PCA and Clustering Reveal Alternate mtDNA Phylogeny of N and M Clades

Phylogenetic trees based on mtDNA polymorphisms are often used to infer the history of recent human migrations. However, there is no consensus on which method to use. Most methods make strong assumptions which may bias the choice of polymorphisms and result in computational complexity which limits the analysis to a few samples/polymorphisms. For example, parsimony minimizes the number of mutations, which biases the results to minimizing homoplasy events. Such biases may miss the global structure of the polymorphisms altogether, with the risk of identifying a "common" polymorphism as ancient without an internal check on whether it either is homoplasic or is identified as ancient because of sampling bias (from oversampling the population with the polymorphism). A signature of this problem is that different methods applied to the same data or the same method applied to different datasets results in different tree topologies. When the results of such analyses are combined, the consensus trees have a low internal branch consensus. We determine human mtDNA phylogeny from 1737 complete sequences using a new, direct method based on principal component analysis (PCA) and unsupervised consensus ensemble clustering. PCA identifies polymorphisms representing robust variations in the data and consensus ensemble clustering creates stable haplogroup clusters. The tree is obtained from the bifurcating network obtained when the data are split into k = 2,3,4,...,kmax clusters, with equal sampling from each haplogroup. Our method assumes only that the data can be clustered into groups based on mutations, is fast, is stable to sample perturbation, uses all significant polymorphisms in the data, works for arbitrary sample sizes, and avoids sample choice and haplogroup size bias. The internal branches of our tree have a 90% consensus accuracy. In conclusion, our tree recreates the standard phylogeny of the N, M, L0/L1, L2, and L3 clades, confirming the African origin of modern humans and showing that the M and N clades arose in almost coincident migrations. However, the N clade haplogroups split along an East-West geographic divide, with a "European R clade" containing the haplogroups H, V, H/V, J, T, and U and a "Eurasian N subclade" including haplogroups B, R5, F, A, N9, I, W, and X. The haplogroup pairs (N9a, N9b) and (M7a, M7b) within N and M are placed in nonnearest locations in agreement with their expected large TMRCA from studies of their migrations into Japan. For comparison, we also construct consensus maximum likelihood, parsimony, neighbor joining, and UPGMA-based trees using the same polymorphisms and show that these methods give consistent results only for the clade tree. For recent branches, the consensus accuracy for these methods is in the range of 1-20%. From a comparison of our haplogroups to two chimp and one bonobo sequences, and assuming a chimp-human coalescent time of 5 million years before present, we find a human mtDNA TMRCA of 206,000 +/- 14,000 years before present.     

Orexin-A-induced feeding is dependent on nitric oxide

Orexin-A is a peptide produced in the lateral hypothalamus/perifornical area, which stimulates feeding. The production of orexin-A is determined by the metabolic state of the animal. We have previously shown that nitric oxide (NO) plays an important role as a mediator of feeding induced by a variety of neuropeptides. This raises the question of whether orexin-A's effects are NO dependent. Here, we first determined that intracerebroventricular administration of 25 ng of orexin-A significantly increased food intake in satiated mice. We next examined the effects of Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, on orexin-A-induced increase in food intake. L-NAME (50 mg/kg; SC) significantly blocked the orexin-A-induced increase in food intake. Orexin-A administration increased the levels of nitric oxide synthase in the hypothalamus. To further verify the importance of NO in the orexin-A-induced increase in food intake, we compared the ability of orexin-A to increase food intake in neuronal nitric oxide synthase knockout (NOS-KO) mice and their wild-type controls. Orexin-A failed to increase food intake in the NOS-KO mice, whereas it did increase food intake in the wild-type controls. This supports the hypothesis that nitric oxide is a central regulator of food consumption.     

DNA microarrays on nanoscale-controlled surface

We have developed new surface to ensure a proper spacing between immobilized biomolecules. While DNA microarray on this surface provided each probe DNA with ample space for hybridization with incoming target DNAs, the microarray showed enhanced discrimination efficiency for various types of single nucleotide polymorphism. The high discrimination efficiency holds for all tested cases (100:<1 for internal mismatched cases; 100:<28 for terminal mismatched ones). In addition, by investigating influence of hybridization temperature and washing condition on the fluorescence intensity and the discrimination efficiency with and without controlled mesospacing, it was observed that the nanoscale-controlled surface showed good discrimination efficiency in a wide range of temperature (37–50°C), and hybridization behavior on the surface was in agreement with the solution one. Intriguingly, it was found that washing process after the hybridization was critical for the high discrimination efficiency. For the particular case, washing process was so efficient that only 30 s washing was sufficient to reach the optimal discrimination ratio.     

