Isomer-sourced structure iteration methods for in silico development of inhibitors: Inducing GTP-bound NRAS-Q61 oncogenic mutations to an "off-like" state.

The NRAS-mutant subset of melanoma represent some of the most aggressive and deadliest types associated with poor overall survival. Unfortunately, for more than 40 years, no therapeutic agent directly targeting NRAS mutations has been clinically approved. In this work, based on microsecond scale molecular dynamics simulations, the effect of Q61 mutations on NRAS conformational characteristics is revealed at the atomic level. The GTP-bound NRAS-Q61R and Q61K mutations show a specific targetable pocket between Switch-II and α-helix 3 whereas the NRAS-Q61L non-polar mutation category shows a different targetable pocket. Moreover, a new isomer-sourced structure iteration method has been developed for the in silico design of potential inhibitor prototypes for oncogenes. We show the possibility of a designed prototype HM-387 to target activated NRAS-Q61R and that it can gradually induce the transition from the activated NRAS-Q61R to an "off-like" state.

Effect of Functionalization of Texturized Polypropylene Surface by Silanization and HBII-RGD Attachment on Response of Primary Abdominal and Vaginal Fibroblasts.

Soft tissue defects, such as incisional hernia or pelvic organ prolapse, are prevalent pathologies characterized by a tissue microenvironment rich in fragile and dysfunctional fibroblasts. Precision medicine could improve their surgical repair, currently based on polymeric materials. Nonetheless, biomaterial-triggered interventions need first a better understanding of the cell-material interfaces that truly consider the patients' biology. Few tools are available to study the interactions between polymers and dysfunctional soft tissue cells in vitro. Here, we propose polypropylene (PP) as a matrix to create microscale surfaces w/wo functionalization with an HBII-RGD molecule, a fibronectin fragment modified to include an RGD sequence for promoting cell attachment and differentiation. Metal mold surfaces were roughened by shot blasting with aluminum oxide, and polypropylene plates were obtained by injection molding. HBII-RGD was covalently attached by silanization. As a proof of concept, primary abdominal and vaginal wall fasciae fibroblasts from control patients were grown on the new surfaces. Tissue-specific significant differences in cell morphology, early adhesion and cytoskeletal structure were observed. Roughness and biofunctionalization parameters exerted unique and combinatorial effects that need further investigation. We conclude that the proposed model is effective and provides a new framework to inform the design of smart materials for the treatment of clinically compromised tissues.

In silico design of a lipid-like compound targeting KRAS4B-G12D through non-covalent bonds.

One of the most common drivers in human cancer is the peripheral membrane protein KRAS4B, able to promote oncogenic signalling. To signal, oncogenic KRAS4B not only requires a sufficient nucleotide exchange, but also needs to recruit effectors by exposing its effector-binding sites while anchoring to the phospholipid bilayer where KRAS4B-mediated signalling events occur. The enzyme phosphodiesterase-δ plays an important role in sequestering KRAS4B from the cytoplasm and targeting it to cellular membranes of different cell species. In this work, we present an in silico design of a lipid-like compound that has the remarkable feature of being able to target both an oncogenic KRAS4B-G12D mutant and the phosphodiesterase-δ enzyme. This double action is accomplished by adding a lipid tail (analogous to the farnesyl group of the KRAS4B protein) to an previously known active compound (2H-1,2,4-benzothiadiazine, 3,4-dihydro-,1,1-dioxide). The proposed lipid-like molecule was found to lock KRAS4B-G12D in its GDP-bound state by adjusting the effector-binding domain to be blocked by the interface of the lipid bilayer. Meanwhile, it can tune GTP-bound KRAS4B-G12D to shift from the active orientation state to the inactive state. The proposed compound is also observed to stably accommodate itself in the prenyl-binding pocket of phosphodiesterase-δ, which impairs KRAS4B enrichment at the lipid bilayer, potentially reducing the proliferation of KRAS4B inside the cytoplasm and its anchoring at the bilayer. In conclusion, we report a potential inhibitor of KRAS4B-G12D with a lipid tail attached to a specific warhead, a compound which has not yet been considered for drugs targeting RAS mutants. Our work provides new ways to target KRAS4B-G12D and can also foster drug discovery efforts for the targeting of oncogenes of the RAS family and beyond.

Titanium Boston keratoprosthesis with corneal cell adhesive and bactericidal dual coating.

