The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another ...important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2−/− model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2−/− mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2−/− mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them.
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•Host genetics and microbiota differentially regulate behaviors in an ASD mouse model•Microbe therapy (L. reuteri) rescues Cntnap2−/− social deficits but not hyperactivity•Microbe-induced metabolite (BH4) selectively rescues Cntnap2−/− social deficits•L. reuteri and BH4 improves Cntnap2−/− social-reward-mediated synaptic transmission
Host genetics and the microbiome interdependently regulate maladaptive behaviors in a mouse model for neurodevelopmental disorders. Additionally, precise microbial therapy, or microbe-induced metabolite-based therapy, selectively rescues social deficits but not hyperactivity.
Proper characterization of cancer cell states within the tumor microenvironment is a key to accurately identifying matching experimental models and the development of precision therapies. To ...reconstruct this information from bulk RNA-seq profiles, we developed the XDec Simplex Mapping (XDec-SM) reference-optional deconvolution method that maps tumors and the states of constituent cells onto a biologically interpretable low-dimensional space. The method identifies gene sets informative for deconvolution from relevant single-cell profiling data when such profiles are available. When applied to breast tumors in The Cancer Genome Atlas (TCGA), XDec-SM infers the identity of constituent cell types and their proportions. XDec-SM also infers cancer cells states within individual tumors that associate with DNA methylation patterns, driver somatic mutations, pathway activation and metabolic coupling between stromal and breast cancer cells. By projecting tumors, cancer cell lines, and PDX models onto the same map, we identify in vitro and in vivo models with matching cancer cell states. Map position is also predictive of therapy response, thus opening the prospects for precision therapy informed by experiments in model systems matched to tumors in vivo by cancer cell state.
Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly ...attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers.
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•GBM EVs and growth factors promote angiogenesis by distinct pathways•EV-exposed endothelial cells show a footprint of gene regulation by EV miRNAs•The EV miRNA pathway explains failure of anti-angiogenic therapy for GBM•The results suggest an angiogenic pathway and liquid biopsy biomarkers for GBM
Extensive intercellular interactions occur within the notoriously heterogeneous tumor microenvironment of GBM. Lucero et al. identify distinct angiogenic gene regulatory responses of brain endothelial cells to growth factors and to extracellular vesicles (EVs) secreted by GBM stem-like cells. The response to EVs shows a footprint of EV-derived miRNAs.
Glucose–insulin models, testing glucose sensors and support systems for health care decisions play an important role in synthesis of glucose control algorithms. In this work we propose an online ...glucose–insulin identification using the Recurrent High Order Neural Network (RHONN). Then, the model obtained is used to predict n-steps forward of glucose levels, also by RHONN. The used data for identification is from a Type 1 Diabetes Mellitus (T1DM) patient, it was collected from the Continuous Monitoring Glucose System (CMGS) by MiniMed Inc ® and an insulin pump by Paradigm Real-time Insulin Pump ®. RHONN is trained online by Extended Kalman Filter (EKF). The results suggest that it is possible to make a prediction of up to 35 min in the future, which it would help to prevent risky events (hypoglycemia and hyperglycemia). Also shows that, it could be directly connected to a CGMS to help the patient improve the glucose control and even an automatic glucose control algorithm. The proposed Neural Network shows good performance compared to baseline methods in terms of evaluation criteria.
•With RHOON we perform identification of the dynamics of blood glucose.•35 min of blood glucose were predicted with RHONN.•Training for the identification and prediction network was done online.•RHONN predictions are compared with three methods.
In response to signals from the embryonic testis, the germ cell intrinsic factor NANOS2 coordinates a transcriptional program necessary for the differentiation of pluripotent-like primordial germ ...cells toward a unipotent spermatogonial stem cell fate. Emerging evidence indicates that genetic risk factors contribute to testicular germ cell tumor initiation by disrupting sex-specific differentiation. Here, using the 129.MOLF-Chr19 mouse model of testicular teratomas and a NANOS2 reporter allele, we report that the developmental phenotypes required for tumorigenesis, including failure to enter mitotic arrest, retention of pluripotency and delayed sex-specific differentiation, were exclusive to a subpopulation of germ cells failing to express NANOS2. Single-cell RNA sequencing revealed that embryonic day 15.5 NANOS2-deficient germ cells and embryonal carcinoma cells developed a transcriptional profile enriched for MYC signaling, NODAL signaling and primed pluripotency. Moreover, lineage-tracing experiments demonstrated that embryonal carcinoma cells arose exclusively from germ cells failing to express NANOS2. Our results indicate that NANOS2 is the nexus through which several genetic risk factors influence tumor susceptibility. We propose that, in the absence of sex specification, signals native to the developing testis drive germ cell transformation.
