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Background The clinical heterogeneity of SLE with its complex pathogenesis remains challenging as we strive to provide optimal management. The contribution of platelets to endovascular homeostasis, inflammation and immune regulation highlights their potential importance in SLE. Prior work from our group showed that the Fcγ receptor type IIa (FcγRIIa)–R/H131 biallelic polymorphism is associated with increased platelet activity and cardiovascular risk in SLE. The study was initiated to investigate the platelet transcriptome in patients with SLE and evaluate its association across FcγRIIa genotypes and distinct clinical features. Methods Fifty-one patients fulfilling established criteria for SLE (mean age = 41.1 ± 12.3, 100% female, 45% Hispanic, 24% black, 22% Asian, 51% white, mean SLEDAI = 4.4 ± 4.2 at baseline) were enrolled and compared with 18 demographically matched control samples. The FCGR2a receptor was genotyped for each sample, and RNA-seq was performed on isolated, leukocyte-depleted platelets. Transcriptomic data were used to create a modular landscape to explore the differences between SLE patients and controls and various clinical parameters in the context of FCGR2a genotypes. Results There were 2290 differentially expressed genes enriched for pathways involved in interferon signaling, immune activation, and coagulation when comparing SLE samples vs controls. When analyzing patients with proteinuria, modules associated with oxidative phosphorylation and platelet activity were unexpectedly decreased. Furthermore, genes that were increased in SLE and in patients with proteinuria were enriched for immune effector processes, while genes increased in SLE but decreased in proteinuria were enriched for coagulation and cell adhesion. A low-binding FCG2Ra allele (R131) was associated with decreases in FCR activation, which further correlated with increases in platelet and immune activation pathways. Finally, we were able to create a transcriptomic signature of clinically active disease that performed significantly well in discerning SLE patients with active clinical disease form those with inactive clinical disease. Conclusions In aggregate, these data demonstrate the platelet transcriptome provides insight into lupus pathogenesis and disease activity, and shows potential use as means of assessing this complex disease using a liquid biopsy.

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared with those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aβ) for growth and survival in the brain parenchyma. Melanoma-secreted Aβ activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacologic inhibition of Aβ decreases brain metastatic burden.Significance:Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer's disease, two previously unrelated pathologies; establish Aβ as a promising therapeutic target for brain metastasis; and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Table from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Supplementary Data from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Supplementary Figure from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared with those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aβ) for growth and survival in the brain parenchyma. Melanoma-secreted Aβ activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacologic inhibition of Aβ decreases brain metastatic burden. Significance: Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer's disease, two previously unrelated pathologies; establish Aβ as a promising therapeutic target for brain metastasis; and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma. This article is highlighted in the In This Issue feature, p. 1171

mRNA isoforms

Pharmacodynamic changes (pathways)

Response markers

Extended Materials and Methods with in depth details about experimental procedures and protocols

Pharmacodynamic markers

Supplementary tables 1 and 2; Clinical information about the patients that the PDX models were established from and pathways that are regulated in the sensitive tumors after buparlisib treatment, respectively.

Response markers (pathways)

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro. A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4–dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy.Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732–46. ©2018 AACR.

Supplementary Figures 1-10 supporting the study Mundt et al., "Proteomic profiling of PI3K inhibitor resistance"

Our previous studies identified a population of stem cell-like proliferating myeloid cells within inflamed tissues that could serve as a reservoir for tissue macrophages to adopt different activation states depending on the microenvironment. By lineage tracing cells derived from CX3CR1+precursors in mice during infection and profiling by scRNA-seq, here we identify a cluster of BIRC5+myeloid cells that expanded in the liver during either chronic infection with the parasiteSchistosoma mansonior the bacterial pathogenStaphylococcus aureus. In the absence of tissue damaging toxins,S. aureusinfection does not elicit these BIRC5+cells. Moreover, deletion of BIRC5 from CX3CR1 expressing cells results in improved survival duringS. aureusinfection. Hence, the combination of scRNA-Seq and genetic fate mapping CX3CR1+cells revealed a toxin dependent pathogenic role for BIRC5 in myeloid cells duringS. aureusinfection.

