Publications by authors named "Mark Keating"

53 Publications

Cell contact guidance via sensing anisotropy of network mechanical resistance.

Proc Natl Acad Sci U S A 2021 Jul;118(29)

Department of Biomedical Engineering, University of Minnesota, Twin Cities, Minneapolis, MN 55455;

Despite the ubiquitous importance of cell contact guidance, the signal-inducing contact guidance of mammalian cells in an aligned fibril network has defied elucidation. This is due to multiple interdependent signals that an aligned fibril network presents to cells, including, at least, anisotropy of adhesion, porosity, and mechanical resistance. By forming aligned fibrin gels with the same alignment strength, but cross-linked to different extents, the anisotropic mechanical resistance hypothesis of contact guidance was tested for human dermal fibroblasts. The cross-linking was shown to increase the mechanical resistance anisotropy, without detectable change in network microstructure and without change in cell adhesion to the cross-linked fibrin gel. This methodology thus isolated anisotropic mechanical resistance as a variable for fixed anisotropy of adhesion and porosity. The mechanical resistance anisotropy |*| - |*| increased over fourfold in terms of the Fourier magnitudes of microbead displacement |*| and |*| at the drive frequency with respect to alignment direction obtained by optical forces in active microrheology. Cells were found to exhibit stronger contact guidance in the cross-linked gels possessing greater mechanical resistance anisotropy: the cell anisotropy index based on the tensor of cell orientation, which has a range 0 to 1, increased by 18% with the fourfold increase in mechanical resistance anisotropy. We also show that modulation of adhesion via function-blocking antibodies can modulate the guidance response, suggesting a concomitant role of cell adhesion. These results indicate that fibroblasts can exhibit contact guidance in aligned fibril networks by sensing anisotropy of network mechanical resistance.
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http://dx.doi.org/10.1073/pnas.2024942118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8307272PMC
July 2021

Clinical evaluation of a novel subcutaneous lactate monitor.

J Clin Monit Comput 2021 Apr 10. Epub 2021 Apr 10.

Beckman Laser Institute and Medical Clinic, University of California Irvine, Irvine, USA.

Lactate levels are commonly used as an indirect measure to assess metabolic stress in clinical conditions like sepsis. Dynamic lactate measurements are recommended to assess and guide treatment in patients with shock and other critical care conditions. A minimally invasive, continuous lactate monitor has potential to improve clinical decisions and patient care. The purpose of the study was to evaluate continuous lactate measurements of a novel enzymatic Continuous Lactate Monitor (CLM) developed in our laboratory. Lactate levels were monitored during incremental cycling exercise challenges as a tool for hyperlactatemia. Six healthy individuals 18-45 y/o (4 males, 2 females) participated in the study. CLM devices were inserted subcutaneously in the postero-lateral trunk below the renal angle, one hour before the exercise challenge. Each exercise challenge consisted of a 3 to 12-min warm up period, followed by up to 7, 4-min incremental workload bouts separated by rest intervals. Continuous lactate measurements obtained from CLM were compared with commercial lactate analyzer (Abbott iSTAT) measurements of venous blood (plasma) drawn from the antecubital vein. Blood was drawn at up to 25 time points spanning the duration of before exercise, during exercise, and up to 120 min post exercise. Area under the curve (AUC), and delay time were calculated to compare the CLM readings with plasma lactate concentration. Average plasma lactate concentration increased from 1.02 to 16.21 mM. Ratio of AUC derived from CLM to plasma lactate was 1.025 (0.990-1.058). Average dynamic delay time of CLM to venous plasma lactate was 5.22 min (2.87-10.35). Insertion sites examined 48 h after CLM removal did not show signs of side effects and none required medical attention upon examination. The newly developed CLM has shown to be a promising tool to continuously measure lactate concentration in a minimally invasive fashion. Results indicate the CLM can provide needed trends in lactate over time. Such a device may be used in the future to improve treatment in clinical conditions such as sepsis.
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http://dx.doi.org/10.1007/s10877-021-00685-1DOI Listing
April 2021

Matrix crosslinking enhances macrophage adhesion, migration, and inflammatory activation.

APL Bioeng 2019 Mar 27;3(1):016103. Epub 2019 Mar 27.

Department of Biomedical Engineering and The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, Irvine, California 92697, USA.

Macrophages are versatile cells of the innate immune system that can adopt a variety of functional phenotypes depending on signals in their environment. In previous work, we found that culture of macrophages on fibrin, the provisional extracellular matrix protein, inhibits their inflammatory activation when compared to cells cultured on polystyrene surfaces. Here, we sought to investigate the role of matrix stiffness in the regulation of macrophage activity by manipulating the mechanical properties of fibrin. We utilize a photo-initiated crosslinking method to introduce dityrosine crosslinks to a fibrin gel and confirm an increase in gel stiffness through active microrheology. We observe that matrix crosslinking elicits distinct changes in macrophage morphology, integrin expression, migration, and inflammatory activation. Macrophages cultured on a stiffer substrate exhibit greater cell spreading and expression of αM integrin. Furthermore, macrophages cultured on crosslinked fibrin exhibit increased motility. Finally, culture of macrophages on photo-crosslinked fibrin enhances their inflammatory activation compared to unmodified fibrin, suggesting that matrix crosslinking regulates the functional activation of macrophages. These findings provide insight into how the physical properties of the extracellular matrix might control macrophage behavior during inflammation and wound healing.
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http://dx.doi.org/10.1063/1.5067301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481736PMC
March 2019

Selective stiffening of fibrin hydrogels with micron resolution via photocrosslinking.

