Publications on this page begin in 2012. Some staff have a record of earlier publications on their personal pages.
Batten disease (neuronal ceroid lipofuscinosis) refers to a group of neurodegenerative lysosomal storage diseases predominantly affecting children. There are currently no effective treatments, and the functions of many of the associated gene products are unknown. Here we characterise fetal neural cultures from two genetically distinct sheep forms of Batten disease, with mutations in the lysosomal protein encoding gene CLN5 and endoplasmic reticulum membrane protein encoding gene CLN6, respectively. We found similar reductions in autophagy, acidic organelles and synaptic recycling in both forms compared to unaffected cells. We then developed a high-throughput screen and tested for correction of deficient cells with lentiviral-mediated CLN5 or CLN6 gene transfer and fibrate drugs, gemfibrozil and fenofibrate in CLN6 deficient neural cultures. These assays provide a simple system to rapidly screen candidate therapies or libraries of drugs prior to in vivo testing.
Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a 'phenotype of interest' (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with 'Fuzzy-Spreader'-like morphologies were also identified through a visual screen. The second, a 'mutant of interest' (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.
The Antarctic nematode, Panagrolaimus sp. DAW1 (formerly called Panagrolaimus davidi), is the best documented example of an organism able to survive intracellular ice formation in all of its compartments. Not only is it able to survive such extreme physiological disruption, but it is able to produce progeny once thawed from such a state. In addition, under slower rates, or less extreme degrees, of cooling, its body remains unfrozen and the vapour pressure difference between the supercooled body fluids and the surrounding ice leads to a process termed cryoprotective dehydration. In contrast to a fairly large body of work in building up our molecular understanding of cryoprotective dehydration, no comparable work has been undertaken on intracellular freezing. This paper describes an experiment subjecting cultures of Panagrolaimus sp. DAW1 to a range of temperatures including a rapid descent to -10 °C, in a medium just prior to, and after, freezing. Through deep sequencing of RNA libraries we have gained a snapshot of which genes are highly abundant when P. sp. DAW1 is undergoing an intracellular freezing event. The onset of freezing correlated with a high production of genes involved in cuticle formation and subsequently, after 24 h in a frozen state, protease production. In addition to the mapping of RNA sequencing, we have focused on a select set of genes arising both from the expression profiles, as well as implicated from other cold tolerance studies, to undertake qPCR. Among the most abundantly represented transcripts in the RNA mapping is the zinc-metalloenzyme, neprilysin, which also shows a particularly strong upregulated signal through qPCR once the nematodes have frozen.
Resistance to hormonal therapies is a major clinical problem in the treatment of estrogen receptor α-positive (ERα(+)) breast cancers. Epigenetic marks, namely DNA methylation of cytosine at specific CpG sites (5mCpG), are frequently associated with ERα(+) status in human breast cancers. Therefore, ERα may regulate gene expression in part via DNA methylation. This hypothesis was evaluated using a panel of breast cancer cell line models of antiestrogen resistance. Microarray gene expression profiling was used to identify genes normally silenced in ERα(+) cells but derepressed upon exposure to the demethylating agent decitabine, derepressed upon long-term loss of ERα expression, and resuppressed by gain of ERα activity/expression. ERα-dependent DNA methylation targets (n = 39) were enriched for ERα-binding sites, basal-up/luminal-down markers, cancer stem cell, epithelial-mesenchymal transition, and inflammatory and tumor suppressor genes. Kaplan-Meier survival curve and Cox proportional hazards regression analyses indicated that these targets predicted poor distant metastasis-free survival among a large cohort of breast cancer patients. The basal breast cancer subtype markers LCN2 and IFI27 showed the greatest inverse relationship with ERα expression/activity and contain ERα-binding sites. Thus, genes that are methylated in an ERα-dependent manner may serve as predictive biomarkers in breast cancer.
IMPLICATIONS:ERα directs DNA methylation-mediated silencing of specific genes that have biomarker potential in breast cancer subtypes. Mol Cancer Res; ©2016 AACR.
ERK1 and ERK2 (ERK1/2) are the primary effector kinases of the RAS-RAF-MEK-ERK signaling pathway. A variety of substrates and regulatory partners associate with ERK1/2 through distinct D-peptide- and DEF-docking sites on their kinase domains. While understanding of D-peptides that bind to ERK1/2 has become increasingly clear over the last decade, only more recently have structures of proteins interacting with other binding sites on ERK1/2 become available. PEA-15 is a 130-residue ERK1/2 regulator that engages both the D-peptide- and DEF-docking sites of ERK kinases, and directly sequesters the ERK2 activation loop in various different phosphorylation states. Here we describe the methods used to derive crystallization-grade complexes of ERK2-PEA-15, which may also be adapted for other regulators that associate with the activation loop of ERK1/2.
