Downregulation of the Wnt inhibitor CXXC5 predicts a better prognosis in acute myeloid leukemia

The gene CXXC5 on 5q31 is frequently deleted in acute myeloid leukemia (AML) with del(5q), suggesting that inactivation of CXXC5 might play a role in leukemogenesis. Here, we investigated the functional and prognostic implications of CXXC5 expression in AML. CXXC5 mRNA was downregulated in AML with MLL rearrangements, t(8;21) and GATA2 mutations. As a mechanism of CXXC5 inactivation, we found evidence for epigenetic silencing by promoter methylation. Patients with CXXC5 expression below the median level had a lower relapse rate (45% vs 59%; P = .007) and a better overall survival (OS, 46% vs 28%; P < .001) and event-free survival (EFS, 36% vs 21%; P < .001) at 5 years, independent of cytogenetic risk groups and known molecular risk factors. In gene-expression profiling, lower CXXC5 expression was associated with upregulation of cell-cycling genes and codownregulation of genes implicated in leukemogenesis (WT1, GATA2, MLL, DNMT3B, RUNX1). Functional analyses demonstrated CXXC5 to inhibit leukemic cell proliferation and Wnt signaling and to affect the p53-dependent DNA damage response. In conclusion, our data suggest a tumor suppressor function of CXXC5 in AML. Inactivation of CXXC5 is associated with different leukemic pathways and defines an AML subgroup with better outcome.

The impact of next generation sequencing technologies on haematological research - A review

Next-generation sequencing (NGS) technologies have begun to revolutionize the field of haematological malignancies through the assessment of a patient's genetic makeup with a minimal cost. Significant discoveries have already provided a unique insight into disease initiation, risk stratification and therapeutic intervention. Sequencing analysis will likely form part of the routine diagnostic testing in the future. However, a number of important issues need to be addressed for that to become a reality with regard to result interpretation, laboratory workflow, data storage and ethical issues. In this review we summarize the contribution that NGS has already made to the field of haematological malignancies. Finally, we discuss the challenges that NGS technologies will bring in relation to data storage, ethical and legal issues and laboratory validation. Despite these challenges, we predict that high-throughput DNA sequencing will redefine haematological malignancies based on individualized genomic analysis.

Biosensor based toxicity dissection of tannery and associated environmental samples

Kenyan tannery and associated environmental samples were selected for ecotoxicological assessment. A tool-kit of techniques was developed, including whole-cell biosensor and chemical assays. A luminescence based bacterial biosensor (Escherichia coli HB101 pUCD607) (via a multi-copy plasmid) was used for toxicity assessment. Samples were manipulated prior to biosensor interrogation to identify the nature of the toxic contaminants. Untreated samples (before any manipulations) showed a strong toxic effect at the discharge point in comparison to other sampling points. Sparging was used to identify toxicity associated with volatile organics. The toxicity of contaminants, removed by treatment with activated charcoal was identified for all the sampling points except for those upstream of effluent discharges. Filtration identified toxicity associated with suspended solids. Changes in availability of toxic contaminants due to pH adjustment of most samples from the tannery effluent treatment pits were also associated with the extreme pH values (4.0 and 8.0). The approach used has highlighted the complexicity of toxic pollutants in effluent from the tanning industry and the dissection of toxicity points to possible remediation strategies for effluents from the tanning industry.

Degradation of the polycyclic aromatic hydrocarbon (PAH) fluorene is retarded in a Scots pine ectomycorrhizosphere

