28,672 research outputs found

    Fatty-acid uptake in prostate cancer cells using dynamic microfluidic raman technology

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    It is known that intake of dietary fatty acid (FA) is strongly correlated with prostate cancer progression but is highly dependent on the type of FAs. High levels of palmitic acid (PA) or arachidonic acid (AA) can stimulate the progression of cancer. In this study, a unique experimental set-up consisting of a Raman microscope, coupled with a commercial shear-flow microfluidic system is used to monitor fatty acid uptake by prostate cancer (PC-3) cells in real-time at the single cell level. Uptake of deuterated PA, deuterated AA, and the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were monitored using this new system, while complementary flow cytometry experiments using Nile red staining, were also conducted for the validation of the cellular lipid uptake. Using this novel experimental system, we show that DHA and EPA have inhibitory effects on the uptake of PA and AA by PC-3 cells

    Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

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    Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.1152sciescopu

    Morphological plasticity of astroglia: Understanding synaptic microenvironment

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    Memory formation in the brain is thought to rely on the remodeling of synaptic connections which eventually results in neural network rewiring. This remodeling is likely to involve ultrathin astroglial protrusions which often occur in the immediate vicinity of excitatory synapses. The phenomenology, cellular mechanisms, and causal relationships of such astroglial restructuring remain, however, poorly understood. This is in large part because monitoring and probing of the underpinning molecular machinery on the scale of nanoscopic astroglial compartments remains a challenge. Here we briefly summarize the current knowledge regarding the cellular organisation of astroglia in the synaptic microenvironment and discuss molecular mechanisms potentially involved in use-dependent astroglial morphogenesis. We also discuss recent observations concerning morphological astroglial plasticity, the respective monitoring methods, and some of the newly emerging techniques that might help with conceptual advances in the area. GLIA 2015

    ์ƒ๋ฆฌํ™œ์„ฑ ์ €๋ถ„์ž ํ™”ํ•ฉ๋ฌผ์˜ ๋ฐœ๊ตด๊ณผ ์„ธํฌ ์ด๋ฏธ์ง•์„ ํ†ตํ•œ ์ž‘์šฉ ๊ธฐ์ „์˜ ์ดํ•ด

