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The gut microbiome and its metabolic processes are dynamic systems. Surprisingly, our understanding of gut microbiome dynamics is limited. Here, we report a metaproteomic workflow that involves protein stable isotope probing (protein-SIP) and identification/quantification of partially labeled peptides. We also developed a package, which we call MetaProfiler, that corrects for false identifications and performs phylogenetic and time series analysis for the study of microbiome dynamics. From the stool sample of five mice that were fed with 15 N hydrolysate from Ralstonia eutropha , we identified 12 326 nonredundant unlabeled peptides, of which 8256 of their heavy counterparts were quantified. These peptides revealed incorporation profiles over time that were different between and within taxa, as well as between and within clusters of orthologous groups (COGs). Our study helps unravel the complex dynamics of protein synthesis and bacterial dynamics in the mouse microbiome. MetaProfiler and the bioinformatic pipeline are available at https://github.com/northomics/MetaProfiler.git.

In vitro culture based approaches are time- and cost-effective solutions for rapidly evaluating the effects of drugs or natural compounds against microbiomes. The nutritional composition of the culture medium is an important determinant for effectively maintaining the gut microbiome in vitro. This study combines orthogonal experimental design and a metaproteomics approach to obtaining functional insights into the effects of different medium components on the microbiome. Our results show that the metaproteomic profile respond differently to medium components, including inorganic salts, bile salts, mucin, and short-chain fatty acids. Multifactor analysis of variance further revealed significant main and interaction effects of inorganic salts, bile salts, and mucin on the different functional groups of gut microbial proteins. While a broad regulating effect was observed on basic metabolic pathways, different medium components also showed significant modulations on cell wall, membrane, and envelope biogenesis and cell motility related functions. In particular, flagellar assembly related proteins were significantly responsive to the presence of mucin. This study provides information on the functional influences of medium components on the in vitro growth of microbiome communities and gives insight on the key components that must be considered when selecting and optimizing media for culturing ex vivo microbiotas.

Background: The gut microbiota has been shown to be closely associated with human health and disease. While next-generation sequencing can be readily used to profile the microbiota taxonomy and metabolic potential, metaproteomics is better suited for deciphering microbial biological activities. However, the application of gut metaproteomics has largely been limited due to the low efficiency of protein identification. Thus, a high-performance and easy-to-implement gut metaproteomic approach is required., Results: In this study, we developed a high-performance and universal workflow for gut metaproteome identification and quantification (named MetaPro-IQ) by using the close-to-complete human or mouse gut microbial gene catalog as database and an iterative database search strategy. An average of 38 and 33 % of the acquired tandem mass spectrometry (MS) spectra was confidently identified for the studied mouse stool and human mucosal-luminal interface samples, respectively. In total, we accurately quantified 30,749 protein groups for the mouse metaproteome and 19,011 protein groups for the human metaproteome. Moreover, the MetaPro-IQ approach enabled comparable identifications with the matched metagenome database search strategy that is widely used but needs prior metagenomic sequencing. The response of gut microbiota to high-fat diet in mice was then assessed, which showed distinct metaproteome patterns for high-fat-fed mice and identified 849 proteins as significant responders to high-fat feeding in comparison to low-fat feeding., Conclusions: We present MetaPro-IQ, a metaproteomic approach for highly efficient intestinal microbial protein identification and quantification, which functions as a universal workflow for metaproteomic studies, and will thus facilitate the application of metaproteomics for better understanding the functions of gut microbiota in health and disease.

The lower risk of coronary artery disease in premenopausal women than in men and postmenopausal women implicates sex steroids in cardioprotective processes. β-Estradiol upregulates liver low-density lipoprotein receptor (LDLR), which, in turn, decreases circulating levels of low-density lipoprotein, which is a risk factor for coronary artery disease. Conversely, LDLR protein is negatively regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9). Herein, we investigated PCSK9 regulation by β-estradiol and its impact on LDLR in human hepatocarcinoma HuH7 cells grown in the presence or absence of β-estradiol. Immunoblot analysis showed upregulation of LDLR at 3 μm β-estradiol (140%), and the upregulation reached 220% at 10 μm β-estradiol; only at the latter dose was an increase in LDLR mRNA detected by qPCR, suggesting post-translational regulation of LDLR. No changes in PCSK9 mRNA or secreted protein levels were detected by qPCR or ELISA, respectively. β-estradiol-conditioned medium devoid of PCSK9 failed to upregulate LDLR. Similarly, PCSK9 knockdown cells showed no upregulation of LDLR by β-estradiol. Together, these results indicate a requirement for PCSK9 in the β-estradiol-induced upregulation of LDLR. A radiolabeling assay showed a significant, dose-dependent decrease in the ratio of secreted phosphoPCSK9 to total secreted PCSK9 with increasing β-estradiol levels, suggesting a change in the functional state of PCSK9 in the presence of β-estradiol. Our results indicate that the protein upregulation of LDLR at subtranscriptionally effective doses of β-estradiol, and its supratranscriptional upregulation at 10 μm β-estradiol, occur through an extracellular PCSK9-dependent mechanism., (© 2015 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.)

