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Abstract Fluidic force microscopy (FluidFM) fuses the force sensitivity of atomic force microscopy with the manipulation capabilities of microfluidics by using microfabricated cantilevers with embedded fluidic channels. This innovation initiated new research and development directions in biology, biophysics, and material science. To acquire reliable and reproducible data, the calibration of the force sensor is crucial. Importantly, the hollow FluidFM cantilevers contain a row of parallel pillars inside a rectangular beam. The precise spring constant calibration of the internally structured cantilever is far from trivial, and existing methods generally assume simplifications that are not applicable to these special types of cantilevers. In addition, the Sader method, which is currently implemented by the FluidFM community, relies on the precise measurement of the quality factor, which renders the calibration of the spring constant sensitive to noise. In this study, the hydrodynamic function of these special types of hollow cantilevers was experimentally determined with different instruments. Based on the hydrodynamic function, a novel spring constant calibration method was adapted, which relied only on the two resonance frequencies of the cantilever, measured in air and in a liquid. Based on these results, our proposed method can be successfully used for the reliable, noise-free calibration of hollow FluidFM cantilevers.

Abstract The invasiveness of cancer cells describes the metastasizing capability of a primary tumor. The straightforward detection and quantification of cancer cell invasion are important to predict the survival rate of a cancer patient and to test how anti-cancer compounds influence cancer progression. Digital holographic microscopy based M4 Holomonitor (HM) is a technique that allows the label-free monitoring of cell morphological and kinetical parameters in real-time. Here, a fully confluent epithelial monolayer derived from the African green monkey kidney (Vero) on a gelatin-coated surface was established, then HeLa cells were seeded on top of the monolayer, and their behavior was monitored for 24 h using HM. Several cancer cells showing invasiveness were detected during this period, while other HeLa cells did not show any signs of aggressivity. It was demonstrated that the invasion of single cancer cells is soundly observable and also quantifiable through monitoring parameters such as phase shift, optical volume, area, and motility, which parameters can easily be obtained and processed automatically. Based on the experimental data, the invasion speed of cancer cells entering the epithelial layer can be defined as the shrinking of detected single-cell volume per unit time. The invasion speed and its correlation with cell migration parameters were analyzed in depth. A clear linear relationship between migration and invasion speed was found, cancer cells with stronger migration have slower invasion speed. These results not only describe the effect of how cancer cells invade the underlying monolayer in contrast to non-invasive HeLa cells, but could help in future research to optimize drugs affecting cell invasibility in a fully automated, label-free and high-throughput manner.

Abstract Single-cell adhesion plays an essential role in biological and biomedical sciences, but its precise measurement for a large number of cells is still a challenging task. At present, typical force measuring techniques usually offer low throughput, a few cells per day, and therefore are unable to uncover phenomena emerging at the population level. In this work, robotic fluidic force microscopy (FluidFM) was utilized to measure the adhesion parameters of cells in a high-throughput manner to study their population distributions in-depth. The investigated cell type was the genetically engineered HeLa Fucci construct with cell cycle-dependent expression of fluorescent proteins. This feature, combined with the high-throughput measurement made it possible for the first time to characterize the single-cell adhesion distributions at various stages of the cell cycle. It was found that parameters such as single-cell adhesion force and energy follow a lognormal population distribution. Therefore, conclusions based on adhesion data of a low number of cells or treating the population as normally distributed can be misleading. Moreover, we found that the cell area was significantly the smallest, and the area normalized maximal adhesion force was significantly the largest for the colorless cells (the mitotic (M) and early G1 phases). Notably, the parameter characterizing the elongation of the cells until the maximum level of force between the cell and its substratum was also dependent on the cell cycle, which quantity was the smallest for the colorless cells. A novel parameter, named the spring coefficient of the cell, was introduced as the fraction of maximal adhesion force and maximal cell elongation during the mechanical detachment, which was found to be significantly the largest for the colorless cells. Cells in the M phase adhere in atypical way, with so-called reticular adhesions, which are different from canonical focal adhesions. We first revealed that reticular adhesion can exert a higher force per unit area than canonical focal adhesions, and cells in this phase are significantly stiffer. The possible biological consequences of these findings were also discussed, together with the practical relevance of the observed population-level adhesion phenomena.

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.

Abstract Lenalidomide maintenance (LM) has shown benefit in progression‐free survival (PFS) and overall survival (OS) in clinical trials. LM is the recommended standard of care in patients with newly diagnosed multiple myeloma (MM) after high‐dose melphalan and autologous stem cell transplantation (HDM‐ASCT). In Denmark, LM has been approved and publicly funded for all patients treated with HDM‐ASCT since June 2019. Patients with newly diagnosed MM treated with their first HDM‐ASCT between June 2019 and March 2022 were included and followed until data cut‐off in June 2023. To compare outcomes, a historical pre‐LM cohort from the Danish MM Registry, consisting of 364 MM patients treated with HDM‐ASCT between June 2015 and June 2019, was used. Among 364 patients treated with HDM‐ASCT after June 2019, 22.3% received consolidation therapy and 3.7% underwent tandem HDM‐ASCT. During follow‐up, 297 patients (81.6%) initiated maintenance therapy, with 277 (76.1%) receiving LM. Overall, 145 patients (52.3%) discontinued LM most commonly due to toxicity 75 (51.7%), with fatigue (30.7%), cytopenia (25.3%), and neuropathy (17.3%) being the main reasons. In a 6‐month landmark analysis, early discontinuation did not negatively impact PFS or OS. The LM cohort had similar PFS, and OS compared to the pre‐LM cohort. The 3‐year PFS and OS rates in the LM cohort were 61% and 86%, respectively, while the pre‐LM cohort had a 3‐year PFS of 55% and a 3‐year OS of 89%. In conclusion, the introduction of LM as a nationwide treatment option in Denmark did not lead to improved clinical outcomes.

Abstract Objectives The study aimed to compare the antibacterial effect of a novel disinfectant, hyper-pure chlorine dioxide (hClO2) to sodium hypochlorite (NaOCl) in various depths of dentin tubules. Materials and methods The distal root of the extracted lower molars was infected artificially with Enterococcus faecalis. The control group was rinsed with saline, and the test groups were irrigated with either 5% NaOCl or 0.12% hClO2. The longitudinally split teeth were stained by viability stain. The coronal third of the root was scanned with a confocal laser scanning microscope. The fluorescent intensities were measured, and the percentage of dead bacteria was calculated at depths up to 950 μm along the dentin tubules. The effect of penetration depth, irrigants, and their interaction on antimicrobial efficacy was determined by the linear mixed model. Results The percentage of dead bacteria was higher both in the NaOCl (45.1 ± 2.3%, p

Abstract Pomalidomide‐dexamethasone (Pd) has been a standard care treatment for relapsed and refractory multiple myeloma since 2013. However, the outcomes of Pd after exposure to CD38 antibodies are not known. Here we describe the real‐world use and efficacy of pomalidomide in a Danish, nationwide cohort of daratumumab‐exposed patients. We identified 328 patients that were treated with pomalidomide. Of these, 137 received Pd, 65 daratumumab‐pomalidomide‐dexamethasone (DPd), 43 pomalidomide‐cyclophosphamide‐dexamethasone (PCd), 19 carfilzomib‐pomalidomide‐dexamethasone (KPD), 11 pomalidomide‐bortezomib‐dexamethasone (PVd), and 52 pomalidomide in other combinations. Patients treated with Pd in this cohort had a partial response or better (≥ PR) rate of 35.8% and median time to next treatment (mTNT) of 4.9 months, almost identical to the results of previous prospective clinical trials. Although treatment with the various pomalidomide‐containing triplet regimens resulted in higher ≥ PR rates (PCd: 46.5%, PVd: 63.6%, DPd: 55.4%, KPd: 63.2%), the mTNT achieved was not significantly better than with Pd in most cases (PCD: 5.4, PVD: 5.3, DPD: 4.7 months). The exception to this was KPd (mTNT 7.4 months), but this regimen was mainly used earlier in the course of the disease (median time from diagnosis 2.3 years vs. 3.7–4.3 years). The most important predictor of outcomes was not the choice of index regimen (p = 0.72), but prior exposure (p = 0.0116). Compared to CD38 antibody‐naïve patients, triple‐class‐exposed patients achieved reduced ≥ PR rate (38.0% vs. 47.3%), shorter mTNT (4.0 vs. 5.9 months), and shorter median overall survival (12.4 vs. 24.2 months) with pomalidomide treatment.

Objectives: Reducing the microbial level in the aerosol created during dental procedures is essential to avoiding infections. The aim of this study was to examine the change in Streptococcus mutans (S. mutans) and the total bacterial load in human saliva in vivo after a single rinse with different mouthwashes. Material and methods: One mL of unstimulated saliva was collected from volunteers with poor oral hygiene at baseline and 5 min after a 1-min rinsing with diluted Solumium Oral® (hyper-pure 0.0015% chlorine dioxide; ClO2), Listerine Total Care®, Corsodyl® (0.2% chlorhexidine-digluconate; CHX), or BioGate Si*CLEAN for bacterial investigation. In a second study, volunteers rinsed with 0.003% ClO2 or CHX for 1 min, and saliva was collected at baseline, after 5 min, and after 90 min. After plating, the total plate and S. mutans colony numbers were determined. Results: In the first study, ClO2 and CHX similarly reduced both total germ and S. mutans numbers, while Listerine Total Care® decreased only the S. mutans counts. BioGate Si*Clean had no effect on either the total germ or S. mutans numbers. In the second study, an increasing tendency toward bacterial regrowth was observed with CHX after 90 min compared to the 5-min value, while no change was measured after ClO2 rinsing. Conclusion: Hyper-pure ClO2 rinsing may be a new promising preventive and therapeutic adjuvant in dental practice, as it is similar in effectiveness to the gold standard CHX-containing mouthwashes, especially in patients concerned with taste or tooth discoloration during oral health therapy.