Acta Biomater 2019 03 17;87:88-96. Epub 2019 Jan 17.

Department of Biomedical Engineering, University of California, Irvine, CA 92697-2730, United States; Center for Complex Biological Systems, University of California, Irvine, CA 92697-2280, United States; Department of Surgery, University of California, Irvine, CA 92697-2730, United States. Electronic address:

Fibrin hydrogels are used as a model system for studying cell-ECM biophysical interactions. Bulk mechanical stiffness of these hydrogels has been correlated to mechanotransduction and downstream signaling. However, stiffness values proximal to cells can vary by orders of magnitude at the length scale of microns. Patterning of matrix stiffness at this spatial scale can be useful in studying such interactions. Here we present and evaluate a technique to selectively stiffen defined regions within a fibrin hydrogel. Laser scanning illumination activates ruthenium-catalyzed crosslinking of fibrin tyrosine residues, resulting in tunable stiffness changes spanning distances as small as a few microns and a localized compaction of the material. As probed by active microrheology, stiffness increases by as much as 25X, similar to previously observed stiffness changes around single cells in 3D culture. In summary, our method allows for selective modification of fibrin stiffness at the micron scale with the potential to create complex patterns, which could be valuable for the investigation of mechanotransduction in a biologically meaningful way. STATEMENT OF SIGNIFICANCE: Fibrin hydrogels are used as a naturally derived model to study interactions between cells and their surrounding extracellular matrix (ECM). ECM stiffness influences cell state. Cells in 3D culture considerably modify the stiffness of their pericellular space, which can be quite heterogeneous at the micron-scale. Here we present and evaluate a method to pattern stiffness within fibrin hydrogels using a laser scanning confocal microscope and selective photo crosslinking. We believe that this technique can aid future studies of cell-ECM interactions by enabling researchers to modify the pericellular distribution of stiffness.
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http://dx.doi.org/10.1016/j.actbio.2019.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684034PMC
March 2019

Sprouting angiogenesis induces significant mechanical heterogeneities and ECM stiffening across length scales in fibrin hydrogels.

Biomaterials 2018 04 6;162:99-108. Epub 2018 Feb 6.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. Electronic address:

Matrix stiffness is a well-established instructive cue in two-dimensional cell cultures. Its roles in morphogenesis in 3-dimensional (3D) cultures, and the converse effects of cells on the mechanics of their surrounding microenvironment, have been more elusive given the absence of suitable methods to quantify stiffness on a length-scale relevant for individual cell-extracellular matrix (ECM) interactions. In this study, we applied traditional bulk rheology and laser tweezers-based active microrheology to probe mechanics across length scales during the complex multicellular process of capillary morphogenesis in 3D, and further characterized the relative contributions of neovessels and supportive stromal cells to dynamic changes in stiffness over time. Our data show local ECM stiffness was highly heterogeneous around sprouting capillaries, and the variation progressively increased with time. Both endothelial cells and stromal support cells progressively stiffened the ECM, with the changes in bulk properties dominated by the latter. Interestingly, regions with high micro-stiffness did not necessarily correlate with remodeled regions of high ECM density as shown by confocal reflectance microscopy. Collectively, these findings, especially the large spatiotemporal variations in local stiffness around cells during morphogenesis in soft 3D fibrin gels, underscore that characterizing ECM mechanics across length scales. provides an opportunity to attain a deeper mechanobiological understanding of the microenvironment's roles in cell fate and tissue patterning.
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http://dx.doi.org/10.1016/j.biomaterials.2018.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831523PMC
April 2018

Vascularization and innervation of slits within polydimethylsiloxane sheets in the subcutaneous space of athymic nude mice.

J Tissue Eng 2017 Jan-Dec;8:2041731417691645. Epub 2017 Jan 30.

Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA; Department of Surgery, School of Medicine, University of California, Irvine, Irvine, CA, USA.

Success of cell therapy in avascular sites will depend on providing sufficient blood supply to transplanted tissues. A popular strategy of providing blood supply is to embed cells within a functionalized hydrogel implanted within the host to stimulate neovascularization. However, hydrogel systems are not always amenable for removal post-transplantation; thus, it may be advantageous to implant a device that contains cells while also providing access to the circulation so retrieval is possible. Here we investigate one instance of providing access to a vessel network, a thin sheet with through-cut slits, and determine if it can be vascularized from autologous materials. We compared the effect of slit width on vascularization of a thin sheet following subcutaneous implantation into an animal model. Polydimethylsiloxane sheets with varying slit widths (approximately 150, 300, 500, or 1500 µm) were fabricated from three-dimensional printed molds. Subcutaneous implantation of sheets in immunodeficient mice revealed that smaller slit widths have evidence of angiogenesis and new tissue growth, while larger slit widths contain native mature tissue squeezing into the space. Our results show that engineered slit sheets may provide a simple approach to cell transplantation by providing a prevascularized and innervated environment.
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http://dx.doi.org/10.1177/2041731417691645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308423PMC
January 2017

Recapitulating the human tumor microenvironment: Colon tumor-derived extracellular matrix promotes angiogenesis and tumor cell growth.