The Boston keratoprosthesis (BKPro) is a medical device used to restore vision in complicated cases of corneal blindness. This device is composed by a front plate of polymethylmethacrylate (PMMA) and a backplate usually made of titanium (Ti). Ti is an excellent biomaterial with numerous applications, although there are not many studies that address its interaction with ocular cells. In this regard, despite the good retention rates of the BKPro, two main complications compromise patients' vision and the viability of the prosthesis: imperfect adhesion of the corneal tissue to the upside of the backplate and infections. Thus, in this work, two topographies (smooth and rough) were generated on Ti samples and tested with or without functionalization with a dual peptide platform. This molecule consists of a branched structure that links two peptide moieties to address the main complications associated with BKPro: the well-known RGD peptide in its cyclic version (cRGD) as cell pro-adherent motif and the first 11 residues of lactoferrin (LF1-11) as antibacterial motif. Samples were physicochemically characterized, and their biological response was evaluated in vitro with human corneal keratocytes (HCKs) and against the gram-negative bacterial strain Pseudomonas aeruginosa. The physicochemical characterization allowed to verify the functionalization in a qualitative and quantitative manner. A higher amount of peptide was anchored to the rough surfaces. The studies performed using HCKs showed increased long-term proliferation on the functionalized samples. Gene expression was affected by topography and peptide functionalization. Roughness promoted α-smooth muscle actin (α-SMA) overexpression, and the coating notably increased the expression of extracellular matrix components (ECM). Such changes may favour the development of unwanted fibrosis, and thus, corneal haze. In contrast, the combination of the coating with a rough topography decreased the expression of α-SMA and ECM components, which would be desirable for the long-term success of the prosthesis. Regarding the antibacterial activity, the functionalized smooth and rough surfaces promoted the death of bacteria, as well as a perturbation in their wall definition and cellular morphology. Bacterial killing values were 58 % for smooth functionalised and 68 % for rough functionalised samples. In summary, this study suggests that the use of the dual peptide platform with cRGD and LF1-11 could be a good strategy to improve the in vitro and in vivo performance of the rough topography used in the commercial BKPro.

Satisfaction with vaccination services and its relationship to emotional responses of service users in Lima. LEGADO's quality management model as a public solution to promote citizen emotional well-being during pandemic.

This article analyzes the levels of citizen satisfaction with LEGADO's quality management model service during the first year of vaccination against SARS-CoV-2 in public spaces administered by LEGADO, and its relationship with the user's emotional responses. To this end, a survey study has been developed from July 2021 until March 2022 at 4 moments to citizens (n = 1,697) who attended 3 vaccination locations administered by LEGADO (VIDENA, Complejo VMT and Polideportivo VES). The results show a high level of satisfaction with LEGADO's quality model service, which is associated with a positive emotional balance. Specifically, the elements that have the greatest effect on positive emotions are the cleanliness and facilities' organization and the agility of service. These results are discussed emphasizing the importance of the role of public institutions in developing inclusive quality public services for all citizens. This strategy of public quality model service according to citizens' necessities should result in confidence towards public institutions and socially responsible behavior among citizens through the reduction of social gaps. The research establishes the urgency to promote this model in order to bring legitimacy and confidence to public institutions in Perú.

Nucleation of helium in pure liquid lithium.

Tritium self-sufficiency in fusion nuclear reactors will be based on the neutron capture by lithium in the so-called breeding blankets of the reactor, a nuclear reaction that will produce helium along with tritium. The low solubility of helium in liquid metals could cause the eventual formation of helium bubbles, which may have a negative impact on the performance of the breeding blanket in a way that has yet to be fully understood. In this work, we provide deep insight into the behavior of lithium and helium mixtures at experimentally operating conditions (800 K and pressures between 1 and 100 bars) using a microscopic model suitable to describe the interactions between helium and lithium at the atomic level, in excellent agreement with available experimental data. The simulations predict the formation of helium bubbles with radii around 10 Å at ambient pressure with surface tension values in the range of 0.6-1.0 N/m. We also report the cohesive energies of helium and the work of formation of the cluster of atoms, as well as a quantitative estimation of the Hildebrand and Kumar cohesion parameters. Our results indicate that the segregation between He and Li atoms is strong, and once a bubble is formed, it never dissociates.

Guiding Fibroblast Activation Using an RGD-Mutated Heparin Binding II Fragment of Fibronectin for Gingival Titanium Integration.

