Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa

Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined. Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium. Mucosal sheets from porcine distal colon were mounted in Ussing chambers. Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively. After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (I(sc)) was measured continuously to assess changes in active ion transport. NE-treated tissues exhibited concentration-dependent increases in I(sc) and EHEC adherence. NE did not alter adherence of a rodent-adapted, noninfectious E. coli strain or two porcine-adapted non-O157 E. coli strains. The actions of NE on EHEC adherence but not I(sc) were prevented by the alpha-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate. Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence. Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase appeared to innervate the colonic epithelium. EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism. Through interactions with alpha(2)-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa. Changes in sympathetic neural outflow may alter intestinal susceptibility to infection.     

Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG

The ends of human chromosomes are protected from the degradation associated with cell division by 15-20 kb long segments of hexameric repeats of 5'-TTAGGG-3' termed telomeres. In normal cells telomeres lose up to 300 bp of DNA per cell division that ultimately leads to senescence; however, most cancer cells bypass this lifespan restriction through the expression of telomerase. hTERT, the catalytic subunit essential for the proper function of telomerase, has been shown to be expressed in approximately 90% of all cancers. In this study we investigated the hTERT inhibiting effects of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea catechins, in MCF-7 breast cancers cells and HL60 promyelocytic leukemia cells. Exposure to EGCG reduced cellular proliferation and induced apoptosis in both MCF-7 and HL60 cells in vitro, although hTERT mRNA expression was decreased only in MCF-7 cells when treated with EGCG. Furthermore, down-regulation of hTERT gene expression in MCF-7 cells appeared to be largely due to epigenetic alterations. Treatment of MCF-7 cells with EGCG resulted in a time-dependent decrease in hTERT promoter methylation and ablated histone H3 Lys9 acetylation. In conjunction with demethylation, further analysis showed an increase in hTERT repressor E2F-1 binding at the promoter. From these findings, we propose that EGCG is effective in causing cell death in both MCF-7 and HL60 cancer cell lines and may work through different pathways involving both anti-oxidant effects and epigenetic modulation.     

Identification of regulatory variants of carboxylesterase 1 (CES1): A proof-of-concept study for the application of the Allele-Specific Protein Expression (ASPE) assay in identifying cis-acting regulatory genetic polymorphisms

It is challenging to study regulatory genetic variants as gene expression is affected by both genetic polymorphisms and non-genetic regulators. The mRNA allele-specific expression (ASE) assay has been increasingly used for the study of cis-acting regulatory variants because cis-acting variants affect gene expression in an allele-specific manner. However, poor correlations between mRNA and protein expressions were observed for many genes, highlighting the importance of studying gene expression regulation at the protein level. In the present study, we conducted a proof-of-concept study to utilize a recently developed allele-specific protein expression (ASPE) assay to identify the cis-acting regulatory variants of CES1 using a large set of human liver samples. The CES1 gene encodes for carboxylesterase 1 (CES1), the most abundant hepatic hydrolase in humans. Two cis-acting regulatory variants were found to be significantly associated with CES1 ASPE, CES1 protein expression, and its catalytic activity on enalapril hydrolysis in human livers. Compared to conventional gene expression-based approaches, ASPE demonstrated an improved statistical power to detect regulatory variants with small effect sizes since allelic protein expression ratios are less prone to the influence of non-genetic regulators (e.g., diseases and inducers). This study suggests that the ASPE approach is a powerful tool for identifying cis-regulatory variants.     

Amazonia trees have limited capacity to acclimate plant hydraulic properties in response to long‐term drought

The fate of tropical forests under future climate change is dependent on the capacity of their trees to adjust to drier conditions. The capacity of trees to withstand drought is likely to be determined by traits associated with their hydraulic systems. However, data on whether tropical trees can adjust hydraulic traits when experiencing drought remain rare. We measured plant hydraulic traits (e.g. hydraulic conductivity and embolism resistance) and plant hydraulic system status (e.g. leaf water potential, native embolism and safety margin) on >150 trees from 12 genera (36 species) and spanning a stem size range from 14 to 68 cm diameter at breast height at the world's only long‐running tropical forest drought experiment. Hydraulic traits showed no adjustment following 15 years of experimentally imposed moisture deficit. This failure to adjust resulted in these drought‐stressed trees experiencing significantly lower leaf water potentials, and higher, but variable, levels of native embolism in the branches. This result suggests that hydraulic damage caused by elevated levels of embolism is likely to be one of the key drivers of drought‐induced mortality following long‐term soil moisture deficit. We demonstrate that some hydraulic traits changed with tree size, however, the direction and magnitude of the change was controlled by taxonomic identity. Our results suggest that Amazonian trees, both small and large, have limited capacity to acclimate their hydraulic systems to future droughts, potentially making them more at risk of drought‐induced mortality.     

RhoA as a mediator of clinically relevant androgen action in prostate cancer cells

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.     

The Fab Conformations in the Solution Structure of Human Immunoglobulin G4 (IgG4) Restrict Access to Its Fc Region: IMPLICATIONS FOR FUNCTIONAL ACTIVITY*

Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser²²²) and a hinge mutant IgG4(Pro²²²) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s_20,w⁰ of 6.6–6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration R_g was unchanged with concentration in 50–250 mm NaCl buffers, whereas the neutron R_g values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser²²²) with extended hinge structures. The IgG4(Pro²²²) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications.     

