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PURPOSE: Cardiac tissue engineering is suggested as a promising approach to overcome problems associated with impaired myocardium. This is the first study to investigate the use of BC and gelatin for cardiomyocyte adhesion and growth. METHODS: Bacterial cellulose (BC) membranes were produced by Komagataeibacter xylinus and coated or mixed with gelatin to make gelatin-coated BC (BCG) or gelatin-mixed BC (mBCG) scaffolds, respectively. BC based-scaffolds were characterized via SEM, FTIR, XRD, and AFM. Neonatal rat-ventricular cardiomyocytes (nr-vCMCs) were cultured on the scaffolds to check the capability of the composites for cardiomyocyte attachment, growth and expansion. RESULTS: The average nanofibrils diameter in all scaffolds was suitable (~ 30-65 nm) for nr-vCMCs culture. Pore diameter (≥ 10 µm), surface roughness (~ 182 nm), elastic modulus (0.075 ± 0.015 MPa) in mBCG were in accordance with cardiomyocyte requirements, so that mBCG could better support attachment of nr-vCMCs with high concentration of gelatin, and appropriate surface roughness. Also, it could better support growth and expansion of nr-vCMCs due to submicron scale of nanofibrils and proper elasticity (~ 0.075 MPa). The viability of nr-vCMCs on BC and BCG scaffolds was very low even at day 2 of culture (~ ≤ 40%), but, mBCG could promote a metabolic active state of nr-vCMCs until day 7 (~ ≥ 50%). CONCLUSION: According to our results, mBCG scaffold was the most suitable composite for cardiomyocyte culture, regarding its physicochemical and cell characteristics. It is suggested that improvement in mBCG stability and cell attachment features may provide a convenient scaffold for cardiac tissue engineering.
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The prevention of biofilm formation plays a pivotal role in managing Helicobacter pylori inside the body and the environment. This study showed in vitro potentials of two recently isolated probiotic strains, Bacillus sp. 1630F and Enterococcus sp. 7C37, to form biofilm and combat H. pylori attachment to the abiotic and biotic surfaces. Lactobacillus casei and Bifidobacterium bifidum were used as the reference probiotics. The biofilm rates were the highest in the solidliquid interface for Lactobacillus and Bifidobacterium and the airliquid interface for Bacillus and Enterococcus. The highest tolerances to the environmental conditions were observed during the biofilm formations of Enterococcus and Bifidobacterium (pH), Enterococcus and Bacillus (bile), and Bifidobacterium and Lactobacillus (NaCl) on the polystyrene and glass substratum, respectively. Biofilms occurred more quickly by Bacillus and Enterococcus strains than reference strains on the polystyrene and glass substratum, respectively. Enterococcus (competition) and Bacillus (exclusion) achieved the most inhibition of H. pylori biofilm formations on the polystyrene and AGS cells, respectively. Expression of luxS was promoted by Bacillus (exclusion, 3.2 fold) and Enterococcus (competition, 2.0 fold). Expression of ropD was decreased when H. pylori biofilm was excluded by Bacillus (0.4 fold) and Enterococcus (0.2 fold) cells. This study demonstrated the ability of Bacillus and Enterococcus probiotic bacteria to form biofilm and combat H. pylori biofilm formation.(AU)
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Humanos , Bacillus , Enterococcus , Helicobacter pylori , Probióticos , Poliestirenos , Biopelículas , Microbiología , Técnicas Microbiológicas , Infecciones por BifidobacterialesRESUMEN
The prevention of biofilm formation plays a pivotal role in managing Helicobacter pylori inside the body and the environment. This study showed in vitro potentials of two recently isolated probiotic strains, Bacillus sp. 1630F and Enterococcus sp. 7C37, to form biofilm and combat H. pylori attachment to the abiotic and biotic surfaces. Lactobacillus casei and Bifidobacterium bifidum were used as the reference probiotics. The biofilm rates were the highest in the solid-liquid interface for Lactobacillus and Bifidobacterium and the air-liquid interface for Bacillus and Enterococcus. The highest tolerances to the environmental conditions were observed during the biofilm formations of Enterococcus and Bifidobacterium (pH), Enterococcus and Bacillus (bile), and Bifidobacterium and Lactobacillus (NaCl) on the polystyrene and glass substratum, respectively. Biofilms occurred more quickly by Bacillus and Enterococcus strains than reference strains on the polystyrene and glass substratum, respectively. Enterococcus (competition) and Bacillus (exclusion) achieved the most inhibition of H. pylori biofilm formations on the polystyrene and AGS cells, respectively. Expression of luxS was promoted by Bacillus (exclusion, 3.2 fold) and Enterococcus (competition, 2.0 fold). Expression of ropD was decreased when H. pylori biofilm was excluded by Bacillus (0.4 fold) and Enterococcus (0.2 fold) cells. This study demonstrated the ability of Bacillus and Enterococcus probiotic bacteria to form biofilm and combat H. pylori biofilm formation.
