Comparison of methodologies used to define the protein quality of human foods and support regulatory claims.

Protein quality (PQ) is the capacity of a protein to meet the amino acid (AA) requirements of an individual. There are several methodologies for determining the PQ of foods. The protein efficiency ratio is an animal growth bioassay. The protein-digestibility-corrected AA score considers the AA requirements of a reference population, and the true nitrogen digestibility coefficient for each ingredient. The digestible indispensable AA score is based on true ileal AA digestibility and better represents bioavailability of AAs. In vitro techniques for assessment of PQ are available but require validation against a greater range of protein sources. Isotopic methods, such as the indicator AA oxidation and dual tracer techniques measure AA relative bioavailability and digestibility, respectively, but require sophisticated equipment, and may not be cost nor time effective for the industry to adopt. The present review discusses advantages and disadvantages of methodologies for determining PQ of food for humans focused on methods that are or could be adopted by regulatory agencies. Understanding the framework and resources available for PQ determination will help in the selection of appropriate methods depending on the application. Novelty Understanding the framework and resources available for PQ determination will help in the selection of appropriate methods depending on the application.

Nonsense mutations in AAGAB cause punctate palmoplantar keratoderma type Buschke-Fischer-Brauer.

Punctate palmoplantar keratodermas (PPKPs) are rare autosomal-dominant inherited skin diseases that are characterized by multiple hyperkeratotic plaques distributed on the palms and soles. To date, two different loci in chromosomal regions 15q22-15q24 and 8q24.13-8q24.21 have been reported. Pathogenic mutations, however, have yet to be identified. In order to elucidate the genetic cause of PPKP type Buschke-Fischer-Brauer (PPKP1), we performed exome sequencing in five affected individuals from three families, and we identified in chromosomal region 15q22.33-q23 two heterozygous nonsense mutations-c.370C>T (p.Arg124(∗)) and c.481C>T (p.Arg161(∗))-in AAGAB in all affected individuals. Using immunoblot analysis, we showed that both mutations result in premature termination of translation and truncated protein products. Analyses of mRNA of affected individuals revealed that the disease allele is either not detectable or only detectable at low levels. To assess the consequences of the mutations in skin, we performed immunofluorescence analyses. Notably, the amount of granular staining in the keratinocytes of affected individuals was lower in the cytoplasm but higher around the nucleus than it was in the keratinocytes of control individuals. AAGAB encodes the alpha-and gamma-adaptin-binding protein p34 and might play a role in membrane traffic as a chaperone. The identification of mutations, along with the results from additional studies, defines the genetic basis of PPKP1 and provides evidence that AAGAB plays an important role in skin integrity.

Dynamic optical imaging of metabolic and NADPH oxidase-derived superoxide in live mouse brain using fluorescence lifetime unmixing.

Superoxide is the single-electron reduction product of molecular oxygen generated by mitochondria and the innate immune enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and its isoforms. Initially identified as critical to the host defense against infection, superoxide has recently emerged as an important signaling molecule and as a proposed mediator of central nervous system injury in stroke, neurodegenerative conditions, and aging itself. Complete understanding of superoxide in central nervous system disease has been hampered by lack of noninvasive imaging techniques to evaluate this highly reactive, short-lived molecule in vivo. Here we describe a novel optical imaging technique to monitor superoxide real time in intact animals using a fluorescent probe compound and fluorescence lifetime contrast-based unmixing. Specificity for superoxide was confirmed using validated mouse models with enhanced or attenuated brain superoxide production. Application of fluorescence lifetime unmixing removed autofluorescence, further enhanced sensitivity and specificity of the technique, permitted visualization of physiologically relevant levels of superoxide, and allowed superoxide in specific brain regions (e.g., hippocampus) to be mapped. Lifetime contrast-based unmixing permitted disease model-specific and brain region-specific differences in superoxide levels to be observed, suggesting this approach may provide valuable information on the role of mitochondrial and Nox-derived superoxide in both normal function and pathologic conditions in the central nervous system.

The digital Emily project: achieving a photorealistic digital actor.

The Digital Emily Project uses advanced face scanning, character rigging, performance capture, and compositing to achieve one of the world's first photorealistic digital facial performances. The project scanned the geometry and reflectance of actress Emily O'Brien's face in 33 poses, showing different emotions, gaze directions, and lip formations in a light stage. These high-resolution scans-accurate to skin pores and fine wrinkles-became the basis for building a blendshape-based facial-animation rig whose expressions closely matched the scans. The blendshape rig drove displacement maps to add dynamic surface detail. A video-based facial animation system animated the face according to the performance in a reference video, and the digital face was tracked onto the video's motion and rendered under the same illumination. The result was a realistic 3D digital facial performance credited as one of the first to cross the "uncanny valley" between animated and fully human performances.