Biomaterials 2017 02 24;116:118-129. Epub 2016 Nov 24.

Department of Biomedical Engineering, The Henry Samueli School of Engineering, UC Irvine, USA; Department of Molecular Biology and Biochemistry, School of Biological Sciences, UC Irvine, USA; The Edwards Lifesciences Center for Advanced Cardiovascular Technology, UC Irvine, USA. Electronic address:

Extracellular matrix (ECM) is an essential and dynamic component of all tissues and directly affects cellular behavior by providing both mechanical and biochemical signaling cues. Changes in ECM can alter tissue homeostasis, potentially leading to promotion of cellular transformation and the generation of tumors. Therefore, understanding ECM compositional changes during cancer progression is vital to the development of targeted treatments. Previous efforts to reproduce the native 3D cellular microenvironment have utilized protein gels and scaffolds that incompletely recapitulate the complexity of native tissues. Here, we address this problem by extracting and comparing ECM from normal human colon and colon tumor that had metastasized to liver. We found differences in protein composition and stiffness, and observed significant differences in vascular network formation and tumor growth in each of the reconstituted matrices, both in vitro and in vivo. We studied free/bound ratios of NADH in the tumor and endothelial cells using Fluorescence Lifetime Imaging Microscopy as a surrogate for the metabolic state of the cells. We observed that cells seeded in tumor ECM had higher relative levels of free NADH, consistent with a higher glycolytic rate, than those seeded in normal ECM. These results demonstrate that the ECM plays an important role in the growth of cancer cells and their associated vasculature.
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http://dx.doi.org/10.1016/j.biomaterials.2016.11.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226635PMC
February 2017

Combination scaffolds of salmon fibrin, hyaluronic acid, and laminin for human neural stem cell and vascular tissue engineering.

Acta Biomater 2016 10 27;43:122-138. Epub 2016 Jul 27.

Department of Biomedical Engineering, University of California, Irvine, Irvine, CA 92697, USA; Sue & Bill Gross Stem Cell Research Center, University of California, Irvine, Irvine, CA 92697, USA; Department of Neurology, University of California, Irvine, Irvine, CA 92697, USA. Electronic address:

Unlabelled: Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVβ1 and α5β1, and several laminin binding integrins (α7β1, α6β1, α3β1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs.

Statement Of Significance: Interest has increased recently in the development of biomaterials as neural stem cell transplantation scaffolds to treat central nervous system (CNS) injury since scaffolds improve survival and integration of transplanted cells. We report here on a novel combination scaffold composed of fibrin, hyaluronic acid, and laminin to support human neural stem/progenitor cell (hNSPC) function. This combined biomaterial scaffold has appropriate physical properties for hNSPCs and the CNS, supports hNSPC proliferation and differentiation, and attenuates rapid cell-mediated scaffold degradation. The hNSPCs and scaffold components synergistically encourage new vessel formation from human endothelial cells. This work marks the first report of a combination scaffold supporting human neural and vascular cells to encourage vasculogenesis, and sets a benchmark for biomaterials to treat CNS injury.
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http://dx.doi.org/10.1016/j.actbio.2016.07.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386322PMC
October 2016

Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Tissue Eng Part A 2016 08;22(15-16):1016-25

1 Department of Molecular Biology and Biochemistry, School of Biological Sciences , UC Irvine, Irvine, California.

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease.
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http://dx.doi.org/10.1089/ten.TEA.2016.0027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991595PMC
August 2016

Novel insights from 3D models: the pivotal role of physical symmetry in epithelial organization.

Sci Rep 2015 Oct 16;5:15153. Epub 2015 Oct 16.

University of California Irvine, Department of Biomedical Engineering, Irvine, USA.

3D tissue culture models are utilized to study breast cancer and other pathologies because they better capture the complexity of in vivo tissue architecture compared to 2D models. However, to mimic the in vivo environment, the mechanics and geometry of the ECM must also be considered. Here, we studied the mechanical environment created in two 3D models, the overlay protocol (OP) and embedded protocol (EP). Mammary epithelial acini features were compared using OP or EP under conditions known to alter acinus organization, i.e. collagen crosslinking and/or ErbB2 receptor activation. Finite element analysis and active microrheology demonstrated that OP creates a physically asymmetric environment with non-uniform mechanical stresses in radial and circumferential directions. Further contrasting with EP, acini in OP displayed cooperation between ErbB2 signalling and matrix crosslinking. These differences in acini phenotype observed between OP and EP highlight the functional impact of physical symmetry in 3D tissue culture models.
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http://dx.doi.org/10.1038/srep15153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608012PMC
October 2015

Spectral pre and post processing for infrared and Raman spectroscopy of biological tissues and cells.

Chem Soc Rev 2016 Apr 14;45(7):1865-78. Epub 2015 Oct 14.

FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.

