Project description:Despite the physiological and pathophysiological significance of microenvironmental gradients, tools for generating such gradients and analysing their impact on cellular phenotypes are lacking. Here we present an integrated microfluidics-based workflow for mimicking extracellular pH gradients characteristic of solid tumors, and studying their multifactorial impact on cancer cells. Our microfluidics device generates a pH gradient across cancer cell 3D cultures in an extracellular matrix. The gradient, validated using pH-sensitive fluorophores can be rapidly controlled to represent spatiotemporal microenvironmental changes, and the device allows high resolution live imaging of, e.g., cell motility and chemotaxis. The device can be reopened, allowing immunofluorescence analysis of phenotypes and spatially resolved analysis of gene expression changes across the pH gradient. The workflow is easily adaptable for other gradients and multiple cell types, making it broadly applicable for integrated analysis of roles of microenvironmental gradients in biology.
Project description:Plasma membrane proton pump maintains proton electrochemical gradient and provides energy to secondary transporters. Arabidopsis mutant plants with reduced proton pump activity grow normal under ideal growth conditions; however their growth are reduced compared with wildtype plants when placed under the conditions that stress on protonmotive force (high external pH or high external potassium).
Project description:The paper describes a model of pH control in tumor.
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This model is described in the article:
Regulation of tumour intracellular pH: A mathematical model examining the interplay between H and lactate
Maymona Al-Husari, Steven D. Webb
Journal of Theoretical Biology 322 (2013) 58–71
Abstract:
Non-invasive measurements of pH have shown that both tumour and normal cells have intracellular pH (pHi) that lies on the alkaline side of neutrality (7.1–7.2). However, extracellular pH (pHe) is reported to be more acidic in some tumours compared to normal tissues. Many cellular processes and therapeutic agents are known to be tightly pH dependent which makes the study of intracellular pH regulation of paramount importance. We develop a mathematical model that examines the role of various membrane-based ion transporters in tumour pH regulation, in particular, with a focus on the interplay between lactate and H ions and whether the lactate/H symporter activity is sufficient to give rise to the observed reversed pH gradient that is seen is some tumours. Using linear stability analysis and H ions. We extend this analysis using perturbation techniques to specifically examine a rapid change in H-ion concentrations relative to variations in lactate. We then perform a parameter sensitivity analysis to explore solution robustness to parameter variations. An important result from our study is that a reversed pH gradient is possible in our system but for unrealistic parameter estimates—pointing to the possible involvement of other mechanisms in cellular pH gradient reversal, for example acidic vesicles, lysosomes, golgi and endosomes.
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Project description:We aim to compare the genomic discrepancies across de novo Ph+ ALL, Ph+ MPAL and Ph+ AML, three diseases characterized by the occurrence of BCR-ABL1 transcripts but showing varied immunophenotypes. The data we are now submitting is the genomic copy number variants of these three groups. The following is the abstract with associated manuscript. The chromosome abnormality of Philadelphia (Ph) is typically seen in de novo acute lymphoblastic leukemia (ALL). It has also been identified in mixed phenotype acute leukemia (MPAL) and acute myeloid leukemia (AML) in the revisions to World Health Organization classification of myeloid neoplasms and actue leukemia. The discrepancies between these patients and potential mechanisms underlying differentiation fate of the leukemia cells remain poorly defined. We evaluated the clinical, genomic and transcriptomic features of Ph+ ALL, Ph+ MPAL and Ph+ AML by taking advantage of high-density genomic analysis, including next-generation sequencing array comparative genomic hybridization and gene expression profiling for transcriptomic analysis. Our results showed that the three cohorts demonstrated diversified clinical features. Ph+ ALL had the best response to induction therapy, with a complete remission (CR) rate of 93.5 and molecular response of 43.5%. Ph+ MPAL had a 90.0% CR rate but only 5.9% of molecular response. The CR rate of Ph+ AML was only 68.8%. Ph+ ALL was characterized by loss and mutations of B-cell development gene IKZF1 and PAX5, and frequent histone H3K36 trimethyltransferase SETD2 mutations. SETD2 mutations were detected in 11.3% of Ph+ ALL patients and predicted higher relapse rate. Ph+ MPAL and Ph+ AML featured high frequency of RUNX1 mutations. Further studies showed RUNX1-R177X mutation inhibited 32D cell differentiation induced by G-Csf, and cooperated with BCR-ABL1 to lead to myeloid differentiation arrest of human cord blood CD34+ cells. It is therefore presumed that these additional mutations work in synergy with BCR-ABL1 fusion gene to facilitate the development of Ph-positive acute leukemia in different immunophenotypic classifications.
Project description:The purpose of this study was to compare changes in translation (using Gradient Encoding, described below) to changes in mRNA abundance. Lysates of wildtype v-Abl transformed pre-B cells harvested before and after 12 hours of treatment either with 2.5 uM imatinib, a v-Abl kinase inhibitor, or 10ng/mL (10.9 nM) rapamycin, an mTOR inhibitor, were fractionated by sedimentation through linear sucrose gradients. Gradient fractions were encoded such that the mRNA from successive fractions was labeled with increasing ratios of Cy5 to Cy3. mRNAs derived from fractions in the lighter portion of the gradient therefore have a lower Cy5 to Cy3 ratio, whereas those deeper in the gradient have a higher Cy5 to Cy3 ratio. The ratio of Cy5 to Cy3 for each mRNA therefore reflects its average position within the gradient. We thus encoded the sedimentation rate of each mRNA across the entire gradient. The resulting ratios were quantitatively measured for each mRNA species by hybridization to DNA microarrays, and related to the 260 nm absorbance peaks representing different numbers of ribosomes bound per mRNA Compound Based Treatment: wildtype v-Abl transformed pre-B cells were treated with imatinib mesylate (IMA), rapamycin (RAP) or nothing (NONE)
Project description:The purpose of this study was to compare changes in translation (using Gradient Encoding, described below) to changes in mRNA abundance. Lysates of wildtype v-Abl transformed pre-B cells harvested before and after 12 hours of treatment either with 2.5 uM imatinib, a v-Abl kinase inhibitor, or 10ng/mL (10.9 nM) rapamycin, an mTOR inhibitor, were fractionated by sedimentation through linear sucrose gradients. Gradient fractions were encoded such that the mRNA from successive fractions was labeled with increasing ratios of Cy5 to Cy3. mRNAs derived from fractions in the lighter portion of the gradient therefore have a lower Cy5 to Cy3 ratio, whereas those deeper in the gradient have a higher Cy5 to Cy3 ratio. The ratio of Cy5 to Cy3 for each mRNA therefore reflects its average position within the gradient. We thus encoded the sedimentation rate of each mRNA across the entire gradient. The resulting ratios were quantitatively measured for each mRNA species by hybridization to DNA microarrays, and related to the 260 nm absorbance peaks representing different numbers of ribosomes bound per mRNA Compound Based Treatment: wildtype v-Abl transformed pre-B cells were treated with imatinib mesylate (IMA), rapamycin (RAP) or nothing (NONE) compound_treatment_design