Intratumoral radiation dose heterogeneity augments antitumor immunity in mice and primes responses to checkpoint blockade.

Radiation therapy (RT) activates multiple immunologic effects in the tumor microenvironment (TME), with diverse dose-response relationships observed. We hypothesized that, in contrast with homogeneous RT, a heterogeneous RT dose would simultaneously optimize activation of multiple immunogenic effects in a single TME, resulting in a more effective antitumor immune response. Using high-dose-rate brachytherapy, we treated mice bearing syngeneic tumors with a single fraction of heterogeneous RT at a dose ranging from 2 to 30 gray. When combined with dual immune checkpoint inhibition in murine models, heterogeneous RT generated more potent antitumor responses in distant, nonirradiated tumors compared with any homogeneous dose. The antitumor effect after heterogeneous RT required CD4 and CD8 T cells and low-dose RT to a portion of the tumor. At the 3-day post-RT time point, dose heterogeneity imprinted the targeted TME with spatial differences in immune-related gene expression, antigen presentation, and susceptibility of tumor cells to immune-mediated destruction. At a later 10-day post-RT time point, high-, moderate-, or low-RT-dose regions demonstrated distinct infiltrating immune cell populations. This was associated with an increase in the expression of effector-associated cytokines in circulating CD8 T cells. Consistent with enhanced adaptive immune priming, heterogeneous RT promoted clonal expansion of effector CD8 T cells. These findings illuminate the breadth of dose-dependent effects of RT on the TME and the capacity of heterogeneous RT to promote antitumor immunity when combined with immune checkpoint inhibitors.

Assessing immune factors in maternal milk and paired infant plasma antibody binding to human rhinoviruses.

Introduction: Before they can produce their own antibodies, newborns are protected from infections by transplacental transfer of maternal IgG antibodies and after birth through breast milk IgA antibodies. Rhinovirus (RV) infections are extremely common in early childhood, and while RV infections often result in only mild upper respiratory illnesses, they can also cause severe lower respiratory illnesses such as bronchiolitis and pneumonia. Methods: We used high-density peptide arrays to profile infant and maternal antibody reactivity to capsid and full proteome sequences of three human RVs - A16, B52, and C11. Results: Numerous plasma IgG and breast milk IgA RV epitopes were identified that localized to regions of the RV capsid surface and interior, and also to several non-structural proteins. While most epitopes were bound by both IgG and IgA, there were several instances where isotype-specific and RV-specific binding were observed. We also profiled 62 unique RV-C protein loop sequences characteristic of this species' capsid VP1 protein. Discussion: Many of the RV-C loop sequences were highly bound by IgG from one-year-old infants, indicating recent or ongoing active infections, or alternatively, a level of cross-reactivity among homologous RV-C sites.

MPAC: a computational framework for inferring cancer pathway activities from multi-omic data.

Fully capturing cellular state requires examining genomic, epigenomic, transcriptomic, proteomic, and other assays for a biological sample and comprehensive computational modeling to reason with the complex and sometimes conflicting measurements. Modeling these so-called multi-omic data is especially beneficial in disease analysis, where observations across omic data types may reveal unexpected patient groupings and inform clinical outcomes and treatments. We present Multi-omic Pathway Analysis of Cancer (MPAC), a computational framework that interprets multi-omic data through prior knowledge from biological pathways. MPAC uses network relationships encoded in pathways using a factor graph to infer consensus activity levels for proteins and associated pathway entities from multi-omic data, runs permutation testing to eliminate spurious activity predictions, and groups biological samples by pathway activities to prioritize proteins with potential clinical relevance. Using DNA copy number alteration and RNA-seq data from head and neck squamous cell carcinoma patients from The Cancer Genome Atlas as an example, we demonstrate that MPAC predicts a patient subgroup related to immune responses not identified by analysis with either input omic data type alone. Key proteins identified via this subgroup have pathway activities related to clinical outcome as well as immune cell compositions. Our MPAC R package, available at https://bioconductor.org/packages/MPAC, enables similar multi-omic analyses on new datasets.

Dietary isoleucine content defines the metabolic and molecular response to a Western diet.