Vibrational spectroscopy, both infrared absorption and Raman spectroscopy, have attracted increasing attention for biomedical applications, from in vivo and ex vivo disease diagnostics and screening, to in vitro screening of therapeutics. There remain, however, many challenges related to the accuracy of analysis of physically and chemically inhomogeneous samples, across heterogeneous sample sets. Data preprocessing is required to deal with variations in instrumental responses and intrinsic spectral backgrounds and distortions in order to extract reliable spectral data. Data postprocessing is required to extract the most reliable information from the sample sets, based on often very subtle changes in spectra associated with the targeted pathology or biochemical process. This review presents the current understanding of the factors influencing the quality of spectra recorded and the pre-processing steps commonly employed to improve on spectral quality. It further explores some of the most common techniques which have emerged for classification and analysis of the spectral data for biomedical applications. The importance of sample presentation and measurement conditions to yield the highest quality spectra in the first place is emphasised, as is the potential of model simulated datasets to validate both pre- and post-processing protocols.
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http://dx.doi.org/10.1039/c5cs00440cDOI Listing
April 2016

Molecular interference of fibrin's divalent polymerization mechanism enables modulation of multiscale material properties.

Biomaterials 2015 May 11;49:27-36. Epub 2015 Feb 11.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, United States; The Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332, United States. Electronic address:

Protein based polymers provide an exciting and complex landscape for tunable natural biomaterials through modulation of molecular level interactions. Here we demonstrate the ability to modify protein polymer structural and mechanical properties at multiple length scales by molecular 'interference' of fibrin's native polymerization mechanism. We have previously reported that engagement of fibrin's polymerization 'hole b', also known as 'b-pockets', through PEGylated complementary 'knob B' mimics can increase fibrin network porosity but also, somewhat paradoxically, increase network stiffness. Here, we explore the possible mechanistic underpinning of this phenomenon through characterization of the effects of knob B-fibrin interaction at multiple length scales from molecular to bulk polymer. Despite its weak monovalent binding affinity for fibrin, addition of both knob B and PEGylated knob B at concentrations near the binding coefficient, Kd, increased fibrin network porosity, consistent with the reported role of knob B-hole b interactions in promoting lateral growth of fibrin fibers. Addition of PEGylated knob B decreases the extensibility of single fibrin fibers at concentrations near its Kd but increases extensibility of fibers at concentrations above its Kd. The data suggest this bimodal behavior is due to the individual contributions knob B, which decreases fiber extensibility, and PEG, which increase fiber extensibility. Taken together with laser trap-based microrheological and bulk rheological analyses of fibrin polymers, our data strongly suggests that hole b engagement increases in single fiber stiffness that translates to higher storage moduli of fibrin polymers despite their increased porosity. These data point to possible strategies for tuning fibrin polymer mechanical properties through modulation of single fiber mechanics.
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http://dx.doi.org/10.1016/j.biomaterials.2015.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4486003PMC
May 2015

Multivariate statistical methodologies applied in biomedical Raman spectroscopy: assessing the validity of partial least squares regression using simulated model datasets.

Analyst 2015 Apr;140(7):2482-92

FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.

Raman spectroscopy is fast becoming a valuable analytical tool in a number of biomedical scenarios, most notably disease diagnostics. Importantly, the technique has also shown increasing promise in the assessment of drug interactions on cellular and subcellular levels, particularly when coupled with multivariate statistical analysis. However, with respect to both Raman spectroscopy and the associated statistical methodologies, an important consideration is the accuracy of these techniques and more specifically, the sensitivities which can be achieved, and ultimately the limits of detection of the various methods. The purpose of this study is thus the construction of a model simulated dataset with the aim of testing the accuracy and sensitivity of the partial least squares regression (PLSR) approach to spectral analysis. The basis of the dataset is the experimental spectral profiles of a previously reported Raman spectroscopic analysis of the interaction of the cancer chemotherapeutic agent cisplatin in an adenocarcinomic human alveolar basal epithelial cell-line, in vitro, and is thus reflective of actual experimental data. The simulated spectroscopic data are constructed by adding known perturbations which are independently linear in drug doses as well as cytological responses experimentally determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. It is demonstrated that, through appropriate choice of dose range, PLSR against the respective targets can differentiate between the spectroscopic signatures of the direct chemical effect of the drug dose and the indirect cytological effect it produces.
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http://dx.doi.org/10.1039/c4an02167cDOI Listing
April 2015

Raman spectroscopy in nanomedicine: current status and future perspective.

Nanomedicine (Lond) 2013 Aug;8(8):1335-51

Focas Research Institute, Dublin Institute of Technology, Camden Row, Dublin 8, Ireland.

Raman spectroscopy is a branch of vibration spectroscopy that is capable of probing the chemical composition of materials. Recent advances in Raman microscopy have significantly added to the range of applications, which now extend from medical diagnostics to exploring the interfaces between biological organisms and nanomaterials. In this review, Raman is introduced in a general context, highlighting some of the areas in which the technique has been successful in the past, as well as some of the potential benefits it offers over other analytical modalities. The subset of Raman techniques that specifically probe the nanoscale, namely surface- and tip-enhanced Raman spectroscopy, will be described and specific applications relevant to nanomedical applications will be reviewed. Progress in the use of traditional label-free Raman for investigation of nanoscale interactions will be described, and recent developments in coherent anti-Stokes Raman scattering will be explored, particularly its applications to biomedical and nanomedical fields.
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http://dx.doi.org/10.2217/nnm.13.108DOI Listing
August 2013

Spectral cross-correlation as a supervised approach for the analysis of complex Raman datasets: the case of nanoparticles in biological cells.

Analyst 2012 Dec;137(24):5792-802

Focas Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.

