Contrast-FEL—A Test for Differences in Selective Pressures at Individual Sites among Clades and Sets of Branches

A number of evolutionary hypotheses can be tested by comparing selective pressures among sets of branches in a phylogenetic tree. When the question of interest is to identify specific sites within genes that may be evolving differently, a common approach is to perform separate analyses on subsets of sequences and compare parameter estimates in a post hoc fashion. This approach is statistically suboptimal and not always applicable. Here, we develop a simple extension of a popular fixed effects likelihood method in the context of codon-based evolutionary phylogenetic maximum likelihood testing, Contrast-FEL. It is suitable for identifying individual alignment sites where any among the K≥2 sets of branches in a phylogenetic tree have detectably different ω ratios, indicative of different selective regimes. Using extensive simulations, we show that Contrast-FEL delivers good power, exceeding 90% for sufficiently large differences, while maintaining tight control over false positive rates, when the model is correctly specified. We conclude by applying Contrast-FEL to data from five previously published studies spanning a diverse range of organisms and focusing on different evolutionary questions.     

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase,UCH-L1

We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.     

Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein

Based on sequence information from tryptic peptides an almost full-size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced. The coding region was cloned into an Escherichia coli expression vector and the protein expressed at high levels. The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays.     

Effects of irradiation level on leaf growth of sunflower

Sunflower, Helianthus annuus L. cv. INRA 6501, plants were grown in a gravel culture subirrigated with Hoagland nutrient solution, at photosynthetically active radiation levels of 15, 30 and 60 W m^-2 at a daylength of 16 h, a temperature of 20°C and a relative humidity of 60% throughout. Development of the plant and growth of the leaves were measured. High irradiance accelerated development proportionally in all phases from germination, through leaf initiation, primordial flower formation and the maturation of all plant organs until anthesis. High irradiance levels stimulated the expansion of the growing shoot, which produced more and larger primordia. Under constant conditions the ratio between leaf initiation rate and mature length of a leaf remained constant, although the growth patterns [relationship between relative growth rate (RGR) and organ age] of successive leaves were not similar. Consequently, it may be assumed that, as in poplar, the increasing size of the growing shoot reflects the increase of the vascular system of sunflower. The growth patterns of the leaves depend on the developmental stage of the plant and, in the young primordial stage, also on irradiance level. In the linear phase of growth the growth pattern is independent of irradiance level.     

Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

RS46(DXS548) genotyping of reproductive cells: approaching preimplantation testing of the fragile-X syndrome

In order to approach preimplantation testing for the fragile-X syndrome, we used genotyping of the polymorphic RS46(DXS548) locus closely linked to the FMR1 gene, in single reproductive cells of females. The RS46(DXS548) amplification was adjusted to the single cell level by a two-round polymerase chain reaction (PCR) procedure. Unfertilized oocytes and extruded polar bodies were subjected to PCR. RS46(DXS548) genotyping at the single cell level was successful in 95% of the samples. In two-third of the metaphase II oocytes and first polar bodies obtained from women who were heterozygous at the RS46(DXS548) locus, both maternal RS46(DXS548) alleles were observed because of crossing over during the first meiotic division. This makes gamete selection by first polar body analysis inefficient. From the allele frequencies found in 56 unrelated individuals, a heterozygote frequency of 51% was estimated, whereas the observed heterozygote frequency was 56%. The whole PCR procedure can be performed within 16 h after blastomere biopsy. Consequently, the selection and transfer of the diagnosed embryos can be carried out within an acceptable time. Therefore, preimplantation testing for the fragile-X syndrome with the RS46(DXS548) AC-repeat may be an alternative choice for prenatal testing for those carrier females who are heterozygous (informative) at the RS46(DXS548) locus.     

Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain

The intracellular transport and location of major histocompatibility complex (MHC) class II molecules and associated invariant chain (Ii) were investigated in a human melanoma cell line. In contrast to the class II molecules, which remain stable for greater than 4 h after synthesis, the associated Ii is proteolytically processed within 2 h. During or shortly after synthesis the NH2-terminal cytoplasmic and membrane-spanning segment is in some of the Ii molecules cleaved off; during intracellular transport, class II associated and membrane integrated Ii is processed from its COOH terminus in distinct steps in endocytic compartments. Immunocytochemical studies at the light and electron microscopic level revealed the presence of class II molecules, but not of Ii on the cell surface. Intracellularly both Ii and class II molecules were localized in three morphologically and kinetically distinct compartments, early endosomes, multivesicular bodies, and prelysosomes. This localization in several distinct endosomal compartments contrasts with the localization of class II molecules in mainly one endocytic compartment in B lymphoblastoid cell lines. As in these lymphoblastoid cell lines Ii is known to be rapidly degraded it is conceivable that the rate of proteolysis of the class II associated Ii and its dissociation from class II molecules modulates the retention of the oligomeric complex in endocytic compartments, and as a consequence the steady-state distribution of these molecules within the endosomal system.     

Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.