Colorimetric approach to high-throughput mutation analysis

High-throughput genomic mutation screening for primary tumors has characteristically been expensive, labor-intensive, and inadequate to detect low levels of mutation in a background of wild-type signal. We present a new, combined PCR and colorimetric approach that is inexpensive, simple, and can detect the presence of 1% mutation in a background of wild-type. We compared manual dideoxy sequencing of p53 for eight lung cancer samples to a novel assay combining a primer extension step and an enzymatic colorimetric step in a 96-well plate with covalently attached oligonucleotide sequences. For every sample, we were able to detect the presence or absence of the specific mutation with a statistically significant difference between the sample optical density (OD) and the background OD, with a sensitivity and specificity of 100%. This assay is straightforward, accurate, inexpensive, and allows for rapid, high-throughput analysis of samples, making it ideal for genomic mutation or polymorphism screening studies in both clinical and research settings.     

Chlamydomonas IFT172 is encoded by FLA11, interacts with CrEB1, and regulates IFT at the flagellar tip

The transport of flagellar precursors and removal of turnover products from the flagellar tip is mediated by intraflagellar transport (IFT) , which is essential for both flagellar assembly and maintenance . Large groups of IFT particles are moved from the flagellar base to the tip by kinesin-2, and smaller groups are returned to the base by cytoplasmic dynein 1b. The IFT particles are composed of two protein complexes, A and B, comprising approximately 16-18 polypeptides. How cargo is unloaded from IFT particles, turnover products loaded, and active IFT motors exchanged at the tip is unknown. We previously showed that the Chlamydomonas microtubule end binding protein 1 (CrEB1) localizes to the flagellar tip and is depleted from the tips of the temperature-sensitive (ts) mutant fla11ts . We demonstrate here that FLA11 encodes IFT protein 172, a component of IFT complex B, and show that IFT172 interacts with CrEB1. Because fla11ts cells are defective in IFT particle turnaround at the tip, our results indicate that IFT172 is involved in regulating the transition between anterograde and retrograde IFT at the tip, perhaps by a mechanism involving CrEB1. Therefore, IFT172 is involved in the control of flagellar assembly/disassembly at the tip.     

Nuclear markers confirm taxonomic status and relationships among highly endangered and closely related right whale species

Right whales (genus: Eubalaena) are among the most endangered mammals, yet their taxonomy and phylogeny have been questioned. A phylogenetic hypothesis based on mitochondrial DNA (mtDNA) variation recently prompted a taxonomic revision, increasing the number of right whale species to three. We critically evaluated this hypothesis using sequence data from 13 nuclear DNA (nuDNA) loci as well as the mtDNA control region. Fixed diagnostic characters among the nuclear markers strongly support the hypothesis of three genetically distinct species, despite lack of any diagnostic morphological characters. A phylogenetics analysis of all data produced a strict consensus cladogram with strong support at nodes that define each right whale species as well as relationships among species. Results showed very little conflict among the individual partitions as well as congruence between the mtDNA and nuDNA datasets. These data clearly demonstrate the strength of using numerous independent genetic markers during a phylogenetics analysis of closely related species. In evaluating phylogenetic support contributed by individual loci, 11 of the 14 loci provided support for at least one of the nodes of interest to this study. Only a single marker (mtDNA control region) provided support at all four nodes. A study using any single nuclear marker would have failed to support the proposed phylogeny, and a strong phylogenetic hypothesis was only revealed by the simultaneous analysis of many nuclear loci. In addition, nu DNA and mtDNA data provided complementary levels of support at nodes of different evolutionary depth indicating that the combined use of mtDNA and nuDNA data is both practical and desirable.     

The case-only odds ratio as a causal parameter

In the simplest case-only design, cases of a disease are cross-classified into a 2 x 2 table describing a genotype attribute and exposure to some environmental agent. In some instances, the genetic attribute has described inherited genes; in other instances, it has described mutations, for instance, damage to proto-oncogenes or tumor suppressor genes leading to cancer. Here, the population case-only odds ratio is written as a causal parameter in terms of potential outcomes with and without exposure to the agent. It is shown that the case-only odds ratio makes sense as a causal parameter with inherited genes, but its magnitude does not have a causal interpretation with mutations, although deviations from 1 do provide information. The difference is that the environmental agent certainly did not cause an individual to inherit particular genes, but it may have caused the mutation.     

