Native Membrane Research Articles

Comparative proteomics reveals Cryptosporidium parvum infection disrupts cellular barriers

Cryptosporidium is a protozoan parasite capable of infecting humans and animals and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms underlying invasive infection and its pathogenesis remain largely unknown. To better understand the molecular mechanism of the interaction between C. parvum and host cells, we profiled the changes of host cells membrane proteins extracted using native membrane protein extraction kit between C. parvum-infected HCT-8 cells and the control group after C. parvum infected 6 h combined with quantitative Tandem Mass Tags (TMT) liquid chromatography-dual mass spectrometry proteomic analysis. Among the 4844 quantifiable proteins identified, the expression levels of 625 were upregulated, and those of 116 were downregulated at 6 h post-infection compared with controls (1.5-fold difference in abundance, p < 0.05). Enrichment analysis of the function, protein domain and Kyoto Encyclopedia of Genes and Genomes pathway of the differentially expressed proteins revealed that the differentially expressed proteins were mainly related to biological functions related to the cytoskeleton and cytoplasmic matrix. We also found that infection with C. parvum may destroy HCT-8 intercellular space adhesion. Six proteins were further verified using quantitative real-time reverse transcription polymerase chain reaction and western blotting. Through systematic analysis of proteomics related to HCT-8 cell membranes infected by C. parvum, we found many host membrane proteins that can serve as potential receptors in C. parvum adhesion or invasion. C. parvum infection destroyed host cell barrier function and caused extensive changes in host cytoskeleton proteins, providing a deeper understanding of the molecules and their functions involved in the host-C. parvum interaction. SignificanceThere is a lack of systematic research on the molecular mechanisms underlying the interaction of C. parvum with host cells. Changes of host cell membrane proteins after C. parvum infection may be used to examine the host cell receptors for parasite adhesion and invasion, and how the parasite interacts with these receptors. It is of great significance that host cells undergo membrane fusion to mediate invasion. Through proteomic studies on the host cell membrane after infection with HCT-8 cells by C. parvum, we observed disruption of the host cell cellular barrier function and widespread alteration of host cytoskeletal proteins caused by C. parvum infection, providing a deeper understanding of the molecules and their functions involved in host-C. parvum interaction.

Functional characterization of the transient receptor potential melastatin 2 (TRPM2) cation channel from Nematostella vectensis reconstituted into lipid bilayer

Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel activation upon oxidative stress is involved in the pathology of stroke and neurodegenerative disorders. Cytosolic Ca2+, ADP-ribose (ADPR) and phosphatidylinositol-4,5-bisphosphate (PIP2) are the obligate activators of the channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality of the purified protein has not been tested. Here we reconstituted overexpressed and purified TRPM2 from Nematostella vectensis (nvTRPM2) into lipid bilayers and found that the protein is fully functional. Consistent with the observations in native membranes, nvTRPM2 in lipid bilayers is co-activated by cytosolic Ca2+ and either ADPR or ADPR-2′-phosphate (ADPRP). The physiological metabolite ADPRP has a higher apparent affinity than ADPR. In lipid bilayers nvTRPM2 displays a large linear unitary conductance, its open probability (Po) shows little voltage dependence and is stable over several minutes. Po is high without addition of exogenous PIP2, but is largely blunted by treatment with poly-l-Lysine, a polycation that masks PIP2 headgroups. These results indicate that PIP2 or some other activating phosphoinositol lipid co-purifies with nvTRPM2, suggesting a high PIP2 binding affinity of nvTRPM2 under physiological conditions.

A Synthetic Bio-Absorbable Membrane in Guided Bone Regeneration in Dehiscence-Type Defects: An Experimental In Vivo Investigation in Dogs.

