Kudo L.C.,Neuroindx Inc. |
Parfenova L.,University of California at Los Angeles |
Ren G.,University of California at Los Angeles |
Ren G.,Shandong University |
And 10 more authors.
Human Molecular Genetics | Year: 2011
Accumulation of neurotoxic hyperphosphorylated TAU protein is a major pathological hallmark of Alzheimer disease and other neurodegenerative dementias collectively called tauopathies. Puromycin-sensitive amino- peptidase (PSA/NPEPPS) is a novel modifier of TAU-induced neurodegeneration with neuroprotective effects via direct proteolysis of TAU protein. Here, to examine the effects of PSA/NPEPPS overexpression in vivo in the mammalian system, we generated and crossed BAC-PSA/NPEPPS transgenic mice with the TAU P301L mouse model of neurodegeneration. PSA/NPEPPS activity in the brain and peripheral tissues of human PSA/NPEPPS (hPSA) mice was elevated by ~2-3-fold with no noticeable deleterious physiological effects. Double-transgenic animals for hPSA and TAU P301L transgenes demonstrated a distinct trend for delayed paralysis and showed significantly improved motor neuron counts, no gliosis and markedly reduced levels of total and hyperphosphorylated TAU in the spinal cord, brain stem, cortex, hippocampus and cerebellum of adult and aged animals when compared with TAU P301L mice. Furthermore, endogenous TAU protein abun- dance in human neuroblastoma SH-SY5Y cells was significantly reduced or augmented by overexpression or knockdown of PSA/NPEPPS, respectively. This study demonstrated that without showing neurotoxic effects, elevation of PSA/NPEPPS activity in vivo effectively blocks accumulation of soluble hyperphosphorylated TAU protein and slows down the disease progression in the mammalian system. Our data suggest that increasing PSA/NPEPPS activity may be a feasible therapeutic approach to eliminate accumulation of unwanted toxic substrates such as TAU. © The Author 2011. Published by Oxford University Press. All rights reserved.
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.
The Foundation For The Promotion Of Industrial Science, Neuroindx Inc. and French National Center for Scientific Research | Date: 2016-09-07
An object is to make a bridge of DNA expanding between a pair of electrodes, and to characterize the bridge of DNA, to thereby detect DNA easily and surely without employing any marker or labeling substances, such as fluorescent reagents. A method of detecting DNA using a detection device with at least a couple of electrodes, the method comprising immobilizing a primer on the electrodes; making a bridge of the DNA expanded between the electrodes, by immersing the electrodes in a solution including circular templates of single stranded DNA, annealing the circular templates, and generating single stranded DNA product utilizing RCA (Rolling Circle Amplification), with impressing a designated voltage between the electrodes; and characterizing the bridge of DNA which includes multiple single stranded DNA molecules between the electrodes.
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.
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.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 190.31K | Year: 2016
DESCRIPTION provided by applicant Micro RNAs are crucial regulators of gene expression in plants and animals Their complex expression patterns are associated with numerous human diseases developmental programs and often appear to be cell and tissue specific However our understanding of miRNA expression and function in specific cell types and tissues is limited because of difficulty in obtaining appropriate cell and region specific specimen Current methods of cell and region specific micro RNA isolation involve two major steps first tissue microdissection or dissociation with a follow up cell sorting and second isolation of micro RNA molecules from acquired cells or tissue regions This approach is time consuming and invasive It results in the destruction of often valuable original tissue sample and demands for the use of costly equipment e g laser based microdissection or flow sorting and prior training Here we will develop a noninvasive method for cell and region specific micro RNA collection from complex heterogeneous tissues The approach is based on the use of polysaccharide resins and their specific regional acquisition Prior pretreatment and even coating of the tissue sections with a resin permits micro RNA absorption directly from the tissue sample Acquisition of cell or region specific micro RNA is performed with our recently developed KuiqpicK technology via collection of the specific resin samples from the desired tissue regions or cells using carefully controlled vacuum pulses Resin samples containing micro RNA are collected in the barrel of the disposable capillary unit DCU and transferred to a test tube where the captured miRNA is eluted and may be used for a variety of downstream applications including large scale gene expression studies The advantage of the proposed method over current approaches is manifold including direct one step acquisition of micro RNA from the specific cells which eliminates the need for cell and tissue collection i e microdissection preservation of the original tissue integrity which permits its use for further experimentation