumes that all amino acid positions function independently, whereas in reality, the amino acids in a protein interact with each other and work cooperatively to produce the optimum structure required for its function. Correlation analysis is a tool that can capture the effect of such interactions. In a previously published study, we made consensus variants of the triosephosphate isomerase (TIM) protein using MSAs that included sequences form both prokaryotic and eukaryotic organisms. These variants were not completely native-like and were also surprisingly different from each other in terms of oligomeric state, structural dynamics, and activity. Extensive correlation analysis of the TIM database has revealed some clues about factors leading to the unusual behavior of the previously constructed consensus proteins. Among other things, we have found that the more ill-behaved consensus mutant had more broken correlations than the better-behaved consensus variant. Moreover, we report three correlation and phylogeny-based consensus variants of TIM. These variants were more native-like than the previous consensus mutants and considerably more stable than a wild-type TIM from a mesophilic organism. This study highlights the importance of choosing the appropriate diversity of MSA for consensus analysis and provides information that can be used to engineer stable enzymes.

Linker engineering in anti-TAG-72 antibody fragments optimizes biophysical properties, serum half-life, and high-specificity tumor imaging.

Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scFV), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scFV binding, stability, quaternary structure, and other biophysical properties. Comprehensive studies of these attributes in a single scaffold have not been reported, making design and optimization of Ab fragments challenging. Here, we constructed libraries of 3E8, an Ab specific to tumor-associated glycoprotein 72 (TAG-72), a mucinous glycoprotein overexpressed in 80% of adenocarcinomas. We cloned, expressed, and characterized scFVs, diabodies, and higher-order multimer constructs with varying linker compositions, linker lengths, and domain orientations. These constructs dramatically differed in their oligomeric states and stabilities, not only because of linker and orientation but also related to the purification method. For example, protein L-purified constructs tended to have broader distributions and higher oligomeric states than has been reported previously. From this library, we selected an optimal construct, 3E8.G4S, for biodistribution and pharmacokinetic studies and in vivo xenograft mouse PET imaging. These studies revealed significant tumor targeting of 3E8.G4S with a tumor-to-background ratio of 29:1. These analyses validated 3E8.G4S as a fast, accurate, and specific tumor-imaging agent.

A 3E8.scFv.Cys-IR800 Conjugate Targeting TAG-72 in an Orthotopic Colorectal Cancer Model.

PURPOSE: Optical surgical navigation (OSN) will be a potent tool to help surgeons more accurately and efficiently remove tumors. The purpose of this study was to evaluate a novel humanized 3E8 antibody (3E8 MAb) fragment site-specifically conjugated with IR800, 3E8.scFv.Cys-IR800, as a potential OSN agent to target colorectal adenocarcinoma. PROCEDURES: An engineered single-chain variable fragment of 3E8 MAb (targeted to TAG-72), appending a C-terminal cysteine residue (3E8.scFv.Cys), was created and reacted with IRDye800-maleimide. 3E8.scFv.Cys-IR800 identity and purity were verified by MALDI-TOF mass spectra and 800 nm detected size exclusion column HPLC. In vitro human colon adenocarcinoma LS-174 T cells binding and competition assay validated biological functionality. We further evaluated the imaging ability and receptor-specific binding of 3E8.scFv.Cys-IR800 in an orthotopic LS-174 T mouse model. RESULTS: A 1:1 dye to protein conjugate was achieved at greater than 90 % HPLC purity. A 1 nmol dose of 3E8.scFv.Cys-IR800 via intraperitoneal injection administration was sufficient to produce high tumor to background fluorescence contrast. Blocking competition studies both in vitro and in vivo using a different blocking protein, 3E8ΔCH2, demonstrated 3E8.scFv.Cys-IR800 binding specificity for TAG-72 antigen. CONCLUSIONS: 3E8.scFv.Cys-IR800 shows properties useful in a clinically viable OSN agent for colorectal cancer.

IgE-activated basophils regulate eosinophil tissue entry by modulating endothelial function.

