Unraveling Antimicrobial Susceptibility of Bacterial Networks on Micropillar Architectures Using Intrinsic Phase-Shift Spectroscopy.

With global antimicrobial resistance becoming increasingly detrimental to society, improving current clinical antimicrobial susceptibility testing (AST) is crucial to allow physicians to initiate appropriate antibiotic treatment as early as possible, reducing not only mortality rates but also the emergence of resistant pathogens. In this work, we tackle the main bottlenecks in clinical AST by designing biofunctionalized silicon micropillar arrays to provide both a preferable solid-liquid interface for bacteria networking and a simultaneous transducing element that monitors the response of bacteria when exposed to chosen antibiotics in real time. We harness the intrinsic ability of the micropillar architectures to relay optical phase-shift reflectometric interference spectroscopic measurements (referred to as PRISM) and employ it as a platform for culture-free, label-free phenotypic AST. The responses of E. coli to various concentrations of five clinically relevant antibiotics are optically tracked by PRISM, allowing for the minimum inhibitory concentration (MIC) values to be determined and compared to both standard broth microdilution testing and clinic-based automated AST system readouts. Capture of bacteria within these microtopologies, followed by incubation of the cells with the appropriate antibiotic solution, yields rapid determinations of antibiotic susceptibility. This platform not only provides accurate MIC determinations in a rapid manner (total assay time of 2-3 h versus 8 h with automated AST systems) but can also be employed as an advantageous method to differentiate bacteriostatic and bactericidal antibiotics.

Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light.

The online monitoring of recombinant protein aggregate inclusion bodies during microbial cultivation is an immense challenge. Measurement of scattered and reflected light offers a versatile and non-invasive measurement technique. Therefore, we investigated two methods to detect the formation of inclusion bodies and monitor their production: (1) online 180° scattered light measurement (λ = 625 nm) using a sensor platform during cultivation in shake flask and (2) online measurement of the light reflective interference using a porous Si-based optical biosensor (SiPA). It could be shown that 180° scattered light measurement allows monitoring of alterations in the optical properties of Escherichia coli BL21 cells, associated with the formation of inclusion bodies during cultivation. A reproducible linear correlation between the inclusion body concentration of the non-fluorescent protein human leukemia inhibitory factor (hLIF) carrying a thioredoxin tag and the shift ("Δamp") in scattered light signal intensity was observed. This was also observed for the glutathione-S-transferase-tagged green fluorescent protein (GFP-GST). Continuous online monitoring of reflective interference spectra reveals a significant increase in the bacterium refractive index during hLIF production in comparison to a non-induced reference that coincide with the formation of inclusion bodies. These online monitoring techniques could be applied for fast and cost-effective screening of different protein expression systems.

Tethered Lipid Bilayers within Porous Si Nanostructures: A Platform for (Optical) Real-Time Monitoring of Membrane-Associated Processes.

The importance of cell membranes in biological systems has prompted the development of artificial lipid bilayers, which can mimic the cellular membrane structure. Supported lipid bilayers (SLBs) have emerged as a promising avenue for studying basic membrane processes and for possible biotechnological applications. Conventional methods for SLB formation involve the spreading of lipid vesicles on hydrophilic solid supports. Herein, a facile approach for the construction of tethered SLB within an oxidized porous Si (pSiO2) nanostructure, avoiding liposome preparation, is presented. We employ a two-step lipid self-assembly process, in which a first lipid layer is tethered to the pore walls resulting in a highly stable monolayer. A subsequent solvent exchange step induces the self-assembly of the unbound lipids into a robust SLB. Formation of pSiO2-SLB is confirmed by fluorescence resonance energy transfer (FRET), and the properties of the confined SLB are characterized by environment-sensitive fluorophores. The unique optical properties of the pSiO2 support are employed to monitor in real time the partitioning of a model amphiphilic molecule within the SLB via reflective interferometric Fourier transform spectroscopy (RIFTS) method. These self-reporting SLB platforms provide a highly generic approach for bottom-up construction of complex lipid architectures for performing biological assays at the micro- and nanoscale.

The uptake of HIV Tat peptide proceeds via two pathways which differ from macropinocytosis.