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    ํ•™์œ„๋…ผ๋ฌธ (๋ฐ•์‚ฌ)-- ์„œ์šธ๋Œ€ํ•™๊ต ๋Œ€ํ•™์› : ์ž์—ฐ๊ณผํ•™๋Œ€ํ•™ ์ƒ๋ฌผ๋ฌผ๋ฆฌ ๋ฐ ํ™”ํ•™์ƒ๋ฌผํ•™๊ณผ, 2019. 2. ๋ฐ•์Šน๋ฒ”.Our body has very sophisticated and complex signaling pathways to maintain homeostasis. Therefore, we could understand growth processes based on the knowledge from researches on biological events and improve the quality of life by the suggestion of disease treatment strategies. Among various approaches to understand new biological events, the chemical biology approaches were used in this researches, especially, discovery of bioactive small molecules and visualization of its mode of action by cellular fluorescence imaging for understanding biological phenomena with various aspects. Two biological systems were covered in this dissertation. First, studies about mechanistic target of Rapamycin complex 1 (mTORC1) regulation via Leucyl-tRNA Synthetase (LRS) and Ras-like GTPase D (RagD) protein-protein interaction was described. Three distinct bioactive small molecules were identified by using target-based screening. New aspects on mTORC1 regulation mechanism via LRS-RagD interaction were inferred from the unique activity patterns and biophysical studies of each molecule. Second, the investigation of lipid droplet (LD) reduction mechanism was explained. To identify LD reducing small molecule, image-based LD-monitoring high-contents screening was carried out and the novel LD reduction mechanism study of the hit compound was described. As researches on both mTORC1 regulation via LRS-RagD interaction and LD reduction mechanism are relatively deficient, new results described in this dissertation has greatly contributed to the academic field. Furthermore, it has been confirmed that it is possible to suggest a new disease treatment strategy based on the newly discovered results. Therefore, it is expected to be applied to the medicinal industry as an anti-cancer drug sensitizer and a steatosis attenuating agent.์šฐ๋ฆฌ ๋ชธ์€ ํ•ญ์ƒ์„ฑ์„ ์œ ์ง€ํ•˜๊ธฐ ์œ„ํ•œ ๋งค์šฐ ์ •๊ตํ•˜๊ณ  ๋ณต์žกํ•œ ์‹ ํ˜ธ์ „๋‹ฌ์ฒด๊ณ„๋ฅผ ๊ฐ–์ถ”๊ณ  ์žˆ๋‹ค. ๊ทธ๋Ÿฌ๋ฏ€๋กœ, ์ƒ์ฒด ๋‚ด ํ˜„์ƒ์„ ์กฐ์ ˆํ•˜๋Š” ์ž‘์šฉ ์›๋ฆฌ๋ฅผ ์—ฐ๊ตฌํ•˜๋ฉด ์ธ์ฒด์˜ ์ƒ์žฅ ๋ฐฉ์‹์„ ์ดํ•ดํ•  ์ˆ˜ ์žˆ๊ณ , ์ด๋ฅผ ๋ฐ”ํƒ•์œผ๋กœ ์งˆ๋ณ‘์˜ ์น˜๋ฃŒ ์ „๋žต์„ ์ œ์‹œํ•จ์œผ๋กœ์จ ์ธ๋ฅ˜์˜ ์‚ถ์˜ ์งˆ์„ ๋†’์ผ ์ˆ˜ ์žˆ๋‹ค. ์ƒˆ๋กœ์šด ์ƒ๋ช… ํ˜„์ƒ์„ ํƒ๊ตฌํ•˜๊ณ  ์ดํ•ดํ•˜๊ธฐ ์œ„ํ•œ ๋ฐฉ๋ฒ•์€ ์—ฌ๋Ÿฌ ๊ฐ€์ง€๊ฐ€ ์žˆ๋Š”๋ฐ, ๋ณธ ํ•™์œ„ ๋…ผ๋ฌธ์—์„œ๋Š” ์ƒ๋ฆฌํ™œ์„ฑ ์ €๋ถ„์ž ํ™”ํ•ฉ๋ฌผ์„ ๋ฐœ๊ตดํ•˜๊ณ , ์ด ํ™”ํ•ฉ๋ฌผ์˜ ์ž‘์šฉ ๊ธฐ์ „์„ ์„ธํฌ ์ด๋ฏธ์ง•์„ ํ†ตํ•ด ์‹œ๊ฐํ™”ํ•˜์—ฌ ๋‹ค์–‘ํ•œ ์ธก๋ฉด์—์„œ ์ƒˆ๋กœ์šด ์ƒ๋ช… ํ˜„์ƒ์„ ์ดํ•ดํ•˜๋Š” ํ™”ํ•™์ƒ๋ฌผํ•™์  ์ ‘๊ทผ ๋ฐฉ๋ฒ•์„ ์ด์šฉํ•˜์˜€๋‹ค. ๋ณธ ํ•™์œ„ ๋…ผ๋ฌธ์—๋Š” ๋‘ ๊ฐ€์ง€์˜ ์ƒ๋ช… ํ˜„์ƒ์„ ์—ฐ๊ตฌ ํ•˜์˜€๋‹ค. ์ฒซ ๋ฒˆ์งธ๋Š” ์„ธํฌ์˜ ์„ฑ์žฅ์„ ์กฐ์ ˆํ•˜๋Š” ์ค‘์š”ํ•œ ๋‹จ๋ฐฑ์งˆ ๋ณตํ•ฉ์ฒด์ธ mechanistic target of Rapamycin complex 1 (mTORC1)์„ ์กฐ์ ˆํ•  ์ˆ˜ ์žˆ๋Š” ๋ฐฉ๋ฒ• ์ค‘ ํ•˜๋‚˜์ธ Leucyl-tRNA Synthetase (LRS)์™€ Ras-like GTPase D (RagD) ๋‹จ๋ฐฑ์งˆ ๊ฐ„์˜ ์ƒํ˜ธ์ž‘์šฉ์— ๋Œ€ํ•œ ์—ฐ๊ตฌ์ด๋‹ค. ํƒ€๊ฒŸ ๊ธฐ๋ฐ˜ ์Šคํฌ๋ฆฌ๋‹์„ ํ†ตํ•ด ์„œ๋กœ ๋‹ค๋ฅธ ์„ธ ๊ฐ€์ง€์˜ LRS-RagD ์ƒํ˜ธ์ž‘์šฉ ์กฐ์ ˆ ๋ฌผ์งˆ์„ ๋ฐœ๊ตดํ•˜๊ณ , ๊ฐ๊ฐ์˜ ํ™”ํ•ฉ๋ฌผ์ด ์ผ์œผํ‚ค๋Š” ๋…ํŠนํ•œ ํ™œ์„ฑ ๋ณ€ํ™” ์–‘์ƒ๊ณผ ์ƒ๋ฌผ๋ฌผ๋ฆฌํ•™ ์—ฐ๊ตฌ ๊ฒฐ๊ณผ๋ฅผ ๋ฐ”ํƒ•์œผ๋กœ LRS-RagD ๋‹จ๋ฐฑ์งˆ๊ฐ„ ์ƒํ˜ธ์ž‘์šฉ์„ ํ†ตํ•œ mTORC1๋ฅผ ์กฐ์ ˆํ•˜๋Š” ์ƒˆ๋กœ์šด ๊ด€์ ์— ๋Œ€ํ•ด ๊ธฐ์ˆ ํ•˜๊ณ  ์žˆ๋‹ค. ๋‘ ๋ฒˆ์งธ๋Š” ์ง€๋ฐฉ ๋Œ€์‚ฌ์— ์ค‘์š”ํ•œ ์„ธํฌ ์†Œ๊ธฐ๊ด€์ธ ์ง€๋ฐฉ ๋ฐฉ์šธ์„ ๊ฐ์†Œ ์‹œํ‚ค๋Š” ๋ฐฉ๋ฒ•์— ๋Œ€ํ•œ ๋‚ด์šฉ์ด๋‹ค. ์ด๋ฏธ์ง• ๊ธฐ๋ฐ˜ ์Šคํฌ๋ฆฌ๋‹์„ ์ˆ˜ํ–‰ํ•˜์—ฌ ์ง€๋ฐฉ ๋ฐฉ์šธ์„ ๊ฐ์†Œ์‹œํ‚ค๋Š” ์ €๋ถ„์ž ํ™”ํ•ฉ๋ฌผ์„ ๋ฐœ๊ตดํ•˜๊ณ , ์ด ํ™”ํ•ฉ๋ฌผ์˜ ์ž‘์šฉ ๊ธฐ์ „์„ ํƒ๊ตฌํ•˜์—ฌ ๊ธฐ์กด์— ๋ณด๊ณ ๋˜์ง€ ์•Š์€ ์ง€๋ฐฉ ๋ฐฉ์šธ์„ ์ค„์ด๋Š” ์ƒˆ๋กœ์šด ์ž‘์šฉ ๊ธฐ์ „์„ ์—ฐ๊ตฌํ•œ ๋‚ด์šฉ์„ ๊ธฐ์ˆ ํ•˜๊ณ  ์žˆ๋‹ค. LRS-RagD์— ์˜ํ•œ mTORC1์˜ ์กฐ์ ˆ ๋ฐฉ๋ฒ•๊ณผ ์ง€๋ฐฉ ๋ฐฉ์šธ์˜ ๊ฐ์†Œ ์ž‘์šฉ ๊ธฐ์ „์— ๋Œ€ํ•œ ์—ฐ๊ตฌ๋Š” ์ƒ๋Œ€์ ์œผ๋กœ ์—ฐ๊ตฌ๊ฐ€ ๋ฏธ์•ฝํ•œ ๋ถ„์•ผ์ด๋ฏ€๋กœ ๋ณธ ํ•™์œ„ ๋…ผ๋ฌธ์— ๊ธฐ์ˆ ๋œ ์ƒˆ๋กœ์šด ์—ฐ๊ตฌ ๊ฒฐ๊ณผ๋Š” ํ•™๋ฌธ์ ์œผ๋กœ ๊ธฐ์—ฌํ•˜๋Š” ๋ฐ”๊ฐ€ ํฌ๋‹ค. ๋” ๋‚˜์•„๊ฐ€, ์ƒˆ๋กœ ๋ฐํ˜€์ง„ ์—ฐ๊ตฌ ๊ฒฐ๊ณผ๋ฅผ ๋ฐ”ํƒ•์œผ๋กœ ์ƒˆ๋กœ์šด ์งˆ๋ณ‘ ์น˜๋ฃŒ ์ „๋žต์„ ์ œ์‹œํ•  ์ˆ˜ ์žˆ๋Š” ๊ฐ€๋Šฅ์„ฑ์„ ํ™•์ธํ•˜์˜€์œผ๋ฏ€๋กœ, ํ•ญ์•” ์ฆ๊ฐ์ œ์™€ ์ง€๋ฐฉ์ฆ ์™„ํ™”์ œ๋กœ์„œ์˜ ์˜์•ฝ ์‚ฐ์—…์— ์ ์šฉ์„ ๊ธฐ๋Œ€ํ•˜๊ณ  ์žˆ๋‹ค.Abstract Table of Contents List of Figures List of Table Chapter 1. Introduction 1.1. Chemical biology approaches for comprehension of new biological phenomena 1.1.1. Discovery of bioactive small molecules 1.1.2. Cellular imaging 1.2. mTORC1 pathway and LRS-RagD interaction 1.3. Lipid droplet and autophagy process 1.4. Aims of the dissertation Chapter 2. A new perspective to modulate mTORC1 through LRS-RagD interaction modulators 2.1. Introduction 2.2. Materials and methods 2.3. Results and Discussion 2.3.1. In-house library screening with ELISA-based high-throughput screening system 2.3.2. Possibility to modulate mTORC1 through LRS-RagD perturbation with small molecules 2.3.3. Distinct perturbation of mTORC1 by LRS-RagD inhibition 2.3.4. Different effects on mTORC1 of serum and LRS-RagD interaction 2.3.5. Sensitizing effects to anti-cancer drug 2.4. Conclusion Chapter 3. Discovery of a novel lipid droplet reduction mechanism via lipophagy inducing compound 3.1. Introduction 3.2. Materials and methods 3.3. Results and Discussion 3.3.1. Image-based LD monitoring high-contents screening and Hit compound selection 3.3.2. Selective autophagy, especially lipophagy activation 3.3.3. Ubiquitination of LD surface proteins 3.3.4. Relationship with Endoplasmic reticulum (ER) stress 3.3.5. LD reduction in drug induced steatosis in vitro model 3.4. Conclusion References Abstract in KoreanDocto

