Escherichia coli response to subinhibitory concentration of Colistin: Insights from study of membrane dynamics and morphology.

Prevalence of wide spread bacterial infections bring forth a critical need in understanding the molecular mechanisms of the antibiotics as well as the bacterial response to those antibiotics. Improper usage of antibiotics, which can be in sub-lethal concentrations is one among the multiple reasons for acquiring antibiotic resistance which makes it vital to understand the bacterial response towards sub-lethal concentrations of antibiotics. In this work, we have used colistin, a well-known membrane active antibiotic used to treat severe bacterial infections and explored the impact of its subminimum inhibitory concentration (MIC) on the lipid membrane dynamics and morphological changes of E. coli. Upon investigation of live cell membrane properties such as lipid dynamics using fluorescence correlation spectroscopy, we observed that colistin disrupts the lipid membrane at sub-MIC by altering the lipid diffusivity. Interestingly, filamentationlike cell elongation was observed upon colistin treatment which led to further exploration of surface morphology with the help of atomic force spectroscopy. The changes in the surface roughness upon colistin treatment provides additional insight on the colistin-membrane interaction corroborating with the altered lipid diffusion. Although altered lipid dynamics could be attributed to an outcome of lipid rearrangement due to direct disruption by antibiotic molecules on the membrane or an indirect consequence of disruptions in lipid biosynthetic pathways, we were able to ascertain that altered bacterial membrane dynamics is due to direct disruptions. Our results provide a broad overview on the consequence of the cyclic polypeptide, colistin on membrane specific lipid dynamics and morphology of a live Gram-negative bacterial cell.

Thermodynamic Modeling of Solvent-Assisted Lipid Bilayer Formation Process

The solvent-assisted lipid bilayer (SALB) formation method provides a simple and efficient, microfluidic-based strategy to fabricate supported lipid bilayers (SLBs) with rich compositional diversity on a wide range of solid supports. While various studies have been performed to characterize SLBs formed using the SALB method, relatively limited work has been carried out to understand the underlying mechanisms of SALB formation under various experimental conditions. Through thermodynamic modeling, we studied the experimental parameters that affect the SALB formation process, including substrate surface properties, initial lipid concentration, and temperature. It was found that all the parameters are critically important to successfully form high-quality SLBs. The model also helps to identify the range of parameter space within which conformal, homogeneous SLBs can be fabricated, and provides mechanistic guidance to optimize experimental conditions for lipid membrane-related applications.

Advances in the Application of Exosomes Identification Using Surface-Enhanced Raman Spectroscopy for the Early Detection of Cancers

Exosomes are small nanoscale vesicles with a double-layered lipid membrane structure secreted by cells, and almost all types of cells can secrete exosomes. Exosomes carry a variety of biologically active contents such as nucleic acids and proteins, and play an important role not only in intercellular information exchange and signal transduction, but also in various pathophysiological processes in the human body. Surface-enhanced Raman Spectroscopy (SERS) uses light to interact with nanostructured materials such as gold and silver to produce a strong surface plasmon resonance effect, which can significantly enhance the Raman signal of molecules adsorbed on the surface of nanostructures to obtain a rich fingerprint of the sample itself or Raman probe molecules with ultra-sensitivity. The unique advantages of SERS, such as non-invasive and high sensitivity, good selectivity, fast analysis speed, and low water interference, make it a promising technology for life science and clinical testing applications. In this paper, we briefly introduce exosomes and the current main detection methods. We also describe the basic principles of SERS and the progress of the application of unlabeled and labeled SERS in exosome detection. This paper also summarizes the value of SERS-based exosome assays for early tumor diagnosis.

Lifetime of actin-dependent protein nanoclusters

Protein nanoclusters (PNCs) are dynamic collections of a few proteins that spatially organize in nanometer length clusters. PNCs are one of the principal forms of spatial organization of membrane proteins and they have been shown or hypothesized to be important in various cellular processes, including cell signaling. PNCs show remarkable diversity in size, shape, and lifetime. In particular, the lifetime of PNCs can vary over a wide range of timescales. The diversity in size and shape can be explained by the interaction of the clustering proteins with the actin cytoskeleton or the lipid membrane, but very little is known about the processes that determine the lifetime of the nanoclusters. In this paper, using mathematical modelling of the cluster dynamics, we model the biophysical processes that determine the lifetime of actin-dependent PNCs. In particular, we investigated the role of actin aster fragmentation, which had been suggested to be a key determinant of the PNC lifetime, and found that it is important only for a small class of PNCs. A simple extension of our model allowed us to investigate the kinetics of protein-ligand interaction near PNCs. We found an anomalous increase in the lifetime of ligands near PNCs, which agrees remarkably well with experimental data on RAS-RAF kinetics. In particular, analysis of the RAS-RAF data through our model provides falsifiable predictions and novel hypotheses that will not only shed light on the role of RAS-RAF kinetics in various cancers, but also will be useful in studying membrane protein clustering in general.

Autophagy and evasion of immune system by SARS-CoV-2. Structural features of the Non-structural protein 6 from Wild Type and Omicron viral strains interacting with a model lipid bilayer.