Polycyclic aromatic hydrocarbons (PAHs) are an important class of persistent organic pollutants (POPs) in the environment and accumulate in forest soils. These soils are often dominated by ectomycorrhizal (EcM) roots, but little is known about how EcM fungi degrade PAHs, or the overall effect of field colonized EcM roots on the fate of PAHs. The ability of eight EcM fungi to degrade PAHs in liquid culture spiked with ¹⁴C labelled PAHs was investigated. Microcosms were used to determine the impact of naturally colonized mycorrhizal pine seedlings on PAH mineralization and volatilization. Only two EcM fungi (Thelephora terrestris and Laccaria laccata) degraded at least one PAH and none were able to mineralize the PAHs in pure culture. Where degradation occurred, the compounds were only mono-oxygenated. EcM pine seedlings did not alter naphthalene mineralization or volatilization but retarded fluorene mineralization by 35% compared with unplanted, ectomycorrhizosphere soil inoculated, microcosms. The EcM fungi possessed limited PAH degrading abilities, which may explain why EcM dominated microcosms retarded fluorene mineralization. This observation is considered in relation to the 'Gadgil-effect', where retarded litter decomposition has been observed in the presence of EcM roots. © New Phytologist (2004).

Biosensing 2,4-dichlorophenol toxicity during biodegradation by Burkholderia sp. RASC c2 in soil

Biodegradation of the model pollutant, 2,4-dichlorophenol (2,4-DCP) by Burkholderia sp. RASC c2, in contaminated soil was assessed by combining chemical analysis with a toxicity test using Escherichia coli HB101 pUCD607. E. coli HB101 pUCD607 was previously marked with luxCDABE genes, encoding bacterial bioluminescence and was used as an alternative to Microtox. Mineralization of ¹⁴C-2,4-DCP (196.2 μg g^-1 dry wt) in soil occurred rapidly after a 24 h lag. Correspondingly, 2,4-DCP concentrations in soil and soil water extracts decreased with time and concentrations in the latter were at background levels (<0.12 μg mL^-1) after day 2. Toxicity of soil water extracts to the lux-based biosensor also decreased with time. Mean light output of E. coli was stimulated by ~1.5 X control values in soil water extracts when concentrations of 2,4-DCP were approaching the limit of detection by HPLC but returned to values equivalent to those of controls when soil water 2,4-DCP concentrations were below the detection limit. No mineralization or microbial growth was detected in noninoculated microcosms. 2,4-DCP concentration in sterile controls decreased significantly with time as did toxicity to E. coli Lux-based E. coli was a sensitive biosensor of 2,4-DCP toxicity during biodegradation and results complemented chemical analysis.

Toxicity of chlorobenzenes to a lux-marked terrestrial bacterium, Pseudomonas fluorescens

Insertion of lux genes, encoding for bioluminescence in naturally bioluminescent marine bacteria, into the genome of Pseudomonas fluorescens resulted in a bioluminescent strain of this terrestrial bacterium. The lux- marked bacterium was used to toxicity test the chlorobenzene series. By correlating chlorobenzenes 50% effective concentration (EC50) values against physiochemical parameters, the physiochemical properties of chlorobenzenes that elicit toxic responses were investigated. The results showed that the more chlorinated the compounds, the more toxic they were to lux-marked P. fluorescens. Furthermore, it was shown that the more symmetrical the compound, the greater its toxicity to P. fluorescens. In general, the toxicity of a chlorobenzene was inversely proportional to its solubility (S) and directly proportional to its lipophilicity (K(ow). By correlating lux- marked P. fluorescens EC50 values, determined for chlorobenzenes, with toxicity values determined using Pimephales promelas (fathead minnow), Cyclotella meneghiniana (diatom), and Vibrio fischeri (marine bacterium), it was apparent that lux-marked P. fluorescens correlated well with freshwater species such as the diatoms and fathead minnow but not with the bioluminescent marine bacterium V. fischeri. The implications of these findings are that a terrestrial bacterium such as P. fluorescens should be used for toxicity testing of soils and freshwaters rather than the marine bacterium V. fischeri.

