Probabilistic cross-species inference of orthologous genomic regions created by whole-genome duplication in yeast

Identification of orthologous genes across species becomes challenging in the presence of a whole-genome duplication (WGD). We present a probabilistic method for identifying orthologs that considers all possible orthology/paralogy assignments for a set of genomes with a shared WGD (here five yeast species). This approach allows us to estimate how confident we can be in the orthology assignments in each genomic region. Two inferences produced by this model are indicative of purifying selection acting to prevent duplicate gene loss. First, our model suggests that there are significant differences (up to a factor of seven) in duplicate gene half-life. Second, we observe differences between the genes that the model infers to have been lost soon after WGD and those lost more recently. Gene losses soon after WGD appear uncorrelated with gene expression level and knockout fitness defect. However, later losses are biased toward genes whose paralogs have high expression and large knockout fitness defects, as well as showing biases toward certain functional groups such as ribosomal proteins. We suggest that while duplicate copies of some genes may be lost neutrally after WGD, another set of genes may be initially preserved in duplicate by natural selection for reasons including dosage.     

Determinants for the subcellular localization and function of a nonessential SEDS protein

The Bacillus subtilis SpoVE integral membrane protein is essential for the heat resistance of spores, probably because of its involvement in spore peptidoglycan synthesis. We found that an SpoVE-yellow fluorescent protein (YFP) fusion protein becomes localized to the forespore during the earliest stages of engulfment, and this pattern is maintained throughout sporulation. SpoVE belongs to a well-conserved family of proteins that includes the FtsW and RodA proteins of B. subtilis. These proteins are involved in bacterial shape determination, although their function is not known. FtsW is necessary for the formation of the asymmetric septum in sporulation, and we found that an FtsW-YFP fusion localized to this structure prior to the initiation of engulfment in a nonoverlapping pattern with SpoVE-cyan fluorescent protein. Since FtsW and RodA are essential for normal growth, it has not been possible to identify loss-of-function mutations that would greatly facilitate analysis of their function. We took advantage of the fact that SpoVE is not required for growth to obtain point mutations in SpoVE that block the development of spore heat resistance but that allow normal protein expression and targeting to the forespore. These mutant proteins will be invaluable tools for future experiments aimed at elucidating the function of members of the SEDS (“shape, elongation, division, and sporulation”) family of proteins.     

Molecular chaperones and the assembly of the prion Ure2p in vitro

The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94-354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3].     

Differential signaling networks induced by mild and lethal hemorrhagic fever virus infections

The family Arenaviridae includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants of Pichinde virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified key intermediates which may be targets for therapeutic intervention.     

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.     

QT interval variability and cardiac norepinephrine spillover in patients with depression and panic disorder

Suggestions were made that increased myocardial sympathetic activity is reflected by elevated QT variability (dynamic changes in QT interval duration). However, the relationship between QT variability and the amount of norepinephrine released from the cardiac sympathetic terminals is unknown. We thus attempted to assess this relationship. The study was performed in 17 subjects (12 with major depressive disorder and 5 with panic disorder). Cardiac norepinephrine spillover (measured by direct catheter technique coupled with norepinephrine isotope dilution methodology) was assessed before and 4 mo after treatment with selective serotonin reuptake inhibitor (SSRI) antidepressants. The distribution of the cardiac norepinephrine spillover was bimodal, with the majority of patients having values of < or =10 ng/min. There was a positive correlation between cardiac norepinephrine spillover and corrected QT interval (r = 0.7, P = 0.03) but not with any of the QT variability measures. However, in a subgroup of five patients who had high levels of cardiac norepinephrine spillover (>20 ng/min) a tendency for a strong positive correlation with variance of QT intervals (r = 0.9, P = 0.08) was observed. There were significant correlations between the severity of depression and QT variability indexes normalized to the heart rate [QTVi and QT interval/R-R interval (QT/RR) coherence] and between the severity of anxiety and the QT/RR residual and regression coefficient, respectively. Treatment with SSRI antidepressants substantially reduced depression score but did not affect any of the QT variability indexes. We conclude that in depression/panic disorder patients with near-normal cardiac norepinephrine levels QT variability is not correlated with cardiac norepinephrine spillover and is not affected by treatment with SSRI.     

Antibody targeting of camptothecin-loaded PLGA nanoparticles to tumor cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immuno-nanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 microg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC 50 of 21.8 ng mL (-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC 50 of 0.37 ng mL (-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.     

Phylogeny of the basal angiosperm genus Pseuduvaria (Annonaceae) inferred from five chloroplast DNA regions, with interpretation of morphological character evolution

Phylogenetic relationships within the magnoliid basal angiosperm genus Pseuduvaria (Annonaceae) are investigated using chloroplast DNA sequences from five regions: psbA-trnH spacer, trnL-F, matK, rbcL, and atpB-rbcL spacer. Over 4000 nucleotides from 51 species (of the total 53) were sequenced. The five cpDNA datasets were analyzed separately and in combination using maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. The phylogenetic trees constructed using all three phylogenetic methods, based on the combined data, strongly support the monophyly of Pseuduvaria following the inclusion of Craibella phuyensis. The trees generated using MP were less well resolved, but relationships are similar to those obtained using the other methods. ML and Bayesian analyses recovered trees with short branch lengths, showing five main clades. This study highlights the evolutionary changes in seven selected morphological characters (floral sex, stamen and carpel numbers, inner petal color, presence of inner petal glands, flowering peduncle length, and monocarp size). Although floral unisexuality is ancestral within the genus, several evolutionary lineages reveal reversal to bisexuality. Other phylogenetic transitions include the evolution of sapromyophily, and fruit-bat frugivory and seed dispersal, thus allowing a wide range of adaptations for species survival.