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Bacillus , Helicobacter pylori , Probióticos , Enterococcus , Poliestirenos , Biopelículas , Lactobacillus , BifidobacteriumRESUMEN
PURPOSE: To investigate the genetic cause of nonobstructive azoospermia (NOA). METHODS: We performed whole exome sequencing (WES) on the proband who had three relatives suffering from NOA. We used a list of candidate genes which have high expression level in testis and their mutations have been reported in NOA. Sanger sequencing verified the identified variant and its structural and functional consequence was evaluated by protein three-dimensional (3D) structure prediction and protein-ligand docking. RESULTS: WES revealed a novel splice-acceptor mutation (c.1832-2A>T) in helicase for meiosis 1 (HFM1) gene, which co-segregated with the NOA in this family. 3D structural models were generated and verified. Molecular docking indicated that the c.1832-2A>T mutation affects not only the ADP binding residues but also the hydrogen bond interactions. The ADP binding site will be lost in the mutant protein, potentially causing defective crossover and synapsis. CONCLUSION: We report that the c.1832-2A>T mutation is the likely cause of NOA in the family studied. Regarding that many reported NOA genes are involved in the formation of crossovers and synapsis and have critical roles in the production of germ cells, we suggest that such genes should be considered for screening of infertility among large cohorts of infertile individuals.
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Azoospermia , Adenosina Difosfato/metabolismo , Azoospermia/diagnóstico , ADN Helicasas/genética , Humanos , Masculino , Simulación del Acoplamiento Molecular , Mutación/genética , Testículo/metabolismoRESUMEN
Azoospermia is a common cause of male infertility without any sperm in the semen and consists of â¼1% of all males and â¼15% of infertile ones. Currently, no accurate non-invasive diagnostic method exists for patients with azoospermia and testis biopsy is mandatory to determine if any spermatozoa exist in the testes. Studies have clarified that the expression of some distinct microRNAs shows alterations in azoospermic patients. MicroRNAs play critical roles during spermatogenesis and their dysregulation can defect this process. Here, we review studied microRNAs involved in the pathogenesis of azoospermia and their target genes. Moreover, we will imply the utility of seminal plasma microRNAs as non-invasive diagnostic biomarkers for azoospermia. We hope such studies could help patients with azoospermia in both diagnosis and treatment, in order that they could father their own biological children.
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Azoospermia/diagnóstico , Biomarcadores/análisis , MicroARNs/análisis , Adulto , Azoospermia/sangre , Azoospermia/genética , Biomarcadores/sangre , Humanos , Infertilidad Masculina/sangre , Infertilidad Masculina/diagnóstico , Infertilidad Masculina/genética , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Testículo/patologíaRESUMEN
Co-infection with other microorganisms can promote the Candida albicans to be invasive. In this study, Escherichia coli and C. albicans were co-isolated from the women with candidiasis symptoms. The in vitro effects of E. coli on C. albicans hypha development, biofilm formation, antibiotic susceptibility, dispersion from the biofilm, expression of Als3, Hwp1, and Tup1 genes, and pathogenesis in Galleria mellonella were investigated. Electron microscopic images revealed that hypha induction was markedly increased in the bacteria-fungi co-culture. Biofilm formation was increased 2.2 fold in the presence of E. coli. The minimum inhibitory concentration of nystatin against Candida was increased from (µg mL-1) 25 to 50 in the dual biofilm. Candida dissemination was increased up to 2.7 fold from the mixed fungi/bacteria biofilm. The expression of ALS3 and HWP1 genes was increased (5.9 and 2.0 fold, respectively) while the TUP1 gene expression was decreased (0.4 fold) when C. albicans was incubated with E. coli. The simultaneous injection of C. albicans and E. coli to the insect larvae increased Galleria mortality up to 40%. This study demonstrated the effects of E. coli to promote fungi virulence factors, which suggest polymicrobial interaction should be considered during treatment of fungal infections.