This study aimed to determine the performance and characteristics of a synthetic barrier membrane of polylactic acid and acetyl butyl citrate (PLAB) for the lateral bone augmentation of peri-implant dehiscence defects (mean height × depth = 3 mm × 1 mm). In eight dogs, three treatment groups were randomly allocated at each chronic peri-implant dehiscence-type defect: (i) a deproteinized bovine bone mineral covered by a synthetic barrier membrane (test group), (ii) a deproteinized bovine bone mineral covered by a natural collagen membrane (positive control), and (iii) a synthetic barrier membrane (negative control). After 4 and 12 weeks of submerged healing, dissected tissue blocks were processed for calcified and decalcified histological analysis. Histometric measurements for tissue and bone width were performed, and bone-to-implant contact and alkaline phosphatase expression where measured. After 4 and 12 weeks of healing, no statistical differences between the groups were observed for the histometric measurements. The expression of alkaline phosphatase was higher in the positive control group after 4 weeks followed by the positive and negative controls (5.25 ± 4.09, 4.46 ± 3.03, and 4.35 ± 2.28%, p > 0.05) and 12 weeks followed by the negative and positive controls (4.3 ± 2.14, 3.21 ± 1.53, and 2.39 ± 1.03%, p > 0.05). Concerning the bone-to-implant contact, after 4 weeks, the test group obtained the highest results (39.54 ± 48.7) vs. (31.24 ± 42.6) and (20.23 ± 36.1), respectively, while after 12 weeks, the positive control group obtained the highest Bone to imaplant contact (BIC) results, followed by the test and negative controls, (35.91 ± 24.9) vs. (18.41 ± 20.5) and (24.3 ± 32.1), respectively; no statistically significant differences were obtained. Within the limitations of the study, new bone formation can be achieved in guided bone regeneration procedures simultaneously with implant placement either with the use of a PLAB membrane or a native collagen membrane, although these differences were not statistically significant.

Two-Dimensional NMR Spectroscopy of the G Protein-Coupled Receptor A2AAR in Lipid Nanodiscs.

Eight hundred and twenty-six human G protein-coupled receptors (GPCRs) mediate the actions of two-thirds of the human hormones and neurotransmitters and over one-third of clinically used drugs. Studying the structure and dynamics of human GPCRs in lipid bilayer environments resembling the native cell membrane milieu is of great interest as a basis for understanding structure-function relationships and thus benefits continued drug development. Here, we incorporate the human A2A adenosine receptor (A2AAR) into lipid nanodiscs, which represent a detergent-free environment for structural studies using nuclear magnetic resonance (NMR) in solution. The [15N,1H]-TROSY correlation spectra confirmed that the complex of [u-15N, ~70% 2H]-A2AAR with an inverse agonist adopts its global fold in lipid nanodiscs in solution at physiological temperature. The global assessment led to two observations of practical interest. First, A2AAR in nanodiscs can be stored for at least one month at 4 °C in an aqueous solvent. Second, LMNG/CHS micelles are a very close mimic of the environment of A2AAR in nanodiscs. The NMR signal of five individually assigned tryptophan indole 15N-1H moieties located in different regions of the receptor structure further enabled a detailed assessment of the impact of nanodiscs and LMNG/CHS micelles on the local structure and dynamics of A2AAR. As expected, the largest effects were observed near the lipid-water interface along the intra- and extracellular surfaces, indicating possible roles of tryptophan side chains in stabilizing GPCRs in lipid bilayer membranes.

Natural clay membranes: A sustainable and affordable solution for treating dye solutions, coal mine washery waste, and aquaculture wastewater

In this study, we fabricated low-cost, sustainable inorganic membranes using a one-step dry compaction process that incorporates natural clay and edible glycerin, followed by sintering at low temperatures. Open porosity and permeability of the membranes were in the 33.6–45.2 % and 621.74–864.7 l/m2.h.bar range, respectively. Regarding molecular weight cut-off (MWCO) and mercury porosity of membranes with 33.6 % and 45.2 % porosity, the average pore size of the former was ~10 nm, while it was increased up to 10–70 nm, 0.9–3 μm, and ~ 10 μm for the latter. The membranes were subjected to filtration for dye solutions, coal mine washery waste, and aquaculture wastewater, resulting in remarkable removal efficiencies. Specifically, the membranes achieved removal rates of 99.8 % for direct blue 71, 92.44 % for disperse red 74, and 99.1 % for red 2G in dye solutions. Furthermore, they demonstrated removal efficiencies of 99.19 % for turbidity and 94.2 % for chemical oxygen demand (COD) in coal mine washery waste. The membranes removed 98.5 % of total suspended solids from the fish farm effluent. During extended periods of operation, the clay membrane exhibited remarkable stability and minimal leaching of metal ions. Superior properties, including low-cost, nontoxic, and accessible precursors, one-step uni-axial fabrication process without the need for lubricant and plasticizer, low sintering temperature compared to commercial ceramic membranes, promising long-term chemical stability in highly acidic and alkaline media, recyclability, and reproducibility, make the as-prepared membranes an affordable and sustainable solution for treating wastewater in various industries, including textile, mining, and aquaculture, thereby mitigating the environmental impact of these industries.