Vertebrate immunity has evolved a modular architecture in response to perturbations. Allergic inflammation represents such a module, with signature features of antigen-specific IgE and tissue eosinophilia, although the cellular and molecular circuitry coupling these responses remains unclear. Here, we use genetic and imaging approaches in models of IgE-dependent eosinophilic dermatitis to demonstrate a requisite role for basophils. After antigenic inflammation, basophils initiate transmigration like other granulocytes but, upon activation via their high-affinity IgE receptor, alter their migratory kinetics to persist at the endothelium. Prolonged basophil-endothelial interactions, in part dependent on activation of focal adhesion kinases, promote delivery of basophil-derived IL-4 to the endothelium and subsequent induction of endothelial vascular cell adhesion molecule-1 (VCAM-1), which is required for eosinophil accumulation. Thus, basophils are gatekeepers that link adaptive immunity with innate effector programs by altering access to tissue sites by activation-induced interactions with the endothelium.

Understanding the sequence requirements of protein families: insights from the BioVis 2013 contests.

INTRODUCTION: In 2011, the BioVis symposium of the IEEE VisWeek conferences inaugurated a new variety of data analysis contest. Aimed at fostering collaborations between computational scientists and biologists, the BioVis contest provided real data from biological domains with emerging visualization needs, in the hope that novel approaches would result in powerful new tools for the community. In 2011 and 2012 the theme of these contests was expression Quantitative Trait Locus analysis, within and across tissues respectively. In 2013 the topic was updated to protein sequence and mutation visualization. METHODS: The contest was framed in the context of a real protein with numerous mutations that had lost function, and the question posed "what minimal set of changes would you propose to rescue function, or how could you support a biologist attempting to answer that question?". The data was grounded in actual experimental results in triosephosphate isomerase(TIM) enzymes. Seven teams composed of 36 individuals submitted entries with proposed solutions and approaches to the challenge. Their contributions ranged from careful analysis of the visualization and analytical requirements for the problem through integration of existing tools for analyzing the context and consequences of protein mutations, to completely new tools addressing the problem. RESULTS: Judges found valuable and novel contributions in each of the entries, including interesting ways to hierarchicalize the protein into domains of informational interaction, tools for simultaneously understanding both sequential and spatial order, and approaches for conveying some types of inter-residue dependencies. In this manuscript we document the problem presented to the contestants, summarize the biological contributions of their entries, and suggest opportunities that this work has highlighted for even more improved tools in the future.

Simplifying protein expression with ligation-free, traceless and tag-switching plasmids.

Synthetic biology and genome-scale protein work both require rapid and efficient cloning, expression and purification. Tools for co-expression of multiple proteins and production of fusion proteins with purification and solubility tags are often desirable. Here we present a survey of plasmid vectors that provide for some of these features with a focus on tools for rapid cloning and traceless tagging - a setup that facilitates removal of fusion tags post-purification leaving behind no 'scar' on the final construct. Key features are reviewed, including plasmid replication origins and resistance markers, transcriptional promoters, cloning methods, and fusion tags and their removal by proteolysis. We describe a vector system called pHLIC, which assembles features for simple cloning, overexpression, facile purification, and traceless cleavage, as well as flexibility in modifying the vector to exchange fusion tags.

Stabilizing proteins from sequence statistics: the interplay of conservation and correlation in triosephosphate isomerase stability.

