Identification of Genes Transcriptionally Responsive to the Loss of MLL Fusions in MLL-Rearranged Acute Lymphoblastic Leukemia

Introduction

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is characterized by high relapse rates and a dismal prognosis. To facilitate the discovery of novel therapeutic targets, we here searched for genes directly influenced by the repression of various MLL fusions.

Methods

For this, we performed gene expression profiling after siRNA-mediated repression of MLL-AF4, MLL-ENL, and AF4-MLL in MLL-rearranged ALL cell line models. The obtained results were compared with various already established gene signatures including those consisting of known MLL-AF4 target genes, or those associated with primary MLL-rearranged infant ALL samples.

Results

Genes that were down-regulated in response to the repression of MLL-AF4 and MLL-ENL appeared characteristically expressed in primary MLL-rearranged infant ALL samples, and often represented known MLL-AF4 targets genes. Genes that were up-regulated in response to the repression of MLL-AF4 and MLL-ENL often represented genes typically silenced by promoter hypermethylation in MLL-rearranged infant ALL. Genes that were affected in response to the repression of AF4-MLL showed significant enrichment in gene expression profiles associated with AF4-MLL expressing t(4;11)+ infant ALL patient samples.

Conclusion

We conclude that the here identified genes readily responsive to the loss of MLL fusion expression potentially represent attractive therapeutic targets and may provide additional insights in MLL-rearranged acute leukemias.     

Scale Adjustments to Facilitate Two-Dimensional Measurements in OCT Images

Purpose

To address the problem of unequal scales for the measurement of two-dimensional structures in OCT images, and demonstrate the use of intra¬ocular objects of known dimensions in the murine eye for the equal calibration of axes.

Methods

The first part of this work describes the mathematical foundation of major distortion effects introduced by X-Y scaling differences. Illustrations were generated with CorelGraph X3 software. The second part bases on image data obtained with a HRA2 Spectralis (Heidelberg Engineering) in SV129 wild-type mice. Subretinally and intravitreally implanted microbeads, alginate capsules with a diameter of 154±5 μm containing GFP-marked mesenchymal stem cells (CellBeads), were used as intraocular objects for calibration.

Results

The problems encountered with two-dimensional measurements in cases of unequal scales are demonstrated and an estimation of the resulting errors is provided. Commonly, the Y axis is reliably calibrated using outside standards like histology or manufacturer data. We show here that intraocular objects like dimensionally stable spherical alginate capsules allow for a two-dimensional calibration of the acquired OCT raw images by establishing a relation between X and Y axis data. For our setup, a correction factor of about 3.3 was determined using both epiretinally and subretinally positioned beads (3.350 ± 0.104 and 3.324 ± 0.083, respectively).

Conclusions

In this work, we highlight the distortion-related problems in OCT image analysis induced by unequal X and Y scales. As an exemplary case, we provide data for a two-dimensional in vivo OCT image calibration in mice using intraocular alginate capsules. Our results demonstrate the need for a proper two-dimensional calibration of OCT data, and we believe that equal scaling will certainly improve the efficiency of OCT image analysis.     

Specific variability in Fe-enriched cultures from the equatorial Pacific

Water collected on the spring equatorial Pacific Joint GlobalOcean Flux Study (JGOFS) was placed in ultra-clean bottles inan incubator on deck under the direction of the team of J.H.Martin.Half of the bottles were enriched with 2.53 mM iron; the otherhalf served as controls. Cell counts increased, while the numberof species was reduced, in both the controls and the treatedsamples over the period of the experiment. Diatoms dominated,and after an early growth spurt in the Fe-enriched bottles,most diatom species showed greater growth in the controls atthe end of the 6 day experiment. However, as the experimentwas terminated, more cells overall were noted in the Fe-enrichedsamples, with the most abundant diatom, Cylindrotheca closterium.principally responsible for the difference. Growth rates werehigh in both the controls and the treated samples, with overallrates of 1.3–3.0 divisions day^–1 in the Fe-treatedsamples and 1.9–3.4 divisions day^–1 in the controls.The group of smaller pennate diatoms averaged 2.9 divisionsday^–1 in the Fe-treated samples and 3.3 divisions day^–1in the controls over the entire experiment.     