PRGF Membrane with Tailored Optical Properties Preserves the Cytoprotective Effect of Plasma Rich in Growth Factors: In Vitro Model of Retinal Pigment Epithelial Cells

The present study evaluates the ability of a novel plasma rich in growth factors (PRGF) membrane with improved optical properties to reduce oxidative stress in retinal pigment epithelial cells (ARPE-19 cells) exposed to blue light. PRGF was obtained from three healthy donors and divided into four main groups: (i) PRGF membrane (M-PRGF), (ii) PRGF supernatant (S-PRGF), (iii) platelet-poor plasma (PPP) membrane diluted 50% with S-PRGF (M-PPP 50%), and (iv) M-PPP 50% supernatant (S-PPP 50%). ARPE-19 cells were exposed to blue light and then incubated with the different PRGF-derived formulations or control for 24 and 48 h under blue light exposure. Mitochondrial and cell viability, reactive oxygen species (ROS) production, and heme oxygenase-1 (HO-1) and ZO-1 expression were evaluated. Mitochondrial viability and cell survival were significantly increased after treatment with the different PRGF-derived formulations. ROS synthesis and HO-1 expression were significantly reduced after cell treatment with any of the PRGF-derived formulations. Furthermore, the different PRGF-derived formulations significantly increased ZO-1 expression in ARPE-19 exposed to blue light. The new PRGF membrane with improved optical properties and its supernatant (M-PPP 50% and S-PPP 50%) protected and reversed blue light-induced oxidative stress in ARPE-19 cells at levels like those of a natural PRGF membrane and its supernatant.

Elucidating interprotein energy transfer dynamics within the antenna network from purple bacteria

In photosynthesis, absorbed light energy transfers through a network of antenna proteins with near-unity quantum efficiency to reach the reaction center, which initiates the downstream biochemical reactions. While the energy transfer dynamics within individual antenna proteins have been extensively studied over the past decades, the dynamics between the proteins are poorly understood due to the heterogeneous organization of the network. Previously reported timescales averaged over such heterogeneity, obscuring individual interprotein energy transfer steps. Here, we isolated and interrogated interprotein energy transfer by embedding two variants of the primary antenna protein from purple bacteria, light-harvesting complex 2 (LH2), together into a near-native membrane disc, known as a nanodisc. We integrated ultrafast transient absorption spectroscopy, quantum dynamics simulations, and cryogenic electron microscopy to determine interprotein energy transfer timescales. By varying the diameter of the nanodiscs, we replicated a range of distances between the proteins. The closest distance possible between neighboring LH2, which is the most common in native membranes, is 25 Å and resulted in a timescale of 5.7 ps. Larger distances of 28 to 31 Å resulted in timescales of 10 to 14 ps. Corresponding simulations showed that the fast energy transfer steps between closely spaced LH2 increase transport distances by ∼15%. Overall, our results introduce a framework for well-controlled studies of interprotein energy transfer dynamics and suggest that protein pairs serve as the primary pathway for the efficient transport of solar energy.

Green natural rubber-based membrane for drug release

Drug release membranes have been developed in various points of view for many decades. They should be flexible responding to body movement for the patient convenience. Most of them are synthetic materials. Since natural rubber (NR) is elastic and flexible, it is an interesting green material to develop as a drug release membrane. However, there is limitation of drug release of NR membrane. Therefore, the development of drug release efficiency of NR membrane is important and challenging. The objective of this work is to develop drug release membrane from NR based as NR is an alternative green material. NR, epoxidized natural rubber (ENR), poly(vinyl alcohol) (PVA), NR/PVA and ENR/PVA membranes were prepared by casting process. Sodium diclofenac was used as a drug in this work. The result from H1 NMR revealed that 25 mol% ENR was obtained. It was proven that there is no formation of chemical bonding between drug and matrix membrane by Fourier transform infrared spectroscopy characterization. The swelling ratios of PVA, ENR/PVA, NR/PVA were about 165%, 120% and 100%, respectively, whereas ENR and NR were not swollen. It is probably due to the high hydrophilicity of PVA. NR and NR/PVA membrane surfaces presented some agglomeration of drug particles shown in scanning electron microscopy micrographs. The drug distribution in NR matrix was very poor whereas obvious agglomeration in ENR and PVA membranes was not present. The polar groups (epoxide and hydroxyl) may assist in ionic drug distribution. The result of drug release showed that neat NR membrane gave the lowest value at about 50%. Therefore, the drug was predicted to be trapped inside the NR matrix. This was similar to the case of NR/PVA membrane, whereas PVA membrane provided the highest and fastest release compared to others. However, in case of ENR, the %drug release increased gradually with the release time increased until it was constant at 7 hours onwards. It can be said that the modification of NR by adding hydrophilic group (as ENR) could enhance the %drug release. Moreover, the addition of hydrophilic part (combination of PVA) also increased the %drug release that obviously seen in ENR/PVA and NR/PVA membranes.

Mechanical Characterization of Soft Membranes with One-Shot Projection Moiré and Metaheuristic Optimization

Mechanical characterization of soft materials is a complicated inverse problem that includes nonlinear constitutive behavior and large deformations. A further complication is introduced by the structural inhomogeneity of tested specimens (for example, caused by thickness variations). Optical methods are very useful in mechanical characterization of soft matter, as they provide accurate full-field information on displacements, strains and stresses regardless of the magnitude and/or gradients of those quantities. In view of this, the present study describes a novel hybrid framework for mechanical characterization of soft membranes, combining (i) inflation tests and preliminary in-plane equi-biaxial tests, (ii) a one-shot projection moiré optical setup with two symmetric projectors that project cross-gratings onto the inflated membrane, (iii) a mathematical model to extract 3D displacement information from moiré measurements, and (iv) metaheuristic optimization hybridizing harmony search and JAYA algorithms. The use of cross-gratings allows us to determine the surface curvature and precisely reconstruct the shape of the deformed object. Enriching metaheuristic optimization with gradient information and elitist strategies significantly reduces the computational cost of the identification process. The feasibility of the proposed approach wassuccessfully tested on a 100 mm diameter natural rubber membrane that had some degree of anisotropy in mechanical response because of its inhomogeneous thickness distribution. Remarkably, up to 324 hyperelastic constants and thickness parameters can be precisely identified by the proposed framework, reducing computational effort from 15% to 70% with respect to other inverse methods.

Conformations of Human Immunodeficiency Virus Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles.

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

Effect of moderate prenatal ethanol exposure on the differential expression of two histamine H3 receptor isoforms in different brain regions of adult rat offspring.