CMT3 alters mitochondrial function in murine osteoclast lineage cells.

Chemically modified tetracyclines (CMTs 1-10) were developed as non-antibiotic inhibitors of matrix metalloproteinases (MMPs). We previously demonstrated that MMP inhibition alone is insufficient to explain the pro-apoptotic action of CMTs in osteoclast lineage cells and we have explored additional mechanisms of action. We compared the characteristics of apoptosis in RAW264.7 murine monocyte and osteoclast cultures treated with pharmacologically relevant concentrations of CMT3 or the bisphosphonate alendronate, which induces osteoclast apoptosis through inhibition of farnesyl diphosphate synthase. CMT3 induced apoptosis rapidly (2-3h), whereas alendronate-induced apoptosis was delayed (>12h). CMT3-treated cells did not accumulate unprenylated Rap1A in contrast to cells treated with alendronate. Importantly, CMT3 induced a rapid loss of mitochondrial stability in RAW264.7 cells measured by loss of Mitotracker((R)) Red fluorescence, while bongkrekic acid protected polykaryons from CMT3-induced apoptosis. Modulation of mitochondrial function is therefore a significant early action of CMT3 that promotes apoptosis in osteoclast lineage cells.

Robust and accurate registration of 2-D electrophoresis gels using point-matching.

Point-matching is a widely applied image registration method and many algorithms have been developed. Registration of 2-D electrophoresis gels is an important problem in biological research that presents many of the technical difficulties that beset point-matching: large numbers of points with variable densities, large nonrigid transformations between point sets, paucity of structural information and large numbers of unmatchable points (outliers) in either set. In seeking the most suitable algorithm for gel registration we have evaluated a number of approaches for accuracy and robustness in the face of these difficulties. Using synthetic images we test combinations of three algorithm components: correspondence assignment, distance metrics and image transformation. We show that a version of the iterated closest point (ICP) algorithm using a non-Euclidean distance metric and a robust estimation of transform parameters provides best performance, equalling SoftAssign in the presence of moderate image distortion, and providing superior robustness against large distortions and high outlier proportions. From this evaluation we develop a gel registration algorithm based on robust ICP and a novel distance metric combining Euclidean, shape context and image-related features. We demonstrate the accuracy of gel matching using synthetic distortions of real gels and show that robust estimation of transform parameters using M-estimators can enforce inverse consistency, ensuring that matching results are independent of the order of the images.

Prediction of pre-eclampsia in early pregnancy.

Pre-eclampsia is a multisystem disorder of pregnancy, usually characterized by the appearance of high blood pressure and the excretion of protein in the urine of a previously healthy woman. Symptoms and signs vary in intensity from woman to woman; from a borderline rise in blood pressure, to convulsions (eclampsia), stroke and death. The disease remits following removal of the placenta and so the mainstay of current treatment is timely delivery. A pathophysiological framework of the disease has been established, beginning with failures in placental development, inducing oxidative stress and release of compounds that lead to endothelial activation, vasoconstriction and glomerular endotheliosis. A combination of epidemiological, biophysical and biochemical tests now allow most patients at-risk to be identified by midpregnancy, whilst minimizing false-positive prediction. It is hoped that earlier classification of patients at-risk of the disease, on the basis of pathophysiological changes, will enable specific therapies to be developed targeting these changes.

Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31.

The whirler mouse mutant (wi) does not respond to sound stimuli, and detailed ultrastructural analysis of sensory hair cells in the organ of Corti of the inner ear indicates that the whirler gene encodes a protein involved in the elongation and maintenance of stereocilia in both inner hair cells (IHCs) and outer hair cells (OHCs). BAC-mediated transgene correction of the mouse phenotype and mutation analysis identified the causative gene as encoding a novel PDZ protein called whirlin. The gene encoding whirlin also underlies the human autosomal recessive deafness locus DFNB31. In the mouse cochlea, whirlin is expressed in the sensory IHC and OHC stereocilia. Our findings suggest that this novel PDZ domain-containing molecule acts as an organizer of submembranous molecular complexes that control the coordinated actin polymerization and membrane growth of stereocilia.