RESUMO
Induced pluripotent stem cells (iPSCs) are a promising cell source for regenerative medicine. However, their feeder-free maintenance in undifferentiated states remains challenging. In recent past extensive studies have been directed using pristine or functionalized carbon nanotube in tissue engineering. Here we proposed thin films of functionalized carbon nanotubes (OH-single-walled CNTs [SWCNTs] and OH-multiwalled CNTs [MWCNTs]), as alternatives for the feeder-free in vitro culture of canine iPSCs (ciPSCs), considered as the cellular model. The ciPSC colonies could maintain their dome-shaped compactness and other characteristics when propagated on CNT films. Concomitantly, high cell viability and upregulation of pluripotency-associated genes and cell adhesion molecules were observed, further supported by molecular docking. Moreover, CNTs did not have profound toxic effects compared to feeder cultures as evident by cytocompatibility studies. Further, cardiac and neuronal differentiation of ciPSCs was induced on these films to determine their influence on the differentiation process. The cells retained differentiation potential and the nanotopographical features of the substrates provided positive cues to enhance differentiation to both lineages as evident by immunocytochemical staining and marker gene expression. Overall, OH-SWCNT provided better cues, maintained pluripotency, and induced the differentiation of ciPSCs. These results indicate that OH-functionalized CNT films could be used as alternatives for the feeder-free maintenance of ciPSCs towards prospective utilization in regenerative medicine.
Assuntos
Células-Tronco Pluripotentes Induzidas , Nanotubos de Carbono , Animais , Diferenciação Celular/fisiologia , Cães , Simulação de Acoplamento Molecular , Nanotubos de Carbono/química , Estudos ProspectivosRESUMO
Some imaging techniques reduce the effect of optical aberrations either by detecting and actively compensating for them or by utilizing interferometry. A microscope based on a Mach-Zehnder interferometer has been recently introduced to allow obtaining sharp images of light-transmitting objects in the presence of strong aberrations. However, the method is not capable of imaging microstructures on opaque substrates. In this work, we use a Michelson interferometer to demonstrate imaging of reflecting and back-scattering objects on any substrate with micrometer-scale resolution. The system is remarkably insensitive to both deterministic and random aberrations that can completely destroy the object's intensity image.
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The role of growth factors in the modulation of ovarian function is an interesting area of research in reproductive biology. Recently, we have shown the expression and role of IGF, EGF, VEGF and FGF in the follicle and CL. Here, we report the presence of Bone Morphogenetic Proteins (BMPs) and their functional receptors in the corpus luteum (CL) of buffalo. The bubaline CL was classified into four stages according to the morphology and progesterone (P4) concentration. The qPCR, immunoblot and immunohistochemistry studies revealed that BMP2 and BMP Receptors (BMPR1A, BMPR1B and BMPR2) were significantly upregulated during the mid stage whereas BMP4 and BMP7 were upregulated during the early stage of CL (P<0.05). Studies on primary luteal cell culture (LCC) using mid CL showed a significant time and concentration dependent effect of BMP4 and BMP7 (P<0.05). At 100ngml-1, the BMPs maximally stimulated the transcripts of StAR, CYP11A1 and 3ßHSD that paralleled with P4 accretion in the media (P<0.05). Further, the BMP4 as well as BMP7 upregulated the transcripts of PCNA and downregulated CASPASE3 in the LCC at the same concentration (P<0.05). Though the combined effect of BMP4 and 7 was significantly higher (P<0.05) than that of individual one, it was not additive. In conclusion, the expression of BMPs and their receptors were dependent on the stages of CL in the buffalo. Treatment of LCC with BMPs in vitro confirmed the presence of functional receptors that stimulated the P4 production and luteal cell survival. Moreover, the results support the concept that the upregulation of P4 and its biosynthetic pathway enzymes such as CYP11A1, StAR and 3ßHSD in the CL is likely due to the autocrine and /or paracrine effects of BMP4 and BMP7 under physiological milieu.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Búfalos/genética , Corpo Lúteo/metabolismo , Regulação da Expressão Gênica , Animais , Apoptose , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Immunoblotting , Imuno-Histoquímica , Progesterona/genética , Progesterona/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Although amyloids are a well-known pathological structure, functional amyloids are beneficial. Functional amyloids can be engineered to cultivate desired functionality that can destroy malicious amyloids. However, not much is known about such amyloid destructors. On the other hand, nearly all approaches to developing a drug against pathogenic amyloidoses have failed. Therefore, novel approaches are needed. Herein, a rationally designed catalytic triad based "proteolytic functional amyloid (PFA)" formed by linking a target protein recognizing unit with a Gly-integrated non-native Glu-His-Cys segment is reported. The proteolytic mechanism of the designed PFA is boosted by thiol/disulfide exchange cleavage. Rigorous MALDI-TOF and FRET-based analyses indicated that the PFAs cleaved Aß12-21 (model Aß) and the pathogenic Aß1-40 site selectively in vitro. PFA significantly inhibited the aggregation of Aß1-40 and broke-down the preformed fibrillar amyloids into non-toxic metabolites. Such a platform may be helpful in not only the on-demand chemical degradation of pathogenic amyloids but also the targeted degradation of other malicious proteins. PFAs are the first "amyloid-destroying" amyloids.
