14 research outputs found
Surface-modified elastomeric nanofluidic devices for single nanoparticle trapping
No full textAbstract Our work focuses on the development of simpler and effective production of nanofluidic devices for high-throughput charged single nanoparticle trapping in an aqueous environment. Single nanoparticle confinement using electrostatic trapping has been an effective approach to study the fundamental properties of charged molecules under a controlled aqueous environment. Conventionally, geometry-induced electrostatic trapping devices are fabricated using SiOx-based substrates and comprise nanochannels imbedded with nanoindentations such as nanopockets, nanoslits and nanogrids. These geometry-induced electrostatic trapping devices can only trap negatively charged particles, and therefore, to trap positively charged particles, modification of the device surface is required. However, the surface modification process of a nanofluidic device is cumbersome and time consuming. Therefore, here, we present a novel approach for the development of surface-modified geometry-induced electrostatic trapping devices that reduces the surface modification time from nearly 5 days to just a few hours. We utilized polydimethylsiloxane for the development of a surface-modified geometry-induced electrostatic trapping device. To demonstrate the device efficiency and success of the surface modification procedure, a comparison study between a PDMS-based geometry-induced electrostatic trapping device and the surface-modified polydimethylsiloxane-based device was performed. The device surface was modified with two layers of polyelectrolytes (1: poly(ethyleneimine) and 2: poly(styrenesulfonate)), which led to an overall negatively charged surface. Our experiments revealed the presence of a homogeneous surface charge density inside the fluidic devices and equivalent trapping strengths for the surface-modified and native polydimethylsiloxane-based geometry-induced electrostatic trapping devices. This work paves the way towards broader use of geometry-induced electrostatic trapping devices in the fields of biosensing, disease diagnosis, molecular analysis, fluid quality control and pathogen detection
Structural centrosome aberrations promote non-cell-autonomous invasiveness
Get PDFCentrosomes are the main microtubule-organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein-like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP-induced structural centrosome aberrations trigger the escape (“budding”) of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP-induced centrosome aberrations trigger a re-organization of the cytoskeleton, which stabilizes microtubules and weakens E-cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non-cell-autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes
Abstract 3926: Nanomechanical profiling of breast cancer: A novel tool for cancer diagnostics and prognostics
No full textAnalysis of the Leukemogenic Potential of the CALM/AF10 Fusion Gene in Patients, Transgenic Mice and Cell Culture Models
No full textThe t(10;11)(p13;q14) is a recurring translocation resulting in the fusion of the CALM and AF10 genes. The leukemogenic CALM/AF10 fusion genes codes for a 1595 amino acids protein. This translocation was first identified in a patient with hystiocytic lymphoma and was subsequently found in patients with AML, T-ALL and malignant lymphoma. This translocation is found in younger patients and is associated with a poor prognosis.
The CALM/AF10-associated leukemias can exhibit myeloid, lymphoid or mixed lymphoid-meyloid features, indicating a stem cell or an early commited progenitor as the target cell of leukemic transformation. At the present time the target cells in CALM/AF10-associated leukemogenesis are unknown. It is also not known which target genes are up or downregulated by the presence of the CALM/AF10 fusion protein.
To answer these questions, the following experiments were performed:
1) Five transgenic mouse lines, two expressing CALM/AF10 under the control of the immunoglobulin heavy chain enhancer promoter and three under the control of the murine proximal Lck promoter were generated. Although the CALM/AF10 expression was confirmed to be present and specific to the cells targeted by the promoters used (B- and T-cell progenitors for IgH and Lck promoters, respectively), the transgenic animals did not show a phenotype that could be detected after meticulous clinical, haematological, immunological, flow cytometrical and immunohistopatological analysis .