The effects of long-term endurance training on the immune and endocrine systems of elderly men: the role of cytokines and anabolic hormones

Background  

a decline in immune and endocrine function occurs with aging. The main purpose of this study was to investigate the impact of long-term endurance training on the immune and endocrine system of elderly men. The possible interaction between these systems was also analysed.     

Calnexin Deficiency Leads to Dysmyelination

Calnexin is a molecular chaperone and a component of the quality control of the secretory pathway. We have generated calnexin gene-deficient mice (cnx^−/−) and showed that calnexin deficiency leads to myelinopathy. Calnexin-deficient mice were viable with no discernible effects on other systems, including immune function, and instead they demonstrated dysmyelination as documented by reduced conductive velocity of nerve fibers and electron microscopy analysis of sciatic nerve and spinal cord. Myelin of the peripheral and central nervous systems of cnx^−/− mice was disorganized and decompacted. There were no abnormalities in neuronal growth, no loss of neuronal fibers, and no change in fictive locomotor pattern in the absence of calnexin. This work reveals a previously unrecognized and important function of calnexin in myelination and provides new insights into the mechanisms responsible for myelin diseases.     

Electrotonic load triggers remodeling of repolarizing current Ito in ventricle

A change in activation sequence electrically remodels ventricular myocardium, causing persistent changes in repolarizing currents (T-wave memory). However, the underlying mechanism for triggering activation sequence-dependent remodeling is unknown. Optical action potentials were mapped with high resolution from the epicardial surface of the arterially perfused canine wedge preparation (n = 23) during 30 min of baseline endocardial stimulation, followed by 40 min of epicardial stimulation, and, finally, restoration of endocardial stimulation. Immediately after the change from endocardial to epicardial stimulation, phase 1 notch amplitude of epicardial cells was attenuated by 74 +/- 8% (P < 0.001) compared with baseline and continued to diminish during the period of epicardial pacing, suggesting progressive remodeling of the transient outward current (Ito). When endocardial pacing was restored, notch amplitude did not immediately recover but remained attenuated by 23 +/- 10% (P < 0.001), also consistent with a remodeling effect. Peak Ito current measured from isolated epicardial myocytes changed by 12 +/- 4% (P < 0.025), providing direct evidence for Ito remodeling occurring on a surprisingly short time scale. The mechanism for triggering remodeling of Ito was a significant reduction (by 14 +/- 4%, P < 0.001) of upstroke amplitude in epicardial cells during epicardial stimulation. Reduction in upstroke amplitude during epicardial pacing was explained by electrotonic load on epicardial cells by fully repolarized downstream endocardial cells. These data suggest a novel mechanism for triggering electrical remodeling in the ventricle. Electrotonic load imposed by a change in activation sequence reduces upstroke amplitude, which, in turn, attenuates Ito according to its known voltage-dependent properties, triggering downregulation of current.     

Minimal similarity in songs suggests limited exchange between humpback whales (Megaptera novaeangliae) in the southern Indian Ocean

Comparing humpback whale song from different breeding assemblages can reveal similarities in song due to acoustically interacting males, and therefore indirectly test whether males from different breeding sites are mixing. Northern Hemisphere song comparisons illustrated that whales within ocean basins share similar songs and are subpopulations within a larger population, whereas whales in different ocean basins are isolated populations and therefore do not share songs. During the 2006 breeding season, recordings were collected in Madagascar and Western Australia, and were compared visually plus aurally. Both regions shared one theme, whereas each region had four and six private themes, respectively. This study had a substantially low number of shared themes. The co‐occurrence of one theme was interpreted as an indication of limited exchange between these breeding assemblages, and we speculate that limited song similarity is due to inter‐oceanic interactions. Male(s) from an Indian Ocean breeding group could be exposed to novel song when they geographically overlap, and acoustically interact, with males from a different ocean basin. Novel song could induce rapid temporal changes as new song content is incorporated, thereby minimizing song similarities between that breeding group and other Indian Ocean breeding groups that were not exposed to the novel song.     

The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3's LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly.