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Grade 4 astrocytoma is incurable due to the diffusely infiltrative nature of the disease. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is not feasible when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources such as bioluminescence that can generate light at cancer cells. This requires a substrate (a luciferin) and an enabling enzyme (a luciferase), neither seen in mammalian cells.. Preliminary studies confirmed that U87 cells (derived from a human grade 4 astrocytoma) could be killed by conventional PDT using the photosensitizers hypericin or mTHPC. U87 cells were then transfected with firefly and other luciferases and light generating cell lines (U87-luc, U87-hRluc, U87-CBG68luc) identified using the appropriate substrate. Reagent doses and conditions were optimized and U87-luc cells incubated with hypericin or mTHPC with d-luciferin added to initiate bioluminescence activated PDT (bPDT). Cell survival was assessed by MTT assay, haemocytometry and growth assay. Control groups included U87-luc cells with no added active reagents, substrate only, photosensitizer only and non-transfected U87 cells. Results were expressed as a percentage of surviving cells compared with untreated U87-luc controls.. There was no bPDT effect on non-transfected cells. The mean survival of treated transfected cells was 36%, (P0.001) using hypericin and 35% (P0.001) using mTHPC, compared with untreated U87-luc cells. bPDT effects were suppressed by the anti-oxidant, lycopene.. bPDT can kill Grade 4 astrocytoma cells transfected with luciferase in vitro. This justifies progression to in vivo studies.

AimIntrauterine growth restriction (IUGR) is associated with poorer outcomes in later life. We used a monochorionic twin model with IUGR in one twin to determine its impact on growth and neurocognitive outcomes.MethodsMonochorionic twins with ≥20% birth weight discordance born in the north of England were eligible. Cognitive function was assessed using the British Ability Scales. The Strength and Difficulties Questionnaire was used to identify behavioural problems. Auxological measurements were collected. Generalised estimating equations were used to determine the effects of birth weight on cognition.ResultsFifty-one monochorionic twin pairs were assessed at a mean age of 6.3 years. Mean birth weight difference was 664 g at a mean gestation of 34.7 weeks. The lighter twin had a General Conceptual Ability (GCA) score that was three points lower (TwinL −105.4 vs TwinH −108.4, 95% CI −0.9 to −5.0), and there was a significant positive association (B 0.59) of within-pair birth weight differences and GCA scores. Mathematics and memory skills showed the largest differences. The lighter twin at school age was shorter (mean difference 2.1 cm±0.7) and lighter (mean difference 1.9 kg±0.6). Equal numbers of lighter and heavier twins were reported to have behavioural issues.ConclusionsIn a monochorionic twin cohort, fetal growth restriction results in lower neurocognitive scores in early childhood, and there remain significant differences in size. Longer term follow-up will be required to determine whether growth or cognitive differences persist in later child or adulthood, and whether there are any associated longer term metabolic sequelae.

BACKGROUND DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing. METHODS We evaluated DNA methylation of promoter regions of TLX3 (T-cell leukemia homeobox) and FOXE3 (forkhead box E3) in bone marrow biopsies from 197 patients classified as leukemic (n = 95) or clear of the disease (n = 102) by MALDI-TOF. Using a single nucleotide extension assay (methylSABER), we tested 10 bone marrow biopsies collected throughout the course of patient chemotherapy. Using reference materials, diagnostic thresholds and limits of detection were characterized for both methods. RESULTS Reliable detection of DNA methylation of TLX3 and FOXE3 segregated ALL from those clear of disease with minimal false-negative and false-positive results. The limit of detection with MALDI-TOF was 1000–5000 copies of methylated allele. For methylSABER, the limit of detection was 10 copies of methylated TLX3, which enabled monitoring of minimal residual disease in ALL patients. CONCLUSIONS Mass spectrometry procedures can be used to regionally multiplex and detect rare DNA methylation events, establish DNA methylation loci as clinically applicable biomarkers for disease diagnosis, and track pediatric ALL.

