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Signal Hill, CA, United States

Tarhan M.C.,Tokyo International University | Orazov Y.,Tokyo International University | Yokokawa R.,Kyoto University | Yokokawa R.,Japan Science and Technology Agency | And 2 more authors.
Analyst | Year: 2013

Microtubule (MT) based intraneuronal transport deficiency is directly linked to neurodegeneration. Hence, the development of a reliable and sensitive in vitro approach permitting efficient analysis of MT-based transport is essential for our understanding of the underlying molecular mechanisms that may lead to novel therapeutic approaches for treating neurodegenerative diseases. Here, based on previously developed reconstructed MT-kinesin assay, we propose its "suspended" modification that shows higher sensitivity and lower experimental variability. This journal is © The Royal Society of Chemistry. Source


Tarhan M.C.,Tokyo International University | Orazov Y.,Tokyo International University | Yokokawa R.,Kyoto University | Yokokawa R.,Japan Science and Technology Agency | And 2 more authors.
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2013

The concept of a reconstructed microtubule kinesin-based transport system was originally introduced for studies of underlying biophysical mechanisms of intracellular transport and its potential applications in bioengineering at micro- and nanoscale levels. However, several technically challenging shortcomings prohibit its use in practical applications. One of them is the propensity of microtubules to bind various protein molecules creating "roadblocks" for kinesin molecule movement and subsequently preventing efficient delivery of the molecular cargo. The interruption in kinesin movement strictly depends on the specific type of "roadblock", i.e. the microtubule associated protein (MAP). Therefore, we propose to use the "roadblock" effect as a molecular sensor that may be used for functional characterization of particular MAPs with respect to their role in MT-based transport and associated pathologies, such as neurodegeneration. Here, we applied a kinesin-based assay using a suspended MT design (sMT assay) to functionally characterize known MAP tau protein isoforms and common mutations found in familial frontotemporal dementia (FTD). The proposed sMT assay is compatible with an on-chip format and may be used for the routine characterization of MT associated molecules applicable to diagnostics and translational research. © 2013 The Royal Society of Chemistry. Source


Winden K.D.,University of California at Los Angeles | Karsten S.L.,University of California at Los Angeles | Bragin A.,University of California at Los Angeles | Kudo L.C.,University of California at Los Angeles | And 5 more authors.
PLoS ONE | Year: 2011

Neither the molecular basis of the pathologic tendency of neuronal circuits to generate spontaneous seizures (epileptogenicity) nor anti-epileptogenic mechanisms that maintain a seizure-free state are well understood. Here, we performed transcriptomic analysis in the intrahippocampal kainate model of temporal lobe epilepsy in rats using both Agilent and Codelink microarray platforms to characterize the epileptic processes. The experimental design allowed subtraction of the confounding effects of the lesion, identification of expression changes associated with epileptogenicity, and genes upregulated by seizures with potential homeostatic anti-epileptogenic effects. Using differential expression analysis, we identified several hundred expression changes in chronic epilepsy, including candidate genes associated with epileptogenicity such as Bdnf and Kcnj13. To analyze these data from a systems perspective, we applied weighted gene co-expression network analysis (WGCNA) to identify groups of co-expressed genes (modules) and their central (hub) genes. One such module contained genes upregulated in the epileptogenic region, including multiple epileptogenicity candidate genes, and was found to be involved the protection of glial cells against oxidative stress, implicating glial oxidative stress in epileptogenicity. Another distinct module corresponded to the effects of chronic seizures and represented changes in neuronal synaptic vesicle trafficking. We found that the network structure and connectivity of one hub gene, Sv2a, showed significant changes between normal and epileptogenic tissue, becoming more highly connected in epileptic brain. Since Sv2a is a target of the antiepileptic levetiracetam, this module may be important in controlling seizure activity. Bioinformatic analysis of this module also revealed a potential mechanism for the observed transcriptional changes via generation of longer alternatively polyadenlyated transcripts through the upregulation of the RNA binding protein HuD. In summary, combining conventional statistical methods and network analysis allowed us to interpret the differentially regulated genes from a systems perspective, yielding new insight into several biological pathways underlying homeostatic anti-epileptogenic effects and epileptogenicity. © 2011 Winden et al. Source


