Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

Transmembrane transport in inorganic colloidal cell-mimics.

A key aspect of living cells is their ability to harvest energy from the environment and use it to pump specific atomic and molecular species in and out of their system-typically against an unfavourable concentration gradient1. Active transport allows cells to store metabolic energy, extract waste and supply organelles with basic building blocks at the submicrometre scale. Unlike living cells, abiotic systems do not have the delicate biochemical machinery that can be specifically activated to precisely control biological matter2-5. Here we report the creation of microcapsules that can be brought out of equilibrium by simple global variables (illumination and pH), to capture, concentrate, store and deliver generic microscopic payloads. Borrowing no materials from biology, our design uses hollow colloids serving as spherical cell-membrane mimics, with a well-defined single micropore. Precisely tunable monodisperse capsules are the result of a synthetic self-inflation mechanism and can be produced in bulk quantities. Inside the hollow unit, a photoswitchable catalyst6 produces a chemical gradient that propagates to the exterior through the membrane's micropore and pumps target objects into the cell, acting as a phoretic tractor beam7. An entropic energy barrier8,9 brought about by the micropore's geometry retains the cargo even when the catalyst is switched off. Delivery is accomplished on demand by reversing the sign of the phoretic interaction. Our findings provide a blueprint for developing the next generation of smart materials, autonomous micromachinery and artificial cell-mimics.

Modulation of α1ß3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix.

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a ß-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABAA α1ß3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the ß+α- interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in ß3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [ß3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1ß3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the ß+α- interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to ß3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the ß+α- interface for the anesthetic propofol.

Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites.

Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-ß, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-ß). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-ß plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

Live-cell lipid biochemistry reveals a role of diacylglycerol side-chain composition for cellular lipid dynamics and protein affinities.

Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol-protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.

A tryptophan synchronous and normal fluorescence study on bacteria inactivation mechanism.

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

Antibacterial Activity of Caffeic Acid Combined with UV-A Light against Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes.

The aim of this study was to evaluate the antibacterial activity of caffeic acid (CA), which is a natural polyphenol, combined with UV-A light against the representative foodborne bacteria Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Data regarding the inactivation of these bacteria and its dependence on CA concentration, light wavelength, and light dose were obtained. E. coli O157:H7 and Salmonella Typhimurium were reduced to the detection limit when treated with 3 mM CA and UV-A for 3 J/cm2 and 4 J/cm2, respectively, and 5 J/cm2 treatment induced 3.10 log reduction in L. monocytogenes. To investigate the mechanism for inactivation of Salmonella Typhimurium and L. monocytogenes, measurement of polyphenol uptake, membrane damage assessment, enzymatic activity assay, and transmission electron microscopy (TEM) were conducted. It was revealed that CA was significantly (P < 0.05) absorbed by bacterial cells, and UV-A light allowed a higher uptake of CA for both pathogens. Additionally, CA plus UV-A treatment induced significant (P < 0.05) cell membrane damage. In the enzymatic activity assay, the activities of both pathogens were reduced by CA, and a greater reduction occurred by use of CA plus UV-A. Moreover, transmission electron microscopy (TEM) images indicated that CA plus UV-A treatment notably destroyed the intercellular structure. In addition, antibacterial activity was also observed in commercial apple juice, which showed results similar to those obtained from phosphate-buffered saline (PBS), resulting in a significant (P < 0.05) reduction for all three pathogens without any changes in color parameters (L*, a*, and b*), total phenolic compounds, and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity. IMPORTANCE Photodynamic inactivation (PDI), which involves photoactivation of a photosensitizer (PS), is an emerging field of study, as it effectively reduces various kinds of microorganisms. Although there are several PSs that have been used for PDI, there is a need to find naturally occurring PSs for safer application in the food industry. Caffeic acid, a natural polyphenol found in most fruits and vegetables, has recently been studied for its potential to act as a novel photosensitizer. However, no studies have been conducted regarding its antibacterial activity depending on treatment conditions and its antibacterial mechanism. In this study, we closely examined the effectiveness of caffeic acid in combination with UV-A light for inactivating representative foodborne bacteria in liquid medium. Therefore, the results of this research are expected to be utilized as basic data for future application of caffeic acid in PDI, especially when controlling pathogens in liquid food processing.

Cellular compartments challenged by membrane photo-oxidation.