such as immunohistology reduction in the time and cost possibility of immediate amplification and labeling of captured micro RNA and highly specific capturing of micro RNA species PUBLIC HEALTH RELEVANCE Micro RNAs miRNAs regulate expression of up to of human protein coding genes Moreover recent data convincingly demonstrate that miRNAs are regulated in a complex cell and tissue specific pattern However our understanding of these complex cell tissue disease specific and developmental regulation patterns is hampered by several technical limitations including difficulties of obtaining micro RNA from cell and tissue specific sources Current methods involving tissue microdissection or dissociation with a follow up micro RNA isolation are cumbersome expensive time consuming and result in the destruction of an original tissue sample Our approach proposed here permits noninvasive single step collection of cell and tissue specific micro RNA via the use of polysaccharide resins and a recently developed sample acquisition technology KuiqpicK utilizing controlled vacuum impulse Briefly the resin is evenly distributed over the surface of a target tissue section to absorb micro RNA The subsequent area specific collection ensures acquisition of micro RNA from the desired tissue regions or cells miRNA is easily extracted from the collected resin samples and may be used directly in a variety of downstream applications including large scale gene expression studies The approach is rapid and simple and importantly permits preservation of potentially valuable tissue sample that may be used for additional immunoassays or protein studies Phase I project will focus on the evaluation of various resins optimization of micro RNA yields quality assessment in comparison to the laser based microdissection techniques and development of a streamlined protocol Developed protocol will be integrated with KuiqpicK system Further refinement and development of the commercial product will be performed in Phase II of this application
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 265.64K | Year: 2016
ABSTRACT Large banks of archived human tumor tissues including cancer biopsies harbor enormous amounts of disease relevant information that may facilitate development of novel therapeutic approaches and assist in our basic understanding of fundamental biological processes However the resource is clearly underused due to a number of technical challenges associated with the retrieval of molecular information hidden in the RNA DNA and proteins locked inside the samples Current microdissection platforms especially those with single cell resolution are typically sample specific complex expensive and overly dependent on manual user intervention at key steps Despite the need these limitations make integration within standard lab clinical sample processing workflows difficult Rapid cell and region specific nucleic acid extraction from formalin fixed tumor biopsies and archived tissue samples is perhaps one of the most desirable and technically challenging applications To facilitate the use of the archived and clinical tissue specimens we propose to develop an automated platform AutopicK MTM for high throughput fixed tissue microdissection Automatic molecular retrieval from archived and clinical FFPE tissue samples will be based on our recently developed cell and tissue acquisition CTAS technology using GEandapos s proprietary approach for rapid extraction of histological regions of interest ROIs from FFPE tissues Importantly the collected FFPE ROIs are optimized for direct molecular interrogation and compatible with most enzymatic reactions including protocols used for Next Generation Sequencing NGS The proposed instrument will feature auto calibration ROI auto recognition collection and dispensing into multiwell plate formats Our preliminary studies convincingly demonstrate that sample collection may be performed with cellular resolution with a tissue dissociation step that ensures compatibility of isolated DNA with a range of downstream protocols including amplification labeling and sequencing As both NeuroInDx and GEGR technologies can be implemented at low cost AutopicK MTM will have a serious price advantage over existing competitors Moreover the new instrument will remain compatible with fresh frozen live cell and fixed single cell samples making the AutopicK MTM the most versatile single cell acquisition and tissue microdissection platform on the market This Fast Track application will test all critical parameters of the proposed approach including tissue processing and initial instrument architecture in Phase I Commercial prototype of the proposed instrument and a complete workflow will be developed and validated in Phase II NARRATIVE The goal of this FastTrack SBIR is to develop and validate a cost effective microdissection system capable of fully automating microdissection and molecular analysis of single cells and ROIs from an array of tissue and culture samples Importantly the instrument will be optimized for microdissection of FFPE tissues such as tumor specimen which are routinely processed for diagnostic purposes The proposed fully automated concept AutopicK MTM will take advantage of the cell and tissue acquisition CTAS technology