Spectral cross-correlation is introduced as a methodology to identify the presence and subcellular distribution of nanoparticles in cells. Raman microscopy is employed to spectroscopically image biological cells previously exposed to polystyrene nanoparticles, as a model for the study of nano-bio interactions. The limitations of previously deployed strategies of K-means clustering analysis and principal component analysis are discussed and a novel methodology of spectral cross-correlation analysis is introduced and compared with the performance of classical least squares analysis, in both unsupervised and supervised modes. The previous study demonstrated the feasibility of using Raman spectroscopy to map cells and identify polystyrene nanoparticles in a lipid rich environment, which is suggestive of the membrane rich endoplasmic reticulum. However, short comings in identification of all nanoparticle signatures in the cell using K-means clustering are apparent, as highlighted by principal component analysis of the identified clusters which demonstrates that K-means clustering does not identify all regions where spectral signatures of the nanoparticles are evident. Thus, two more sophisticated analytical approaches to the extraction of the nanoparticle signatures from the Raman spectral datasets, namely classical least squares analysis and cross-correlation analysis, were employed and are demonstrated to improve the identification of spectroscopic signatures characteristic of polystyrene nanoparticles in a cellular environment. Additionally, to investigate the local biochemical environment in which the nanoparticles are trafficked, a pure spectrum of 3-sn-phosphatidyl ethanolamine was cross-correlated against the Raman dataset, further suggesting the particles are indeed localized in a lipid rich environment. Furthermore, to demonstrate the robustness and versatility of the analysis method, a spectrum of pure RNA was used to demonstrate that a differentiation could be made between DNA of the nucleus and RNA of the nucleolus using the supervised spectral cross-correlation technique.
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http://dx.doi.org/10.1039/c2an36169hDOI Listing
December 2012

Mending broken hearts: the future of drug-based cardiac regeneration.

Authors:
Mark Keating

Future Cardiol 2007 Sep;3(5):493-5

Coronary heart disease is a massive health problem worldwide. In the USA alone, 1.2 million people suffer a coronary attack every year, and approximately 40% of these individuals die as a result. As damaged cardiac muscle forms scar tissue and is unable to function properly, those that do survive the attack are often left severely ill. Consequently, the potential for a therapy that allows healthy cardiac muscle to proliferate at the site of injury is enormous. Dr Mark Keating is a leading researcher in this area of regenerative medicine and his group has recently identified a compound that aids in the process of cardiac regeneration. Although currently focussing on ophthalmic drug research at Novartis, Dr Keating still has a great interest in this area. Here he discusses the potential of drug-based cardiac regeneration and the problems that must be overcome before this treatment can reach the clinic.
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http://dx.doi.org/10.2217/14796678.3.5.493DOI Listing
September 2007

Activin-betaA signaling is required for zebrafish fin regeneration.

Curr Biol 2007 Aug 2;17(16):1390-5. Epub 2007 Aug 2.

Department of Cardiology, Children's Hospital Boston, Boston, Massachusetts 02115, USA.

Vertebrate limb regeneration occurs in anamniotes such as newts, salamanders, and zebrafish. After appendage amputation, the resection site is covered by a wound epidermis capping the underlying mature tissues of the stump from which the blastema emerges. The blastema is a mass of progenitor cells that constitute an apical growth zone. During outgrowth formation, the proximal blastemal cells progressively leave the zone and undergo the differentiation that results in the replacement of the amputated structures. Little is known about the mechanisms triggering regenerative events after injury. The zebrafish caudal fin provides a valuable model to study the mechanisms of regeneration. Zebrafish blastemal cells express specific genes, such as the homeobox-containing transcription factors msxB and msxC, and secreted signal FGF20a. In this study, we set out to identify signals that are transcriptionally upregulated after fin amputation and before blastema formation. Accordingly, a gene encoding a TGFbeta-related ligand, activin-betaA (actbetaA), was found to be strongly induced within 6 hr after fin amputation at the wound margin, and later in the blastema. Inhibition of Activin signaling through two specific chemical inhibitors, SB431542 and SB505124, lead to the early and complete block of regeneration. The morpholino knockdown of actbetaA and its receptor alk4 impaired the progression of regeneration. Closer examination of the phenotype revealed that Activin signaling is necessary for cell migration during wound healing and blastemal proliferation. These findings reveal a role of Activin-betaA signaling in the tissue repair after injury and subsequent outgrowth formation during epigenetic regeneration of the vertebrate appendage.
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http://dx.doi.org/10.1016/j.cub.2007.07.019DOI Listing
August 2007

Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair.

Nat Med 2007 Aug 15;13(8):962-9. Epub 2007 Jul 15.

Department of Cardiology, Children's Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115, USA.

