Molecular dynamics studies of peptide-membrane interactions: insights from coarse-grained models

Peptide-membrane interactions play an important role in a number of biological processes, such as antimicrobial defence mechanisms, viral translocation, membrane fusion and functions ofmembrane proteins. In particular, amphipathic α-helical peptides comprise a large family of membrane-active peptides that could exhibit a broad range of biological activities. A membrane, interacting with an amphipathic α-helical peptide, may experience a number of possible structural transitions, including stretching, reorganization of lipid molecules, formation of defects, transient and stable pores, formation of vesicles, endo- and pinocytosis and other phenomena. Naturally, theoretical and experimental studies of these interactions have been an intense on-going area of research. However, complete understanding of the relationship between the structure of the peptide and themechanismof interaction it induces, as well asmolecular details of this process, still remain elusive. Lack of this knowledge is a key challenge in our efforts to elucidate some of the biological functions of membrane active peptides or to design peptides with tailored functionalities that can be exploited in drug delivery or antimicrobial strategies. In principle,molecular dynamics is a powerful research tool to study peptide-membrane interactions, which can provide a detailed description of these processes on molecular level. However, a model operating on the appropriate time and length scale is imperative in this description. In this study, we adopt a coarse-grained approach where the accessible simulation time and length scales reach microseconds and tens of nanometers, respectively. Thus, the two key objectives of this study are to validate the applicability of the adopted coarse-grained approach to the study of peptide-membrane interactions and to provide a systematic description of these interactions as a function of peptide structure and surface chemistry. We applied the adopted strategy to a range of peptide systems, whose behaviour has been well established in either experiments or detailed atomistic simulations and outlined the scope and applicability of the coarse-grained model. We generated some useful insights on the relationship between the structure of the peptides and themechanism of peptide-membrane interactions. Particularly interesting results have been obtained for LS3, a membrane spanning peptide, with a propensity to self-assembly into ion-conducting channels. Firstly, we captured, for the first time, the complete process of self-assembly of LS3 into a hexameric ion-conducting channel and explored its properties. The channel has structure of a barrel-stave pore with peptides aligned along the lipid tails. However, we discovered that a shorter version of the peptide leads to a more disordered, less stable structure often classified as a toroidal pore. This link between two types of pores has been established for the first time and opens interesting opportunities in tuning peptide structures for a particular pore-inducing mechanism. We also established that different classes of peptides can be uniquely characterized by the distinct energy profile as they cross the membrane. Finally, we extended this investigation to the internalization mechanisms of more complex entities such as peptide complexes and nanoparticles. Coarse-grained steered molecular dynamics simulations of these model systems are performed and some preliminary results are presented in this thesis. To summarize, in this thesis, we demonstrate that coarse-grained models can be successfully used to underpin peptide interaction and self-assembly processes in the presence of membranes in their full complexity. We believe that these simulations can be used to guide the design of peptides with tailored functionalities for applications such as drug delivery vectors and antimicrobial systems. This study also suggests that coarse-grained simulations can be used as an efficient way to generate initial configurations for more detailed atomistic simulations. These multiscale simulation ideas will be a natural future extension of this work

Autoencoders for dimensionality reduction in molecular dynamics: collective variable dimension, biasing and transition states

The heat shock protein 90 (Hsp90) is a molecular chaperone that controls the folding and activation of client proteins using the free energy of ATP hydrolysis. The Hsp90 active site is in its N-terminal domain (NTD). Our goal is to characterize the dynamics of NTD using an autoencoder-learned collective variable (CV) in conjunction with adaptive biasing force (ABF) Langevin dynamics. Using dihedral analysis, we cluster all available experimental Hsp90 NTD structures into distinct native states. We then perform unbiased molecular dynamics (MD) simulations to construct a dataset that represents each state and use this dataset to train an autoencoder. Two autoencoder architectures are considered, with one and two hidden layers respectively, and bottlenecks of dimension $k$ ranging from 1 to 10. We demonstrate that the addition of an extra hidden layer does not significantly improve the performance, while it leads to complicated CVs that increases the computational cost of biased MD calculations. In addition, a 2D bottleneck can provide enough information of the different states, while the optimal bottleneck dimension is five. For the 2D bottleneck, the two-dimensional CV is directly used in biased MD simulations. For the 5D bottleneck, we perform an analysis of the latent CV space and identify the pair of CV coordinates that best separates the states of Hsp90. Interestingly, selecting a 2D CV out of the 5D CV space leads to better results than directly learning a 2D CV, and allows to observe transitions between native states when running free energy biased dynamics

