Biophysical characterisation of SMALPs

Membrane proteins such as receptors, ion channels and transport proteins are important drug targets. The structure-based study of membrane proteins is challenging, especially when the target protein contains both soluble and insoluble domains. Most membrane proteins are insoluble in aqueous solvent and embedded in the plasma membrane lipid bilayer, which significantly complicates biophysical studies. Poly(styrene-co-maleic acid) (SMA) and other polymer derivatives are increasingly common solubilisation agents, used to isolate membrane proteins stabilised in their native lipid environment in the total absence of detergent. Since the initial report of SMA-mediated solubilisation, and the formation of SMA lipid particles (SMALPs), this technique can directly isolate therapeutic targets from biological membranes, including G-protein coupled receptors (GPCRs). SMA now allows biophysical and structural analyses of membrane proteins in solution that was not previously possible. Here, we critically review several existing biophysical techniques compatible with SMALPs, with a focus on hydrodynamic analysis, microcalorimetric analysis and optical spectroscopic techniques.

Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit

Shewanella oneidensis exchanges electrons between cellular metabolism and external redox partners in a process that attracts much attention for production of green electricity (microbial fuel cells) and chemicals (microbial electrosynthesis). A critical component of this pathway is the outer membrane spanning MTR complex, a biomolecular wire formed of the MtrA, MtrB, and MtrC proteins. MtrA and MtrC are decaheme cytochromes that form a chain of close-packed hemes to define an electron transfer pathway of 185 Å. MtrA is wrapped inside MtrB for solubility across the outer membrane lipid bilayer; MtrC sits outside the cell for electron exchange with external redox partners. Here, we demonstrate tight and spontaneous in vitro association of MtrAB with separately purified MtrC. The resulting complex is comparable with the MTR complex naturally assembled by Shewanella in terms of both its structure and rates of electron transfer across a lipid bilayer. Our findings reveal the potential for building bespoke electron conduits where MtrAB combines with chemically modified MtrC, in this case, labeled with a Ru-dye that enables light-triggered electron injection into the MtrC heme chain.

Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

Kinetic Study of 17α-Estradiol Activity in Comparison with 17β-Estradiol and 17α-Ethynylestradiol

17α-estradiol (αE2), an endogenous stereoisomer of the hormone 17β-estradiol (E2), is capable of binding to estrogen receptors (ER). We aimed to mathematically describe, using experimental data, the possible interactions between αE2 and sperm ER during the process of sperm capacitation and to develop a kinetic model. The goal was to compare the suggested kinetic model with previously published results of ER interactions with E2 and 17α-ethynylestradiol (EE2). The HPLC-MS/MS method was developed to monitor the changes of αE2 concentration during capacitation. The calculated relative concentrations Bt were used for kinetic analysis. Rate constants k and molar ratio n were optimized and used for the construction of theoretical B(t) curves. Modifications in αE2–ER interactions were discovered during comparison with models for E2 and EE2. These new interactions displayed autocatalytic formation of an unstable adduct between the hormone and the cytoplasmic receptors. αE2 accumulates between the plasma membrane lipid bilayer with increasing potential, and when the critical level is reached, αE2 penetrates through the inner layer of the plasma membrane into the cytoplasm. It then rapidly reacts with the ER and creates an unstable adduct. The revealed dynamics of αE2–ER action may contribute to understanding tissue rejuvenation and the cancer-related physiology of αE2 signaling.

Cytoskeletal Filaments Deep Inside a Neuron Are Not Silent: They Regulate the Precise Timing of Nerve Spikes Using a Pair of Vortices

Hodgkin and Huxley showed that even if the filaments are dissolved, a neuron’s membrane alone can generate and transmit the nerve spike. Regulating the time gap between spikes is key to the brain’s cognitive function; however, the time modulation mechanism is still a mystery. By inserting a coaxial probe deep inside a neuron, we repeatedly show that the filaments transmit electromagnetic signals of ~200 μs before an ionic nerve spike sets in. To understand its origin, here, we mapped the electromagnetic vortex produced by a filamentary bundle deep inside a neuron, regulating the nerve spike’s electrical-ionic vortex. We used monochromatic polarized light to measure the transmitted signals beating from the internal components of a cultured neuron. A nerve spike is a 3D ring of the electric field encompassing the perimeter of a neural branch. Several such vortices flow sequentially to keep precise timing for the brain’s cognition. The filaments hold millisecond order time gaps between membrane spikes with microsecond order signaling of electromagnetic vortices. Dielectric resonance images revealed that ordered filaments inside neural branches instruct the ordered grid-like network of actin–beta-spectrin just below the membrane. That layer builds a pair of electric field vortices, which coherently activates all ion-channels in a circular area of the membrane lipid bilayer when a nerve spike propagates. When biomaterials vibrate resonantly with microwave and radio-wave, simultaneous quantum optics capture ultra-fast events in a non-demolition mode, revealing multiple correlated time-domain operations beyond the Hodgkin–Huxley paradigm. Neuron holograms pave the way to understanding the filamentary circuits of a neural network in addition to membrane circuits.