We have reported that prenatal alcohol exposure (PAE) elevates histamine H3 receptor (H3R) agonist-mediated inhibition of glutamatergic neurotransmission in the dentate gyrus. Here, we hypothesized that PAE alters the expression of two prominent H3R isoforms namely, the rH3A and rH3C isoforms, which have differing intrinsic activities for H3R agonists, in a manner that may contribute to heightened H3R function in PAE rats. In contrast to our predictions, we found different effects of sex and PAE in various brain regions with significant interactions between sex and PAE in dentate gyrus and entorhinal cortex for both isoforms. Subsequently, to confirm the PAE-and sex-induced differences on H3R isoform mRNA expression, we developed a polyclonal antibody selective for the rH3A inform. Western blots of rH3A mRNA-transfected HEK-293 cells identified a ~ 48 kDa band of binding consistent with the molecular weight of rH3A, thus confirming antibody sensitivity for rH3A protein. In parallel, we also established a pan-H3R knockout mice line to confirm antibody specificity in rodent brain membranes. Both qRT-PCR and H3R agonist-stimulated [35S]-GTPγS binding confirmed the absence of mH3A mRNA and H3 receptor-effector coupling in H3R knockout (KO) mice. Subsequent western blotting studies in both rat and mouse brain membranes were unable to detect rH3A antibody binding at ~48 kDa. Rather, the H3RA antibody bound to a ~ 55 kDa band in both rat and mouse membranes, including H3R KO mice, suggesting H3RA binding was not specific for H3Rs in rodent membranes. Subsequent LC/MS analysis of the ~55 kDa band in frontal cortical membranes identified the highly abundant beta subunit of ATPase in both WT and KO mice. Finally, LC/MS analysis of the ~48 kDa band from rH3A mRNA-transfected HEK-293 cell membranes was able to detect rH3A protein, but its presence was below the limits of quantitative reliability. We conclude that PAE alters rH3A and rH3C mRNA expression in some of the same brain regions where we have previously reported PAE-induced alterations in H3R-effector coupling. However, interpreting the functional consequences of altered H3R isoform expression was limited given the technical challenges of measuring the relatively low abundance of rH3A protein in native membrane preparations.

An Extracellular miRNA‐Responsive Artificial Receptor via Dynamic DNA Nano‐assembly for Biomarker‐Driven Therapy

AbstractMicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers and therapeutic targets in various diseases. However, there is currently a lack of molecular strategies that can effectively use disease‐associated extracellular miRNAs as input signals to drive therapeutic functions. Herein, we present a modular and programmable miRNA‐responsive chimeric DNA receptor (miRNA‐CDR) capable of biomarker‐driven therapy. By grafting a miRNA‐responsive DNA nanodevice on a natural membrane receptor via aptamer anchoring, miRNA‐CDR can sense extracellular miRNA levels and autonomously induce dimerization‐mediated receptor activation via the complementary‐mediated strand displacement reaction‐induced dynamic DNA assembly. The sequence programmability of miRNA‐CDR allows it to sense and respond to a user‐defined miRNA with tunable sensitivity. Moreover, the miRNA‐CDR is versatile and customizable to reprogram desirable signaling output via adapting a designated receptor, such as MET and FGFR1. Using a mouse model of drug‐induced acute liver injury (DILI), we demonstrate the functionality of a designer miRNA‐CDR in rewiring the recognition of the DILI‐elevated miR‐122 to promote MET signaling of hepatocytes for biomarker‐driven in situ repair and liver function restoration. Our synthetic miRNA‐CDR platform provides a novel molecular device enabling biomarker‐driven therapeutic cellular response, potentially paving the way for improving the precision of cell therapy in regenerative medicine.

An Extracellular miRNA-Responsive Artificial Receptor via Dynamic DNA Nano-assembly for Biomarker-Driven Therapy.

MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers and therapeutic targets in various diseases. However, there is currently a lack of molecular strategies that can effectively use disease-associated extracellular miRNAs as input signals to drive therapeutic functions. Herein, we present a modular and programmable miRNA-responsive chimeric DNA receptor (miRNA-CDR) capable of biomarker-driven therapy. By grafting a miRNA-responsive DNA nanodevice on a natural membrane receptor via aptamer anchoring, miRNA-CDR can sense extracellular miRNA levels and autonomously induce dimerization-mediated receptor activation via the complementary-mediated strand displacement reaction-induced dynamic DNA assembly. The sequence programmability of miRNA-CDR allows it to sense and respond to a user-defined miRNA with tunable sensitivity. Moreover, the miRNA-CDR is versatile and customizable to reprogram desirable signaling output via adapting a designated receptor, such as MET and FGFR1. Using a mouse model of drug-induced acute liver injury (DILI), we demonstrate the functionality of a designer miRNA-CDR in rewiring the recognition of the DILI-elevated miR-122 to promote MET signaling of hepatocytes for biomarker-driven in situ repair and liver function restoration. Our synthetic miRNA-CDR platform provides a novel molecular device enabling biomarker-driven therapeutic cellular response, potentially paving the way for improving the precision of cell therapy in regenerative medicine.