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Biopelículas/crecimiento & desarrollo , Candida albicans/patogenicidad , Candidiasis Vulvovaginal/microbiología , Coinfección/microbiología , Escherichia coli/fisiología , Interacciones Microbianas , Factores de Virulencia , Animales , Candida albicans/genética , Femenino , Humanos , Hifa/genética , Hifa/crecimiento & desarrollo , Larva/microbiología , Mariposas Nocturnas/microbiologíaRESUMEN
Different factors may trigger arrhythmias in diseased hearts, including fibrosis, cardiomyocyte hypertrophy, hypoxia, and inflammation. This makes it difficult to establish the relative contribution of each of them to the occurrence of arrhythmias. Accordingly, in this study, we used an in vitro model of pathological cardiac hypertrophy (PCH) to investigate its proarrhythmic features and the underlying mechanisms independent of fibrosis or other PCH-related processes. Neonatal rat ventricular cardiomyocyte (nr-vCMC) monolayers were treated with phorbol 12-myristate 13-acetate (PMA) to create an in vitro model of PCH. The electrophysiological properties of PMA-treated and control monolayers were analyzed by optical mapping at day 9 of culture. PMA treatment led to a significant increase in cell size and total protein content. It also caused a reduction in sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 level (32%) and an increase in natriuretic peptide A (42%) and α1-skeletal muscle actin (34%) levels, indicating that the hypertrophic response induced by PMA was, indeed, pathological in nature. PMA-treated monolayers showed increases in action potential duration (APD) and APD dispersion, and a decrease in conduction velocity (CV; APD30 of 306 ± 39 vs. 148 ± 18 ms, APD30 dispersion of 85 ± 19 vs. 22 ± 7 and CV of 10 ± 4 vs. 21 ± 2 cm/s in controls). Upon local 1-Hz stimulation, 53.6% of the PMA-treated cultures showed focal tachyarrhythmias based on triggered activity (n = 82), while the control group showed 4.3% tachyarrhythmias (n = 70). PMA-treated nr-vCMC cultures may, thus, represent a well-controllable in vitro model for testing new therapeutic interventions targeting specific aspects of hypertrophy-associated arrhythmias.NEW & NOTEWORTHY Phorbol 12-myristate 13-acetate (PMA) treatment of neonatal rat ventricular cardiomyocytes (nr-vCMCs) led to induction of many significant features of pathological cardiac hypertrophy (PCH), including action potential duration prolongation and dispersion, which provided enough time and depolarizing force for formation of early afterdepolarization (EAD)-induced focal tachyarrhythmias. PMA-treated nr-vCMCs represent a well-controllable in vitro model, which mostly resembles to moderate left ventricular hypertrophy (LVH) rather than severe LVH, in which generation of a reentry is the putative mechanism of its arrhythmias.
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Cardiomegalia , Miocitos Cardíacos , Animales , Animales Recién Nacidos , Células Cultivadas , Ventrículos Cardíacos , RatasRESUMEN
BACKGROUND: Skeletal development and its cellular function are regulated by various transcription factors. The T-box (Tbx) family of transcription factors have critical roles in cellular differentiation as well as heart and limbs organogenesis. These factors possess activator and/or repressor domains to modify the expression of target genes. Despite the obvious effects of Tbx20 on heart development, its impact on bone development is still unknown. METHODS: To investigate the consequence by forced Tbx20 expression in the osteogenic differentiation of human mesenchymal stem cells derived from adipose tissue (Ad-MSCs), these cells were transduced with a bicistronic lentiviral vector encoding Tbx20 and an enhanced green fluorescent protein. RESULTS: Tbx20 gene delivery system suppressed the osteogenic differentiation of Ad-MSCs, as indicated by reduction in alkaline phosphatase activity and Alizarin Red S staining. Consistently, reverse transcription-polymerase chain reaction analyses showed that Tbx20 gain-of-function reduced the expression levels of osteoblast marker genes in osteo-inductive Ad-MSCs cultures. Accordingly, Tbx20 negatively affected osteogenesis through modulating expression of key factors involved in this process. CONCLUSION: The present study suggests that Tbx20 could inhibit osteogenic differentiation in adipose-derived human mesenchymal stem cells.
