The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel''s cartilage, nasal glands, tongue, and ear.     

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS)

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases the accuracy of current delivery to the brain by using arrays of smaller "high-definition" electrodes, instead of the larger pad-electrodes of conventional tDCS. Targeting is achieved by energizing electrodes placed in predetermined configurations. One of these is the 4x1-ring configuration. In this approach, a center ring electrode (anode or cathode) overlying the target cortical region is surrounded by four return electrodes, which help circumscribe the area of stimulation. Delivery of 4x1-ring HD-tDCS is capable of inducing significant neurophysiological and clinical effects in both healthy subjects and patients. Furthermore, its tolerability is supported by studies using intensities as high as 2.0 milliamperes for up to twenty minutes.Even though 4x1 HD-tDCS is simple to perform, correct electrode positioning is important in order to accurately stimulate target cortical regions and exert its neuromodulatory effects. The use of electrodes and hardware that have specifically been tested for HD-tDCS is critical for safety and tolerability. Given that most published studies on 4x1 HD-tDCS have targeted the primary motor cortex (M1), particularly for pain-related outcomes, the purpose of this article is to systematically describe its use for M1 stimulation, as well as the considerations to be taken for safe and effective stimulation. However, the methods outlined here can be adapted for other HD-tDCS configurations and cortical targets.     

Evaluating the Potential for the Environmentally Sustainable Control of Foot and Mouth Disease in Sub-Saharan Africa

Strategies to control transboundary diseases have in the past generated unintended negative consequences for both the environment and local human populations. Integrating perspectives from across disciplines, including livestock, veterinary and conservation sectors, is necessary for identifying disease control strategies that optimise environmental goods and services at the wildlife-livestock interface. Prompted by the recent development of a global strategy for the control and elimination of foot-and-mouth disease (FMD), this paper seeks insight into the consequences of, and rational options for potential FMD control measures in relation to environmental, conservation and human poverty considerations in Africa. We suggest a more environmentally nuanced process of FMD control that safe-guards the integrity of wild populations and the ecosystem dynamics on which human livelihoods depend while simultaneously improving socio-economic conditions of rural people. In particular, we outline five major issues that need to be considered: 1) improved understanding of the different FMD viral strains and how they circulate between domestic and wildlife populations; 2) an appreciation for the economic value of wildlife for many African countries whose presence might preclude the country from ever achieving an FMD-free status; 3) exploring ways in which livestock production can be improved without compromising wildlife such as implementing commodity-based trading schemes; 4) introducing a participatory approach involving local farmers and the national veterinary services in the control of FMD; and 5) finally the possibility that transfrontier conservation might offer new hope of integrating decision-making at the wildlife-livestock interface.     

Vitamin D and maternal and child health: Overview and implications for dietary requirements

The essentiality of vitamin D for normal growth and development has been recognized for over 80 years, and vitamin D fortification programs have been in place in the United States for more than 70 years. Despite the above, vitamin D deficiency continues to be a common finding in certain population groups. Vitamin D deficiency has been suggested as a potential risk factor for the development of preeclampsia, and vitamin D deficiency during infancy and early childhood is associated with an increased risk for numerous skeletal disorders, as well as immunological and vascular abnormalities. Vitamin D deficiency can occur through multiple mechanisms including the consumption of diets low in this vitamin and inadequate exposure to environmental ultraviolet B rays. The potential value of vitamin D supplementation in high‐risk pregnancies and during infancy and early childhood is discussed. Currently, there is vigorous debate concerning what constitutes appropriate vitamin D intakes during early development as exemplified by differing recommendations from the Institute of Medicine Dietary Reference Intake report and recent recommendations by the Endocrine Society. As is discussed, a major issue that needs to be resolved is what key biological endpoint should be used when making vitamin D recommendations for the pregnant woman and her offspring. Birth Defects Research (Part C) 99:24–44, 2013. © 2013 Wiley Periodicals, Inc.     