Adult mammalian hearts respond to injury with scar formation and not with cardiomyocyte proliferation, the cellular basis of regeneration. Although cardiogenic progenitor cells may maintain myocardial turnover, they do not give rise to a robust regenerative response. Here we show that extracellular periostin induced reentry of differentiated mammalian cardiomyocytes into the cell cycle. Periostin stimulated mononucleated cardiomyocytes to go through the full mitotic cell cycle. Periostin activated alphaV, beta1, beta3 and beta5 integrins located in the cardiomyocyte cell membrane. Activation of phosphatidylinositol-3-OH kinase was required for periostin-induced reentry of cardiomyocytes into the cell cycle and was sufficient for cell-cycle reentry in the absence of periostin. After myocardial infarction, periostin-induced cardiomyocyte cell-cycle reentry and mitosis were associated with improved ventricular remodeling and myocardial function, reduced fibrosis and infarct size, and increased angiogenesis. Thus, periostin and the pathway that it regulates may provide a target for innovative strategies to treat heart failure.
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http://dx.doi.org/10.1038/nm1619DOI Listing
August 2007

Impaired interaction between the slide helix and the C-terminus of Kir2.1: a novel mechanism of Andersen syndrome.

Cardiovasc Res 2007 Sep 10;75(4):748-57. Epub 2007 May 10.

Nora Eccles Harrison CVRTI and Department of Physiology, University of Utah, Salt Lake City, UT 84112, USA.

Objective: Andersen syndrome (AS) is a rare genetic disease caused by mutations of the potassium channel Kir2.1 (KCNJ2). We identified two unrelated patients with mutations in the slide helix of Kir2.1 leading to AS. The functional consequences of these two mutations, Y68D and D78Y, were studied and compared with previously reported slide helix mutations.

Methods: Channel function and surface expression were studied by voltage clamp recordings and a chemiluminescence assay in Xenopus laevis oocytes and by patch clamp recordings and fluorescence microscopy in HEK293 cells. In addition, a phosphatidylinositol bisphosphate (PIP(2)) binding assay and a yeast-two-hybrid assay were used to characterize the molecular mechanisms by which slide helix mutations cause AS.

Results: Neither mutant channel produced any current, but both had dominant negative effects on Kir2.2, Kir2.3, and Kir2.4 channels. We show that Y68D, D78Y, and previously reported AS mutations are clustered on the hydrophilic, cytosolic side of the slide helix and traffic normally to the plasma membrane. The in vitro lipid binding assay indicated that Y68D or D78Y N-terminal peptides bind PIP(2) similar to wild-type peptides. Yeast-two-hybrid assays showed that AS-associated mutations disturb the interaction between the slide helix and the C-terminal domain of the channel protein.

Conclusion: Our experiments indicate a new disease-causing mechanism independent of trafficking and PIP(2) binding defects. Our findings suggest that the hydrophilic side of the slide helix interacts with a specific domain of the C-terminus facing the membrane. This interaction, which may be required for normal gating both in homomeric and heteromeric Kir2 channels, is disturbed by several mutations causing AS.
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http://dx.doi.org/10.1016/j.cardiores.2007.05.010DOI Listing
September 2007

Transcriptional profiling of caudal fin regeneration in zebrafish.

ScientificWorldJournal 2006 Jun 2;6 Suppl 1:38-54. Epub 2006 Jun 2.

Howard Hughes Medical Institute, Department of Cardiology, Harvard Medical School, 320 Longwood Avenue, Boston, MA 02115, USA.

Regeneration of severed limbs in adult animals is restricted to urodele amphibians. Mammals, including humans, have very limited regenerative capabilities and even with proper treatment, only the tips of our digits can grow back. Teleost fish can regenerate amputated fins, the evolutionary ancestors of limbs. To elucidate the principles of limb-fin regeneration, we performed an Affymetrix microarray screen on regenerating caudal fins 12, 24, 48, and 72 h post amputation. Approximately 15,000 zebrafish transcripts were analyzed, identifying 829 transcripts as differentially expressed during regeneration. Of those, 563 were up-regulated and 266 were down-regulated. We constructed a comprehensive database containing expression data, functional assignment, and background information from the literature for each differentially expressed transcript. In order to validate our findings, we employed three approaches: (1) microarray expression analysis of genes previously implicated in fin regeneration, (2) RT-PCR analysis of genes newly identified as differentially expressed during regeneration, and (3) in situ hybridization of the up-regulated genes bambi, dlx5A, and her6. Moreover, we show that Smad 1/5/8 proteins, effector molecules of Bmp signaling, are phosphorylated during fin regeneration. Taken together, we provide a comprehensive database of fin regeneration that will serve as an important tool for understanding the molecular mechanisms of regeneration.
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http://dx.doi.org/10.1100/tsw.2006.326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917411PMC
June 2006

FGF1/p38 MAP kinase inhibitor therapy induces cardiomyocyte mitosis, reduces scarring, and rescues function after myocardial infarction.

Proc Natl Acad Sci U S A 2006 Oct 10;103(42):15546-51. Epub 2006 Oct 10.

Department of Pediatrics, Harvard Medical School, Children's Hospital, 320 Longwood Avenue, Boston, MA 02115, USA.

Mammalian cardiomyocytes have limited proliferation potential, and acutely injured mammalian hearts do not regenerate adequately. Instead, injured myocardium develops fibrosis and scarring. Here we show that FGF1/p38 MAP kinase inhibitor treatment after acute myocardial injury in 8- to 10-week-old rats increases cardiomyocyte mitosis. At 3 months after injury, 4 weeks of FGF1/p38 MAP kinase inhibitor therapy results in reduced scarring and wall thinning, with markedly improved cardiac function. In contrast, p38 MAP kinase inhibition alone fails to rescue heart function despite increased cardiomyocyte mitosis. FGF1 improves angiogenesis, possibly contributing to the survival of newly generated cardiomyocytes. Our data indicate that FGF1 and p38 MAP kinase, proteins involved in cardiomyocyte proliferation and angiogenesis during development, may be delivered therapeutically to enhance cardiac regeneration.
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http://dx.doi.org/10.1073/pnas.0607382103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1622860PMC
October 2006

The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes.

