Laser-based tissue microdissection is an important tool for the molecular evaluation of histological sections. The technology has continued to advance since its initial commercialization in the ...1990s, with improvements in many aspects of the process. More recent developments are tailored toward an automated, operator-independent mode that relies on antibodies as targeting probes, such as immuno–laser capture microdissection or expression microdissection (xMD). Central to the utility of expression-based dissection techniques is the effect of the staining process on the biomolecules in histological sections. To investigate this issue, the authors analyzed DNA, RNA, and protein in immunostained, microdissected samples. DNA was the most robust molecule, exhibiting no significant change in quality after immunostaining but a variable 50% to 75% decrease in the total yield. In contrast, RNA in frozen and ethanol-fixed, paraffin-embedded samples was susceptible to hydrolysis and digestion by endogenous RNases during the initial steps of staining. Proteins from immunostained tissues were successfully analyzed by one-dimensional electrophoresis and mass spectrometry but were less amenable to solution phase assays. Overall, the results suggest investigators can use immunoguided microdissection methods for important analytic techniques; however, continued improvements in staining protocols and molecular extraction methods are key to further advancing the capability of these methods.
Understanding the pattern of linkage disequilibrium (LD) in the human genome is important both for successful implementation of disease-gene mapping approaches and for inferences about human ...demographic histories. Previous studies have examined LD between loci within single genes or confined genomic regions, which may not be representative of the genome; between loci separated by large distances, where little LD is seen; or in population groups that differ from one study to the next. We measured LD in a large set of locus pairs distributed throughout the genome, with loci within each pair separated by short distances (average 124 bp). Given current models of the history of the human population, nearly all pairs of loci at such short distances would be expected to show complete LD as a consequence of lack of recombination in the short interval. Contrary to this expectation, a significant fraction of pairs showed incomplete LD. A standard model of recombination applied to these data leads to an estimate of effective human population size of 110,000. This estimate is an order of magnitude higher than most estimates based on nucleotide diversity. The most likely explanation of this discrepancy is that gene conversion increases the apparent rate of recombination between nearby loci.
In humans, kappa opioid receptor agonists produce, among other effects, sedation and difficulty concentrating, suggesting that they may disrupt attention. The purpose of the present studies was ...therefore to evaluate the effects of kappa opioid receptor agonists on attention as assessed by a 5-choice serial reaction time task in rats. The kappa opioid receptor agonists (+)-U69,593 (0.1–0.56
mg/kg), (±)-U50,488 (1.0–5.6
mg/kg) and racemic GR89,696 (0.0003–0.01
mg/kg) all produced dose-related decreases in the percentage of trials terminated by a correct or incorrect response and increases in the percentage of omissions. In contrast, the peripherally restricted opioid agonist ICI-204,448 was ineffective (1.0–10
mg/kg). Moreover, the effects of GR89,696 were stereoselective in that (R)-GR89,696 was approximately equipotent to racemic GR89,696 and approximately 100-fold more potent than (S)-GR89,696. The opioid receptor antagonist naltrexone (0.3–3
mg/kg) administered alone had no effects on performance. However, naltrexone, over the dose-range of 0.03–1.0
mg/kg, produced a dose-related antagonism of the disruption produced by U69,593 (0.56
mg/kg). In contrast, naltrexone, over the dose-range of 0.01–0.3
mg/kg produced a dose-related antagonism of morphine (5.6
mg/kg). Recent evidence has suggested that kappa opioid receptor agonists decrease dopaminergic and noradrenergic neurotransmission in prefrontal cortex and locus coeruleus. Together with previous findings, the present data indicate that kappa opioid receptor agonists disrupt performance of this attention task by decreasing the probability of responding by specific actions at central kappa opioid receptors, perhaps by decreasing dopaminergic and noradrenergic neurotransmission.
Adult-onset cerebellar ataxias are a group of neurodegenerative conditions that challenge both genetic discovery and molecular diagnosis. In this study, we identified an intronic (GAA) repeat ...expansion in fibroblast growth factor 14 (FGF14). Genetic analysis of 95 Australian individuals with adult-onset ataxia identified four (4.2%) with (GAA)>300 and a further nine individuals with (GAA)>250. PCR and long-read sequence analysis revealed these were pure (GAA) repeats. In comparison, no control subjects had (GAA)>300 and only 2/311 control individuals (0.6%) had a pure (GAA)>250. In a German validation cohort, 9/104 (8.7%) of affected individuals had (GAA)>335 and a further six had (GAA)>250, whereas 10/190 (5.3%) control subjects had (GAA)>250 but none were (GAA)>335. The combined data suggest (GAA)>335 are disease causing and fully penetrant (p = 6.0 × 10−8, OR = 72 95% CI = 4.3–1,227), while (GAA)>250 is likely pathogenic with reduced penetrance. Affected individuals had an adult-onset, slowly progressive cerebellar ataxia with variable features including vestibular impairment, hyper-reflexia, and autonomic dysfunction. A negative correlation between age at onset and repeat length was observed (R2 = 0.44, p = 0.00045, slope = −0.12) and identification of a shared haplotype in a minority of individuals suggests that the expansion can be inherited or generated de novo during meiotic division. This study demonstrates the power of genome sequencing and advanced bioinformatic tools to identify novel repeat expansions via model-free, genome-wide analysis and identifies SCA27B/ATX-FGF14 as a frequent cause of adult-onset ataxia.
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Pathogenic repeat expansions (RE) cause an array of neurogenetic disorders including cerebellar ataxia. While traditionally difficult to identify, new genomic tools and bioinformatic analyses are enabling rapid RE discovery and diagnosis. Here we characterize SCA27B, an adult-onset ataxia caused by a pathogenic GAA repeat expansion within intron one of FGF14.