BackgroundStaphylococcus aureus and S. pseudintermedius are the two most common coagulase positive staphylococci (CPS). S. aureus is more prevalent among humans, whereas S. pseudintermedius is more commonly isolated from dogs, however, both can cause various community and hospital acquired diseases in humans.MethodsIn the current study we screened 102 dogs and 84 owners in Hungary. We tested the antibiotic susceptibility of the strains and in order to get a better picture of the clonal relationship of the strains, we used pulsed-field gel electrophoresis. In addition, three pairs of isolates with identical PFGE patterns were whole genome sequenced, MLST and spa types were established.ResultsCarriage rate of S. aureus was 23.8% in humans and 4.9% in dogs and two cases of co-carriage were found among dogs and owners. S. pseudintermedius carriage rate was 2.4% and 34.3%, respectively, with only one co-carriage. The isolates were generally rather susceptible to the tested antibiotics, but high tetracycline resistance of S. pseudintermedius strains was noted. The co-carried isolates shared almost the same resistance genes (including tet(K), bla(Z), norA, mepR, lmrS, fosB) and virulence gene pattern. Apart from the common staphylococcal enzymes and cytotoxins, we found enterotoxins and exfoliative toxins as well. The two S. aureus pairs belonged to ST45-t630, ST45-t671 and ST15-t084, ST15-t084, respectively. The co-carried S. pseudintermedius isolates shared the same housekeeping gene alleles determining a novel sequence type ST1685.ConclusionsBased on the genomic data, dog-owner co-carried strains displayed only insignificant differences therefore provided evidence for potential human-to-dog and dog-to-human transmission.

Most patients cannot be included in randomized clinical trials. We report real-world outcomes of all Danish patients with multiple myeloma (MM) treated with daratumumab-based regimens until 1 January 2019.MethodsInformation of 635 patients treated with daratumumab was collected retrospectively and included lines of therapy (LOT), hematologic responses according to the International Myeloma Working Group recommendations, time to next treatment (TNT) and the cause of discontinuation of treatment. Baseline characteristics were acquired from the validated Danish Multiple Myeloma Registry (DMMR).ResultsDaratumumab was administrated as monotherapy (Da-mono) in 27.7%, in combination with immunomodulatory drugs (Da-IMiD) in 57.3%, in combination with proteasome inhibitors (Da-PI) in 11.2% and in other combinations (Da-other) in 3.8% of patients. The median number of lines of therapy given before daratumumab was 5 for Da-mono, 3 for Da-IMiD, 4 for Da-PI, and 2 for Da-other. In Da-mono, overall response rate (ORR) was 44.9% and median time to next treatment (mTNT) was 4.9 months. In Da-IMiD, ORR was 80.5%, and mTNT was 16.1 months. In Da-PI, OOR was 60.6% and mTNT was 5.3 months. In patients treated with Da-other, OOR was 54,2% and mTNT was 5.6 months. The use of daratumumab in early LOT was associated with longer TNT (pConclusionThe real-world outcomes of multiple myeloma patients treated with daratumumab are worse than the results of clinical trials. Outcomes achieved with daratumumab were best when daratumumab was used in combination with IMIDs and in early LOT. Patients with high-risk CA had worse outcomes, but patients with amp1q had similar outcomes to standard-risk patients.

Background: Preventing infectious diseases has become particularly relevant in the past few years. Therefore, antiseptics that are harmless and insusceptible to microbial resistance mechanisms are desired in medicine and public health. In our recent work, a poly(ethylene oxide)-based nanofibrous mat loaded with sodium chlorite was formulated. Methods: We tested the chlorine dioxide production and bacterial inactivation of the fibers in a medium, modeling the parameters of human exhaled air (ca. 5% (v/v) CO2, T = 37 °C, RH > 95%). The morphology and microstructure of the fibers were investigated via scanning electron microscopy and infrared spectroscopy. Results: Smooth-surfaced, nanoscale fibers were produced. The ClO2-producing ability of the fibers decreased from 65.8 ppm/mg to 4.8 ppm/mg with the increase of the sample weight from 1 to 30 mg. The effect of CO2 concentration and exposure time was also evaluated. The antibacterial activity of the fibers was tested in a 24 h experiment. The sodium-chlorite-loaded fibers showed substantial antibacterial activity. Conclusions: Chlorine dioxide was liberated into the gas phase in the presence of CO2 and water vapor, eliminating the bacteria. Sodium-chlorite-loaded nanofibers can be sources of prolonged chlorine dioxide production and subsequent pathogen inactivation in a CO2-rich and humid environment. Based on the results, further evaluation of the possible application of the formulation in face-mask filters as medical devices is encouraged.