Assuntos
Amiloidose , Fluorocarbonos , Amiloide/química , Proteínas Amiloidogênicas/química , Amiloidose/metabolismo , Amiloidose/patologia , Humanos , Peptídeo HidrolasesRESUMO
Orchestration of differential architectures of designer peptidomimetics that modulate metal oxidation states to perform multiple chemical transformations remains a challenge. Cu-chelation and self-assembly properties of amyloid ß (Aß14-23) peptide were tuned by the incorporation of cyclic dipeptide (CDP) and pyrene (Py) as the assembly directing and reporting units, respectively. We explore the molecular architectonics of Aß14-23 derived peptidomimetics (AkdNMCPy) to form differential architectures that stabilize distinct Cu oxidation states. The fibrillar self-assembly of AkdNMCPy is modulated to form nanosheets by the one-off addition of CuII. Notably, the serial addition of CuII resulted in the formation of micelle-like core-shell architectures. The micelle-like and nanosheet architectures were found to differentially stabilize CuII and CuI states and catalyze tandem oxidative-hydrolysis and alkyne-azide cycloaddition reactions, respectively.
RESUMO
Induced pluripotent stem cells (iPSCs) have attracted an interest for personalized cell based therapy along with various other applications. There have been few studies that effective nanomaterial based scaffolds act as alternative to the commonly used feeder dependent in vitro maintenance of iPSCs. The present study provides the fundamental information on ex vivo behavior of canine iPSC (ciPSCs) maintained on carboxylic acid (COOH) functionalized single-walled carbon nanotubes (COOH-SWCNTs) and multi-walled carbon nanotubes (COOH-MWCNTs) substrates. Here in we evaluated the comparative colony morphology, propagation, characterization, cytocompatibility and differentiation capability of ciPSC cultured on MEF feeder taken as control, and COOH-SWCNTs and COOH-MWCNTs substrates. We observed a healthy growth of ciPSCs on both the types of carbon nanotubes (CNTs) similar to feeder. The ciPSC colonies grown on both CNTs were positive for alkaline phosphatase staining and expressed pluripotent markers with notable significance. Further, the ciPSC colonies grew on these CNTs retained the in vitro differentiation ability into three germ layers as well as cardiac cell. Cytotoxicity analysis revealed that (COOH) functionalized CNTs provided a culture condition of low cytotoxicity. The results of the present study indicated that (COOH) functionalized CNTs could be used as xeno-free substrate to support the maintenance of iPSCs.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Teste de Materiais , Miócitos Cardíacos/metabolismo , Nanotubos de Carbono/química , Animais , Cães , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologiaRESUMO
One of the main challenges of the current ultrasonic transducers for powering brain implants is the complexity of focusing ultrasonic waves in various axial and lateral directions. The available transducers usually use electrically controlled phased array for beamforming the ultrasonic waves, which increases the complexity of the system even further. In this article, we propose a straightforward solution for selective powering of brain implants to remove the complexity of conventional phased arrays. Our approach features a Sectored-Multiring Ultrasonic Transducer (S-MRUT) on a single piezoelectric sheet, specifically designed for powering implantable devices for optogenetics in freely moving animals. The proposed unidirectional S-MRUT is capable of focusing the ultrasonic waves on brain implants located at different depths and regions of the brain. The S-MRUT is designed based on Fresnel Zone Plate (FZP) theory, simulated in COMSOL, and fabricated with the microfabrication process. The acoustic profile of the seven different configurations of the S-MRUT was measured using a hydrophone with the total number of 7436 grid points. The measurements show the ability of the proposed S-MRUT to sweep the focus point of the acoustic waves in the axial direction in depths of 1 - 3 mm, which is suitable for powering implants in the striatum of the mouse. Furthermore, the proposed S-MRUT demonstrates a steering area with an average radius of 0.862 mm and 0.678 mm in experiments and simulations, respectively. The S-MRUT is designed with the size of 3.8×3.8×0.5 mm3 and the weight of 0.054gr , showing that it is compact and light enough to be worn by a mouse. Finally, the S-MRUT was tested in our measurement setup, where it successfully transfers sufficient power to a 2.8-mm3 optogentic stimulator to turn on a micro-LED on the stimulator.
Assuntos
Transdutores , Ultrassom , Animais , Encéfalo/diagnóstico por imagem , Desenho de Equipamento , Camundongos , Próteses e ImplantesRESUMO
A miniaturized mimic of the active site of a protease, chymotrypsin, was linked to a target recognition unit to generate "Miniature Artificial Proteases" (mAPs). Time-resolved MALDI-TOF data analyses indicated that mAPs cleaved every amide bond between Lys16-Phe20 of the amyloid ß fragment (Aß12-21) and Aß1-40, resulting in inhibition of fibrillization and disruption of the preformed amyloid. Such a platform may offer not only new therapeutic options against various amyloidoses but also novel routes for the selective knockdown of specific proteins.