Similar to most cancers, genome-wide DNA methylation profiles are commonly altered in pediatric acute lymphoblastic leukemia (ALL); however, recent observations highlight that a large portion of malignancy-associated DNA methylation alterations are not accompanied by related gene expression changes. By analyzing and integrating the methylome and transcriptome profiles of pediatric B-cell ALL cases and primary tissue controls, we report 325 genes hypermethylated and downregulated and 45 genes hypomethylated and upregulated in pediatric B-cell ALL, irrespective of subtype. Repressed cation channel subunits and cAMP signaling activators and transducers are overrepresented, potentially indicating a reduced cellular potential to receive and propagate apoptotic signals. Furthermore, we report specific DNA methylation alterations with concurrent gene expression changes within individual ALL subtypes. The ETV6-RUNX1 translocation was associated with downregulation of ASNS and upregulation of the EPO-receptor, while Hyperdiploid patients (> 50 chr) displayed upregulation of B-cell lymphoma (BCL) members and repression of PTPRG and FHIT. In combination, these data indicate genetically distinct B-cell ALL subtypes contain cooperative epimutations and genome-wide epigenetic deregulation is common across all B-cell ALL subtypes.

We investigated the changes in intracortical neuronal circuits of the hand motor cortex following sensory stimulation of the fingers in 11 healthy subjects. Motor evoked potentials (MEPs) were recorded from intrinsic hand muscles (right first dorsal interosseous and abductor digiti minimi muscles). Electrical stimulation was applied to a digit near (homotopic) or distant (heterotopic stimulation) from each muscle. The right index or little finger was stimulated electrically, followed by single- or paired-pulse transcranial magnetic stimulation (TMS) at an interval of 25, 200, 600, 1,000 or 1,400 ms. Paired-pulse TMS was applied with interstimuli intervals of 2 ms or 12 ms and was expected to stimulate inhibitory or facilitatory intracortical circuits, respectively. MEPs induced by single-pulse TMS were significantly suppressed 200, 600, and 1,000 ms after heterotopic and homotopic stimuli. Intracortical facilitation was significantly enhanced only after homotopic stimuli and such enhancement was maximal 200 ms after digit stimulation. Intracortical inhibition was slightly weakened after homotopic stimulation but this effect did not reach statistical significance ( P=0.25). Our results show that sensory feedback can modify intracortical and corticospinal motor excitability and that intracortical facilitation can be enhanced in a topographic-specific way especially at long latencies. These findings suggest that indirect pathways, probably through somatosensory cortex and other areas, enhance intracortical motor excitability in a somatotopically organized manner.

Effects of nitrogen (N) and phosphorus (P) fertilizers on average fibre radial diameter, tangential diameter, wall thickness and number of fibres per unit area were studied over 7 years of growth in radiata pine. Stands were in mid-rotation, thinned and treatments replicated 4 times at 3 sites. Compared to a thinned, unfertilized control, N decreased and P increased fibre radial diameter. Only N decreased fibre radial diameter, thereby increasing the number of fibres per unit area. All fertilizer treatments decreased fibre wall thickness. Responses in ring width and fibre properties, other than fibre tangential diameter peaked 2 years following fertiliser application, before gradually disappearing after 4–5 years. Fertilizer effects on fibre properties diminished at different rates, causing significant interactions with time. Ring width had little effect on density. Changes in density and fibre coarseness were attributed to changes in fibre wall thickness more than fibre radial diameter and tangential diameter. The largest decrease in density and fibre coarseness in the combined N and P treatment was explained mostly by thinner walls.

Growth or ring width responses to nitrogen, phosphorus and the two combined fertilizers were significant over five out of seven years that followed treatment (post-treatment period) in thinned, mid-rotation radiata pine (Pinus radiata D. Don). Responses were similar between nitrogen and phosphorus treatments and largest in the nitrogen plus phosphorus (NP) treatment. Fertilizer increased the proportion of wood formed in mid-rotation relative to juvenile wood thereby increasing tree density in comparison to the control. Fertilizers, other than nitrogen, significantly lowered density compared to the control in the posttreatment period only. Density was higher in nitrogen than phosphorus and similar between phosphorus and NP. Density responses lasted for up to 5 years and were inconsistent between treatments. Ring width and density were weakly correlated. Ring width and density responded differently to climatic variables within and between sites, signalling independent physiological controls and highlighting the importance of site specific differences.

