Shotgun metagenomics and marker gene amplicon sequencing can be used to directly measure or predict the functional repertoire of the microbiota en masse, but current methods do not readily estimate the functional capability of individual microorganisms. Here we present BugBase, an algorithm that predicts organism-level coverage of functional pathways as well as biologically interpretable phenotypes such as oxygen tolerance, Gram staining and pathogenic potential, within complex microbiomes using either whole-genome shotgun or marker gene sequencing data. We find BugBase's organism-level pathway coverage predictions to be statistically higher powered than current 'bag-of-genes' approaches for discerning functional changes in both host-associated and environmental microbiomes.

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743 -7481. To investigate whether this stimulation was due to a soluble c1 subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein By dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that By subunits, purified from bovine retinal transducin (By,), markedly stimulated the hydrolysis of phosphatidylinositol4,Sbisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by by, was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin a subunit and was additive to activation of phospholipase C by guanosine 5'-[3-0-thio]triphosphate. byt also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to By,, but was also observed with highly purified subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in granulocytes. Only one of these forms was sensitive to stimulation by by,, demonstrating that stimulation of phospholipase C by by subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein By subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.The hydrolysis by phospholipase C of phosphatidylinositol 4,5-bisphosphate (PtdInsPJ to inositol 1,4,5-trisphosphate (InsP,) and diacylglycerol is a key mechanism by which many extracellular signalling molecules (hormones, growth factors and neurotransmitters) regulate the functions of their target cells [l, 21. There is ample evidence to suggest that many of the receptors interacting with these mediators stimulate phospholipase C via a guanine-nucleotide-binding protein (G protein) [3-51. In certain cell types, e.g.

Fecal microbiota transplantation (FMT) is now widely used to treat recurrent Clostridium difficile infection, but has been less studied as a means to restore microbiome diversity and composition following antibiotic or chemotherapy treatments. The purpose of our study was to assess the efficacy of FMT to reverse antibiotic- and chemotherapy-induced gut dysbiosis in a mouse model. C57BL/6J mice were treated with ampicillin for 1 week and/or received a single intraperitoneal injection of 5-Fluorouracil. Fresh stool was collected and analyzed using shotgun metagenomics and the Illumina sequencing platform. Ampicillin caused a significant and immediate decrease in bacterial species richness and diversity that persisted for one week. In mice that received FMT, disruption of the intestinal microbiota was reversed immediately. Antibiotic and chemotherapy administration caused significant alteration in species distribution, including a decrease in the relative proportions of Clostridium scindens and Faecalibacterium prausnitzii, and an increase in known pathogenic species. In mice receiving FMT, we observed a significant increase in species known to exhibit anti-inflammatory properties. Moreover, chemotherapy led to a critical decrease in key ‘health-promoting’ species and to an altered functional profile, especially when chemotherapy was administered in tandem with antibiotics, and that FMT can ameliorate these effects.

Bovine peripheral neutrophils contain high levels of a 40-kDa pertussis toxin substrate, which was found highly enriched in a light membrane fraction upon subcellular fractionation of neutrophil homogenates. The 40-kDa pertussis toxin substrate, referred to as a,, was purified to near homogeneity from t h s fraction by sequential ion-exchange, gel-filtration and hydrophobic chromatography. Purified a, was shown to interact with p,, subunits, undergo ADP-ribosylation by pertussis toxin, and bind guanine nucleotides with high affinity. The mobility of purified a, on SDS/polyacrylamide gels was intermediate between those of the a subunits of Gi and Go, purified from bovine brain, and slightly lower than the mobility of the a subunit of transducin (G,). Several polyclonal antisera against the a subunits of bovine GI and G, did not react with a, on immunoblots. CW 6, a polyclonal antiserum reactive against the bovine ai, reacted only minimally with a,.These results suggest that the major pertussis toxin substrate of bovine neutrophils, designated G,, is structurally different from previously identified pertussis toxin substrates and may represent a novel guanine-nucleotide-binding protein.

In neutrophils and several other phagocytic cell types, a pertussis-and cholera-toxin-sensitive form of the guanine-nucleotide-binding protein (G-protein) G, couples receptors for N-formylmethionine-containing chemotactic peptides to stimulation of phospholipase C. Using membranes of myeloid differentiated HL 60 cells, we have examined the role of Mg2+ and guanine nucleotides in regulating (a) the interaction of the formylpeptide receptor with the chemotactic agonist N-formylmethionyl-leucyl-phenylalanine (Met-Leu-Phe) and (b) the receptor-mediated activation of G,. Mg2+ markedly enhanced the number of receptors with high affinity for the radiolabeled oligopeptide fMet-Le~-[~H]Phe. At the same time, Mg" largely increased the potency of guanosine-5'-(3-@thio)triphosphate, but not of GDP or guanosine-5'-(2-O-thio)diphosphate, to inhibit binding of the peptide. Comparison of the potency of Mgz+ in eliciting these two effects and analysis of the specificities of the relevant divalent cation sites revealed that Mg2+ interacts with at least two independent sites on the receptor-G, complex. One site is specific for Mg2+ and exhibits affinity in the micromolar range, the other site interacts with millimolar concentrations of several divalent cations in a non-selective fashion. It is suggested that the former site is located on G, and that interaction of Mg2+ with this site is necessary for the receptor-mediated G-protein activation, whereas interaction of divalent cations with the latter site is necessary for high affinity agonist binding. The regulation of the formyl-peptide receptor binding properties by guanine nucleotides is independent of G, activation, since inhibition of peptide binding is achieved by addition of both guanine nucleoside diphosphates and triphosphates and is readily seen both in the presence and in the absence of Mg2+. The latter finding, together with the observation that, at micromolar concentrations of Mg2+, high-affinity GTPase activity is stimulated by Met-Leu-Phe primarily via low affinity receptors, suggests that, contrary to widely held opinions, (a) divalent cations are not required for a functional receptor -G-protein interaction and (b) high-affinity agonist binding is not a prerequisite for the receptor-mediated activation of the G-protein.

Therapeutic combinations to alter immunosuppressive, solid tumor microenvironments (TME), such as in breast cancer, are essential to improve responses to immune checkpoint inhibitors (ICI). Entinostat, an oral histone deacetylase inhibitor, has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs. The precise and comprehensive alterations to the TME induced by entinostat remain unknown. Here, we employed single-cell RNA sequencing on HER2-overexpressing breast tumors from mice treated with entinostat and ICIs to fully characterize changes across multiple cell types within the TME. This analysis demonstrates that treatment with entinostat induced a shift from a protumor to an antitumor TME signature, characterized predominantly by changes in myeloid cells. We confirmed myeloid-derived suppressor cells (MDSC) within entinostat-treated tumors associated with a less suppressive granulocytic (G)-MDSC phenotype and exhibited altered suppressive signaling that involved the NFκB and STAT3 pathways. In addition to MDSCs, tumor-associated macrophages were epigenetically reprogrammed from a protumor M2-like phenotype toward an antitumor M1-like phenotype, which may be contributing to a more sensitized TME. Overall, our in-depth analysis suggests that entinostat-induced changes on multiple myeloid cell types reduce immunosuppression and increase antitumor responses, which, in turn, improve sensitivity to ICIs. Sensitization of the TME by entinostat could ultimately broaden the population of patients with breast cancer who could benefit from ICIs.

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