Model Membranes Research Articles

3D Tumor-Engineered Model Replicating the Osteosarcoma Stem Cell Niche and In Vivo Tumor Complexity.

Osteosarcoma, among all bone sarcomas, remains a challenge despite the unwavering efforts of medical professionals and scientists. To address this, the scientific community is actively pursuing the development of three-dimensional (3D) in vitro models to faithfully replicate the heterogeneity of osteosarcoma, thereby facilitating the reliable preclinical screening of potential therapies. In this study, we present the latest advancements in engineering an in vitro 3D osteosarcoma model comprising enriched Cancer Stem Cells (CSCs) and a hybrid hydroxyapatite-based scaffold (MgHA/CoII). The improvement of the model occurred through two primary steps: (1) serial passaging of sarcospheres as the CSCs enrichment system and (2) the optimization of the structural configuration of the niche in the scaffold. Two injection-mediated approaches of sarcosphere seeding were designed and extensively characterized in vitro and in vivo Chorioallantoic Membrane (CAM) models to explore their biological properties and tumorigenic potential. The combination of the selected enriched-CSCs and custom-made seeding into the scaffold resulted in the development of 3D osteosarcoma models exhibiting tumor-like features in vitro and tumorigenic properties in vivo. The outcomes of this study offer prospects for future endeavors involving more complex systems capable of replicating specific malignant tumor behaviors (metastatic process and drug resistance), pushing the discovery of new therapeutic strategies for clinical applications.

The impact of acyl-CoA:cholesterol transferase (ACAT) inhibitors on biophysical membrane properties depends on membrane lipid composition

Acyl-coenzyme A: cholesterol acyltransferases are enzymes which are involved in the homeostasis of cholesterol. Impaired enzyme activity is associated with the occurrence of various diseases like Alzheimer’s disease, atherosclerosis, and cancers. At present, mitotane is the only inhibitor of this class of enzymes in clinical use for the treatment of adrenocortical carcinoma but associated with common and severe adverse effects. The therapeutic effect of mitotane depends on its interaction with cellular membranes. The search for less toxic but equally effective compounds is hampered by an incomplete understanding of these biophysical properties. In the present study, the interaction of the three ACAT inhibitors nevanimibe, Sandoz 58-035, and AZD 3988 with membranes has been investigated using lipid model membranes in conjunction with biophysical experimental (NMR, ESR, fluorescence) and theoretical (MD simulations) approaches. The data show, that the drugs (i) incorporate into lipid membranes, (ii) differently influence the structure of lipid membranes; (iii) affect membrane structure depending on the lipid composition; and (iv) do not cause hemolysis of red blood cells. The results are discussed with regard to the use of the drugs, in particular to better understand their efficacy and possible side effects.

Recovery of Whey Protein by Using Microfiltration: Artificial Neural Network–Based Modeling and Effects of Different Operating Parameters

ABSTRACTMicrofiltration is one of the most suitable processes for protein recovery from whey due to its low energy consumption and lack of use of heat and chemicals. However, membrane fouling is one of the limiting factors in the microfiltration process, preventing its commercial use. In this study, an artificial neural network (ANN) based model was employed to study the effects of different operating parameters on membrane fouling in whey concentration. Trans‐membrane pressure, Reynolds number, and feed temperature were selected as the input parameters. Experimental data from the available studies were used to train the ANN. The ANN with 23 neurons gave a minimum mean squared error (MSE) for trans‐membrane pressure and Reynolds number. The ANN with seven neurons gave the minimum MSE for feed temperature. The predicted values from both ANNs well fitted with the experimental results with R2 < 0.99. Simulations showed that membrane fouling increased as flux reduction increased from 36.3% to 76.39% when trans‐membrane pressure increased from 0.5 to 2 bar. In contrast, a 19.96% reduction in flux was observed by increasing the Reynolds number from 750 to 2500. An increment of 77.37% of flux reduction was observed with increasing feed temperature from 30°C to 40°C. Simulations confirmed that transmembrane pressure, Reynolds number, and feed temperature strongly influence membrane fouling. An ANN‐based approach was the most accurate method to model membrane fouling for whey protein separation.

