Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds

status: publishe

Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N(3)-Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

The 6-anilinouracils (AUs) constitute a new class of bactericidal antibiotics selective against gram-positive (Gr(+)) organisms. The AU family of compounds specifically inhibits a novel target, replicative DNA polymerase Pol IIIC. Like other antibiotics, AUs can be expected to engender the development of resistant bacteria. We have used a representative AU and clinically relevant strains of Staphylococcus aureus and Enterococcus to determine the frequency and mechanism(s) of resistance development. The frequency of resistance was determined by using N(3)-hydroxybutyl 6-(3′-ethyl-4′-methylanilino) uracil (HBEMAU) and commercially available antibiotics at eight times the MICs. For all five Gr(+) organisms tested, the frequency of resistance to HBEMAU ranged from 1 × 10(−8) to 3 × 10(−10). The frequencies of resistance to the antibiotics tested, including rifampin, gentamicin, and ciprofloxacin, were either greater than or equal to those for HBEMAU. In order to understand the mechanism of resistance, HBEMAU-resistant organisms were isolated. MIC assays showed that the organisms had increased resistance to AU inhibitors but not to other families of antibiotics. Inhibition studies with DNA polymerases from HBEMAU-sensitive and -resistant strains demonstrated that the resistance was associated with Pol IIIC. DNA sequence analysis of the entire polC genes from both wild-type and resistant organisms revealed that the resistant organisms had a sequence change that mapped to a single amino acid codon in all strains examined

The Effects of Physical Activity on Arterial Stiffness during Pregnancy: An Observational Study

The objective of the present study was to investigate the relationship between moderate-to-vigorous physical activity (MVPA) and arterial stiffness in pregnancy. Thirty-nine women participated in this study resulting in 68 measurements in non-pregnant (NP; n=21), first (TM1; n=8), second (TM2; n=20), and third trimesters (TM3; n=19). Compliance, distensibility, elasticity, β-stiffness, and carotid to femoral (central) and carotid to finger (peripheral) pulse wave velocity (PWV) were assessed. MVPA was measured using accelerometry. Multilevel linear regressions adjusted for multiple tests per participant using random effects to generate β coefficients and 95% confidence intervals (CI) were performed. Distensibility, elasticity, β-stiffness, central- and peripheral-PWV did not differ between pregnant and non-pregnant assessments. Carotid artery compliance was higher in TM2 compared to NP. Central PWV (β Coef: -0.14, 95% CI: -0.27, -0.02) decreased from early to mid-pregnancy and increased in late pregnancy. Meeting the MVPA guidelines was significantly associated with central-PWV (Adj. β Coef: -0.34, 95% CI: -0.62, -0.06, p=0.016), peripheral-PWV (Adj. β Coef: -0.54, 95% CI: -0.91, -0.16, p=0.005), and distensibility (Adj. β Coef: -0.001, 95% CI: -0.002, -0.0001, p=0.018), in pregnancy. These results suggest that MVPA may be associated with improved (i.e. reduced) arterial stiffness in pregnancy. Novelty Bullets • Central PWV, distensibility, compliance, elasticity, and -stiffness, but not peripheral PWV, exhibited curvilinear relationships with gestational age • Central and peripheral PWV were lower in pregnant women who met the physical activity guidelines of 150 minutes of moderate-to-vigorous physical activity per weekThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Antibacterial Activity and Mechanism of Action of a Novel Anilinouracil-Fluoroquinolone Hybrid Compound

The anilinouracils (AUs) such as 6-(3-ethyl-4-methylanilino)uracil (EMAU) are a novel class of gram-positive, selective, bactericidal antibacterials which inhibit pol IIIC, the gram-positive-specific replicative DNA polymerase. We have linked various fluoroquinolones (FQs) to the N-3 position of EMAU to generate a variety of AU-FQ “hybrids” offering the potential for targeting two distinct steps in DNA replication. In this study, the properties of a hybrid, “251D,” were compared with those of representative AUs and FQs in a variety of in vitro assays, including pol IIIC and topoisomerase/gyrase enzyme assays, antibacterial, bactericidal, and mammalian cytotoxicity assays. Compound 251D potently inhibited pol IIIC and topoisomerase/gyrase, displayed gram-positive antibacterial potency at least 15 times that of the corresponding AU compound, and as expected, acted selectively on bacterial DNA synthesis. Compound 251D was active against a broad panel of antibiotic-resistant gram-positive pathogens as well as several gram-negative organisms and was also active against both AU- and FQ-resistant gram-positive organisms, demonstrating its capacity for attacking both of its potential targets in the bacterium. 251D also was bactericidal for gram-positive organisms and lacked toxicity in vitro. Although we obtained strains of Staphylococcus aureus resistant to the individual parent compounds, spontaneous resistance to 251D was not observed. We obtained 251D resistance in multiple-passage experiments, but resistance developed at a pace comparable to those for the parent compounds. This class of AU-FQ hybrids provides a promising new pharmacophore with an unusual dual mechanism of action and potent activity against antibiotic-sensitive and -resistant gram-positive pathogens

