Abstract Because of the advantages of dynamic, non-invasive live cell imaging techniques (LCIT) over static visualisation methods, the former are increasingly applied in various disciplines such as biomedicine, cell biology, pharmacology, and developmental biology, to obtain more reliable results than with fixed cells. We prove and discuss the advantages of LCIT via its application in real-time visualization of sarcomeres, Ca 2+ changes and ROS in cardiomyocytes from human induced pluri-potent stem cells.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
FHC related mutations have been reported to interfere with myofibrillar responses to cAMP‐PKA signaling. We have previously shown that a deletion mutation in the C‐terminus of cTnI (cTnI‐ΔK184) increased Ca 2+ ‐sensitivity, slowed relaxation and increased passive stiffness of cardiac myofibrils (Mf) isolated from transgenic mice (Tg) carrying this mutation. The impaired relaxation, which likely underlies diastolic dysfunction, could be ascribed to incomplete inactivation of crossbridges at resting pCa (Iorga et al., 2007). Here, we tested the hypothesis that these biomechanical effects are modulated by β‐adrenoceptor mediated phosphorylation of sarcomeric proteins. To alter phosphorylation in vivo , Tg and WT mice were treated with i.p. injection of propranolol (30 mg/kg b.w.) 1 hr before sacrifice (propranolol treated, PT) in accordance with the Directive 2010/63/EU and German Animal Welfare Act. Skinned fibers (Sf) and Mf were isolated from hearts of such treated mice and untreated control mice and analyzed for mechanical performance. We further tested whether in vitro treatment of these preparations with PKA reversed the PT mediated effects on biomechanics. ProQ Diamond staining and cTnI‐pS22/pS23 phosphospecific antibodies detected no significant difference in cTnI and MyBP‐C phosphorylation between Tg and WT untreated mice. In such untreated mice, Ca 2+ ‐sensitivity of Tg‐Sf from was higher compared to WT (pCa 50 ‐values: WT 5.48, Tg 5.65, p<0.01). PT significantly lowered phosphorylation in both lines by a similar extent which was associated with a right‐ward shift in the force‐pCa relation in Tg‐Sf by 0.07 units but unexpectedly not in WT‐Sf. While PKA had no effect on the force‐pCa relation in Sf from untreated mice, it decreased Ca 2+ ‐ sensitivity by ~ 0.11 pCa‐units in WT‐Sf and ~0.21 pCa‐units in Tg‐Sf from PT mice. The changes in Ca 2+ ‐sensitivity were associated with respective changes in cTnI phosphorylation. The passive force‐sarcomere length (SL) relation was shifted upward in WT‐Mf from PT but surprisingly, this effect was not reversed by PKA. In contrast, PT had no effect on this relation in Tg‐Mf but subsequent incubation with PKA shifted it downward. PT slowed relaxation kinetics only in WT and treatment with PKA only reversed the slowing effect on the duration of the initial phase of relaxation. However, we cannot exclude possible confounding effects of PKA buffer. Neither PT nor PKA altered relaxation kinetics in Tg‐Mf. In summary, our results suggest that propranolol modulates phosphorylation sites in addition to PKA sites, which appear to manifest differently in WT and Tg. Still, our results indicate that the Ca 2+ ‐desensitizing effect of PKA is not abrogated by the FHC related cTnI‐D184 mutation. Importantly, propranolol slows relaxation kinetics in WT but not in Tg suggesting that propranolol does not worsen the diastolic dysfunction. Support or Funding Information German Research Foundation (SFB 612 to GP and RS). DM was supported by the graduate program in Pharmacology and Experimental Therapeutics at the University of Cologne which is financially and scientifically supported by Bayer and Medical Faculty of the Universty of Cologne
Abstract Background Atrial fibrillation (AF) is a prevalent diagnosis among individuals with interatrial shunts, and the prevalence increases with age (1). Since the 1990s, interventionists have introduced transcatheter techniques, to close interatrial shunts, while surgical procedures reserved in cases of technical complexity (2). Despite the potential reduction in AF incidence post interatrial shunt closure, discernible modifications in the cardiac conduction system may persist (3). Notwithstanding, reports indicate an incidence of new-onset AF in approximately 10-25% of patients after interatrial shunt closure, with a pronounced prevalence among elderly (4). This study aims to delineate the incidence of AF subsequent to interatrial shunt, surgical och transcatheter, closure within a national cohort in Sweden. Methods The study includes all patients diagnosed with an interatrial