Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein ​CrkL, a major substrate of the oncogenic tyrosine kinase ​BCR-​ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <104 cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-​CrkL and the protein tyrosine phosphatase ​PTPRC/​CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents

Reconstruction of cell population dynamics using CFSE

Background: Quantifying cell division and death is central to many studies in the biological sciences. The fluorescent dye CFSE allows the tracking of cell division in vitro and in vivo and provides a rich source of information with which to test models of cell kinetics. Cell division and death have a stochastic component at the single-cell level, and the probabilities of these occurring in any given time interval may also undergo systematic variation at a population level. This gives rise to heterogeneity in proliferating cell populations. Branching processes provide a natural means of describing this behaviour. Results: We present a likelihood-based method for estimating the parameters of branching process models of cell kinetics using CFSE-labeling experiments, and demonstrate its validity using synthetic and experimental datasets. Performing inference and model comparison with real CFSE data presents some statistical problems and we suggest methods of dealing with them. Conclusion: The approach we describe here can be used to recover the (potentially variable) division and death rates of any cell population for which division tracking information is available

Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition.

UNLABELLED: A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)-persistent leukemic stem cells (LSC). These are BCR-ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specific inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused significant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in significant loss of LSCs compared to TKI alone, in vitro, and in long-term bone marrow murine xenografts. Our findings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs. SIGNIFICANCE: In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a significant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57. ©2016 AACR.See related article by Xie et al., p. 1237This article is highlighted in the In This Issue feature, p. 1197

Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells

e Glasgow and Manchester Experimental Cancer Medicine Centres (ECMC), which are funded by CR-UK and the Chief Scientist’s Office (Scotland). We acknowledge the funders who have contributed to this work: MRC stratified medicine infrastructure award (A.D.W.), CR-UK C11074/A11008 (F.P., L.E.M.H., T.L.H., A.D.W.); LLR08071 (S.A.A., E.C.); LLR11017 (M.C.); SCD/04 (M.C.); LLR13035 (S.A.A., K.D., A.D.W., and A.P.); LLR14005 (M.T.S., D.V.); KKL690 (L.E.P.); KKL698 (P.B.); LLR08004 (A.D.W., A.P. and A.J.W.); MRC CiC (M.E.D.); The Howat Foundation (FACS support); Friends of Paul O’Gorman (K.D. and FACS support); ELF 67954 (S.A.A.); BSH start up fund (S.A.A.); MR/K014854/1 (K.D.)

Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance

Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenanc

Aspects of mitochondrial DNA mutations in relation to human male fertilising potential

Fertilisation is a chance event dependent on, amongst other things, the number, motility, and physical characteristics of sperm. There is a strong link: between ATP production and sperm motility. The mitochondrially encoded OXPHOS ATP production pathway provides ATP for sperm motility. Thirteen polypeptides involved in OXPHOS are encoded in mtDNA. Therefore, the aim of this study was to assess the influence of point mutations and small deletions in mtDNA on human male fertilising potential. Two hundred and fifty two semen samples were screened for mutations in the ATPase 6 and 8, and COII genes using polymerase chain reaction (PCR), followed by single-strand conformation polymorphism (SSCP) analysis of restriction digests of two overlapping amplicons. ATPase 6 and 8 gene analysis of 210 semen samples identified three singly occurring known homoplasmic polymorphisms (G → A 8860, G → A 8856, and G → A 8839); one polymorphism found in 13 samples that created a known HaeII RFLP; and a novel heteroplasmic point mutation (T → C 8821) found in the immature sperm from two semen samples from an oligo-astheno-teratozoospermic individual. Based on amino acid homologies and protein structure this new heteroplasmic mutation is predicted to be potentially pathogenic. COII gene analysis of 223 semen samples identified two previously described, singly occurring homoplasmic polymorphisms (C → T 7476, and G → A 7853); three novel singly occurring homoplasmic polymorphisms (T → C 8077, G → A 7789, and G → A 7754); two known homoplasmic polymorphisms found in multiple samples (9 bp deletion, 7 samples; G → A 8251, 11 samples) and one novel heteroplasmic homopolymeric tract insertion in the COII/tRNALys intergenic spacer. Of the multiply occurring variants found in these genes only a 9 bp deletion from the COII/RNALys intergenic spacer of 7 semen samples could be associated with asthenozoospermia. This association was negated when disease and cultural factors were considered. In addition to the characterisation of true heteroplasmy, one case of pseudogenecreated heteroplasmy was characterised in the COII amplicon from one semen sample. During the analysis of this known pseudogene-created heteroplasmy, two other novel pseudogenes paralogous to the mitochondrial ND4/ND5 genes and the HV1 region of the D-loop were isolated. All three pseudogenes were characterised and the times of pseudogene insertion were calculated. The COII and ND4/ND5 pseudogenes are predicted to have inserted into nuclear DNA since the time of the last common ancestor of humans and chimpanzees, whilst the HV1 pseudogene is predicted to have an ancient origin. These pseudogenes may act as useful markers for molecular evolution

