Phylogenetic Comparative Methods on Phylogenetic Networks with Reticulations.

The goal of phylogenetic comparative methods (PCMs) is to study the distribution of quantitative traits among related species. The observed traits are often seen as the result of a Brownian Motion (BM) along the branches of a phylogenetic tree. Reticulation events such as hybridization, gene flow or horizontal gene transfer, can substantially affect a species' traits, but are not modeled by a tree. Phylogenetic networks have been designed to represent reticulate evolution. As they become available for downstream analyses, new models of trait evolution are needed, applicable to networks. We develop here an efficient recursive algorithm to compute the phylogenetic variance matrix of a trait on a network, in only one preorder traversal of the network. We then extend the standard PCM tools to this new framework, including phylogenetic regression with covariates (or phylogenetic ANOVA), ancestral trait reconstruction, and Pagel's $\lambda$ test of phylogenetic signal. The trait of a hybrid is sometimes outside of the range of its two parents, for instance because of hybrid vigor or hybrid depression. These two phenomena are rather commonly observed in present-day hybrids. Transgressive evolution can be modeled as a shift in the trait value following a reticulation point. We develop a general framework to handle such shifts and take advantage of the phylogenetic regression view of the problem to design statistical tests for ancestral transgressive evolution in the evolutionary history of a group of species. We study the power of these tests in several scenarios and show that recent events have indeed the strongest impact on the trait distribution of present-day taxa. We apply those methods to a data set of Xiphophorus fishes, to confirm and complete previous analysis in this group. All the methods developed here are available in the Julia package PhyloNetworks.

Computerized Cognitive Testing for Use in Clinical Trials: A Comparison of the NIH Toolbox and Cogstate C3 Batteries.

BACKGROUND: As prevention trials for Alzheimer's disease move into asymptomatic populations, identifying older individuals who manifest the earliest cognitive signs of Alzheimer's disease is critical. Computerized cognitive testing has the potential to replace current gold standard paper and pencil measures and may be a more efficient means of assessing cognition. However, more empirical evidence about the comparability of novel computerized batteries to paper and pencil measures is required. OBJECTIVES: To determine whether two computerized IPad batteries, the NIH Toolbox Cognition Battery and Cogstate-C3, similarly predict subtle cognitive impairment identified using the Preclinical Alzheimer Cognitive Composite (PACC). DESIGN, SETTING, PARTICIPANTS: A pilot sample of 50 clinically normal older adults (Mage=68.5 years±7.6, 45% non-Caucasian) completed the PACC assessment, and the NIH Toolbox and Cogstate-C3 at research centers of Massachusetts General and Brigham and Women's Hospitals. Participants made 3-4 in-clinic visits, receiving the PACC first, then the NIH Toolbox, and finally the Cogstate-C3.>= 0.5SD), versus subtle cognitive impairment (<0.5SD). Composites for each computerized battery were created using principle components analysis, and compared with the PACC using non-parametric Spearman correlations. Logistic regression analyses were used to determine which composite was best able to classify subtle cognitive impairment from typical performance. RESULTS: The NIH Toolbox formed one composite and exhibited the strongest within-battery alignment, while the Cogstate-C3 formed two distinct composites (Learning-Memory and Processing Speed-Attention). The NIH Toolbox and C3 Learning-Memory composites exhibited positive correlations with the PACC (ρ=0.49, p<0.001; ρ=0.58, p<0.001, respectively), but not the C3 Processing Speed-Attention composite, ρ=-0.18, p=0.22. The C3 Learning-Memory was the only composite that classified subtle cognitive impairment, and demonstrated the greatest sensitivity (62%) and specificity (81%) for that subtle cognitive impairment. CONCLUSIONS: Preliminary findings suggest that the NIH Toolbox has the advantage of showing the strongest overall clustering and alignment with standardized paper-and-pencil tasks. By contrast, Learning-Memory tasks within the Cogstate-C3 battery have the greatest potential to identify cross-sectional, subtle cognitive impairment as defined by the PACC.

Genetic predictors of cardiovascular morbidity in Bardet-Biedl syndrome.