    Monitoring and Modelling the Glutamine Metabolic Pathway: A Review and Future Perspectives

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    In this review, the contribution of NMR to the metabolomics field will be discussedโ€”particularly as it relates to cancer and drug metabolism research. An overview of the typical NMR metabolomic experiment will be presented while emphasizing important caveats ranging from experimental design to data interpretation

    Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography

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    We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 {\mu}m axial and 0.4 {\mu}m lateral resolution maintained over a depth of 40 {\mu}m, while preserving the advantages of Fourier domain OCM. Our method uses an ultra-broad spectrum from a super- continuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the method visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo mouse brain tissue.Comment: 15 pages, 7 figure

    Monitoring the dynamics of Src activity in response to anti-invasive dasatinib treatment at a subcellular level using dual intravital imaging

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    Optimising response to tyrosine kinase inhibitors in cancer remains an extensive field of research. Intravital imaging is an emerging tool, which can be used in drug discovery to facilitate and fine-tune maximum drug response in live tumors. A greater understanding of intratumoural delivery and pharmacodynamics of a drug can be obtained by imaging drug target-specific fluorescence resonance energy transfer (FRET) biosensors in real time. Here, we outline our recent work using a Src-FRET biosensor as a readout of Src activity to gauge optimal tyrosine kinase inhibition in response to dasatinib treatment regimens in vivo. By simultaneously monitoring both the inhibition of Src using FRET imaging, and the modulation of the surrounding extracellular matrix using second harmonic generation (SHG) imaging, we were able to show enhanced drug penetrance and delivery to live pancreatic tumors. We discuss the implications of this dual intravital imaging approach in the context of altered tumor-stromal interactions, while summarising how this approach could be applied to assess other combination strategies or tyrosine kinase inhibitors in a preclinical setting

    Dynamic hypoxic pre-conditioning of cells seeded in tissue-engineered scaffold to improve neovascularisation