Synthesis and antibacterial activity of 3-substituted-6-(3-ethyl-4-methylanilino)uracils

Numerous 3-substituted-6-(3-ethyl-4-methylanilino)uracils (EMAU) have been synthesized and screened for their capacity to inhibit the replication-specific bacterial DNA polymerase IIIC (pol IIIC) and the growth of Gram+ bacteria in culture. Direct alkylation of 2-methoxy-6-amino-4-pyrimidone produced the N3-substituted derivatives, which were separated from the byproduct 4-alkoxy analogues. The N3-substituted derivatives were heated with a mixture of 3-ethyl-4-methylaniline and its hydrochloride to effect displacement of the 6-amino group and simultaneous demethylation of the 2-methoxy group to yield target compounds in good yields. Certain intermediates, e.g. the 3-(iodoalkyl) compounds, were converted to a variety of (3-substituted-alkyl)-EMAUs by displacement. Most compounds were potent competitive inhibitors of pol IIIC (K(i)s 0.02-0.5 microM), and those with neutral, moderately polar 3-substituents had potent antibacterial activity against Gram+ organisms in culture (MICs 0.125-10 microg/mL). Several compounds protected mice from lethal intraperitoneal (ip) infections with S. aureus (Smith) when given by the ip route. A water soluble derivative, 3-(4-morpholinylbutyl)-EMAU hydrochloride, given subcutaneously, prolonged the life of infected mice in a dose dependent manner

A GPBAR1 (TGR5) small molecule agonist shows specific inhibitory effects on myeloid cell activation in vitro and reduces experimental autoimmune encephalitis (EAE) in vivo.

GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases

GPBAR1 agonism reduces EAE disease severity.

<p>(A) EAE clinical scores are shown for both vehicle and BIX02694 groups (n = 10 mice per group). (B) Cell counts of CNS immune cells assessed on day 13 post-immunization are shown (n = 5 mice). (C) Percentages of blood monocytes (n = 5) or (C) lymphocytes (n = 5) are shown for naive mice (n = 8), along with EAE mice treated with either vehicle or BIX02694. Protein expression of activation markers on both CNS-infiltrating monocytes (E) and microglia (F) are shown (n = 5 mice per group). (G) Protein expression of activation markers on blood monocytes from EAE mice treated with either vehicle or BIX02694 (n = 5 mice per group).</p

Agonism of GPBAR1 on human monocytes modulates a subset of LPS activated genes and blocks M1 differentiation.

<p>Monocytes from 5 donors were primed with IFNγ and stimulated with LPS ± agonist. RNA was isolated from these samples and subjected to RNA-Seq. (A) Heatmap of genes whose activation or repression by LPS at 6 hours was inhibited by the GPBAR1 agonist EX00000246. (B) Cellular function data from Ingenuity Systems was mapped onto the GPBAR1 regulated genes. (C) Heatmap of genes annotated as cytokine or chemokine whose activation or repression by LPS at 6 hours, 24 hours, or both times was inhibited by the GPBAR1 agonist EX00000246. (D) Individual plots of <i>Ccr7</i>, <i>Il12b</i> expression and ratio of <i>Il12/Il10</i> expression after stimulation with LPS treated with EX00000246.</p

Mouse monocytes express GPBAR1 and its agonism reduces LPS-induced cytokine production.

<p>(A) <i>Gpbar1</i> and <i>18s</i> mRNA levels were assessed by Taqman in C57BL/6 mouse whole blood and isolated leukocytes (n = 2 for leukocytes, n = 5 for blood). (B–C) GPBAR1 protein levels were assessed on mouse blood cells by flow cytometry using CD115 to stain monocytes. (B) Gating scheme and representative histograms of lymphocytes, granulocytes and monocytes are shown (n = 1 is shown, representative of 3 mice). (C) Median fluorescence intensity of GPBAR1 on monocytes, granulocytes, and lymphocytes is shown (n = 3, averaged). Monocytes stained with an isotype control are the control sample (B) and (C). (D–F) Impact of GPBAR1 agonism on LPS induced cytokines. Blood from C57BL/6 mice (n = 5, averaged) was stimulated ex vivo with 100 ng LPS in the presence of BIX02694 and intracellular TNFα in monocytes was measured by flow cytometry (D). Balb/c mice were challenged with 2 µg LPS, in the presence of vehicle, Prednisolone (3 mg/kg), or BIX02694 (0.3, 3, 10 mg/kg) and serum cytokine levels were measured; TNFα at 1 hour (E) IL-12p40 (F) and CCL2 (G) both at 4 hours post LPS administration.</p