Assuntos
Amiloide/metabolismo , Quimotripsina/metabolismo , Amiloide/química , Domínio Catalítico , Quimotripsina/química , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
This paper presents an ultrasonically powered microsystem for deep tissue optogenetic stimulation. All the phases in developing the prototype starting from modelling the piezoelectric crystal used for energy harvesting, design, simulation and measurement of the chip, and finally testing the whole system in a mimicking setup are explained. The developed system is composed of a piezoelectric harvesting cube, a rectifier chip, and a micro-scale custom-designed light-emitting-diode (LED), and envisioned to be used for freely moving animal studies. The proposed rectifier chip with a silicon area of [Formula: see text] is implemented in standard TSMC [Formula: see text] CMOS technology, for interfacing the piezoelectric cube and the microLED. Experimental results show that the proposed microsystem produces an available electrical power of [Formula: see text] while loaded by a microLED, out of an acoustic intensity of [Formula: see text] using a [Formula: see text] crystal as the receiver. The whole system including the tested rectifier chip, a piezoelectric cube with the dimensions of [Formula: see text], and a µLED of [Formula: see text] have been integrated on a [Formula: see text] glass substrate, encapsulated inside a bio-compatible PDMS layer and tested successfully for final prototyping. The total volume of the fully-packaged device is estimated around [Formula: see text].
Assuntos
Optogenética/instrumentação , Semicondutores , Ultrassom/instrumentação , Animais , Desenho de Equipamento , Próteses e ImplantesRESUMO
BACKGROUND AND AIM: Veterinary health care is an emergent area in animal sciences and innovative therapeutic approaches happen to be imperative in the present days. In view of the importance of cattle health and production, it is necessary to take up contemporary approach of stem cell therapy in this sector also. This study aimed to standardize an explant culture method of bovine umbilical tissue offcut to isolate mesenchymal stem cells (MSCs) because considerable efforts are required for ensuring easy accessibility and availability of MSCs in bulk quantity, as well as in establishing and characterizing the cell lines. MATERIALS AND METHODS: The umbilical cord (UC) tissue matrix offcut was collected after calving. A simplified in vitro cell isolation technique was followed to collect the emerged out cells from the explants of UC. Further, we expanded these isolated cells in vitro, observed its growth kinetics, and characterized to confirm as per the criterion of bovine MSCs. RESULTS: A considerable exponential growth rate of the UC-derived cells was noticed. In addition to their confirmation as MSCs, the cells also exhibited plastic adherent property and maintained the spindle-shaped morphology throughout the in vitro culture. The cultured cells were found positive MSC-specific surface markers CD105, CD90, and CD73 and were negative for hematopoietic cell marker CD45. Cytochemical studies revealed the ability of the cells to differentiate into osteogenic, chondrogenic, and adipogenic lineages. CONCLUSION: This simplified method of isolation and culture of bovine multipotent MSCs from the UC offcut collected after calving could be extrapolated for the greater availability of the cells for prospective therapeutic applications.
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Inspired by the unique mechanism of proteolytic maturation of host cell factor-1, we designed small peptide-based constructs that selectively recognized amyloid ß (Aß) and cleaved it in a non-catalytic manner initially around the α-secretase cleavage-site, thus termed as "artificial α-secretases". "Artificial α-secretases" also cleaved Aß on the cleavage-sites of other Aß processing enzymes by prolonged treatment, as evidenced by time-resolved MALDI-TOF-mass analyses.
Assuntos
Peptídeos beta-Amiloides/análise , Engenharia de Proteínas , Peptídeos beta-Amiloides/síntese química , Peptídeos beta-Amiloides/metabolismo , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de TempoRESUMO
Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) is the receptor for diphtheria toxin (DT). Mutated or truncated, non-toxic DT has been used earlier for HB-EGF-targeted drug delivery and to modulate HB-EGF signaling. In the present work, we have synthesized a peptide corresponding to a 26 amino acid long stretch of the receptor-binding domain of DT. This region of DT makes multiple contacts with HB-EGF and has residues critical for binding to HB-EGF. We show that this peptide and two of its mutants bind to HB-EGF. We have also created recombinant proteins by fusing Maltose-binding Protein (MBP) with these peptides. These recombinant MBP-tagged peptides bind to HB-EGF with affinities in the range of 10-7 to 10-8â¯M. We have observed that these MBP-tagged peptides can modulate molecular signaling of HB-EGF. Therefore, this 26 amino acid long stretch of DT can be considered as an independent functional segment for binding to HB-EGF. Peptides corresponding to this region may be used for HB-EGF targeted cellular delivery of molecular cargo or to modulate HB-EGF signaling.
Assuntos
Toxina Diftérica/química , Família de Proteínas EGF/química , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Insertion of an anthranilic acid in an amyloidogenic peptide sequence generates a novel conformationally restricted α/ß-hybrid peptide that inhibits amyloid formation of Aß(1-40) and disrupts preformed fibrillar aggregates in vitro. Such ß-sheet breaker hybrid peptides (BSBHps) may be useful for designing novel physiologically important compounds relevant to diverse amyloidoses and for studying the process of aggregation.