DNA methylation is a stable epigenetic mechanism that has important roles in the normal function of a cell and therefore also in disease etiology. Accurate measurements of normal and altered DNA methylation patterns are important to understand its role in regulating gene expression and cell phenotype. Remarkable progress has been made over the last decade in developing methodologies to investigate DNA methylation. The availability of next-generation sequencing has enabled the profiling of methylation marks at an unprecedented scale. Several methods that were previously used to profile locus-specific methylation have now been upgraded to a genome-wide scale using high-throughput sequencing or array platforms. However, because there are so many techniques available, researchers are faced with the challenge of assessing the potential merits or limitations of each technique and selecting the appropriate method for their analysis. In this review we discuss the strengths and weaknesses of genome-wide and gene-specific analysis tools for interrogating DNA methylation. We particularly focus on the design and analysis strategies involved. This review will provide a guideline for selecting the appropriate methods and tools for large-scale and locus-specific DNA methylation analysis.
Reduced representation bisulfite sequencing (RRBS) is an effective technique for profiling genome-wide DNA methylation patterns in eukaryotes. RRBS couples size selection, bisulfite conversion, and second-generation sequencing to enrich for CpG-dense regions of the genome. The progressive improvement of second-generation sequencing technologies and reduction in cost provided an opportunity to examine the DNA methylation patterns of multiple genomes. Here, we describe a protocol for sequencing multiple RRBS libraries in a single sequencing reaction to generate base-resolution methylomes. Furthermore, we provide a brief guideline for base-calling and data analysis of multiplexed RRBS libraries. These strategies will be useful to perform large-scale, genome-wide DNA methylation analysis.
Physicochemical (zeta potential (ζ), conductivity, surface hydrophobicity (H0), protein solubility (PS)) and emulsifying (emulsion capacity (EC), droplet size, polydispersity (PDI), emulsifying activity (EAI), and stability (ESI) indexes) properties of alkali-(A-FPC), enzymatic-(E-FPC), and enzymatic-solvent-(ES-FPC) extracted protein concentrates from flaxseed meal (FM) were investigated and compared to commercial pea protein concentrate (PPC). The yield, composition, and properties of the protein concentrates were significantly influenced by the methods of extraction. All emulsions were similar in polydispersity with mono-modal droplet distribution and size of ⩽0.43μm that carried a net negative charge at neutral conditions (pH 7.0). A-FPC showed significantly higher H0 (66.14) than that of ES-FPC (52.63), and E-FPC (43.27) and was comparable to PPC (68.47). The highest solubility was found for E-FPC followed by A-FPC at neutral pH. A-FPC displayed significantly (p<0.05) the highest EC (87.91%), EAI (87.18m(2)/g) and ESI (12.51min) compared to the other protein concentrates.
Adherence to urate-lowering therapy (ULT) in people with gout is often poor. A recent systematic review revealed 10%–46% of people with gout adhere to treatment.1 Among chronic diseases, gout has particularly low adherence rates.2 Adherence in clinical trials of ULT is a particularly important issue, as the primary efficacy endpoint for most studies (including phase III studies that form the basis of regulatory approval) is the ability of the agent to reduce serum urate (SU). Pill count-based adherence ≥80% is frequently regarded as an appropriate cut-off for good adherence; however, this is an indirect measure. Measurement of drug concentration may be an improved measure of the adherence.3 The aim of this study was to establish the relationship between two different measures of adherence and SU endpoints in a clinical trial of allopurinol in gout.
RATIONALE:Lipoprotein(a) [Lp(a)] is a low density lipoprotein (LDL)-like lipoprotein and important cardiovascular risk factor whose cognate receptor and intracellular fate remains unknown.
OBJECTIVE:Our study aimed to determine the intracellular trafficking pathway for Lp(a) and the receptor responsible for its uptake in liver cells.
METHODS AND RESULTS:Human hepatoma (HepG2) cells were treated with Lp(a) purified from human plasma and Lp(a) uptake studied using western blot analysis and intracellular localisation of Lp(a) by confocal microscopy. Lp(a) was maximally internalised by 2 hours and was detected by an anti-apo(a) antibody to be localised to Rab5-positive early endosomes, the trans-Golgi network and subsequently Rab11-positive recycling endosomes. In HepG2 cells, the apo(a) component from the internalised Lp(a) was resecreted back into the cellular media, while the LDL component was localised to the lysosomal compartment. Lp(a) internalisation was reduced 0.35 fold in HAP1 and 0.33 fold in HepG2 cells in which the plasminogen receptor (KT) (PlgRKT) was knocked out. Conversely, Lp(a) internalisation was enhanced 2-fold in HAP1 and 1.6-fold in HepG2 cells in which PlgRKT was overexpressed, showing for the first time the role of a specific plasminogen receptor in Lp(a) uptake.
CONCLUSIONS:The finding that Lp(a) is internalised by the plasminogen receptor, PlgRKT and the apo(a) component is recycled are novel findings which may have important implications for the catabolism and function of Lp(a).