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1.
Artigo em Inglês | MEDLINE | ID: mdl-30479120

RESUMO

The mitochondria have emerged as a novel target for cancer chemotherapy primarily due to their central roles in energy metabolism and apoptosis regulation. Here we report a new molecular approach to achieve high levels of tumor- and mitochondria-selective delivery of the anticancer drug doxorubicin. This is achieved by molecular engineering which functionalizes doxorubicin with a hydrophobic lipid tail conju-gated by a solubility-promoting polyethylene glycol polymer (amphiphilic Doxorubicin or amph-DOX). In vivo, the amphiphile conjugated to doxorubicin exhibits a dual function: i) it binds avidly to serum albumin and hijacks albumin's circulating and transporting pathways, resulting in prolonged circulation in blood, increased accumulation in tumor, and reduced exposure to the heart; ii) it also redirects doxorubicin to mi-tochondria by altering the drug molecule's intracellular sorting and transportation routes. Efficient mito-chondrial targeting with amph-DOX causes a significant increase of reactive oxygen species (ROS) levels in tumor cells, resulting in markedly improved antitumor efficacy than the unmodified doxorubicin. Am-phiphilic modification provides a simple strategy to simultaneously increase the efficacy and safety of doxorubicin in cancer chemotherapy.

2.
Sci Rep ; 8(1): 3294, 2018 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-29459640

RESUMO

Efforts for tissue engineering vascular grafts focuses on the tunica media and intima, although the tunica adventitia serves as the primary structural support for blood vessels. In surgery, during endarterectomies, surgeons can strip the vessel, leaving the adventitia as the main strength layer to close the vessel. Here, we adapted our recently developed technique of forming vascular tissue rings then stacking the rings into a tubular structure, to accommodate human fibroblasts to create adventitia vessels in 8 days. Collagen production and fibril cross-linking was augmented with TGF-ß and ascorbic acid, significantly increasing tensile strength to 57.8 ± 3.07 kPa (p = 0.008). Collagen type I gel was added to the base fibrin hydrogel to further increase strength. Groups were: Fibrin only; 0.7 mg/ml COL; 1.7 mg/ml COL; and 2.2 mg/ml COL. The 0.7 mg/ml collagen rings resulted in the highest tensile strength at 77.0 ± 18.1 kPa (p = 0.015). Culture periods of 1-2 weeks resulted in an increase in extracellular matrix deposition and significantly higher failure strength but not ultimate tensile strength. Histological analysis showed the 0.7 mg/ml COL group had significantly more, mature collagen. Thus, a hydrogel of 0.7 mg/ml collagen in fibrin was ideal for creating and strengthening engineered adventitia vessels.

3.
Stem Cell Res Ther ; 8(1): 230, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29037258

RESUMO

BACKGROUND: Application of cardiac stem cells combined with biomaterial scaffold is a promising therapeutic strategy for heart repair after myocardial infarction. However, the optimal cell types and biomaterials remain elusive. METHODS: In this study, we seeded Isl1+ embryonic cardiac progenitor cells (CPCs) into decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM) to assess the therapeutic potential of Isl1+ CPCs and the biocompatibility of SIS-ECM with these cells. RESULTS: We observed that SIS-ECM supported the viability and attachment of Isl1+ CPCs. Importantly, Isl1+ CPCs differentiated into cardiomyocytes and endothelial cells 7 days after seeding into SIS-ECM. In addition, SIS-ECM with CPC-derived cardiomyocytes showed spontaneous contraction and responded to ß-adrenergic stimulation. Next, patches of SIS-ECM seeded with CPCs for 7 days were transplanted onto the outer surface of infarcted myocardium in mice. Four weeks after transplantation, the patches were tightly attached to the surface of the host myocardium and remained viable. Transplantation of patches improved cardiac function, decreased the left ventricular myocardial scarring area, and reduced fibrosis and heart failure. CONCLUSIONS: Transplantation of Isl1+ CPCs seeded in SIS-ECM represents an effective approach for cell-based heart therapy.


