Metal-binding motifs appear on protein scaffolds throughout nature and are critical for a vast array of functions that span structure, electron transfer, and catalysis. In an effort to reproduce and exploit this activity in vitro, described herein are versatile bacteriophage Qbeta particles bearing metal-binding motifs polyvalently. The three motifs, His(6), His(6)-His(6), and Cys-His(6), were incorporated into the capsid via a coexpression methodology at ratios of 1.1:1, 1.1:1, and 2.3:1 for wild-type to modified coat protein. Size-exclusion chromatography yielded elution profiles identical to wild-type particles, while Ni-NTA affinity chromatography resulted in retention times that increase according to Qbeta-His(6)< Qbeta-Cys-His(6)< Qbeta-His(6)-His(6). In addition to interacting with metal-derivatized surfaces, Qbeta-Cys-His(6) and Qbeta-His(6)-His(6) bind heme as evidenced by the appearance of new absorbances at 416 and 418 nm, respectively, upon addition of hemin-Cl. The heme-bearing particles were also found to be electrochemically active as a surface-confined system. While both constructs yield similar E(1/2) values anaerobically and with carbon monoxide present, and both display similar pH dependences, a standard rate constant k degrees could only be measured for Qbeta-Cys-His(6)(83 s(-1)), as electron transfer for Qbeta-His(6)-His(6) was too rapid to estimate. Experiments with rotated-disk electrodes yielded significant activity of the constructs toward dioxygen reduction. The versatility of the particles is further underscored by their multivalent nature, permitting simultaneously a range of activities for applications demanding polyfunctionality.

Biological molecules can be used as versatile templates for assembling nanoscale materials because of their unique structures and chemical diversities. Supramolecular organization of molecular pigments, as is found in the natural light-harvesting antenna, has drawn attention for its potential applications to sensors, photocatalytic systems, and photonic devices. Here we show the arrangement of molecular pigments into a one-dimensional light-harvesting antenna using M13 viruses as scaffolds. Chemical grafting of zinc porphyrins to M13 viruses induces distinctive spectroscopic changes, including fluorescence quenching, the extensive band broadening and small red shift of their absorption spectrum, and the shortened lifetime of the excited states. Based on these optical signatures we suggest a hypothetical model to explain the energy transfer occurring in the supramolecular porphyrin structures templated with the virus. We expect that further genetic engineering of M13 viruses can allow us to coassemble other functional materials (e.g., catalysts and electron transfer mediators) with pigments, implying potential applications to photochemical devices.

PURPOSE: The purposes of this study were to develop an educational program to reduce the use of physical restraints for caregivers in geriatric hospitals and to evaluate the effects of the program on cargivers' knowledge, attitude and nursing practice related to the use of physical restraints. METHODS: A quasi experimental study with a non-equivalent control group pretest-posttest design was used. Participants were recruited from two geriatric hospitals. Eighteen caregivers were assigned to the experimental group and 20 to the control group. The data were collected prior to the intervention and at 6 weeks after the intervention through the use of self-administered questionnaires. Descriptive statistics, X(2) test, Fisher's exact probability test, and Mann-Whitney U test were used to analyze the data. RESULTS: After the intervention, knowledge about physical restraints increased significantly in experimental group compared to the control group. However, there were no statistically significant differences between the groups for attitude and nursing practice involving physical restraints. CONCLUSION: Findings indicate that it is necessary to apply knowledge acquired through educational programs to nursing practice to reduce the use of physical restraints. User friendly guidelines for physical restraints, administrative support of institutions, and multidisciplinary approaches are required to achieve this goal.