Understanding the determinants of protein stability remains one of protein science's greatest challenges. There are still no computational solutions that calculate the stability effects of even point mutations with sufficient reliability for practical use. Amino acid substitutions rarely increase the stability of native proteins; hence, large libraries and high-throughput screens or selections are needed to stabilize proteins using directed evolution. Consensus mutations have proven effective for increasing stability, but these mutations are successful only about half the time. We set out to understand why some consensus mutations fail to stabilize, and what criteria might be useful to predict stabilization more accurately. Overall, consensus mutations at more conserved positions were more likely to be stabilizing in our model, triosephosphate isomerase (TIM) from Saccharomyces cerevisiae. However, positions coupled to other sites were more likely not to stabilize upon mutation. Destabilizing mutations could be removed both by removing sites with high statistical correlations to other positions and by removing nearly invariant positions at which "hidden correlations" can occur. Application of these rules resulted in identification of stabilizing mutations in 9 out of 10 positions, and amalgamation of all predicted stabilizing positions resulted in the most stable yeast TIM variant we produced (+8 °C). In contrast, a multimutant with 14 mutations each found to stabilize TIM independently was destabilized by 2 °C. Our results are a practical extension to the consensus concept of protein stabilization, and they further suggest the importance of positional independence in the mechanism of consensus stabilization.

Fluorescent in vivo detection reveals that IgE(+) B cells are restrained by an intrinsic cell fate predisposition.

IgE antibodies may be protective in parasite immunity, but their aberrant production can lead to allergic disease and life-threatening anaphylaxis. Despite the importance of IgE regulation, few studies have directly examined the B cells that express IgE, because these cells are rare and difficult to detect. Here, we describe fluorescent IgE reporter mice and validate a flow cytometry procedure to allow sensitive and specific identification of IgE-expressing B cells in vivo. Similar to IgG1(+) cells, IgE(+) B cells differentiated into germinal center (GC) B cells and plasma cells (PCs) during primary immune responses to a T cell-dependent hapten-protein conjugate and the helminth Nippostrongylus brasiliensis. However, the participation of IgE(+) B cells in GCs was transient. IgE(+) B cells had an atypical propensity to upregulate the transcription factor Blimp-1 and undergo PC differentiation. Most IgE(+) PCs were short lived and showed reduced affinity maturation, revealing intrinsic mechanisms that restrict the IgE antibody response.

Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity.

Interleukin 4 (IL-4) and IL-13 are critical for responses to parasitic helminthes. We used genetically engineered reporter mice to assess the temporal and spatial production of these cytokines in vivo. In lymph nodes, IL-4, but not IL-13, was made by follicular helper T cells (T(FH) cells). In contrast, tissue type 2 helper T cells (T(H)2 cells) produced both cytokines. There was also divergent production of IL-4 and IL-13 among cells of the innate immune system, whereby basophils produced IL-4, whereas innate helper type 2 cells (Ih2 cells) produced IL-13. IL-13 production by T(H)2 and Ih2 cells was dependent on the transcription factor GATA-3, which was present in large amounts in these cells, and in contrast to the small amount of GATA-3 in T(FH) cells and basophils. The distinct localization and cellular expression of IL-4 and IL-13 explains their unique roles during allergic immunity.

Deletion of Syk in neutrophils prevents immune complex arthritis.

The K/BxN serum transfer model of arthritis is critically dependent on FcγR signaling events mediated by spleen tyrosine kinase (Syk). However, the specific cell types in which this signaling is required are not known. We report that deletion of Syk in neutrophils, achieved using syk(f/f) MRP8-cre(+) mice, blocks disease development in serum transfer arthritis. The syk(f/f) MRP8-cre(+) mice display absent joint disease and reduced deposition of pathogenic anti-glucose-6-phosphate isomerase Abs in the joint (with a reciprocal accumulation of these Abs in the peripheral circulation). Additionally, syk(f/f) MRP8-cre(+) mice manifest poor edema formation within 3 h after formation of cutaneous immune complexes (Arthus reaction). Together, this suggests that neutrophil-dependent recognition of immune complexes contributes significantly to changes in vascular permeability during the early phases of immune complex disease. Using mixed chimeric mice, containing both wild-type and syk(f/f) MRP8-cre(+) neutrophils, we find no impairment in recruitment of Syk-deficient neutrophils to the inflamed joint, but they fail to become primed, demonstrating lower cytokine production after removal from the joint. They also display an increased apoptotic rate compared with wild-type cells in the same joint. Mast cell-deficient c-kit(sh/sh) mice developed robust arthritis after serum transfer whereas c-kit(W/Wv) mice did not, suggesting that previous conclusions concerning the central role of mast cells in this model may need to be revised. Basophil-deficient mice also responded normally to K/BxN serum transfer. These results demonstrate that Syk-dependent signaling in neutrophils alone is critically required for arthritis development in the serum transfer model.

