Generation of <em>Escherichia coli</em> nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954

Escherichia coli nitroreductase (NTR) activates the prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or tumors to the prodrug. This is one of several enzyme-prodrug combinations that are under development for cancer gene therapy, and the system has now entered clinical trials. Enhancing the catalytic efficiency of NTR for CB1954 could improve its therapeutic potential. From the crystal structure of an enzyme-ligand complex, we identified nine amino acid residues within the active site that could directly influence prodrug binding and catalysis. Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to CB1954. Amino acid substitutions at six positions conferred markedly greater sensitivity to CB1954 than did the WT enzyme; the best mutants, at residue F124, resulted in ∼5-fold improvement. Using an adenovirus vector, we introduced the F124K NTR mutant into human SK-OV-3 ovarian carcinoma cells and showed it to be ∼5-fold more potent in sensitizing the cells to CB1954 at the clinically relevant prodrug concentration of 1 μM than was the WT enzyme. Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/CB1954 combination in cancer gene therapy

Barriers to effective discharge planning: a qualitative study investigating the perspectives of frontline healthcare professionals

<p>Abstract</p> <p>Background</p> <p>Studies have shown that effective discharge planning is one of the key factors related to the quality of inpatient care and unnecessary hospital readmission. The perception and understanding of hospital discharge by health professionals is important in developing effective discharge planning. The aims of this present study were to explore the perceived quality of current hospital discharge from the perspective of health service providers and to identify barriers to effective discharge planning in Hong Kong.</p> <p>Methods</p> <p>Focus groups interviews were conducted with different healthcare professionals who were currently responsible for coordinating the discharge planning process in the public hospitals. The discussion covered three main areas: current practice on hospital discharge, barriers to effective hospital discharge, and suggested structures and process for an effective discharge planning system.</p> <p>Results</p> <p>Participants highlighted that there was no standardized hospital-wide discharge planning and policy-driven approach in public health sector in Hong Kong. Potential barriers included lack of standardized policy-driven discharge planning program, and lack of communication and coordination among different health service providers and patients in both acute and sub-acute care provisions which were identified as mainly systemic issues. Improving the quality of hospital discharge was suggested, including a multidisciplinary approach with clearly identified roles among healthcare professionals. Enhancement of health professionals' communication skills and knowledge of patient psychosocial needs were also suggested.</p> <p>Conclusions</p> <p>A systematic approach to develop the structure and key processes of the discharge planning system is critical in ensuring the quality of care and maximizing organization effectiveness. In this study, important views on barriers experienced in hospital discharge were provided. Suggestions for building a comprehensive, system-wide, and policy-driven discharge planning process with clearly identified staff roles were raised. Communication and coordination across various healthcare parties and provisions were also suggested to be a key focus.</p

Habitat and Host Indicate Lineage Identity in Colletotrichum gloeosporioides s.l. from Wild and Agricultural Landscapes in North America

Understanding the factors that drive the evolution of pathogenic fungi is central to revealing the mechanisms of virulence and host preference, as well as developing effective disease control measures. Prerequisite to these pursuits is the accurate delimitation of species boundaries. Colletotrichum gloeosporioides s.l. is a species complex of plant pathogens and endophytic fungi for which reliable species recognition has only recently become possible through a multi-locus phylogenetic approach. By adopting an intensive regional sampling strategy encompassing multiple hosts within and beyond agricultural zones associated with cranberry (Vaccinium macrocarpon Aiton), we have integrated North America strains of Colletotrichum gloeosporioides s.l. from these habitats into a broader phylogenetic framework. We delimit species on the basis of genealogical concordance phylogenetic species recognition (GCPSR) and quantitatively assess the monophyly of delimited species at each of four nuclear loci and in the combined data set with the genealogical sorting index (gsi). Our analysis resolved two principal lineages within the species complex. Strains isolated from cranberry and sympatric host plants are distributed across both of these lineages and belong to seven distinct species or terminal clades. Strains isolated from V. macrocarpon in commercial cranberry beds belong to four species, three of which are described here as new. Another species, C. rhexiae Ellis & Everh., is epitypified. Intensive regional sampling has revealed a combination of factors, including the host species from which a strain has been isolated, the host organ of origin, and the habitat of the host species, as useful indicators of species identity in the sampled regions. We have identified three broadly distributed temperate species, C. fructivorum, C. rhexiae, and C. nupharicola, that could be useful for understanding the microevolutionary forces that may lead to species divergence in this important complex of endophytes and plant pathogens