This study assessed the clinical management and impact of diarrhoea aetiology (rotavirus positive/negative) and rotavirus genotype on diarrhoeal disease severity. Of 7391 diarrhoea admissions less than 5 years of age over a 2-year period, 80% of patients were tested for rotavirus, 87% were cultured for bacterial pathogens and 78% were assessed for both. Diarrhoeal severity scores were greatest in those children with mixed rotavirus and bacterial infections. Between 1.3 and 8.4% of infants were considered dehydrated yet intravenous fluids were used for 48% of infants (69% rotavirus positive, 72% mixed infection). These findings support the promotion of oral rehydration therapy over intravenous fluids.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family of cytokines, causes apoptosis by caspase activation in various cell types, particularly in transformed cells. Numerous types of tumors are relatively resistant to TRAIL-induced cytotoxicity; however, the reasons for this are not yet fully understood. We report here a new signal transduction pathway involving protein kinase Cdelta (PKCdelta), NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS), that inhibits caspase-dependent cell death induced by TRAIL ligation in human malignant astrocytoma cells. In our experiments, TRAIL ligation-induced generation of intracellular ROS through caspase-dependent proteolytic activation of PKCdelta and subsequent activation of the NOX4 complex. Suppression of intracellular ROS induction using various pharmacological inhibitors or PKCdelta- or NOX4-specific RNA interference enhanced the enzymatic activity of caspase-3 by blocking the oxidative modification of its catalytic cysteine residue, resulting in marked augmentation of TRAIL-mediated cell death. These results collectively indicate that TRAIL-induced activation of PKCdelta and NOX4 can modulate TRAIL-mediated apoptosis by promoting oxidative modification of active caspase-3 in a negative-feedback manner.Cell Death and Differentiation advance online publication, 30 October 2009; doi:10.1038/cdd.2009.154.

Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.

RATIONALE: Ineffective repair of a damaged alveolar epithelium has been postulated to be a cause of pulmonary fibrosis. In support of this theory, epithelial cell abnormalities including hyperplasia of type II cells, apoptosis, and persistent denudation of the alveolar basement membrane are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar epithelial cells have been identified in familial forms of pulmonary fibrosis. Although these findings are compelling, there are no studies that demonstrate a direct role for the alveolar epithelium or, more specifically, type II cells in the scarring process. OBJECTIVES: We sought to determine if a targeted injury to type II cells would result in pulmonary fibrosis. METHODS: A transgenic mouse was generated to express the human diphtheria toxin receptor on type II alveolar epithelial cells. Diphtheria toxin was then administered to these animals in order to specifically target the type II epithelium for injury. Lung fibrosis was assessed both by histology and by hydroxyproline measurement. Measurements and RESULTS: Transgenic mice treated with diphtheria toxin developed an approximate 2-fold increase in their lung hydroxyproline content on day 21 and day 28 after diphtheria toxin treatment. The fibrosis developed in conjunction with type II cell injury. Histological evaluation revealed diffuse collagen deposition with patchy areas of more confluent scarring and associated alveolar contraction. CONCLUSIONS: The development of lung fibrosis in the setting of type II cell injury in our model provides evidence for a causal link between the epithelial defects seen in IPF and the corresponding areas of scarring.

Using a technique similar to chirped pulse compression, we have compressed the 50-mW cw output of a diode laser into pulses of greater than 500-mW peak power and less than 400-ps duration. By applying a small current modulation to the diode, we induced a small wavelength modulation in the vicinity of the 6s(1/2)-to-6p(3/2) cesium resonance transition at 852 nm. Group-velocity dispersion on propagation through a cesium vapor cell then led to pulse compression. We developed a simple model to make predictions of output pulse shapes by using different modulation waveforms.

BACKGROUND:: Up to 15% of infants with asymptomatic congenital cytomegalovirus (CMV) infection will experience some degree of sensorineural hearing loss. Many infants who fail newborn hearing screening (NHS) are likely to have congenital CMV infection, but may escape definitive virologic identification because diagnostic evaluation may not commence until several weeks or months of age, making differentiation between congenital and postnatal CMV infection difficult. Early diagnosis linking virologic identification of congenital CMV infection to infants failing NHS may improve diagnostic precision and enhance opportunities for therapeutic intervention. METHODS:: The goal of this study was to compare newborn dried blood spots from Minnesota infants who had failed NHS, and were designated for referral, with control infants who passed NHS, for the presence of CMV DNA by real-time PCR, using hybridization probes for the CMV gene UL54. RESULTS:: Of 479 infants with a failed NHS (bilateral failure), 13 had CMV DNA present in the blood spot (2.7%). This compared with only 2/479 positive results from a control group of infants who passed the NHS (0.4%; P = 0.007, Fisher exact test). Analysis of the glycoprotein B (gB) genotype revealed that these PCR positive samples represented separate, distinct clinical isolates. The mean viral load among the 13 positive samples was 1.8 x 10 genomes/microgram of total DNA. CONCLUSIONS:: Newborn bloodspot CMV screening by real-time PCR may be a useful and rapid adjunct to functional NHS and may enable more rapid etiologic diagnosis of sensorineural hearing loss in newborns.