Determination of the surface density of polyethylene glycol on gold nanoparticles by use of microscale thermogravimetric analysis.

The widespread integration of nanoparticle technologies into biomedicine will depend on the ability to repeatedly create particles with well-defined properties and predictable behaviors. For this to happen, fast, reliable, inexpensive, and widely available techniques to characterize nanomaterials are needed. Characterization of the surface molecules is particularly important since the surface, including the surface molecule density, plays a dominant role in determining how nanoparticles interact with their surroundings. Here, 10 and 30 nm gold nanoparticle NIST Standard Reference Materials were functionalized with fluorescently labeled polyethylene glycol (PEG) with either thiolate or lipoic acid anchoring groups to evaluate analytical techniques for determining surface coverage. The coating of the nanoparticles was confirmed with dynamic light scattering, microscale thermogravimetric analysis (µ-TGA), and ultraviolet-visible (UV-vis) spectroscopy. A UV-vis method for determining gold nanoparticle concentrations that takes into account spectral broadening upon functionalization was developed. The amount of bound PEG was quantified with µ-TGA, a technique analogous to thermogravimetric analysis that uses quartz crystal microbalances, and fluorescence spectroscopy of displaced ligands. It is shown that µ-TGA is a convenient technique for the quantification of ligands bound to inorganic particles while sacrificing a minimal amount of sample, and the treatment of the functionalized nanoparticle dispersions with dithiothreitol may be insufficient to achieve complete displacement of the surface ligands for quantification by fluorescence measurements. The µ-TGA and fluorescence results were used to determine ligand footprint sizes-average areas occupied by each ligand on the particles' surface. The lipoic acid bound ligands had footprint sizes of 0.21 and 0.25 nm(2) on 10 and 30 nm particles, respectively while the thiolate ligands had footprint sizes of 0.085 and 0.18 nm(2).

Industrial innovation in Japan and the United States.

Japanese firms tend to be quicker and more economical than U.S.firms at developing and introducing new products and processes, but this advantage seems to exist only among innovations based on external technology, rather than internal technology. Whereas U.S.firms put more emphasis on marketing start-up, they put much less emphasis on tooling, equipment, and manufacturing facilities than do Japanese firms. Applied R&D in Japan, which focuses more on processes than in the United States, seems to have yielded a handsome return; but there is no evidence that the rate of return from basic research has been relatively high in Japan. In robotics, the Japanese edge seems to increase as one moves from R&D toward the market.

Tax policy and innovation.

Tax policy should be formulated with recognition of its effects on research and development and innovation. Many changes in tax policy designed to stimulate innovation have been proposed in recent years. Some of these changes were embodied in the 1981 tax bill. Basic economic analysis and rudimentary statistics enable economists to make some useful statements about the effects of recent and proposed tax changes but, because practically no studies have been conducted in this area, there is little or no dependable information concerning the quantitative impact of particular changes of this sort on the rate of innovation.

Research and development, productivity, and inflation.

R & D, through its effects on the rate of productivity increase, can significantly restrain the rate of inflation in the medium and long run. High rates of inflation damage the workings of the price system and impair the efficiency of practically all economic activities, including R & D. Findings suggest that the percentage increase between 1969 and 1979, in total real R & D expenditures, has been exaggerated due to the inadequacy of the gross national product deflator as applied to R & D.

Contribution of r&d to economic growth in the United States.

Technological change has certainly contributed in a very important way to economic growth in the United States. Although existing studies have not been able to estimate this contribution with great accuracy, they have certainly indicated that this contribution has been large. Moreover, although econometric studies of the relationship between R&D and productivity increase have been subject to many limitations, they provide reasonably persuasive evidence that R&D has an important effect on productivity increase in the industries and time periods that have been studied. Turning to the adequacy of the nation's investment in R&D, there is too little evidence to support a very confident judgment as to whether or not we are underinvesting in certain types of R&D. However, practically all of the studies addressed to this question seem to conclude, with varying degrees of confidence, that we may be underinvesting in particular types of R&D in the civilian sector of the economy, and the estimated marginal rates of return from certain types of civilian R&D seem very high. Additional research is badly needed to determine more adequately the relationship of R&D to economic growth. I have indicated a number of specific areas where work is needed.

Walking in the shoes of caregivers of children with obesity: supporting caregivers in paediatric weight management.

To incorporate the perspectives and experiences of family caregivers of children with obesity, the KidFit Health and Wellness Clinic, a paediatric weight management programme, embedded feedback opportunities into various stages of programme development. Caregivers were eligible to participate if their children had completed initial 4-week group-based pilot programming or were currently receiving treatment in 10 or 12 week group-based programming. Data were collected through feedback session discussions, audio-recorded, transcribed verbatim and analysed thematically. In total, 6 caregivers participated in the pilot group feedback session and 32 caregivers participated in the structured group feedback sessions. Caregivers reported that healthy lifestyle strategies first communicated by clinic staff to children during group sessions provided expert validation and reinforcement when discussing similar messages at home. Caregivers reported feeling isolated and blamed for causing their children's obesity and appreciated the supportive forum that group-based programming provided for sharing experiences. Since experiences of blame and isolation can burden caregivers of children with obesity, paediatric weight management programmes might consider including peer support opportunities and discussion forums for ongoing social support in addition to education about lifestyle change.

Iron content and molecular weight of uteroferrin and a comparison of its iron and copper forms.

The molecular weight of the purple, iron-containing glycoprotein uteroferrin has been determined to be 35140 +/- 1610 by sedimentation equilibrium centrifugation. This is consistent with measurements carried out by other procedures. The dry weight of a solution of uteroferrin with an absorption of 1.00 cm -1 at 280 nm was 0.87 mg/ml. Highly purified uteroferrin has a ratio of absorbance at 280 nm to 545 nm of about 13.2 and a revised extinction coefficient at 545 nm of 3.1 . 10(3) M -1 is presented. The iron content of uteroferrin has been determined both colorimetrically and by atomic absorption spectroscopy. One iron atom is present per polypeptide chain. Reduction with dithionite leads to loss of iron, allowing the apoprotein to be prepared. Enzymatic activity can be restored to the apoprotein with Fe(III) or with Cu(II). The copper enzyme has no visible color and has a higher pH optimum than the Fe-uteroferrin. Whereas the phosphatase activity of the latter is increased several-fold by beta-mercaptoethanol, reduction inactivates Cu-uteroferrin. Both forms of uteroferrin have similar Km values for p--nitrophenyl phosphate and are inhibited by molybdate but not by tartrate. Excess Cu(II) and Fe(III) strongly inhibit uteroferrin phosphatase activity and these results may explain the failure of other to restore activity to the apoprotein using Cu(II) and Fe(III) salts.

Parallel molecular genetic analysis.

We describe recent progress in parallel molecular genetic analyses using DNA microarrays, gel-based systems, and capillary electrophoresis and utilization of these approaches in a variety of molecular biology assays. These applications include use of polymorphic markers for mapping of genes and disease-associated loci and carrier detection for genetic diseases. Application of these technologies in molecular diagnostics as well as fluorescent technologies in DNA analysis using immobilized oligonucleotide arrays on silicon or glass microchips are discussed. The array-based assays include sequencing by hybridization, cDNA expression profiling, comparative genome hybridization and genetic linkage analysis. Developments in non microarray-based, parallel analyses of mutations and gene expression profiles are reviewed. The promise of and recent progress in capillary array electrophoresis for parallel DNA sequence analysis and genotyping is summarized. Finally, a framework for decision making in selecting available technology options for specific molecular genetic analyses is presented.

Linkage of DFNB1 to non-syndromic neurosensory autosomal-recessive deafness in Mediterranean families.

Recent studies show a susceptibility locus (DFNB1) responsible for non-syndromic neurosensory autosomal-recessive deafness (NSRD) mapping to the pericentromeric region of chromosome 13q. In order to better understand the frequency with which DFNB1 is the gene for deafness in our patient population and the role of DFNB1 in Caucasians, we performed a genetic linkage study with four microsatellite markers linked to DFNB1 in a total of 48 independent Mediterranean families, of which 30 and 18 were of Italian and Spanish descent, respectively. A maximum two-point lod score of 7.28 was found with marker D13S115 at a recombination frequency of theta 0.1. Significant lod scores were also obtained for D13S143, D13S292 and D13S175. Genetic heterogeneity was confirmed using the HOMOG program which indicated absence of linkage to DFNB1 in approximately 21% of the sample. This study clearly demonstrates that DFNB1 plays an important role in 79% of Mediterranean families with NSRD. Furthermore, results from multipoint analysis predict that the DFNB1 gene maps between markers D13S175 and D13S115 which are separated by approximately 14.2 cM.

Alternative labeling techniques for automated fluorescence based analysis of PCR products.

We present here convenient and simple methods that utilize two new fluorescent tags, TOTO-1 and fluorescein-12-dUTP, in conjunction with analysis of PCR products on automated, fluorescence-based electrophoretic instruments. These methods should prove valuable in those applications wherein the effort or expense of covalently attaching a fluorescent tag onto one or both of the PCR primers is not justified. Advantages and disadvantages of the new labeling methods are then reviewed.

Fluorescence imaging in human identity testing.

We have investigated the use of fluorescence detection and the FluorImager S1 System (Molecular Dynamics) for analyzing a comprehensive set of human DNA typing tests. We used an alkaline phosphatase-conjugated YNH24 oligonucleotide probe to the repeat-containing D2S44 locus to detect both alleles in 50 ng of human genomic DNA (0.025 amol) by Southern hybridization using a chemifluorescent substrate. We used a similar approach to quantify human DNA using an enzyme-conjugated oligonucleotide probe to the D17Z1 locus. Both fluorescent nucleic acid gel staining and direct fluorescent labeling methods were tested to detect PCR-based D1S80 and short tandem repeat (STR) multiplex allele profiles. The fluorescent staining method sensitively detected these allelic profiles in both denaturing and non-denaturing acrylamide gels using a simple, 10-min procedure. Fluorescent primers eliminate the doublet band patterns often seen with staining methods, which label both strands of the amplified products. This complicates interpretation of STR typing tests. Only one primer for each locus is labeled, so only one strand of the DNA product is detected. Fluorescein end-labeled primers were used in multiplex PCR to amplify, detect and type STRs.

Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A by multicolor in situ hybridization.

Genetic heterogeneity within the most common genetic neuropathy, Charcot-Marie-Tooth disease (CMT) results in about 70% slow nerve conduction CMT1 and 30% normal nerve conduction CMT2. Autosomal dominant CMT1A on chromosome 17p11.2 represents about 70% of CMT1 cases and about 50% of all CMT cases. Three different size CMT1A duplications with variable flanking breakpoints were characterized by multicolor in situ hybridization and confirmed by pulsed field gel electrophoresis and quantitative polymerase chain reaction (PCR) amplification. These different size duplications result in the same CMT1A phenotype confirming that trisomy of a normal gene region results in CMT1A. The smallest duplication does not include the 409 locus used previously to screen for CMT1A duplications. Direct analysis of interphase nuclei from fetuses and at-risk patients by multicolor in situ hybridization to a commonly duplicated CMT1A probe is informative more often than polymorphic PCR analysis, faster than pulsed field gel electrophoresis (PFGE), and faster, more informative, and more reliable than restriction enzyme analysis. CMT1B restriction enzyme analysis of CMT pedigrees without CMT1A is expected to diagnose another 8% of at-risk CMT1 patients (total: 78%).

Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2'-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis.

Infundibulopelvic stenosis, multicystic kidney, and calyectasis in a kindred: clinical observations and genetic analysis.

Congenital obstructive anomalies of the urinary tract usually occur sporadically. We describe inheritance in a three-generation kindred of a spectrum of kidney anomalies consistent with an autosomal-dominant mode of transmission, with incomplete penetrance, calyectasis (maternal grandmother), infundibulopelvic stenosis (uncle), and multicystic kidney (male proband, age 4 years). The proband's mother, father and half sister had normal renal imaging studies. Inheritance of informative polymorphic markers (3'-HVR, GGG1, GGG9, SM-7, KG8, and CW3) mapping close to the adult polycystic kidney disease type 1 (PKD-1) and tuberous sclerosis (TSC-2) loci on chromosome 16p was evaluated by Southern blot studies and by PCR-based, fluorescent genotyping for linkage to phenotype. The 3 affected individuals, as well as the unaffected mother (obligate carrier) and unaffected half-sister, inherit a common chromosome haplotype linked to the PKD1 locus. Our findings support the hypothesis that these anomalies may be part of a spectrum of obstructive renal dysplasia which are inherited as a simple Mendelian trait exhibiting an autosomal-dominant mode of transmission with variable expression and incomplete penetrance.

