Improving genome assemblies by sequencing PCR products with PacBio

Advances in sequencing technologies have dramatically reduced costs in producing high-quality draft genomes. However, there are still many contigs and possible misassembled regions in those draft genomes. Improving the quality of these genomes requires an efficient and economical means to close gaps and resequence some regions. Sequencing pooled gap region PCR products with Pacific Biosciences (PacBio) provides a significantly less expensive means for this need. We have developed a genome improvement pipeline with this strategy after decreasing a loading bias against larger PCR products in the PacBio process. Compared with Sanger technology, this approach is not only cost-effective but also can close gaps greater than 2.5 kb in a single round of reactions, and sequence through high GC regions as well as difficult secondary structures such as small hairpin loops.     

Estimating the infectivity of CCR5-tropic simian immunodeficiency virus SIV(mac251) in the gut

CD4+ T-cell depletion during acute human immunodeficiency virus infection occurs predominantly in the gastrointestinal mucosa. Using experimental data on SIV(mac251) viral load in blood and CD4+ T cells in the jejunum, we modeled the kinetics of CD4+ T-cell infection and death and estimated the viral infectivity. The infectivity of SIV(mac251) is higher than previously estimated for SHIV89.6P infection, but this higher infectivity is offset by a lower average peak viral load in SIV(mac251). Thus, the dynamics of target cell infection and death are remarkably similar between a CXCR4- and a CCR5-tropic infection in vivo.     

Independent roles of Rho-GTPases in growth cone and axonal behavior

Many external signals influence growth cone motility, pathfinding, and the formation of synapses that lead to the final map formation of the retinotectal system. Chick temporal retinal ganglion cell axons (RGCs) collapse and retract after encountering posterior tectal cells in vitro. During this process lateral extensions appear along the RGC axonal shaft. Lateral extensions appear as nascent interstitial axonal branches and also as defasciculating growth cones that are trailing along the pioneer axon. RGC branching controlled by repellent tectal cues has recently been shown to be the critical event in retinotectal map development. The intracellular mechanism underlying this phenomenon, however, is not understood. Inhibiting RhoA with either C3 toxin or inhibiting p160Rock kinase, an effector of RhoA, with Y27632 inhibited collapse, retraction, and the number of axons that showed lateral extensions. Lateral extension length increased significantly. Inhibiting Rac1A and cdc42 with cell permeable peptide inhibitors did not inhibit collapse of growth cones, but did inhibit axon retraction. In addition, the number of axons that showed lateral extensions and lateral extension length were significantly reduced. A dynamic cytoskeleton is necessary to react to incoming guidance information. This study addresses the problems of how growth cone motility and branching or defasciculation are affected by Rho-GTPases as extracellular signals are transmitted to the cytoskeleton.     

The mitotic checkpoint gene BubR1 has two distinct functions in mitosis

BubR1 is one of two putative vertebrate homologs of the yeast spindle checkpoint protein Bub1. We have used deletion and point mutants to elucidate the functions of BubR1 in mitosis. The nocodazole-activated spindle checkpoint of HeLa cells was disrupted by expression of a 39 amino acid fragment (residues 382-420) of BubR1 containing the Bub3-binding GLEBS motif. In contrast, we observed normal checkpoint function in a truncation mutant comprising residues 1-477, despite the lack of the C-terminal BubR1 kinase domain. In the absence of nocodazole, expression of the 477 amino acid fragment slowed progress through prometaphase of mitosis, causing accumulation of mitotic cells. This accumulation was also seen in a kinase dead mutant. The prolongation of mitosis required both kinetochore binding and an intact, functional spindle checkpoint. The prolongation of mitosis by kinase deficient BubR1 constructs indicates a crucial role for the BubR1 C-terminal kinase domain in chromosome movement, in addition to the role of the N-terminus in the checkpoint.     

Effects of antibody on viral kinetics in simian/human immunodeficiency virus infection: implications for vaccination

Passive antibody treatment of macaques prior to simian/human immunodeficiency virus infection produces "sterilizing immunity" in some animals and long-term reductions in viral loads in others. Analysis of viral kinetics suggests that antibody mediates sterilizing immunity by its effects on the initial viral inoculum. By contrast, reduction in peak viral load later in infection prevents CD4 depletion and contributes to long-term viral control.     

Trabecular architecture and joint loading of the proximal humerus in extant hominoids,Ateles, and Australopithecus africanus

Objectives

Several studies have investigated potential functional signals in the trabecular structure of the primate proximal humerus but with varied success. Here, we apply for the first time a “whole‐epiphyses” approach to analysing trabecular bone in the humeral head with the aim of providing a more holistic interpretation of trabecular variation in relation to habitual locomotor or manipulative behaviors in several extant primates and Australopithecus africanus.

Materials and methods

We use a “whole‐epiphysis” methodology in comparison to the traditional volume of interest (VOI) approach to investigate variation in trabecular structure and joint loading in the proximal humerus of extant hominoids, Ateles and A. africanus (StW 328).