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BACKGROUND: Mesenchymal stem cells (MSCs) are attractive choices in regenerative medicine and can be genetically modified to obtain better results in therapeutics. Bone development and metabolism are controlled by various factors including microRNAs (miRs) interference, which are small non-coding endogenous RNAs. METHODS: In the current study, the effects of forced miR-148b expression was evaluated on osteogenic activity. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with bicistronic lentiviral vector encoding hsa-miR-148b-3p or -5p and the enhanced green fluorescent protein. Fourteen days post-transduction, immunostaining as well as Western blotting were used to analyze osteogenesis. RESULTS: Overexpression of miR-148b-3p increased the osteogenic differentiation of human BM-MSCs as demonstrated by anenhancement of mineralized nodular formation and an increase in the levels of osteoblastic differentiation biomarkers, alkaline phosphatase and collagen type I. CONCLUSIONS: Since lentivirally overexpressed miR-148b-3p increased osteogenic differentiation capability of BM-MSCs, this miR could be applied as a therapeutic modulator to optimize bone function.
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Médula Ósea/metabolismo , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Osteogénesis/genética , Fosfatasa Alcalina , Secuencia de Bases , Biomarcadores , Médula Ósea/crecimiento & desarrollo , Médula Ósea/patología , Diferenciación Celular , Colágeno Tipo I , Vectores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Células Madre Mesenquimatosas/citología , Transducción GenéticaRESUMEN
Osteogenic differentiation is a controlled developmental process in which external and internal factors including cytokines, growth factors, transcription factors (TFs), signaling pathways and microRNAs (miRNAs) play important roles. Various stimulatory and inhibitory TFs contribute to osteogenic differentiation and are responsible for bone development. In addition, cross-talk between several complex signaling pathways regulates the osteogenic differentiation of some stem cells. Although much is known about regulatory genes and signaling pathways in osteogenesis, the role of miRNAs in osteogenic differentiation still needs to be explored. miRNAs are small, approximately 22 nucleotides, single-stranded nonprotein coding RNAs which are abundant in many mammalian cell types. They paly significant regulated roles in various biological processes and serve as promising biomarkers for disease states. Recently, emerging evidence have shown that miRNAs are the key regulators of osteogenesis of stem cells. They may endogenously regulate osteogenic differentiation of stem cells through direct targeting of positive or negative directors of osteogenesis and depending on the target result in the promotion or inhibition of osteogenic differentiation. This review aims to provide a general overview of miRNAs participating in osteogenic differentiation of stem cells and explain their regulatory effect based on the genes targeted with these miRNAs.
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MicroARNs/genética , Osteogénesis/genética , Células Madre/metabolismo , Animales , Diferenciación Celular/genética , Humanos , MicroARNs/metabolismo , Osteoblastos/citología , Vía de Señalización WntRESUMEN
Ischemic heart disease often results in myocardial infarction and is the leading cause of mortality and morbidity worldwide. Improvement in the function of infarcted myocardium is a main purpose of cardiac regenerative medicine. One possible way to reach this goal is via stem cell therapy. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types but display limited cardiomyogenic differentiation potential. Members of the T-box family of transcription factors including Tbx20 play important roles in heart development and cardiomyocyte homeostasis. Therefore, in the current study, we investigated the potential of Tbx20 to enhance the cardiomyogenic differentiation of human adipose-derived MSCs (ADMSCs). Human ADMSCs were transduced with a bicistronic lentiviral vector encoding Tbx20 (murine) and the enhanced green fluorescent protein (eGFP) and analyzed 7 and 14 days post transduction. Transduction of human ADMSCs with this lentiviral vector increased the expression of the cardiomyogenic differentiation markers ACTN1, TNNI3, ACTC1, NKX2.5, TBX20 (human), and GATA4 as revealed by RT-qPCR. Consistently, immunocytological results showed elevated expression of α-actinin and cardiac troponin I in these cells in comparison to the cells transduced with control lentiviral particles coding for eGFP alone. Accordingly, forced expression of Tbx20 exerts cardiomyogenic effects on human ADMSCs by increasing the expression of cardiomyogenic differentiation markers at the RNA and protein level.