The amino acid composition of the diet has recently emerged as a critical regulator of metabolic health. Consumption of the branched-chain amino acid isoleucine is positively correlated with body mass index in humans, and reducing dietary levels of isoleucine rapidly improves the metabolic health of diet-induced obese male C57BL/6J mice. However, it is unknown how sex, strain, and dietary isoleucine intake may interact to impact the response to a Western Diet (WD). Here, we find that although the magnitude of the effect varies by sex and strain, reducing dietary levels of isoleucine protects C57BL/6J and DBA/2J mice of both sexes from the deleterious metabolic effects of a WD, while increasing dietary levels of isoleucine impairs aspects of metabolic health. Despite broadly positive responses across all sexes and strains to reduced isoleucine, the molecular response of each sex and strain is highly distinctive. Using a multi-omics approach, we identify a core sex- and strain- independent molecular response to dietary isoleucine, and identify mega-clusters of differentially expressed hepatic genes, metabolites, and lipids associated with each phenotype. Intriguingly, the metabolic effects of reduced isoleucine in mice are not associated with FGF21 - and we find that in humans plasma FGF21 levels are likewise not associated with dietary levels of isoleucine. Finally, we find that foods contain a range of isoleucine levels, and that consumption of dietary isoleucine is lower in humans with healthy eating habits. Our results demonstrate that the dietary level of isoleucine is critical in the metabolic and molecular response to a WD, and suggest that lowering dietary levels of isoleucine may be an innovative and translatable strategy to protect from the negative metabolic consequences of a WD.

Administration of intratumoral GD2-directed interleukin-2 immunocytokine and local radiation therapy to activate immune rejection of spontaneous canine melanoma.

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral-immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral-immunocytokine at 12 mg/m 2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral-immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients.

MerTK Drives Proliferation and Metastatic Potential in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG's role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.

Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells for treatment of acute radiation syndrome.

BACKGROUND: Hematopoietic acute radiation syndrome (H-ARS) occurring after exposure to ionizing radiation damages bone marrow causing cytopenias, increasing susceptibility to infections and death. We and others have shown that cellular therapies like human mesenchymal stromal cells (MSCs), or monocytes/macrophages educated ex-vivo with extracellular vesicles (EVs) from MSCs were effective in a lethal H-ARS mouse model. However, given the complexity of generating cellular therapies and the potential risks of using allogeneic products, development of an "off-the-shelf" cell-free alternative like EVs may have utility in conditions like H-ARS that require rapid deployment of available therapeutics. The purpose of this study was to determine the feasibility of producing MSC-derived EVs at large scale using a bioreactor and assess critical quality control attributes like identity, sterility, and potency in educating monocytes and promoting survival in a lethal H-ARS mouse model. METHODS: EVs were isolated by ultracentrifugation from unprimed and lipopolysaccharide (LPS)-primed MSCs grown at large scale using a hollow fiber bioreactor and compared to a small scale system using flasks. The physical identity of EVs included a time course assessment of particle diameter, yield, protein content and surface marker profile by flow-cytometry. Comparison of the RNA cargo in EVs was determined by RNA-seq. Capacity of EVs to generate exosome educated monocytes (EEMos) was determined by qPCR and flow cytometry, and potency was assessed in vivo using a lethal ARS model with NSG mice. RESULTS: Physical identity of EVs at both scales were similar but yields by volume were up to 38-fold more using a large-scale bioreactor system. RNA-seq indicated that flask EVs showed upregulated let-7 family and miR-143 micro-RNAs. EEMos educated with LPS-EVs at each scale were similar, showing increased gene expression of IL-6, IDO, FGF-2, IL-7, IL-10, and IL-15 and immunophenotyping consistent with a PD-L1 high, CD16 low, and CD86 low cell surface expression. Treatment with LPS-EVs manufactured at both scales were effective in the ARS model, improving survival and clinical scores through improved hematopoietic recovery. EVs from unprimed MSCs were less effective than LPS-EVs, with flask EVs providing some improved survival while bioreactor EVs provide no survival benefit. CONCLUSIONS: LPS-EVs as an effective treatment for H-ARS can be produced using a scale-up development manufacturing process, representing an attractive off-the-shelf, cell-free therapy.

Farm animal exposure, respiratory illnesses, and nasal cell gene expression.

