Force and kinetics of fast and slow muscle myosin determined with a synthetic sarcomere-like nanomachine.

Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and shortening by cyclic ATP-driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform-dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle. Data fitting with a stochastic model provides a self-consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin-attached motors and the rate of transition through the attachment-detachment cycle. The achievements in this paper set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules either engineered or purified from mutant animal models or human biopsies.

Adsorption Mass Transfer Zone of Vancomycin in Cartridges With Styrene-Divinylbenzene Sorbent.

Cartridges for hemoadsorption containing styrene-divinylbenzene sorbent are used for multiple conditions, such as intoxication. The mass transfer zone comprises the extension along the longitudinal span of the cartridge where adsorption occurs. The aim of this experiment is to evaluate the mass transfer zone for vancomycin in the HA380 cartridge. The experiment was carried out twice. A saline solution with vancomycin passed through a HA380-modified cartridge at 100 ml/min in a single-pass fashion. The cartridge had four openings along its longitudinal dimension, at 3, 6, 9, and 12 cm. In both experiments, the collection of aliquots occurred at minute 4, in the four openings and pre- and post-cartridge, and an additional sample from the effluent bag at the end of each experiment. In the second experiment, an additional sampling of the same six sites occurred at minute 14. The sigmoidal shape of the curve for the mass transfer zone of vancomycin was similar to the theoretical one. In experiment one, at minute 4, vancomycin clearance was 98.75 ml/min. In experiment two, vancomycin clearance at minutes 4 and 14 was 93.76 and 93.20 ml/min, respectively. This implies an adequate and optimal design of the HA380 cartridge.

Effect of Mechanical Vibration on Kinetics of Solute Adsorption.

INTRODUCTION: Hemadsorption with new sorbent cartridges is an emerging extracorporeal blood purification technique. Flow distribution inside the sorbent is one of the main issues concerning the device's performance and optimal sorbent utilization. In this experiment, we aimed to investigate the efficacy of vibration during adsorption by measuring the removal of vancomycin. METHODS: In this experimental study, 1,000 mL of saline with 10 g of vancomycin was circulated in a closed circuit (set flow of 250 mL/min) simulating a hemadsorption blood run using HA380 minimodule cartridge containing 75 g of wet resin. This vibration model was implemented with a damping head device installed in front of the adsorption cartridge during the experiment. The kinetics of the vancomycin were assessed by removal ratio over 120 min. RESULTS: We found no difference between the two models. Adsorption with and without vibration did not differ significantly for partial reduction ratios, overall amount of adsorbed molecule, or adsorption kinetics. CONCLUSION: The current design and structure of the minimodule cartridge demonstrated no difference in small-middle solute removal. Further improvement with the addition of mechanical vibration to the device was not observed.

Sarcomere level mechanics of the fast skeletal muscle of the medaka fish larva.

The medaka fish (Oryzias latipes) is a vertebrate model used in developmental biology and genetics. Here we explore its suitability as a model for investigating the molecular mechanisms of human myopathies caused by mutations in sarcomeric proteins. To this end, the relevant mechanical parameters of the intact skeletal muscle of wild-type medaka are determined using the transparent tail at larval stage 40. Tails were mounted at sarcomere length of 2.1 µm in a thermoregulated trough containing physiological solution. Tetanic contractions were elicited at physiological temperature (10°C-30°C) by electrical stimulation, and sarcomere length changes were recorded with nanometer-microsecond resolution during both isometric and isotonic contractions with a striation follower. The force output has been normalized for the actual fraction of the cross section of the tail occupied by the myofilament lattice, as established with transmission electron microscopy (TEM), and then for the actual density of myofilaments, as established with X-ray diffraction. Under these conditions, the mechanical performance of the contracting muscle of the wild-type larva can be defined at the level of the half-thick filament, where ∼300 myosin motors work in parallel as a collective motor, allowing a detailed comparison with the established performance of the skeletal muscle of different vertebrates. The results of this study point out that the medaka fish larva is a suitable model for the investigation of the genotype/phenotype correlations and therapeutic possibilities in skeletal muscle diseases caused by mutations in sarcomeric proteins.NEW & NOTEWORTHY The suitability of the medaka fish as a model for investigating the molecular mechanisms of human myopathies caused by mutations of sarcomeric proteins is tested by combining structural analysis and sarcomere-level mechanics of the skeletal muscle of the tail of medaka larva. The mechanical performance of the medaka muscle, scaled at the level of the myosin-containing thick filament, together with its reduced genome duplication makes this model unique for investigations of the genotype/phenotype correlations in human myopathies.

[Contrast Induced Encephalopathy after carotid percutaneous transluminal angioplasty in a patient with end stage renal disease undergoing peritoneal Dialysis].

Introduction. Contrast Induced Encephalopathy (CIE) belongs to Major Adverse Renal and Cardiovascular Events (MARCE) after iodinated contrast medium (IOCM), especially for high-risk patients with several comorbidities such as hypertension, diabetes, heart failure, and Chronic Kidney Disease (CKD). We report a case of CIE in a Peritoneal Dialysis (PD)-patient. Case report. A 78-year-old, affected by diabetes, hypertension, chronic heart failure, and End Stage Renal Disease (ESRD) treated with PD, underwent a carotid Percutaneous Angioplasty (PTA). Immediately after the exam, he developed mental confusion and aphasia. Encephalic CT scan and MRI excluded acute ischemia or hemorrhage but showed cerebral oedema. Mannitol and steroids were administered and additional PD exchange was performed with depurative aim. Within 2 days the patient completely recovered. Discussion. CIE mimics severe neurological diseases. It should be considered as a differential diagnosis if symptoms occur immediately after administration of IOCM, especially in high-risk patients and in case of intra-arterial injection. Clinical presentation includes transient cortical blindness, aphasia, focal neurological defects, and confusion. CIE is often a diagnosis of exclusion, and imaging plays a significant role. Symptoms generally resolve spontaneously within 24-48h, rarely in few days. Symptomatic therapy, including mannitol and steroids could be considered. In literature, CIE is reported only in a few patients affected by ESRD treated with chronic HD, and our is the first available case of a patient treated with chronic PD who developed this rare complication.

The Cytotoxic Effect of Septic Plasma on Healthy RBCs: Is Eryptosis a New Mechanism for Sepsis?

Sepsis is a life-threatening multiple-organ dysfunction induced by infection and is one of the leading causes of mortality and critical illness worldwide. The pathogenesis of sepsis involves the alteration of several biochemical pathways such as immune response, coagulation, dysfunction of endothelium and tissue damage through cellular death and/or apoptosis. Recently, in vitro and in vivo studies reported changes in the morphology and in the shape of human red blood cells (RBCs) causing erythrocyte death (eryptosis) during sepsis. Characteristics of eryptosis include cell shrinkage, membrane blebbing, and surface exposure to phosphatidylserine (PS), which attract macrophages. The aim of this study was to evaluate the in vitro induction of eryptosis on healthy RBCs exposed to septic plasma at different time points. Furthermore, we preliminary investigated the in vivo levels of eryptosis in septic patients and its relationship with Endotoxin Activity Assay (EAA), mortality and other biological markers of inflammation and oxidative stress. We enrolled 16 septic patients and 16 healthy subjects (no systemic inflammation in the last 3 months) as a control group. At diagnosis, we measured Interleukin-6 (IL-6) and Myeloperoxidase (MPO). For in vitro study, healthy RBCs were exposed to the plasma of septic patients and CTR for 15 min, 1, 2, 4 and 24 h. Morphological markers of death and eryptosis were evaluated by flow cytometric analyses. The cytotoxic effect of septic plasma on RBCs was studied in vitro at 15 min, 1, 2, 4 and 24 h. Healthy RBCs incubated with plasma from septic patients went through significant morphological changes and eryptosis compared to those exposed to plasma from the control group at all time points (all, p < 0.001). IL-6 and MPO levels were significantly higher in septic patients than in controls (both, p < 0.001). The percentage of AnnexinV-binding RBCs was significantly higher in septic patients with EAA level ≥0.60 (positive EAA: 32.4%, IQR 27.6-36.2) compared to septic patients with EAA level <0.60 (negative EAA: 14.7%, IQR 5.7-30.7) (p = 0.04). Significant correlations were observed between eryptosis and EAA levels (Spearman rho2 = 0.50, p < 0.05), IL-6 (Spearman rho2 = 0.61, p < 0.05) and MPO (Spearman rho2 = 0.70, p < 0.05). In conclusion, we observed a quick and great cytotoxic effect of septic plasma on healthy RBCs and a strong correlation with other biomarkers of severity of sepsis. Based on these results, we confirmed the pathological role of eryptosis in sepsis and we hypothesized its use as a biomarker of sepsis, potentially helping physicians to face important treatment decisions.

Contrast-Induced Encephalopathy: A Rare Complication in a Patient on Peritoneal Dialysis with Several Risk Factors.

Major adverse renal and cardiovascular events are reported for high-risk patients undergoing intra-arterial procedures, even if performed with iso-osmolar contrast media (CM). We report a case of contrast-induced encephalopathy (CIE) in a peritoneal dialysis (PD) patient, affected by diabetes, hypertension, and chronic heart failure. A 78-year-old PD patient (diuresis 1,000 mL) underwent a percutaneous angioplasty of the carotid. Immediately after the exam, he developed mental confusion and aphasia. Encephalic computed tomography scan and magnetic resonance imaging excluded ischemia or hemorrhage, but both showed cerebral edema; EEG showed right hemisphere abnormalities, sequelae of recent ischemia. Mannitol and steroids were administered to reduce edema, and additional PD exchange was performed with depurative aim. Within 2 days the patient completely recovered. CIE mimics severe neurological diseases, and it should be considered as differential diagnosis if symptoms come out soon after intra-arterial administration of CM, especially in high-risk patients. Our patient suffered from diabetes, chronic kidney disease, hypertension, chronic heart failure, which are possible contributing factors to the development of CIE. Moreover, this clinical scenario is noteworthy because the development in a patient who underwent PD had never been described before.

Hemoadsorption: Research Agenda and Potential Future Applications.

After initial tentative steps with bioincompatible sorbents, hemoadsorption is making a comeback. This has been fueled by improved coating technology and improved sorbent technology. Both have markedly increased the safety, biocompatibility, and efficiency of hemoadsorption. Despite such development and an emerging body of evidence, the research agenda for hemoadsorption is substantial and, in most ways, unfulfilled. In this chapter, we highlight the need for more extensive and sophisticated work to understand the biological effect of hemoadsorption in key areas (especially sepsis). We also explain why more technical research needs to be conducted ex vivo and in large animals to understand the performance characteristics of hemoadsorption sorbent cartridge, including optimal blood flow, optimal anticoagulation, and optimal duration of application. Finally, we focus on the need to develop registries of the use of this technique so that more extensive information can be obtained about current use and real-world performance.

Bilirubin Adsorption with DPMAS: Mechanism of Action and Efficacy of Anion Exchange Resin.

Acute liver failure and acute-on-chronic liver failure are conditions in which the loss of metabolic function of the liver leads to the accumulation of several toxins such as bilirubin. Patients with sepsis or multiple organ dysfunction syndrome have a greater risk of developing liver failure, and hyperbilirubinemia is associated with poor prognosis. Bilirubin removal may not only alleviate signs and symptoms of liver dysfunction but also act as an index of removal of albumin-bound toxins. Conjugated and unconjugated bilirubin, due to their molecular weight and albumin-binding capacity, respectively, cannot be removed by classic dialysis; therefore, different extracorporeal techniques have been developed to remove bilirubin from the blood. Plasma adsorption perfusion is an extracorporeal liver support technique in which bilirubin is removed from the plasma through a specific adsorbing cartridge. Double plasma molecular adsorption system adds a broad-spectrum adsorption column for the removal of inflammatory mediators and antibodies and other medium toxins. Their use in the treatment of hyperbilirubinemia has been established with several emerging data indicating their efficacy when compared to other extracorporeal techniques. However, bilirubin adsorption kinetics has not been sufficiently elucidated, and more studies are needed to improve the quality of treatment in terms of timing and prescriptions.

Clostridium difficile colitis and peritoneal dialysis associated peritonitis: 'Difficile' treatment considerations.

Peritoneal dialysis (PD) associated peritonitis is the leading cause of PD discontinuation and haemodialysis transfer. Current guidelines strongly recommend prompt initiation of empiric broad-spectrum intraperitoneal antibiotics, with suspected peritonitis. Clostridium difficile colitis is one of the most common healthcare-associated infections, with increased morbidity and mortality among end-stage kidney disease patients. Clinical presentation is mainly characterised by diarrhoea of varying severity, which may eventually evolve into toxic megacolon and paralytic ileus. However, PD patients with Clostridium difficile infection (CDI) may also have colitis-triggered peritonitis, presenting challenging scenario for antibiotic treatment strategy, since broad-spectrum antibiotics against peritonitis may worsen CDI-related colitis, while inappropriate or discontinuation of antibiotic therapy may worsen peritonitis. Currently, guidelines on peritonitis management do not include such challenging clinical situations, although increasingly common. We herein describe a case of a patient, with culture-negative PD associated peritonitis and CDI, presenting with diarrhoea, abdominal pain and cloudy effluent.

Bilirubin Removal by Plasmafiltration-Adsorption: Ex vivo Adsorption Kinetics Model and Single Case Report.

BACKGROUND: Extracorporeal removal of bilirubin in patients with severe liver dysfunction is a key blood purification strategy. We conducted an ex vivo study to assess the quantitative capacity to remove bilirubin from plasma of a novel adsorptive cartridge. METHODS: We studied a downscaled module of the BS330 Plasma Bilirubin Adsorption Column Cartridge (Jafron Biomedical, Zhuhai City, China) to minimize the plasma requirement in an ex vivo circulation using a solution of hyperbilirubinemic plasma. We measured the bilirubin concentration gap (ΔC) between inlet (Cpin) and outlet (Cpout) of the unit and we calculated the removal ratio (RR) as mass adsorbed at different time points. Moreover, we compared the ex vivo model with the bilirubin adsorption kinetics in a patient with acute on chronic liver failure treated with the BS330 cartridge. RESULTS: Bilirubin concentration change across the cartridge at 30 min was 16.5%, and cartridge saturation was reached at 750 min. We used a minimodule downscaled to 1:3 and containing approximately 131 g of BS330 sorbent beads: the device retained 759 mg of bilirubin with a RR of 78.1% and a RR of 42.6% at 120 min. Thus, the adsorption capacity was 5.76 mg of bilirubin per gram of sorbent. Bilirubin adsorption kinetics in our clinical case with a full-scale unit shows a coherent trend with a total bilirubin mass adsorbed after 180 min of 470 mg. DISCUSSION: Our findings provide the first assessment of bilirubin adsorption in an ex vivo model of plasma perfusion and can be used to design interventional studies in humans, providing guidance for an adequate prescription of treatment frequency and duration.

Vancomycin Adsorption during in vitro Model of Hemoperfusion with Mini-Module of HA380 Cartridge.

