Trans-centromere effects on meiotic recombination in the zebrafish

We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm's centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.     

S6 kinase localizes to the presynaptic active zone and functions with PDK1 to control synapse development

The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. In this paper, we demonstrate that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. We then provide evidence that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. We further demonstrate that PDK1 is also a presynaptic protein, though it is distributed more broadly. We present a model in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release.     

The cream dilution gene, responsible for the palomino and buckskin coat colours, maps to horse chromosome 21

The colour locus historically referred to as C in the horse is linked to microsatellites markers on horse chromosome 21. Preliminary results demonstrated linkage of Ccr, thought to be the cream dilution variant of the C locus, to HTG10. An analysis of horse chromosome 21 using additional families confirmed and established a group of markers linked to Ccr. This work also improved the resolution of previously reported linkage maps for this chromosome. Linkage analysis unambiguously produced the map order: SGCV16-(19.1 cM)-HTG10-(3.8 cM)-LEX60/COR73-(1.3 cM)-COR68-(4.5 cM)- Ccr-(11.9 cM)-LEX31. Comparative and synteny data suggested that the horse C locus is not tyrosinase (TYR).     

Effectiveness of Spayvac for reducing white-tailed deer fertility

Overabundant white-tailed deer (Odocoileus virginianus) populations have been reported in many urban and suburban communities across the United States. Large populations of deer can potentially increase the risk of human-wildlife conflicts, such as deer-vehicle collisions, transmission of disease to humans, and vegetation damage. In 2003, efforts to control white-tailed deer numbers were initiated at the National Aeronautical and Space Agency's (NASA) Lyndon B. Johnson Space Center (JSC) in Houston, Texas, using the long-lasting, single-dose contraceptive SpayVac. Our objectives were to evaluate the effectiveness of SpayVac for reducing white-tailed deer fertility and determine the partial cost for treatment. Between 2003 and 2004, we monitored 45 adult female deer (34 treated with SpayVac, 11 controls treated with a placebo). Fawning rate over 2 yr for deer treated with SpayVac >30 days prior to the rut was 0% (n=31), whereas the fawning rate for control deer was 78% (n=11). Inoculation 1 mo prior to the breeding season was sufficient time to achieve fertility control. We conclude that SpayVac can effectively reduce the fertility of urban white-tailed deer.     

A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling

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A Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst

There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R² = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R² = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas.     

Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.     

Mosquito Feeding Assays to Determine the Infectiousness of Naturally Infected Plasmodium falciparum Gametocyte Carriers

Introduction

In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes.

Methods

We compared two commonly used mosquito feeding assay procedures: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum.

Results

930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94–2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68–2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52–0.70).

Conclusions

Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.     

Altered patterns of multiple recombinant events are associated with nondisjunction of chromosome 21

We have previously examined characteristics of maternal chromosomes 21 that exhibited a single recombination on 21q and proposed that certain recombination configurations are risk factors for either meiosis I (MI) or meiosis II (MII) nondisjunction. The primary goal of this analysis was to examine characteristics of maternal chromosomes 21 that exhibited multiple recombinant events on 21q to determine whether additional risk factors or mechanisms are suggested. In order to identify the origin (maternal or paternal) and stage (MI or MII) of the meiotic errors, as well as placement of recombination, we genotyped over 1,500 SNPs on 21q. Our analyses included 785 maternal MI errors, 87 of which exhibited two recombinations on 21q, and 283 maternal MII errors, 81 of which exhibited two recombinations on 21q. Among MI cases, the average location of the distal recombination was proximal to that of normally segregating chromosomes 21 (35.28 vs. 38.86 Mb), a different pattern than that seen for single events and one that suggests an association with genomic features. For MII errors, the most proximal recombination was closer to the centromere than that on normally segregating chromosomes 21 and this proximity was associated with increasing maternal age. This pattern is same as that seen among MII errors that exhibit only one recombination. These findings are important as they help us better understand mechanisms that may underlie both age-related and nonage-related meiotic chromosome mal-segregation.     

Facilitated diffusion of myoglobin and creatine kinase and reaction-diffusion constraints of aerobic metabolism under steady-state conditions in skeletal muscle

The roles of creatine kinase (CK) and myoglobin (Mb) on steady-state facilitated diffusion and temporal buffering of ATP and oxygen, respectively, are assessed within the context of a reaction-diffusion model of muscle energetics. Comparison of the reaction-diffusion model with experimental data from a wide range of muscle fibers shows that the experimentally observed skeletal muscle fibers are generally not limited by diffusion, and the model further indicates that while some muscle fibers operate near the edge of diffusion limitation, no detectable effects of Mb and CK on the effectiveness factor, a measure of diffusion constraints, are observed under steady-state conditions. However, CK had a significant effect on average ATP concentration over a wide range of rates and length scales within the reaction limited regime. The facilitated diffusion functions of Mb and CK become observable in the model for larger size cells with low mitochondrial volume fraction and for low boundary O(2) concentration and high ATP demand, where the fibers may be limited by diffusion. From the transient analysis it may be concluded that CK primarily functions to temporally buffer ATP as opposed to facilitating diffusion while Mb has a small temporal buffering effect on oxygen but does not play any significant role in steady-state facilitated diffusion in skeletal muscle fibers under most physiologically relevant regions.