Highlights d Robust method automatically adapting to various unseen experimental scenarios d Deep learning solution for accurate nucleus segmentation without user interaction d Accelerates, improves quality, and reduces complexity of bioimage analysis tasks
Key points• Cardiac repolarization, through which heart-cells return to their resting state after having fired, is a delicate process, susceptible to disruption by common drugs and clinical conditions. • Animal models, particularly the dog, are often used to study repolarization properties and responses to drugs, with the assumption that such findings are relevant to humans. However, little is known about the applicability of findings in animals to man.• Here, we studied the contribution of various ion-currents to cardiac repolarization in canine and human ventricle.• Humans showed much greater repolarization-impairing effects of drugs blocking the rapid delayed-rectifier current I Kr than dogs, because of lower repolarization-reserve contributions from two other important repolarizing currents (the inward-rectifier I K1 and slow delayed-rectifier I Ks ).• Our findings clarify differences in cardiac repolarization-processes among species, highlighting the importance of caution when extrapolating results from animal models to man.Abstract The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective I Kr block (50-100 nmol l −1 dofetilide) lengthened AP duration at 90% of repolarization (APD 90 ) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective I K1 block (10 μmol l −1 BaCl 2 ) and I Ks block (1 μmol l −1 HMR-1556) increased APD 90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that I K1 and I Ks densities were 3-and 4.5-fold larger in dogs than humans, respectively. I Kr density and kinetics were similar in human versus dog. I Ca and I to were respectively ∼30% larger and ∼29% smaller in human, and Na + -Ca 2+ exchange current was comparable. Cardiac mRNA levels for the main I K1 ion channel subunit Kir2.1 and the I Ks accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (I Kr and I Ks α-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. I K1 and I Ks inhibition increased the APD-prolonging effect of I Kr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human-canine ion current differences confirmed the role of I K1 and I Ks in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of I Kr block than dogs, because of lower repolarization reserve contributions from I K1 and I Ks , emphasizing species-specific determinants of repolarization and the limitations of animal models fo...
Uncoupling of GJs prior to ischaemia either by PC or CBX preserves the electrical coupling of cells and results in an antiarrhythmic effect during a subsequent ischaemic insult, indicating that a partial closure of gap junctions may play a trigger role in the protection. In contrast, when CBX is administered in PC dogs the protection both against GJ uncoupling and arrhythmias is markedly attenuated, suggesting that the antiarrhythmic protection, at least in part, is mediated through GJs.
To determine whether herpes simplex virus type 1 (HSV-1) infection causes oxidative stress and lipid peroxidation in cultured neural cells, mouse P19 embryonal carcinoma cells were differentiated into cells with neural phenotypes (P19N cells) by retinoic acid and were then infected with HSV-1. Cellular levels of reactive oxygen species (ROS) and the release of lipid peroxidation by-products into the tissue culture medium were then measured by the generation of fluorescent markers hydroxyphenyl fluorescein and a stable chromophore produced by lipid peroxidation products, malondialdehyde (MDA) and hydroxyalkenals (4-HAEs; predominantly 4-hydroxy-2-nonenal [HNE]), respectively. HSV-1 infection increased ROS levels in neural cells as early as 1 h post infection (p.i.) and ROS levels remained elevated at 24 h p.i. This viral effect required viral entry and replication as heat- and ultraviolet light-inactivated HSV-1 were ineffective. HSV-1 infection also was associated with increased levels of MDA/HAE in the culture medium at 2 and 4 h p.i., but MDA/HAE levels were not different from those detected in mock infected control cultures at 1, 6, and 24 h p.i. HSV-1 replication in P19N cells was inhibited by the antioxidant compound ebselen and high concentrations of HNE added to the cultures, but was increased by low concentrations of HNE. These findings indicate that HSV-1 infection of neural cells causes oxidative stress that is required for efficient viral replication. Furthermore, these observations raise the possibility that soluble, bioactive lipid peroxidation by-products generated in infected neural cells may be important regulators of HSV-1 pathogenesis in the nervous system.
HSV-1 entry into HCjE cells is a pH-dependent process that is aided by targeted virus travel on filopodia. HCjE cells express all three major entry receptors, with nectin-1 and HVEM playing the predominant role in mediating entry.
Herpes simplex virus 1 (HSV‐1) demonstrates a unique ability to infect a variety of host cell types. Retinal pigment epithelial (RPE) cells form the outermost layer of the retina and provide a potential target for viral invasion and permanent vision impairment. Here we examine the initial cellular and molecular mechanisms that facilitate HSV‐1 invasion of human RPE cells. High‐resolution confocal microscopy demonstrated initial interaction of green fluorescent protein (GFP)‐tagged virions with filopodia‐like structures present on cell surfaces. Unidirectional movement of the virions on filopodia to the cell body was detected by live cell imaging of RPE cells, which demonstrated susceptibility to pH‐dependent HSV‐1 entry and replication. Use of RT‐PCR indicated expression of nectin‐1, herpes virus entry mediator (HVEM) and 3‐O‐sulfotransferase‐3 (as a surrogate marker for 3‐O‐sulfated heparan sulfate). HVEM and nectin‐1 expression was subsequently verified by flow cytometry. Nectin‐1 expression in murine retinal tissue was also demonstrated by immunohistochemistry. Antibodies against nectin‐1, but not HVEM, were able to block HSV‐1 infection. Similar blocking effects were seen with a small interfering RNA construct specifically directed against nectin‐1, which also blocked RPE cell fusion with HSV‐1 glycoprotein‐expressing Chinese hamster ovary (CHO‐K1) cells. Anti‐nectin‐1 antibodies and F‐actin depolymerizers were also successful in blocking the cytoskeletal changes that occur upon HSV‐1 entry into cells. Our findings shed new light on the cellular and molecular mechanisms that help the virus to enter the cells of the inner eye.
Exosomes are small extracellular vesicles (sEVs), playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here, we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions, including cytostatic, heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance, melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study, we concluded that (i) molecular patterns of tumour-derived sEVs, dictated by the microenvironmental conditions, resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response, with a potential influence on treatment efficacy.
Heparan sulfate (HS) proteoglycans are commonly exploited by multiple viruses for initial attachment to host cells. Herpes simplex virus-1 (HSV-1) is unique because it can use HS for both attachment and penetration, provided specific binding sites for HSV-1 envelope glycoprotein gD are present. The interaction with gD is mediated by specific HS moieties or 3-O sulfated HS (3-OS HS), which are generated by all but one of the seven isoforms of 3-O sulfotransferases (3-OSTs). Here we demonstrate that several common experimental cell lines express unique sets of 3-OST isoforms. While the isoforms 3-OST-3, -5 and -6 were most commonly expressed, isoforms 3-OST-2 and -4 were undetectable in the cell lines examined. Since most cell lines expressed multiple 3-OST isoforms, we addressed the significance of 3-OS HS in HSV-1 entry by down-regulating 2-O-sulfation, a prerequisite for 3-OS HS formation, by knocking down 2-OST expression by RNA interference (RNAi). 2-OST knockdown was verified by reverse-transcriptase PCR and Western blot analysis, while 3-OS HS knockdown was verified by immunofluorescence. Cells showed a significant decrease in viral entry, suggesting an important role for 3-OS HS. Implicating 3-OS HS further, cells knocked-down for 2-OST expression also demonstrated decreased cell-cell fusion when cocultivated with effector cells transfected with HSV-1 glycoproteins. Our findings suggest that 3-OS HS may play an important role in HSV-1 entry into many different cell lines.
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