Chem Biol 2006 Sep;13(9):957-63

Howard Hughes Medical Institute, Department of Cardiology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

The maintenance of self-renewal in stem cells appears to be distinct from the induction of proliferation of the terminally differentiated mammalian cardiomyocytes because it is believed that the latter are unable to divide. However, proliferation is a necessary step in both processes. Interestingly, the small molecule 6-bromoindirubin-3'-oxime (BIO) is the first pharmacological agent shown to maintain self-renewal in human and mouse embryonic stem cells. To determine whether a molecule that can maintain stem cell properties can also participate in controlling the proliferative capability of the highly differentiated cardiomyocytes, we examine the effect of BIO in postmitotic cardiac cells. Here, we show that BIO promotes proliferation in mammalian cardiomyocytes. Our demonstration of a second role for BIO suggests that the maintenance of stem cell self-renewal and the induction of proliferation in differentiated cardiomyocytes may share common molecular pathways.
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http://dx.doi.org/10.1016/j.chembiol.2006.08.004DOI Listing
September 2006

Anillin localization defect in cardiomyocyte binucleation.

J Mol Cell Cardiol 2006 Oct 4;41(4):601-12. Epub 2006 Aug 4.

Department of Pediatrics, Harvard Medical School, and Department of Cardiology, Children's Hospital, 320 Longwood Avenue, Boston, MA 02115, USA.

Heart growth is augmented during early development by cardiomyocyte proliferation. In contrast, heart growth during postnatal life occurs by increasing cell size. Postnatal cardiomyocytes can undergo DNA synthesis, mitosis and binucleation. However, they lose the ability to complete cytokinesis. The underlying mechanism is poorly understood. It has been suggested that incomplete disassembly of contractile elements prohibits cytokinesis. Here, we show that serum-induced binucleation results in the normal disassembly of the contractile apparatus. In contrast, analysis of Aurora B and Anillin localization demonstrates that binucleation is characterized by asymmetric constriction, delay of furrow constriction and defective mid-body formation. Anillin fails to focus at the cortex in anaphase and shows an expanded localization around the mid-body during cytokinesis. p38 inhibition rescues the mid-body formation defect. We show that p38 accumulates during cytokinesis at the mid-body and suggest that p38 activity has a regulatory role in cytokinesis. Microarray analysis reveals that p38 inhibition upregulates core components of the central spindle. Taken together, our results demonstrate that postnatal cardiomyocytes form a cleavage furrow and that binucleation is associated with an Anillin localization defect.
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http://dx.doi.org/10.1016/j.yjmcc.2006.06.012DOI Listing
October 2006

Gene expression analysis of zebrafish heart regeneration.

PLoS Biol 2006 Aug;4(8):e260

Department of Cardiology, Children's Hospital, Boston, Massachusetts, USA.

Mammalian hearts cannot regenerate. In contrast, zebrafish hearts regenerate even when up to 20% of the ventricle is amputated. The mechanism of zebrafish heart regeneration is not understood. To systematically characterize this process at the molecular level, we generated transcriptional profiles of zebrafish cardiac regeneration by microarray analyses. Distinct gene clusters were identified based on temporal expression patterns. Genes coding for wound response/inflammatory factors, secreted molecules, and matrix metalloproteinases are expressed in regenerating heart in sequential patterns. Comparisons of gene expression profiles between heart and fin regeneration revealed a set of regeneration core molecules as well as tissue-specific factors. The expression patterns of several secreted molecules around the wound suggest that they play important roles in heart regeneration. We found that both platelet-derived growth factor-a and -b (pdgf-a and pdgf-b) are upregulated in regenerating zebrafish hearts. PDGF-B homodimers induce DNA synthesis in adult zebrafish cardiomyocytes. In addition, we demonstrate that a chemical inhibitor of PDGF receptor decreases DNA synthesis of cardiomyocytes both in vitro and in vivo during regeneration. Our data indicate that zebrafish heart regeneration is associated with sequentially upregulated wound healing genes and growth factors and suggest that PDGF signaling is required.
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http://dx.doi.org/10.1371/journal.pbio.0040260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1523227PMC
August 2006

CACNA1H mutations in autism spectrum disorders.

J Biol Chem 2006 Aug 5;281(31):22085-22091. Epub 2006 Jun 5.

Howard Hughes Medical Institute, Department of Cardiology, Children's Hospital, Departments of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115; Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type Ca(V)1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type Ca(V)3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p = 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce Ca(V)3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype.
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http://dx.doi.org/10.1074/jbc.M603316200DOI Listing
August 2006

Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.