/SummaryTo maintain blood homeostasis, millions of terminally differentiated effector cells are produced every day. At the apex of this massive and constant blood production lie hematopoietic stem cells (HSCs), a rare cell type harboring unique self-renewal and multipotent properties. A key feature of HSCs is their ability to temporarily exit the cell cycle in a state termed quiescence. Defective control of cell cycle progression can eventually lead to bone marrow failure or malignant transformation. It is thought that HSCs must re-enter the cell cycle in order to commit to terminal differentiation. However, the molecular mechanisms tying cell cycle re-entry to cell fate commitment in HSCs remain elusive. Here, we identify the chromatin-associated Sin3B protein as a molecular link between cell cycle progression and differentiation in HSCs. We demonstrate that Sin3B is necessary for HSCs’ commitment to differentiation, but dispensable for their self-renewal or survival. Single cell transcriptional profiling of hematopoietic stem and progenitor cells (HSPCs) inactivated for Sin3B reveals aberrant cell cycle gene expression, consistent with the observed aberrant progression through the G1phase of the cell cycle. The defective cell cycle control elicited upon Sin3B inactivation correlates with the engagement of discrete signaling programs, including aberrant expression of cell adhesion molecules and essential components of the interferon signaling cascade in LT-HSCs. Additionally, chromatin accessibility profiling in LT-HSCs reveals the Sin3B-dependent accessibility of genomic elements controlling HSC differentiation, suggesting a functional link between cell cycle progression, and priming of hematopoietic stem cells for differentiation. Together, these results point to controlled progression through the G1phase of the cell cycle as a likely regulator of HSC lineage commitment through the modulation of chromatin features.

Objective Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID‐19 patients. Given that platelets are key regulators of thrombosis, inflammation, and immunity they represent prime candidates as mediators of COVID‐19‐associated pathogenesis. The objective of this study was to understand the contribution of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) to the platelet phenotype via phenotypic (activation, aggregation) and transcriptomic characterization. Approach and Results In a cohort of 3915 hospitalized COVID‐19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication, and biomarkers of inflammation and thrombosis, we find platelet count, size, and immaturity are associated with increased critical illness and all‐cause mortality. Bone marrow, lung tissue, and blood from COVID‐19 patients revealed the presence of SARS‐CoV‐2 virions in megakaryocytes and platelets. Characterization of COVID‐19 platelets found them to be hyperreactive (increased aggregation, and expression of P‐selectin and CD40) and to have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. In vitro mechanistic studies highlight that the interaction of SARS‐CoV‐2 with megakaryocytes alters the platelet transcriptome, and its effects are distinct from the coronavirus responsible for the common cold (CoV‐OC43). Conclusions Platelet count, size, and maturity associate with increased critical illness and all‐cause mortality among hospitalized COVID‐19 patients. Profiling tissues and blood from COVID‐19 patients revealed that SARS‐CoV‐2 virions enter megakaryocytes and platelets and associate with alterations to the platelet transcriptome and activation profile.

Objective:Peripheral artery disease (PAD) is an atherothrombotic disease of the lower limbs with substantial morbidity and mortality. We used next-generation sequencing to identify genome-wide expression signatures associated with prevalent PAD and its outcomes.Approach and Results:We performed whole blood RNA sequencing among patients with severe symptomatic PAD undergoing lower extremity revascularization and controls. Dysregulated pathways and blood transcriptional modules were identified by comparing patients with PAD (n=42) to age- and sex-matched controls (N=29). The identified signature was compared in patients with PAD before LER with or without incident major adverse cardiac or limb events (MACLE). A novel microRNA associated with prevalent PAD and incident MACLE was then evaluated in a mouse hindlimb ischemia model. One hundred twenty-seven transcripts were differentially expressed (77 upregulated and 50 downregulated; adjustedP0.5) and analyzed using weighted gene co-expression network analysis. Weighted gene co-expression network analysis revealed blood modules enriched for immune activation, secretory granules, and coagulation in patients with PAD. Of these 127 differentially expressed transcripts, 40 were significantly associated with MACLE (log-rank false discovery rate Conclusions:A whole blood transcript signature identified patients with symptomatic PAD and PAD patients at increased risk of MACLE. A previously uncharacterized transcript microRNA-4477b was overexpressed in prevalent PAD, incident MACLE, and in a mouse hindlimb ischemia model. Our novel transcriptomic signature provides insight into potential mechanisms of patients with severe symptomatic PAD.