INTRODUCTION: Sepsis is a frequent complication in critically ill patients. Patients may require control of the source of infection, removal of pathogens and damaged cells, and organ support. Often, these targets can be achieved through the utilization of extracorporeal therapies including hemoperfusion for the adsorption of cytokines and other circulating mediators. On extracorporeal organ support, patients are generally treated with antibiotic therapy, and vancomycin is one of the most commonly used antibiotics. Because of the aspecific nature of adsorption, antibiotics can be removed from the circulation, leading to altered plasma levels and requiring prescription adjustment. The aim was to define the amount of vancomycin adsorbed by a sorbent cartridge (HA380, Jafron, China) during hemoperfusion and to establish possible strategies to maintain an effective plasma level in critically ill patients undergoing extracorporeal therapies. METHODS: In vitro experiments with incremental concentrations of vancomycin in the test solution (500 and 1,000 mL) were carried out in a recirculation circuit until sorbent saturation was observed. A maximum of 10 g of vancomycin were injected and mini-modules containing 25 g of dry resin were utilized. RESULTS: In different experiments with various concentration of vancomycin, a maximum amount of 244 mg/g of sorbent was adsorbed reaching saturation between 60 and 80 min from the beginning of the experiments. The kinetics of adsorption appears to be governed by a Langmuir-like isotherm with maximal removal speed in the early minutes and a plateau after 60 min. DISCUSSION/CONCLUSION: HA380 adsorbs significant amounts of vancomycin. Adjusting the achieved results with the experimental mini-module to a full-scale cartridge, a total of 25 g of antibiotic can be removed. This might have affected outcome results in clinical trials. This suggests prescribing administration to critically ill patients requiring hemoperfusion, immediately after or in the inter-session time window. In case of administration during hemoperfusion, adequate adjustment and plasma level monitoring is strongly recommended.

The force of the myosin motor sets cooperativity in thin filament activation of skeletal muscles.

Contraction of striated muscle is regulated by a dual mechanism involving both thin, actin-containing filament and thick, myosin-containing filament. Thin filament is activated by Ca2+ binding to troponin, leading to tropomyosin displacement that exposes actin sites for interaction with myosin motors, extending from the neighbouring stress-activated thick filaments. Motor attachment to actin contributes to spreading activation along the thin filament, through a cooperative mechanism, still unclear, that determines the slope of the sigmoidal relation between isometric force and pCa (-log[Ca2+]), estimated by Hill coefficient nH. We use sarcomere-level mechanics in demembranated fibres of rabbit skeletal muscle activated by Ca2+ at different temperatures (12-35 °C) to show that nH depends on the motor force at constant number of attached motors. The definition of the role of motor force provides fundamental constraints for modelling the dynamics of thin filament activation and defining the action of small molecules as possible therapeutic tools.

Use of animal models to understand titin physiology and pathology.

In recent years, increasing attention has been paid to titin (TTN) and its mutations. Heterozygous TTN truncating variants (TTNtv) increase the risk of a cardiomyopathy. At the same time, TTNtv and few missense variants have been identified in patients with mainly recessive skeletal muscle diseases. The pathogenic mechanisms underlying titin-related diseases are still partly unknown. Similarly, the titin mechanical and functional role in the muscle contraction are far from being exhaustively clarified. In the last few years, several animal models carrying variants in the titin gene have been developed and characterized to study the structural and mechanical properties of specific titin domains or to mimic patients' mutations. This review describes the main animal models so far characterized, including eight mice models and three fish models (Medaka and Zebrafish) and discusses the useful insights provided by a thorough characterization of the cell-, tissue- and organism-phenotypes in these models.

Subclinical AKI and Clinical Outcomes in Elderly Patients Undergoing Cardiac Surgery: Diagnostic Utility of NGAL versus Standard Creatinine Increase Criteria.