Triosephosphate isomerase by consensus design: dramatic differences in physical properties and activity of related variants.

Consensus design, the selection of mutations based on the most common amino acid in each position of a multiple sequence alignment, has proven to be an efficient way to engineer stabilized mutants and even to design entire proteins. However, its application has been limited to small motifs or small families of highly related proteins. Also, we have little idea of how information that specifies a protein's properties is distributed between positional effects (consensus) and interactions between positions (correlated occurrences of amino acids). Here, we designed several consensus variants of triosephosphate isomerase (TIM), a large, diverse family of complex enzymes. The first variant was only weakly active, had molten globular characteristics, and was monomeric at 25 °C despite being based on nearly all dimeric enzymes. A closely related variant from curation of the sequence database resulted in a native-like dimeric TIM with near-diffusion-controlled kinetics. Both enzymes vary substantially (30-40%) from any natural TIM, but they differ from each other in only a relatively small number of unconserved positions. We demonstrate that consensus design is sufficient to engineer a sophisticated protein that requires precise substrate positioning and coordinated loop motion. The difference in oligomeric states and native-like properties for the two consensus variants is not a result of defects in the dimerization interface but rather disparate global properties of the proteins. These results have important implications for the role of correlated amino acids, the ability of TIM to function as a monomer, and the ability of molten globular proteins to carry out complex reactions.

Genetic analysis of basophil function in vivo.

Contributions by basophils to allergic and helminth immunity remain incompletely defined. Using sensitive interleukin 4 (Il4) reporter alleles, we demonstrate here that basophil IL-4 production occurs by a CD4(+) T cell-dependent process restricted to the peripheral tissues affected. We genetically marked and achieved specific deletion of basophils and found that basophils did not mediate T helper type 2 (T(H)2) priming in vivo. Two-photon imaging confirmed that basophils did not interact with antigen-specific T cells in lymph nodes but engaged in prolonged serial interactions with T cells in lung tissues. Although targeted deletion of IL-4 and IL-13 in either CD4(+) T cells or basophils had a minimal effect on worm clearance, deletion from both lineages demonstrated a nonredundant role for basophil cytokines in primary helminth immunity.

Protein stability by number: high-throughput and statistical approaches to one of protein science's most difficult problems.

Most proteins are only barely stable, which impedes research, complicates therapeutic applications, and makes proteins susceptible to pathologically destabilizing mutations. Our ability to predict the thermodynamic consequences of even single point mutations is still surprisingly limited, and established methods of measuring stability are slow. Recent advances are bringing protein stability studies into the high-throughput realm. Some methods are based on inferential read-outs such as activity, proteolytic resistance or split-protein fragment reassembly. Other methods use miniaturization of direct measurements, such as intrinsic fluorescence, H/D exchange, cysteine reactivity, aggregation and hydrophobic dye binding (DSF). Protein engineering based on statistical analysis (consensus and correlated occurrences of amino acids) is promising, but much work remains to understand and implement these methods.

Systemically dispersed innate IL-13-expressing cells in type 2 immunity.

Type 2 immunity is a stereotyped host response to allergens and parasitic helminths that is sustained in large part by the cytokines IL-4 and IL-13. Recent advances have called attention to the contributions by innate cells in initiating adaptive immunity, including a novel lineage-negative population of cells that secretes IL-13 and IL-5 in response to the epithelial cytokines IL-25 and IL-33. Here, we use IL-4 and IL-13 reporter mice to track lineage-negative innate cells that arise during type 2 immunity or in response to IL-25 and IL-33 in vivo. Unexpectedly, lineage-negative IL-25 (and IL-33) responsive cells are widely distributed in tissues of the mouse and are particularly prevalent in mesenteric lymph nodes, spleen, and liver. These cells expand robustly in response to exogenous IL-25 or IL-33 and after infection with the helminth Nippostrongylus brasiliensis, and they are the major innate IL-13-expressing cells under these conditions. Activation of these cells using IL-25 is sufficient for worm clearance, even in the absence of adaptive immunity. Widely dispersed innate type 2 helper cells, which we designate Ih2 cells, play an integral role in type 2 immune responses.