The formation of a biological seal around the neck of titanium (Ti) implants is critical for ensuring integration at the gingival site and for preventing bacterial colonization that may lead to periimplantitis. This process is guided by activated fibroblasts, named myofibroblasts, which secrete extracellular matrix (ECM) proteins and ECM-degrading enzymes resolving the wound. However, in some cases, Ti is not able to attract and activate fibroblasts to a sufficient extent, which may compromise the success of the implant. Fibronectin (FN) is an ECM component found in wounds that is able to guide soft tissue healing through the adhesion of cells and attraction of growth factors (GFs). However, clinical use of FN functionalized Ti implants is problematic because FN is difficult to obtain, and is sensitive to degradation. Herein, functionalizing Ti with a modified recombinant heparin binding II (HBII) domain of FN, mutated to include an Arg-Gly-Asp (RGD) sequence for promoting both fibroblast adhesion and GF attraction, is aimed at. The HBII-RGD domain is able to stimulate fibroblast adhesion, spreading, proliferation, migration, and activation to a greater extent than the native HBII, reaching values closer to those of full-length FN suggesting that it might induce the formation of a biological sealing.

Functionalization of 3D-Printed Titanium Scaffolds with Elastin-like Recombinamers to Improve Cell Colonization and Osteoinduction.

The 3D printing of titanium (Ti) offers countless possibilities for the development of personalized implants with suitable mechanical properties for different medical applications. However, the poor bioactivity of Ti is still a challenge that needs to be addressed to promote scaffold osseointegration. The aim of the present study was to functionalize Ti scaffolds with genetically modified elastin-like recombinamers (ELRs), synthetic polymeric proteins containing the elastin epitopes responsible for their mechanical properties and for promoting mesenchymal stem cell (MSC) recruitment, proliferation, and differentiation to ultimately increase scaffold osseointegration. To this end, ELRs containing specific cell-adhesive (RGD) and/or osteoinductive (SNA15) moieties were covalently attached to Ti scaffolds. Cell adhesion, proliferation, and colonization were enhanced on those scaffolds functionalized with RGD-ELR, while differentiation was promoted on those with SNA15-ELR. The combination of both RGD and SNA15 into the same ELR stimulated cell adhesion, proliferation, and differentiation, although at lower levels than those for every single moiety. These results suggest that biofunctionalization with SNA15-ELRs could modulate the cellular response to improve the osseointegration of Ti implants. Further investigation on the amount and distribution of RGD and SNA15 moieties in ELRs could improve cell adhesion, proliferation, and differentiation compared to the present study.

Comparación entre la administración endovenosa y la administración periarticular de ácido tranexámico en cirugía de reemplazo total de rodilla: estudio de dos cohortes consecutivas / Comparison between intravenous and periarticular administration of tranexamic acid in total knee replacement surgery: a study of two consecutive cohorts

Introducción. Se ha reportado que el ácido tranexámico (AT) es útil en el manejo del sangrado posoperatorio de pacientes sometidos a artroplastia total de rodilla (ATR), pero no hay consenso sobre su dosificación y vía de administración. Objetivo. Evaluar y comparar la eficacia para reducir el sangrado posoperatorio de la administración endovenosa y la administración periarticular de AT en pacientes sometidos a ATR. Metodología. Estudio observacional realizado con 72 pacientes operados entre 2021 y 2022 en un centro hospitalario de segundo nivel de atención en Barcelona (España). Los pacientes se dividieron en 2 grupos según la vía de administración del AT (grupo 1: administración endovenosa de dos dosis, una intraoperatoria de 15mg/kg y una posoperatoria de 10mg/kg; grupo 2: una infiltración periarticular de 2g). Se realizó un análisis bivariado con la prueba t de Student para evaluar las diferencias entre grupos. Resultados. El promedio de edad fue 73,72 años en el grupo 1 y 72,96 años en el grupo 2. Se observó una disminución en los niveles de hemoglobina preoperatorio y a las 24 horas en ambos grupos (grupo 1: 2,1 mg/dL; grupo 2: 2,14 mg/dL). En el grupo 2, un paciente presentó embolia pulmonar y otro requirió de transfusión de glóbulos rojos. Conclusiones. El uso de AT, tanto por vía endovenosa como en administración local, permite reducir el sangrado posoperatorio en pacientes sometidos a ATR. No se evidenciaron diferencias estadísticamente significativas en la disminución de los niveles de hemoglobina posoperatorios entre los grupos.