Identification of genes differentially expressed in wild type and Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice are characterized by death of virtually all cerebellar Purkinje cells by postnatal day 30. In this study, we used DNA microarray analysis to investigate differences in gene expression between the brains of wild type and pcd mice on postnatal day 20, before the appearance of clear-cut phenotypic abnormalities. We identified 300 differentially expressed genes, most of which were involved in metabolic and physiological processes. Among the differentially expressed genes were several calcium binding proteins including calbindin-28k, paravalbumin, matrix gamma-carboxyglutamate protein and synaptotagamins 1 and 13, suggesting the involvement of abnormal Ca2+ signaling in the pcd phenotype.     

Left ventricular geometry immediately following defibrillation: shock-induced relaxation

A previous two-dimensional (2D) ultrasound study suggested that there is relaxation of the myocardium after defibrillation. The 2D study could not measure activity occurring within the first 33 ms after the shock, a period that may be critical for discriminating between shock- and excitation-induced relaxation. The objective of our study was to determine the left ventricular (LV) geometry during the first 33 ms after defibrillation. Biphasic defibrillation shocks were delivered 5-50 s after the induction of ventricular fibrillation in each of the seven dogs. One-dimensional, short-axis ultrasound images of the LV cavity were acquired at a rate of 250 samples/s. The LV cavity diameter was computed from 32 ms before to 32 ms after the shock. Preshock and postshock percent changes in LV diameter were analyzed as a function of time with the use of regression analysis. The normalized mean pre- and postshock slopes (0.2 +/- 2.2 and 3.3 +/- 7.9% per 10 ms) were significantly different (P < 0.01). The postshock slope was positive (P < 0.005). Our results confirm that the bulk of the myocardium is relaxing immediately after defibrillation.     

Pam and its ortholog highwire interact with and may negatively regulate the TSC1.TSC2 complex

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder associated with mutations in TSC1, which codes for hamartin, or TSC2, which codes for tuberin. The brain is one of the most severely affected organs, and CNS lesions include cortical tubers and subependymal giant cell astrocytomas, resulting in mental retardation and seizures. Tuberin and hamartin function together as a complex in mammals and Drosophila. We report here the association of Pam, a protein identified as an interactor of Myc, with the tuberin-hamartin complex in the brain. The C terminus of Pam containing the RING zinc finger motif binds to tuberin. Pam is expressed in embryonic and adult brain as well as in cultured neurons. Pam has two forms in the rat CNS, an approximately 450-kDa form expressed in early embryonic stages and an approximately 350-kDa form observed in the postnatal period. In cortical neurons, Pam co-localizes with tuberin and hamartin in neurites and growth cones. Although Pam function(s) are yet to be defined, the highly conserved Pam homologs, HIW (Drosophila) and RPM-1 (Caenorhabditis elegans), are neuron-specific proteins that regulate synaptic growth. Here we show that HIW can genetically interact with the Tsc1.Tsc2 complex in Drosophila and could negatively regulate Tsc1.Tsc2 activity. Based on genetic studies, HIW has been implicated in ubiquitination, possibly functioning as an E3 ubiquitin ligase through the RING zinc finger domain. Therefore, we hypothesize that Pam, through its interaction with tuberin, could regulate the ubiquitination and proteasomal degradation of the tuberin-hamartin complex particularly in the CNS.     

TorsinB--perinuclear location and association with torsinA

The torsins comprise a four-member family of AAA+ chaperone proteins, including torsinA, torsinB, torp2A and torp3A in humans. Mutations in torsinA underlie early onset torsion dystonia, an autosomal dominant, neurologically based movement disorder. TorsinB is highly homologous to torsinA with its gene adjacent to that for torsinA on human chromosome 9q34. Antibodies have been generated which can distinguish torsinA and torsinB from each other, and from the torps in human and rodent cells. TorsinB (approximately MW 38 kDa), like torsinA ( approximately MW 37 kDa), is an N-glycosylated protein and both reside primarily in the endoplasmic reticulum (ER) and nuclear envelope in cultured cells. Immunoprecipitation studies in cultured cells and human brain tissue indicate that torsinA and torsinB are associated with each other in cells. Overexpression of both wild-type torsinB and mutant torsinA lead to enrichment of the protein in the nuclear envelope and formation of large cytoplasmic inclusions. We conclude that torsinB and torsinA are localized in overlapping cell compartments within the same protein complex, and thus may carry out related functions in vivo.