Microfluidic arrays in genetic analysis.

The goal of genetic analysis is to discover genetic markers that are informative for providing high confidence, positive predictive value in managing phenotypic outcomes. Primary consensus sequence data, genetic polymorphism databases and associated phenotype data are rapidly making genetic analysis more useful. Therefore, genetic analysis applications are gradually becoming more mainstream. The diversity and complexity of genetic analysis currently requires an array of analytical techniques, instrument platforms and software to support all the steps from data acquisition to interpretation. As supporting research technologies mature, they are incorporating increasing levels of automation, system integration and miniaturization. Microfluidic arrays are positioned to play a key role in routine genetic analysis, particularly as they begin to appear in more fully integrated analytical platforms.

Rapid sizing of polymorphic microsatellite markers by capillary array electrophoresis.

Genetic mapping and DNA sequencing projects could potentially be completed more rapidly by using capillary array electrophoresis (CAE) systems running 48-96 capillaries simultaneously. Currently, multiplex polymerase chain reaction (PCR) and multicolor fluorescent dye-labeling strategies are used to generate DNA profiles containing 18-24 genotypes per sample. By using 4-color fluorescence detection and these multiplex PCR strategies, a CAE system has the capacity to generate up to 5.5 million genotypes per year. CAE offers extremely fast, high-resolution separation of DNA and more automated sample processing than conventional systems because the labor-intensive slab-gel pouring and sample-loading steps are eliminated. We used a prototype CAE system in an ongoing linkage analysis study of inherited deafness in Mediterranean families. CA-repeat markers linked to deafness susceptibility genes on chromosomes 7, 11 and 13 were analyzed and DNA profiles generated which contain 6 markers per color. Fragment sizes of over 28,000 short tandem repeat alleles and 3200 CA-repeat alleles have been determined by CAE. An average sizing precision of +/- 0.12 base pairs (bp) for fragments up to 350 bp was realized in 1-h runs. In addition, a versatile non-denaturing matrix was used to separate DNA sizing standards, restriction digests, and multiplex PCR samples. Application of this matrix to Duchenne muscular dystrophy exon deletion screening is also described. These CAE approaches should facilitate rapid genotyping of microsatellite markers and subsequent identification of disease-causing mutations.

Diet and waist-to-hip ratio: important predictors of lipoprotein levels in sedentary and active young men with no evidence of cardiovascular disease.

OBJECTIVE: Healthy, young men were studied to determine the relationship of energy and nutrient intake and physical activity to concentrations of plasma lipoprotein and cholesteryl ester transfer protein. DESIGN: A cross-sectional study compared active and sedentary male subjects (17 to 35 years old) with no personal or family history of coronary heart disease. Participants kept 20-day food and activity journals. Individual intakes of energy, protein, carbohydrate, fat, saturated fat, monounsaturated fatty acids, polyunsaturated fatty acids, dietary fiber, and alcohol were evaluated. Measurements of blood lipids (total cholesterol and triglycerides, high- and low-density lipoprotein cholesterol); apolipoproteins; cholesteryl ester transfer protein; anthropometric variables (body mass index, waist-to-hip ratio, percentage of body fat); and aerobic capacity were taken during fall and spring data collection periods. SUBJECT SELECTION: Subjects were selected on the basis of normal blood lipid levels, absence of underlying disease, and willingness to comply with their current level of physical activity for the duration of the study. Minimal sample size for statistical power was 12 men per group: 12 of 15 subjects who exercised and 13 of 15 subjects who were sedentary completed all phases of the study. STATISTICAL ANALYSES: Statistical analyses consisted of 2-way analysis of variance (activity level and season). Pearson product moment correlations and multiple regression analyses were conducted to assess whether energy and nutrient intakes, physical activity status, and/or anthropometric variables predicted plasma concentrations of lipids and apolipoproteins. RESULTS: Lower waist-to-hip ratio, and not specifically activity level, was associated with higher levels of high-density lipoprotein cholesterol (HDL-C) and lower levels of low-density lipoprotein cholesterol (LDL-C). Dietary intake of saturated and monounsaturated fats and alcohol predicted changes in some apolipoprotein and lipoprotein levels. APPLICATIONS: Use of waist-to-hip ratio in the primary prevention of coronary heart disease is a simple and cost-effective measure to predict development of abnormal lipoprotein profiles in young men. Specific dietary recommendations include adoption of a heart-healthy diet with emphasis on monounsaturated fatty acids (10% to 12% of energy or one third of total fat intake) and the suggestion that small amounts of alcohol (< 3 drinks per week) may, indeed, be beneficial. Because alcohol and waist-to-hip ratio were both important predictors of LDL-C level, even in active young men, the consumption of low levels of alcohol may be beneficial only if waist-to-hip ratio is maintained within the healthful range by achieving an appropriate balance of physical activity and macronutrient intake.