2) We performed molecular characterization of several CALM/AF10 patient samples: A group of 13 patients with different types of leukemia: case 1 (AML M2), case 2 (Acute Biphetnotypic leukemia), case 3 (Pre T-ALL), case 4 (Acute Undifferentiated Leukemia), case 5 (PreT-ALL), cases 6 and 7 (ProT-ALL), case 8 (T-ALL), case 9 (AML), case 14 (T-ALL), case 15, 16 and 17 (AML) with a t(10;11) translocation detected by cytogenetic analysis suggesting a CALM/AF10-rearrangement. The samples were analyzed for the presence of the CALM/AF10 and AF10/CALM fusion transcripts by RT-PCR and sequence analysis. All these patients were found to be positive for the CALM/AF10 fusion. In addition, we analyzed a series of twenty-nine patients with T-ALL with T-cell receptor ≥¥ rearrangement. Among these patients, four (case 10 to 13) were positive for the CALM/AF10 fusion transcript, indicating a high incidence of CALM/AF10 fusions in this group of leukemia.
Three different breakpoints in CALM at nucleotide 1926, 2091 and a new exon, with 106 bases inserted after nt 2064 of CALM in patient 4 were found. In AF10 four breakpoints were identified: at nucleotide position 424, 589, 883 and 979. In patient 16 we found an extra exon before nt 424 of AF10. In seven patients it was also possible to amplify the reciprocal AF10/CALM fusion transcript (case 1, 3, 4, 8, 9, 10 and 14). There was no correlation between disease phenotype and breakpoint location. Ten CALM/AF10 positive patients were analyzed using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix). Analysis of microarray gene expression signatures of these patients revealed high expression levels of the polycomb group gene BMI1, the homeobox gene MEIS1 and the HOXA cluster genes HOXA1, HOXA4, HOXA5, HOXA7, HOXA9, and HOXA10. The overexpression of HOX genes seen in these CALM/AF10 positive leukemias is reminiscent to the pattern seen in leukemias with rearrangements of the MLL gene, normal karyotypes and complex aberrant karyotypes suggesting a common effector pathway (i.e. HOX gene deregulation) for these diverse leukemias. In addition, the general pattern of gene expression of CALM/AF10 patients when compared to other leukemia subtypes and to normal bone marrow was dominated by a global downregulation of genes some of them with function identified as related to important molecular mechanisms, such as membrane trafficking, cell growth regulation, proliferation, differentiation and tumor suppression.
3) We cloned CALM/AF10 fusion gene into a vector that allowed us to induce the expression of CALM/AF10 using doxycycline in transiently and stably-transfected NIH3T3 and HEK293 cells. This system will be an important tool to identify direct CALM/AF10 target genes and to answer the question whether continued CALM/AF10 expression is necessary to maintain the CALM/AF10-associated expression pattern
Systematic review and economic modelling of the effectiveness and cost-effectiveness of non-surgical treatments for women with stress urinary incontinence
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Investigating crosstalk in lipid rafts between the glucocorticoid receptor and gonadotropin-releasing hormone receptor signaling pathways in a gonadotrope cell line
No full textIncludes abstract.Includes bibliographical references (leaves 121-158).A recent study from the Hapgood laboratory demonstrated the presence of a novel crosstalk mechanism between the glucocorticoid receptor (GR) and gonadotropin-releasing hormone receptor (GnRHR), indicating an additional direct mechanism for the effects of stress on reproduction. The present study investigated whether this crosstalk between the GR and GnRHR involves the co-localization of these receptors to lipid rafts, providing a specialized distinct region where the receptors can be in close proximity and reciprocally modulate each other’s signaling pathways
The impact of illness and the impact of school closure on social contact patterns.
