Simple and efficient CRISPR/Cas9‐mediated targeted mutagenesis in Xenopus tropicalis

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers. genesis 51:835–843. © 2013 Wiley Periodicals, Inc.     

Partial Deletion of Chromosome 8 β-defensin Cluster Confers Sperm Dysfunction and Infertility in Male Mice

β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.     

The Effect of Factor VIII Deficiencies and Replacement and Bypass Therapies on Thrombus Formation under Venous Flow Conditions in Microfluidic and Computational Models

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5–30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1–5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.     

Maternal weight affects placental DNA methylation of genes involved in metabolic pathways in the common marmoset monkey (Callithrix jacchus)

Accumulating evidence suggests that dysregulation of placental DNA methylation (DNAm) is a mechanism linking maternal weight during pregnancy to metabolic programming outcomes. The common marmoset, Callithrix jaccus, is a platyrrhine primate species that has provided much insight into studies of the primate placenta, maternal condition, and metabolic programming, yet the relationships between maternal weight and placental DNAm are unknown. Here, we report genome-wide DNAm from term marmoset placentas using reduced representation bisulfite sequencing. We identified 74 genes whose DNAm pattern is associated with maternal weight during gestation. These genes are predominantly involved in energy metabolism and homeostasis, including the regulation of glycolytic and lipid metabolic processes pathways.     

Linear skin markings in common bottlenose dolphins (Tursiops truncatus) from the Indian River Lagoon,Florida

A previously undescribed skin abnormality, referred to as “linear skin markings” (LSM), has been identified in free-ranging common bottlenose dolphins (Tursiops truncatus) in the Indian River Lagoon, Florida (IRL). The lesions were identified during photo-identification surveys conducted from 2002 and 2015. LSM presented as distinct, parallel lines running dorso-ventrally on the torso and varied in length and width. The goals of this study were to determine (1) prevalence of the condition in IRL dolphins, (2) age and sex distribution of affected animals, (3) spatial and temporal distribution patterns, (4) duration of the condition, and (5) development of hypotheses regarding the etiology of the condition. Among 1,357 individual dolphins identified during the study period, 96 (7.0%) showed evidence of LSM. Nearly all (98.8%) cases with an established home range occurred in the northern and central regions of the IRL. The majority of cases of known sex were female (85%), of which 100% had given birth to one or more calves. The mean age of animals with LSM when first observed was 7.3 with a range of 1–20 years. The maximum observed duration of LSM was 15 years. Once observed, the condition persisted indefinitely. The etiology of LSM has not been established.     

A Prospective Study of Seasonal Variation in Shift‐Work Tolerance

Seasonal effects on shift‐work tolerance were assessed using the Standardized Shiftwork Index and the 21‐item Hamilton Depression Scale. Participants (N=88) mainly worked a two‐day, two‐night, four‐off rotation with 12 h shifts changing at 06∶00 and 18∶00 h in Vancouver, Canada. At this latitude (～49° N), daylength varies seasonally from ～16 to ～8 h, and both daily commutes occur in the dark in mid‐winter and in sunlight in mid‐summer. Questionnaires were completed twice, near the summer and winter solstices (order counterbalanced). Outcome variables were mood, general psychological health, sleep quality, chronic fatigue, physical health, job satisfaction, and social and domestic disruption. Of these, general psychological health and mood were significantly worse in winter, while sleep was more disturbed in summer. In winter, 31% exceeded the cutoff for psychological distress, and >70% scored in the higher than normal range for depressive symptoms. In summer, the proportions dropped to 19% and 53%, respectively. Measures of physical health and psychosocial well‐being showed no seasonal effects. Relationships among explanatory and outcome variables, assessed by linear regression and canonical correlations, were also stable across season. Neuroticism was the strongest predictor of tolerance to shift work. Age was predictive only of sleep disturbance in both summer and winter. These results indicate that time of year can affect important outcome measures in shift‐work assessment and intervention studies. The high average scores on measures of psychological distress and depression in winter suggest that at northern latitudes, some shift schedules may increase the risk of seasonal‐type depression.     

Body mass growth in common marmosets: Toward a model of pediatric obesity

While much is known about adult obesity in nonhuman primates, very little is known regarding development of childhood adiposity. As small monkeys that are easy to handle and have a relatively fast life history, common marmoset monkeys (Callithrix jacchus) offer interesting opportunities to examine the question of fat versus lean mass growth in a nonhuman primate. This article provides an overview of our understanding of early life growth in mass in marmoset monkeys, based primarily upon our past 20 years of research, culminating in our recent findings on early life obesity in this species. Common marmosets display variance in early life growth patterns that is related to both pre‐ and postnatal factors and the marmoset uterine environment is exquisitely designed to reflect resources available for the gestation of multiple offspring, making them an interesting model of developmental programming. We have demonstrated that obesity can be generated in very early life in captive marmosets, with excess adiposity evident by one month of age, making this species a potentially valuable model in which to study pediatric obesity and its sequelae. Birth weight is associated with adiposity in animals vulnerable to obesity. Early life exposure to higher fat diets enhances the chances of postweaning obesity development. However, overall higher food consumption is also associated with obesity development at later ages. One unexpected finding in our studies has been the relatively high body fat percentage of neonatal (12–18%) marmosets suggesting that hypotheses regarding the uniqueness of high human neonatal adiposity merit further examination. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.