shunt, atrial septal defect and patent foramen ovale, classified using the International Classification of Diseases, 10th edition (ICD-10), as documented in the Swedish National Patient Register, along with data sourced from the Cause of Death Register. Patients with multiple diagnoses of other congenital heart diseases are excluded, and interventions performed between 1997 and 2017 are identified using the NOMESCO surgical classification. Comorbidities such as chronic ischemic heart disease, myocardial infarction, heart failure, hypertension, and diabetes are considered, with the primary endpoint being the incidence of AF post-intervention. Results A total of 3,426 patients with interatrial shunts underwent intervention between 1997 and 2017, with a mean follow-up duration of 6.2 years. Among these, 49% (n=1,725) underwent transcatheter closure, while 41 patients underwent both transcatheter and surgical procedures. The mean age was 36 years, with 9% presenting with heart failure at baseline. Before the intervention, 14% (n=483) had AF, increasing to 22% (n=755) post-intervention. Of those developing AF post-intervention, 16% (n=119) experienced the AF event within 60 days, evenly distributed between transcatheter and surgical procedures. Over 40% of patients with post-intervention AF had an average age of 62. The incidence of AF post-intervention was 1.6 per 100 patient-years (see Figure). Stroke incidence was 3.6 per 100 patient-years and the mortality rate was 7% (n=257) during follow up (see Table). Conclusions Patients with interatrial shunts exhibit a high incidence of AF, which further escalates following closure of the interatrial shunt, not only within the first few days post-intervention, particularly among the elderly population. This can relate to high incidence of ischemic stroke.Figure
This work explored the mechanism of augmented stress-induced vascular reactivity of senescent murine femoral arteries (FAs). Mechanical and pharmacological reactivity of young (12-25 weeks, y-FA) and senescent (>104 weeks, s-FAs) femoral arteries was measured by wire myography. Expression and protein phosphorylation of selected regulatory proteins were studied by western blotting. Expression ratio of the Exon24 in/out splice isoforms of the regulatory subunit of myosin phosphatase, MYPT1 (MYPT1-Exon24 in/out), was determined by polymerase chain reaction (PCR). While the resting length-tension relationship showed no alteration, the stretch-induced-tone increased to 8.3 ± 0.9 mN in s-FA versus only 4.6 ± 0.3 mN in y-FAs. Under basal conditions, phosphorylation of the regulatory light chain of myosin at S19 was 19.2 ± 5.8% in y-FA versus 49.2 ± 12.6% in s-FA. Inhibition of endogenous NO release raised tone additionally to 10.4 ± 1.2 mN in s-FA, whereas this treatment had a negligible effect in y-FAs (4.8 ± 0.3 mN). In s-FAs, reactivity to NO donor was augmented (pD2 = -4.5 ± 0.3 in y-FA vs. -5.2 ± 0.1 in senescent). Accordingly, in s-FAs, MYPT1-Exon24-out-mRNA, which is responsible for expression of the more sensitive to protein-kinase G, leucine-zipper-positive MYPT1 isoform, was increased. The present work provides evidence that senescent murine s-FA undergoes vascular remodelling associated with increases in stretch-activated contractility and sensitivity to NO/cGMP/PKG system.
Deep learning techniques have been successfully applied in Synthetic Aperture Radar (SAR) target recognition in static scenarios relying on predefined datasets. However, in real-world scenarios, models must incrementally learn new information without forgetting previously learned knowledge. Models' tendency to forget old knowledge when learning new tasks, known as catastrophic forgetting, remains an open challenge. In this paper, an incremental learning framework, called IncSAR, is proposed to mitigate catastrophic forgetting in SAR target recognition. IncSAR comprises a Vision Transformer (ViT) and a custom-designed Convolutional Neural Network (CNN) in individual branches combined through a late-fusion strategy. A denoising module, utilizing the properties of Robust Principal Component Analysis (RPCA), is introduced to alleviate the speckle noise present in SAR images. Moreover, a random projection layer is employed to enhance the linear separability of features, and a Linear Discriminant Analysis (LDA) approach is proposed to decorrelate the extracted class prototypes. Experimental results on the MSTAR and OpenSARShip benchmark datasets demonstrate that IncSAR outperforms state-of-the-art approaches, leading to an improvement from $98.05\%$ to $99.63\%$ in average accuracy and from $3.05\%$ to $0.33\%$ in performance dropping rate.