The viral cycle of SARS-CoV-2 is based on a complex interplay with the cellular machinery, which is mediated by specific proteins eluding or hijacking the cellular defense mechanisms. Among the complex pathways called by the viral infection autophagy is particularly crucial and is strongly influenced by the action of the non-structural protein 6 (Nsp6) interacting with the endoplasmic reticulum membrane. Importantly, differently from other non-structural proteins Nsp6 is mutated in the recently emerged Omicron variant, suggesting a possible different role of autophagy. In this contribution we explore, for the first time, the structural property of Nsp6 thanks to long-time scale molecular dynamic simulations and machine learning analysis, identifying the interaction patterns with the lipid membrane. We also show how the mutation brought by the Omicron variant may indeed modify some of the specific interactions, and more particularly help anchoring the viral protein to the lipid bilayer interface.

The amphoteric cartilage surface tested in the pH Range from 2.0 to 9.0

Background: Phospholipids adsorbed to negatively-charged proteoglycan matrix form phospholipid (membrane), have negatively charged surface (-PO4-) and are hydrophilic. Strong adsorption and strong cohesion are necessary for phospholipids to provide a good lubricant. The surface energy of spherical lipid bilayers have "bell-curve" shaped has amphoteric character and lowest surface energy at a pH 7.4 ± 1 of the natural joint. Objectives: The amphoteric character of the natural surface of the articular cartilage was determined by measuring the surface energy of the model spherical bilayer lipid membrane. It was found that the friction (f) vs. pH 2.0 to 9.0 of the pair (cartilage/cartilage) has the amphoteric character by exposing "bell-curve" shaped with an isoelectric point (IEP). Methods: The friction coefficient (f) was measured with the sliding pin-on-disc tribotester the friction between two surfaces (cartilage/cartilage) pair. The method of interfacial tension measurements of the spherical lipid bilayer model vs the pH over the range 0.2 to 9.0 was used. Results: The dependence of friction coefficient between two cartilage surfaces on the pH over the range 2.0 to 9.0 is demonstrated by a “bell - curve” in Fig. 2(A). The surface energy of a model spherical bilayer lipid membrane vs. the pH has the character of a “bell - curve” with an (IEP) is shown in Fig. 2(B). Conclusion: The amphoteric effect on friction between the bovine cartilage/cartilage contacts has been found to be highly sensitive to the pH of an aqueous solution. In this paper we demonstrate experimentally that the pH sensitivity of cartilage to friction provides a novel concept in joint lubrication on charged surfaces. The change in friction was consistently related to the change of charge density of an amphoteric surface.

On crystallization of water confined in liposomes and cryoprotective action of DMSO

Ice-free phase formed by DMSO acting as a protective layer of lipid membrane.

Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol

The need to gain insights into the molecular details of peripheral membrane proteins’ specificity towards phosphatidic acid (PA) is undeniable. The variety of PA species classified in terms of acyl chain length and saturation translates into a complicated, enigmatic network of functional effects that exert a critical influence on cell physiology. As a consequence, numerous studies on the importance of phosphatidic acid in human diseases have been conducted in recent years. One of the key proteins in this context is mTOR, considered to be the most important cellular sensor of essential nutrients while regulating cell proliferation, and which also appears to require PA to build stable and active complexes. Here, we investigated the specific recognition of three physiologically important PA species by the mTOR FRB domain in the presence or absence of cholesterol in targeted membranes. Using a broad range of methods based on model lipid membrane systems, we elucidated how the length and saturation of PA acyl chains influence specific binding of the mTOR FRB domain to the membrane. We also discovered that cholesterol exerts a strong modulatory effect on PA-FRB recognition. Our data provide insight into the molecular details of some physiological effects reported previously and reveal novel mechanisms of fine-tuning the signaling cascades dependent on PA.

The Role of Extracellular Vesicles in the Progression of Tumors towards Metastasis

Extracellular vesicles (EVs) are cell-derived lipid membrane bound vesicles that serve as mediators of intercellular communication. EVs have been found to regulate a wide range of cellular processes through the transference of genetic, protein and lipid messages from the host cell to the recipient cell. Unsurprisingly, this major mode of intracellular communication would be abrogated in cancer. Ever increasing evidence points towards a key role of EVs in promoting tumor development and in contributing to the various stages of metastasis. Tumor released EVs have been shown to facilitate the transference of oncogenic proteins and nucleic acids to other tumor cells and to the surrounding stromal cells, thereby setting up a tumor permissive microenvironment. EVs released from tumor cells have been shown to promote extracellular matrix (ECM) remodeling through the modulation of neighboring tumor cells and stromal cells. EVs released from disseminated tumor cells have been reported to attract circulating tumor cells (CTCs) via chemotaxis and induce the production of specific extracellular matrix components from neighboring stromal cells so as to support the growth of metastatic cells at the secondary tumor site. Circulating levels of tumor derived EVs of patients have been correlated with incidence of metastasis and disease relapse.

Reconstitution of Prenyltransferase Activity on Nanodiscs by Components of the Rubber Synthesis Machinery of the Para Rubber Tree and Guayule

Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity. The proteins HRT1, HRT2, and HRBP have been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Co-expression of HRT1 and HRBP in the presence of nanodiscs yielded marked polyisoprene synthesis activity. By contrast, neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP manifested such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (PaCPT1–3) manifested prenyltransferase activity only if co-expressed with PaCBP, the homolog of HRBP. Our results thus indicate that two heterologous subunits form the core prenyltransferase of the rubber biosynthetic machinery. A recently developed structure modeling program predicted the structure of such heterodimer complexes including HRT1/HRBP and PaCPT2/PaCBP. HRT and PaCPT proteins were also found to possess affinity for a lipid membrane in the absence of HRBP or PaCBP, and structure modeling implicated an amphipathic α-helical domain of HRT1 and PaCPT2 in membrane binding of these proteins.