Establishment of DsRed.T3_S4T as an improved autofluorescent marker for microbial ecology applications

Autofluorescent proteins (AFPs), such as green fluorescent protein (GFP) and DsRed, are valuable tools for studying plant-microbe interactions. Nevertheless, because of some limitations, efforts are ongoing to generate improved AFP variants. Several groups have generated variants of GFP with altered spectral characteristics, and faster maturing and brighter variants of DsRed. In this study we used plasmid and chromosomal constructs to test the efficacy of a new variant of DsRed, DsRed.T3_S4T, in Pseudomonas fluorescens F113rif. In addition, we compared the ecological fitness of strains carrying chromosomal copies of EGFP, DsRed or DsRed.T3_S4T. Strains expressing DsRed.T3_S4T fluoresced significantly brighter than strains expressing DsRed. Furthermore, it was found that although all strains grew equally well in vitro, only strains carrying DsRed.T3_S4T functioned as well as wild type in a competitive rhizosphere colonization assay. In particular, it was observed that DsRed.T3_S4T is an improved marker over DsRed for microbial ecology studies in this strain.     

Metabolic effect of telmisartan and losartan in hypertensive patients with metabolic syndrome

Background

Metabolic syndrome is a cluster of common cardiovascular risk factors that includes hypertension and insulin resistance. Hypertension and diabetes mellitus are frequent comorbidities and, like metabolic syndrome, increase the risk of cardiovascular events. Telmisartan, an antihypertensive agent with evidence of partial peroxisome proliferator-activated receptor activity-gamma (PPARγ) activity, may improve insulin sensitivity and lipid profile in patients with metabolic syndrome.

Methods

In a double-blind, parallel-group, randomized study, patients with World Health Organization criteria for metabolic syndrome received once-daily doses of telmisartan (80 mg, n = 20) or losartan (50 mg, n = 20) for 3 months. At baseline and end of treatment, fasting and postprandial plasma glucose, insulin sensitivity, glycosylated haemoglobin (HBA_1c) and 24-hour mean systolic and diastolic blood pressures were determined.

Results

Telmisartan, but not losartan, significantly (p < 0.05) reduced free plasma glucose, free plasma insulin, homeostasis model assessment of insulin resistance and HbA_ic. Following treatment, plasma glucose and insulin were reduced during the oral glucose tolerance test by telmisartan, but not by losartan. Telmisartan also significantly reduced 24-hour mean systolic blood pressure (p < 0.05) and diastolic blood pressure (p < 0.05) compared with losartan.

Conclusion

As well as providing superior 24-hour blood pressure control, telmisartan, unlike losartan, displayed insulin-sensitizing activity, which may be explained by its partial PPARγ activity.     

Effects of i.c.v. losartan on the angiotensin II-mediated vasopressin release and hypothalamic fos expression in near-term ovine fetuses

Our previous studies have shown that central administration of angiotensin (ANG II) causes arginine vasopressin (AVP) release in the fetus at 70-90% gestation. This is evidence that the hypothalamic-neurohypophysial system is relatively mature before birth. However, few data exist regarding central ANG receptor mechanisms-mediated AVP response during fetal life. To determine roles of brain ANG receptor subtypes in this response, AT1 and AT2 receptor antagonists, losartan and PD123319, were investigated in the brain in chronically prepared ovine fetuses at the last third of gestation. Application of losartan intracerebroventricularly (i.c.v.) at 0.5 mg/kg suppressed central ANG II-stimulated plasma AVP release. Losartan at 5 mg/kg (i.c.v.) demonstrated a significant enhancement of AVP increase to i.c.v. ANG II. Associated with the increase of plasma vasopressin levels, c-fos expression in the hypothalamic neurons was significantly different between the low and high doses of losartan. The low dose losartan markedly reduced the dual immunoreactivity for FOS and AVP in the supraoptic nuclei and paraventricular nuclei after i.c.v. ANG II, whereas the high dose losartan together with ANG II, significantly increased the co-localization of positive FOS in the AVP-containing neurons than that induced by i.c.v. ANG II alone. Central ANG II induced fetal plasma vasopressin increase was not altered by PD123319. The data suggest that losartan in the fetal brain has remarkably different effects based on the doses administrated on central ANG II-related neuroendocrine effects at the late gestation, and that the AT1 mechanism is critical in the regulation of fetal body fluid homeostasis related to plasma AVP levels.     