The lipid composition impacts directly on the structure and function of the cytoplasmic as well as organelle membranes. Depending on the type of membrane, specific lipids are required to accommodate, intercalate, or pack membrane proteins to the proper functioning of the cells/organelles. Rather than being only a physical barrier that separates the inner from the outer spaces, membranes are responsible for many biochemical events such as cell-to-cell communication, protein-lipid interaction, intracellular signaling, and energy storage. Photochemical reactions occur naturally in many biological membranes and are responsible for diverse processes such as photosynthesis and vision/phototaxis. However, excessive exposure to light in the presence of absorbing molecules produces excited states and other oxidant species that may cause cell aging/death, mutations and innumerable diseases including cancer. At the same time, targeting key compartments of diseased cells with light can be a promising strategy to treat many diseases in a clinical procedure called Photodynamic Therapy. Here we analyze the relationships between membrane alterations induced by photo-oxidation and the biochemical responses in mammalian cells. We specifically address the impact of photosensitization reactions in membranes of different organelles such as mitochondria, lysosome, endoplasmic reticulum, and plasma membrane, and the subsequent responses of eukaryotic cells.

Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells.

The purpose of this study was to verify whether the nanosecond pulsed electric field, not eliciting thermal effects, permanently changes the molecular processes and gene expression of Leydig TM3 cells. The cells were exposed to a moderate electric field (80 quasi-rectangular shape pulses, 60 ns pulse width, and an electric field of 14 kV/cm). The putative disturbances were recorded over 24 h. After exposure to the nanosecond pulsed electric field, a 19% increase in cell diameter, a loss of microvilli, and a 70% reduction in cell adhesion were observed. Some cells showed the nonapoptotic externalization of phosphatidylserine through the pores in the plasma membrane. The cell proportion in the subG1 phase increased by 8% at the expense of the S and G2/M phases, and the DNA was fragmented in a small proportion of the cells. The membrane mitochondrial potential and superoxide content decreased by 37% and 23%, respectively. Microarray's transcriptome analysis demonstrated a negative transient effect on the expression of genes involved in oxidative phosphorylation, DNA repair, cell proliferation, and the overexpression of plasma membrane proteins. We conclude that nanosecond pulsed electric field affected the physiology and gene expression of TM3 cells transiently, with a noticeable heterogeneity of cellular responses.

Expression Level of Membrane Markers CD5, CD19, and CD20 in B Cells after UV-Irradiation and Incubation in the Presence of Autologous Plasma.

The effect of UV-light (240-390 nm) in doses of 151 and 755 J/m2 on the expression of membrane markers CD5, CD19, CD20 in human peripheral blood B cells was studied by flow cytometry. In 24 h after exposure to UV light, we observed activation of processes accompanied by structural rearrangements of B-cell membranes leading to changes in the expression of receptor molecules: the content of of CD19 and CD20 increased due to activation of the synthesis of these proteins, while the content of CD5 decreased. The percentage of CD5+ cells decreased over 24 h after UV-irradiation of lymphocytes, while addition of autologous plasma to the incubation medium produced a photoprotective effect on CD5+ cells.

Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles.

Light-driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS-g-PEO and diblock copolymer PBd-PEO. Activity of more than 90 % in lipid/polymer-based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long-term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer-based hybrids.

Synergistic bactericidal effect and mechanism of X-ray irradiation and citric acid combination against food-borne pathogens on spinach leaves.

In this study, we investigated the antimicrobial activity of the X-ray irradiation and citric acid (CA) combination against Escherichia coli O157:H7 and Listeria monocytogenes on the surface of spinach leaves and elucidated the mechanisms underlying their synergistic interaction. Upon treatment with 0.3 kGy X-ray irradiation and 1% CA combination, the cell counts of E. coli O157:H7 and L. monocytogenes reduced by 4.23 and 3.69 log CFU/mL on spinach leaves, respectively. The synergistic reduction in the cell counts of E. coli O157:H7 and L. monocytogenes by the combination treatment was 0.95 and 1.14 log units, respectively. The X-ray and CA combination exerts its antimicrobial effect by damaging the bacterial cell membrane and enhancing the generation of intracellular reactive oxygen species in the pathogens. The enhanced bactericidal effect of the combination treatment may not be due to the loss of intracellular enzyme activity. We also evaluated the effect of the combination treatment on the quality attributes of spinach leaves. The combination treatment did not result in adverse changes in color and texture of spinach leaves. These results demonstrate the potential of citric acid and X-ray irradiation combination for decontaminating foodborne pathogens on fresh produce.