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    Introduction: Tissue engineering (TE) is the potential solution to the global shortage of tissue and organs. However, the lack of adequate angiogenesis to TE scaffolds during the initial stages of implantation has hindered its success in vivo. Mesenchymal stem cells (MSC) have the most established track record for translational regenerative therapy and have been widely used in combination with TE scaffolds. Hypoxia is one of the main potentiators for upregulating angiogenic factors in MSC. However, fine-tuning their cellular function and behaviour is still not fully understood. This study aims to help increase the understanding of this process by determining the effects of in vitro hypoxic conditioning on enhancement of angiogenesis of MSC for the purpose of pre-clinical translational for TE application. Methods: The angiogenic potential of 3 different tissue sources (bone marrow, umbilical cord and adipose) MSC were initially determined for downstream pre-clinical application. We established the appropriate regime for in vitro dynamic hypoxia conditions in 2D and 3D hydrogel to enhance MSC angiogenic pathway using real-time continuous oxygen sensors and angiogenic cytokine profiling. Cell metabolism and proliferation effects were also evaluated using intravital Realtime-glo, D-luciferin (on transduced MSC) and microscopic Live-Dead stain techniques. We optimised seeding of cells on the tissue engineered dermal (INTEGRAยฎ) for in vivo translational purpose and used targeted in vitro and ex vivo angiogenesis assays, which helped to determine aspects of the MSC conditioned media on endothelial migration, proliferation, morphogenesis and matrix degradation. Finally, the functional reproducibility of the in vitro angiogenic response was assessed using in vivo angiogenesis CAM assay and murine diabetic wound healing models. Results: Adipose derived MSC (adMSC) were found to have the most angiogenic potential in response to hypoxic conditioning. Dynamic hypoxia (DH) regime of changing oxygen levels from 21% to 1% when transitioning from T-flask subculture to multiwell plate seeding was most effective at eliciting pro-angiogenic response from adMSC for both in vitro 2D and 3D models compared to controls using static normoxia (21% oxygen) and static hypoxia (1%). Low seeding density of adMSC was found to be the most appropriate to ensure optimised cell adherence and survival post-seeding on TE dermal scaffold (INTEGRAยฎ). It also minimised on localised hypoxic gradient induced oxidative stress by the seeded cells when compared to high seeding density techniques found on non-invasive oxygen monitoring. Conditioned media from DH seeded adMSC was shown to have enhanced angiogenic proteomic profile compared to the controls. In vitro angiogenesis assays showed better human endothelial cell migration and morphogenesis in scratch assay and tubular formation assay compared to controls. Preliminary ex vivo organ assay results using novel human umbilical arterial rings showed better endothelial out-sprouting and migration through embedded matrix compared to controls. Results from in vivo transplantation of adMSC seeded INTEGRAยฎ scaffold showed a mixed response in the CAM assay, highlighting an unaccounted scaffold effect from INTEGRAยฎ from the host. Histological sections showed increased vascular and host tissue infiltration into the scaffold. When evaluating the functional angiogenesis in murine wound healing models, although DH adMSC seeded scaffolds showed non-statistically significant increased rate of wound closure, there was significantly greater vessel density within the scaffold on histological evaluation in this group compared to controls. Conclusion: The results provide a better comprehension of how cells behave in 2D and 3D environments when cultured in dynamically changing oxygen environments. The study addresses important issues, such as the effects of chronic hypoxia on MSC, and how dynamic hypoxia can enhance angiogenic signalling. It also offers a crucial understanding of the in vitro oxygen culture environments for future research applications. Further insight into cell-scaffold interaction during in vivo transplantation was also established. The importance of having an appropriate in vivo model to determine if such in vitro angiogenic enhancement would translate to functionally improving neoangiogenesis and subsequent tissue regeneration in vivo was also highlighted in this study. Improving and advancing research into optimising and evaluating the in vitro environment for clinical application will undoubtedly have a huge impact on the future of cell therapy for regenerative medicine purposes

    Label-free characterization of ultra violet-radiation-induced changes in skin fibroblasts with Raman spectroscopy and quantitative phase microscopy

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    Minimizing morbidities and mortalities associated with skin cancers requires sustained research with the goal of obtaining fresh insights into disease onset and progression under specific stimuli, particularly the influence of ultraviolet rays. In the present study, label-free profiling of skin fibroblasts exposed to time-bound ultra-violet radiation has been performed using quantitative phase imaging and Raman spectroscopy. Statistically significant differences in quantifiable biophysical parameters, such as matter density and cell dry mass, were observed with phase imaging. Accurate estimation of changes in the biochemical constituents, notably nucleic acids and proteins, was demonstrated through a combination of Raman spectroscopy and multivariate analysis of spectral patterns. Overall, the findings of this study demonstrate the promise of these non-perturbative optical modalities in accurately identifying cellular phenotypes and responses to external stimuli by combining molecular and biophysical information.National Institutes of Health (U.S.) (Grant P41-EB015871-30)National Institutes of Health (U.S.) (Grant U01-NS090438-03)National Institutes of Health (U.S.) (Grant R21-NS091982-01)National Institutes of Health (U.S.) (Grant R01-HL121386-03
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