BACKGROUND: Farm exposures in early life reduce the risks for childhood allergic diseases and asthma. There is less information about how farm exposures relate to respiratory illnesses and mucosal immune development. OBJECTIVE: We hypothesized that children raised in farm environments have a lower incidence of respiratory illnesses over the first 2 years of life than nonfarm children. We also analyzed whether farm exposures or respiratory illnesses were related to patterns of nasal cell gene expression. METHODS: The Wisconsin Infant Study Cohort included farm (n = 156) and nonfarm (n = 155) families with children followed to age 2 years. Parents reported prenatal farm and other environmental exposures. Illness frequency and severity were assessed using illness diaries and periodic surveys. Nasopharyngeal cell gene expression in a subset of 64 children at age 2 years was compared to farm exposure and respiratory illness history. RESULTS: Farm versus nonfarm children had nominally lower rates of respiratory illnesses (rate ratio 0.82 [95% CI, 0.69, 0.97]) with a stepwise reduction in illness rates in children exposed to 0, 1, or ≥2 animal species, but these trends were nonsignificant in a multivariable model. Farm exposures and preceding respiratory illnesses were positively related to nasal cell gene signatures for mononuclear cells and innate and antimicrobial responses. CONCLUSIONS: Maternal and infant exposure to farms and farm animals was associated with nonsignificant trends for reduced respiratory illnesses. Nasal cell gene expression in a subset of children suggests that farm exposures and respiratory illnesses in early life are associated with distinct patterns of mucosal immune expression.

Development of a next-generation sequencing protocol for the canine T cell receptor beta chain repertoire.

Profiling the T cell receptor (TCR) repertoire using next-generation sequencing has become common in both human and translational research. Companion dogs with spontaneous tumors, including canine melanoma, share several features, e.g., natural occurrence, shared environmental exposures, natural outbred population, and immunocompetence. T cells play an important role in the adaptive immune system by recognizing specific antigens via a surface TCR. As such, understanding the canine T cell response to vaccines, cancer, immunotherapies, and infectious diseases is critically important for both dog and human health. Off-the-shelf commercial reagents, kits and services are readily available for human, non-human primate, and mouse in this context. However, these resources are limited for the canine. In this study, we present a cost-effective protocol for analysis of canine TCR beta chain genes. Workflow can be accomplished in 1-2 days starting with total RNA and resulting in libraries ready for sequencing on Illumina platforms.

Novel and unique rheumatoid factors cross-react with viral epitopes in COVID-19.

Rheumatoid factors (RFs), polyreactive antibodies canonically known to bind two conformational epitopes of IgG Fc, are a hallmark of rheumatoid arthritis but also can arise in other inflammatory conditions and infections. Also, infections may contribute to the development of rheumatoid arthritis and other autoimmune diseases. Recently, RFs only in rheumatoid arthritis were found to bind novel linear IgG epitopes as well as thousands of other rheumatoid arthritis autoantigens. Specific epitopes recognized by infection-induced polyreactive RFs remain undefined but could provide insights into loss of immune tolerance. Here, we identified novel linear IgG epitopes bound by RFs in COVID-19 but not rheumatoid arthritis or other conditions. The main COVID-19 RF was polyreactive, binding two IgG and multiple viral peptides with a tripeptide motif, as well as IgG Fc and SARS-CoV-2 spike proteins. In contrast, a rheumatoid arthritis-specific RF recognized IgG Fc, but not tripeptide motif-containing peptides or spike. Thus, RFs have disease-specific IgG reactivity and distinct polyreactivities that reflect the broader immune response. Moreover, the polyreactivity of a virus-induced RF appears to be attributable to a very short peptide motif. These findings refine our understanding of RFs and provide new insights into how viral infections may contribute to autoimmunity.

Antibody landscape of C57BL/6 mice cured of B78 melanoma via a combined radiation and immunocytokine immunotherapy regimen.

Sera of immune mice that were previously cured of their melanoma through a combined radiation and immunocytokine immunotherapy regimen consisting of 12 Gy of external beam radiation and the intratumoral administration of an immunocytokine (anti-GD2 mAb coupled to IL-2) with long-term immunological memory showed strong antibody-binding against melanoma tumor cell lines via flow cytometric analysis. Using a high-density whole-proteome peptide array (of 6.090.593 unique peptides), we assessed potential protein-targets for antibodies found in immune sera. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by these 6 mice and exhibited strong antibody binding only by immune (after successful cure and rechallenge), not naïve (before tumor implantation) sera and developed a robust method to detect these differentially targeted peptides. Confirmatory studies were done to validate these results using 2 separate systems, a peptide ELISA and a smaller scale peptide array utilizing a slightly different technology. To the best of our knowledge, this is the first study of the full set of germline encoded linear peptide-based proteome epitopes that are recognized by immune sera from mice cured of cancer via radio-immunotherapy. We furthermore found that although the generation of B-cell repertoire in immune development is vastly variable, and numerous epitopes are identified uniquely by immune serum from each of these 6 immune mice evaluated, there are still several epitopes and proteins that are commonly recognized by at least half of the mice studied. This suggests that every mouse has a unique set of antibodies produced in response to the curative therapy, creating an individual "fingerprint." Additionally, certain epitopes and proteins stand out as more immunogenic, as they are recognized by multiple mice in the immune group.