Assessment of toxicological interactions of benzene and its primary degradation products (catechol and phenol) using a lux-modified bacterial bioassay

A bacterial bioassay has been developed to assess the relative toxicities of xenobiotics commonly found in contaminated soils, rivers, waters, and ground waters. The assay utilized decline in luminescence of lux- marked Pseudomonas fluorescens on exposure to xenobiotics. Pseudomonas fluorescens is a common bacterium in the terrestrial environment, providing environmental relevance to soil, river, and ground water systems. Three principal environmental contaminants associated with benzene degradation were exposed to the luminescence-marked bacterial biosensor to assess their toxicity individually and in combination. Median effective concentration (EC50) values for decline in luminescence were determined for benzene, catechol, and phenol and were found to be 39.9, 0.77, and 458.6 mg/L, respectively. Catechol, a fungal and bacterial metabolite of benzene, was found to be significantly more toxic to the biosensor than was the parent compound benzene, showing that products of xenobiotic biodegradation may be more toxic than the parent compounds. Combinations of parent compounds and metabolites were found to be significantly more toxic to the bioassay than were the individual compounds themselves. Development of this bioassay has provided a rapid screening system suitable for assessing the toxicity of xenobiotics commonly found in contaminated soil, river, and ground-water environments. The assay can be utilized over a wide pH range and is therefore more applicable to such environmental systems than bioluminescence-based bioassays that utilize marine organisms and can only be applied over a limited pH and salinity range.

Alpha particles induce pan-nuclear phosphorylation of H2AX in primary human lymphocytes mediated through ATM

The use of high linear energy transfer radiations in the form of carbon ions in heavy ion beam lines or alpha particles in new radionuclide treatments has increased substantially over the past decade and will continue to do so due to the favourable dose distributions they can offer versus conventional therapies. Previously it has been shown that exposure to heavy ions induces pan-nuclear phosphorylation of several DNA repair proteins such as H2AX and ATM in vitro. Here we describe similar effects of alpha particles on ex vivo irradiated primary human peripheral blood lymphocytes. Following alpha particle irradiation pan-nuclear phosphorylation of H2AX and ATM, but not DNA-PK and 53BP1, was observed throughout the nucleus. Inhibition of ATM, but not DNA-PK, resulted in the loss of pan-nuclear phosphorylation of H2AX in alpha particle irradiated lymphocytes. Pan-nuclear gamma-H2AX signal was rapidly lost over 24h at a much greater rate than foci loss. Surprisingly, pan-nuclear gamma-H2AX intensity was not dependent on the number of alpha particle induced double strand breaks, rather the number of alpha particles which had traversed the cell nucleus. This distinct fluence dependent damage signature of particle radiation is important in both the fields of radioprotection and clinical oncology in determining radionuclide biological dosimetry and may be indicative of patient response to new radionuclide cancer therapies.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV) several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question, and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. Additionally, endo-lysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable over-expression of p62 significantly suppressed DENV replication suggesting a novel role for p62 as viral restriction factor. Overall our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an anti-viral role, which is countered by DENV.

IMPORTANCE: Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we employed high-content, imaging-based flow cytometry to quantify autophagic flux and endo-lysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endo-lysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a pro- to an antiviral cellular process, which is counteracted by the virus.

Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer

Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I), there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs), such as Per ARNT SIM (PAS) domain containing 1 (PASD1), are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I (n = 164) and stage II (n = 14) disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.

Living long and ageing well: is epigenomics the missing link between nature and nurture?

Human longevity is a complex trait and increasingly we understand that both genes and lifestyle interact in the longevity phenotype. Non-genetic factors, including diet, physical activity, health habits, and psychosocial factors contribute approximately 50 % of the variability in human lifespan with another 25 % explained by genetic differences. Family clusters of nonagenarian and centenarian siblings, who show both exceptional age-span and health-span, are likely to have inherited facilitatory gene groups, but also have nine decades of life experiences and behaviours which have interacted with their genetic profiles. Identification of their shared genes is just one small step in the link from genes to their physical and psychological profiles. Behavioural genomics is beginning to demonstrate links to biological mechanisms through regulation of gene expression, which directs the proteome and influences the personal phenotype. Epigenetics has been considered the missing link between nature and nurture. Although there is much that remains to be discovered, this article will discuss some of genetic and environmental factors which appear important in good quality longevity and link known epigenetic mechanisms to themes identified by nonagenarians themselves related to their longevity. Here we suggest that exceptional 90-year old siblings have adopted a range of behaviours and life-styles which have contributed to their ageing-well-phenotype and which link with important public health messages.