Project description:This dataset was obtained from the authors of Starke et al. (2020, Journal of Proteomics 222: 103791, https://doi.org/10.1016/j.jprot.2020.103791). Sample labels: 1 - High fiber diet inoculum 2 - High protein diet inoculum 3,4,5 - High fiber + unlabeled water 6,7,8 - High protein + unlabeled water 9,10,11 - High fiber + 25% of 99.9 atom% D2O 12,13,14 - High protein + 25% of 99.9 atom% D2O 15,16,17 - High fiber + 25% of 99.0 atom% H218O 18,19,20 - High protein + 25% of 99.0 atom% H218O Direct quote from the original article describing the dataset. “…the impact of specific diets on a defined microbial community derived from a human fecal sample was to be determined. The defined community was chosen to provide a stable in vitro setup that could be useful for future microbiome research. Briefly, the microbial community was grown in a heavy fiber and a heavy protein diet in addition to 25% heavy water, either as D2O or H218O. A dosage of 25% isotopically labeled water was chosen by trial-and-error to yield sufficient incorporation into protein but avoid the reduction of activity of individual organisms. A difference in growth medium formulation, representing a high-fiber diet and a high-protein diet, was utilized to evaluate shifts in microbial activity. We chose to assess these diets because high-protein, low-carbohydrate interventions represent a popular weight-loss strategy and because we expected a strong effect on the synthesis of amino acids by this comparison. After an incubation time of 12 h, the proteins were analyzed for label-free quantitation (metaproteomics) and incorporation of isotopes (protein-SIP), each in triplicates….”
Project description:Sipuleucel-T (sip-T) is the only FDA-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). Here, we report the first high-dimensional analyses of sip-T using mass cytometry (CyTOF) and show a lymphoid predominance, with CD3+ T cells constituting the highest proportion (median ~60%) of sip-T, followed by B-cells, and natural killer (NK) and NKT cells. We hypothesized that treatment of sip-T with homeostatic cytokines known to activate/expand effector lymphocytes could augment efficacy against prostate cancer. Of cytokines tested, IL-15 treated sip-T showed the most significant activation/proliferation of effector lymphocytes in an antigen-specific manner, as well as augmentation of tumor cytotoxicity in-vitro. Adoptive transfer of IL-15 treated sip-T into NSG mice resulted in potent prostate tumor growth inhibition compared with control sip-T. Evaluation of tumor-infiltrating lymphocytes revealed a two-to-fourteen-fold higher influx of sip-T and a significant increase in interferon (IFN)-γ producing CD8+ T and NKT cells within the tumor microenvironment (TME) in the IL-15 group.
Project description:Sipuleucel-T (sip-T) is the only FDA-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). Here, we report the first high-dimensional analyses of sip-T using mass cytometry (CyTOF) and show a lymphoid predominance, with CD3+ T cells constituting the highest proportion (median ~60%) of sip-T, followed by B-cells, and natural killer (NK) and NKT cells. We hypothesized that treatment of sip-T with homeostatic cytokines known to activate/expand effector lymphocytes could augment efficacy against prostate cancer. Of cytokines tested, IL-15 treated sip-T showed the most significant activation/proliferation of effector lymphocytes in an antigen-specific manner, as well as augmentation of tumor cytotoxicity in-vitro. Adoptive transfer of IL-15 treated sip-T into NSG mice resulted in potent prostate tumor growth inhibition compared with control sip-T. Evaluation of tumor-infiltrating lymphocytes revealed a two-to-fourteen-fold higher influx of sip-T and a significant increase in interferon (IFN)-γ producing CD8+ T and NKT cells within the tumor microenvironment (TME) in the IL-15 group.
Project description:Previous research has linked perceived social isolation (loneliness) to reduced antiviral immunity, but the immunologic effects of the objective social isolation imposed by pandemic “shelter in place” (SIP) policies is unknown. We assessed the immunologic impact of SIP by relocating 21 adult male rhesus macaques from 2000 sq-m field cage communities of 70-132 other macaques to 2 wks of individual housing in indoor shelters. SIP was associated with down-regulation of Type I interferon (IFN) antiviral gene expression. This effect emerged within the first 48 hrs of SIP, persisted for at least 2 wks, and abated within 4 wks of return to social housing. A subsequent round of SIP in the presence of a novel juvenile macaque abrogated this effect. These results identify a significant adverse effect of SIP social isolation on antiviral immune regulation in circulating immune cells and they suggest a potential behavioral strategy for ameliorating such effects by promoting pro-social engagement during SIP.
Project description:Stable isotope probing (SIP) has been used to track nu- trient flows in microbial communities, but existing pro- tein-based SIP methods capable of quantifying the degree of label incorporation into peptides and proteins have been demonstrated only by targeting usually less than 100 proteins per sample. Our method automatically (i) identi- fies the sequence of and (ii) quantifies the degree of heavy atom enrichment for thousands of proteins from microbial community proteome samples. These features make our method suitable for comparing isotopic differences be- tween closely related protein sequences, and for detect- ing labeling patterns in low-abundance proteins or pro- teins derived from rare community members. The proteomic SIP method was validated using proteome samples of known stable isotope incorporation levels at 0.4%, 50%, and 98%. The method was then used to monitor incorporation of 15 N into established and regrow- ing microbial biofilms. The results indicate organism-spe- cific migration patterns from established communities into regrowing communities and provide insights into me- tabolism during biofilm formation. The proteomic SIP method can be extended to many systems to track fluxes of 13 C or 15 N in microbial communities. Molecular and Cellular Proteomics 10: 10.1074/mcp.M110.006049, 1–11, 2011.
Project description:Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil contaminated aquifer in Germany, where a specialized community of contaminant degraders co-dominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase alpha-subunit, bssA) genes detected in situ appeared more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae for sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This, and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy amongst anaerobic toluene degraders on site.
Project description:We developed an adaptation to Split-Pool Recognition of Interactions by Tag Extension (SPRITE) called SPRITE-immunoprecipitation (SIP), which enables us to map genome-wide higher-order interactions that are coupled with the protein of interest. In this study we generated SIP data fo H3K4me3 and pan-promoter mark. We generated SIP maps in two mammalian cell types – mouse embryonic stem cells (mES) and mouse bone marrow derived dendritic cells.
Project description:Chromatin loops are a major componant of 3D nuclear organization that appear as intense point-to-point interactions in Hi-C maps. Identification of these loops is an important part of Hi-C analysis. We present SIP, Significant Interaction Peak caller, a platform independent program to identify these loops in a time and memory efficient manner and which is resistant to noise and sequencing depth. We also present a companion tool, SIPMeta, to create more visually accurate average plots of Hi-C and HiChIP data. We then demonstrate that use of SIP and SIPMeta can lead to biological insight through characterizing the contribution of several transcription factors to CTCF loops in human cells. We then use SIP and SIPMeta to discover loops associated with condensin IDCC in C. elegans and confirm these loops by HiChIP. These loops form a network of interactions and likely explain the partial condensation of dosage compensated X chromosomes in hermaphrodites.
Project description:Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil contaminated aquifer in Germany, where a specialized community of contaminant degraders co-dominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase alpha-subunit, bssA) genes detected in situ appeared more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae for sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This, and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy amongst anaerobic toluene degraders on site. 2 samples examined from the different electron-acceptors (sulphate or ferric iron) incubates at the time point of maximal toluene degradation.