Restoring full biological activity to the isolated ectodomain of an integral membrane protein

Integral membrane proteins, which include many cellular effector proteins and drug targets, can be difficult to produce, purify, and manipulate. Although the isolated ectodomains of many membrane proteins can be expressed as water soluble proteins, biological activity is frequently lost when these proteins are released from the membrane surface. An example is tissue factor, the integral membrane protein that triggers the blood clotting cascade and for which membrane anchoring is essential. Its isolated ectodomain (soluble tissue factor) can be expressed with high yield in bacteria but is orders of magnitude less active than the intact, membrane-anchored protein. We now report full restoration of biological activity to the isolated tissue factor ectodomain via the engineering of a hexahistidine tag onto its C-terminus and its use in combination with membrane bilayers containing nickel-chelating lipids. When soluble tissue factor was tethered to the membrane surface via such metal-chelating lipids, it bound factor VIIa with the same high affinity as wild-type tissue factor, and the resulting factor VIIa-tissue factor complexes supported factor X activation and factor VII autoactivation with essentially wild-type enzyme kinetic constants. Furthermore, when such bilayers were immobilized onto solid supports, they efficiently captured histidine-tagged soluble tissue factor directly from crude culture supernatants, with full biological activity, obviating the need for purification or laborious membrane reconstitution procedures. This strategy is rapid, efficient, scalable, and automatable and should be applicable to other integral membrane proteins, especially those with a single transmembrane domain. Applications include high-throughput screening of mutants or drugs, flow reactors, clinical assays, and point-of-care instrumentation.     

Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation

Pseudomonas spp. that can colonise the roots of crop plants and produce antifungal metabolites represent a real alternative to the application of chemical fungicides. Presently, much research is aimed at understanding, at the molecular level, the mechanisms that enable Pseudomonas strains to act as efficient biological control agents. This approach is facilitating the development of novel strains with modified traits for enhanced biocontrol efficacy. However, without solving some inherent problems associated with the effective delivery of microbial inoculants to seeds and without knowledge on the biosafety aspects of novel biocontrol agents, the commercial potential of Pseudomonas spp. for plant disease control will not be realised.     

Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-aryloxy substituents of 2,6-diphenoxypyridines

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.     

Metabolomic profiling of regulatory lipid mediators in sputum from adult cystic fibrosis patients

Retained respiratory tract (RT) secretions, infection, and exuberant inflammatory responses are core abnormalities in cystic fibrosis (CF) lung disease. Factors contributing to the destructive CF airway inflammatory processes remain incompletely characterized. The pro-oxidative inflammatory CF RT milieu is known to contain enzymatically and nonenzymatically produced regulatory lipid mediators, a panel of structurally defined oxidized metabolites of polyunsaturated fatty acids known to play a role in pathology related to inflammation. Using an extraction protocol that maximizes recoveries of sputum-spiked deuterated standards, coupled with an LC/MS/MS detection system, this study presents a metabolomic method to assess a broad spectrum of regulatory lipid mediators in freshly obtained sputum from CF patients. A broad range of both proinflammatory and anti-inflammatory lipid mediators was detected, including PGE2, PGD2, TXB2, LTB4, 6-trans-LTB4, 20-OH-LTB4, 20-COOH-LTB4, 20-HETE, 15-HETE, 11-HETE, 12-HETE, 8-HETE, 9-HETE, 5-HETE, EpETrEs, diols, resolvin E1, 15-deoxy-PGJ2, and LXA4. The vast majority of these oxylipins have not been reported previously in CF RT secretions. Whereas direct associations of individual proinflammatory lipid mediators with compromised lung function (FEV-1) were observed, the relationships were not robust. However, multiple statistical analyses revealed that the regulatory lipid mediators profile taken in aggregate proved to have a stronger association with lung function in relatively stable outpatient adult CF patients. Our data reveal a relative paucity of the anti-inflammatory lipid mediator lipoxin A4 in CF sputum. Patients displaying detectable levels of the anti-inflammatory lipid mediator resolvin E1 demonstrated a better lung function compared to those patients with undetectable levels. Our data suggest that comprehensive metabolomic profiling of regulatory lipid mediators in CF sputum should contribute to a better understanding of the molecular mechanisms underlying CF RT inflammatory pathobiology. Further studies are required to determine the extent to which nutritional or pharmacological interventions alter the regulatory lipid mediators profile of the CF RT and the impact of potential modulations of RT regulatory lipid mediators on the clinical progression of CF lung disease.     