Staphylococcus aureusis an opportunistic pathogen and chief among bloodstream-infecting bacteria.S. aureusproduces an array of human-specific virulence factors that may contribute to immune suppression. Here, we defined the response of primary human phagocytes following infection withS. aureususing RNA-sequencing (RNA-Seq). We found that the overall transcriptional response toS. aureuswas weak both in the number of genes and in the magnitude of response. Using an ex vivo bacteremia model with fresh human blood, we uncovered that infection withS. aureusresulted in the down-regulation of genes related to innate immune response and cytokine and chemokine signaling. This muted transcriptional response was conserved across diverseS. aureusclones but absent in blood exposed to heat-killedS. aureusor blood infected with the less virulent staphylococcal speciesStaphylococcus epidermidis. Notably, this signature was also present in patients withS. aureusbacteremia. We identified the master regulatorS. aureusexoprotein expression (SaeRS) and the SaeRS-regulated pore-forming toxins as key mediators of the transcriptional suppression. TheS. aureus–mediated suppression of chemokine and cytokine transcription was reflected by circulating protein levels in the plasma. Wild-typeS. aureuselicited a soluble milieu that was restrictive in the recruitment of human neutrophils compared with strains lackingsaeRS. Thus,S. aureusblunts the inflammatory response resulting in impaired neutrophil recruitment, which could promote the survival of the pathogen during invasive infection.

Purpose The functional composition and diversity of the gut microbiome (estrobolome) may affect breast cancer risk by modulation of systemic sex hormones. Gut bacteria with β-glucuronidase enzymatic activity may deconjugate estrogens, leading to increased estrogen reabsorption into the circulation thereby increasing breast cancer risk. We investigated the relationship between the gut bacterial microbiome and endogenous estrogens and related sex hormones in women with hormone receptor-positive breast cancer compared to healthy control women. Methods In this prospective case-control study, postmenopausal women (n = 46) with newly diagnosed stage I-III estrogen and/or progesterone receptor-positive breast cancer were compared with healthy postmenopausal female controls (n = 22). Bacterial composition of the gut microbiome was analyzed by 16S rRNA gene sequencing from fecal specimens. Plasma and urine sex hormones were quantified using high-performance liquid chromatography/mass spectrometry. Results We found evidence that some β-glucuronidase positive bacteria were enriched in the breast cancer patients compared to healthy controls, whereas abundances of some β-glucuronidase negative bacteria were reduced. There was also a wide distribution of prevalence of β-glucuronidase positive taxa in both the breast cancer subjects and healthy controls, as well as higher probability of breast cancer subjects having higher average β-glucuronidase levels. Significant differences were found in endogenous progesterone levels between the breast cancer patients and healthy controls. Conclusion This pilot study showed differences in the gut microbiome and endogenous progesterone levels among postmenopausal women with hormone receptor-positive breast cancer compared with healthy controls. These interesting findings may have implications for breast cancer risk and prevention and warrant further exploration.

Soil-transmitted intestinal worms known as helminths colonize over 1.5 billion people worldwide. Although helminth colonization has been associated with altered composition of the gut microbiota, such as increases in Clostridia, individual species have not been isolated and characterized. Here, we isolate and sequence the genome of 13 Clostridia from the Orang Asli, an indigenous population in Malaysia with a high prevalence of helminth infections. Metagenomic analysis of 650 fecal samples from urban and rural Malaysians confirm the prevalence of species corresponding to these isolates and reveal a specific association between Peptostreptococcaceae family members and helminth colonization. Remarkably, Peptostreptococcaceae isolated from the Orang Asli display superior capacity to promote the life cycle of whipworm species, including hatching of eggs from Trichuris muris and Trichuris trichiura. These findings support a model in which helminths select for gut colonization of microbes that support their life cycle.