Biofilms of As(III)-oxidising bacteria: formation and activity studies for bioremediation process development

The formation and activity of an As(III)-oxidising biofilm in a bioreactor, using pozzolana as bacterial growth support, was studied for the purpose of optimising fixed-bed bioreactors for bioremediation. After 60 days of continuous functioning with an As(III)-contaminated effluent, the active biofilm was found to be located mainly near the inflow rather than homogeneously distributed. Biofilm development by the CAsO1 bacterial consortium and by Thiomonas arsenivorans was then studied both on polystyrene microplates and on pozzolana. Extra-cellular polymeric substances (EPS) and yeast extract were found to enhance bacteria attachment, and yeast extract also appears to increase the kinetics of biofilm formation. Analysis of proteins, sugars, lipids and uronic acids indicate that sugars were the main EPS components. The specific As(III)-oxidase activity of T. arsenivorans was higher (by ninefold) for planktonic cells than for sessile ones and was induced by As(III). All the results suggest that the biofilm structure is a physical barrier decreasing As(III) access to sessile cells and thus to As(III)-oxidase activity induction. The efficiency of fixed-bed reactors for the bioremediation of arsenic-contaminated waters can be thus optimised by controlling different factors such as temperature and EPS addition and/or synthesis to increase biofilm density and activity.     

Street heroin induces mitochondrial dysfunction and apoptosis in rat cortical neurons

Cortical function has been suggested to be highly compromised by repeated heroin self-administration. We have previously shown that street heroin induces apoptosis in neuronal-like PC12 cells. Thus, we analysed the apoptotic pathways involved in street heroin neurotoxicity using primary cultures of rat cortical neurons. Our street heroin sample was shown to be mainly composed by heroin, 6-monoacetylmorphine and morphine. Exposure of cortical neurons to street heroin induced a slight decrease in metabolic viability, without loss of neuronal integrity. Early activation of caspases involved in the mitochondrial apoptotic pathway was observed, culminating in caspase 3 activation, Poly-ADP Ribose Polymerase (PARP) cleavage and DNA fragmentation. Apoptotic morphology was completely prevented by the non-selective caspase inhibitor z-VAD-fmk, indicating an important role for caspases in neurodegeneration induced by street heroin. Ionotropic glutamate receptors, opioid receptors and oxidative stress were not involved in caspase 3 activation. Interestingly, street heroin cytotoxicity was shown to be independent of a functional mitochondrial respiratory chain, as determined using NT-2 rho(0) cells. Nonetheless, in street heroin-treated cortical neurons, cytochrome c was released, accompanied by a decrease in mitochondrial potential and Bcl-2/Bax. Pure heroin hydrochloride similarly decreased metabolic viability but only slightly activated caspase 3. Altogether, our data suggest an important role for mitochondria in mediating street heroin neurotoxic effects.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

T cells are crucial for the anti-metastatic effect of anti-epidermal growth factor receptor antibodies

Experimental evidences supporting the epidermal growth factor receptor (EGFR) as an important molecule for tumor metastasis had been accumulated. Currently, anti-EGFR monoclonal antibodies (mAbs) constitute a promising approach for the treatment of patients with metastatic tumors. However, the mechanisms associated with the potent anti-metastatic effect of these mAbs have not been completely elucidated due to the lack of appropriate syngeneic preclinical models. In this paper, we have investigated the effects of 7A7, an antibody specific to murine EGFR, on the metastatic properties of D122 murine lung carcinoma. 7A7 mAb significantly impaired metastatic spread of D122 cells in C57BL/6 mice by direct anti-proliferative and pro-apoptotic effects on tumor metastasis. 7A7 mAb capacity to inhibit EGFR activation on D122 cells could contribute to its anti-metastatic effect. In addition, 7A7 mAb was able to induce in vitro antibody-dependent cell-mediated cytotoxicity on D122 cells. Interestingly, 7A7 mAb treatment increased the number of natural killer cells, T lymphocytes and dendritic cells infiltrating the metastatic sites. More strikingly, depletion of CD8⁺ and CD4⁺ T cells in vivo completely abrogated the 7A7 mAb anti-metastatic activity whereas function of natural killer cells was irrelevant. This study supports an in vivo role for T cell response in the mechanism of action of anti-EGFR mAbs, suggesting the induction of an adjuvant effect. This work was supported by the Cuban Government.     