Unexpected asymmetric distribution of cholesterol and phospholipids in equilibrium model membranes

Lipid compositional asymmetry across the leaflets of the plasma membrane is a ubiquitous feature in eukaryotic cells. How this asymmetry is maintained is thought to be primarily controlled by active transport of lipids between leaflets. This strategy is facilitated by the fact that long tail phospholipids and sphingolipids diffuse through the lipid bilayer slowly – taking many hours or days. However, a lipid like cholesterol – which is the most abundant lipid in the plasma membrane of animal cells – has been harder to pin-point in terms of its favored side. In the present work we show that when a saturated lipid is added to a mix of the unsaturated lipid palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol, both cholesterol and the long tail phospholipids organize asymmetrically across the membrane’s leaflets naturally. In these extruded unilamellar vesicles, most cholesterol as well as the saturated lipid – dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) – segregated to the inner leaflet while POPC preferentially localized in the outer leaflet. This asymmetric arrangement generated a slight phospholipid number imbalance favoring the outer leaflet and thus opposite to where cholesterol and the saturated lipids preferentially partitioned. These results were obtained using Magic Angle Spinning (MAS) NMR in combination with Small Angle Neutron Scattering (SANS) using isotope labeling to differentiate lipid species. We suggest that sidedness in membranes can be driven by thermodynamic processes. In addition, our MAS NMR results show that the lower bound for cholesterol’s flip-flop half-time at 45°C is 10ms, which is at least two orders of magnitude slower than current MD simulations predict. This result stands in stark contrast to previous work that suggested that cholesterol’s flip-flop half-time at 37°C has an upper bound of 10ms.

Understanding mechanotransduction in the distal colon and rectum via multiscale and multimodal computational modeling

Visceral pain in the large bowel is a defining symptom of irritable bowel syndrome (IBS) and the primary reason that patients visit gastroenterologists. This pain is reliably triggered by mechanical distension of the distal colon and rectum (colorectum). Consequently, the process of mechanotransduction by sensory afferents, responsible for translating mechanical colorectal stimuli into neural action potentials, plays a central role in IBS-related bowel pain. In this study, we aim to enhance our understanding of colorectal mechanotransduction by combining experimental findings in colorectal biomechanics and afferent neural encoding within a comprehensive computational simulation framework. To achieve this, we implemented a three-layered, fiber-reinforced finite element model that accurately replicates the nonlinear, heterogeneous, and anisotropic mechanical characteristics of the mouse colorectum. This model facilitates the computation of local mechanical stresses and strains around individual afferent endings, which have diameters on the micron-scale. We then integrated a neural membrane model to simulate the encoding of action potentials by afferent nerves in response to microscopic stresses and strains along the afferent endings. Our multiscale simulation framework enables the assessment of three hypotheses regarding the mechanical gating of action potential generation: (1) axial stress dominates mechanical gating of mechanosensitive channels, (2) both axial and circumferential stresses contribute, and (3) membrane shear stress dominates. Additionally, we explore how the orientation of afferent endings impacts neural encoding properties. This computational framework not only allows for the virtual investigation of colorectal mechanotransduction in the context of prolonged visceral hypersensitivity but can also guide the development of new experimental studies aimed at uncovering the neural and biomechanical mechanisms underlying IBS-related bowel pain.