IL-18 and IL-33 elicit Th2 cytokines from basophils via a MyD88- and p38alpha-dependent pathway.

IL-4 and IL-13 are instrumental in the development and progression of allergy and atopic disease. Basophils represent a key source of these cytokines and produce IL-4 and IL-13 when stimulated with IL-18, a member of the IL-1 family of cytokines. Comparative analyses of the effects of caspase-1-dependent IL-1 family cytokines on basophil IL-4 and IL-13 production have not been performed, and the signaling pathway proteins required for FcepsilonRI-independent Th2 cytokine production from basophils remain incompletely defined. Using mouse bone marrow-derived cultured basophils, we found that IL-4 and IL-13 are produced in response to IL-18 or IL-33 stimulation. IL-18- or IL-33-mediated Th2 cytokine production is dependent on MyD88 and p38alpha signaling proteins. In addition, basophil survival increased in the presence of IL-18 or IL-33 as a result of increased Akt activation. Studies in vivo confirmed the potency of IL-18 and IL-33 in activating cytokine release from mouse basophils.

High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering.

The low stability of natural proteins often limits their use in therapeutic, industrial, and research applications. The scale and throughput of methods such as circular dichroism, fluorescence spectroscopy, and calorimetry severely limit the number of variants that can be examined. Here we demonstrate a high-throughput thermal scanning (HTTS) method for determining the approximate stabilities of protein variants at high throughput and low cost. The method is based on binding to a hydrophobic dye akin to ANS, which fluoresces upon binding to molten globules and thermal denaturation intermediates. No inherent properties of the protein, such as enzymatic activity or presence of an intrinsic fluorophore, are required. Very small sample sizes are analyzed using a real-time PCR machine, enabling the use of high-throughput purification. We show that the apparent T(M) values obtained from HTTS are approximately linearly related to those from CD thermal denaturation for a series of four-helix bundle hydrophobic core variants. We demonstrate similar results for a small set of TIM barrel variants. This inexpensive, general, and scaleable approach enables the search for conservative, stable mutants of biotechnologically important proteins and provides a method for statistical correlation of sequence-stability relationships.

Basophils: a nonredundant contributor to host immunity.

The role of basophils, the rarest of blood granulocytes, in host immunity has been a mystery. Long considered the poor relative of mast cells, basophils have received much recent attention because of the availability of new reagents and models that reveal unique properties of these cells. Basophils are known to have distinct roles in allergic hypersensitivity reactions and in the immune response to intestinal helminthes. In this review, we highlight these advances and summarize our current understanding of the repertoire of functions attributed to these cells. Despite these recent insights, we are likely only beginning to gain a full understanding of how and where these cells lend effector functions to vertebrate immunity. Advances are likely to come only with the development of specific reagents that enable the finer study of basophil lineage and function. Although many fundamental aspects of basophil biology remain unanswered, the prospects remain bright for unmasking new contributions by these unusual cells.

Normal development and activation but altered cytokine production of Fyn-deficient CD4+ T cells.

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4(+) T cell responses. In Fyn-deficient mice, CD4(+) T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10(+)CD4(+) T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323-339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10(+) Fyn-deficient CD4(+) T cells, was virtually indistinguishable from the response of DO11.10(+) wild-type CD4(+) T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4(+) Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10(+) Fyn-deficient CD4(+) T cells were more potent cytokine-producing cells than DO11.10(+) wild-type CD4(+) T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4(+) T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4(+) T cells.