NK cells propagate T cell immunity following in situ tumor vaccination.

We report an in situ vaccination, adaptable to nearly any type of cancer, that combines radiotherapy targeting one tumor and intratumoral injection of this site with tumor-specific antibody and interleukin-2 (IL-2; 3xTx). In a phase I clinical trial, administration of 3xTx (with an immunocytokine fusion of tumor-specific antibody and IL-2, hu14.18-IL2) to subjects with metastatic melanoma increases peripheral CD8+ T cell effector polyfunctionality. This suggests the potential for 3xTx to promote antitumor immunity against metastatic tumors. In poorly immunogenic syngeneic murine melanoma or head and neck carcinoma models, 3xTx stimulates CD8+ T cell-mediated antitumor responses at targeted and non-targeted tumors. During 3xTx treatment, natural killer (NK) cells promote CTLA4+ regulatory T cell (Treg) apoptosis in non-targeted tumors. This is dependent on NK cell expression of CD86, which is upregulated downstream of KLRK1. NK cell depletion increases Treg infiltration, diminishing CD8+ T cell-dependent antitumor response. These findings demonstrate that NK cells sustain and propagate CD8+ T cell immunity following 3xTx.

Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice.

Low-protein diets promote health and longevity in diverse species. Restriction of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine recapitulates many of these benefits in young C57BL/6J mice. Restriction of dietary isoleucine (IleR) is sufficient to promote metabolic health and is required for many benefits of a low-protein diet in C57BL/6J males. Here, we test the hypothesis that IleR will promote healthy aging in genetically heterogeneous adult UM-HET3 mice. We find that IleR improves metabolic health in young and old HET3 mice, promoting leanness and glycemic control in both sexes, and reprograms hepatic metabolism in a sex-specific manner. IleR reduces frailty and extends the lifespan of male and female mice, but to a greater degree in males. Our results demonstrate that IleR increases healthspan and longevity in genetically diverse mice and suggests that IleR, or pharmaceuticals that mimic this effect, may have potential as a geroprotective intervention.

MASH Native: a unified solution for native top-down proteomics data processing.

MOTIVATION: Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. RESULTS: We have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a "one-stop shop" for characterizing both native protein complexes and proteoforms. AVAILABILITY AND IMPLEMENTATION: The MASH Native app, video tutorials, written tutorials, and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHSoftware.php. All data files shown in user tutorials are included with the MASH Native software in the download .zip file.

Proteogenomic and V(D)J Analysis of Human Decidual T Cells Highlights Unique Transcriptional Programming and Clonal Distribution.

Immunological tolerance toward the semiallogeneic fetus is one of many maternal adaptations required for a successful pregnancy. T cells are major players of the adaptive immune system and balance tolerance and protection at the maternal-fetal interface; however, their repertoire and subset programming are still poorly understood. Using emerging single-cell RNA sequencing technologies, we simultaneously obtained transcript, limited protein, and receptor repertoire at the single-cell level, from decidual and matched maternal peripheral human T cells. The decidua maintains a tissue-specific distribution of T cell subsets compared with the periphery. We find that decidual T cells maintain a unique transcriptome programming, characterized by restraint of inflammatory pathways by overexpression of negative regulators (DUSP, TNFAIP3, ZFP36) and expression of PD-1, CTLA-4, TIGIT, and LAG3 in some CD8 clusters. Finally, analyzing TCR clonotypes demonstrated decreased diversity in specific decidual T cell populations. Overall, our data demonstrate the power of multiomics analysis in revealing regulation of fetal-maternal immune coexistence.

Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C.

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. SIGNIFICANCE: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

SOX18-enforced expression diverts hemogenic endothelium-derived progenitors from T towards NK lymphoid pathways.

Hemogenic endothelium (HE) is the main source of blood cells in the embryo. To improve blood manufacturing from human pluripotent stem cells (hPSCs), it is essential to define the molecular determinants that enhance HE specification and promote development of the desired blood lineage from HE. Here, using SOX18-inducible hPSCs, we revealed that SOX18 forced expression at the mesodermal stage, in contrast to its homolog SOX17, has minimal effects on arterial specification of HE, expression of HOXA genes and lymphoid differentiation. However, forced expression of SOX18 in HE during endothelial-to-hematopoietic transition (EHT) greatly increases NK versus T cell lineage commitment of hematopoietic progenitors (HPs) arising from HE predominantly expanding CD34+CD43+CD235a/CD41a-CD45- multipotent HPs and altering the expression of genes related to T cell and Toll-like receptor signaling. These studies improve our understanding of lymphoid cell specification during EHT and provide a new tool for enhancing NK cell production from hPSCs for immunotherapies.