Cm-p5, a molluscan-derived antifungal peptide exerts its activity by a membrane surface covering in a non-penetrating mode

Amidst the health crisis caused by the rise of multi-resistant pathogenic microorganisms, Antimicrobial Peptides (AMPs) have emerged as a potential alternative to traditional antibiotics. In this sense, Cm-p5 is an AMP with fungistatic activity against the yeast Candida albicans. Its antimicrobial activity and selectivity have been well characterized; however, the mechanism of action is still unknown. This study used biophysical approaches to gain insight into how this peptide exerts its activity. Stability and fluidity of lipid membrane were explored by liposome leakage and Laurdan generalized polarization (GP) respectively, suggesting that Cm-p5 does not perturb lipid membranes even at very high concentrations (≥100µm.L-1). Likewise, no depolarizing action was observed using 3,3'-propil-2,2'-thyodicarbocianine, a potential membrane fluorescent reporter, with C. albicans cells or the corresponding liposome models. Changes in liposome size were analyzed by Dynamic Light Scattering (DLS) data, indicating that Cm-p5 covers the vesicular surface slightly increasing liposome hydrodynamic size, without liposome rupture. These results were further corroborated with Langmuir monolayer isotherms, where no significant changes in lateral pressure or area per lipid were detected, indicating little or no insertion. Finally, data obtained from molecular dynamics simulations aligned with in vitro observations, whereby Cm-p5 slightly interacted with the fungal membrane model surface without causing significant perturbation. These results suggest Cm-p5 is not a pore-forming anti-fungal peptide and that other mechanisms of action on the membrane as some limitation of fungal nutrition or receptor-dependent transduction for depressing growth development should be explored.

Exploiting WEE1 Kinase Activity as FUS::DDIT3-Dependent Therapeutic Vulnerability in Myxoid Liposarcoma.

The pathognomonic FUS::DDIT3 fusion protein drives myxoid liposarcoma (MLS) tumorigenesis via aberrant transcriptional activation of oncogenic signaling. As FUS::DDIT3 has so far not been pharmacologically tractable to selectively target MLS cells, this study investigated the functional role of the cell cycle regulator WEE1 as novel FUS::DDIT3-dependent therapeutic vulnerability in MLS. Immunohistochemical evaluation of the cell cycle regulator WEE1 was performed in a large cohort of MLS specimens. FUS::DDIT3 dependency and biological function of the G1/S cell cycle checkpoint were analyzed in a mesenchymal stem cell model and liposarcoma cell lines in vitro. WEE1 activity was modulated by RNAi-mediated knockdown and the small molecule inhibitor MK-1775 (adavosertib). An established MLS cell line-based chicken chorioallantoic membrane model was employed for in vivo confirmation. We demonstrate that enhanced WEE1 pathway activity represents a hallmark of FUS::DDIT3-expressing cell lines as well as MLS tissue specimens and that WEE1 is required for MLS cellular survival in vitro and in vivo. Pharmacologic inhibition of WEE1 activity results in DNA damage accumulation and cell cycle progression forcing cells to undergo apoptotic cell death. In addition, our results uncover FUS::DDIT3-dependent WEE1 expression as an oncogenic survival mechanism to tolerate high proliferation and resulting replication stress in MLS. Fusion protein-driven G1/S cell cycle checkpoint deregulation via overactive Cyclin E/CDK2 complexes thereby contributes to enhanced WEE1 inhibitor sensitivity in MLS. Our preclinical study identifies WEE1-mediated replication stress tolerance as molecular vulnerability in FUS::DDIT3-driven MLS tumorigenesis that could represent a novel target for therapeutic intervention.

Curvature-Assisted Vesicle Explosion Under Light-Induced Asymmetric Oxidation.

Exposure of cell membranes to reactive oxygen species can cause oxidation of membrane lipids. Oxidized lipids undergo drastic conformational changes, compromising the mechanical integrity of the membrane and causing cell death. For giant unilamellar vesicles, a classic cell mimetic system, a range of mechanical responses under oxidative assault has been observed including formation of nanopores, transient micron-sized pores, and total sudden catastrophic collapse (i.e., explosion). However, the physical mechanism regarding how lipid oxidation causes vesicles to explode remains elusive. Here, with light-induced asymmetric oxidation experiments, the role of spontaneous curvature on vesicle instability and its link to the conformational changes of oxidized lipid products is systematically investigated. A comprehensive membrane model is proposed for pore-opening dynamics incorporating spontaneous curvature and membrane curling, which captures the experimental observations well. The kinetics of lipid oxidation are further characterized and how light-induced asymmetric oxidation generates spontaneous curvature in a non-monotonic temporal manner is rationalized. Using the framework, a phase diagram with an analytical criterion to predict transient pore formation or catastrophic vesicle collapse is provided. The work can shed light on understanding biomembrane stability under oxidative assault and strategizing release dynamics of vesicle-based drug deliverysystems.