Assuntos
Mucosa Intestinal/metabolismo , Mioblastos Cardíacos/transplante , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Transplante de Células-Tronco/métodos , Animais , Diferenciação Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Intestino Delgado/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Mioblastos Cardíacos/citologia , Mioblastos Cardíacos/metabolismo , Miócitos Cardíacos/metabolismo , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
J Vis Exp ; (121)2017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28447994

RESUMO

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.


Assuntos
Prótese Vascular , Impressão Tridimensional , Engenharia Tecidual , Enxerto Vascular/métodos , Animais , Vasos Coronários/cirurgia , Humanos
5.
Plast Reconstr Surg Glob Open ; 4(9): e864, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27757329

RESUMO

Efforts have been made to engineer knee meniscus tissue for injury repair, yet most attempts have been unsuccessful. Creating a cell source that resembles the complex, heterogeneous phenotype of the meniscus cell remains difficult. Stem cell differentiation has been investigated, mainly using bone marrow mesenchymal cells and biochemical means for differentiation, resulting in no solution. Mechanical stimulation has been investigated to an extent with no conclusion. Here, we explore the potential for and effectiveness of mechanical stimulation to induce the meniscal phenotype in adipose-derived stromal cells. METHODS: Human adipose-derived stromal cells were chosen for their fibrogenic nature and conduciveness for chondrogenesis. Biochemical and mechanical stimulation were investigated. Biochemical stimulation included fibrogenic and chondrogenic media. For mechanical stimulation, a custom-built device was used to apply constant, cyclical, uniaxial strain for up to 6 hours. Strain and frequency varied. RESULTS: Under biochemical stimulation, both fibrogenic (collagen I, versican) and chondrogenic (collagen II, Sox9, aggrecan) genes were expressed by cells exposed to either fibrogenic or chondrogenic biochemical factors. Mechanical strain was found to preferentially promote fibrogenesis over chondrogenesis, confirming that tensile strain is an effective fibrogenic cue. Three hours at 10% strain and 1 Hz in chondrogenic media resulted in the highest expression of fibrochondrogenic genes. Although mechanical stimulation did not seem to affect protein level expression, biochemical means did affect protein level presence of collagen fibers. CONCLUSION: Mechanical stimulation can be a useful differentiation tool for mechanoresponsive cell types as long as biochemical factors are also integrated.

7.
Methods ; 99: 20-7, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26732049

RESUMO

Current techniques for tissue engineering blood vessels are not customizable for vascular size variation and vessel wall thickness. These critical parameters vary widely between the different arteries in the human body, and the ability to engineer vessels of varying sizes could increase capabilities for disease modeling and treatment options. We present an innovative method for producing customizable, tissue engineered, self-organizing vascular constructs by replicating a major structural component of blood vessels - the smooth muscle layer, or tunica media. We utilize a unique system combining 3D printed plate inserts to control construct size and shape, and cell sheets supported by a temporary fibrin hydrogel to encourage cellular self-organization into a tubular form resembling a natural artery. To form the vascular construct, 3D printed inserts are adhered to tissue culture plates, fibrin hydrogel is deposited around the inserts, and human aortic smooth muscle cells are then seeded atop the fibrin hydrogel. The gel, aided by the innate contractile properties of the smooth muscle cells, aggregates towards the center post insert, creating a tissue ring of smooth muscle cells. These rings are then stacked into the final tubular construct. Our methodology is robust, easily repeatable and allows for customization of cellular composition, vessel wall thickness, and length of the vessel construct merely by varying the size of the 3D printed inserts. This platform has potential for facilitating more accurate modeling of vascular pathology, serving as a drug discovery tool, or for vessel repair in disease treatment.