Ranking Antibody Binding Epitopes and Proteins Across Samples from Whole Proteome Tiled Linear Peptides.

Ultradense peptide binding arrays that can probe millions of linear peptides comprising the entire proteomes or immunomes of human or mouse, or numerous microbes, are powerful tools for studying the abundance of different antibody repertoire in serum samples to understand adaptive immune responses. There are few statistical analysis tools for exploring high-dimensional, significant and reproducible antibody targets for ultradense peptide binding arrays at the linear peptide, epitope (grouping of adjacent peptides), and protein level across multiple samples/subjects (I.e. epitope spread or immunogenic regions within each protein) for understanding the heterogeneity of immune responses. We developed HERON (Hierarchical antibody binding Epitopes and pROteins from liNear peptides), an R package, which allows users to identify immunogenic epitopes using meta-analyses and spatial clustering techniques to explore antibody targets at various resolution and confidence levels, that can be found consistently across a specified number of samples through the entire proteome to study antibody responses for diagnostics or treatment. Our approach estimates significance values at the linear peptide (probe), epitope, and protein level to identify top candidates for validation. We test the performance of predictions on all three levels using correlation between technical replicates and comparison of epitope calls on 2 datasets, which shows HERON's competitiveness in estimating false discovery rates and finding general and sample-level regions of interest for antibody binding. The code is available as an R package downloadable from http://github.com/Ong-Research/HERON.

Genome-wide association study (GWAS) of circulating vitamin D outcomes among individuals of African ancestry.

BACKGROUND: Vitamin D deficiency is more common among African-ancestry individuals and may be associated with adverse health outcomes. Vitamin D binding protein (VDBP) regulates concentrations of biologically active vitamin D. OBJECTIVE: We conducted genome-wide association study (GWAS) of VDBP and 25-hydroxyvitamin D among African-ancestry individuals. METHODS: Data were collected from 2,602 African American adults from the Southern Community Cohort Study (SCCS) and 6,934 African- or Caribbean-ancestry adults from the UK Biobank. Serum VDBP concentrations were available only in the SCCS and were measured by using the Polyclonal Human VDBP ELISA kit. Serum 25-hydroxyvitamin D concentrations for both study samples were measured by using Diasorin Liason, a chemiluminescent immunoassay. Participants were genotyped for single nucleotide polymorphisms (SNPs) with genome-wide coverage by using Illumina or Affymetrix platforms. Fine-mapping analysis was performed by using forward stepwise linear regression models including all variants with P value < 5 × 10-8 and within 250 kbps of a lead SNP. RESULTS: We identified 4 loci notably associated with VDBP concentrations in the SCCS population: rs7041 (per allele ß = 0.61 µg/mL, SE = 0.05, P = 1.4 × 10-48) and rs842998 (per allele ß = 0.39 µg/mL, SE = 0.03, P = 4.0 × 10-31) in GC, rs8427873 (per allele ß = 0.31 µg/mL, SE = 0.04, P = 3.0 × 10-14) near GC and rs11731496 (per allele ß = 0.21 µg/mL, SE = 0.03, P = 3.6 × 10-11) in between GC and NPFFR2. In conditional analyses, which included the above-mentioned SNPs, only rs7041 remained notable (P = 4.1 × 10-21). SNP rs4588 in GC was the only GWAS-identified SNP associated with 25-hydroxyvitamin D concentration. Among UK Biobank participants: per allele ß = -0.11 µg/mL, SE = 0.01, P = 1.5 × 10-13; in the SCCS: per allele ß = -0.12 µg/mL, SE = 0.06, P = 2.8 × 10-02). rs7041 and rs4588 are functional SNPs that influence the binding affinity of VDBP to 25-hydroxyvitamin D. CONCLUSIONS: Our results were in line with previous studies conducted in European-ancestry populations, showing that GC, the gene that directly encodes for VDBP, would be important for VDBP and 25-hydroxyvitamin D concentrations. The current study extends our knowledge of the genetics of vitamin D in diverse populations.

MASH Native: A Unified Solution for Native Top-Down Proteomics Data Processing.

Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. Herein, we have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a one-stop shop for characterizing both native protein complexes and proteoforms. The MASH Native app, video tutorials, written tutorials and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHNativeSoftware.php . All data files shown in user tutorials are included with the MASH Native software in the download .zip file.