Bardet-Biedl syndrome is a rare ciliopathy characterized by retinal dystrophy, obesity, intellectual disability, polydactyly, hypogonadism and renal impairment. Patients are at high risk of cardiovascular disease. Mutations in BBS1 and BBS10 account for more than half of those with molecular confirmation of the diagnosis. To elucidate genotype-phenotype correlations with respect to cardiovascular risk indicators 50 patients with mutations in BBS1 were compared with 19 patients harbouring BBS10 mutations. All patients had truncating, missense or compound missense/truncating mutations. The effect of genotype and mutation type was analysed. C-reactive protein was higher in those with mutations in BBS10 and homozygous truncating mutations (p = 0.013 and p = 0.002, respectively). Patients with mutations in BBS10 had higher levels of C peptide than those with mutations in BBS1 (p = 0.043). Triglyceride levels were significantly elevated in patients with homozygous truncating mutations (p = 0.048). Gamma glutamyl transferase was higher in patients with homozygous truncating mutations (p = 0.007) and heterozygous missense and truncating mutations (p = 0.002) than those with homozygous missense mutations. The results are compared with clinical cardiovascular risk factors. Patients with missense mutations in BBS1 have lower biochemical cardiovascular disease markers compared with patients with BBS10 and other BBS1 mutations. This could contribute to stratification of the clinical service.

Ethanol isotope method (EIM) for uncovering illegal wine.

Isotopic methods have proven to be a powerful analytical tool for the determination of origin and authenticity of wine. In addition, measuring the stable isotope ratio provides useful information for the detection of many illegal practices in the production of wine. The determinations of the stable isotope composition of compounds are based on measuring the relative ratios using isotope ratio mass spectrometry. This article describes a new isotopic method for measuring the δD value of non-exchangeable hydrogen stable isotopes in ethanol for investigating adulteration practices in wine making. With this new method, we are able to determine the addition of water and sugar in wine with higher accuracy, repeatability and reliability.

A self-correcting method for improving the precision of beam blocks.

A technique for manufacturing precise custom blocks is described. Using the tracing stylus of the mold making machine, reference markers are cut into the lateral borders of the polystyrene mold after the cavities for the blocks have been made. These markers are aligned with the central ray cross hair of the shadow tray when the blocks are mounted on the tray. The ability of the technique to enhance precision has been verified in laboratory tests by intentionally introducing small imperfections into a mold making machine and checking the positional accuracy of the mounted blocks. The clinical performance has been tested by evaluating 47 check films of blocks for 16 randomly selected patients. The average positional error of individual blocks, projected to the isocenter, was less than one mm. The average time needed to cut the reference markers was 25 seconds. Implementing the technique required only minor modifications of a commercial mold making machine.

A truncated cytoplasmic topoisomerase IIalpha in a drug-resistant lung cancer cell line is encoded by a TOP2A allele with a partial deletion of exon 34.

To study the problem of acquired resistance to widely used anti-cancer drugs that target the 170 kDa topoisomerase IIalpha (topo IIalpha), a drug-resistant human small-cell lung cancer cell line, H209/VP, was selected in VP-16. H209/VP cells express reduced levels of the 170 kDa topo IIalpha that is localized normally in the nucleus and also express lower levels of a 160 kDa topo IIalpha-related protein that is located predominantly in the cytoplasm. Band depletion immunoblotting experiments suggest that the H209/VP nuclear 170 kDa topo IIalpha is able to form ternary complexes with DNA and VP-16 in intact cells, but the ability of the cytoplasmic 160 kDa protein to do so is greatly diminished. Sequence analysis of the 3; end of the H209/VP mutant topo IIalpha mRNA and the TOP2A gene indicates that the mRNA is missing 200 nt that corresponds to exon 34 because the partial loss of the minimal 3; splice-acceptor sequence at the beginning of exon 34 results in splicing of exon 33 to exon 35. The protein predicted to be encoded by this mutant mRNA does not contain the COOH-terminal 109 amino acids of the wild-type enzyme that we have demonstrated contain a strongly functional nuclear localization signal sequence. Consequently, our data explain both the size and the cytoplasmic localization of the H209/VP mutant topo IIalpha. The mutant TOP2A allele in H209/VP cells differs from those in previously characterized cell lines with cytoplasmic topo IIalpha and extends the number of types of resistance-associated deletions in this region to 4. These findings indicate that this region of the TOP2A gene may be a hot spot for mutations.

A novel depot preparation of desferrioxamine-B: development of formulation principles.