The Impact of Increased Food Availability on Reproduction in a Long-Distance Migratory Songbird: Implications for Environmental Change?

Many populations of migratory songbirds are declining or shifting in distribution. This is likely due to environmental changes that alter factors such as food availability that may have an impact on survival and/or breeding success. We tested the impact of experimentally supplemented food on the breeding success over three years of northern wheatears (Oenanthe oenanthe), a species in decline over much of Europe. The number of offspring fledged over the season was higher for food-supplemented birds than for control birds. The mechanisms for this effect were that food supplementation advanced breeding date, which, together with increased resources, allowed further breeding attempts. While food supplementation did not increase the clutch size, hatching success or number of chicks fledged per breeding attempt, it did increase chick size in one year of the study. The increased breeding success was greater for males than females; males could attempt to rear simultaneous broods with multiple females as well as attempting second broods, whereas females could only increase their breeding effort via second broods. Multiple brooding is rare in the study population, but this study demonstrates the potential for changes in food availability to affect wheatear breeding productivity, primarily via phenotypic flexibility in the number of breeding attempts. Our results have implications for our understanding of how wheatears may respond to natural changes in food availability due to climate changes or changes in habitat management.     

Adiposity in Early,Middle and Later Adult Life and Cardiometabolic Risk Markers in Later Life; Findings from the British Regional Heart Study

Objectives: This research investigates the associations between body mass index (BMI) at 21, 40–59, 60–79 years of age on cardiometabolic risk markers at 60–79 years. Methods: A prospective study of 3464 British men with BMI measured at 40–59 and 60–79 years, when cardiometabolic risk was assessed. BMI at 21 years was ascertained from military records, or recalled from middle-age (adjusted for reporting bias); associations between BMI at different ages and later cardiometabolic risk markers were examined using linear regression. Sensitive period, accumulation and mobility life course models were devised for high BMI (defined as BMI≥75^th centile) and compared with a saturated BMI trajectory model. Results: At ages 21, 40–59 and 60–79 years, prevalences of overweight (BMI≥25 kg/m²) were 12%, 53%, 70%, and obesity (≥30 kg/m²) 1.6%, 6.6%, and 17.6%, respectively. BMI at 21 years was positively associated with serum insulin, blood glucose, and HbA1c at 60–79 years, with increases of 1.5% (95%CI 0.8,2.3%), 0.4% (0.1,0.6%), 0.3% (0.1,0.4%) per 1 kg/m², respectively, but showed no associations with blood pressure or blood cholesterol. However, these associations were modest compared to those between BMI at 60–79 years and serum insulin, blood glucose and HbA1c at 60–79 years, with increases of 8.6% (8.0,9.2%), 0.7% (0.5,0.9%), and 0.5% (0.4,0.7%) per 1 kg/m², respectively. BMI at 60–79 years was also associated with total cholesterol and blood pressure. Associations for BMI at 40–59 years were mainly consistent with those of BMI at 60–79 years. None of the life course models fitted the data as well as the saturated model for serum insulin. A sensitive period at 50 years for glucose and HbA1c and sensitive period at 70 years for blood pressure were identified. Conclusions: In this cohort of men who were thin compared to more contemporary cohorts, BMI in later life was the dominant influence on cardiovascular and diabetes risk. BMI in early adult life may have a small long-term effect on diabetes risk.     

Into the deep: the functionality of mesopelagic excursions by an oceanic apex predator

Comprehension of ecological processes in marine animals requires information regarding dynamic vertical habitat use. While many pelagic predators primarily associate with epipelagic waters, some species routinely dive beyond the deep scattering layer. Actuation for exploiting these aphotic habitats remains largely unknown. Recent telemetry data from oceanic whitetip sharks (Carcharhinus longimanus) in the Atlantic show a strong association with warm waters (>20°C) less than 200 m. Yet, individuals regularly exhibit excursions into the meso‐ and bathypelagic zone. In order to examine deep‐diving behavior in oceanic whitetip sharks, we physically recovered 16 pop‐up satellite archival tags and analyzed the high‐resolution depth and temperature data. Diving behavior was evaluated in the context of plausible functional behavior hypotheses including interactive behaviors, energy conservation, thermoregulation, navigation, and foraging. Mesopelagic excursions (n = 610) occurred throughout the entire migratory circuit in all individuals, with no indication of site specificity. Six depth‐versus‐time descent and ascent profiles were identified. Descent profile shapes showed little association with examined environmental variables. Contrastingly, ascent profile shapes were related to environmental factors and appear to represent unique behavioral responses to abiotic conditions present at the dive apex. However, environmental conditions may not be the sole factors influencing ascents, as ascent mode may be linked to intentional behaviors. While dive functionality remains unconfirmed, our study suggests that mesopelagic excursions relate to active foraging behavior or navigation. Dive timing, prey constituents, and dive shape support foraging as the most viable hypothesis for mesopelagic excursions, indicating that the oceanic whitetip shark may regularly survey extreme environments (deep depths, low temperatures) as a foraging strategy. At the apex of these deep‐water excursions, sharks exhibit a variable behavioral response, perhaps, indicating the presence or absence of prey.