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Tejido Adiposo/citología , Diferenciación Celular , Vectores Genéticos/administración & dosificación , Lentinula/genética , Células Madre Mesenquimatosas/citología , Miocitos Cardíacos/citología , Proteínas de Dominio T Box/metabolismo , Tejido Adiposo/metabolismo , Animales , Biomarcadores/metabolismo , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Proteínas de Dominio T Box/genéticaRESUMEN
Since the adult mammalian heart has limited regenerative capacity, cardiac trauma, disease, and aging cause permanent loss of contractile tissue. This has fueled the development of stem cell-based strategies to provide the damaged heart with new cardiomyocytes. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of self-renewal and differentiation into cardiomyocytes, albeit inefficiently. MicroRNAs (miRNAs, miRs) are non-coding RNAs that have the potential to control stem cell fate decisions and are employed in cardiac regeneration and repair. In this study, we tested the hypothesis that overexpression of miR-499a induces cardiomyogenic differentiation in BM-MSCs. Human BM-MSCs (hBM-MSCs) were transduced with lentiviral vectors encoding miR-499a-3p or miR-499a-5p and analyzed by immunostaining and western blotting methods 14 days post-transduction. MiR-499a-5p-transduced cells adopted a polygonal/rod-shaped (myocyte-like) phenotype and showed an increase in the expression of the cardiomyocyte markers α-actinin and cTnI, as cardiogenic differentiation markers. These results indicate that miR-499a-5p overexpression promotes the cardiomyogenic differentiation of hBM-MSCs and may thereby increase their therapeutic efficiency in cardiac regeneration.
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Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/citología , MicroARNs/fisiología , Miocitos Cardíacos/citología , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Vectores Genéticos , VIH-1/genética , Humanos , Lentivirus/genética , MicroARNs/genética , Proteínas Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Regeneración , Transducción GenéticaRESUMEN
Electrical cardioversion (ECV), a mainstay in atrial fibrillation (AF) treatment, is unsuccessful in up to 10-20% of patients. An important aspect of the remodeling process caused by AF is the constitutive activition of the atrium-specific acetylcholine-dependent potassium current (IK,ACh â IK,ACh-c), which is associated with ECV failure. This study investigated the role of IK,ACh-c in ECV failure and setting the atrial defibrillation threshold (aDFT) in optically mapped neonatal rat cardiomyocyte monolayers. AF was induced by burst pacing followed by application of biphasic shocks of 25-100 V to determine aDFT. Blocking IK,ACh-c by tertiapin significantly decreased DFT, which correlated with a significant increase in wavelength during reentry. Genetic knockdown experiments, using lentiviral vectors encoding a Kcnj5-specific shRNA to modulate IK,ACh-c, yielded similar results. Mechanistically, failed ECV was attributed to incomplete phase singularity (PS) removal or reemergence of PSs (i.e. re-initiation) through unidirectional propagation of shock-induced action potentials. Re-initiation occurred at significantly higher voltages than incomplete PS-removal and was inhibited by IK,ACh-c blockade. Whole-heart mapping confirmed our findings showing a 60% increase in ECV success rate after IK,ACh-c blockade. This study provides new mechanistic insight into failing ECV of AF and identifies IK,ACh-c as possible atrium-specific target to increase ECV effectiveness, while decreasing its harmfulness.
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Acetilcolina/metabolismo , Fibrilación Atrial/metabolismo , Cardioversión Eléctrica/efectos adversos , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/metabolismo , Potenciales de Acción , Animales , Fibrilación Atrial/genética , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/terapia , Técnicas de Silenciamiento del Gen , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Técnicas de Placa-Clamp , Canales de Potasio/genética , Canales de Potasio de Rectificación Interna/genética , RatasRESUMEN
AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia and often involves reentrant electrical activation (e.g. spiral waves). Drug therapy for AF can have serious side effects including proarrhythmia, while electrical shock therapy is associated with discomfort and tissue damage. Hypothetically, forced expression and subsequent activation of light-gated cation channels in cardiomyocytes might deliver a depolarizing force sufficient for defibrillation, thereby circumventing the aforementioned drawbacks. We therefore investigated the feasibility of light-induced spiral wave termination through cardiac optogenetics. METHODS AND RESULTS: Neonatal rat atrial cardiomyocyte monolayers were transduced with lentiviral vectors encoding light-activated Ca(2+)-translocating channelrhodopsin (CatCh; LV.CatChâ¼eYFP↑) or eYFP (LV.eYFP↑) as control, and burst-paced to induce spiral waves rotating around functional cores. Effects of CatCh activation on reentry were investigated by optical and multi-electrode array (MEA) mapping. Western blot analyses and immunocytology confirmed transgene expression. Brief blue light pulses (10 ms/470 nm) triggered action potentials only in LV.CatChâ¼eYFP↑-transduced cultures, confirming functional CatCh-mediated current. Prolonged light pulses (500 ms) resulted in reentry termination in 100% of LV.CatChâ¼eYFP↑-transduced cultures (n = 31) vs. 0% of LV.eYFP↑-transduced cultures (n = 11). Here, CatCh activation caused uniform depolarization, thereby decreasing overall excitability (MEA peak-to-peak amplitude decreased 251.3 ± 217.1 vs. 9.2 ± 9.5 µV in controls). Consequently, functional coresize increased and phase singularities (PSs) drifted, leading to reentry termination by PS-PS or PS-boundary collisions. CONCLUSION: This study shows that spiral waves in atrial cardiomyocyte monolayers can be terminated effectively by a light-induced depolarizing current, produced by the arrhythmogenic substrate itself, upon optogenetic engineering. These results provide proof-of-concept for shockless defibrillation.