Background Patient-derived tumour xenografts are an attractive model for preclinical testing of anti-cancer drugs. Insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. As a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale DNA methylation and gene expression profiling on a panel of 10 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) tumours that were stratified by prednisolone response. Results We found high correlations in DNA methylation and gene expression profiles between matching primary and xenograft tumour samples with Pearson’s correlation coefficients ranging between 0.85 and 0.98. In order to demonstrate the potential utility of epigenetic analyses in BCP-ALL xenografts, we identified DNA methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. Differential methylation of CAPS2, ARHGAP21, ARX and HOXB6 were confirmed by locus specific analysis. We identified 20 genes showing an inverse relationship between DNA methylation and gene expression in association with prednisolone response. Pathway analysis of these genes implicated apoptosis, cell signalling and cell structure networks in prednisolone responsiveness. Conclusions The findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric BCP-ALL propagated in mouse xenograft models. Further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-416) contains supplementary material, which is available to authorized users.

BackgroundLittle is currently known about how maternal depression symptoms and unhealthy nutrition during pregnancy may developmentally interrelate to negatively affect child cognitive function.AimsTo test whether prenatal maternal depression symptoms predict poor prenatal nutrition, and whether this in turn prospectively associates with reduced postnatal child cognitive function.MethodIn 6979 mother–offspring pairs participating in the Avon Longitudinal Study of Parents and Children (ALSPAC) in the UK, maternal depression symptoms were assessed five times between 18 weeks gestation and 33 months old. Maternal reports of the nutritional environment were assessed at 32 weeks gestation and 47 months old, and child cognitive function was assessed at age 8 years.ResultsDuring gestation, higher depressive symptoms were related to lower levels of healthy nutrition and higher levels of unhealthy nutrition, each of which in turn was prospectively associated with reduced cognitive function. These results were robust to postnatal depression symptoms and nutrition, as well as a range of potential prenatal and postnatal confounds (i.e. poverty, teenage mother, low maternal education, parity, birth complications, substance use, criminal lifestyle, partner cruelty towards mother).ConclusionsPrenatal interventions aimed at the well-being of children of parents with depression should consider targeting the nutritional environment.

Illumina Infinium Human Methylation (HM) BeadChips are widely used for measuring genome-scale DNA methylation, particularly in relation to epigenome-wide association studies (EWAS) studies. The methylation profile of human samples can be assessed accurately and reproducibly using the HM27 BeadChip (27,578 CpG sites) or its successor, the HM450 BeadChip (482,421 CpG sites). To date no mouse equivalent has been developed, greatly hindering the application of this methodology to the wide range of valuable murine models of disease and development currently in existence. We found 1308 and 13,715 probes from HM27 and HM450 BeadChip respectively, uniquely matched the bisulfite converted reference mouse genome (mm9). We demonstrate reproducible measurements of DNA methylation at these probes in a range of mouse tissue samples and in a murine cell line model of acute myeloid leukaemia. In the absence of a mouse counterpart, the Infinium Human Methylation BeadChip arrays have utility for methylation profiling in non-human species.

Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line “age,” given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1 −/− ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1 −/−, allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1 −/− ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway., Author Summary In mammalian cells, DNA methylation is required for the maintenance of genome stability. Recent studies have shown that the genome-wide levels of DNA methylation can be reduced in DICER1-deficient mouse embryonic stem (ES) cells, suggesting that the activity of DNA methylating enzymes (DNMTs) may be regulated by small RNA molecules. The enzyme DICER1 catalyses the production of these small RNAs that serve as sequence-specific guides for modifying chromatin or transcription. However, these observations of defective DNA methylation have been inconsistent. We surmised that this inconsistency could be due to cell line “age,” because it can take many cell divisions before reduced DNMT activity may result in loss of DNA methylation. To test this idea, we rigorously assayed the functional level of DNMT activity in DICER1-deficient ES cells. First, we tested their ability to maintain DNA methylation over prolonged culture. Second, we tested their ability to rebound in DNA methylation after first stripping it from the genome. In both cases functional DNMT activity was entirely normal. We suggest that losses of DNA methylation sometimes seen in DICER1-deficient ES cells is stochastic and could involve cell line adaptation.

In an experimental model of fetal growth retardation which involves the reduction of placental mass in ewes, we have investigated the effects of intrauterine deprivation on aspects of structural development of the trachea and lungs of fetal sheep (140 days gestation). We have also measured the volume of luminal liquid aspirated from the lungs and the phospholipid content of this liquid as an index of pulmonary surfactant production. The effects of growth retardation are evident in the trachea where the structural development of the mucosal and submucosal layers has been affected. Abnormal aspects of development include the frequent lack of a ciliated border on epithelial cells in the mucosal layer and the reduction in the extent of the folds usually characteristic of this layer in near term fetal sheep. Although the fetal lungs are smaller in growth retardation (P less than 0.01) they are appropriate for fetal weight and their structural development does not appear to have been retarded. In contrast, lung liquid volume is significantly reduced in relation to lung weight in growth retarded fetuses and the concentration of phospholipids in lung liquids is also reduced (P less than 0.01).