Introduction: Tranexamic acid (TA) has been reported as useful in the management of postoperative bleeding in patients undergoing total knee arthroplasty (TKA), but there is no consensus on its dosage and route of administration. Objective: To evaluate and compare the efficacy of intravenous and periarticular administration of TA in patients undergoing TKA to reduce postoperative bleeding.Methodology: Observational study conducted with 72 patients operated on between 2021 and 2022 in a secondary care center in Barcelona (Spain). Patients were divided into 2 groups depending on the route of TA administration (group 1: intravenous administration of two doses, an intraoperative dose of 15mg/kg and a postoperative dose of 10mg/kg; group 2: periarticular infiltration of 2g). A bivariate analysis was performed using Student's t-test to evaluate the differences between groups.Results: The mean age of the patients was 73.72 years in group 1 and 72.96 years in group 2. A decrease in preoperative and 24-hour hemoglobin levels was observed in both groups (group 1: 2.1 mg/dL; group 2: 2.14 mg/dL). One patient in group 2 presented pulmonary embolism and another required red blood cell transfusion.Conclusions: The use of TA, both intravenous and locally administered, reduces postoperative bleeding in patients undergoing TKA. There were no statistically significant differences in the decrease of postoperative hemoglobin levels between groups

Discovering and Targeting Dynamic Drugging Pockets of Oncogenic Proteins: The Role of Magnesium in Conformational Changes of the G12D Mutated Kirsten Rat Sarcoma-Guanosine Diphosphate Complex.

KRAS-G12D mutations are the one of most frequent oncogenic drivers in human cancers. Unfortunately, no therapeutic agent directly targeting KRAS-G12D has been clinically approved yet, with such mutated species remaining undrugged. Notably, cofactor Mg2+ is closely related to the function of small GTPases, but no investigation has been conducted yet on Mg2+ when associated with KRAS. Herein, through microsecond scale molecular dynamics simulations, we found that Mg2+ plays a crucial role in the conformational changes of the KRAS-GDP complex. We located two brand new druggable dynamic pockets exclusive to KRAS-G12D. Using the structural characteristics of these two dynamic pockets, we designed in silico the inhibitor DBD15-21-22, which can specifically and tightly target the KRAS-G12D-GDP-Mg2+ ternary complex. Overall, we provide two brand new druggable pockets located on KRAS-G12D and suitable strategies for its inhibition.

Reducing bacterial adhesion to titanium surfaces using low intensity alternating electrical pulses.

BACKGROUND: Orthopedic implant-related infection remains one of the most serious complications after orthopedic surgery. In recent years, there has been an increased scientific interest to improve prevention and treatment strategies. However, many of these strategies have focused on chemical measures. AIM: To analyze the effect of alternating current electrical fields on bacterial adherence to titanium surfaces. METHODS: Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were exposed to 6.5 V electrical currents at different frequencies: 0.5 Hz, 0.1 Hz, and 0.05 Hz. After exposure, a bacterial count was then performed and compared to the control model. Other variables registered included the presence of electrocoagulation of the medium, electrode oxidation and/or corrosion, and changes in pH of the medium. RESULTS: The most effective electrical model for reducing S. aureus adhesion was 6.5 V alternating current at 0.05 Hz achieving a 90% adhesion reduction rate. For E. coli, the 0.05 Hz frequency model also showed the most effective results with a 53% adhesion reduction rate, although these were significantly lower than S. aureus. Notable adhesion reduction rates were observed for S. aureus and E.coli in the studied conditions. However, the presence of electrode oxidation makes us presume these conditions are not optimal for in vivo use. CONCLUSION: Although our findings suggest electrical currents may be useful in preventing bacterial adhesion to metal surfaces, further research using other electrical conditions must be examined to consider their use for in vivo trials.

Modeling and Simulation of Lipid Membranes.

Cell membranes separate the interior of cells and the exterior environment, providing protection, controlling the passage of substances, and governing the interaction with other biomolecules and signalling processes [...].

Nucleation of Helium in Liquid Lithium at 843 K and High Pressures.