Assuntos
Prótese Vascular , Engenharia Tecidual , Materiais Biocompatíveis/química , Células Cultivadas , Expressão Gênica , Humanos , Hidrogéis/química , Teste de Materiais , Contração Muscular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/fisiologia , Impressão Tridimensional , Resistência à Tração
8.
Endocrinology ; 155(8): 2942-52, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24693964

RESUMO

Evidence suggests that when presented with novel acute stress, animals previously exposed to chronic homotypic or heterotypic stressors exhibit normal or enhanced hypothalamic-pituitary-adrenal (HPA) response compared with animals exposed solely to that acute stressor. The molecular mechanisms involved in this effect remain unknown. The extracellular signal-regulated kinase (ERK) is one of the key pathways regulated in the hippocampus in both acute and chronic stress. The aim of this study was to examine the interaction of prior chronic stress, using the chronic variable stress model (CVS), with exposure to a novel acute stressor (2,5-dihydro-2,4,5-trimethyl thiazoline; TMT) on ERK activation, expression of the downstream protein BCL-2, and the glucocorticoid receptor co-chaperone BAG-1 in control and chronically stressed male rats. TMT exposure after chronic stress resulted in a significant interaction of chronic and acute stress in all 3 hippocampus subregions on ERK activation and BCL-2 expression. Significantly, acute stress increased ERK activation, BCL-2 and BAG-1 protein expression in the dentate gyrus (DG) of CVS-treated rats compared with control, CVS-treated alone, and TMT-only animals. Furthermore, CVS significantly increased ERK activation in medial prefrontal cortex, but acute stress had no significant effect. Inhibition of corticosterone synthesis with metyrapone had no significant effect on ERK activation in the hippocampus; therefore, glucocorticoids alone do not mediate the molecular effects. Finally, because post-translational modifications of histones are believed to play an important role in the stress response, we examined changes in histone acetylation. We found that, in general, chronic stress decreased K12H4 acetylation, whereas acute stress increased acetylation. These results indicate a molecular mechanism by which chronic stress-induced HPA axis plasticity can lead to neurochemical alterations in the hippocampus that influence reactivity to subsequent stress exposure. This may represent an important site of dysfunction that contributes to stress-induced pathology such as depression, anxiety disorders, and posttraumatic stress disorder.


Assuntos
Giro Denteado/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema de Sinalização das MAP Quinases , Sistema Hipófise-Suprarrenal/metabolismo , Estresse Psicológico/metabolismo , Acetilação , Acetiltransferases/metabolismo , Animais , Histonas/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Estresse Fisiológico , Tiazóis
9.
Tissue Eng Part A ; 19(5-6): 738-47, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23072446

RESUMO

Wound healing is one of the most complex biological processes and occurs in all tissues and organs of the body. In humans, fibrotic tissue, or scar, hinders function and is aesthetically unappealing. Stem cell therapy offers a promising new technique for aiding in wound healing; however, current findings show that stem cells typically die and/or migrate from the wound site, greatly decreasing efficacy of the treatment. Here, we demonstrate effectiveness of a stem cell therapy for improving wound healing in the skin and reducing scarring by introducing stem cells using a natural patch material. Adipose-derived stromal cells were introduced to excisional wounds created in mice using a nonimmunogenic extracellular matrix (ECM) patch material derived from porcine small-intestine submucosa (SIS). The SIS served as an attractive delivery vehicle because of its natural ECM components, including its collagen fiber network, providing the stem cells with a familiar structure. Experimental groups consisted of wounds with stem cell-seeded patches removed at different time points after wounding to determine an optimal treatment protocol. Stem cells delivered alone to skin wounds did not survive post-transplantation as evidenced by bioluminescence in vivo imaging. In contrast, delivery with the patch enabled a significant increase in stem cell proliferation and survival. Wound healing rates were moderately improved by treatment with stem cells on the patch; however, areas of fibrosis, indicating scarring, were significantly reduced in wounds treated with the stem cells on the patch compared to untreated wounds.


Assuntos
Cicatriz/patologia , Matriz Extracelular/metabolismo , Pele/patologia , Transplante de Células-Tronco , Células-Tronco/citologia , Cicatrização , Animais , Proliferação de Células , Sobrevivência Celular , Feminino , Fibrose , Imunofluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indóis/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Medições Luminescentes , Camundongos , Células Estromais/patologia , Sus scrofa
10.
Expert Rev Cardiovasc Ther ; 10(8): 1039-49, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23030293