developed and commercialized by NeuroInDx and GE Global Research GEGR proprietary technologies permitting efficient region specific nucleic acid extraction from FFPE tissues and their direct use for downstream molecular analysis including Next Generation Sequencing NGS The proposed instrument will close existing gaps in clinical and pharmaceutical service workflows facilitating tumor specific analysis for diagnostic and drug development purposes Moreover because both NeuroInDx and GEGR technologies can be implemented at low cost AutopicK MTM will have a serious price advantage over existing competitors This taken together with unprecedented applicability of AutopicK MTM to nearly the whole range of research and clinical applications involving the acquisition and analysis of single cells ensure successful commercialization of the proposed instrument
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.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.59M | Year: 2013
DESCRIPTION provided by applicant A In order to advance todayandapos s basic and translational research in neuroscience it is imperative that cell region specific studies can be conducted Complexity of brain tissues poses a significant challenge when cell specific information has to be derived Currently there is no commercially available low cost instrument that would permit complex tissue microdissection at cellular resolution and more importantly collection of live cells from both native brain tissues and cell culture dishes Furthermore existing laser assisted microdissection instruments are sophisticated and limited by the specific sample pretreatment protocol generally prohibiting its use on native brain tissues During Phase I NeuroInDx developed a method for live cell and tissue acquisition that is based on the use of disposable capillary units DCU Live and short carefully regulated vacuum impulses Proof of principle experiments demonstrated that the approach is extremely efficient for the microdissection of native and pretreated brain tissues The cells collected with CTAS Live are suited for reculturing Collected samples yield high quality RNA DNA and protein for downstream applications Phase I of this project resulted in the development of a CTAS Live prototype that features cellular resolution microdissection capabilities from both tissues and cultures without affecting the viability of collected samples Phase II of this SBIR project will develop CTAS Live into two commercial models a base model featuring semiautomatic functions and capability for both microdissection and live cell collection and a fully automated version of CTAS Live capable of fast cell and tissue acquisition from multiple specimen via computer controlled calibration process target recognition and continuous collection controlled via touch screen interface An important advantage of the proposed instruments is their low price that permits efficient market entrance and commercialization The base model is priced under $ and the high end automatic version would be priced in the range of $ demonstrating at least four and two times price advantage respectively versus existing laser based systems Both base and automated models of CTAS Live will be thoroughly tested for its performance with a variety of tissue samples and cell cultures focusing on the viability of the acquired cells and hig quality of the macromolecules e g RNA and protein The epifluorescent module will be tested for the collection of fluorescently labeled cells from tissues Microdissection of human postmortem tissues will be optimized and the quality of RNA and protein will be evaluated for genomics and proteomics applications Therefore aside from initial testing of CTAS Live prototypes several Application Notes will be developed offering andquot sample to resultandquot protocols for the potential customers Developed and andquot in houseandquot tested CTAS Live instruments will be sent to the beta testing sites to ensure end user satisfaction and obtain valuable feedback The result of these studies will be successful commercialization of CTAS Live in Phase III of this project PUBLIC HEALTH RELEVANCE NeuroInDx will develop and commercialize a novel cell and tissue acquisition system CTAS Live capable of microdissection at cellular resolution and collection of live cells from both native brain tissues and cell cultures Proprietary CTAS Live technology offers a highly efficient and minimally invasive microdissection method permitting collection of viable cells Development of a base and advanced fully automated version of CTAS Live instruments will be performed in this Phase II work The effort will be made to keep the cost low to ensure instrumentandapos s affordability Both instruments will be thoroughly tested for the collection of live cells and microdissection Specific end to end protocols will be developed for the collection of neural progenitor zones individual cells for recultivation fluorescently labeled cells from tissue sections and microdissections for genomics and proteomics applications Both base and automated CTAS Live instruments will be subjected to beta testing to obtain feedback from the community and identify potential improvement areas
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.