In this retrospective study we reviewed the clinical course of every patient with multiple myeloma treated from 2006 to 2016 at Vejle Hospital: 303 patients with a median age of 69 years at diagnosis received a median of four (range 1–18) lines of therapy; 149 in a 2006–2010 cohort and 154 in a 2011–2016 cohort. After initiation of treatment, the median decrease in the number of patients per each subsequent line of therapy was 22%. Lenalidomide-dexamethasone (n = 156), bortezomib-dexamethasone (n = 107), and bortezomib-lenalidomide-dexamethasone (n = 84) were the most commonly used regimens. The partial response or better rate was 78%, 58%, 55%, and 44% in lines of therapy one to four, respectively. The median (95% confidence interval [CI]) progression-free survival was 18 (15–22), 10 (8–13), 8 (7–10), and 6 (4–8) months in lines of therapy one to four, respectively. The median (95% CI) overall survival (OS) was 4.1 (3.7–4.8) years. Compared with the 2006–2010 cohort, patients in the 2011–2016 cohort had longer OS; 5.3 (4.7 to not reached) versus 3.4 (2.7–4.0) years, p < 0.0001. This was especially true in patients not treated with high-dose therapy and autologous stem cell transplantation; 4.7 (3.2–5.9) versus 2.6 (2.0–3.3) years, p = 0.0052. Patients in the 2011–2016 cohort were on treatment during a greater part of their life and had higher exposure to high-dose melphalan with autologous stem cell transplantation, lenalidomide, pomalidomide, daratumumab, and carfilzomib.

Human faces play a special role in social cognition, since as a core signal of interpersonal communication, they convey various kinds of information (e.g., about sex, age, race, emotions, intentions). Study 1 aimed to explore how this specialization manifests itself in eye movements when looking at neutral, static, female faces. We monitored the gaze pattern of 23 adult participants using eye-tracking method. To test if template-driven processes are involved in face perception, and to see how inversion affects fixations on special facial stimuli, we presented vertically cut half-faces in upright and inverted positions (so half of each stimulus represented a half-face, whereas the other half was left blank). Our results corroborate prior findings consistently demonstrating the dominance of the triangular area marked by the eyes and the mouth, measured by the number and duration of fixations. In addition, we found evidence for so-called complementary fixations, targeted at the non-informative parts (i.e., the half that does not contain any facial information) of the pictures, suggesting that other mechanisms beyond purely stimulus-driven ones might drive looking behavior when scanning faces. Study 2 was intended to test if these systematic eye movements are face-specific or occur in case of other visual objects as well.

Non-invasive polysomnography recording on dogs has been claimed to produce data comparable to those for humans regarding sleep macrostructure, EEG spectra and sleep spindles. While functional parallels have been described relating to both affective (e.g., emotion processing) and cognitive (e.g., memory consolidation) domains, methodologically relevant questions about the reliability of sleep stage scoring still need to be addressed. In Study 1, we analyzed the effects of different coders and different numbers of visible EEG channels on the visual scoring of the same polysomnography recordings. The lowest agreement was found between independent coders with different scoring experience using full (3 h-long) recordings of the whole dataset, and the highest agreement within-coder, using only a fraction of the original dataset (randomly selected 100 epochs (i.e., 100 × 20 s long segments)). The identification of drowsiness was found to be the least reliable, while that of non-REM (rapid eye movement, NREM) was the most reliable. Disagreements resulted in no or only moderate differences in macrostructural and spectral variables. Study 2 targeted the task of automated sleep EEG time series classification. Supervised machine learning (ML) models were used to help the manual annotation process by reliably predicting if the dog was sleeping or awake. Logistic regression models (LogREG), gradient boosted trees (GBT) and convolutional neural networks (CNN) were set up and trained for sleep state prediction from already collected and manually annotated EEG data. The evaluation of the individual models suggests that their combination results in the best performance: ~0.9 AUC test scores.

The domestic dog (Canis familiaris) has been shown to both excel in recognising human emotions and produce emotion-related vocalisations and postures that humans can easily recognise. However, little is known about the effect of emotional experiences on subsequent sleep physiology, a set of phenomena heavily interrelated with emotions in the case of humans. The present paper examines heart rate (HR) and heart rate variability (HRV) during dogs’ sleep, measures that are influenced by both positive and negative emotions in awake dogs. In Study I, descriptive HR and HRV data is provided on N = 12 dogs about the different sleep stages (wake, drowsiness, non-rapid eye movement (non-REM), REM; scoring based on electroencephalogram (EEG) data). We conclude that wakefulness is characterised by higher HR and lower HRV compared to all sleep stages. Furthermore, drowsiness is characterised by higher HR and lower HRV than non-REM and REM, but only if the electrocardiogram (ECG) samples are taken from the first occurrence of a given sleep stage, not when the longest periods of each sleep stage are analysed. Non-REM and REM sleep were not found to be different from each other in either HR or HRV parameters. In Study II, sleep HR and HRV measures are compared in N = 16 dogs after a positive versus negative social interaction (within-subject design). The positive social interaction consisted of petting and ball play, while the negative social interaction was a mixture of separation, threatening approach and still face test. Results are consistent with the two-dimensional emotion hypothesis in that following the intense positive interaction more elevated HR and decreased HRV is found compared to the mildly negative (lower intensity) interaction. However, although this trend can be observed in all sleep stages except for REM, the results only reach significance in the wake stage. In sum, the present findings suggest that HR and HRV are possible to measure during dogs’ sleep, and can potentially be used to study the effect of emotions not only during but also after such interactions.

Background: Although the number of point-of-care ultrasound devices available in Hungarian primary care practices are increasing due to government funding, their use in day-to-day patient care is limited and unregulated. Our study aimed to evaluate the attitudes and needs of general practitioners (GPs) and patients in Hungary regarding the introduction of bedside ultrasonography in primary care practices., Methods: As a part of a cross-sectional study, an anonymous, self-administered questionnaire was distributed to GPs and patients on a social media platform. Data collection was carried out from August 2023 to October 2023. Chi-square test was used to determine the associations between categorical variables., Results: The survey was completed by 415 GPs (mean age 53.8 ± 11.1 years, 54.9% female, mean 19.5 ± 11.9 years of practice) and 693 patients (mean age 45.5 ± 12.3 years, 95.2% female). There was a statistically significant increase in interest in PoCUS among young and middle-aged GPs (age 28-59; p = 0.02). In addition, this population of GPs was also more likely to undertake training in PoCUS than their older colleagues (p < 0.0001). An inverse relationship was found between the duration of practice and training willingness (p = 0.0011). Even with the government's financial support, only 8.2% of GPs currently use PoCUS in a daily basis, and 59.5% of GPs are unfamiliar with the indications and the ways of using it. Patients would even pay to have the examination done in a primary care setting, even though only 45.9% of patients would pay a GP who is not certified in PoCUS, but the willingness to pay increased to 99.4% for those with formal training (p = 0.024)., Conclusion: Our findings indicate a significant interest in adapting PoCUS in primary care from both GPs and patients. Based on the fact that a significant proportion of Hungarian GPs are unaware of PoCUS and its indications, it is particularly important to develop educational frameworks, and practical guidelines for the effective incorporation of PoCUS in Hungary., (© 2024. The Author(s).)

The health benefits of regular physical exercise are well known. Even so, there is increasing evidence that the exercise regimes of elite athletes can evoke cardiac arrhythmias including ventricular fibrillation and even sudden cardiac death (SCD). The mechanism of exercise-induced arrhythmia and SCD is poorly understood. Here, we show that chronic training in a canine model (12 sedentary and 12 trained dogs) that mimics the regime of elite athletes induces electrophysiological remodeling (measured by ECG, patch-clamp, and immunocytochemical techniques) resulting in increases of both the trigger and the substrate for ventricular arrhythmias. Thus, 4 months sustained training lengthened ventricular repolarization (QTc: 237.1±3.4 ms vs. 213.6±2.8 ms, n=12; APD90: 472.8±29.6 ms vs. 370.1±32.7 ms, n=29 vs. 25), decreased transient outward potassium current (6.4±0.5 pA/pF vs. 8.8±0.9 pA/pF at 50 mV, n=54 vs. 42), and increased the short-term variability of repolarization (29.5±3.8 ms vs. 17.5±4.0 ms, n=27 vs. 18). Left ventricular fibrosis and HCN4 protein expression were also enhanced. These changes were associated with enhanced ectopic activity (number of escape beats from 0/hr to 29.7±20.3/hr) in vivo and arrhythmia susceptibility (elicited ventricular fibrillation: 3 of 10 sedentary dogs vs. 6 of 10 trained dogs). Our findings provide in vivo, cellular electrophysiological and molecular biological evidence for the enhanced susceptibility to ventricular arrhythmia in an experimental large animal model of endurance training., Competing Interests: AP, LT, NZ, NT, JP, ZK, SD, VD, PH, VV, GÁ, ZH, PG, JS, TÁ, MN, AS, NJ, LV, NN, IB, AF, AV No competing interests declared, (© 2023, Polyák, Topal et al.)

