Marginal proportional hazards models for clustered interval-censored data with time-dependent covariates

The Botswana Combination Prevention Project was a cluster-randomized HIV prevention trial whose follow-up period coincided with Botswana's national adoption of a universal test and treat strategy for HIV management. Of interest is whether, and to what extent, this change in policy modified the preventative effects of the study intervention. To address such questions, we adopt a stratified proportional hazards model for clustered interval-censored data with time-dependent covariates and develop a composite expectation maximization algorithm that facilitates estimation of model parameters without placing parametric assumptions on either the baseline hazard functions or the within-cluster dependence structure. We show that the resulting estimators for the regression parameters are consistent and asymptotically normal. We also propose and provide theoretical justification for the use of the profile composite likelihood function to construct a robust sandwich estimator for the variance. We characterize the finite-sample performance and robustness of these estimators through extensive simulation studies. Finally, we conclude by applying this stratified proportional hazards model to a re-analysis of the Botswana Combination Prevention Project, with the national adoption of a universal test and treat strategy now modeled as a time-dependent covariate.     

Optimization of peptidic HIV‐1 fusion inhibitor T20 by phage display

The HIV fusion inhibitor T20 has been approved to treat those living with HIV/AIDS, but treatment gives rise to resistant viruses. Using combinatorial phage‐displayed libraries, we applied a saturation scan approach to dissect the entire T20 sequence for binding to a prefusogenic five‐helix bundle (5HB) mimetic of HIV‐1 gp41. Our data set compares all possible amino acid substitutions at all positions, and affords a complete view of the complex molecular interactions governing the binding of T20 to 5HB. The scan of T20 revealed that 12 of its 36 positions were conserved for 5HB binding, which cluster into three epitopes: hydrophobic epitopes at the ends and a central dyad of hydrophilic residues. The scan also revealed that the T20 sequence was highly adaptable to mutations at most positions, demonstrating a striking structural plasticity that allows multiple amino acid substitutions at contact points to adapt to conformational changes, and also at noncontact points to fine‐tune the interface. Based on the scan result and structural knowledge of the gp41 fusion intermediate, a library was designed with tailored diversity at particular positions of T20 and was used to derive a variant (T20v1) that was found to be a highly effective inhibitor of infection by multiple HIV‐1 variants, including a common T20‐escape mutant. These findings show that the plasticity of the T20 functional sequence space can be exploited to develop variants that overcome resistance of HIV‐1 variants to T20 itself, and demonstrate the utility of saturation scanning for rapid epitope mapping and protein engineering.     

Potential feedback between coral presence and farmerfish collective behavior promotes coral recovery

Stable between‐group differences in collective behavior have been documented in a variety of social taxa. Here we evaluate the effects of such variation, often termed collective or colony‐level personality, on coral recovery in a tropical marine farmerfish system. Groups of the farmerfish Stegastes nigricans cultivate and defend gardens of palatable algae on coral reefs in the Indo‐Pacific. These gardens can promote the recruitment, growth, and survival of corals by providing a refuge from coral predation. Here we experimentally evaluate whether the collective response of farmerfish colonies is correlated across intruder feeding guilds – herbivores, corallivores and egg‐eating predators. Further, we evaluate if overall colony responsiveness or situation‐specific responsiveness (i.e. towards herbivores, corallivores, or egg‐eaters in particular) best predicts the growth of outplanted corals. Finally, we experimentally manipulated communities within S. nigricans gardens, adding either macroalgae or large colonies of coral, to assess if farmerfish behavior changes in response to the communities they occupy. Between‐group differences in collective responsiveness were repeatable across intruder guilds. Despite this consistency, responsiveness towards corallivores (porcupinefish and ornate butterflyfish) was a better predictor of outplanted coral growth than responsiveness towards herbivores or egg‐eaters. Adding large corals to farmerfish gardens increased farmerfish attacks towards intruders, pointing to possible positive feedback loops between their aggression towards intruders and the presence of corals whose growth they facilitate. These data provide evidence that among‐group behavioral variation could strongly influence the ecological properties of whole communities.     