Results

There are important differences in the quantification of trabecular parameters using a “whole‐epiphysis” versus a VOI‐based approach. Variation in trabecular structure across knuckle‐walking African apes, suspensory taxa, and modern humans was generally consistent with predictions of load magnitude and inferred joint posture during habitual behaviors. Higher relative trabecular bone volume and more isotropic trabeculae in StW 328 suggest A. africanus may have still used its forelimbs for arboreal locomotion.

Discussion

A whole‐epiphysis approach to analysing trabecular structure of the proximal humerus can help distinguish functional signals of joint loading across extant primates and can provide novel insight into habitual behaviors of fossil hominins.

     

Predicting the impact of a nonsterilizing vaccine against human immunodeficiency virus

Studies of human immunodeficiency virus (HIV) vaccines in animal models suggest that it is difficult to induce complete protection from infection (sterilizing immunity) but that it is possible to reduce the viral load and to slow or prevent disease progression following infection. We have developed an age-structured epidemiological model of the effects of a disease-modifying HIV vaccine that incorporates the intrahost dynamics of infection, a transmission rate and host mortality that depend on the viral load, the possible evolution and transmission of vaccine escape mutant viruses, a finite duration of vaccine protection, and possible changes in sexual behavior. Using this model, we investigated the long-term outcome of a disease-modifying vaccine and utilized uncertainty analysis to quantify the effects of our lack of precise knowledge of various parameters. Our results suggest that the extent of viral load reduction in vaccinated infected individuals (compared to unvaccinated individuals) is the key predictor of vaccine efficacy. Reductions in viral load of about 1 log(10) copies ml(-1) would be sufficient to significantly reduce HIV-associated mortality in the first 20 years after the introduction of vaccination. Changes in sexual risk behavior also had a strong impact on the epidemic outcome. The impact of vaccination is dependent on the population in which it is used, with disease-modifying vaccines predicted to have the most impact in areas of low prevalence and rapid epidemic growth. Surprisingly, the extent to which vaccination alters disease progression, the rate of generation of escape mutants, and the transmission of escape mutants are predicted to have only a weak impact on the epidemic outcome over the first 25 years after the introduction of a vaccine.     

Exploring human interaction and diet effects on the behavior of dogs in a public animal shelter

This study examined the effects of 2 manipulations-a brief, regular period of human contact and diet-on the behavior of dogs confined in a public animal shelter. A behavioral battery designed to assess reactions to novel situations, and a test of responsiveness to an unfamiliar human were administered both prior to (pretest) and immediately following (posttest) the 8-week intervention period. Overall, the regular periods of increased human contact together with a diet that contained augmented levels of digestible protein, fat, calories, and animal-derived ingredients reduced signs of behavioral reactivity from pretest to posttest. In some cases, the comparison diet appeared more effective, but only for dogs receiving minimal human interaction. The results indicate that a combination of human interaction and high quality diet may positively affect the behavior of dogs in animal shelters.     

Amphibious fish: why do they leave water?

Summary Amphibious behaviour in fish has evolved separately many times since the first amphibious fishes, the rhipidistian crossopterygians, ventured onto land about 350 million years ago. This behaviour has resulted in the colonization and eventual domination by vertebrates of the terrestrial habitat. It is generally proposed that aquatic hypoxia, owing to metabolic oxygen consumption and organic decay, was the most important selective force in the evolution of air-breathing vertebrates (e.g. Randall et al., 1981). Modern amphibious fish species give an insight into the reasons for leaving and eventually abandoning the aquatic habitat. Amphibious fishes today leave the water for a variety of reasons associated with degradation of their aquatic habitat, or biotic factors within it.The possible causal factors which may elicit an emergence response are summarized in Fig. 1(a) and (b). Amphibious fish inhabiting closed systems, as typified by freshwater or intertidal pools, may leave water for any of the reasons detailed in Fig. 1(a). The relative importance of any one stimulus is likely to vary between different species. However, it is possible that in closed systems, adverse fluctuations in physico-chemical parameters will have a more important effect in eliciting amphibious behaviour than will biotic factors. In open systems, such as coastal waters or large freshwater bodies, effectively two routes of escape from adverse aquatic conditions are available to amphibious fish. They may move onto land, or alternatively they may move underwater to find better conditions. In such a system, where physico-chemical parameters remain relatively constant, abiotic factors are unlikely to have a significant influence on amphibious behaviour. The dominant stimulus in open systems is possibly the three-way interaction between predation, competition, and short-or long-term food availability (Fig. 1(b)).It is unlikely that any one of the factors discussed in this review will act alone in causing amphibious behaviour, and in this respect the available literature on fish leaving water is lacking. Much of it is fragmentary and partly anecdotal, and the limited amount of experimental work tends to concentrate on individual causal factors. There is evidently scope for detailed examination of emersion in a number of amphibious fishes, testing a matrix of environmental and biotic stimuli, in an attempt to determine in more detail the reasons for such behaviour.