Aims: Volume overload (VO) induced hypertrophy is one of the hallmarks to the development of heart diseases. Understanding the compensatory mechanisms involved in this process might help preventing the disease progression., Methods and Results: Therefore, the present study used 2 months old Wistar rats, which underwent an aortocaval fistula to develop VO-induced hypertrophy. The animals were subdivided into four different groups, two sham operated animals served as age-matched controls and two groups with aortocaval fistula. Echocardiography was performed prior termination after 4- and 8-month. Functional and molecular changes of several sarcomeric proteins and their signalling pathways involved in the regulation and modulation of cardiomyocyte function were investigated., Results: The model was characterized with preserved ejection fraction in all groups and with elevated heart/body weight ratio, left/right ventricular and atrial weight at 4- and 8-month, which indicates VO-induced hypertrophy. In addition, 8-months groups showed increased left ventricular internal diameter during diastole, RV internal diameter, stroke volume and velocity-time index compared with their age-matched controls. These changes were accompanied by increased Ca 2+ sensitivity and titin-based cardiomyocyte stiffness in 8-month VO rats compared with other groups. The altered cardiomyocyte mechanics was associated with phosphorylation deficit of sarcomeric proteins cardiac troponin I, myosin binding protein C and titin, also accompanied with impaired signalling pathways involved in phosphorylation of these sarcomeric proteins in 8-month VO rats compared with age-matched control group. Impaired protein phosphorylation status and dysregulated signalling pathways were associated with significant alterations in the oxidative status of both kinases CaMKII and PKG explaining by this the elevated Ca 2+ sensitivity and titin-based cardiomyocyte stiffness and perhaps the development of hypertrophy., Conclusions: Our findings showed VO-induced cardiomyocyte dysfunction via deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative state of CaMKII and PKG and this might contribute to the development of hypertrophy., (© 2022 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Hypoxia upregulates PCSK9 expression in the heart, and PCSK9 affects the function of myocytes. This study aimed to investigate the impact of PCSK9 on reperfusion injury in rats and mice fed normal or high-fat diets. Either the genetic knockout of PCSK9 (mice) or the antagonism of circulating PCSK9 via Pep2-8 (mice and rats) was used. Isolated perfused hearts were exposed to 45 min of ischemia followed by 120 min of reperfusion. In vivo, mice were fed normal or high-fat diets (2% cholesterol) for eight weeks prior to coronary artery occlusion (45 min of ischemia) and reperfusion (120 min). Ischemia/reperfusion upregulates PCSK9 expression (rats and mice) and releases it into the perfusate. The inhibition of extracellular PCSK9 does not affect infarct sizes or functional recovery. However, genetic deletion largely reduces infarct size and improves post-ischemic recovery in mice ex vivo but not in vivo. A high-fat diet reduced the survival rate during ischemia and reperfusion, but in a PCSK9-independent manner that was associated with increased plasma matrix metalloproteinase (MMP)9 activity. PCSK9 deletion, but not the inhibition of extracellular PCSK9, reduces infarct sizes in ex vivo hearts, but this effect is overridden in vivo by factors such as MMP9.

Background: Aortic stenosis (AS) is the most common primary valve lesion requiring intervention in Europe and North America. It has a prolonged subclinical period during which, as AS worsens, left ventricular adaptation becomes inadequate and impaired systolic and/or diastolic dysfunction may lead to overt heart failure (HF). The development of HF is an inflexion point in the natural history of AS. Pulmonary congestion is a cardinal feature in HF, and lung ultrasound (LUS) evaluation of B-lines has been proposed as a simple, noninvasive tool to assess pulmonary congestion. Aim: To assess the presence and the prognostic value of sonographic pulmonary congestion in patients with moderate or severe AS. Methods: 75 consecutive patients (39 women, mean age 73.85 ± 7.7 years) with moderate or severe AS were enrolled. All patients underwent comprehensive echocardiography and LUS with the 28 scanning-site assessment. Patients were followed-up for 13.4 ± 6 months to establish the prognostic value of LUS. A composite endpoint of death (of any cause), hospitalization for HF and intensification of loop diuretic therapy was considered. Results: We found a severe degree of B-lines (≥30) in 29.33% of patients. The number of B-lines correlated with the estimated pulmonary artery systolic pressure ( p < 0.001, r = 0.574) and increased along with NYHA class ( p < 0.05, rho = 0.383). At multivariable analysis, B-lines ≥30, and mean gradient were the independent predictors of events [B-lines: 2.79 (CI 1.03-7.54), p = 0.04; mean gradient: 1.04 (CI 1.01-1.07), p = 0.004]. Conclusion: Evaluation of B-lines is a simple, highly feasible method to detect pulmonary congestion in AS. The number of B-lines correlates with the hemodynamic changes caused by AS and with the functional status of patients. A severe degree of sonographic pulmonary congestion is associated with an increased risk of adverse events., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Szabó, Gargani, Morvai-Illés, Polestyuk-Németh, Frigy, Varga and Ágoston.)