Fusion energy stands out as a promising alternative for a future decarbonised energy system. In order to be sustainable, future fusion nuclear reactors will have to produce their own tritium. In the so-called breeding blanket of a reactor, the neutron bombardment of lithium will produce the desired tritium, but also helium, which can trigger nucleation mechanisms owing to the very low solubility of helium in liquid metals. An understanding of the underlying microscopic processes is important for improving the efficiency, sustainability and reliability of the fusion energy conversion process. The spontaneous creation of helium droplets or bubbles in the liquid metal used as breeding material in some designs may be a serious issue for the performance of the breeding blankets. This phenomenon has yet to be fully studied and understood. This work aims to provide some insight on the behaviour of lithium and helium mixtures at experimentally corresponding operating conditions (843 K and pressures between 108 and 1010 Pa). We report a microscopic study of the thermodynamic, structural and dynamical properties of lithium-helium mixtures, as a first step to the simulation of the environment in a nuclear fusion power plant. We introduce a new microscopic model devised to describe the formation of helium droplets in the thermodynamic range considered. Our model predicts the formation of helium droplets at pressures around 109 Pa, with radii between 1 and 2 Å. The diffusion coefficient of lithium (2 Å2/ps) is in excellent agreement with reference experimental data, whereas the diffusion coefficient of helium is in the range of 1 Å2/ps and tends to decrease as pressure increases.

In Silico Drug Design of Benzothiadiazine Derivatives Interacting with Phospholipid Cell Membranes.

The use of drugs derived from benzothiadiazine, a bicyclic heterocyclic benzene derivative, has become a widespread treatment for diseases such as hypertension, low blood sugar or the human immunodeficiency virus, among others. In this work we have investigated the interactions of benzothiadiazine and four of its derivatives designed in silico with model zwitterionic cell membranes formed by dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoserine and cholesterol at the liquid-crystal phase inside aqueous potassium chloride solution. We have elucidated the local structure of benzothiadiazine by means of microsecond molecular dynamics simulations of systems including a benzothiadiazine molecule or one of its derivatives. Such derivatives were obtained by the substitution of a single hydrogen site of benzothiadiazine by two different classes of chemical groups, one of them electron-donating groups (methyl and ethyl) and another one by electron-accepting groups (fluorine and trifluoromethyl). Our data have revealed that benzothiadiazine derivatives have a strong affinity to stay at the cell membrane interface although their solvation characteristics can vary significantly-they can be fully solvated by water in short periods of time or continuously attached to specific lipid sites during intervals of 10-70 ns. Furthermore, benzothiadiazines are able to bind lipids and cholesterol chains by means of single and double hydrogen-bonds of characteristic lengths between 1.6 and 2.1 Å.

Predicting the conformational variability of oncogenic GTP-bound G12D mutated KRas-4B proteins at zwitterionic model cell membranes.

KRas proteins are the largest family of mutated Ras isoforms, participating in a wide variety of cancers. Due to their importance, large effort is being carried out on drug development by small-molecule inhibitors. However, understanding protein conformational variability remains a challenge in drug discovery. In the case of the Ras family, their multiple conformational states can affect the binding of potential drug inhibitors. To overcome this challenge, we propose a computational framework based on combined all-atom Molecular Dynamics and Metadynamics simulations in order to accurately access conformational variants of the target protein. We tested the methodology using a G12D mutated GTP bound oncogenic KRas-4B protein located at the interface of a DOPC/DOPS/cholesterol model anionic cell membrane. Two main orientations of KRas-4B at the anionic membrane have been determined. The corresponding torsional angles are taken as reliable reaction coordinates so that free-energy landscapes are obtained by well-tempered metadynamics simulations, revealing local and global minima of the free-energy hypersurface and unveiling reactive paths of the system between the two preferential orientations. We have observed that GTP-binding to KRas-4B has huge influence on the stabilisation of the protein and it can potentially help to open Switch I/II druggable pockets, lowering energy barriers between stable states and resulting in cumulative conformers of KRas-4B. This may highlight new opportunities for targeting the unique meta-stable states through the design of new efficient drugs.

On-Growth and In-Growth Osseointegration Enhancement in PM Porous Ti-Scaffolds by Two Different Bioactivation Strategies: Alkali Thermochemical Treatment and RGD Peptide Coating.