RESUMO

Cardiovascular disease physically damages the heart, resulting in loss of cardiac function. Medications can help alleviate symptoms, but it is more beneficial to treat the root cause by repairing injured tissues, which gives patients better outcomes. Besides heart transplants, cardiac surgeons use a variety of methods for repairing different areas of the heart such as the ventricular septal wall and valves. A multitude of biomaterials are used in the repair and replacement of impaired heart tissues. These biomaterials fall into two main categories: synthetic and natural. Synthetic materials used in cardiovascular applications include polymers and metals. Natural materials are derived from biological sources such as human donor or harvested animal tissues. A new class of composite materials has emerged to take advantage of the benefits of the strengths and minimize the weaknesses of both synthetic and natural materials. This article reviews the current and prospective applications of biomaterials in cardiovascular therapies.


Assuntos
Materiais Biocompatíveis/uso terapêutico , Doenças Cardiovasculares/terapia , Fenômenos Fisiológicos Cardiovasculares , Regeneração , Animais , Bioprótese , Cardiologia/métodos , Cardiologia/tendências , Doenças Cardiovasculares/cirurgia , Humanos , Medicina Regenerativa/métodos , Medicina Regenerativa/tendências , Engenharia Tecidual/métodos , Engenharia Tecidual/tendências
11.
J Tissue Eng Regen Med ; 6 Suppl 3: s80-6, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22610948

RESUMO

As actual stem cell application quickly approaches tissue engineering and regenerative medicine, aspects such as cell attachment to scaffolds and biomaterials become important and are often overlooked. Here, we compare the effects of several attachment proteins on the adhesion, proliferation and stem cell identity of three promising human stem cell types: human adipose-derived stem cells (hASCs), human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Traditional tissue culture polystyrene plates (TCPS), Matrigel (Mat), laminin (Lam), fibronectin (FN) and poly-L-lysine (PLL) were investigated as attachment protein surfaces. For hASCs typically cultured on TCPS, laminin resulted in the greatest cell attachment and proliferation with largest cell areas, indicating favourability by cell spreading. However, mesenchymal stem cell markers indicative of hASCs were slightly more expressed on surfaces with lowest cell attachment, corresponding to increased cell roundness, a newly observed attribute in hASCs possibly indicating a more stem cell-like character. hESCs preferred Matrigel as a feeder-free culture surface. Interestingly, hiPSCs favoured laminin over Matrigel for colony expansion, shown by larger cell colony area and perimeter lengths, although cell numbers and stem cell marker expression level remained highest on Matrigel. These data provide a practical reference guide for selecting a suitable attachment method for using human induced pluripotent, embryonic or adipose stem cells in tissue engineering and regenerative medicine applications.


Assuntos
Tecido Adiposo/citologia , Adesão Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Engenharia Tecidual , Humanos , Tecidos Suporte
12.
Tissue Eng Part A ; 17(3-4): 495-502, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20807017

RESUMO

The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.


Assuntos
Fibroblastos/citologia , Fibroblastos/fisiologia , Microscopia de Força Atômica/métodos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Módulo de Elasticidade/fisiologia , Humanos
13.
Tissue Eng Part C Methods ; 16(5): 999-1009, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20001822

RESUMO

BACKGROUND: The objective of this study was to develop a physiological model of longitudinal smooth muscle tissue from isolated longitudinal smooth muscle cells arranged in the longitudinal axis. METHODS: Longitudinal smooth muscle cells from rabbit sigmoid colon were isolated and expanded in culture. Cells were seeded at high densities onto laminin-coated Sylgard surfaces with defined wavy microtopographies. A highly aligned cell sheet was formed, to which addition of fibrin resulted in delamination. RESULTS: (1) Acetylcholine (ACh) induced a dose-dependent, rapid, and sustained force generation. (2) Absence of extracellular calcium attenuated the magnitude and sustainability of ACh-induced force by 50% and 60%, respectively. (3) Vasoactive intestinal peptide also attenuated the magnitude and sustainability of ACh-induced force by 40% and 60%, respectively. These data were similar to force generated by longitudinal tissue. (4) Bioengineered constructs also maintained smooth muscle phenotype and calcium-dependence characteristics. SUMMARY: This is a novel physiologically relevant in vitro three-dimensional model of colonic longitudinal smooth muscle tissue. Bioengineered three-dimensional longitudinal smooth muscle presents the ability to generate force, and respond to contractile agonists and relaxant peptides similar to native longitudinal tissue. This model has potential applications to investigate the underlying pathophysiology of dysfunctional colonic motility. It also presents as a readily implantable band-aid colonic longitudinal muscle tissue.