Simplified discrete model for axisymmetric dielectric elastomer membranes with robotic applications

Soft robots utilizing inflatable dielectric membranes can realize intricate functionalities through the application of non-mechanical fields. However, given the current limitations in simulations, including low computational efficiency and difficulty in dealing with complex external interactions, the design and control of such soft robots often require trial and error. Thus, a novel one-dimensional (1D) discrete differential geometry (DDG)-based numerical model is developed for analyzing the highly nonlinear mechanics in axisymmetric inflatable dielectric membranes. The model captures the intricate dynamics of these membranes under both inflationary pressure and electrical stimulation. Comprehensive validations using hyperelastic benchmarks demonstrate the model’s accuracy and reliability. Additionally, the focus on the electro-mechanical coupling elucidates critical insights into the membrane’s behavior under varying internal pressures and electrical loads. The research further translates these findings into innovative soft robotic applications, including a spherical soft actuator, a soft circular fluid pump, and a soft toroidal gripper, where the snap-through of electroelastic membrane plays a crucial role. Our analyses reveal that the functional ranges of soft robots are amplified by the snap-through of an electroelastic membrane upon electrical stimuli. This study underscores the potential of DDG-based simulations to advance the understanding of the nonlinear mechanics of electroelastic membranes and guide the design of electroelastic actuators in soft robotics applications.

3D Covalent Organic Framework Membranes for Molecular Separations.

Membrane separation has become an indispensable separation technology in social production, playing an important role in drug production, water purification, etc. The key core of membranes lies in achieving efficient and precise sieving between substances. As a result, a typical trade-off arises: highly permeable membranes usually sacrifice selectivity and vice versa. To address this dilemma, long-term research has focused on comprehensive understanding and modelling of synthetic membranes at various scales. A significant advancement in this arena is the advent of three-dimensional covalent organic framework (3D COF) membranes, a novel category of long-range ordered porous organic polymer materials. Characterized by an abundance of interconnected channels, diverse pore wall properties, tunable structures, and robust thermal and chemical resilience, 3D COF membranes offer a promising approach for efficient substance separation. This review undertakes a meticulous investigation of the synthesis and physicochemical properties of 3D COF membranes, accentuating the underlying design principles, fabrication methods, and application attempts. A comprehensive assessment of their research trajectory and current standing in the field of membrane processes is provided. The review culminates in a forward-looking outlook, summarizing future research directions and highlighting the substantial potential of this innovative work to shape the future of efficient membrane separation processes.

Time-dependent mechanical behavior analysis using Weibull models of cellulose hollow fiber membranes produced by green solvent