Endoscopic enucleation of the prostate with Thulium Fiber Laser (ThuFLEP). A retrospective single-center study

Purpose: The aim of the present, retrospective study was to describe our initial experience and early outcomes of Thulium Fiber Laser enucleation of the prostate (ThuFLEP) with the use of the FiberDust™ (Quanta System, Samarate, Italy) in patients with benign prostate hyperplasia. Methods: From June 2022 to April 2023, all patients who underwent endoscopic enucleation of the prostate at Urology Department of the University Hospital of Patras were included. A single surgeon utilizing the same standardized operative technique performed all the surgeries. The primary endpoints included the uneventful completion of the operation, the surgical time and any minor or major complication observed intra- or post-operatively. Results: Twenty patients with benign prostate hyperplasia were treated with ThuFLEP. All the surgeries were completed successfully and uneventfully. The enucleation phase of the operation was completed in a mean time of 45 ± 9.1 min, while the average time needed for the morcellation was 17.65 ± 3.42 min. No significant complications were observed intra- or post-operatively. The average hemoglobin drop was calculated to be 0.94 ± 0.71 g/dL. Conclusions: All the operations were successfully and efficiently completed with the use of the FiberDust™ (Quanta System, Samarate, Italy) in ThuFLEP. Significant blood loss or major complications were not observed

Feasibility study of a novel robotic system for transperitoneal partial nephrectomy: An in vivo experimental animal study

Purpose: To evaluate the safety and feasibility of partial nephrectomy with the use of the novel robotic system in an in vivo animal model. Methods: Right partial nephrectomy was performed in female pigs by a surgical team consisting of one surgeon and one bedside assistant. Both were experienced in laparoscopic surgery and trained in the use of the novel robotic system. The partial nephrectomies were performed using four trocars (three trocars for the robotic arms and one as an assistant trocar). The completion of the operations, set-up time, operation time, warm ischemia time (WIT) and complication events were recorded. The decrease in all variables between the first and last operation was calculated. Results: In total, eight partial nephrectomies were performed in eight female pigs. All operations were successfully completed. The median set-up time was 19.5 (range, 15-30) minutes, while the estimated median operative time was 80.5 minutes (range, 59-114). The median WIT was 23.5 minutes (range, 17-32) and intra- or postoperative complications were not observed. All variables decreased in consecutive operations. More precisely, the decrease in the set-up time was calculated to 15 minutes between the first and third attempts. The operative time was reduced by 55 minutes between the first and last operation, while the WIT was decreased by 15 minutes during the consecutive attempts. No complications were noticed in any operation. Conclusions: Using the newly introduced robotic system, all the advantages of robotic surgery are optimized and incorporated, and partial nephrectomies can be performed in a safe and effective manner

Molecular dynamics studies of peptide-membrane interactions : insights from coarse-grained models

