Sleeve sensor versus high-resolution manometry for the detection of transient lower esophageal sphincter relaxations

Transient lower esophageal sphincter relaxations (TLESRs) are the most important mechanism by which gastroesophageal reflux occurs, and sleeve sensor manometry is the gold standard for detection of TLESRs. The aim of this study was to evaluate manometry with closely spaced sideholes (high-resolution manometry) for the detection of TLESRs as an alternative. In 12 patients with gastroesophageal reflux disease, a 90-min postprandial manometry was performed by using a catheter incorporating both a sleeve sensor and closely spaced sideholes in the esophagogastric junction. TLESRs recorded with both techniques were scored. Reflux during TLESRs was detected by using manometry (common cavity), intraluminal impedance, and pH monitoring. A total of 145 TLESRs were detected by using both techniques, 117 with high-resolution manometry and 108 with sleeve sensor manometry [not significant (NS)]. Manometric signs of reflux during TLESRs detected with high-resolution and sleeve sensor manometry were found in 62.4 and 56.5%, NS, respectively, versus 38.5 and 35.2%, NS on pH-metry and 70.1 and 60.2%, NS on impedance monitoring. TLESRs recognized only with high-resolution manometry were more often accompanied by reflux, as detected with manometry (59.5%) and impedance monitoring (67.6%), than TLESRs recognized only with sleeve sensor manometry (32.1 and 28.6%). High-resolution manometry is at least as accurate as sleeve sensor manometry for the detection of TLESRs.     

Biomass and nutrient dynamics of Chinese fir seedlings under conventional and exponential fertilization regimes

Containerized Chinese fir (Cunninghamia lanceolata (Lamb) Hook) were reared from seed at four fertilizer levels (0, 15, 45, 75 mg N seedling-1 season-1) and two topdressing schedules (conventional or exponential) for a 22-week greenhouse rotation to assess growth, nutrition and nutrient loading capacity of seedlings. Extra P supplemented high fertilization (or nutrient loading) treatments to test for induced deficiency of this element. The schedule and rate of fertilization significantly affected growth and nutrient dynamics of the seedlings. Steady-state nutrition and superior growth performance were achieved by seedlings fertilized exponentially at the operational dose (15 mg N), yielding 23, 72 and 52% more in respective biomass, N uptake and P uptake than seedlings fertilized conventionally at the equivalent dose. The improved growth and fertilizer efficiency were attributed to close synchronization of exponential nutrient supply with exponential growth and nutrient demand of plants. High dose exponential fertilization (45 and 75 mg N) induced steady state-nutrition late in the season, increasing seedling N and P uptake by 72–83% and 50–96% compared to low dose exponential fertilization, demonstrating effective nutrient loading of plants without changing biomass. The extra P stimulated P uptake without altering growth or N uptake, thus P was probably not limiting during the greenhouse culture despite high N additions.     

Methyl group reorientation under ligand binding probed by pseudocontact shifts

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1–3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.     

Model-based extension of high-throughput to high-content data

Background  

High-quality quantitative data is a major limitation in systems biology. The experimental data used in systems biology can be assigned to one of the following categories: assays yielding average data of a cell population, high-content single cell measurements and high-throughput techniques generating single cell data for large cell populations. For modeling purposes, a combination of data from different categories is highly desirable in order to increase the number of observable species and processes and thereby maximize the identifiability of parameters.     

PI3K‐p110‐alpha‐subtype signalling mediates survival,proliferation and neurogenesis of cortical progenitor cells via activation of mTORC2

Development of the cerebral cortex is controlled by growth factors among which transforming growth factor beta (TGFβ) and insulin‐like growth factor 1 (IGF1) have a central role. The TGFβ‐ and IGF1‐pathways cross‐talk and share signalling molecules, but in the central nervous system putative points of intersection remain unknown. We studied the biological effects and down‐stream molecules of TGFβ and IGF1 in cells derived from the mouse cerebral cortex at two developmental time points, E13.5 and E16.5. IGF1 induces PI3K, AKT and the mammalian target of rapamycin complexes (mTORC1/mTORC2) primarily in E13.5‐derived cells, resulting in proliferation, survival and neuronal differentiation, but has small impact on E16.5‐derived cells. TGFβ has little effect at E13.5. It does not activate the PI3K‐ and mTOR‐signalling network directly, but requires its activity to mediate neuronal differentiation specifically at E16.5. Our data indicate a central role of mTORC2 in survival, proliferation as well as neuronal differentiation of E16.5‐derived cortical cells. mTORC2 promotes these cellular processes and is under control of PI3K‐p110‐alpha signalling. PI3K‐p110‐beta signalling activates mTORC2 in E16.5‐derived cells but it does not influence cell survival, proliferation and differentiation. This finding indicates that different mTORC2 subtypes may be implicated in cortical development and that these subtypes are under control of different PI3K isoforms.

     

Parameter estimation in stochastic biochemical reactions

Gene regulatory, signal transduction and metabolic networks are major areas of interest in the newly emerging field of systems biology. In living cells, stochastic dynamics play an important role; however, the kinetic parameters of biochemical reactions necessary for modelling these processes are often not accessible directly through experiments. The problem of estimating stochastic reaction constants from molecule count data measured, with error, at discrete time points is considered. For modelling the system, a hidden Markov process is used, where the hidden states are the true molecule counts, and the transitions between those states correspond to reaction events following collisions of molecules. Two different algorithms are proposed for estimating the unknown model parameters. The first is an approximate maximum likelihood method that gives good estimates of the reaction parameters in systems with few possible reactions in each sampling interval. The second algorithm, treating the data as exact measurements, approximates the number of reactions in each sampling interval by solving a simple linear equation. Maximising the likelihood based on these approximations can provide good results, even in complex reaction systems.     