The T-cell receptor (TCR) plays a central role in the immune system, and >90% of human T cells present a receptor that consists of the α TCR subunit (TCRA) and the β subunit (TCRB). Here we report an ...analysis of 63 variable genes (BV), spanning 553 kb of
TCRB that yielded 279 single-nucleotide polymorphisms (SNPs). Samples were drawn from 10 individuals and represent four populations—African American, Chinese, Mexican, and Northern European. We found nine variants that produce nonfunctional BV segments, removing those genes from the
TCRB genomic repertoire. There was significant heterogeneity among population samples in SNP frequency (including the BV-inactivating sites), indicating the need for multiple-population samples for adequate variant discovery. In addition, we observed considerable linkage disequilibrium (LD) (
r
2>0.1) over distances of ∼30 kb in
TCRB, and, in general, the distribution of
r
2 as a function of physical distance was in close agreement with neutral coalescent simulations. LD in
TCRB showed considerable spatial variation across the locus, being concentrated in “blocks” of LD; however, coalescent simulations of the locus illustrated that the heterogeneity of LD we observed in
TCRB did not differ markedly from that expected from neutral processes. Finally, examination of the extended genotypes for each subject demonstrated homozygous stretches of >100 kb in the locus of several individuals. These results provide the basis for optimization of locuswide SNP typing in
TCRB for studies of genotype-phenotype association.
T cell receptors (TR), through their interaction with the major histocompatibility complex, play a central role in immune responsiveness and potentially immune-related disorders. We resequenced all ...57 variable (V) genes in the human T cell receptor alpha and delta (TRA/TRD) locus in 40 individuals of Northern European, Mexican, African-American and Chinese descent. Two hundred and eighty-four single nucleotide polymorphisms (SNPs) were identified. The distribution of SNPs between V genes was heterogeneous, with an average of five SNPs per gene and a range of zero to 15. We describe the patterns of linkage disequilibrium for these newly discovered SNPs and compare these patterns with other emerging large-scale datasets (e.g. Perlegen and HapMap projects) to place our findings into a framework for future analysis of genotype-phenotype associations across this locus. Furthermore, we explore signatures of natural selection across V genes. We find evidence of strong directional selection at this locus as evidenced by unusually high values of Fst.
Hydrogen is a promising energy carrier in future energy systems. However, storage of hydrogen is a substantial challenge, especially for applications in vehicles with fuel cells that use ...proton-exchange membranes (PEMs). Different methods for hydrogen storage are discussed, including high-pressure and cryogenic-liquid storage, adsorptive storage on high-surface-area adsorbents, chemical storage in metal hydrides and complex hydrides, and storage in boranes. For the latter chemical solutions, reversible options and hydrolytic release of hydrogen with off-board regeneration are both possible. Reforming of liquid hydrogen-containing compounds is also a possible means of hydrogen generation. The advantages and disadvantages of the different systems are compared.
•A new method for estimating the bending stiffness curve of a non-uniform Euler-Bernoulli beam from static data was developed.•Instead of a complex and time-consuming optimization problem, only a ...simple boundary value problem needs to be solved.•With the new method, it is no problem to determine the bending stiffness on the hinges of the bending test.•The new method was validated both numerically and experimentally.
The static Euler-Bernoulli beam is a simplified model from linear elasticity theory for small deformations, with which the stresses and deflections of slender beams can be determined very well. The simplicity of Euler-Bernoulli beam theory makes it still an important tool in the sciences, particularly in construction and mechanical engineering. The behavior and the deflection of a static Euler-Bernoulli beam is completely given by the bending stiffness curve. The bending stiffness curve of a real beam structure can be determined using measured data from a bending test. However, the determination of the bending stiffness curve for non-uniform beams leads to a so-called inverse problem, which is ill-posed. In this work, a robust and computationally effective method for determining the bending stiffness curve of a non-uniform Euler-Bernoulli beam based on measured data from a static bending test and the calculus of variations is presented. With the calculus of variations, the complex optimization problem of the inverse problem is transformed into a boundary value problem, which is then solved by collocation with lower computational costs. In addition, the new method does not require the second derivative of the beam deflection and the problem of determining the bending stiffness at the boundaries does not arise, as with the traditional method. The new method to determine the bending stiffness curve of non-uniform beams is demonstrated in several test examples.
Identifying common sequence variations known as single nucleotide polymorphisms (SNPs) in human populations is one of the current objectives of the human genome project. Nearly 3 million SNPs have ...been identified. Analysis of the relative allele frequency of these markers in human populations and the genetic associations between these markers, known as linkage disequilibrium, is now underway to generate a high‐density genetic map. Because of the central role T cells play in immune reactivity, the T‐cell receptor (TCR) loci have long been considered important candidates for common disease susceptibility within the immune system (e.g., asthma, atopy and autoimmunity). Over the past two decades, hundreds of SNPs in the TCR loci have been identified. Most studies have focused on defining SNPs in the variable gene segments which are involved in antigenic recognition. On average, the coding sequence of each TCR variable gene segment contains two SNPs, with many more found in the 5′, 3′ and intronic sequences of these segments. Therefore, a potentially large repertoire of functional variants exists in these loci. Association between SNPs (linkage disequilibrium) extends approximately 30 kb in the TCR loci, although a few larger regions of disequilibrium have been identified. Therefore, the SNPs found in one variable gene segment may or may not be associated with SNPs in other surrounding variable gene segments. This suggests that meaningful association studies in the TCR loci will require the analysis and typing of large marker sets to fully evaluate the role of TCR loci in common disease susceptibility in human populations.
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