Nat Chem Biol 2006 May 26;2(5):265-73. Epub 2006 Mar 26.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

We examined the role of angiogenesis and the need for receptor signaling using chemical inhibition of the vascular endothelial growth factor receptor in the adult zebrafish tail fin. Using a small-molecule inhibitor, we were able to exert precise control over blood vessel regeneration. An angiogenic limit to tissue regeneration was determined, as avascular tissue containing skin, pigment, neuronal axons and bone precursors could regenerate up to about 1 mm. This indicates that tissues can regenerate without direct interaction with endothelial cells and at a distance from blood supply. We also investigated whether the effects of chemical inhibition could be enhanced in zebrafish vascular mutants. We found that adult zebrafish, heterozygous for a mutation in the critical receptor effector phospholipase Cgamma1, show a greater sensitivity to chemical inhibition. This study illustrates the utility of the adult zebrafish as a new model system for receptor signaling and chemical biology.
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http://dx.doi.org/10.1038/nchembio778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1534118PMC
May 2006

fgf20 is essential for initiating zebrafish fin regeneration.

Science 2005 Dec;310(5756):1957-60

Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Department of Cardiology, Children's Hospital, Boston, MA 02115, USA.

Epimorphic regeneration requires the presence or creation of pluripotent cells capable of reproducing lost organs. Zebrafish fin regeneration is mediated by the creation of blastema cells. Here, we characterize the devoid of blastema (dob) mutant that fails fin regeneration during initial steps, forms abnormal regeneration epithelium, and does not form blastema. This mutation has no impact on embryonic survival. Dob results from an fgf20a null mutation, Y148S. Fgf20a is expressed during initiation of fin regeneration at the epithelial-mesenchymal boundary and later overlaps with the blastema marker msxb. Thus, fgf20a has a regeneration-specific requirement, initiating fin regeneration, and controlling blastema formation.
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http://dx.doi.org/10.1126/science.1117637DOI Listing
December 2005

Tissue inhibitor of metalloproteinase 1 regulates matrix metalloproteinase activity during newt limb regeneration.

Dev Dyn 2006 Mar;235(3):606-16

Department of Internal Medicine, Division of Cardiology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA.

Matrix metalloproteinase (MMP) activity is important for newt limb regeneration. In most biological processes that require MMP function, MMP activity is tightly controlled by a variety of mechanisms, including the coexpression of natural inhibitors. Here, we show that gene expression of one such inhibitor, tissue inhibitor of metalloproteinase 1 (NvTIMP1), is upregulated during the wound healing and dedifferentiation stages of regeneration when several MMPs are at their maximal expression levels. Newt MMPs and NvTIMP1 also exhibit similar spatial expression patterns during the early stages of limb regeneration. NvTIMP1 inhibits the proteolytic activity of regeneration-related newt MMPs and, like human TIMP1, can induce a weak mitogenic response in certain cell types. These results suggest that NvTIMP1 may be functioning primarily to maintain optimal levels of MMP activity during the early stages of limb regeneration, while possibly serving a secondary role as a mitogen.
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http://dx.doi.org/10.1002/dvdy.20654DOI Listing
March 2006

Generation and characterization of Sca2 (ataxin-2) knockout mice.

Biochem Biophys Res Commun 2006 Jan 8;339(1):17-24. Epub 2005 Nov 8.

Rose Moss Laboratory for Parkinson and Neurodegenerative Diseases, Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.

Ataxin-2, the gene product of the Spinocerebellar Ataxia Type 2 (SCA2) gene, is a protein of unknown function with abundant expression in embryonic and adult tissues. Its interaction with A2BP1/Fox-1, a protein with an RNA recognition motif, suggests involvement of ataxin-2 in mRNA translation or transport. To study the effects of in vivo ataxin-2 function, we generated an ataxin-2 deficient mouse strain. Ataxin-2 deficient mice were viable. Genotypic analysis of litters from mating of heterozygous mice showed segregation distortion with a significant reduction in the birth of Sca-/- females. Detailed macroscopic and microscopic analysis of surviving nullizygous Sca2 knockout mice showed no major histological abnormalities. On a fat-enriched diet, ataxin-2 deficient animals had increased weight gain. Our results demonstrate that ataxin-2, although widely expressed, is not essential in development or during adult survival in the mouse, but leads to adult-onset obesity.
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http://dx.doi.org/10.1016/j.bbrc.2005.10.186DOI Listing
January 2006

Heat-shock protein 60 is required for blastema formation and maintenance during regeneration.

Proc Natl Acad Sci U S A 2005 Oct 4;102(41):14599-604. Epub 2005 Oct 4.

Howard Hughes Medical Institute, Department of Cardiology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Zebrafish fin regeneration requires the formation and maintenance of blastema cells. Blastema cells are not derived from stem cells but behave as such, because they are slow-cycling and are thought to provide rapidly proliferating daughter cells that drive regenerative outgrowth. The molecular basis of blastema formation is not understood. Here, we show that heat-shock protein 60 (hsp60) is required for blastema formation and maintenance. We used a chemical mutagenesis screen to identify no blastema (nbl), a zebrafish mutant with an early fin regeneration defect. Fin regeneration failed in nbl due to defective blastema formation. nbl also failed to regenerate hearts. Positional cloning and mutational analyses revealed that nbl results from a V324E missense mutation in hsp60. This mutation reduced hsp60 function in binding and refolding denatured proteins. hsp60 expression is increased during formation of blastema cells, and dysfunction leads to mitochondrial defects and apoptosis in these cells. These data indicate that hsp60 is required for the formation and maintenance of regenerating tissue.
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http://dx.doi.org/10.1073/pnas.0507408102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1253607PMC
October 2005
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