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Fibrilación Atrial/terapia , Luz , Miocitos Cardíacos/efectos de la radiación , Optogenética , Potenciales de Acción , Animales , Animales Recién Nacidos , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/fisiopatología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Estimulación Cardíaca Artificial , Células Cultivadas , Channelrhodopsins , Estudios de Factibilidad , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Atrios Cardíacos/efectos de la radiación , Lentivirus/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Ratas Wistar , Factores de Tiempo , Transducción Genética , Transfección , Imagen de Colorante Sensible al VoltajeRESUMEN
Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS-) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS- both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS- while at low acceptor-to-donor cell ratios FLPeNLS- was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying cell-to-cell fusion progression based on GpLuc has been developed.
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Fusión Celular/métodos , Genes Reporteros/genética , Vectores Genéticos/genética , Mediciones Luminiscentes/métodos , Animales , Secuencia de Bases , Western Blotting , Luciérnagas/enzimología , Inmunohistoquímica , Lentivirus , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Mioblastos/metabolismo , Señales de Localización Nuclear/metabolismo , Oligodesoxirribonucleótidos/genética , Plásmidos/genética , Alineación de SecuenciaRESUMEN
Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin(-)/Sca-1(+)/cKit(+) hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin(-)/Sca-1(+)/cKit(+) cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin(-)/Sca-1(+)/cKit(+) cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.
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Lesión Renal Aguda/prevención & control , Células Madre Hematopoyéticas/fisiología , Riñón/irrigación sanguínea , MicroARNs/fisiología , Neovascularización Fisiológica/fisiología , Daño por Reperfusión/prevención & control , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Movimiento Celular/fisiología , Quimiocina CXCL12/metabolismo , Riñón/metabolismo , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Receptores CXCR4/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patologíaRESUMEN
OBJECTIVE(S): Adeno-associated virus type 2 (AAV2) vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple YâF mutations. MATERIALS AND METHODS: A one-step, high-fidelity polymerase chain reaction (PCR) cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with YâF substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant), subcloning (Y444730F and Y500730F mutants) or a combination of both techniques (Y444500730F mutant). RESULTS: Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping. CONCLUSION: PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.