BACKGROUND: Acute kidney injury (AKI) is a common and serious postoperative complication in patients undergoing cardiac surgery and its incidence is particularly high among elderly patients. Cardiac surgery-associated AKI (CSA-AKI) represents the second most common cause of AKI in the intensive care unit but its true incidence could be underestimated, especially in elderly population. The current biomarkers of AKI are unreliable and delayed during acute changes in kidney function. In the setting of subclinical AKI (SAKI), biomarkers of tubular damage, such as NGAL, seem to be an early indicator of kidney damage. The aim of this study was to investigate NGAL utility in the SAKI diagnosis in the first 48 h after cardiac surgery and its helpfulness in predicting adverse clinical outcomes in comparison to current criteria for AKI. METHODS: This is an observational study of 72 patients admitted to San Bortolo's cardiac surgery department for elective cardiosurgical procedure enrolled over a 5-months period. All patients underwent peripheral venous sample 48 h after cardiac surgery to assess plasmatic creatinine (48Cr) and NGAL (48pNGAL) in addition to exams already foreseen by clinical practice. For each patient we studied renal, respiratory and cardiovascular outcome during hospitalization as well as 30 days and 6 months mortality. Creatinine Increase AKI (CrIAKI) was defined by 48CrI ≥0.3 mg/dL and SAKI was defined by 48pNGAL ≥100 pg/dL. We also assessed Respiratory (ArespO) as well as Cardiovascular (ACvO) outcome. RESULTS: Thirty days mortality was 8.3% (6 patients) and 6 months mortality was 12.5% (9 patients). A total of 27 patients (37.5%) presented AKI according to KDIGO (4) and 4 (5.5%) needed renal replacement therapy (RRT). SAKI was significantly associated with 30 days mortality (p = 0.0238), 6 months mortality (p = 0.002), Adverse renal outcome (ARenO) (p = 0.004) and need for RRT (p = 0.005). CrIAKI was significantly associated with 30 days mortality (p = 0.009) and ARenO (p = 0.0001), but not with 6 months mortality nor need for RRT.

Peritoneal Vicenza "Short" Catheter Outcomes and Comparison with International Society for Peritoneal Dialysis Guidelines.

INTRODUCTION: A well-functioning peritoneal catheter is key to success of peritoneal dialysis (PD). The Vicenza "short" catheter is a modified Tenckhoff catheter with a shorter intraperitoneal segment. The aim of this study was to evaluate the incidence of catheter-related complications and catheter survival rate using the Vicenza "short" catheter, according to the goals suggested by the International Society for Peritoneal Dialysis (ISPD) guidelines. Second, we compared insertion techniques used in our center. METHODS: This is a retrospective cohort, single-center study analyzing incident PD patients undergoing Vicenza "short" peritoneal catheter placement between January 1, 2015, and December 31, 2019. As clinical outcomes, we evaluated catheter patency at 12 months, exit-site/tunnel infection and peritonitis within 30 days of catheter insertion, visceral injury, or significant hemorrhage during the procedure, in accordance with ISPD guidelines. RESULTS: The percentage of patency at 12 months for all catheter insertion methods was 88.91%, and the percentage for laparoscopic placement was 93.75%. The exit-site/tunnel infection and peritonitis occurring within 30 days of catheter insertion were, respectively, 0.75% and 2.2%; the visceral injury leading to intervention was 0.75%. We did not have any case of significant hemorrhage. All results were in line with ISPD guidelines. CONCLUSION: We conclude that the Vicenza "short" catheter is a suitable device for peritoneal access. The implantation procedure is safe and easy to perform, and both nephrologists and surgeons can do it. A confident use and a proper implantation of the Vicenza "short" catheter help achieve the clinical ISPD goals for the PD access procedure in terms of catheter survival and complication rates.

[Slowing progression of chronic kidney disease in polycistic kidney disease patients with tolvaptan: from guidelines to clinical practice].

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and accounts for∼10% of patients on renal replacement therapy. In the last decade, no specific treatment was available and only preventive measures could be put in place to delay the onset of ESRD. Following the results of the TEMPO 3:4 study, tolvaptan was approved in many countries, for the purpose of slowing the progression of renal insufficiency. In Italy tolvaptan is available since 2016 for patients with chronic kidney disease (CKD) stage 1-3, and since 2020 for patients with CKD stage 4, who fulfil the criteria of "rapid disease progression", according to the European recommendations. After this approval, Italian nephrology units have had to change their organization to be able to identify the patients eligible for the drug and to guarantee frequent patient monitoring. In this paper, we present our three-year experiences with tolvaptan, focusing on its safety profile and tolerability, but also on the high burden of care that such therapy represents not only for doctors, but also for patients. Strategies to implement remote monitoring may be useful to reduce the burden of assistance on one side, and the medicalization of ADPKD patients in the early stage of the disease, on the other.