The mechanical behavior of sustainable (“green”), bio-based cellulose hollow fiber membranes (hereafter, hollow fiber) produced using green solvent, which is potential for desalination and solvent-resistant nanofiltration, can be evaluated either in dry or water-conditioned (wet) states. However, the effect of conditioning time on the mechanical behavior of hollow fibers has not been conclusive. In this work, an extensive evaluation involving an experimental campaign and rigorous application of statistical models for cellulose hollow fibers produced using green solvent (1-ethyl-3-methylimidazolium diethyl phosphate or [EMIM][DEP]) via a dry-jet spinning technique is reported. First, we evaluated the microstructures and separation performance (water permeation, solute rejection) of hollow fibers made of different cellulose concentrations (10–15 wt%) in both pristine and conditioned states. Then, the strength, stiffness, and ductility of hollow fiber samples of different gauge lengths (25 and 50 mm) were measured by a standard tensile test device. Here, the effect of conditioning time on mechanical properties was evaluated after the hollow fiber samples were water-immersed at different times (from 30 s to 2 months). The strength distribution of conditioned hollow fibers was modeled and analyzed using two-parameter and three-parameter Weibull models. Our results showed that the cellulose content of 15 wt% in the hollow fibers produced a denser microstructure, providing the lowest molecular weight cut-off (MWCO). The cellulose content and gauge length slightly modified the mechanical properties of hollow fibers when they were evaluated in pristine, dry conditions. The mechanical properties of hollow fibers were drastically reduced after 10 min of water immersion, beyond which their properties were stabilized. Weibull modeling of mechanical reliability showed that two- or three-parameter Weibull could estimate the failure probability of hollow fibers quite well. Nonetheless, the three-parameter Weibull is better than the two-parameter one in terms of accuracy in determining the threshold strength located at the “lower-end tail.” Our analysis provides a rigorous demonstration of Weibull modeling in evaluating the time-dependent mechanical behavior of ductile, cellulose-based hollow fiber membranes, which is essential to support the design and development of future hollow fiber membranes in desalination industries.

Effects of Yishentongluo Recipe on Nrf2 and HO-1 pathway activation leading to reduction of oxidative stress and kidney fibrosis in membranous nephropathy rat model

This study investigated Yishentongluo Recipe (YSTLF) effects on renal oxidative stress and fibrosis in membranous nephropathy (MN) rats. MN was induced by cationized bovine serum albumin injection. Rats were divided intocontrol, MN, YSTLF, and benazepril groups. After four weeks of treatment, urine protein levels (UTP), serum total cholesterol (TC), triglycerides (TG), total protein (TP), and albumin (ALB) were assessed. Kidney microstructure, IgG immune complex deposition, and protein expressions of superoxide dismutase (SOD), malondialdehyde (MDA), transforming growth factor β1 (TGF-β1), collagen I (Collagen-I), α-smooth muscle actin (α-SMA), nuclear factor E2-related factor (Nrf2), haem oxygenase 1 (HO-1), and NADPH oxidase 4 (NOX4) were evaluated. YSTLF and BNPL treatments reduced UTP, TC, TG, increased TP and ALB levels, downregulated TGF-β1, Collagen-I, and α-SMA, and upregulated Nrf2, HO-1, and NOX4. YSTLF partially reversed SOD reduction and MDA elevation, suggesting its efficacy in alleviating renal oxidative stress and fibrosis in MN rats via Nrf2/HO-1 signaling pathway activation.

Critical appraisal of the chorioallantoic membrane model for studying angiogenesis in preclinical research.

Angiogenesis, the biological mechanism by which new blood vessels are generated from existing ones, plays a vital role in growth and development. Effective preclinical screening is necessary for the development of medications that may enhance or inhibit angiogenesis in the setting of different disorders. Traditional in vitro and, in vivo models of angiogenesis are laborious and time-consuming, necessitating advanced infrastructure for embryo culture. A challenge encountered by researchers studying angiogenesis is the lack of appropriate techniques to evaluate the impact of regulators on the angiogenic response. An ideal test should possess reliability, technical simplicity, easy quantifiability, and, most importantly, physiological relevance. The CAM model, leveraging the extraembryonic membrane of the chicken embryo, offers a unique combination of accessibility, low cost, and rapid development, making it an attractive option for angiogenesis assays. This review evaluates the strengths and limitations of the CAM model in the context of its anatomical and physiological properties, and its relevance to human pathophysiological conditions. Its abundant capillary network makes it a common choice for studying angiogenesis. The CAM assay serves as a substitute for animal models and offers a natural setting for developing blood vessels and the many elements involved in the intricate interaction with the host. Despite its advantages, the CAM model's limitations are notable. These include species-specific responses that may not always extrapolate to humans and the ethical considerations of using avian embryos. We discuss methodological adaptations that can mitigate some of these limitations and propose future directions to enhance the translational relevance of this model. This review underscores the CAM model's valuable role in angiogenesis research and aims to guide researchers in optimizing its use for more predictive and robust preclinical studies. The highly vascularized chorioallantoic membrane (CAM) of fertilized chicken eggs is a cost-effective and easily available method for screening angiogenesis, in comparison to other animal models.