Peptide-membrane interactions play an important role in a number of biological processes, such as antimicrobial defence mechanisms, viral translocation, membrane fusion and functions ofmembrane proteins. In particular, amphipathic α-helical peptides comprise a large family of membrane-active peptides that could exhibit a broad range of biological activities. A membrane, interacting with an amphipathic α-helical peptide, may experience a number of possible structural transitions, including stretching, reorganization of lipid molecules, formation of defects, transient and stable pores, formation of vesicles, endo- and pinocytosis and other phenomena. Naturally, theoretical and experimental studies of these interactions have been an intense on-going area of research. However, complete understanding of the relationship between the structure of the peptide and themechanismof interaction it induces, as well asmolecular details of this process, still remain elusive. Lack of this knowledge is a key challenge in our efforts to elucidate some of the biological functions of membrane active peptides or to design peptides with tailored functionalities that can be exploited in drug delivery or antimicrobial strategies. In principle,molecular dynamics is a powerful research tool to study peptide-membrane interactions, which can provide a detailed description of these processes on molecular level. However, a model operating on the appropriate time and length scale is imperative in this description. In this study, we adopt a coarse-grained approach where the accessible simulation time and length scales reach microseconds and tens of nanometers, respectively. Thus, the two key objectives of this study are to validate the applicability of the adopted coarse-grained approach to the study of peptide-membrane interactions and to provide a systematic description of these interactions as a function of peptide structure and surface chemistry. We applied the adopted strategy to a range of peptide systems, whose behaviour has been well established in either experiments or detailed atomistic simulations and outlined the scope and applicability of the coarse-grained model. We generated some useful insights on the relationship between the structure of the peptides and themechanism of peptide-membrane interactions. Particularly interesting results have been obtained for LS3, a membrane spanning peptide, with a propensity to self-assembly into ion-conducting channels. Firstly, we captured, for the first time, the complete process of self-assembly of LS3 into a hexameric ion-conducting channel and explored its properties. The channel has structure of a barrel-stave pore with peptides aligned along the lipid tails. However, we discovered that a shorter version of the peptide leads to a more disordered, less stable structure often classified as a toroidal pore. This link between two types of pores has been established for the first time and opens interesting opportunities in tuning peptide structures for a particular pore-inducing mechanism. We also established that different classes of peptides can be uniquely characterized by the distinct energy profile as they cross the membrane. Finally, we extended this investigation to the internalization mechanisms of more complex entities such as peptide complexes and nanoparticles. Coarse-grained steered molecular dynamics simulations of these model systems are performed and some preliminary results are presented in this thesis. To summarize, in this thesis, we demonstrate that coarse-grained models can be successfully used to underpin peptide interaction and self-assembly processes in the presence of membranes in their full complexity. We believe that these simulations can be used to guide the design of peptides with tailored functionalities for applications such as drug delivery vectors and antimicrobial systems. This study also suggests that coarse-grained simulations can be used as an efficient way to generate initial configurations for more detailed atomistic simulations. These multiscale simulation ideas will be a natural future extension of this work.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Μελέτη δεικτών προστατευτικού αερισμού, σε ασθενείς που νοσηλεύονται στη Μονάδα Εντατικής Θεραπείας, κατά τη μετάβασή τους από ελεγχόμενο σε υποβοηθούμενο μοντέλο μηχανικής υποστήριξης αναπνοής