Directional selection in temporally replicated studies is remarkably consistent

Temporal variation in selection is a fundamental determinant of evolutionary outcomes. A recent paper presented a synthetic analysis of temporal variation in selection in natural populations. The authors concluded that there is substantial variation in the strength and direction of selection over time, but acknowledged that sampling error would result in estimates of selection that were more variable than the true values. We reanalyze their dataset using techniques that account for the necessary effect of sampling error to inflate apparent levels of variation and show that directional selection is remarkably constant over time, both in magnitude and direction. Thus we cannot claim that the available data support the existence of substantial temporal heterogeneity in selection. Nonetheless, we conject that temporal variation in selection could be important, but that there are good reasons why it may not appear in the available data. These new analyses highlight the importance of applying techniques that estimate parameters of the distribution of selection, rather than parameters of the distribution of estimated selection (which will reflect both sampling error and "real" variation in selection); indeed, despite availability of methods for the former, focus on the latter has been common in synthetic reviews of the aspects of selection in nature, and can lead to serious misinterpretations.     

The prediction of adaptive evolution: empirical application of the secondary theorem of selection and comparison to the breeder's equation

Adaptive evolution occurs when fitness covaries with genetic merit for a trait (or traits). The breeder's equation (BE), in both its univariate and multivariate forms, allows us to predict this process by combining estimates of selection on phenotype with estimates of genetic (co)variation. However, predictions are only valid if all factors causal for trait-fitness covariance are measured. Although this requirement will rarely (if ever) be met in practice, it can be avoided by applying Robertson's secondary theorem of selection (STS). The STS predicts evolution by directly estimating the genetic basis of trait-fitness covariation without any explicit model of selection. Here we apply the BE and STS to four morphological traits measured in Soay sheep (Ovis aries) from St. Kilda. Despite apparently positive selection on heritable size traits, sheep are not getting larger. However, although the BE predicts increasing size, the STS does not, which is a discrepancy that suggests unmeasured factors are upwardly biasing our estimates of selection on phenotype. We suggest this is likely to be a general issue, and that wider application of the STS could offer at least a partial resolution to the common discrepancy between naive expectations and observed trait dynamics in natural populations.     

Pyrosequencing Reveals Diverse and Distinct Sponge-Specific Microbial Communities in Sponges from a Single Geographical Location in Irish Waters

Marine sponges are host to numerically vast and phylogenetically diverse bacterial communities, with 26 major phyla to date having been found in close association with sponge species worldwide. Analyses of these microbial communities have revealed many sponge-specific novel genera and species. These endosymbiotic microbes are believed to play significant roles in sponge physiology including the production of an array of bioactive secondary metabolites. Here, we report on the use of culture-based and culture-independent (pyrosequencing) techniques to elucidate the bacterial community profiles associated with the marine sponges Raspailia ramosa and Stelligera stuposa sampled from a single geographical location in Irish waters and with ambient seawater. To date, little is known about the microbial ecology of sponges of these genera. Culture isolation grossly underestimated sponge-associated bacterial diversity. Four bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria) were represented amongst ~200 isolates, compared with ten phyla found using pyrosequencing. Long average read lengths of ~430 bp (V1-V3 region of 16S rRNA gene) allowed for robust resolution of sequences to genus level. Bacterial OTUs (2,109 total), at 95% sequence similarity, from ten bacterial phyla were recovered from R. ramosa, 349 OTUs were identified in S. stuposa representing eight phyla, while 533 OTUs from six phyla were found in surrounding seawater. Bacterial communities differed significantly between sponge species and the seawater. Analysis of the data for sponge-specific taxa revealed that 2.8% of classified reads from the sponge R. ramosa can be defined as sponge-specific, while 26% of S. stuposa sequences represent sponge-specific bacteria. Novel sponge-specific clusters were identified, whereas the majority of previously reported sponge-specific clusters (e.g. Poribacteria) were absent from these sponge species. This deep and robust analysis provides further evidence that the microbial communities associated with marine sponge species are highly diverse and divergent from one another and appear to be host-selected through as yet unknown processes.