Targeted mass spectrometry (MS)-based proteomic assays, such as multiplexed multiple reaction monitoring (MRM)-MS assays, enable sensitive and specific quantification of proteotypic peptides as ...stoichiometric surrogates for proteins. Efforts are underway to expand the use of MRM-MS assays in clinical environments, which requires a reliable strategy to monitor proteolytic digestion efficiency within individual samples. Towards this goal, extended stable isotope-labeled standard (SIS) peptides (hE), which incorporate native proteolytic cleavage sites, can be spiked into protein lysates prior to proteolytic (trypsin) digestion, and release of the tryptic SIS peptide (hT) can be monitored. However, hT measurements alone cannot monitor the extent of digestion and may be confounded by matrix effects specific to individual patient samples; therefore, they are not sufficient to monitor sample-to-sample digestion variability. We hypothesized that measuring undigested hE, along with its paired hT, would improve detection of digestion issues compared to only measuring hT. We tested the ratio of the SIS pair measurements, or hE/hT, as a quality control (QC) metric of trypsin digestion for two MRM assays: a direct-MRM (398 targets) and an immuno-MRM (126 targets requiring immunoaffinity peptide enrichment) assay, with extended SIS peptides observable for 54% (216) and 62% (78) of the targets, respectively. We evaluated the quantitative bias for each target in a series of experiments that adversely affected proteolytic digestion (e.g., variable digestion times, pH, and temperature). We identified a subset of SIS pairs (36 for the direct-MRM, 7 for the immuno-MRM assay) for which the hE/hT ratio reliably detected inefficient digestion that resulted in decreased assay sensitivity and unreliable endogenous quantification. The hE/hT ratio was more responsive to a decrease in digestion efficiency than a metric based on hT measurements alone. For clinical-grade MRM-MS assays, this study describes a ready-to-use QC panel and also provides a road map for designing custom QC panels.
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•Extended SIS peptides can control for trypsin digestion in targeted MS-based assays.•Monitoring extended SIS along with tryptic SIS peptides is the basis of a QC metric.•The ratio of extended to tryptic SIS is a useful in-sample QC metric for digestion.•The QC metric also correlates with assay sensitivity and quantification fidelity.•Additional assays can apply this ready-to-use QC panel or identify custom panels.
Extended stable isotope-labeled standard (SIS) peptides (hE), proteolytically digested to release the targeted tryptic SIS peptide (hT) form, have been shown to control for trypsin. We found that by monitoring hE along with hT, the resulting hE/hT ratio is a useful in-sample QC metric for digestion efficiency that correlates with endogenous measurement sensitivity and quantification fidelity. This study describes a ready-to-use QC panel as well as a path for identifying custom QC panels that can be applied to additional mass spectrometry-based assays.
Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and ...immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens.
Here, we developed a panel of novel monoclonal antibodies and incorporated them into a novel, multiplexed, immuno-multiple reaction monitoring mass spectrometry (MRM-MS)-based proteomic assay targeting 49 proteotypic peptides representing 43 immunomodulatory proteins.
The multiplex assay was validated in human tissue and plasma matrices, where the linearity of quantification was >3 orders of magnitude with median interday CVs of 8.7% (tissue) and 10.1% (plasma). Proof-of-principle demonstration of the assay was conducted in plasma samples collected in clinical trials from lymphoma patients receiving an immune checkpoint inhibitor. We provide the assays and novel monoclonal antibodies as a publicly available resource for the biomedical community.
To improve the understanding of chemo-refractory high-grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one ...discovery and two validation cohorts across two biospecimen types (formalin-fixed paraffin-embedded and frozen). We identified a 64-protein signature that predicts with high specificity a subset of HGSOCs refractory to initial platinum-based therapy and is validated in two independent patient cohorts. We detected significant association between lack of Ch17 loss of heterozygosity (LOH) and chemo-refractoriness. Based on pathway protein expression, we identified 5 clusters of HGSOC, which validated across two independent patient cohorts and patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic vulnerabilities.
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•A comprehensive proteogenomic analysis of 242 HGSOC tumors was performed•A lack of Chr17-LOH was observed to be associated with refractoriness•A 64-protein signature predicts refractoriness in multiple tumor cohorts•Pathway-based clustering reveals 5 subtypes validated in independent cohorts
Patients with high-grade serous ovarian cancers (HGSOCs) have a poor outcome, with the standard of care not having changed over the decades. A detailed characterization of the proteogenomic landscape of HGSOCs across multiple cohorts and validation studies identifies a distinct signature that predicts with high specificity a subset of patients with chemotherapy-refractory cancers and implicates potential therapeutic vulnerabilities.
Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are ...not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.
Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and costly clinical ...validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Previously, mass spectrometry-based approaches have been used to fill this gap but feature poor quantitative performance and were generally limited to hundreds of proteins. Here, we demonstrate the capability of an internal standard triggered-parallel reaction monitoring (IS-PRM) assay to greatly expand the numbers of candidates that can be tested with improved quantitative performance. The assay couples immunodepletion and fractionation with IS-PRM and was developed and implemented in human plasma to quantify 5176 peptides representing 1314 breast cancer biomarker candidates. Characterization of the IS-PRM assay demonstrated the precision (median % CV of 7.7%), linearity (median R 2 > 0.999 over 4 orders of magnitude), and sensitivity (median LLOQ < 1 fmol, approximately) to enable rank-ordering of candidate biomarkers for validation studies. Using three plasma pools from breast cancer patients and three control pools, 893 proteins were quantified, of which 162 candidate biomarkers were verified in at least one of the cancer pools and 22 were verified in all three cancer pools. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.
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