Probing the Influence of Hydrophobicity of Modified Gold Nanoparticles in Modulating the Lipid Surface Behavior Using Vibrational Sum Frequency Generation Spectroscopy.

A deep understanding of how the surface modifications of nanoparticles impact their interactions with cell membranes is vital for advancing safe and effective biomedical applications. Among the pivotal factors governing these interactions, the hydrophobicity of nanoparticles plays a crucial role, predominantly driven by the hydrophobic interactions with the cell membrane. Herein, we study the influence of the hydrophobic alkyl chain length of thiol-capped gold nanoparticles (GNPs) on lipid surfaces with the help of vibrational sum frequency generation spectroscopy. We have utilized the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid monolayer as a representative model of cell membranes on the water surface. Our findings revealed that GNPs capped with the thiol ligand having a shorter alkyl chain such as heptanethiol (HT, C7) show minimal changes in the C-H stretching vibrations while interacting with the lipid monolayer. These observations could be attributed to the perturbation of the lipid chain due to hydrophobic-hydrophobic interactions between the alkyl chain of thiol-capped GNPs and the hydrophobic group of the lipid membrane or simply by the adsorption of GNPs at the interface without disrupting the monolayer structure. However, with increasing the chain length of thiol-capped GNPs from decanethiol (DDT, C10) to octadecanethiol (ODT, C18), the extent of spectral change in the C-H stretching vibration is increased. The controlled experiment performed with the deuterated lipids conforms that the changes observed in the C-H stretching vibration after adding HT (C7) GNPs are only because of their presence in the surface without altering the monolayer structure. However, in the case of DT (C10) and DDT (C12) GNPs, the strong hydrophobic interactions between the monolayer and the alkyl chain of the thiol-capped GNPs result in the increased orientational order of the monolayer. Moreover, in the case of ODT (C18) GNPs, the very long alkyl chain induces pronounced perturbations in the monolayer structure with net disordering of the monolayer. These observations are further supported by the spectral changes observed in the O-H vibration of the interfacial water molecules. Our findings reveal the crucial role of the hydrophobic nature of GNPs in influencing the interface. Understanding these effects is crucial for drug delivery applications and improving the stability and effectiveness of lipid-based systems.

Magnetomechanical Behaviors of Hard-Magnetic Elastomer Membranes Placed in Uniform Magnetic Field.

This paper aims to develop a theoretical model for a viscoelastic hard-magnetic elastomer membrane (HMEM) actuated by pressure and uniform magnetic field. The HMEM is initially a flat, circular film with a fixed boundary. The HMEM undergoes nonlinear large deformations in the transverse direction. The viscoelastic behaviors are characterized by using a rheological model composed of a spring in parallel with a Maxwell unit. The governing equations for magneto-visco-hyperelastic membrane under the axisymmetric large deformation are constructed. The Zeeman energy, which is related to the magnetization of the HMEM and the magnetic flux density, is employed. The governing equations are solved by the shooting method and the improved Euler method. Several numerical examples are implemented by varying the magnitude of the pre-stretch, pressure, and applied magnetic field. Under different magnetic fields, field variables such as latitudinal stress exhibit distinct curves in the radial direction. It is observed that these varying curves intersect at a point. The position of the intersection point is independent of the applied magnetic field and only controlled by pressure and pre-stretch. On the left side of the intersection point, the field variables increase as magnetic field strength increases. However, on the other side, this trend is reversed. During viscoelastic evolution, one can find that the magnetic field can be used to modulate the instability behaviors of the HMEM. These findings may provide valuable insights into the design of the hard-magnetic elastomer membrane structures and actuators.

