Characterization of mechanical behavior of an epithelial monolayer in response to epidermal growth factor stimulation

Cell signaling often causes changes in cellular mechanical properties. Knowledge of such changes can ultimately lead to insight into the complex network of cell signaling. In the current study, we employed a combination of atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) to characterize the mechanical behavior of A431 cells in response to epidermal growth factor receptor (EGFR) signaling. From AFM, which probes the upper portion of an individual cell in a monolayer of cells, we observed increases in energy dissipation, Young's modulus, and hysteresivity. Increases in hysteresivity imply a shift toward a more fluid-like mechanical ordering state in the bodies of the cells. From QCM-D, which probes the basal area of the monolayer of cells collectively, we observed decreases in energy dissipation factor. This result suggests a shift toward a more solid-like state in the basal areas of the cells. The comparative analysis of these results indicates a regionally specific mechanical behavior of the cell in response to EGFR signaling and suggests a correlation between the time-dependent mechanical responses and the dynamic process of EGFR signaling. This study also demonstrates that a combination of AFM and QCM-D is able to provide a more complete and refined mechanical profile of the cells during cell signaling.     

Continuous exposure to l-arginine induces oxidative stress and physiological tolerance in cultured human endothelial cells

The therapeutic benefits of L-arginine (ARG) supplementation in humans, often clearly observed in short-term studies, are not evident after long-term use. The mechanisms for the development of ARG tolerance are not known and cannot be readily examined in humans. We have developed a sensitive in vitro model using a low glucose/low arginine culture medium to study the mechanisms of ARG action and tolerance using two different human endothelial cells, i.e., Ea.hy926 and human umbilical venous endothelial cells. Cultured cells were incubated with different concentrations of ARG and other agents to monitor their effects on endothelial nitric oxide synthase (eNOS) expression and function, as well as glucose and superoxide (O2(·-) ) accumulation. Short-term (2 h) exposure to at least 50 μM ARG moderately increased eNOS activity and intracellular glucose (p < 0.05), with no change in eNOS mRNA or protein expression. In contrast, 7-day continuous ARG exposure suppressed eNOS expression and activity. This was accompanied by increase in glucose and O2(·-) accumulation. Co-incubation with 100 μM ascorbic acid, 300 U/ml polyethylene glycol-superoxide dismutase (PEG-SOD), 100 μM L-lysine or 30 μM 5-chloro-2-(N-2,5-dichlorobenenesulfonamido)-benzoxazole (a fructose-1,6-bisphosphatase inhibitor) prevented the occurrence of cellular ARG tolerance. Short-term co-incubation of ARG with PEG-SOD improved cellular nitrite accumulation without altering cellular ARG uptake. These studies suggest that ARG-induced oxidative stress may be a primary causative factor for the development of cellular ARG tolerance.     

Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.     

Characterization of Oral Disintegrating Film Containing Donepezil for Alzheimer Disease

The aim of this study was to develop a taste-masked oral disintegrating film (ODF) containing donepezil, with fast disintegration time and suitable mechanical strength, for the treatment of Alzheimer’s disease. Hydroxypropyl methylcellulose, corn starch, polyethylene glycol, lactose monohydrate and crosspovidone served as the hydrophilic polymeric bases of the ODF. The uniformity, in vitro disintegration time, drug release and the folding endurance of the ODF were examined. The in vitro results showed that 80% of donepezil hydrochloride was released within 5 minutes with mean disintegration time of 44 seconds. The result of the film flexibility test showed that the number of folding time to crack the film was 40 times, an indication of sufficient mechanical property for patient use. A single-dose, fasting, four-period, eight-treatment, double-blind study involving 16 healthy adult volunteers was performed to evaluate the in situ disintegration time and palatability of ODF. Five parameters, namely taste, aftertaste, mouthfeel, ease of handling and acceptance were evaluated. The mean in situ disintegration time of ODF was 49 seconds. ODF containing 7 mg of sucralose were more superior than saccharin and aspartame in terms of taste, aftertaste, mouthfeel and acceptance. Furthermore, the ODF was stable for at least 6 months when stored at 40°C and 75% relative humidity.     

Long-term mortality of patients with septic ocular or central nervous system complications from pyogenic liver abscess: a population-based study

Background

Taiwan is endemic for pyogenic liver abscess (PLA). Septic ocular or central nervous system (CNS) complications derived from PLA can result in catastrophic disability. We investigated the epidemiology and long-term prognosis of PLA patients with septic ocular or CNS complications over an 8-year period.

Methodology/Principal Findings

We extracted 21,307 patients with newly diagnosed PLA from a nationwide health registry in Taiwan between 2000 and 2007. The frequency of and risk factors for PLA with septic ocular or CNS complications were determined. The 2-year survival of these patients was compared between those with and without septic ocular or CNS complications. Septic ocular or CNS complications accounted for 2.1% of all PLA patients. Age and the Charlson comorbidity index were significantly lower in PLA patients with ocular or CNS complications than those without. Diabetes and age <65 years were independent predictors of septic ocular or CNS complications. The 2-year mortality of patients with septic ocular or CNS complications was similar to those without complications (24.8% vs. 27.5%, p = 0.502). However, among patients <65 years old and a Charlson index ≤1, the 2-year mortality was significantly higher in those with than without complications (18.6% vs. 11.8%, p = 0.001).

Conclusions/Significance

Physicians should recognize that catastrophic disability due to ocular or neurological complications from PLA could lead to a poor long-term prognosis, and should follow-up these patients more closely.     

Whole slide images and digital media in pathology education, testing, and practice: the Oklahoma experience

Examination of glass slides is of paramount importance in pathology training. Until the introduction of digitized whole slide images that could be accessed through computer networks, the sharing of pathology slides was a major logistic issue in pathology education and practice. With the help of whole slide images, our department has developed several online pathology education websites. Based on a modular architecture, this program provides online access to whole slide images, still images, case studies, quizzes and didactic text at different levels. Together with traditional lectures and hands-on experiences, it forms the back bone of our histology and pathology education system for residents and medical students. The use of digitized whole slide images has a.lso greatly improved the communication between clinicians and pathologist in our institute.     

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3 mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen > dog fibrinogen ≈ human fibrinogen > goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.     

Microbial Distribution and Abundance in the Digestive System of Five Shipworm Species (Bivalvia: Teredinidae)

Marine bivalves of the family Teredinidae (shipworms) are voracious consumers of wood in marine environments. In several shipworm species, dense communities of intracellular bacterial endosymbionts have been observed within specialized cells (bacteriocytes) of the gills (ctenidia). These bacteria are proposed to contribute to digestion of wood by the host. While the microbes of shipworm gills have been studied extensively in several species, the abundance and distribution of microbes in the digestive system have not been adequately addressed. Here we use Fluorescence In-Situ Hybridization (FISH) and laser scanning confocal microscopy with 16S rRNA directed oligonucleotide probes targeting all domains, domains Bacteria and Archaea, and other taxonomic groups to examine the digestive microbiota of 17 specimens from 5 shipworm species (Bankia setacea, Lyrodus pedicellatus, Lyrodus massa, Lyrodus sp. and Teredo aff. triangularis). These data reveal that the caecum, a large sac-like appendage of the stomach that typically contains large quantities of wood particles and is considered the primary site of wood digestion, harbors only very sparse microbial populations. However, a significant number of bacterial cells were observed in fecal pellets within the intestines. These results suggest that due to low abundance, bacteria in the caecum may contribute little to lignocellulose degradation. In contrast, the comparatively high population density of bacteria in the intestine suggests a possible role for intestinal bacteria in the degradation of lignocellulose.