Get PDFBACKGROUND: Mathematical models, based on data describing normal patterns of social mixing, are used to understand epidemics in order to predict patterns of disease spread and plan interventions and responses. However, individuals who are ill show behavioural changes that affect their social mixing patterns and predictive models should take into account these changes if they are to be effective. OBJECTIVES: To describe and quantify the changes in (1) social contact behaviour experienced by individuals when they are ill with pandemic H1N1 influenza (swine flu) and (2) mixing patterns of school children that take place as a result of swine flu-related school closures. METHODS: For the first part of the study, a self-completed questionnaire-based study was carried out in the autumn/winter of 2009-10. The study population was individuals who had been diagnosed with swine flu and who received a swine flu antiviral prescription from an antiviral distribution centre (ADC). It consisted of an initial survey to be filled in when participants were symptomatic with swine flu and a follow-up survey to be filled in when they had recovered. Each part of the questionnaire had two sections: patient details and a contact diary. The second part of the study was adapted to quantify the difference in mixing patterns of pupils between the school term and the half-term holiday as school closures did not occur during the study period. Eight schools participated and questionnaire packs were distributed to them, containing two surveys: one to be filled in during the school term and one during the spring half-term holiday. RESULTS: For the patient study, approximately 3800 surveys were distributed by 31 ADCs. Overall, 317 responses to the initial survey were received and 179 participants returned the follow-up survey. For all types of a contact, except contacts made at home, there were highly significant differences in contact behaviour (Wilcoxon signed-rank test, p < 0.001). Individuals made substantially fewer contacts when they were ill than when they were well. Analysis showed that returning to work was the most significant predictor of increased numbers of contacts. Also, the greater the change in the number of symptoms reported, the greater the change in the number of contacts. For the school study, approximately 1100 questionnaire packs were distributed and 134 responses were received, with 119 paired contact diaries. Pupils reported on average 18.51 contacts each day during term time and 9.24 during the half-term holiday - a reduction of over 50% and a highly significant change (Wilcoxon signed-rank test, p < 0.0001). CONCLUSIONS: The evidence from this study suggests that ill individuals make substantial changes to their social contact patterns. These changes are strongly linked to absence from work and the severity of the reported illness. Epidemiological modellers should therefore consider the implications of illness-related behavioural changes on model predictions. Future studies to measure the extent of behavioural change in a broader cross-section of infected cases could be valuable, along with more detailed studies of the social contact patterns of school children, focusing on differences between school terms and school holidays
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Effects of mitochondrial dysfunction on neurofilament turnover and distribution in human neuroblastoma cells
No full textA common feature of neurodegenerative conditions including Parkinson’s disease (PD) is the presence of intracytoplasmic proteinacious inclusions. In PD these inclusions are called Lewy bodies (LBs) and contain a number of proteins including α-synuclein, ubiquitin and neurofilaments (NFs). NFs, the intermediate filaments expressed in neuronal cells are responsible for the maintenance of axonal structure. Although NFs were the first proteins identified in LBs their role in PD pathogenesis has not been fully explored. The work presented here attempts to address some of the gaps in the current knowledge concerningNF turnover and the role of NFs in PD using the human SH-SY5Y neuroblastoma cell line, commonly used as a cellular model of neurodegeneration. Mitochondrial dysfunction, dopamine (DA) mediated oxidative stress and impaired protein degradation have all been implicated in PD pathogenesis. The complex I inhibitor MPTP and its active metabolite (MPP+) induce Parkinsonism in humans and other primates and have been extensively used as PD mimetics in both cellular and animal models. Addition of specific protease inhibitors in the presence of cycloheximide (an inhibitor of new protein synthesis) revealed that NF-heavy chains are degraded by macroautophagy and cathepsin D, possibly with some involvement of cysteine cathepsin, but not calpain or the ubiquitin proteasome system (UPS). This is in contrast to α-synuclein which was degraded by macroautophagy, the UPS and calpain. Treatment with MPP+ did not increase NF halflife despite a reduction in the activity of the 20S proteasome, cathepsin D and macroautophagy
Methods of prediction and prevention of pre-eclampsia: Systematic reviews of accuracy and effectiveness literature with economic modelling