Pore-forming toxins in infection and immunity

The integrity of the plasma membranes is extremely crucial for the survival and proper functioning of the cells. Organisms from all kingdoms of life employ specialized pore-forming proteins and toxins (PFPs and PFTs) that perforate cell membranes, and cause detrimental effects. PFPs/PFTs exert their damaging actions by forming oligomeric pores in the membrane lipid bilayer. PFPs/PFTs play important roles in diverse biological processes. Many pathogenic bacteria secrete PFTs for executing their virulence mechanisms. The immune system of the higher vertebrates employs PFPs to kill pathogen-infected cells and transformed cancer cells. The most obvious consequence of membrane pore-formation by the PFPs/PFTs is the killing of the target cells due to the disruption of the permeability barrier function of the plasma membranes. PFPs/PFTs can also activate diverse cellular processes that include activation of the stress-response pathways, induction of programmed cell death, and inflammation. Upon attack by the PFTs, host cells may also activate pathways to repair the injured membranes, restore cellular homeostasis, and trigger inflammatory immune responses. In this article, we present an overview of the diverse cellular responses that are triggered by the PFPs/PFTs, and their implications in the process of pathogen infection and immunity.

pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery

Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new range of applications in image-guided surgery. In this paper, we report a translational study of a new fluorescent agent for use in surgery, pHLIP ICG, where ICG (indocyanine green) is a surgical fluorescent dye used widely for imaging blood flow. We studied pHLIP ICG interaction with the cell membrane lipid bilayer, the pharmacology and toxicology in vitro and in vivo (mice and dogs), and the biodistribution and clearance of pHLIP ICG in mice. The pHLIP ICG tumor targeting and imaging efficacy studies were carried out in several murine and human mouse tumor models. Blood vessels were imaged in mice and pigs. Clinical Stryker imaging instruments for endoscopy and open surgery were used in the study. Intravenously administered pHLIP ICG exhibits a multi-hour circulation half-life, offering protracted delineation of vasculature. As it clears from the blood, pHLIP ICG targets tumors and tumor stroma, marking them for surgical removal. pHLIP ICG is non-toxic, marks blood flow for hours after injection, and effectively delineates tumors for improved resection on the day after administration.

Exosomes: Insights from Retinoblastoma and Other Eye Cancers

Exosomes, considered as cell debris or garbage bags, have been later characterized as nanometer-sized extracellular double-membrane lipid bilayer bio-vesicles secreted by the fusion of vesicular bodies with the plasma membrane. The constituents and the rate of exosomes formation differ in different pathophysiological conditions. Exosomes are also observed and studied in different parts of the eye, like the retina, cornea, aqueous, and vitreous humor. Tear fluid consists of exosomes that are shown to regulate various cellular processes. The role of exosomes in eye cancers, especially retinoblastoma (RB), is not well explored, although few studies point towards their presence. Retinoblastoma is an intraocular tumor that constitutes 3% of cases of cancer in children. Diagnosis of RB may require invasive procedures, which might lead to the spread of the disease to other parts. Due to this reason, better ways of diagnosis are being explored. Studies on the exosomes in RB tumors and serum might help designing better diagnostic approaches for RB. In this article, we reviewed studies on exosomes in the eye, with a special emphasis on RB. We also reviewed miRNAs expressed in RB tumor, serum, and cell lines and analyzed the targets of these miRNAs from the proteins identified in the RB tumor exosomes. hsa-miR-494 and hsa-miR-9, upregulated and downregulated, respectively in RB, have the maximum number of targets. Although oppositely regulated, they share the same targets in the proteins identified in RB tumor exosomes. Overall this review provides the up-to-date progress in the area of eye exosome research, with an emphasis on RB.

The role of sterols in plant response to abiotic stress

Sterols are integral components of the membrane lipid bilayer and they are involved in many processes occurring in plants, ranging from regulation of growth and development to stress resistance. Maintenance of membrane homeostasis represents one of the principal functions of sterols in plant cells. Plant cell membranes are important sites of perception of environmental abiotic factors, therefore, it can be surmised that sterols may play an important role in the plant stress response. The aim of this review was to discuss the most representative trends in recent studies regarding the role of sterols in plant defense reactions to environmental factors, such as UV radiation, cold and drought stress. Some correlations were observed between changes in the sterol profile, referring to the ratios of individual compounds (including 24-methyl/ethyl sterols and sitosterol/stigmasterol) as well as the relative proportions of conjugated sterols (ASGs, SGs and SEs) and the nature of the stress response. Diversity of sterols and their conjugated forms may allow sessile plants to adapt to environmental stress conditions.