A lack of primary stability and osteointegration in metallic implants may result in implant loosening and failure. Adding porosity to metallic implants reduces the stress shielding effect and improves implant performance, allowing the surrounding bone tissue to grow into the scaffold. However, a bioactive surface is needed to stimulate implant osteointegration and improve mechanical stability. In this study, porous titanium implants were produced via powder sintering to create different porous diameters and open interconnectivity. Two strategies were used to generate a bioactive surface on the metallic foams: (1) an inorganic alkali thermochemical treatment, (2) grafting a cell adhesive tripeptide (RGD). RGD peptides exhibit an affinity for integrins expressed by osteoblasts, and have been reported to improve osteoblast adhesion, whereas the thermochemical treatment is known to improve titanium implant osseointegration upon implantation. Bioactivated scaffolds and control samples were implanted into the tibiae of rabbits to analyze the effect of these two strategies in vivo regarding bone tissue regeneration through interconnected porosity. Histomorphometric evaluation was performed at 4 and 12 weeks after implantation. Bone-to-implant contact (BIC) and bone in-growth and on-growth were evaluated in different regions of interest (ROIs) inside and outside the implant. The results of this study show that after a long-term postoperative period, the RGD-coated samples presented higher quantification values of quantified newly formed bone tissue in the implant's outer area. However, the total analyzed bone in-growth was observed to be slightly greater in the scaffolds treated with alkali thermochemical treatment. These results suggest that both strategies contribute to enhancing porous metallic implant stability and osteointegration, and a combination of both strategies might be worth pursuing.

Mating Disruption of the Olive Moth Prays oleae (Bernard) in Olive Groves Using Aerosol Dispensers.

The olive moth (OM), Prays oleae (Bern.) (Lepidoptera: Yponomeutidae), is a major olive grove pest worldwide; however, until now, very few studies have investigated the effectiveness of mating disruption (MD) techniques against this pest. Experiments were carried out for two successive years (2019 and 2020) in three different olive groves in Andalucía (Southern Spain) to evaluate mating disruption's efficacy in controlling the OM from the first to the third generation. The effectiveness of MD formulations against the three generations of OM was assessed by determining the percentage of infested olive fruits, the reduction of pheromone trap catches, and the number of affected inflorescences in both MD-treated and untreated control olive groves. The number of release points (one or two aerosol devices per ha) was also evaluated. In all years and trials, the mean number of males caught in traps placed in the MD-treated plots was significantly lower than untreated sites. Mating disruption registered a high suppression of male captures (>75%) in treated plots for two consecutive seasons. Concerning infested olive fruits, substantial reductions (about 80%) were observed in the MD plots of locations B and C, and a reduction of about 40% was detected in location A, compared to the control plot. Results showed that the installation of two aerosol devices/ha reduced fruit damage below 20% of infested olive fruits except for one site where a reduction of about 71% in the MD plot was recorded in 2019. Although few significant differences were associated with OM male catches and infested olive fruits between plots treated with one aerosol/ha and two aerosols/ha in most of the comparisons, significant differences in the number of olive inflorescences infested by P. oleae were found, suggesting a similar performance between the two tested aerosol densities. Results of two-year field trials in Andalucía demonstrated the potential of Mister P X841 aerosol devices as an effective tool for controlling the olive moth, P. oleae.

Structure of benzothiadiazine at zwitterionic phospholipid cell membranes.

The use of drugs derived from benzothiadiazine, which is a bicyclic heterocyclic benzene derivative, has become a widespread treatment for diseases such as hypertension (treated with diuretics such as bendroflumethiazide or chlorothiazide), low blood sugar (treated with non-diuretic diazoxide), or the human immunodeficiency virus, among others. In this work, we have investigated the interactions of benzothiadiazine with the basic components of cell membranes and solvents, such as phospholipids, cholesterol, ions, and water. The analysis of the mutual microscopic interactions is of central importance to elucidate the local structure of benzothiadiazine as well as the mechanisms responsible for the access of benzothiadiazine to the interior of the cell. We have performed molecular dynamics simulations of benzothiadiazine embedded in three different model zwitterionic bilayer membranes made by dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoserine, and cholesterol inside aqueous sodium-chloride solution in order to systematically examine microscopic interactions of benzothiadiazine with the cell membrane at liquid-crystalline phase conditions. From data obtained through radial distribution functions, hydrogen-bonding lengths, and potentials of mean force based on reversible work calculations, we have observed that benzothiadiazine has a strong affinity to stay at the cell membrane interface although it can be fully solvated by water in short periods of time. Furthermore, benzothiadiazine is able to bind lipids and cholesterol chains by means of single and double hydrogen-bonds of different characteristic lengths.

Microscopic Interactions of Melatonin, Serotonin and Tryptophan with Zwitterionic Phospholipid Membranes.

The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10-20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10-7 cm2/s for the cholesterol-free and cholesterol-rich setups.

Membrane perturbation, altered morphology and killing of Staphylococcus epidermidis upon contact with a cytocompatible peptide-based antibacterial surface.

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1-18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ∼50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4-5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1-18) on osteoblast cells.