Relative Contribution of Th1 and Th17 Cells in Adaptive Immunity to Bordetella pertussis: Towards the Rational Design of an Improved Acellular Pertussis Vaccine

Whooping cough caused by Bordetella pertussis is a re-emerging infectious disease despite the introduction of safer acellular pertussis vaccines (Pa). One explanation for this is that Pa are less protective than the more reactogenic whole cell pertussis vaccines (Pw) that they replaced. Although Pa induce potent antibody responses, and protection has been found to be associated with high concentrations of circulating IgG against vaccine antigens, it has not been firmly established that host protection induced with this vaccine is mediated solely by humoral immunity. The aim of this study was to examine the relative contribution of Th1 and Th17 cells in host immunity to infection with B. pertussis and in immunity induced by immunization with Pw and Pa and to use this information to help rationally design a more effective Pa. Our findings demonstrate that Th1 and Th17 both function in protective immunity induced by infection with B. pertussis or immunization with Pw. In contrast, a current licensed Pa, administered with alum as the adjuvant, induced Th2 and Th17 cells, but weak Th1 responses. We found that IL-1 signalling played a central role in protective immunity induced with alum-adsorbed Pa and this was associated with the induction of Th17 cells. Pa generated strong antibody and Th2 responses, but was fully protective in IL-4-defective mice, suggesting that Th2 cells were dispensable. In contrast, Pa failed to confer protective immunity in IL-17A-defective mice. Bacterial clearance mediated by Pa-induced Th17 cells was associated with cell recruitment to the lungs after challenge. Finally, protective immunity induced by an experimental Pa could be enhanced by substituting alum with a TLR agonist that induces Th1 cells. Our findings demonstrate that alum promotes protective immunity through IL-1β-induced IL-17A production, but also reveal that optimum protection against B. pertussis requires induction of Th1, but not Th2 cells.     

Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.     

Low Frequency Variants,Collapsed Based on Biological Knowledge,Uncover Complexity of Population Stratification in 1000 Genomes Project Data

Analyses investigating low frequency variants have the potential for explaining additional genetic heritability of many complex human traits. However, the natural frequencies of rare variation between human populations strongly confound genetic analyses. We have applied a novel collapsing method to identify biological features with low frequency variant burden differences in thirteen populations sequenced by the 1000 Genomes Project. Our flexible collapsing tool utilizes expert biological knowledge from multiple publicly available database sources to direct feature selection. Variants were collapsed according to genetically driven features, such as evolutionary conserved regions, regulatory regions genes, and pathways. We have conducted an extensive comparison of low frequency variant burden differences (MAF<0.03) between populations from 1000 Genomes Project Phase I data. We found that on average 26.87% of gene bins, 35.47% of intergenic bins, 42.85% of pathway bins, 14.86% of ORegAnno regulatory bins, and 5.97% of evolutionary conserved regions show statistically significant differences in low frequency variant burden across populations from the 1000 Genomes Project. The proportion of bins with significant differences in low frequency burden depends on the ancestral similarity of the two populations compared and types of features tested. Even closely related populations had notable differences in low frequency burden, but fewer differences than populations from different continents. Furthermore, conserved or functionally relevant regions had fewer significant differences in low frequency burden than regions under less evolutionary constraint. This degree of low frequency variant differentiation across diverse populations and feature elements highlights the critical importance of considering population stratification in the new era of DNA sequencing and low frequency variant genomic analyses.     

Single Cell Analysis of Lymph Node Tissue from HIV-1 Infected Patients Reveals that the Majority of CD4+ T-cells Contain One HIV-1 DNA Molecule

Genetic recombination contributes to the diversity of human immunodeficiency virus (HIV-1). Productive HIV-1 recombination is, however, dependent on both the number of HIV-1 genomes per infected cell and the genetic relationship between these viral genomes. A detailed analysis of the number of proviruses and their genetic relationship in infected cells isolated from peripheral blood and tissue compartments is therefore important for understanding HIV-1 recombination, genetic diversity and the dynamics of HIV-1 infection. To address these issues, we used a previously developed single-cell sequencing technique to quantify and genetically characterize individual HIV-1 DNA molecules from single cells in lymph node tissue and peripheral blood. Analysis of memory and naïve CD4⁺ T cells from paired lymph node and peripheral blood samples from five untreated chronically infected patients revealed that the majority of these HIV-1-infected cells (>90%) contain only one copy of HIV-1 DNA, implying a limited potential for productive recombination in virus produced by these cells in these two compartments. Phylogenetic analysis revealed genetic similarity of HIV-1 DNA in memory and naïve CD4⁺ T-cells from lymph node, peripheral blood and HIV-1 RNA from plasma, implying exchange of virus and/or infected cells between these compartments in untreated chronic infection.     

Multi-organ Abnormalities and mTORC1 Activation in Zebrafish Model of Multiple Acyl-CoA Dehydrogenase Deficiency

Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa^vu463) that has an inactivating mutation in the etfa gene. dxa^vu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa^vu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa^vu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.