ER Stress Induces Anabolic Resistance in Muscle Cells through PKB-Induced Blockade of mTORC1

Anabolic resistance is the inability to increase protein synthesis in response to an increase in amino acids following a meal. One potential mediator of anabolic resistance is endoplasmic reticulum (ER) stress. The purpose of the present study was to test whether ER stress impairs the response to growth factors and leucine in muscle cells

The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

Cellular RNA polymerases RNAPs can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP amp; 948; subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP amp; 948; HelD complexes. HelD has two long arms a Gre cleavage factor like coiled coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the amp; 946; and amp; 946; amp; 8242; subunits apart and, aided by amp; 948;, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP dependent manner. HelD abundance during slow growth and a dimeric RNAP amp; 948; HelD 2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cue

When Music and Long-Term Memory Interact: Effects of Musical Expertise on Functional and Structural Plasticity in the Hippocampus

The development of musical skills by musicians results in specific structural and functional modifications in the brain. Surprisingly, no functional magnetic resonance imaging (fMRI) study has investigated the impact of musical training on brain function during long-term memory retrieval, a faculty particularly important in music. Thus, using fMRI, we examined for the first time this process during a musical familiarity task (i.e., semantic memory for music). Musical expertise induced supplementary activations in the hippocampus, medial frontal gyrus, and superior temporal areas on both sides, suggesting a constant interaction between episodic and semantic memory during this task in musicians. In addition, a voxel-based morphometry (VBM) investigation was performed within these areas and revealed that gray matter density of the hippocampus was higher in musicians than in nonmusicians. Our data indicate that musical expertise critically modifies long-term memory processes and induces structural and functional plasticity in the hippocampus

Callous-unemotional traits moderate the relation between prenatal testosterone (2D:4D) and externalising behaviours in children

Children who exhibit callous-unemotional (CU) traits are identified as developing particularly severe forms of externalising behaviours (EB). A number of risk factors have been identified in the development of CU traits, including biological, physiological, and genetic factors. However, prenatal testosterone (PT) remains un-investigated, yet could signal fetal programming of a combination of CU/EB. Using the 2D:4D digit ratio, the current study examined whether CU traits moderated the relationship between PT and EB. Hand scans were obtained from 79 children aged between 5 and 6 years old whose parents completed the parent report ICU (Inventory of Callous Unemotional Traits) and SDQ (Strengths and Difficulties Questionnaire). CU traits were found to moderate the relationship between PT and EB so that children who were exposed to increased PT and were higher in CU traits exhibited more EB. Findings emphasize the importance of recognising that vulnerability for EB that is accompanied by callousness may arise before birth

Radiation chemistry of solid-state carbohydrates using EMR

We review our research of the past decade towards identification of radiation-induced radicals in solid state sugars and sugar phosphates. Detailed models of the radical structures are obtained by combining EPR and ENDOR experiments with DFT calculations of g and proton HF tensors, with agreement in their anisotropy serving as most important criterion. Symmetry-related and Schonland ambiguities, which may hamper such identification, are reviewed. Thermally induced transformations of initial radiation damage into more stable radicals can also be monitored in the EPR (and ENDOR) experiments and in principle provide information on stable radical formation mechanisms. Thermal annealing experi-ments reveal, however, that radical recombination and/or diamagnetic radiation damage is also quite important. Analysis strategies are illustrated with research on sucrose. Results on dipotassium glucose-1-phosphate and trehalose dihydrate, fructose and sorbose are also briefly discussed. Our study demonstrates that radiation damage is strongly regio-selective and that certain general principles govern the stable radical formation

Does the gender of the subject affect perceived smile aesthetics when varying the dimensions of maxillary lateral incisors?

Objective To assess whether subject gender influences aesthetic opinion when altering the width of maxillary lateral incisors. Method Photographs of a male and a female smile, displaying only the lips and teeth, were digitally altered to produce images where the maxillary lateral incisor was proportioned 52%, 57%, 62%, 67%, 72% and 77% in relation to the width of the maxillary central incisor. The image was then made symmetrical. One hundred participants (50 male and 50 female) were asked to rank each set of photographs from 'most' to 'least attractive'. Result The 57% lateral incisor was considered the 'most attractive' with the 77% lateral incisor the 'least attractive' however no statistically significant difference existed with relation to subject or rater gender. Conclusion Neither the 'golden proportion' nor the 'Recurrent Aesthetic Dental' ('RED') proportion was deemed the most attractive. As subject gender did not have a significant effect, dentists should work to create aesthetic results on an individual basis, operating within a so-called 'golden range'