Cell Membrane Nanostructure is Altered by Heat-Induced Antigen Retrieval: A Possible Consequence for Immunocytochemical Detection of Membranous Antigens.

Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.

Single-Molecule Detection with Lightguiding Nanowires: Determination of Protein Concentration and Diffusivity in Supported Lipid Bilayers.

Determining the surface concentration and diffusivity of cell-membrane-bound molecules is central to the understanding of numerous important biochemical processes taking place at cell membranes. Here we use the high aspect ratio and lightguiding properties of semiconductor nanowires (NWs) to detect the presence of single freely diffusing proteins bound to a lipid bilayer covering the NW surface. Simultaneous observation of light-emission dynamics of hundreds of individual NWs occurring on the time scale of only a few seconds is interpreted using analytical models and employed to determine both surface concentration and diffusivity of cholera toxin subunit B (CTxB) bound to GM1 gangliosides in supported lipid bilayer (SLB) at surface concentrations down to below one CTxB per µm2. In particular, a decrease in diffusivity was observed with increasing GM1 content in the SLB, suggesting increasing multivalent binding of CTxB to GM1. The lightguiding capability of the NWs makes the method compatible with conventional epifluorescence microscopy, and it is shown to work well for both photostable and photosensitive dyes. These features make the concept an interesting complement to existing techniques for studying the diffusivity of low-abundance cell-membrane-bound molecules, expanding the rapidly growing use of semiconductor NWs in various bioanalytical sensor applications and live cell studies.

Efficient Photodynamic Killing of Gram-Positive Bacteria by Synthetic Curcuminoids.

In our previous study, we have demonstrated that curcumin can efficiently kill the anaerobic bacterium Propionibacterium acnes by irradiation with low-dose blue light. The curcuminoids present in natural plant turmeric mainly include curcumin, demethoxycurcumin, and bisdemethoxycurcumin. However, only curcumin is commercially available. Eighteen different curcumin analogs, including demethoxycurcumin and bisdemethoxycurcumin, were synthesized in this study. Their antibacterial activity against Gram-positive aerobic bacteria Staphylococcus aureus and Staphylococcus epidermidis was investigated using the photodynamic inactivation method. Among the three compounds in turmeric, curcumin activity is the weakest, and bisdemethoxycurcumin possesses the strongest activity. However, two synthetic compounds, (1E,6E)-1,7-bis(5-methylthiophen-2-yl)hepta-1,6-diene-3,5-dione and (1E,6E)-1,7-di(thiophen-2-yl)hepta-1,6-diene-3,5-dione, possess the best antibacterial activity among all compounds examined in this study. Their chemical stability is also better than that of bisdemethoxycurcumin, and thus has potential for future clinical applications.

Surface Functionalization with Polyethylene Glycol and Polyethyleneimine Improves the Performance of Graphene-Based Materials for Safe and Efficient Intracellular Delivery by Laser-Induced Photoporation.

Nanoparticle mediated laser-induced photoporation is a physical cell membrane disruption approach to directly deliver extrinsic molecules into living cells, which is particularly promising in applications for both adherent and suspension cells. In this work, we explored surface modifications of graphene quantum dots (GQD) and reduced graphene oxide (rGO) with polyethylene glycol (PEG) and polyethyleneimine (PEI) to enhance colloidal stability while retaining photoporation functionality. After photoporation with FITC-dextran 10 kDa (FD10), the percentage of positive HeLa cells (81% for GQD-PEG, 74% for rGO-PEG and 90% for rGO-PEI) increased approximately two-fold compared to the bare nanomaterials. While for Jurkat suspension cells, the photoporation efficiency with polymer-modified graphene-based nanomaterial reached as high as 80%. Cell viability was >80% in all these cases. In addition, polymer functionalization proved to be beneficial for the delivery of larger macromolecules (FD70 and FD500) as well. Finally, we show that rGO is suitable for photoporation using a near-infrared laser to reach 80% FD10 positive HeLa cells at 80% cell viability. We conclude that modification of graphene-based nanoparticles with PEG and especially PEI provide better colloidal stability in cell medium, resulting in more uniform transfection and overall increased efficiency.

The Lorentz force on ions in membrane channels of neurons as a mechanism for transcranial static magnetic stimulation.