An aluminum cantilever beam bonded with 1-3 piezocomposite dampers is modeled by means of ANSYS finite element and SIMULINK simulation softwares. ANSYS currently cannot account for heat dissipation in piezoelectric materials. As such, ANSYS is used to obtain strain energies to be input into the SIMULINK model to investigate the dynamic behavior of the system and calculate the damping ratio. The impact of two different shunting arrangements, a damper in conjunction with a simple resistive electrical circuit in series and parallel, is investigated. In addition, a simply supported beam and a simply supported straight pipe are also analyzed for their wide applications in industry, and as an indication of the utility of this methodology to analyze complex structural configurations. For a typical cantilever beam, energy dissipation and transient analysis are used to calculate the tip displacement as a function of time and the damping ratio. Then using ANSYS, with the parameter BETAD to incorporate damping as a stiffness multiplier, a comparison of the transient results is used to quantify the damping response of aluminum beams with bonded 1-3 piezocomposite dampers. The system loss factor due to the piezoelectric damping is also compared to the inherent loss factor of different beam materials. The results show that circuits in series provides a better damping ratio (0.000581) as compared to circuits in parallel (0.000374). In addition, for different boundary conditions (cantilever, simply supported), the damping ratios (0.000581, 0.000202) and the BETAD values (6.3 E-6, 0.7 E-6), respectively, are functions of the boundary conditions and are not directly related to each other. Finally, damping using 1-3 piezocomposites effectively increases the overall system loss factor by at least 100% to almost 300% as compared to the inherent material damping. In general, this methodology of combining finite element method (ANSYS) and transient modeling tools (SIMULINK) can be used to study damping characteristics of any structural system damped with 1-3 piezocomposites.Copyright © 2005 by ASME

Mexiletine is widely used for the treatment of neuropathic pain although its site(s) of action remain unclear. Here we have studied the effect of spinal administration of mexiletine (10-1000 microg) on the spontaneous and peripherally evoked responses of spinal neurones of nerve injured (selective ligation of spinal nerves L5-L6; SNL) rats. Sham controls for the surgical intervention were performed. A high proportion of the spinal neurones of SNL rats exhibited de novo spontaneous activity (mean frequency of firing 4+/-1 Hz), this activity was highly sensitive to spinal mexiletine (F5,55 = 2.5, P < or = 0.05). The spinal neurones of the sham operated rats exhibited negligible spontaneous activity. The electrically evoked Abeta-fibre neuronal responses of SNL and sham operated rats were not significantly influenced by spinal mexiletine. In contrast, the Adelta-fibre and C-fibre evoked neuronal responses of the SNL rats, but not sham operated rats, were significantly reduced by spinal mexiletine (F5.52 = 4.9, P < or = 0.001 and F5,48 = 12, P < or = 0.0001, respectively). In addition, the mechanical punctate von Frey 9 and 50 g evoked neuronal responses of the SNL rats, but not sham operated rats, were significantly reduced by spinal mexiletine (F5,57 = 4.3, P < or = 0.002 and F5,52 = 6.1, P < or = 0.001). This pharmacological study suggests that following nerve injury there is a novel mexiletine sensitive spinal substrate which contributes to Adelta-fibre and C-fibre, but not Abeta-fibre, somatosensory transmission. This central action may underlie some of the clinical efficacy of mexiletine in the treatment of neuropathic pain states.

Objective: To report the quantitative assessment of visual fields (VF) in patients with pituitary macroadenomas, and the time course and predictive factors for recovery of vision. Methods: Retrospective study of 41 patients with pituitary adenomas and visual disturbance. Patients underwent pre- and postoperative VF assessment at one week, three to six months, one year, two years, and five years using the Humphrey field analyser, which gives a quantitative measure of VF in each quadrant. Results: 36/41 patients (88%) presented with a visual disturbance. Mean (SEM) duration of symptoms was 94 (50) weeks (range 0.5 to 1500); 12 (29%) had optic atrophy at presentation. Impairment of VF was greatest in the upper temporal quadrant, followed by the lower temporal. VF recovery was progressive and apparent even at the five year follow up (p