Effect of Cross-Linking Methods on Structure and Properties of Poly(ε-caprolactone) Stabilized Hydrogels Containing Biopolymers

Different dense and porous biodegradable matrices based on solely atelocollagen, or with different atelocollagen and hyaluronic acid derivative ratios, were obtained by varying feeding formulations, cross-linking reaction parameters, and preparative protocols. The compositions and methods for forming hydrogels through a combination of physical and chemical cross-linking processes are provided. The chemical cross-linking was mainly mediated by a synthetic component, a poly(ε-caprolactone) reactive derivative, aiming the development of new hybrid hydrogels with tailored characteristics by an appropriate use of the advantages offered by the included natural and synthetic components and the selection of the preparative procedure. The structure and morphology of the 3D hybrid materials were comparatively investigated by means of Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and environmental scanning electron microscopy (ESEM). FTIR and XRD analysis showed no signs of collagen denaturation during the formation of 3D structures. The influence of various factors, such as the chemical composition of the resulted hydrogels and their morphology, on water uptake and water vapor sorption, mechanical behavior, as well as on in vitro degradation characteristics, was systematically investigated. The experimental results point on the advantage offered by the high and modular physicochemical stability of the ternary hydrogels cross-linked by combined approaches. All newly developed materials show no hemolytic effect, which recommends them for potential biomedical applications.     

Silybin combined with phosphatidylcholine and vitamin E in patients with nonalcoholic fatty liver disease: a randomized controlled trial

The only currently recommended treatment for nonalcoholic fatty liver disease (NAFLD) is lifestyle modification. Preliminary studies of silybin showed beneficial effects on liver function. Realsil (RA) comprises the silybin phytosome complex (silybin plus phosphatidylcholine) coformulated with vitamin E. We report on a multicenter, phase III, double-blind clinical trial to assess RA in patients with histologically documented NAFLD. Patients were randomized 1:1 to RA or placebo (P) orally twice daily for 12 months. Prespecified primary outcomes were improvement over time in clinical condition, normalization of liver enzyme plasma levels, and improvement of ultrasonographic liver steatosis, homeostatic model assessment (HOMA), and quality of life. Secondary outcomes were improvement in liver histologic score and/or decrease in NAFLD score without worsening of fibrosis and plasma changes in cytokines, ferritin, and liver fibrosis markers. We treated 179 patients with NAFLD; 36 were also HCV positive. Forty-one patients were prematurely withdrawn and 138 patients analyzed per protocol (69 per group). Baseline patient characteristics were generally well balanced between groups, except for steatosis, portal infiltration, and fibrosis. Adverse events (AEs) were generally transient and included diarrhea, dysgeusia, and pruritus; no serious AEs were recorded. Patients receiving RA but not P showed significant improvements in liver enzyme plasma levels, HOMA, and liver histology. Body mass index normalized in 15% of RA patients (2.1% with P). HCV-positive patients in the RA but not the P group showed improvements in fibrogenesis markers. This is the first study to systematically assess silybin in NAFLD patients. Treatment with RA but not P for 12 months was associated with improvement in liver enzymes, insulin resistance, and liver histology, without increases in body weight. These findings warrant further investigation.     

Hypoxia sensitivity of a voltage-gated potassium current in porcine intrapulmonary vein smooth muscle cells

Hypoxia contracts the pulmonary vein, but the underlying cellular effectors remain unclear. Utilizing contractile studies and whole cell patch-clamp electrophysiology, we report for the first time a hypoxia-sensitive K(+) current in porcine pulmonary vein smooth muscle cells (PVSMC). Hypoxia induced a transient contractile response that was 56 ± 7% of the control response (80 mM KCl). This contraction required extracellular Ca(2+) and was sensitive to Ca(2+) channel blockade. Blockade of K(+) channels by tetraethylammonium chloride (TEA) or 4-aminopyridine (4-AP) reversibly inhibited the hypoxia-mediated contraction. Single-isolated PVSMC (typically 159.1 ± 2.3 μm long) had mean resting membrane potentials (RMP) of -36 ± 4 mV with a mean membrane capacitance of 108 ± 3.5 pF. Whole cell patch-clamp recordings identified a rapidly activating, partially inactivating K(+) current (I(KH)) that was hypoxia, TEA, and 4-AP sensitive. I(KH) was insensitive to Penitrem A or glyburide in PVSMC and had a time to peak of 14.4 ± 3.3 ms and recovered in 67 ms following inactivation at +80 mV. Peak window current was -32 mV, suggesting that I(KH) may contribute to PVSMC RMP. The molecular identity of the potassium channel is not clear. However, RT-PCR, using porcine pulmonary artery and vein samples, identified Kv(1.5), Kv(2.1), and BK, with all three being more abundant in the PV. Both artery and vein expressed STREX, a highly conserved and hypoxia-sensitive BK channel variant. Taken together, our data support the hypothesis that hypoxic inhibition of I(KH) would contribute to hypoxic-induced contraction in PVSMC.     