Transcranial static magnetic stimulation is a novel noninvasive method of reduction of the cortical excitability in certain neurological diseases that makes use of static magnetic fields generated by permanent magnets. By contrast, ordinary transcranial magnetic stimulation makes use of pulsed magnetic fields generated by strong currents. Whereas the physical principle underlying ordinary transcranial magnetic stimulation is well known, that is, the Faraday´s law, the physical mechanism that explains the interaction between neurons and static magnetic fields in transcranial static magnetic stimulation remains unclear. In the present work, it is discussed the possibility that this mechanism might be the Lorentz force exerted on the ions flowing along the membrane channels of neurons. The overall effect of the static magnetic field would be to introduce an additional friction between the ions and the walls of the membrane channels, thus reducing its conductance. Calculations performed by using a Hodgkin-Huxley model demonstrate that even a slight reduction of the conductance of the membrane channels can lead to the suppression of the action potential, thus inhibiting neuronal activity.

Photoswitchable Conjugated Oligoelectrolytes for a Light-Induced Change of Membrane Morphology.

The synthesis of a new conjugated oligoelectrolyte (COE), namely DSAzB, is described, which contains a conjugated core bearing a diazene moiety in the center of its electronically delocalized structure. Similar to structurally related phenylenevinylene-based COEs, DSAzB readily intercalates into model and natural lipid bilayer membranes. Photoinduced isomerization transforms the linear trans COE into a bent or C-shape form. It is thereby possible to introduce DSAzB into the bilayer of a cell and disrupt its integrity by irradiation with light. This leads to controlled permeabilization of membranes, as demonstrated by the release of calcein from DMPG/DMPC vesicles and by propidium iodide influx experiments on S. epidermidis. Both experiments support that the permeabilization is selective for the light stimulus, highly efficient, and repeatable. Target-selective and photoinduced actions demonstrated by DSAzB may have broad applications in biocatalysis and related biotechnologies.

Single-Cell Bacterial Electrophysiology Reveals Mechanisms of Stress-Induced Damage.

An electrochemical gradient of protons, or proton motive force (PMF), is at the basis of bacterial energetics. It powers vital cellular processes and defines the physiological state of the cell. Here, we use an electric circuit analogy of an Escherichia coli cell to mathematically describe the relationship between bacterial PMF, electric properties of the cell membrane, and catabolism. We combine the analogy with the use of bacterial flagellar motor as a single-cell "voltmeter" to measure cellular PMF in varied and dynamic external environments (for example, under different stresses). We find that butanol acts as an ionophore and functionally characterize membrane damage caused by the light of shorter wavelengths. Our approach coalesces noninvasive and fast single-cell voltmeter with a well-defined mathematical framework to enable quantitative bacterial electrophysiology.

Inactivation of Bacteria by γ-Irradiation to Investigate the Interaction with Antimicrobial Peptides.

The activity of antimicrobial peptides (AMPs) has been investigated extensively using model membranes composed of phospholipids or lipopolysaccharides in aqueous environments. However, from a biophysical perspective, there is a large scientific interest regarding the direct interaction of membrane-active peptides with whole bacteria. Working with living bacteria limits the usability of experimental setups and the interpretation of the resulting data because of safety risks and the overlap of active and passive effects induced by AMPs. We killed or inactivated metabolic-active bacteria using γ-irradiation or sodium azide, respectively. Microscopy, flow cytometry, and SYTOX green assays showed that the cell envelope remained intact to a high degree at the minimal bactericidal dose. Furthermore, the tumor-necrosis-factor-α-inducing activity of the lipopolysaccharides and the chemical lipid composition was unchanged. Determining the binding capacity of AMPs to the bacterial cell envelope by calorimetry is difficult because of an overlapping of the binding heat and metabolic activities of the bacteria-induced by the AMPs. The inactivation of all active processes helps to decipher the complex thermodynamic information. From the isothermal titration calorimetry (ITC) results, we propose that the bacterial membrane potential (Δψ) is possibly an underestimated modulator of the AMP activity. The negative surface charge of the outer leaflet of the outer membrane of Gram-negative bacteria is already neutralized by peptide concentrations below the minimal inhibitory concentration. This proves that peptide aggregation on the bacterial membrane surface plays a decisive role in the degree of antimicrobial activity. This will not only enable many biophysical approaches for the investigation between bacteria and membrane-active peptides in the future but will also make it possible to compare biophysical parameters of active and inactive bacteria. This opens up new possibilities to better understand the active and passive interaction processes between AMPs and bacteria.