High-Speed Imaging of Giant Unilamellar Vesicle Formation in cDICE.

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE), which make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating >30 μm-sized droplets and only in some cases GUV-sized (∼15 μm) satellite droplets. According to existing theoretical models, the oil-water interface should allow for the crossing of all droplets, but based on our observations and scaling arguments on the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. The origin of these droplets remains partly unclear; we hypothesize that some small GUVs might be formed from large droplets sitting at the second interface. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering the lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE but ultimately also aid in the development of more reliable and efficient methods for GUV production.

Topological Heterogeneity of Protein Kinase C Modulators in Human T-Cells Resolved with In-Cell Dynamic Nuclear Polarization NMR Spectroscopy.

Phorbol ester analogs are a promising class of anticancer therapeutics and HIV latency reversing agents that interact with cellular membranes to recruit and activate protein kinase C (PKC) isoforms. However, it is unclear how these esters interact with membranes and how this might correlate with the biological activity of different phorbol ester analogs. Here, we have employed dynamic nuclear polarization (DNP) NMR to characterize phorbol esters in a native cellular context. The enhanced NMR sensitivity afforded by DNP and cryogenic operation reveals topological heterogeneity of 13C-21,22-phorbol-myristate-acetate (PMA) within T cells utilizing 13C-13C correlation and double quantum filtered NMR spectroscopy. We demonstrate the detection of therapeutically relevant amounts of PMA in T cells down to an upper limit of ∼60.0 pmol per million cells and identify PMA to be primarily localized in cellular membranes. Furthermore, we observe distinct 13C-21,22-PMA chemical shifts under DNP conditions in cells compared to model membrane samples and homogenized cell membranes, that cannot be accounted for by differences in conformation. We provide evidence for distinct membrane topologies of 13C-21,22-PMA in cell membranes that are consistent with shallow binding modes. This is the first of its kind in-cell DNP characterization of small molecules dissolved in the membranes of living cells, establishing in-cell DNP-NMR as an important method for the characterization of drug-membrane interactions within the context of the complex heterogeneous environment of intact cellular membranes. This work sets the stage for the identification of the in-cell structural interactions that govern the biological activity of phorbol esters.

Classical Simulations on Quantum Computers: Interface-Driven Peptide Folding on Simulated Membrane Surfaces

Background:Antimicrobial peptides (AMPs) are crucial in the fight against infections and play significant roles in various health contexts, including cancer, autoimmune diseases, and aging. A key aspect of AMP functionality is their selective interaction with pathogen membranes, which often exhibit altered lipid compositions. These interactions are thought to induce a conformational shift in AMPs from random coil to alpha-helical structures, essential for their lytic activity. Traditional computational approaches have faced challenges in accurately modeling these structural changes, especially in membrane environments, thereby opening and opportunity for more advanced approaches. Method:This study extends an existing quantum computing algorithm, initially designed for peptide folding simulations in homogeneous environments, to address the complexities of AMP interactions at interfaces. Our approach enables the prediction of the optimal conformation of peptides located in the transition region between hydrophilic and hydrophobic phases, resembling lipid membranes. The new method was tested on three 10-amino-acid-long peptides, each characterized by distinct hydrophobic, hydrophilic, or amphipathic properties, across different media and at interfaces between solvents of different polarity. Results:The developed method successfully modeled the structure of the peptides without increasing the number of qubits required compared to simulations in homogeneous media, making it more feasible with current quantum computing resources. Despite the current limitations in computational power and qubit availability, the findings demonstrate the significant potential of quantum computing in accurately characterizing complex biomolecular processes, particularly AMP folding at membrane models. Conclusions:This research highlights the promising applications of quantum computing in biomolecular simulations, paving the way for future advancements in the development of novel therapeutic agents. We aim to offer a new perspective on enhancing the accuracy and applicability of biomolecular simulations in the context of AMP interactions with membrane models.

