Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach

BACKGROUND: Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems. RESULTS: We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method. CONCLUSION: This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions

Onset of meiosis in the chicken embryo; evidence of a role for retinoic acid

Background: Meiosis in higher vertebrates shows a dramatic sexual dimorphism: germ cells enter meiosis and arrest at prophase I during embryogenesis in females, whereas in males they enter mitotic arrest during embryogenesis and enter meiosis only after birth. Here we report the molecular analysis of meiosis onset in the chicken model and provide evidence for conserved regulation by retinoic acid

Demonstration of a switchable damping system to allow low-noise operation of high-Q low-mass suspension systems

Low mass suspension systems with high-Q pendulum stages are used to enable quantum radiation pressure noise limited experiments. Utilising multiple pendulum stages with vertical blade springs and materials with high quality factors provides attenuation of seismic and thermal noise, however damping of these high-Q pendulum systems in multiple degrees of freedom is essential for practical implementation. Viscous damping such as eddy-current damping can be employed but introduces displacement noise from force noise due to thermal fluctuations in the damping system. In this paper we demonstrate a passive damping system with adjustable damping strength as a solution for this problem that can be used for low mass suspension systems without adding additional displacement noise in science mode. We show a reduction of the damping factor by a factor of 8 on a test suspension and provide a general optimisation for this system.Comment: 5 pages, 5 figure

Amino acid availability acts as a metabolic rheostat to determine the magnitude of ILC2 responses

Group 2 innate lymphoid cells (ILC2) are functionally poised, tissue-resident lymphocytes that respond rapidly to damage and infection at mucosal barrier sites. ILC2 reside within complex microenvironments where they are subject to cues from both the diet and invading pathogens—including helminths. Emerging evidence suggests ILC2 are acutely sensitive not only to canonical activating signals but also perturbations in nutrient availability. In the context of helminth infection, we identify amino acid availability as a nutritional cue in regulating ILC2 responses. ILC2 are found to be uniquely preprimed to import amino acids via the large neutral amino acid transporters Slc7a5 and Slc7a8. Cell-intrinsic deletion of these transporters individually impaired ILC2 expansion, while concurrent loss of both transporters markedly impaired the proliferative and cytokine-producing capacity of ILC2. Mechanistically, amino acid uptake determined the magnitude of ILC2 responses in part via tuning of mTOR. These findings implicate essential amino acids as a metabolic requisite for optimal ILC2 responses within mucosal barrier tissues

A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development

Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6-8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development

sgsR: a structurally guided sampling toolbox for LiDAR-based forest inventories

Establishing field inventories can be labor intensive, logistically challenging and expensive. Optimizing a sample to derive accurate forest attribute predictions is a key management-level inventory objective. Traditional sampling designs involving pre-defined, interpreted strata could result in poor selection of within-strata sampling intensities, leading to inaccurate estimates of forest structural variables. The use of airborne laser scanning (ALS) data as an applied forest inventory tool continues to improve understanding of the composition and spatial distribution of vegetation structure across forested landscapes. The increased availability of wall-to-wall ALS data is promoting the concept of structurally guided sampling (SGS), where ALS metrics are used as an auxiliary data source driving stratification and sampling within management-level forest inventories. In this manuscript, we present an open-source R package named sgsR that provides a robust toolbox for implementing various SGS approaches. The goal of this package is to provide a toolkit to facilitate better optimized allocation of sample units and sample size, as well as to assess and augment existing plot networks by accounting for current forest structural conditions. Here, we first provide justification for SGS approaches and the creation of the sgsR toolbox. We then briefly describe key functions and workflows the package offers and provide two reproducible examples. Avenues to implement SGS protocols according to auxiliary data needs are presented