Cellular Membrane Components-mediated Cancer ImmunotherapeuticPlatforms.

Immune cell engineering is an active field of ongoing research that can be easily applied to nanoscale biomedicine as an alternative to overcoming limitations of nanoparticles. Cell membrane coating and artificial nanovesicle technology have been reported as representative methods with an advantage of good biocompatibility for biomimetic replication of cell membrane characteristics. Cell membrane-mediated biomimetic technique provides properties of natural cell membrane and enables membrane-associated cellular/molecular signaling. Thus, coated NPs and artificial nanovesicles can achieve effective and extended in vivo circulation, enabling execution of target functions. While coated NPs and artificial nanovesicles provide clear advantages, much work remains before clinical application. In this review, we first provide a comprehensive overview of cell membrane coating techniques and artificial nanovesicles. Next, we summarize the function and application of various immune cell membrane types. This article is protected by copyright. All rights reserved.

Revealing Low Amplitude Signals of Neuroendocrine Cells through Disordered Silicon Nanowires-Based Microelectrode Array.

Today, the key methodology to study in vitro or in vivo electrical activity in a population of electrogenic cells, under physiological or pathological conditions, is by using microelectrode array (MEA). While significant efforts have been devoted to develop nanostructured MEAs for improving the electrophysiological investigation in neurons and cardiomyocytes, data on the recording of the electrical activity from neuroendocrine cells with MEA technology are scarce owing to their weaker electrical signals. Disordered silicon nanowires (SiNWs) for developing a MEA that, combined with a customized acquisition board, successfully capture the electrical signals generated by the corticotrope AtT-20cells as a function of the extracellular calcium (Ca2+ ) concentration are reported. The recorded signals show a shape that clearly resembles the action potential waveform by suggesting a natural membrane penetration of the SiNWs. Additionally, the generation of synchronous signals observed under high Ca2+ content indicates the occurrence of a collective behavior in the AtT-20cell population. This study extends the usefulness of MEA technology to the investigation of the electrical communication in cells of the pituitary gland, crucial in controlling several essential human functions, and provides new perspectives in recording with MEA the electrical activity of excitable cells.

Alveolar ridge alterations after lateral GBR with and withouthyaluronic acid (xHyA) - a prospective randomized trial withmorphometric and histomorphometric evaluation.

To clinically and histologically evaluate the potential effect of a cross-linked high molecular weight hyaluronic acid (xHyA) on the outcomes of guided bone regeneration (GBR) performed with a demineralized bovine bone mineral (DBBM) covered with a natural collagen membrane (NCM). Eleven patients (eight females and three males, mean age 53 years) with a total of 27 surgical sites were treated. Treatments were performed with either DBBM and NCM fixated with tacks (group A) or DBBM mixed with xHyA and subsequently covered with NCM (group B). Clinical evaluations were made at Baseline (T1), immediately after GBR (T2) and at the time of implant placement (T3). Additionally, at the time of implant placement, core biopsies were retrieved and submitted for histological analysis. Healing was uneventful in all cases. At six months, group B revealed a statistically significantly higher crestal ridge dimension compared to group A (P=0.007). The histologic analysis revealed a tendency for higher mineralized tissue formation in group B compared to group A (i.e., 67.5% versus 41.6%) and contained a higher amount of new bone (37.2%) and less DBBM residues (20.9%) than group A (i.e., 12.8% new bone and 28.8% DBBM residues, respectively). Within their limits, the present data indicate that, during GBR with NCM, the combination of DBBM and xHyA may improve the quality and quantity of bone formed with DBBM alone.

Patch-clamp technique to study mitochondrial membrane biophysics.