Evaluation of the Regenerative Capacity of Demineralized Bone Matrix vs Fat Graft in Alveolar Cleft Model in Albino Rats.

This study was performed to evaluate the regenerative capacity of demineralized bone matrix vs fat graft, both guided by pericardium membrane in alveolar cleft model in albino rats. A total of 72 rats were required in this study. A surgical bone defect with a 7 mm length × 4 mm width × 3 mm depth was created as a model of an alveolar cleft, then the rats were divided randomly into four equal groups each group contained 18 rats: control group (defect only), the membrane group (the defect was covered by the pericardium membrane), the demineralized bone matrix (DBM) group (the defect was filled with DBM guided by pericardium membrane) and fat group (the defect was filled with a fat graft guided by the pericardium membrane). Around 6 rats from each group were euthanized after 2, 4, and 8 weeks. Skulls were scanned with cone beam computed tomography (CBCT) and harvested for histological evaluation with routine H&E immunohistochemical stains (Anti-osteocalcin and Anti-Wnt5a). The data was recorded and statistically analyzed by a two-way ANOVA. The study showed a notable formation of new bone, and expression of OCN and Wnt5a were notably increased by time in the fat group. However, the density of bone grafts and OCN and Wnt5a expression decreased with time in the DBM group. Control and membrane groups showed negative OCN and Wnt5a immune-reactivity in the cleft site. Fat graft results were superior to DBM results with regard to mucosal closure and accelerated bone regeneration, and may represent an effective treatment for alveolar cleft reconstruction. Finding an inexpensive, accessible, biocompatible and easily manipulated treatment for craniofacial reconstruction and fat graft fulfilled the desired aims. Further investigations with prolonged evaluation periods are needed. How to cite this article: Abdel Raouf E, Elsherbini AM, Abdel Salam Yousef Y, et al. Evaluation of the Regenerative Capacity of Demineralized Bone Matrix vs Fat Graft in Alveolar Cleft Model in Albino Rats. J Contemp Dent Pract 2024;25(6):554-562.

Exploring the influence of acetylenic acetogenin unsaturation patterns on lipid interface interactions: Insights from surface chemistry, rheology, and spectroscopy

This study explores how the unsaturation patterns of three acetogenins from Porcelia macrocarpa (2S,3R,4R)-3-hydroxy-4-methyl-2-(eicos-11′-yn-19′-enyl)-butanolide (1), (2S,3R,4R)-3-hydroxy-4-methyl-2-(eicos-11′-ynyl)-butanolide (2), and (2S,3R,4R)-3-hydroxy-4-methyl-2-eicosyl-butanolide (3)—affect their interactions with lipid interfaces, using Langmuir monolayers of dipalmitoyl-phosphoethanolamine (DPPE) as a model for protozoal membranes. The hypothesis posits that unsaturated acetogenins (1 and 2) will exhibit distinct behaviors compared to the saturated acetogenin (3) due to structural differences. Experiments incorporated each acetogenin into DPPE monolayers, employing techniques like tensiometry, dilatational rheology, infrared spectroscopy, and Brewster angle microscopy (BAM) to assess interactions and changes in film properties. Results showed that unsaturated acetogenin 1, active against Leishmania infantum and Trypanosoma cruzi, destabilized the film and induced rapid molecular reorganization, while acetogenin 2, inactive against these parasites, uniquely increased the viscous contribution to viscoelasticity. Saturated acetogenin 3 did not integrate into the film. All acetogenins enhanced fluidity, reducing elasticity and viscosity, with BAM revealing phase separation only in acetogenin 2. Infrared spectroscopy underscored the impact of drug-lipid interactions, leading to the conclusion that unsaturated acetogenins 1 and 2 have a more significant influence on thermodynamic and structural properties than acetogenin 3, offering insights into their potential therapeutic mechanisms.