RESUMEN
BACKGROUND: Atrial fibrillation is the most common cardiac arrhythmia. Ventricular proarrhythmia hinders pharmacological atrial fibrillation treatment. Modulation of atrium-specific Kir3.x channels, which generate a constitutively active current (I(K,ACh-c)) after atrial remodeling, might circumvent this problem. However, it is unknown whether and how I(K,ACh-c) contributes to atrial fibrillation induction, dynamics, and termination. Therefore, we investigated the effects of I(K,ACh-c) blockade and Kir3.x downregulation on atrial fibrillation. METHODS AND RESULTS: Neonatal rat atrial cardiomyocyte cultures and intact atria were burst paced to induce reentry. To study the effects of Kir3.x on action potential characteristics and propagation patterns, cultures were treated with tertiapin or transduced with lentiviral vectors encoding Kcnj3- or Kcnj5-specific shRNAs. Kir3.1 and Kir3.4 were expressed in atrial but not in ventricular cardiomyocyte cultures. Tertiapin prolonged action potential duration (APD; 54.7±24.0 to 128.8±16.9 milliseconds; P<0.0001) in atrial cultures during reentry, indicating the presence of I(K,ACh-c). Furthermore, tertiapin decreased rotor frequency (14.4±7.4 to 6.6±2.0 Hz; P<0.05) and complexity (6.6±7.7 to 0.6±0.8 phase singularities; P<0.0001). Knockdown of Kcnj3 or Kcnj5 gave similar results. Blockade of I(K,ACh-c) prevented/terminated reentry by prolonging APD and changing APD and conduction velocity restitution slopes, thereby altering the probability of APD alternans and rotor destabilization. Whole-heart mapping experiments confirmed key findings (e.g., >50% reduction in atrial fibrillation inducibility after I(K,ACh-c) blockade). CONCLUSIONS: Atrium-specific Kir3.x controls the induction, dynamics, and termination of fibrillation by modulating APD and APD/conduction velocity restitution slopes in atrial tissue with I(K,ACh-c). This study provides new molecular and mechanistic insights into atrial tachyarrhythmias and identifies Kir3.x as a promising atrium-specific target for antiarrhythmic strategies.
Asunto(s)
Fibrilación Atrial/fisiopatología , Regulación hacia Abajo/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Atrios Cardíacos/fisiopatología , Miocitos Cardíacos/fisiología , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Venenos de Abeja/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/efectos de los fármacos , Atrios Cardíacos/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas Wistar , Factores de Tiempo , Imagen de Colorante Sensible al VoltajeRESUMEN
Recent studies have shown that the use of biomaterials and new biodegradable scaffolds for repair or regeneration of damaged tissues is of vital importance. Scaffolds used in tissue engineering should be biodegradable materials with three-dimensional structures which guide the growth and differentiation of the cells. They also tune physical, chemical and biological properties for efficient supplying of the cells to the selected tissues and have proper porosity along with minimal toxic effects. In this manner, the study of these characteristics is a giant stride towards scaffold design. In this study, Gelatin/Siloxane/Hydroxyapatite (GS-Hyd) scaffold was synthesized and its morphology, in vivo biodegradability, cytotoxic effects and ability for cell adhesion were investigated using mesenchymal stem cells (MSCs). The cells were treated with different volumes of the scaffold suspension for evaluation of its cytotoxic effects. The MSCs were also seeded on scaffolds and cultured for 2 weeks to evaluate the ability of the scaffold in promoting of cell adhesion and growth. To check the biodegradability of the scaffold in vivo, scaffolds were placed in the rat body for 21 days in three different positions of thigh muscle, testicle, and liver and they were analyzed by scanning electron microscopy (SEM) and weight changes. According to the results of the viability of this study, no cytotoxic effects of GS-Hyd scaffold was found on the cells and MSCs could adhere on the scaffold with expanding their elongations and forming colonies. The rate of degradation as assessed by weight loss was significant within each group along with significant differences between different tissues at the same time point. SEM micrographs also indicated the obvious morphological changes on the surface of the particles and diameter of the pores through different stages of implantation. The greatest amount of degradation happened to the scaffold particles implanted into the muscle, followed by testicle and liver, respectively.
RESUMEN
Mesenchymal stem cells (MSCs) are considered as promising candidates for new clinical trials of cell therapies. Bone marrow (BM) was the first source reported to contain MSCs; however, using it may be detrimental due to the highly invasive aspiration procedures. More recently, adipose tissue, attainable by a less invasive method, has been introduced as an alternative source of MSCs. So far, MSCs derived from these two sources have been compared in different characters; however, one of the main properties, i.e., the expression of chemokine receptors, has been ignored in these comparisons. In the present study, human MSCs were derived from bone marrow and adipose tissues and characterized by their expression of some cell surface antigens and also differentiation capacity. The expression of five selected chemokine receptors, which seems to be important in cell homing, was also compared. Semiquantitative reverse transcription-polymerase chain reaction method was used to assess gene expression levels of these chemokine receptors. Our results indicate that expression of these receptors in human MSCs, derived from adipose tissue, was higher than MSCs from bone marrow. Chemokine receptors and their ligands and adhesion molecules play an important role in tissue-specific homing of leukocytes and have also been implicated in trafficking of hematopoietic precursors into and through tissues. Therefore, MSCs from adipose tissue may show a better migration and homing capacity and they might be a better candidate for therapeutic purposes.