Soil transmitted intestinal worms known as helminths colonize over 1.5 billion people worldwide. Although helminth colonization has been associated with altered composition of the gut microbiota, such as increases in Clostridia, individual species have not been isolated and characterized. Here, we isolated and sequenced the genome of 13 Clostridia from the Orang Asli, an indigenous population in Malaysia with high prevalence of helminth infections. Metagenomic analysis of 650 fecal samples from urban and rural Malaysians revealed higher prevalence and abundance of these isolates compared to individuals in the United States, with Peptostreptococcaceae family members displaying a specific association with helminth colonization. Remarkably, Peptostreptococcaceae isolated from the Orang Asli displayed superior capacity to induce hatching of eggs from the murine helminth Trichuris muris. These findings support a model in which helminths select for gut colonization of microbes that in turn support their life cycle by promoting egg hatching.

Aberrant phospho-signaling is a hallmark of cancer. We investigated kinase-substrate regulation of 33,239 phosphorylation sites (phosphosites) in 77 breast tumors and 24 breast cancer xenografts. Our search discovered 2134 quantitatively correlated kinase-phosphosite pairs, enriching for and extending experimental or binding-motif predictions. Among the 91 kinases with auto-phosphorylation, elevated EGFR, ERBB2, PRKG1, and WNK1 phosphosignaling were enriched in basal, HER2-E, Luminal A, and Luminal B breast cancers, respectively, revealing subtype-specific regulation. CDKs, MAPKs, and ataxia-telangiectasia proteins were dominant, master regulators of substrate-phosphorylation, whose activities are not captured by genomic evidence. We unveiled phospho-signaling and druggable targets from 113 kinase-substrate pairs and cascades downstream of kinases, including AKT1, BRAF and EGFR. We further identified kinase-substrate-pairs associated with clinical or immune signatures and experimentally validated activated phosphosites of ERBB2, EIF4EBP1, and EGFR. Overall, kinase-substrate regulation revealed by the largest unbiased global phosphorylation data to date connects driver events to their signaling effects.

Given the evidence for a hyperactive platelet phenotype in COVID-19, we investigated effector cell properties of COVID-19 platelets on endothelial cells (ECs). Integration of EC and platelet RNA sequencing revealed that platelet-released factors in COVID-19 promote an inflammatory hypercoagulable endotheliopathy. We identified S100A8 and S100A9 as transcripts enriched in COVID-19 platelets and were induced by megakaryocyte infection with SARS-CoV-2. Consistent with increased gene expression, the heterodimer protein product of S100A8/A9, myeloid-related protein (MRP) 8/14, was released to a greater extent by platelets from COVID-19 patients relative to controls. We demonstrate that platelet-derived MRP8/14 activates ECs, promotes an inflammatory hypercoagulable phenotype, and is a significant contributor to poor clinical outcomes in COVID-19 patients. Last, we present evidence that targeting platelet P2Y12 represents a promising candidate to reduce proinflammatory platelet-endothelial interactions. Together, these findings demonstrate a previously unappreciated role for platelets and their activation-induced endotheliopathy in COVID-19.

Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury.

