scholarly journals Susceptibility to Ventricular Arrhythmias Resulting from Mutations in FKBP1B, PXDNL, and SCN9A Evaluated in hiPSC Cardiomyocytes

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Hector Barajas-Martinez ◽  
Maya Smith ◽  
Dan Hu ◽  
Robert J. Goodrow ◽  
Colleen Puleo ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Spontaneous Activity ◽  
Induced Pluripotent Stem Cell ◽  
Significant Loss ◽  
Loss Of Function ◽  
Cardiac Events ◽  
Bundle Branch Block ◽  
Microscopy Analysis ◽  
Induced Pluripotent ◽  
Confocal Microscopy Analysis

Background. We report an inherited cardiac arrhythmia syndrome consisting of Brugada and Early Repolarization Syndrome associated with variants in SCN9A, PXDNL, and FKBP1B. The proband inherited the 3 mutations and exhibited palpitations and arrhythmia-mediated syncope, whereas the parents and sister, who carried one or two of the mutations, were asymptomatic. Methods and Results. We assessed the functional impact of these mutations in induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) derived from the proband and an unaffected family member. Current and voltage clamp recordings, as well as confocal microscopy analysis of Ca2+ transients, were evaluated in hiPSC-CMs from the proband and compared these results with hiPSC-CMs from undiseased controls. Genetic analysis using next-generation DNA sequencing revealed heterozygous mutations in SCN9A, PXDNL, and FKBP1B in the proband. The proband displayed right bundle branch block and exhibited episodes of syncope. The father carried a mutation in FKBP1B, whereas the mother and sister carried the SCN9A mutation. None of the 3 family members screened developed cardiac events. Action potential recordings from control hiPSC-CM showed spontaneous activity and a low upstroke velocity. In contrast, the hiPSC-CM from the proband showed irregular spontaneous activity. Confocal microscopy of the hiPSC-CM of the proband revealed low fluorescence intensity Ca2+ transients that were episodic in nature. Patch-clamp measurements in hiPSC-CM showed no difference in INa but reduced ICa in the proband compared with control. Coexpression of PXDNL-R391Q with SCN5A-WT displayed lower INa density compared to PXDNL-WT. In addition, coexpression of PXDNL-R391Q with KCND3-WT displayed significantly higher Ito density compared to PXDNL-WT. Conclusion. SCN9A, PXDNL, and FKBP1B variants appeared to alter spontaneous activity in hiPSC-CM. Only the proband carrying all 3 mutations displayed the ERS/BrS phenotype, whereas one nor two mutations alone did not produce the clinical phenotype. Our results suggest a polygenic cause of the BrS/ERS arrhythmic phenotype due to mutations in these three gene variants caused a very significant loss of function of INa and ICa and gain of function of Ito.

scholarly journals Effects of acids used in the microabrasion technique: Microhardness and confocal microscopy analysis

2015 ◽  
pp. e506-e512 ◽  
Author(s):  
NIP Pini ◽  
DANL Lima ◽  
GMB Ambrosano ◽  
WJ da Silva ◽  
FHB Aguiar ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Microscopy Analysis ◽  
Confocal Microscopy Analysis

Epi-LASIK: A Confocal Microscopy Analysis of the Corneal Epithelium and Anterior Stroma

2012 ◽  
Vol 43 (4) ◽  
pp. 319-322 ◽  
Author(s):  
Manuel Ramírez ◽  
Everardo Hernández-Quintela ◽  
Ramón Naranjo-Tackman
Keyword(s):  
Confocal Microscopy ◽  
Corneal Epithelium ◽  
Microscopy Analysis ◽  
Confocal Microscopy Analysis

scholarly journals Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy.

1991 ◽  
Vol 112 (3) ◽  
pp. 385-395 ◽  
Author(s):  
R M Lynch ◽  
K E Fogarty ◽  
F S Fay
Keyword(s):  
Glucose Metabolism ◽  
Confocal Microscopy ◽  
Cell Metabolism ◽  
Three Dimensional ◽  
Metabolic State ◽  
Metabolic Conditions ◽  
Microscopy Analysis ◽  
Immunocytochemical Studies ◽  
Confocal Microscopy Analysis

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.

scholarly journals A Bioengineered Nisin Derivative to Control Biofilms of Streptococcus uberis .

2021 ◽  
Author(s):  
Mariana Pérez-Ibarreche ◽  
Des Field ◽  
R. Paul Ross ◽  
Colin Hill
Keyword(s):  
Confocal Microscopy ◽  
Biofilm Formation ◽  
Bovine Mastitis ◽  
Multidrug Resistant ◽  
Biofilm Inhibition ◽  
Promising Alternative ◽  
Streptococcus Uberis ◽  
Microscopy Analysis ◽  
The Impact ◽  
Confocal Microscopy Analysis

Antimicrobial peptides are evolving as novel therapeutic options against the increasing problem of multidrug-resistant microorganisms, and nisin is one such avenue. However, some bacteria possess a specific nisin resistance system (NSR) which cleaves the peptide reducing its bactericidal efficacy. NSR-based resistance was identified in strains of Streptococcus uberis , a ubiquitous pathogen that causes mastitis in dairy cattle. Previous studies have demonstrated that a nisin A derivative termed nisin PV, featuring S29P and I30V, exhibits enhanced resistance to proteolytic cleavage by NSR. Our objective was to investigate the ability of this nisin derivative to eradicate and inhibit biofilms of S. uberis DPC 5344 and S. uberis ATCC 700407 ( nsr+ ) using crystal violet (biomass), XTT (viability) assays and confocal microscopy (viability and architecture). When pre-established biofilms were assessed, over 60% of the biofilm biomass was reduced by both peptides compared to the untreated controls. However, a 42% higher reduction in viability was observed following treatment with nisin PV compared to nisin A. Accordingly, confocal microscopy analysis revealed significantly more dead cells on the biofilm upper surface and a reduced thickness following treatment with nisin PV. When biofilm inhibition was assessed, nisin PV inhibited biofilm formation and decrease viability up to 56% and 85% more than nisin A, respectively. Confocal microscopy analysis revealed a lack of biofilm for S. uberis ATCC 700407 and only dead cells for S. uberis DPC 5344. These results suggest that nisin PV is a promising alternative to effectively reduce the biofilm formation of S. uberis strains carrying NSR. IMPORTANCE. One of the four most prevalent species of bovine mastitis-causing pathogens is S. uberis. Its ability to form biofilms confers on the bacteria greater resistance to antibiotics requiring higher doses to be more effective. In a bid to limit antibiotic resistance development, the need for alternative antimicrobials is paramount. Bacteriocins such as nisin represent one such alternative that could alleviate the impact of mastitis caused by S. uberis. However, many strains of S. uberis have been shown to possess nisin resistance determinants such as the nisin resistance protein (NSR). In this study, we demonstrate the ability of nisin and a nisin derivative termed PV that is insensitive to NSR to prevent and remove biofilms of NSR-producing S. uberis strains. These findings will add new information to the antimicrobial, bacteriocins and control of S. uberis research fields specifically in relation to biofilms and nsr + mastitis-associated strains.

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