Get PDF© Queen's Printer and Controller of HMSO 2008. This monograph may be freely reproduced for the purposes of private research and study and may be included in professional journals provided
that suitable acknowledgement is made and the reproduction is not associated with any form of advertising.Objectives: To investigate the accuracy of predictive tests for pre-eclampsia and the effectiveness of preventative interventions for pre-eclampsia. Also to assess the cost-effectiveness of strategies (test-intervention combinations) to predict and prevent pre-eclampsia. Data sources: Major electronic databases were searched to January 2005 at least. Review methods: Systematic reviews were carried out for test accuracy and effectiveness. Quality assessment was carried out using standard tools. For test accuracy, meta-analyses used a bivariate approach. Effectiveness reviews were conducted under the auspices of the Cochrane Pregnancy and Childbirth Group and used standard Cochrane review methods. The economic evaluation was from an NHS perspective and used a decision tree model. Results: For the 27 tests reviewed, the quality of included studies was generally poor. Some tests appeared to have high specificity, but at the expense of compromised sensitivity. Tests that reached specificities above 90% were body mass index > 34, α-foetoprotein and uterine artery Doppler (bilateral notching). The only Doppler test with a sensitivity of over 60% was resistance index and combinations of indices. A few tests not commonly found in routine practice, such as kallikreinuria and SDS-PAGE proteinuria, seemed to offer the promise of high sensitivity, without compromising specificity, but these would require further investigation. For the 16 effectiveness reviews, the quality of included studies was variable. The largest review was of antiplatelet agents, primarily low-dose aspirin, and included 51 trials (36,500 women). This was the only review where the intervention was shown to prevent both preeclampsia and its consequences for the baby. Calcium supplementation also reduced the risk of preeclampsia, but with some uncertainty about the impact on outcomes for the baby. The only other intervention associated with a reduction in RR of pre-eclampsia was rest at home, with or without a nutritional supplement, for women with normal blood pressure. However, this review included just two small trials and its results should be interpreted with caution. The cost of most of the tests was modest, ranging from £5 for blood tests such as serum uric acid to approximately £20 for Doppler tests. Similarly, the cost of most interventions was also modest. In contrast, the best estimate of additional average cost associated with an average case of pre-eclampsia was high at approximately £9000. The results of the modelling revealed that prior testing with the test accuracy sensitivities and specificities identified appeared to offer little as a way of improving cost-effectiveness. Based on the evidence reviewed, none of the tests appeared sufficiently accurate to be clinically useful and the results of the model favoured no-test/treat-all strategies. Rest at home without any initial testing appeared to be the most cost-effective 'test-treatment' combination. Calcium supplementation to all women, without any initial testing, appeared to be the second most cost-effective. The economic model provided little support that any form of Doppler test has sufficiently high sensitivity and specificity to be cost-effective for the early identification of pre-eclampsia. It also suggested that the pattern of cost-effectiveness was no different in high-risk mothers than the low-risk mothers considered in the base case. Conclusions: The tests evaluated are not sufficiently accurate, in our opinion, to suggest their routine use in clinical practice. Calcium and antiplatelet agents, primarily low-dose aspirin, were the interventions shown to prevent pre-eclampsia. The most cost-effective approach to reducing pre-eclampsia is likely to be the provision of an effective, affordable and safe intervention applied to all mothers without prior testing to assess levels of risk. It is probably premature to suggest the implementation of a treat-all intervention strategy at present, however the feasibility and acceptability of this to women could be explored. Rigorous evaluation is needed of tests with modest cost whose initial assessments suggest that they may have high levels of both sensitivity and specificity. Similarly, there is a need for high-quality, adequately powered randomised controlled trials to investigate whether interventions such as advice to rest are indeed effective in reducing pre-eclampsia. In future, an economic model should be developed that considers not just pre-eclampsia, but other related outcomes, particularly those relevant to the infant such as perinatal death, preterm birth and small for gestational age. Such a modelling project should make provision for primary data collection on the safety of interventions and their associated costs.National Institute for Health Researc