Body composition prediction in university football players.

This study was intended to determine if previously-developed body composition prediction equations were valid for use with a Division I university football team. A sample of 68 Division I football players with a mean age of 19.7 yr, was assessed for body density (BD) by underwater weighing (UWW), residual volume by helium dilution, and 26 selected anthropometric measures. A predicted BD was obtained by using two sets of equations developed from college football players and from three generalized equations. The differences between predicted and observed body densities were analyzed. Seven of the nine models examined failed to accurately predict the BD for this population of university football players. One sport-specific equation of White, Mayhew, and Piper for individuals in the backfield and a generalized model of Jackson and Pollock (JP) containing two circumferences performed well when considering the mean of differences and the magnitude of total error relative to the published standard error. However, both of these models overestimate body density for players with low BD and underestimate BD when actual BD is high. Using the JP model for a player whose actual BD is near the sample mean of 1.070, the estimated mean is very close at 1.069. However, for players with actual BD of 1.050, the estimated mean is 1.054, and if actual BD is 1.085, the JP estimated mean is 1.078. The bias is linear between these points.

Validation of body composition models for high school wrestlers.

This study investigates the utility of two equations for predicting minimum wrestling weight and three equations for predicting body density for the population of high school wrestlers. A sample of 54 wrestlers was assessed for body density by underwater weighing, residual volume by helium dilution, and selected anthropometric measures. The differences between observed and predicted responses were analyzed for the five models. Four statistical tests were used to validate the equations, including tests for the mean of differences, proportion of positive differences, equality of standard errors from regression, and equivalence of regression coefficients between original and second sample data. The Michael and Katch equation and two Forsyth and Sinning equations (FS1 and FS21) for body density did not predict as well as expected. The Michael and Katch equation tends to overpredict body density while FS1 underpredicts. The FS2 equation, consisting of a constant adjustment to FS1, predicts well near the mean but not at the ends of the sample range. The two Tcheng and Tipton equations produce estimates which slightly but consistently overpredict minimum wrestling weight, the long form equation by 2.5 pounds and the short form by 3.8 pounds. As a result the proportion of positive differences is less than would be expected. But based on the tests for the standard errors and regression coefficients, the evidence does not uniformly reject these two equations.

Strength, muscle symmetry, and flexibility in young female idiopathic scoliotics.

Application of previous findings regarding muscle strength and function in scoliotics suggests that curvature might result from unbalanced pull of spinal muscles oriented transversely. This study investigated the role of muscle strength and strength symmetry in 48 young female scoliotics and 48 age-matched controls. The subjects were divided into three age levels: 11 and 12, 13, and 14 and 15 years of age. Scoliotics were weaker than the nonscoliotics only for shoulder strength in the two older age levels. For all subjects combined, as well as each group and age level, the dominant versus nondominant strength differences were significantly greater than zero; however, the magnitudes of the differences were not different between any of the groups. Nonscoliotics displayed significantly greater trunk flexibility than scoliotics. J Orthop Sports Phys Ther 1991;14(4):144-148.