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.     

Effect of periodic letters on evidence-based drug therapy on prescribing behaviour: a randomized trial

Background

The effect of regular and expected printed educational materials on physician prescribing behaviour has not been studied. We sought to measure the impact of a series of evidence-based drug therapy letters mailed to physicians in British Columbia on prescribing to newly treated patients.

Methods

A paired, cluster randomized community design was used. The study population included 499 physicians from 24 local health areas in British Columbia. Local health areas were paired by number of physicians, and 1 of each pair was randomly selected and its physicians assigned to an intervention group or a control group. The intervention was 12 issues of an evidence-based series called Therapeutics Letter. Physicians in the control group (n = 241) received the letters 3–8 months after physicians in the intervention group (n = 258). The impact on prescribing to newly treated patients (defined as patients who had not previously made a claim for any medication from the class of drugs profiled in the letter) was analyzed using the drug claims database of BC Pharmacare, a publicly funded drug benefits program that covered all seniors and people receiving social assistance.

Results

The probability of prescribing a drug recommended in the Therapeutics Letter rather than another drug in the same class increased by 30% in the 3 months after the mailing of the letter relative to the preceding 3 months, adjusted for any before–after changes in the control group (relative risk 1.30; 95% confidence interval 1.13–1.52). No letter achieved statistical significance on its own. However, 11 of the 12 letters produced prescribing changes in the predicted direction such that the overall result was significant when their effect was combined.

Interpretation

The combined effect of an ongoing series of printed letters distributed from a credible and trusted source can have a clinically significant effect on prescribing to newly treated patients. Printed letters are regarded as a relatively ineffective method of changing behaviour.^1,2 A systematic review in The Cochrane Library concluded that printed educational materials for health care professionals had negligible impact and were of uncertain clinical significance.³ These conclusions were not based on a quantitative meta-analysis, however, in part because of the poor quality of analysis and reporting of results and in part because the different outcomes could not be easily combined. Although changes in prescribing behaviour as a result of printed materials have tended to be small, printed materials have the potential to be a cost-effective method of education.^4,5 There is also a paucity of studies in Canada on initiatives to change prescribing behaviour.⁶Between 1994 and 1997 the Therapeutics Initiative of the University of British Columbia conducted a randomized controlled trial with its Therapeutics Letter (www.ti.ubc.ca), which is distributed to most practising physicians in British Columbia. We aimed to quantify the publication''s success in producing changes in prescribing behaviour. In 1998 we reported that 2 letters on the treatment of primary hypertension did not produce a statistically significant change in prescribing.⁷ Here we report findings from a combined randomized trial of the impact of 12 of the first 20 issues of the Therapeutics Letter, which were distributed between October 1994 and December 1997. This combined analysis was undertaken to measure the series'' overall impact on prescribing to newly treated patients and to provide evidence of the effectiveness of these interventions in a Canadian setting.     

PEVK domain of titin: an entropic spring with actin-binding properties

The PEVK domain of the giant muscle protein titin is a proline-rich sequence with unknown secondary/tertiary structure. Here we compared the force-extension behavior of cloned cardiac PEVK titin measured by single-molecule atomic force spectroscopy with the extensibility of the PEVK domain measured in intact cardiac muscle sarcomeres. The analysis revealed that cardiac PEVK titin acts as an entropic spring with the properties of a random coil exhibiting mechanical conformations of different flexibility. Since in situ, titin is in close proximity to the thin filaments, we also studied whether the PEVK domain of cardiac or skeletal titin may interact with actin filaments. Interaction was indeed found in the in vitro motility assay, in which recombinant PEVK titin constructs slowed down the sliding velocity of actin filaments over myosin. Skeletal PEVK titin affected the actin sliding to a lesser degree than cardiac PEVK titin. The cardiac PEVK effect was partially suppressed by physiological Ca(2+) concentrations, whereas the skeletal PEVK effect was independent of [Ca(2+)]. Cosedimentation assays confirmed the Ca(2+)-modulated actin-binding propensity of cardiac PEVK titin, but did not detect interaction between actin and skeletal PEVK titin. In myofibrils, the relatively weak actin-PEVK interaction gives rise to a viscous force component opposing filament sliding. Thus, the PEVK domain contributes not only to the extensibility of the sarcomere, but also affects contractile properties.     

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.