Stretch-induced vascular tone is an important element of autoregulatory adaptation of cerebral vasculature to maintain cerebral flow constant despite changes in perfusion pressure. Little is known as to the regulation of tone in senescent basilar arteries. We tested the hypothesis, that thin filament mechanisms in addition to smooth muscle myosin-II regulatory-light-chain-(MLC20)-phosphorylation and non-muscle-myosin-II, contribute to regulation of stretch-induced tone. In young BAs (y-BAs) mechanical stretch does not lead to spontaneous tone generation. Stretch-induced tone in y-BAs appeared only after inhibition of NO-release by L-NAME and was fully prevented by treatment with 3 μmol/L RhoA-kinase (ROK) inhibitor Y27632. L-NAME-induced tone was reduced in y-BAs from heterozygous mice carrying a point mutation of the targeting-subunit of the myosin phosphatase, MYPT1 at threonine696 (MYPT1-T696A/+). In y-BAs, MYPT1-T696A-mutation also blunted the ability of L-NAME to increase MLC20-phosphorylation. In contrast, senescent BAs (s-BAs; >24 months) developed stable spontaneous stretch-induced tone and pharmacological inhibition of NO-release by L-NAME led to an additive effect. In s-BAs the MYPT1-T696A mutation also blunted MLC20-phosphorylation, but did not prevent development of stretch-induced tone. In s-BAs from both lines, Y27632 completely abolished stretch- and L-NAME-induced tone. In s-BAs phosphorylation of non-muscle-myosin-S1943 and PAK1-T423, shown to be down-stream effectors of ROK was also reduced by Y27632 treatment. Stretch- and L-NAME tone were inhibited by inhibition of non-muscle myosin (NM-myosin) by blebbistatin. We also tested whether the substrate of PAK1 the thin-filament associated protein, caldesmon is involved in the regulation of stretch-induced tone in advanced age. BAs obtained from heterozygotes Cald1+/- mice generated stretch-induced tone already at an age of 20-21 months old BAs (o-BA). The magnitude of stretch-induced tone in Cald1+/- o-BAs was similar to that in s-BA. In addition, truncation of caldesmon myosin binding Exon2 (CaD-▵Ex2-/-) did not accelerate stretch-induced tone. Our study indicates that in senescent cerebral vessels, mechanisms distinct from MLC20 phosphorylation contribute to regulation of tone in the absence of a contractile agonist. While in y-and o-BA the canonical pathways, i.e., inhibition of MLCP by ROK and increase in pMLC20, predominate, tone regulation in senescence involves ROK regulated mechanisms, involving non-muscle-myosin and thin filament linked mechanisms involving caldesmon.
Recent advances in content generation technologies (widely known as DeepFakes) along with the online proliferation of manipulated media content render the detection of such manipulations a task of increasing importance. Even though there are many DeepFake detection methods, only a few focus on the impact of dataset preprocessing and the aggregation of frame-level to video-level prediction on model performance. In this paper, we propose a pre-processing step to improve the training data quality and examine its effect on the performance of DeepFake detection. We also propose and evaluate the effect of video-level prediction aggregation approaches. Experimental results show that the proposed pre-processing approach leads to considerable improvements in the performance of detection models, and the proposed prediction aggregation scheme further boosts the detection efficiency in cases where there are multiple faces in a video.
We have investigated the previously published 'metabolon hypothesis' postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1. In these doubly transfected tsA201 cells, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Förster resonance energy transfer measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag-antibody against Flag-tagged AE1 expressed in tsA201 cells, which does not co-precipitate native CAII but co-precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellular HCO3(-) permeability (P(HCO3(-))) sensitive to inhibition by 4,4-Diisothiocyano-2,2-stilbenedisulfonic acid. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N-terminally truncated CAII lacking the proposed site of interaction with the C-terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild-type CAII. These variously co-transfected tsA201 cells exhibit a positive correlation between cellular P(HCO3(-)) and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII-AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII-AE1 metabolon to erythroid AE1-mediated HCO3(-) transport was further tested in normal red cells and red cells from CAII-deficient patients that retain substantial CA activity associated with the erythroid CAI protein lacking the proposed AE1-binding sequence. Erythroid P(HCO3(-)) was indistinguishable in these two cell types, providing no support for the proposed functional importance of the physical interaction of CAII and AE1. A theoretical model predicts that homogeneous cytoplasmic distribution of CAII is more favourable for cellular transport of HCO3(-) and CO2 than is association of CAII with the cytoplasmic surface of the plasma membrane. This is due to the fact that the relatively slow intracellular transport of H(+) makes it most efficient to place the CA in the vicinity of the haemoglobin molecules, which are homogeneously distributed over the cytoplasm.