Summary We have established experimental systems to assess the effects of early-life exposures to antibiotics on the intestinal microbiota and gene expression in the brain. This model system is highly relevant to human exposure and may be developed into a preclinical model of neurodevelopmental disorders in which the gut–brain axis is perturbed, leading to organizational effects that permanently alter the structure and function of the brain. Exposing newborn mice to low-dose penicillin led to substantial changes in intestinal microbiota population structure and composition. Transcriptomic alterations implicate pathways perturbed in neurodevelopmental and neuropsychiatric disorders. There also were substantial effects on frontal cortex and amygdala gene expression by bioinformatic interrogation, affecting multiple pathways underlying neurodevelopment. Informatic analyses established linkages between specific intestinal microbial populations and the early-life expression of particular affected genes. These studies provide translational models to explore intestinal microbiome roles in the normal and abnormal maturation of the vulnerable central nervous system., Graphical abstract, Highlights • Low-dose antibiotic exposure perturbs the infant gut mouse microbiome to PND10 • Frontal cortex and amygdala gene expression were substantially affected • Multiple pathways underlying neurodevelopment were affected • Specific gut microbial populations were linked with expression of particular genes, Developmental neuroscience; Microbiome

Unbiased assays such as shotgun proteomics and RNA-seq provide high-resolution molecular characterization of tumors. These assays measure molecules with highly varied distributions, making interpretation and hypothesis testing challenging. Samples with the most extreme measurements for a molecule can reveal the most interesting biological insights, yet are often excluded from analysis. Furthermore, rare disease subtypes are, by definition, underrepresented in cancer cohorts. To provide a strategy for identifying molecules aberrantly enriched in small sample cohorts, we present BlackSheep--a package for non-parametric description and differential analysis of genome-wide data, available at https://github.com/ruggleslab/blackSheep. BlackSheep is a complementary tool to other differential expression analysis methods that may be underpowered when analyzing small subgroups in a larger cohort.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.Importance:The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

Both SARS-CoV-2 infection and vaccination elicit potent immune responses. A number of studies have described immune responses to SARS-CoV-2 infection. However, beyond antibody production, immune responses to COVID-19 vaccines remain largely uncharacterized. Here, we performed multimodal single-cell sequencing on peripheral blood of patients with acute COVID-19 and healthy volunteers before and after receiving the SARS-CoV-2 BNT162b2 mRNA vaccine to compare the immune responses elicited by the virus and by this vaccine. Phenotypic and transcriptional profiling of immune cells, coupled with reconstruction of the B and T cell antigen receptor rearrangement of individual lymphocytes, enabled us to characterize and compare the host responses to the virus and to defined viral antigens. While both infection and vaccination induced robust innate and adaptive immune responses, our analysis revealed significant qualitative differences between the two types of immune challenges. In COVID-19 patients, immune responses were characterized by a highly augmented interferon response which was largely absent in vaccine recipients. Increased interferon signaling likely contributed to the observed dramatic upregulation of cytotoxic genes in the peripheral T cells and innate-like lymphocytes in patients but not in immunized subjects. Analysis of B and T cell receptor repertoires revealed that while the majority of clonal B and T cells in COVID-19 patients were effector cells, in vaccine recipients clonally expanded cells were primarily circulating memory cells. Importantly, the divergence in immune subsets engaged, the transcriptional differences in key immune populations, and the differences in maturation of adaptive immune cells revealed by our analysis have far-ranging implications for immunity to this novel pathogen.

Advances in mass-spectrometry have generated increasingly large-scale proteomics datasets containing tens of thousands of phosphorylation sites (phosphosites) that require prioritization. We develop a bioinformatics tool called HotPho and systematically discover 3D co-clustering of phosphosites and cancer mutations on protein structures. HotPho identifies 474 such hybrid clusters containing 1255 co-clustering phosphosites, including RET p.S904/Y928, the conserved HRAS/KRAS p.Y96, and IDH1 p.Y139/IDH2 p.Y179 that are adjacent to recurrent mutations on protein structures not found by linear proximity approaches. Hybrid clusters, enriched in histone and kinase domains, frequently include expression-associated mutations experimentally shown as activating and conferring genetic dependency. Approximately 300 co-clustering phosphosites are verified in patient samples of 5 cancer types or previously implicated in cancer, including CTNNB1 p.S29/Y30, EGFR p.S720, MAPK1 p.S142, and PTPN12 p.S275. In summary, systematic 3D clustering analysis highlights nearly 3,000 likely functional mutations and over 1000 cancer phosphosites for downstream investigation and evaluation of potential clinical relevance., Dysregulated phosphorylation is well-known in cancers, but it has largely been studied in isolation from mutations. Here the authors introduce HotPho, a tool that can discover spatial interactions between phosphosites and mutations, which are associated with activating mutation and genetic dependencies in cancer.