Mitochondria are double-membrane organelles crucial for oxidative phosphorylation, enabling efficient ATP synthesis by eukaryotic cells. Both of the membranes, the highly selective inner mitochondrial membrane (IMM) and a relatively porous outer membrane (OMM), harbor a number of integral membrane proteins that help in the transport of biological molecules. These transporters are especially enriched in the IMM, where they help maintain transmembrane gradients for H+, K+, Ca2+, PO43-, and metabolites like ADP/ATP, citrate, etc. Impaired activity of these transporters can affect the efficiency of energy-transducing processes and can alter cellular redox state, leading to activation of cell-death pathways or metabolic syndromes in vivo. Although several methodologies are available to study ion flux through membrane proteins, the patch-clamp technique remains the gold standard for quantitatively analyzing electrogenic ion exchange across membranes. Direct patch-clamp recordings of mitoplasts (mitochondria devoid of outer membrane) in different modes, such as whole-mitoplast or excised-patch mode, allow researchers the opportunity to study the biophysics of mitochondrial transporters in the native membrane, in real time, in isolation from other fluxes or confounding factors due to changes in ion gradients, pH, or mitochondrial potential (ΔΨ). Here, we summarize the use of patch clamp to investigate several membrane proteins of mitochondria. We demonstrate how this technique can be reliably applied to record whole-mitoplast Ca2+ currents mediated via mitochondrial calcium uniporter or H+ currents mediated by uncoupling protein 1 and discuss critical considerations while recording currents from these small vesicles of the IMM (mitoplast diameter = 2-5 µm).

Increased delivery and cytotoxicity of doxorubicin in HeLa cells using the synthetic cationic peptide pEM-2 functionalized liposomes

HypothesisDue to the inability of nano-carriers to passively cross the cell membrane, cell penetration enhancers are used to accelerate cytoplasmic delivery of antineoplastic drugs. In this regard, snake venom phospholipase A2 peptides are known for their ability to destabilize natural and artificial membranes. In this context, functionalized liposomes with peptide pEM-2 should favor the incorporation of doxorubicin and increase its cytotoxicity in HeLa cells compared to free doxorubicin, and doxorubicin encapsulated in non-functionalized liposomes. ExperimentsSeveral characteristics were monitored, including doxorubicin loading capacity of the liposomes, as well as the release and uptake before and after functionalization. Cell viability and half-maximal inhibition concentrations were determined in HeLa cells. FindingsIn vitro studies showed that functionalization of doxorubicin-loaded PC-NG liposomes with pEM-2 not only improved the amount of doxorubicin delivered compared to free doxorubicin or other doxorubicin-containing formulations, but also showed enhanced cytotoxicity against HeLa cells. The PC-NG liposomes loaded with doxorubicin improved treatment efficacy by reducing the IC50 value and incubation time. This increase in cell toxicity was directly related to the concentration of pEM-2 peptide bound to the liposomes. We conclude that the cytotoxicity observed in HeLa cells due to the action of doxorubicin was strongly favored when encapsulated in synthetic liposomes and functionalized with the pEM-2 peptide.

Bio-enabled Engineering of Multifunctional "Living" Surfaces.

Through the magic of "active matter"─matter that converts chemical energy into mechanical work to drive emergent properties─biology solves a myriad of seemingly enormous physical challenges. Using active matter surfaces, for example, our lungs clear an astronomically large number of particulate contaminants that accompany each of the 10,000 L of air we respire per day, thus ensuring that the lungs' gas exchange surfaces remain functional. In this Perspective, we describe our efforts to engineer artificial active surfaces that mimic active matter surfaces in biology. Specifically, we seek to assemble the basic active matter components─mechanical motor, driven constituent, and energy source─to design surfaces that support the continuous operation of molecular sensing, recognition, and exchange. The successful realization of this technology would generate multifunctional, "living" surfaces that combine the dynamic programmability of active matter and the molecular specificity of biological surfaces and apply them to applications in biosensors, chemical diagnostics, and other surface transport and catalytic processes. We describe our recent efforts in bio-enabled engineering of living surfaces through the design of molecular probes to understand and integrate native biological membranes into synthetic materials.

Unleashing the potential of cell biomimetic nanoparticles: Strategies and challenges in their design and fabrication for therapeutic applications.

In this contribution to the Orations - New Horizons of the Journal of Controlled Release, I present design strategies for the two main biomimetic nanoparticles (BNP) groups: BNP composed of isolated cell membrane proteins, and BNP composed of the native cell membrane. I furthermore describe BNP fabrication methods and analyze their advantages and challenges. Finally, I suggest future therapeutic applications of each BNP group and propose a new revolutionary concept for their use.