Generation of Fbn1 conditional null mice implicates the extracellular microfibrils in osteoprogenitor recruitment

Loss-of-function experiments in mice have yielded invaluable mechanistic insights into the pathogenesis of Marfan syndrome (MFS) and implicitly, into the multiple roles fibrillin-1 microfibrils play in the developing and adult organism. Unfortunately, neonatal death from aortic complications of mice lacking fibrillin-1 (Fbn1(-/-) mice) has limited the scope of these studies. Here, we report the creation of a conditional mutant allele (Fbn1(fneo) ) that contains loxP sites bordering exon1 of Fbn1 and an frt-flanked neo expression cassette downstream of it. Fbn1(fneo/+) mice were crossed with FLPeR mice and the resulting Fbn1(Lox/+) progeny were crossed with Fbn1(+/-) ;CMV-Cre mice to generate Fbn1(CMV-/-) mice, which were found to phenocopy the vascular abnormalities of Fbn1(-/-) mice. Furthermore, mating Fbn1(Lox/+) mice with Prx1-Cre or Osx-Cre mice revealed an unappreciated role of fibrillin-1 microfibrils in restricting osteoprogenitor cell recruitment. Fbn1(Lox/+) mice are, therefore, an informative genetic resource to further dissect MFS pathogenesis and the role of extracellular fibrillin-1 assemblies in organ development and homeostasis.     

Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms

Background and Aims

Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns.

Methods

Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden.

Key Results

The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden.

Conclusions

Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchids.Key words: Biogeography, Cypripedium calceolus, genome size, plastid microsatellites, population genetics, seed dispersal     

The low-affinity phosphate transporter PitA is dispensable for in vitro growth of Mycobacterium smegmatis

Background  

Mycobacteria have been shown to contain an apparent redundancy of high-affinity phosphate uptake systems, with two to four copies of such systems encoded in all mycobacterial genomes sequenced to date. In addition, all mycobacteria also contain at least one gene encoding the low-affinity phosphate transporter, Pit. No information is available on a Pit system from a Gram-positive microorganism, and the importance of this system in a background of multiple other phosphate transporters is unclear.     

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule–cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.     

Local filters limit species diversity,but species pools determine composition

In a series of three experiments, we tested for effects of species pools, resource stress, and species interactions on four aspects of community structure: species richness, evenness, species composition, and functional group composition. We also examined whether the impacts of species interactions on the community varied with resource availability or species pool. Communities of sand dune annuals grew from seed bank samples collected from two sites in three different years, so that the species pool differed at two levels: the source site and the year of seed bank collection. Communities experienced one of three irrigation treatments and a range of sowing densities, which varied resource supply (stress) and the potential for species interactions, respectively.Species richness and evenness were most affected by local factors: higher densities and lower water availability decreased species diversity. In contrast, species composition was influenced most by the species pool. Functional group composition had an intermediate response, and was affected by both species pools and local filters.Resource stress and species interactions strongly filtered species from the community, but the identity of species remaining was variable. Furthermore, the magnitude of species interaction effects on richness and evenness varied with species pools. Thus, the outcome of species sorting among biotic and abiotic environments was dependent on the pool of available species. Contrary to predictions from theory, the effects of species interactions on the community did not vary consistently with resource levels.     

The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects.     

Role of tumor necrosis factor-alpha in E1A oncogene-induced susceptibility of neoplastic cells to lysis by natural killer cells and activated macrophages

NIH-3T3 cells transfected with adenovirus E1A oncogene cDNA were found to exhibit cytolytic susceptibility to murine NK cells and activated macrophages associated with a threshold level of oncogene product expression exceeding that required for morphological transformation. A similar correlation was observed between threshold levels of E1A gene product expression and target cell susceptibility to direct cytotoxicity by rTNF. Inhibition of splenic NK cell and peritoneal macrophage cytolysis by antisera specific for murine rTNF confirmed the importance of E1A-induced TNF susceptibility as one determinant of target cell cytolytic susceptibility. Anti-TNF antibody was, however, unable to block killing of E1A-expressing targets by the NK cell line, NKB61A2. These results suggest a direct link between the functions of E1A oncogene products and cellular mechanisms of action of TNF elaborated by host effector cells and indicate that E1A expression also affects target cell susceptibility to TNF-independent cytolytic mechanisms.