Within the last decade, numerous studies have demonstrated changes in the gut microbiome associated with specific autoimmune diseases. Due to differences in study design, data quality control, analysis and statistical methods, many results of these studies are inconsistent and incomparable. To better understand the relationship between the intestinal microbiome and autoimmunity, we have completed a comprehensive re-analysis of 42 studies focusing on the gut microbiome in twelve autoimmune diseases to identify a microbial signature predictive of multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis (RA) and general autoimmune disease using both 16S rRNA sequencing data and shotgun metagenomics data. To do this, we used four machine learning algorithms, random forest, eXtreme Gradient Boosting (XGBoost), ridge regression and support vector machine with radial kernel and recursive feature elimination to rank disease predictive taxa comparing disease vs. healthy participants and pairwise comparisons of each disease. Comparing the performance of these models, we found XGBoost and random forest, tree-based methods capable of handling sparse multidimensional data, to consistently produce the best results. Through this modeling, we identified a number of taxa consistently identified as dysregulated in a general autoimmune disease model including Odoribacter, Lachnospiraceae Clostridium and Mogibacteriaceae implicating all as potential factors connecting the gut microbiome and to autoimmune response. Further, we computed pairwise comparison models to identify disease specific taxa signatures highlighting a role for Peptostreptococcaceae and Ruminococcaceae Gemmiger in IBD and Akkermansia, Butyricicoccus and Mogibacteriaceae in MS. We then connected a subset of these taxa with potential metabolic alterations based on metagenomic/metabolomic correlation analysis, identifying 250 metabolites associated with autoimmunity-predictive taxa.

Over-accumulation of oxalate in humans may lead to nephrolithiasis and nephrocalcinosis. Humans lack endogenous oxalate degradation pathways (ODP), but intestinal microbes can degrade oxalate using multiple ODPs and protect against its absorption. The exact oxalate-degrading taxa in the human microbiota and their ODP have not been described. We leverage multi-omics data (>3000 samples from >1000 subjects) to show that the human microbiota primarily uses the type II ODP, rather than type I. Furthermore, among the diverse ODP-encoding microbes, an oxalate autotroph, Oxalobacter formigenes, dominates this function transcriptionally. Patients with inflammatory bowel disease (IBD) frequently suffer from disrupted oxalate homeostasis and calcium oxalate nephrolithiasis. We show that the enteric oxalate level is elevated in IBD patients, with highest levels in Crohn’s disease (CD) patients with both ileal and colonic involvement consistent with known nephrolithiasis risk. We show that the microbiota ODP expression is reduced in IBD patients, which may contribute to the disrupted oxalate homeostasis. The specific changes in ODP expression by several important taxa suggest that they play distinct roles in IBD-induced nephrolithiasis risk. Lastly, we colonize mice that are maintained in the gnotobiotic facility with O. formigenes, using either a laboratory isolate or an isolate we cultured from human stools, and observed a significant reduction in host fecal and urine oxalate levels, supporting our in silico prediction of the importance of the microbiome, particularly O. formigenes in host oxalate homeostasis.

Background Among adolescents, the pathway from being offered drugs to engaging in drug use has been established. The prevalence of drug transactions specifically in schools is less understood. The purpose of this study was to identify the prevalence of adolescents who have reported drug transaction experiences (being offered, sold, or given an illegal drug) on school property and subsequently identify behavioral correlates associated with these experiences. Methods This study analyzed YRBS data from 2001 to 2015 (N = 117,815). We used 95% confidence intervals to evaluate differences in prevalence across years and demographic subgroups. Odds ratios determined the likelihood of engaging in risk behaviors among those youth also experiencing drug transactions on school property. Results Every survey year, at least 1 in 5 youth reported having drug transaction experiences at school. Boys consistently reported significantly higher rates of drug transaction experiences in comparison to girls, with Hispanic boys representing the highest rates. Youth reporting these experiences with drug transactions were also more likely to carry a weapon to school and to be bullied at school. Conclusions Implications for the reduction of drug transactions in high schools are discussed and recommendations for prevention efforts addressing youth engagement in multiple risk behaviors are identified.