Enhanced growth of cabbage seedlings by a Paenibacillus isolate in the presence of Xanthomonas campestris pv. campestris

Paenibacillus spp. are rhizobacteria that can promote plant growth through a rangeof mechanisms. A New Zealand isolate of Paenibacillus, P16, has reduced the incidence of black rot, caused by Xanthamonas campestris pv. campestris (Xcc), in brassicas. To investigate if this response was provided through plant growth promotion, isolate P16 was co-applied with Xcc as a seed treatment. In the presence of Xcc, P16-treated seedlings had significantly greater root length, leaf area, and root and shoot dry weight compared to the positive control (Xcc alone). There were no significant differences in plant growth parameters between P16-treated seedlings in the absence of the pathogen and the negative control (seeds without Xcc or P16). Isolate P16 enabled plants to survive and grow normally by preventing disease development; the mechanism of disease suppression requires further investigation

Using next-generation sequencing to analyse the diet of a highly endangered land snail (Powelliphanta augusta) feeding on endemic earthworms

Predation is often difficult to observe or quantify for species that are rare, very small, aquatic or nocturnal. The assessment of such species’ diet can be conducted using molecular methods that target prey DNA remaining in predators’ guts and faeces. These techniques do not require high taxonomic expertise, are applicable to soft-bodied prey and allow for identification at the species level. However, for generalist predators, the presence of mixed prey DNA in guts and faeces can be a major impediment as it requires development of specific primers for each potential prey species for standard (Sanger) sequencing. Therefore, next generation sequencing methods have recently been applied to such situations. In this study, we used 454-pyrosequencing to analyse the diet of Powelliphanta augusta, a carnivorous landsnail endemic to New Zealand and critically endangered after most of its natural habitat has been lost to opencast mining. This species was suspected to feed mainly on earthworms. Although earthworm tissue was not detectable in snail faeces, earthworm DNA was still present in sufficient quantity to conduct molecular analyses. Based on faecal samples collected from 46 landsnails, our analysis provided a complete map of the earthworm-based diet of P. augusta. Predated species appear to be earthworms that live in the leaf litter or earthworms that come to the soil surface at night to feed on the leaf litter. This indicates that P. augusta may not be selective and probably predates any earthworm encountered in the leaf litter. These findings are crucial for selecting future translocation areas for this highly endangered species. The molecular diet analysis protocol used here is particularly appropriate to study the diet of generalist predators that feed on liquid or soft-bodied prey. Because it is non-harmful and non-disturbing for the studied animals, it is also applicable to any species of conservation interest

Something in the water: biosecurity monitoring of ornamental fish imports using environmental DNA

The international trade in ornamental aquatic organisms represents an important vector in the spread of invasive species worldwide, but the accurate identification of imported organisms as part of a biosecurity surveillance program offers an opportunity to mitigate potential problems. Species level identification is historically conducted visually, and more recently, with the use of DNA barcoding. However, new diagnostic methods targeting extracellular environmental DNA (eDNA) can offer advantages over these approaches, being non-destructive and potentially more sensitive at low population densities of target organisms (e.g. in mixed consignments). Despite their recent introduction, techniques utilising eDNA are quickly becoming recognised as an important tool for invasion biologists and ecosystem managers. Here, we present a model for the development of a biosecurity protocol for ornamental fish identification using degraded eDNA molecules in water. We demonstrate how a DNA barcode reference library can be mined for informative short-length markers, and report repeatable and accurate detection at low densities of the target species. This study represents a framework for biosecurity agencies to develop eDNA procedures as an innovative management technique for routine surveillance of high risk imports. Future up-scaling of the method will open up prospects for long term monitoring of entire quarantine facilities for a variety of harmful specie

Noninvasive recovery and detection of possum Trichosurus vulpecula DNA from bitten bait interference devices (WaxTags)

The brushtail possum is a major agricultural and ecological pest in New Zealand. A novel noninvasive DNA sampling tool for detecting its presence (WaxTags, or WT) was tested. DNA was recovered from saliva left on WT, and two lengths (407 bp and 648 bp) of the cytochrome c oxidase I (COI) barcoding region were amplified by polymerase chain reaction (PCR). PCR products were considered (+) when a DNA band was clearly visible by electrophoresis. Different factors that might affect PCR (+) were investigated with captive possums: (i) both extraction protocols of the QIAGEN DNeasy Blood and Tissue Kit, (ii) effect of an overnight or longer delay of up to 3 weeks before DNA extraction on both COI amplicons, and (iii) effect of the individual, order and magnitude of the bite. Extraction protocols were not significantly different. The effect of the overnight delay was not significant, and amplification of the short amplicon was significantly higher (100%) than for the long fragment (48%). After a two or 3-week delay, the short amplicon had 94% and 56% PCR (+), success rates, respectively. Individual, order and magnitude of a bite had no significant effect. The delay trial was repeated with WT from the wild, for which PCR (+) rate of the short amplicon was 63%, regardless of freshness. Four microsatellites were amplified from captive WT samples. We conclude that DNA from saliva traces can be recovered from WT, a potential new tool for noninvasive monitoring of possums and other wildlife