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 700.00K | Year: 2011

DESCRIPTION (provided by applicant): Tissue heterogeneity of central nervous system (CNS) is a serious limiting factor for sound cell-specific molecular studies of the disease including genomic or proteomic analysis. This is especially challenging when cell and region specific primary neural progenitor cultures have to be established. Although tissue microdissection and cell sorting technologies have advanced tremendously over the last decade from simple manual tissue dissection to sophisticated laser capture microdissecting (LCM) instruments and high speed fluorescence assisted cell sorting systems (FACS), no reliable integrated methods or instruments are available that would allow isolation and subsequent culturing of cells. LCM is typically performed on fixed stained or unstained tissues. With the advancement of neural stem cell technologies there is a tremendous need for a low- cost and simple-to-use device that would offer microdissection of unfixed brain tissues and manipulation in vitro. The overall goal of this SBIR project is to develop a new low-cost microdissection instrument with cellular resolution that would allow procurement and follow up cultivation of specific live cells. Here we propose to build a prototype and test the feasibility of a novelcapillary-based vacuum-assisted cell and tissue acquisition system (CTAS) that is envisioned as an attachment to inverted microscopes. The proposed CTAS would be able to dissect fresh tissues at cellular resolution and use these cells for downstream applications (e.g. primary cell cultures). We developed a proof of principle functional prototype of CTAS and demonstrated its use for collection of specific cell types from mouse central nervous system (spinal cord and brain). Phase I specific aims include 1) development of the critical components of CTAS; 2) development of CTAS operational parameters; 3) testing of CTAS on tissue sections and cell cultures. After completion of this work, CTAS will be commercialized in phase II of this project. PUBLICHEALTH RELEVANCE: Cell specific sorting/capture technology is a prerequisite for precise characterization of the specific cell classes and types for understanding their function and regulation of the metabolism, as well as for preclinical translational research. However, isolation of live brain cells for the purpose of their culturing and in vitro manipulation is still challenging. This is especially demanding when region specific neural progenitors are targeted. In phase I of this project, we will developa low-cost vacuum-assisted capillary-based cell and tissue acquisition system (CTAS) and demonstrate its feasibility and applicability for collection of live cells from various brain regions. Collected live cells will be used to establish primary cell cultures including neural progenitor cultures (NPCs). It is a simple, non-invasive (unlike LCM it does not require tissue fixing and drying) technology that can be easily automated and offers a wide range of cell- and tissue-specific separation parameters. Inphase I of this SBIR application, we propose the development of the instrument's critical components, optimization and testing for the range of applications including region specific NPCs and cell specific collection from heterogeneous cell cultures and subsequent molecular characterization of the cells. This low-cost microdissection instrument will be affordable for virtually any research laboratory, and therefore, the demand will likely be very high given the growing need for rapid cell specific culturingmethods in neural stem cell biology. It is also a versatile instrument that can be applied to fixed tissue sections and used to collect larger tissue areas in lieu to LCM. Unlike fluorescence-activated cell sorting (FACS), which requires dissociation of tissue, CTAS preserves tissue integrity and microenvironment of the cells to be isolated.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.05M | Year: 2010

DESCRIPTION (provided by applicant): Tissue heterogeneity is a serious limiting factor for sound cell-specific molecular studies of the disease including genomic or proteomic analysis. Tissue microdissection and cell sorting technologies have advanced tremendously over the last decade from simple manual tissue dissection to sophisticated laser capture microdissecting (LCM) instruments and high speed fluorescence assisted cell sorting systems (FACS). In combination with genomics and proteomics technologies it is now possible to generate cell specific transcriptome/proteome data, advancing the identification of disease biomarkers and novel therapeutic targets. Currently, LCM and FACS are the two main technologies for the isolation of specific tissues and cell types. However, due to their high costs and often sophisticated interface, these technologies are not sufficient to fully support the growing need for cell specific molecular data. Therefore, there is a tremendous need for a low-cost and simple-to-use microdissection device that would offer capabilities similar to LCM and FACS. The overall goal of this SBIR project is to develop a new low-cost microdissection instrument with cellular resolution. In phase I of this project we proposed to build a prototype and test the feasibility of a novel capillary- based vacuum-assisted cell and tissue acquisition system (CTAS) that was envisioned as an attachment to inverted microscopes. The proposed CTAS would be able to dissect tissues at cellular resolution and collect material (RNA or protein) for downstream applications (e.g. expression microarrays). Phase I of this project was highly successful. We developed a fully functional prototype and demonstrated its use for collection of specific cell types from mouse central nervous system (spinal cord and brain). The architecture and major components of CTAS, including the capillary holder, collector, vacuum source, CTAS holder and light source, were developed, tested and optimized. Phase II specific aims include 1) further development of the critical components of CTAS; 2) development of control unit and adjustable parameters; 3) further testing of CTAS on tissue sections; and cell cultures. In addition, the prototype will be tested in different laboratory settings including tissue dissection and cell specific collection from heterogeneous cell culture sources. NeuroInDx will complete this work, which will be necessary to successfully evaluate proposed CTAS, and will commercialize the instrument in phase III of this project. PUBLIC HEALTH RELEVANCE: Cell specific sorting/capture technology is a prerequisite for precise characterization of the specific cell types for understanding their function and regulation of the metabolism, as well as for preclinical translational research. Currently two major approaches for the acquisition of specific cells are available: fluorescence assisted cell sorting (FACS) and laser-capture microdissection (LCM). These technologies are sophisticated and the instruments are not only very expensive but have high maintenance costs. In phase I of this project, we developed a low-cost vacuum-assisted capillary-based cell and tissue acquisition system (CTAS) and demonstrated its feasibility and applicability for tissue microdissection and downstream applications. It is a simple, non-invasive (unlike LCM it does not require tissue fixing and drying) technology that can be easily automated and offers a wide range of cell- and tissue-specific separation parameters. We estimate that CTAS will be at least 5-10 times cheaper than LCM or FACS instruments. In phase II of this SBIR application, we propose further development of the instrument, its optimization and testing for the range of applications including tissue microdissection and cell specific collection from heterogeneous cell cultures. As part of Phase II, beta testing of CTAS will be carried out in several sites including academic laboratories and industry. This work will result in its full commercialization in the following phase III. This low-cost microdissection instrument will be affordable for virtually any research laboratory, and therefore, the demand will likely be very high given the growing need for cell specific analysis.

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