Eισαγωγή: Το σύνδρομο οξείας αναπνευστικής δυσχέρειας των ενηλίκων χαρακτηρίζεται από διάχυτη φλεγμονώδη βλάβη του πνευμονικού ιστού που οδηγεί σε αύξηση της πνευμονικής διαπερατότητας, πνευμονικό οίδημα και απώλεια αεριζόμενων περιοχών του πνεύμονα. Η στρατηγική του προστατευτικού αερισμού του πνεύμονα, η οποία, όπως αποδείχθηκε βελτιώνει την επιβίωση στο ARDS, έγκειται στη χρήση μικρών αναπνεόμενων όγκων και χαμηλών τελοεισπνευστικών πιέσεων ώστε να περιορίζεται η κυψελιδική υπερδιάταση, και στην εφαρμογή PEEP ώστε να αποφεύγεται το διαρκές άνοιγμα και κλείσιμο των κυψελίδων στο τέλος της εκπνοής. Οι γενικά αποδεκτές οδηγίες συνίστανται στη χρήση αναπνεόμενων όγκων (Tidal Volume, VT) 4-6 mL/kg ιδανικού βάρους σώματος και ικανών επιπέδων PEEP, ενώ η στατική τελοεισπνευστική πίεση (Pplateau, που θεωρείται ότι εκφράζει το βαθμό της διάτασης του πνεύμονα στο τέλος της εισπνοής) πρέπει να είναι < 30 cmH2O, καθώς επίσης και η οδηγός πίεση- Driving Pressure (δηλαδή, το άθροισμα της χορηγούμενης πίεσης από τον αναπνευστήρα και της πίεσης που γεννά η εισπνευστική προσπάθεια του ασθενούς) dP<14cmH2O. Σκοπός: Σκοπός της έρευνας μας ήταν να παρακολουθήσουμε την εφαρμογή ή μη της στρατηγικής προστατευτικού αερισμού κατά την μετάβαση των ασθενών από ελεγχόμενο σε υποβοηθούμενο μοντέλο μηχανικής υποστήριξης. Υλικό - Μέθοδος: Πρόκειται για μια μελέτη παρατήρησης. Το δείγμα της μελέτης αποτέλεσαν 50 ασθενείς με ARDS, που νοσηλεύονταν διασωληνωμένοι σε ΜΕΘ του Γ.Ν.Ν.Θ.Α «Σωτηρία». Κατά τη διάρκεια της νοσηλείας τους καταγράφηκαν τα δημογραφικά χαρακτηριστικά τους, τα συνοδά νοσήματα, καθώς και πλήθος κλινικών και βιοχημικών παραμέτρων. Για τη συλλογή δεδομένων χρησιμοποιήθηκε φόρμα καταγραφής. Στατιστική ανάλυση: Η ανάλυση των δεδομένων πραγματοποιήθηκε με SPSS – 26. Επίσης, εφαρμόστηκε η μέθοδος Kolmogorov-Smirnov και Shapiro-Wilk για τη μέτρηση της κανονικότητας όλων των μεταβλητών. Στη συνέχεια έγινε ο έλεγχος των υποθέσεων με την εφαρμογή του MANN_WHITNEY με επίπεδο σημαντικότητας ALPHA=0.05. Εφαρμόστηκε το στατιστικό κριτήριο (T test) ανεξάρτητων δειγμάτων με ανεξάρτητη μεταβλητή τη θνησιμότητα των υποκειμένων. Καταγράφηκε σε κάθε μεταβλητή η τιμή της F, το κριτήριο (t – test), οι βαθμοί ελευθερίας και η σημαντικότητα σε επίπεδο P< 0,005. Αποτελέσματα: 82% των ασθενών ήταν άνδρες και η μέση ηλικία τους ήταν 61 χρόνια (από 29 έως 84 έτη, Std=14,2). Το 88% των ασθενών είχε λόγο PaO2/FiO2 < 200mmHg κι επομένως μέτριο/βαρύ ARDS. Η ενδοτικότητα των ασθενών είχε μέση τιμή 37ml/cmH20 (από 15 έως 73 ml/cmH20, Std = 12,4) με υψηλές τιμές ενδοτικότητας για τους ασθενείς που είχαν ιστορικό Χρόνιας αποφρακτικής Πνευμονοπάθειας όπως αναμένεται. Το μέσο APACHE II score των ασθενών ήταν 12 (Std = 4,3). Κατά την εισαγωγή των ασθενών, η μέση τιμή του αναπνεόμενου όγκου που εφαρμόσθηκε ήταν 6,4ml (Std = 0,9) και η μέση τιμή των Driving pressure και Plateau pressure ήταν 11,9 cmH20 (Std = 3,7) και 24,1cmH20 (Std = 4,7). Όσον αφορά την PEEP η μέση τιμή της ήταν 11,6 cmH20 (Std = 4,03). Μετά την αφύπνιση των ασθενών και την εφαρμογή υποβοηθούμενου μοντέλου μηχανικής υποστήριξης της αναπνοής το 45% των τιμών της Driving pressure που μετρήθηκαν στο πρώτο 48ωρο,ήταν πάνω από το όριο των 14cmH20 το οποίο σύμφωνα με τη αναδρομική μελέτη του Amato et al () αποτελεί το ασφαλές όριο για την ελαχιστοποίηση του VILI. Αντίθετα μόνο το 10% των τιμών της plateau pressure ήταν πάνω από το όριο των 30cmH2O. Οσον αφορά τον αναπνεόμενο όγκο, το 80% των τιμών που καταγράφηκαν το πρώτο 48ωρο, ήταν πάνω από 6ml/kg IBW. Από τους 50 ασθενείς που μελετήθηκαν 37 εξήλθαν της μονάδας και 13 κατέληξαν (θνητότητα 26%). Συμπεράσματα: Κατά τη διάρκεια του πρώτου 48ωρου μετά τη μετάβαση σε υποβοηθούμενο μοντέλο μηχανικής υποστήριξης της αναπνοής σε ασθενείς με ARDS, οι δείκτες προστατευτικού αερισμού, σε μεγάλο ποσοστό ασθενών είναι πάνω από