This report describes the feasibility of simple oil-based depot formulations of a novel n-decanesulfonate salt of the iron chelator desferrioxamine-B. After subcutaneous administration in rodents, desferrioxamine-B n-decanesulfonate depot induces both (a) prolonged release of drug and (b) an increase of at least threefold to fourfold in iron excretion efficiency compared with the parent compound Desferal (desferrioxamine-B mesylate). Optimization experiments probing vehicle composition, surfactant loading, drug loading, and particle size distribution of the depot preparation are described, and the physiochemical stability of an identified pilot formulation is assessed.

Regulation of poly(A) site choice of several yeast mRNAs.

Several yeast genes produce multiple transcripts with different 3'-ends. Of these, four genes are known to produce truncated transcripts that end within the coding sequence of longer transcripts: CBP1 , AEP2 / ATP13 , RNA14 and SIR1 . It has been shown that the level of the truncated CBP1 transcript increases during the switch to respiratory growth while that of the full-length transcript decreases. To determine whether this phenomenon is unique to CBP1 , northern analysis was used to determine whether the levels of other truncated transcripts are regulated similarly by carbon source. The levels of the shortest transcripts of AEP2 / ATP13 and RNA14 increased during respiration while the shortest SIR1 transcript remained constant. However, two longer SIR1 transcripts were regulated reciprocally by carbon source. Mapping the 3'-ends of each transcript by sequencing partial cDNA clones revealed multiple 3'-ends for each transcript. Examination of the sequences surrounding the 3'-ends of the induced transcripts failed to identify a consensus sequence but did reveal weak putative 3'-end formation signals in all of the transcripts. Similarly, no consensus sequence was found when the sequences surrounding the 3'-ends of the longest transcripts were compared, but again weak putative 3'-end formation signals were identified. These data are suggestive of carbon source regulation of alternative poly(A) site choice in yeast.

Premature 3'-end formation of CBP1 mRNA results in the downregulation of cytochrome b mRNA during the induction of respiration in Saccharomyces cerevisiae.

The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes. Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes. One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the nuclear gene CBP1 (cytochrome b processing). CBP1 produces two types of transcripts with different 3' ends: full-length 2.2-kb transcripts and 1.2-kb transcripts truncated within the coding sequence of Cbp1. Upon induction of respiration, the steady-state level of the long transcripts decreases while that of the short transcripts increases reciprocally, an unexpected result since the product of the long transcripts is required for COB mRNA stability and thus for respiration. Here we have tested the hypothesis that the short transcripts, or proteins translated from the short transcripts, are also required for respiration. A protein translated from the short transcripts was not detected by Western analysis, although polysome gradient fractions were shown to contain both long and short CBP1 transcripts. A mutant strain in which production of the short transcripts was abolished showed wild-type growth properties, indicating that the short transcripts are not required for respiration. Due to mutation of the carbon source-responsive element, the long transcript level in the mutant strain did not decrease during induction of respiration. The mutant strain had increased levels of COB RNA, suggestive that production of short CBP1 transcripts is a mechanism for downregulation of the levels of long CBP1 transcripts, Cbp1, and COB mRNA during the induction of respiration.

Two COOH-terminal truncated cytoplasmic forms of topoisomerase II alpha in a VP-16-selected lung cancer cell line result from partial gene deletion and alternative splicing.