Background: Heart failure with preserved ejection fraction (HFpEF) is a growing healthcare burden, and its prevalence is steadily increasing. Lung ultrasound (LUS) is a promising screening and prognostic tool in the heart failure population. However, more information on its value in predicting outcome is needed. Aims: The aim of our study was to assess the prognostic performance of LUS B-lines compared to traditional and novel clinical and echocardiographic parameters and natriuretic peptide levels in patients with newly diagnosed HFpEF in an ambulatory setting. Methods: In our prospective cohort study, all ambulatory patients with clinical suspicion of HFpEF underwent comprehensive echocardiography, lung ultrasound and NT-proBNP measurement during their first appointment at our cardiology outpatient clinic. Our endpoint was a composite of worsening heart failure symptoms requiring hospitalization or loop diuretic dose escalation and death. Results: We prospectively enrolled 75 consecutive patients with HFpEF who matched our inclusion and exclusion criteria. We detected 11 events on a 26 ± 10-months follow-up. We found that the predictive value of B-lines is similar to the predictive value of NT-proBNP (AUC 0.863 vs. 0.859), with the best cut-off at >15 B-lines. Having more B-lines than 15 significantly increased the likelihood of adverse events with a hazard ratio of 20.956 ( p = 0.004). The number of B-lines remained an independent predictor of events at multivariate modeling. Having more than 15 B-lines lines was associated with a significantly worse event-free survival (Log-rank: 16.804, p < 0.001). Conclusion: The number of B-lines seems to be an independent prognostic factor for adverse outcomes in HFpEF. Since it is an easy-to-learn, feasible and radiation-free method, it may add substantial value to the commonly used diagnostic and risk stratification models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Morvai-Illés, Polestyuk-Németh, Szabó, Monoki, Gargani, Picano, Varga and Ágoston.)

Background: The effort-reward imbalance (ERI) model by Siegrist encouraged numerous scientific investigations that reported particular ties between psychosocial risks and poor self-reported health (SRH), while psychosocial work-related stress has also been linked to musculoskeletal disorders (MSDs). The aim of this study was to examine the health status and the perceived levels of occupational stress of university employees and to analyse the findings according to the employees' effort and reward structure of work, perceived overall stress, SRH and the presence of MSDs.   Design and methods: 398 employees - including healthcare professionals, academic personnel and workers with administrative or other jobs - employed at the University of Szeged, Faculty of Medicine were investigated with a self-administered questionnaire including the Effort-Reward Imbalance Questionnaire (ERI-Q) and Perceived Stress Scale 4 (PSS-4)., Results: More than half of the investigated subjects (54.8%) reported some forms of MSDs. Low self-reported health (p<0.001) and presence of MSDs (p=0.015) were significantly associated with the level of perceived stress and effort-reward imbalance (ERI), moreover increased level of perceived stress was independently associated with the likelihood of MSDs (AOR=1.13) and low self-reported health (AOR=1.30). ERI well predicted low self-reported health (AOR=2.05) as well. Increased level of perceived stress positively correlated with high work-related effort (r=0.247, p<0.001) and over-commitment (r=0.387, p<0.001) while with work-related reward (r=-0.181, p=0.011) perceived stress showed a negative connection., Conclusion: Our results suggest that addressing the burden of effort-reward imbalance and MSDs would likely lessen employees' perceived level of overall stress and affect their self-reported overall state of health.

Objectives: Cardiopulmonary exercise test (CPET) is a widely used examination to predict the prognosis of many chronic pulmonary diseases, and it has also been tested in systemic sclerosis (SSc) with a focus on the development of pulmonary hypertension. CPET is a highly informative non-invasive tool that provides a more complex information than conventional lung function tests to predict the course of cardiopulmonary diseases, as it provides a general overview of the aerobic metabolism, influenced by pulmonary, cardiovascular and peripheral muscle function. The purpose of this investigation was to assess if the progression and the development of poor overall disease outcome in SSc can be predicted by this method., Methods: Twenty-nine SSc patients were investigated prospectively with standard follow-up plus CPET for a mean of 3.7 years to match the results of conventional evaluation modalities and CPET. A composite end-point of several serious outcomes reflecting SSc-related vascular and cardiopulmonary damage was set up, and the predictive value of and correlations between the CPET parameters and resting lung function and echocardiography variables were assessed., Results: None of the clinical parameters, resting lung function or echocardiographic test results proved to be predictive of the development of the endpoint of poor prognosis in this cohort. In contrast, several CPET parameters were found to discriminate between SSc patients with or without adverse outcome. The detection of desaturation (at any CPET test) was associated with a higher risk of poor prognosis (OR:5.265). VO2 and VE/VCO2 at baseline correlated with the annual decrease in FVC, anaerobic threshold with the development of digital ulcers, and VE/VO2 with the increase in pulmonary arterial pressure., Conclusions: Several CPET parameters obtained at the beginning of follow-up are informative of the appearance of various adverse end-points. CPET is a feasible examination in the care of SSc patients and provides excess information to current standard follow-up examinations.