τα όρια που θέτει η στρατηγική του προστατευτικού αερισμού. Κλινικές τυχαιοποιημένες μελέτες θα μπορούσαν να δείξουν αν και κατά πόσο είναι κλινικά σημαντική αυτή η παρατήρηση.Introduction: Adult acute respiratory distress syndrome is characterized by diffuse inflammatory damage to lung tissue leading to increased pulmonary permeability, pulmonary edema, and loss of ventilated areas of the lung. The strategy of protective lung ventilation, which has been shown to improve survival in ARDS, is to use small respiratory volumes and low end- inspiratory pressures to reduce alveolar hypertension, and to apply PEEP to prevent permanent opening and closing of the cells at the end of the exhalation. The generally accepted guidelines are the use of respiratory volumes (Tidal Volume, VT) 4-6 mL / kg of ideal body weight and adequate PEEP levels, while static end-inspiratory pressure (Pplateau, which expresses the degree of dilation of the lung at the end of inhalation) should be <30 cmH2O, as well as the Driving Pressure (the sum of the pressure delivered by the ventilator and the pressure generated by the patient's effort) dP <14cmH2O. Purpose: The purpose of our research was to observe the implementation or not of the protective ventilation strategy during the transition of patients from a controlled to an assisted model of mechanical ventilation. Material – Method: This is an observational study. The sample of the study consisted of 50 patients with ARDS, who were intudated in the ICU of the General Hospital "Sotiria". During their treatment, their demographic characteristics, concomitant diseases, as well as a number of clinical and biochemical parameters were recorded. A registration form was used for data collection. Statistical analysis: Data analysis was performed with SPSS – 26. Also, the Kolmogorov-Smirnov and Shapiro-Wilk method were applied to measure the regularity of all variables. Then the hypotheses were checked with MANN_WHITNEY with significance level ALPHA = 0.05. The statistical criterion (T test) of independent samples with independent variable mortality of subjects was applied. The value of F, the criterion (t - test), the degrees of freedom and the significance at level P <0.005 were recorded in each variable. Results: 82% of patients were male and their mean age was 61 years (29 to 84 years, Std = 14.2). 88% of patients had a ratio of PaO2 / FiO2 <200mmHg and therefore moderate / severe ARDS. Patient compliance had a mean value of 37ml / cmH20 (from 15 to 73 ml / cmH20, Std = 12.4) with high compliance rates for patients with a history of Chronic Obstructive Pulmonary Disease as expected. The mean APACHE II score of the patients was 12 (Std = 4.3). On the admission of patients, the mean value of respiratory volume applied was 6.4 ml (Std = 0.9) and the mean value of Driving pressure and Plateau pressure was 11.9 cmH20 (Std = 3.7) and 24,1 cmH2O (Std = 4.7). The average value of PEEP was 11.6 cmH2 O (Std = 4.03). After awakening the patients and applying an assisted mechanical support model, 45% of the Driving pressure values measured in the first 48 hours were above 14cmH20, a limit, which according to the retrospective study of Amato et al. is the safe limit to reduce VILI. On the contrary, only 10% of the plateau pressure values were above the 30cmH2O limit. In terms of respiratory volume, 80% of the values recorded in the first 48 hours were over 6ml / kg IBW. Of the 50 patients studied, 37 left the unit and 13 ended up (26% mortality). Conclusions: During the first 48 hours, after the ARDS patients’ transition to an assisted ventilation model, protective ventilation indices are above the limits of protective ventilation strategy in a large amount of patients. Randomized clinical trials could indicate whether and to what extent this observation is clinically relevant