ObjectiveHydroxychloroquine (HCQ) is a mainstay of therapy in the treatment of SLE. The effect of HCQ on platelets and vascular health is uncertain. We investigated the relationship between HCQ use and dose with platelet activity, platelet transcriptomics and vascular health in patients with SLE.MethodsPlatelet aggregation, platelet mRNA expression and vascular health (sublingual capillary perfused boundary region (PBR), red blood cell filling (RBCF) and brachial artery reactivity testing) were analysed by HCQ use and dose.ResultsAmong 132 subjects with SLE (age: 39.7±12.9 years, 97% female), 108 were on HCQ. SLE disease activity was similar between subjects on and off HCQ. Platelet aggregation in response to multiple agonists was significantly lower in patients on HCQ. There were inverse relationships between HCQ dose and gene expression pathways of platelet activity. Gene expression of P-selectin (SELP) was inversely correlated with HCQ dose (r=−0.41, p=0.003), which was validated at the protein level. Subjects on HCQ had improved vascular function correlating with HCQ dose as measured by lower PBR (r=−0.52, p=0.007), higher RBCF (r=0.55, p=0.004) and greater brachial artery reactivity (r=0.43, p=0.056).ConclusionHCQ use was associated with decreased platelet activation and activation-related transcripts and improved vascular health in SLE.

Both SARS-CoV-2 infection and vaccination elicit potent immune responses. A number of studies have described immune responses to SARS-CoV-2 infection. However, beyond antibody production, immune responses to COVID-19 vaccines remain largely uncharacterized. Here, we performed multimodal single-cell sequencing on peripheral blood of patients with acute COVID-19 and healthy volunteers before and after receiving the SARS-CoV-2 BNT162b2 mRNA vaccine to compare the immune responses elicited by the virus and by this vaccine. Phenotypic and transcriptional profiling of immune cells, coupled with reconstruction of the B and T cell antigen receptor rearrangement of individual lymphocytes, enabled us to characterize and compare the host responses to the virus and to defined viral antigens. While both infection and vaccination induced robust innate and adaptive immune responses, our analysis revealed significant qualitative differences between the two types of immune challenges. In COVID-19 patients, immune responses were characterized by a highly augmented interferon response which was largely absent in vaccine recipients. Increased interferon signaling likely contributed to the observed dramatic upregulation of cytotoxic genes in the peripheral T cells and innate-like lymphocytes in patients but not in immunized subjects. Analysis of B and T cell receptor repertoires revealed that while the majority of clonal B and T cells in COVID-19 patients were effector cells, in vaccine recipients clonally expanded cells were primarily circulating memory cells. Importantly, the divergence in immune subsets engaged, the transcriptional differences in key immune populations, and the differences in maturation of adaptive immune cells revealed by our analysis have far-ranging implications for immunity to this novel pathogen.

Early-life antibiotic exposure perturbs the intestinal microbiota, alters innate intestinal immunity, and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here we found that maternal cecal microbiota transfer (CMT) to NOD mice with early-life antibiotic perturbation partially rescued the induced T1D acceleration. The restoration effects on the intestinal microbiome were substantial and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed cecal and serum metabolites and normalized innate and adaptive immune effectors. CMT restored patterns of ileal microRNA and histone regulation of gene expression and exon-splicing. Based on analyses of experimental data, we propose an innate intestinal immune network involving CD44, TLR2, and Reg3γ, as well as their multiple microRNA and epigenetic regulators that sense intestinal signaling by the gut microbiota. This regulation affects downstream immunological tone, leading to protection against the tissue-specific T1D injury.