Background: Sympathetic dysfunction can be evaluated by heart rate reserve (HRR) with exercise test., Objectives: To determine the value of HRR in predicting outcome of patients with hypertrophic cardiomyopathy (HCM)., Methods: We enrolled 917 HCM patients (age = 49 ± 15 years, 516 men) assessed with exercise stress echocardiography (ESE) in 11 centres. ESE modality was semi-supine bicycle in 51 patients (6%), upright bicycle in 476 (52%), and treadmill in 390 (42%). During ESE, we assessed left ventricular outflow tract obstruction (LVOTO), stress-induced new regional wall motion abnormalities (RWMA), and HRR (peak/rest heart rate, HR). By selection, all patients completed the follow-up. Mortality was the predetermined outcome measure Results: During ESE, RWMA occurred in 22 patients (2.4%) and LVOTO (≥50 mmHg) in 281 (30.4%). HRR was 1.90 ± 0.40 (lowest quartile ≤ 1.61, highest quartile > 2.13). Higher resting heart rate (odds ratio 1.027, 95% CI: 1.018-1.036, p < 0.001), older age (odds ratio 1.021, 95% CI: 1.009-1.033, p < 0.001), lower exercise tolerance (mets, odds ratio 0.761, 95% CI: 0.708-0.817, p < 0.001) and resting LVOTO (odds ratio 1.504, 95% CI: 1.043-2.170, p = 0.029) predicted a reduced HRR. During a median follow-up of 89 months (interquartile range: 36-145 months), 90 all-cause deaths occurred. At multivariable analysis, lowest quartile HRR (Hazard ratio 2.354, 95% CI 1.116-4.968 p = 0.025) and RWMA (Hazard ratio 3.279, 95% CI 1.441-7.461 p = 0.004) independently predicted death, in addition to age (Hazard ratio 1.064, 95% CI 1.043-1.085 p < 0.001) and maximal wall thickness (Hazard ratio 1.081, 95% CI 1.037-1.128, p < 0.001)., Conclusions: A blunted HRR during ESE predicts survival independently of RWMA in HCM patients.

Two methods are currently available for left atrial (LA) strain measurement by speckle tracking echocardiography, with two different reference timings for starting the analysis: QRS (QRS-LASr) and P wave (P-LASr). The aim of MASCOT HIT study was to define which of the two was more reproducible, more feasible, and less time consuming. In 26 expert centers, LA strain was analyzed by two different echocardiographers (young vs senior) in a blinded fashion. The study population included: healthy subjects, patients with arterial hypertension or aortic stenosis (LA pressure overload, group 2) and patients with mitral regurgitation or heart failure (LA volume-pressure overload, group 3). Difference between the inter-correlation coefficient (ICC) by the two echocardiographers using the two techniques, feasibility and analysis time of both methods were analyzed. A total of 938 subjects were included: 309 controls, 333 patients in group 2, and 296 patients in group 3. The ICC was comparable between QRS-LASr (0.93) and P-LASr (0.90). The young echocardiographers calculated QRS-LASr in 90% of cases, the expert ones in 95%. The feasibility of P-LASr was 85% by young echocardiographers and 88% by senior ones. QRS-LASr young median time was 110 s (interquartile range, IR, 78-149) vs senior 110 s (IR 78-155); for P-LASr, 120 s (IR 80-165) and 120 s (IR 90-161), respectively. LA strain was feasible in the majority of patients with similar reproducibility for both methods. QRS complex guaranteed a slightly higher feasibility and a lower time wasting compared to the use of P wave as the reference.

Noninvasive estimation of systolic pulmonary artery pressure (SPAP) during exercise stress echocardiography (ESE) is recommended for pulmonary hemodynamics evaluation but remains flow-dependent. Our aim was to assess the feasibility of pulmonary vascular reserve index (PVRI) estimation during ESE combining SPAP with cardiac output (CO) or exercise-time and compare its value in three group of patients: with invasively confirmed pulmonary hypertension (PH), at risk of PH development (PH risk) mainly with systemic sclerosis and in controls (C) without clinical risk factors for PH, age-matched with PH risk patients. We performed semisupine ESE in 171 subjects: 31 PH, 61 PH at risk and 50 controls as well as in 29 young, healthy normals. Rest and stress assessment included: tricuspid regurgitant flow velocity (TRV), pulmonary acceleration time (ACT), CO (Doppler-estimated). SPAP was calculated from TRV or ACT when TRV was not available. We estimated PVRI based on CO (peak CO/SPAP*0.1) or exercise-time (ESE time/SPAP*0.1). During stress, TRV was measurable in 44% patients ACT in 77%, either one in 95%. PVRI was feasible in 65% subjects with CO and 95% with exercise-time (p < 0.0001). PVRI was lower in PH compared to controls both for CO-based PVRI (group 1 = 1.0 ± 0.95 vs group 3 = 4.28 ± 2.3, p < 0.0001) or time-based PVRI estimation (0.66 ± 0.39 vs 3.95 ± 2.26, p < 0.0001). The proposed criteria for PH detection were for CO-based PVRI ≤ 1.29 and ESE-time based PVRI ≤ 1.0 and for PH risk ≤ 1.9 and ≤ 1.7 respectively. Noninvasive estimation of PVRI can be obtained in near all patients during ESE, without contrast administration, integrating TRV with ACT for SPAP assessment and using exercise time as a proxy of CO. These indices allow for comparison of pulmonary vascular dynamics in patients with varied exercise tolerance and clinical status.