Physiological adaptations to head-out aquatic exercises with different levels of body immersion

The purpose of this study was to compare the physiological adaptations to basic head-out aquatic exercises with different levels of body immersion. Sixteen young and clinically healthy subjects (9 women and 7 men) volunteered to participate in this study. Each subject performed 3 repetitions (on land, immersed to the hip, and immersed to the breast) of the aquatic exercise "rocking horse" for 6 minutes. The rating of perceived effort (RPE), the maximal heart rate achieved during the exercitation (HRmax), the percentage of the maximal theoretical heart rate estimated (%HRmax), the peak of oxygen uptake during the exercise (V(.-)O2peak), and the energy expenditure (EE) were evaluated. The RPE was significantly higher when exercising immersed to the hip than on land (p < 0.01) and immersed to the breast (p = 0.03). The HRmax and %HRmax were significantly lower when exercising with immersion to the breast than on land (p < 0.01) and with immersion to the hip (p < 0.01). The V(.-)O2peak was significantly different between all conditions. The lower mean value was verified when exercising immersed to the breast, followed by immersion to the hip and on land. The EE was significantly higher when performing aquatic exercises on land than when immersed to the hip (p = 0.02) and the breast (p < 0.01). So, physiological responses when exercising immersed to the hip are higher than when immersed to the breast. The physiological responses when exercising on land are higher than when exercising with immersion to the hip and to the breast.     

Environmental evaluation of single-use and reusable cups

Goal, Scope and Background The objective of the study was to determine the environmental effects of the resuable cup used during a major event (which took place in Barcelona, Universal Forum of Cultures, 2004), compared with a single-use cup of the same composition (polypropylene) but with different physical characteristics such as mass, shape and capacity. Methods To perform the environmental evaluations and the comparison of both types of cups, the SimaPro software developed and marketed by PRé Consultants was used. The environmental evaluation of the reusable cup was compared with that of a single-use cup using the LCA methodology [6]. The functional unit used was: ‘Serving 1000 liters of draught beverages’. The objective of the study was to find the minimum number of cycles the reusable cup has to do so that its environmental impact is smaller than that of the single-use cup. Results and Conclusions Taking into account all the hypotheses put forward, the study drew the conclusion that the minimum number of uses of the reusable cup necessary for it to have a smaller environmental impact than the single-use cup is 10. The contribution of each process taking part in the entire life cycle of the cups was also studied in detail. In the case of the single-use cup, the most important contribution to all the impact categories is due to the production of polypropylene and the fabrication of the cup, except for the heavy metals category where it is due to the management of the waste coming from the cup’s use. In the case of the reusable cup being used 10 times, the contribution to the different impact categories of the waste generated by the cup’s use is negligible compared to the contribution of the fabrication and washing processes. In addition, the washing process is the one which contributes most to the ozone layer depletion, heavy metals and carcinogens categories. As the number of uses of the reusable cup increases, the contribution to all the environmental impact categories decreases. However, this reduction is not as significant for the ozone layer depletion, heavy metals and carcinogens categories. This is due to the washing process and the fact that the electrical consumption associated with it increases with the number of washings and, consequently, of uses. Recommendations and Perspectives From the environmental point of view, the reusable cup must be used at least 10 times to have less impact than the single-use cup. This is mainly due to the higher weight of the reusable cup and, therefore, the greater amount of raw material needed for its fabrication. If the LCA methodology had been introduced during the design of the reusable cup, its weight would have been lower. This modification would have resulted in a reduction of the environmental impact associated with the use of the reusable cup and, consequently, a smaller number of uses would have been necessary to attain the same level of impact as the single-use cup.