Assuntos
Colo/fisiologia , Músculo Liso/fisiologia , Engenharia Tecidual , Animais , Cálcio/metabolismo , Células Cultivadas , Colo/citologia , Meios de Cultura , Imunofluorescência , Músculo Liso/citologia , Coelhos
14.
Biomaterials ; 30(6): 1150-5, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19064284

RESUMO

Engineering tissue similar in structure to their natural equivalents is a major challenge and crucial to function. Despite attempts to engineer skeletal muscle, it is still difficult to effectively mimic tissue architecture. Rigid scaffolds can guide cell alignment but have the critical drawback of hindering mechanical function of the resultant tissue. We present a method for creating highly ordered tissue-only constructs by using rigid microtopographically patterned surfaces to first guide myoblast alignment, followed by transfer of aligned myotubes into a degradable hydrogel and self-organization of the ordered cells into a functional, 3-dimensional, free-standing construct independent of the initial template substrate. Histology revealed an intracellular organization resembling that of native muscle. Aligned cell constructs exhibited a 2-fold increase in peak force production compared to controls. Effective specific force, or force normalized over cross-sectional area, was increased by 23%. This template, transfer, and self-organization strategy is envisioned to be broadly useful in improving construct function and clinical applicability for highly ordered tissues like muscle.


Assuntos
Músculo Esquelético/fisiologia , Engenharia Tecidual , Tecidos Suporte , Animais , Células Cultivadas , Feminino , Músculo Esquelético/citologia , Ratos , Ratos Sprague-Dawley
15.
Biomaterials ; 29(11): 1705-12, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18192004

RESUMO

Traditional cell culture substrates consist of static, flat surfaces although in vivo, cells exist on various dynamic topographies. We report development of a reconfigurable microtopographical system compatible with cell culture that is comprised of reversible wavy microfeatures on poly(dimethylsiloxane). Robust reversibility of the wavy micropattern is induced on the cell culture customized substrate by first plasma oxidizing the substrate to create a thin, brittle film on the surface and then applying and releasing compressive strain, to introduce and remove the microfeatures, respectively. The reversible topography was able to align, unalign, and realign C2C12 myogenic cell line cells repeatedly on the same substrate within 24 h intervals, and did not inhibit cell differentiation. The flexibility and simplicity of the materials and methods presented here provide a broadly applicable capability by which to investigate and compare dynamic cellular processes not yet easily studied using conventional in vitro culture substrates.


Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Animais , Linhagem Celular , Camundongos
16.
Biomaterials ; 27(24): 4340-7, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16650470

RESUMO

Tissue-engineered muscle is a viable option for tissue repair, though presently technologies are not developed enough to produce tissue in vitro identical to that in vivo. One important step in generating accurate engineered muscle is to mimic natural muscle architecture. Skeletal muscle is composed of fibrils whose organization defines functionality. In musculoskeletal myogenesis, aligning myoblasts in preparation for myotube formation is a crucial step. The ability to efficiently organize myoblasts to form aligned myotubes in vitro would greatly benefit efforts in muscle tissue engineering. This paper reports alignment of prefused and differentiated skeletal muscle cells in vitro by use of continuous micropatterned wavy silicone surfaces, with features sized 3, 6 and 12 microm in periodicity. Wave features with 6 microm periodicity produced the most healthy, aligned myoblasts. Alignment was found to be a function of plating density. Further growth on these substrates with aligned myoblasts promoted fusion, yielding healthy aligned myotubes. This method will be useful for applications in which differentiated myogenic cells need to be aligned unidirectionally as in the development of engineered muscle.


Assuntos
Materiais Biocompatíveis , Fibras Musculares Esqueléticas , Mioblastos Esqueléticos , Silicones , Animais , Técnicas de Cultura de Células , Linhagem Celular , Camundongos
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