Topoisomerase II alpha is a nuclear enzyme involved in chromosome segregation and other essential cellular processes. It is also the target of several clinically important antineoplastic agents such as the epipodophyllotoxin, VP-16 (etoposide). We have previously described a VP-16-selected lung cancer cell line, H209/V6, that expresses reduced levels of two species of topoisomerase II alpha-related mRNAs and a catalytically active, predominantly cytoplasmic topoisomerase II alpha-related protein that is 10 kDa smaller than the wild-type protein [Mirski, S. E. L., et al. (1993) Cancer Res. 53, 4866-4873; Feldhoff, P. W. et al. (1994) Cancer Res. 54, 756-762]. The smaller H209/V6 4.8 kb mRNA is missing 988 nucleotides of contiguous coding and non-coding sequence at its 3' end resulting in an mRNA predicted to encode a truncated polypeptide missing three previously unrecognized potential COOH-proximal bipartite nuclear localization signals [Mirski, S. E. L., & Cole, S. P. C. (1995) Cancer Res. 55, 2129-2134]. We have now determined the structure of the larger 6.2 kb topoisomerase II alpha-related mRNA and show that it is missing 684 nucleotides of contiguous 3' coding and non-coding sequence between nucleotide positions 4267 and 4951. This sequence is replaced by 847 nucleotides of new sequence, containing an in-frame stop codon after 41 nucleotides. The translation product of the 6.2 kb mRNA is predicted to contain 13 new amino acids replacing the COOH-terminal 109 residues of wild-type topoisomerase II alpha, producing a truncated polypeptide of approximately 160 kDa. Immunoblot analyses using antisera against the unique COOH-terminal 13 and 34 amino acids encoded by H209/V6 6.2 kb and 4.8 kb mRNAs, respectively, confirmed that both mRNAs are translated. Restriction enzyme analysis and sequencing of the 3'-proximal region of the TOP2A gene in the H209 and H209/V6 DNA revealed that a partial deletion has occurred in H209/V6 and the novel sequence identified in the H209/V6 6.2 kb mRNA is derived from the adjacent 3' intron as a consequence of read-through at a concensus splice donor site. These observations suggest a mechanism for the generation of the two mutant topoisomerase II alpha mRNAs in H209/V6 cells and provide the first reported example of a drug resistant cell line containing two different cytoplasmic forms of topoisomerase II alpha.

Isolation and characterization of apolipophorin-III from the giant water bug (Lethocerus medius).

Upon injection of synthetic adipokinetic hormone, lipophorin from Lethocerus medius decreased in density and became associated with apolipophorin-III (apoLp-III). ApoLp-III isolated from hemolymph of Lethocerus medius had a M(r) = 19,000 and an amino acid composition high in methionine, in comparison with other apoLp-IIIs. Its circular dichroism spectrum was consistent with a protein with secondary structure of predominantly alpha-helix. NH2-terminal sequence alignment with apoLp-III sequences from other species showed a conservation of the hydrophobic or hydrophilic properties of residues at each position rather than of specific amino acids. ApoLp-III from Lethocerus medius has the potential to form amphipathic alpha-helices, similar to those found in the three-dimensional structure of Locusta migratoria apoLp-III. A portion of the apoLp-III molecules that are not associated with lipophorin contained the blue chromophore, biliverdin.

Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells.

We have previously identified and characterized a novel member of the ATP-binding cassette superfamily of transport proteins, multidrug resistance protein (MRP), and subsequently demonstrated that its overexpression is sufficient to confer multidrug resistance on previously sensitive cells (Cole et al., Science (Washington DC), 258: 1650-1654, 1992; Grant et al., Cancer Res. 54: 357-361, 1994). In the present study, we have transfected two different eukaryotic expression vectors containing MRP complementary DNA into HeLa cells to study the pharmacological phenotype produced exclusively by overexpression of human MRP. The drug resistance patterns of the two MRP-transfected cell populations were similar. They were characterized by a moderate (5- to 15-fold) level of resistance to doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide, and a low (< or = 3-fold) level of resistance to taxol, vinblastine, and colchicine. The transfectants were not resistant to 9-alkyl anthracyclines, mitoxantrone, or cisplatin. The MRP-transfected cells were also resistant to some heavy metal anions including arsenite, arsenate, and trivalent and pentavalent antimonials but were not resistant to cadmium chloride. Accumulation of radiolabeled vincristine was reduced by 45% in the MRP-transfected cells and could be restored to the levels found in sensitive cells by depletion of ATP. Rates of vincristine efflux did not differ greatly in the sensitive and resistant cells. The cytotoxic effects of vincristine and doxorubicin could be enhanced in a dose-dependent fashion by coadministration of verapamil. Cyclosporin A also increased vincristine toxicity but had less effect on doxorubicin toxicity. The degree of chemosensitization by verapamil and cyclosporin A was similar in MRP-transfected cells and in cells transfected with the vector alone, suggesting that sensitization involved mechanisms independent of MRP expression. Verapamil and cyclosporin A caused a modest increase in vincristine accumulation in the resistant cells but did not restore levels to those of the sensitive cells. Taken together, these data indicate that drug-resistant cell lines generated by transfection with MRP complementary DNA display some but not all of the characteristics of MRP-overexpressing cell lines produced by drug selection in vitro. They further demonstrate that the multidrug resistance phenotype conferred by MRP is similar but not identical to that conferred by P-glycoprotein and includes resistance to arsenical and antimonial oxyanions.