Photoconversion and nuclear trafficking cycles determine phytochrome A's response profile to far-red light

Phytochrome A (phyA) is the only photoreceptor in plants, initiating responses in far-red light and, as such, essential for survival in canopy shade. Although the absorption and the ratio of active versus total phyA are maximal in red light, far-red light is the most efficient trigger of phyA-dependent responses. Using a joint experimental-theoretical approach, we unravel the mechanism underlying this shift of the phyA action peak from red to far-red light and show that it relies on specific molecular interactions rather than on intrinsic changes to phyA's spectral properties. According to our model, the dissociation rate of the phyA-FHY1/FHL nuclear import complex is a principle determinant of the phyA action peak. The findings suggest how higher plants acquired the ability to sense far-red light from an ancestral photoreceptor tuned to respond to red light.     

Effects of alcohol septal ablation on coronary microvascular function and myocardial energetics in hypertrophic obstructive cardiomyopathy

This study investigated the effects of alcohol septal ablation (ASA) on microcirculatory function and myocardial energetics in patients with hypertrophic cardiomyopathy (HCM) and left ventricular outflow tract (LVOT) obstruction. In 15 HCM patients who underwent ASA, echocardiography was performed before and 6 mo after the procedure to assess the LVOT gradient (LVOTG). Additionally, [(15)O]water PET was performed to obtain resting myocardial blood flow (MBF) and coronary vasodilator reserve (CVR). Changes in LV mass (LVM) and volumes were assessed by cardiovascular magnetic resonance imaging. Myocardial oxygen consumption (MVo(2)) was evaluated by [(11)C]acetate PET in a subset of seven patients to calculate myocardial external efficiency (MEE). After ASA, peak LVOTG decreased from 41 ± 32 to 23 ± 19 mmHg (P = 0.04), as well as LVM (215 ± 74 to 169 ± 63 g; P < 0.001). MBF remained unchanged (0.94 ± 0.23 to 0.98 ± 0.15 ml·min(-1)·g(-1); P = 0.45), whereas CVR increased (2.55 ± 1.23 to 3.05 ± 1.24; P = 0.05). Preoperatively, the endo-to-epicardial MBF ratio was lower during hyperemia compared with rest (0.80 ± 0.18 vs. 1.18 ± 0.15; P < 0.001). After ASA, the endo-to-epicardial hyperemic (h)MBF ratio increased to 1.03 ± 0.26 (P = 0.02). ΔCVR was correlated to ΔLVOTG (r = -0.82; P < 0.001) and ΔLVM (r = -0.54; P = 0.04). MEE increased from 15 ± 6 to 20 ± 9% (P = 0.04). Coronary microvascular dysfunction in obstructive HCM is at least in part reversible by relief of LVOT obstruction. After ASA, hMBF and CVR increased predominantly in the subendocardium. The improvement in CVR was closely correlated to the absolute reduction in peak LVOTG, suggesting a pronounced effect of LV loading conditions on microvascular function of the subendocardium. Furthermore, ASA has favorable effects on myocardial energetics.     

Myeloid Cell Sirtuin-1 Expression Does Not Alter Host Immune Responses to Gram-Negative Endotoxemia or Gram-Positive Bacterial Infection

The role of sirtuin-1 (SIRT1) in innate immunity, and in particular the influence of SIRT1 on antimicrobial defense against infection, has yet to be reported but is important to define since SIRT1 inhibitors are being investigated as therapeutic agents in the treatment of cancer, Huntington’s disease, and autoimmune diseases. Given the therapeutic potential of SIRT1 suppression, we sought to characterize the role of SIRT1 in host defense. Utilizing both pharmacologic methods and a genetic knockout, we demonstrate that SIRT1 expression has little influence on macrophage and neutrophil antimicrobial functions. Myeloid SIRT1 expression does not change mortality in gram-negative toxin-induced shock or gram-positive bacteremia, suggesting that therapeutic suppression of SIRT1 may be done safely without suppression of myeloid cell-specific immune responses to severe bacterial infections.     

Biophysical characterization of a riboflavin-conjugated dendrimer platform for targeted drug delivery

The present study describes the biophysical characterization of generation-five poly(amidoamine) (PAMAM) dendrimers conjugated with riboflavin (RF) as a cancer-targeting platform. Two new series of dendrimers were designed, each presenting the riboflavin ligand attached at a different site (isoalloxazine at N-3 and d-ribose at N-10) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the binding activity for riboflavin binding protein (RfBP) in a cell-free solution. The ITC data shows dendrimer conjugates have K(D) values of ≥ 465 nM on a riboflavin basis, an affinity ~93-fold lower than that of free riboflavin. The N-3 series showed greater binding affinity in comparison with the N-10 series. Notably, the affinity is inversely correlated with ligand valency. These findings are also corroborated by DSC, where greater protein-conjugate stability is achieved with the N-3 series and at lower ligand valency.