The objective of this retrospective clinical study was to determine the frequency and pathogenicity of unintentional retained tooth root fragments after extraction of the maxillary fourth premolar (108 and 208) and mandibular first molar teeth (309 and 409) in 74 canine and 42 feline client-owned patients. Radiographs of client-owned animals with historical evidence of extraction of teeth 309 and 409 were reviewed. All patients had dental extraction(s) for clinical reasons, and all extractions were deemed successful by the practitioners. Extraction sites were radiographed to identify tooth root fragments and pathology. Twenty-five canine and 25 feline patients that had extractions utilizing preoperative and postoperative radiography were also included. Sixty-one of 74 canine patients (82.4%; P < 0.0001) and 39 of 42 feline patients (92.8%; P < 0.0001) had evidence of retained tooth root fragments. In total, 100 of 116 cases (86.4%; P < 0.0001) had retained tooth root fragments, and periapical pathology was found in 66 of 116 (56.8%; P = 0.000000743) radiographs, including 39 of 74 canine cases (52.7%; P = 0.00002765) and 27 of 42 feline cases (64.3%; P = 0.01589). The control group had no evidence of retained root fragments. Further veterinary dental training and routine use of pre- and postoperative dental radiology are recommended.

From experience with placental pathology and autopsies, we hypothesize that infected amniotic fluid may produce umbilical cord and placental surface vasospasm, reduced fetal perfusion, and perinatal asphyxia, morbidity, and mortality. We tested the hypothesis by a preliminary investigation with group B beta-hemolytic streptococcus, a common cause of perinatal morbidity and mortality. Our methods included suspension of isolated human umbilical vein segments in a 30-mL organ bath with modified Krebs solution, pH 7.3-7.4, under continuous aeration by 95% O2 and 5% CO2 at 37C. The vessels were attached to an isometric transducer which was connected to a polygraph. We tested Krebs solution, sterile culture medium, group B streptococcus spent medium, and washed whole group B streptococcus bacteria. Contractility was compared with results produced by 10(-5) M serotonin. The average contraction produced by 30 microL/mL group B streptococcus spent medium was 40.8% of that produced by serotonin. Whole washed bacteria produced 8.6% of the serotonin contraction. Krebs solution and sterile culture medium did not stimulate vascular contraction. Intraperitoneal injection of group B streptococcus spent medium in mice at four times the maximal experimental dose had little effect. Boiling group B streptococcus spent medium failed to affect its vasoactive stimulatory properties. We conclude that in vitro, group B streptococcus synthesizes a heat-stable, non-protein exotoxin that causes human umbilical vein contraction.

The isolated working guinea-pig heart was used to compare the cardiostimulatory effects of acetylcholine and nicotine observed in the presence of atropine. Both agonists increased aortic pressure, left ventricular pressure, left ventricular dP/dt, cardiac output, and ventricular cyclic AMP levels. These responses were qualitatively and quantitatively similar to the effects of exogenous norepinephrine. Hexamethonium treatment abolished the responses to acetylcholine and to nicotine. However, several differences in the responses of the two agonists were also observed with respect to: 1) the effect of propranolol pretreatment, 2) selective effects on coronary and aortic flow rates, 3) coefficients of correlation between ventricular cyclic AMP and changes in dP/dt, and 4) the "autoinhibition" effect. The results support the view that the cardiostimulatory effects of acetylcholine are due entirely to endogenous catecholamine release, but that the effects of nicotine may involve an additional action.

The topical application of carbon dioxide water to the rat hindpaw produced a concentration-dependent increase of skin blood flow as measured by a laser Doppler flowmeter. About a 100% increase of skin blood flow occurred in response to CO2 when the bath temperature was at 23 degrees C or 34 degrees C, but there was no significant effect of CO2 at 41 degrees C. Carbon dioxide exposure also produced about the same increase of skin blood flow in the acutely or chronically denervated paw as in the control. These findings give experimental support for the clinical use of CO2 bathing in the treatment of disturbances of skin circulation as well as skin ulcers and wounds.

The ionophore X-537A increased heart rate and contractility of the isolated, working rat heart preparation. The increased heart rate appeared to be caused solely by release of catecholamines as the response was completely eliminated by reserpine pretreatment or addition of propranolol to the perfusate. The inotropic response, however, had an apparent catecholamine-independent component as neither propranolol, nor propranol in combination with phentolamine, completely eliminated the inotropic response to X-537A. On the other hand, reserpine pretreatment did abolish the inotropic effect of the ionophore but this action appeared to be a nonspecific one as the responses to norepinephrine and to CaCl2 were substantially diminished.