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic molecules, yet the identity of their uptake routes remained unclear and is still under debate. In this study we provide new insights into CPP entry routes by quantitatively measuring the intracellular uptake of FAM-labeled Tat-peptide under rigorous kinetic and thermal conditions. The uptake of Tat-peptide between 4 and 15°C corresponds to Q10=1.1, proceeding through a prompt (<5 min), temperature-independent process, suggesting direct membrane translocation. At longer durations, Tat rate of uptake shows linear dependence on temperature with Q10=1.44, accompanied by activation energy Ea=4.45 Kcal/mole. These values are significantly lower than those we found for the macropinocytosis probe dextran (Q10=2.2 and Ea=7.2 Kcal/mole) which possesses an exponential dependence on temperature, characteristic of endocytosis processes. Tat-peptide and dextran do not interfere with each other's uptake rate and the ratio of Tat-peptide uptake to its extracellular concentration is ~15 times higher than that for dextran. In addition, Phloretin, a modulator of cell membrane dipole potential, is shown to increase dextran uptake but to reduce that of Tat. We conclude that the uptake of Tat differs from that of dextran in all parameters. Tat uptake proceeds by dual entry routes which differ by their energy dependence.

Trap and track: designing self-reporting porous Si photonic crystals for rapid bacteria detection.

The task of rapid detection and identification of bacteria remains a major challenge in both medicine and industry. This work introduces a new concept for the design of self-reporting optical structures that can detect and quantify bacteria in real-time. The sensor is based on a two-dimensional periodic structure of porous Si photonic crystals in which the pore size is adjusted to fit the target bacteria cells (Escherichia coli). Spontaneous bacteria capture within the pores induces measurable changes in the zero-order reflectivity spectrum collected from the periodic structure. Confocal laser microscopy and electron microscopy confirm that the Escherichia coli cells are individually imprisoned within the porous array. A simple model is suggested to correlate the optical readout and the bacteria concentration and its predictions are found to be in good agreement with experimental results. In addition, we demonstrate that sensing scheme can be easily modified to potentially allow monitoring of concentration, growth and physiological state of bacteria cells. This generic platform can be tailored to target different microorganisms by tuning the array periodicity and its surface chemistry for rapid and label-free detection outside the laboratory environment.

Proton-induced endocytosis is dependent on cell membrane fluidity, lipid-phase order and the membrane resting potential.

Recently it has been shown that decreasing the extracellular pH of cells stimulates the formation of inward membrane invaginations and vesicles, accompanied by an enhanced uptake of macromolecules. This type of endocytosis was coined as proton-induced uptake (PIU). Though the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the dependence of the phenomenon on plasma membrane characteristics is still unknown. The present study shows that depolarization of the membrane resting potential elevates PIU by 25%, while hyperpolarization attenuates it by 25%. Comparison of uptake in suspended and adherent cells implicates that the resting-potential affects PIU through remodeling the actin-cytoskeleton. The pH at the external interface of the cell membrane rather than the pH gradient across it determines the extent of PIU. PIU increases linearly upon temperature increase in the range of 4-36°C, in correlation with the membrane fluidity. The plasma membrane fluidity and the lipid phase order are modulated by enriching the cell's membrane with cholesterol, tergitol, dimethylsulfoxide, 6-ketocholestanol and phloretin and by cholesterol depletion. These treatments are shown to alter the extent of PIU and are better correlated with membrane fluidity than with the lipid phase order. We suggest that the lipid phase order and fluidity influence PIU by regulating the lipid order gradient across the perimeter of the lipid-condensed microdomains (rafts) and alter the characteristic tension line that separates the higher ordered lipid-domains from the lesser ordered ones.

Low electric fields induce ligand-independent activation of EGF receptor and ERK via electrochemical elevation of H(+) and ROS concentrations.

Physiological electric fields are involved in many biological processes and known to elicit their effects during long exposures ranging from a few hours to days. Following exposure to electric fields of physiological amplitude, epidermal growth factor receptor (EGFR) was demonstrated to be redistributed and upregulated with further intracellular signaling such as the MAPK signaling cascade. In our study we demonstrated EGFR activation and signaling induced by short train of pulsed low electric field (LEF) (10V/cm, pulse-width 180µs, 500Hz, 2min) in serum-free medium, following 24-hour starvation, and in the absence of exogenous EGF ligand, suggesting a ligand-independent pathway for EGFR activation. This ligandless activation was further confirmed by using neutralizing antibodies (LA1) that block the EGFR ligand-binding site. EGFR activation was found to be EGFR kinase dependent, yet with no dimerization following exposure to LEF. ERK activation was found to be mainly a result of EGFR downstream signaling though it partially occurred via EGFR-independent way. We demonstrate that reactive oxygen species and especially decrease in pH generated during exposure to LEF are involved in EGFR ligandless activation. We propose a possible mechanism for the LEF-induced EGFR ligand-independent activation and show activation of other receptor tyrosine kinases following exposure to LEF.

Actin-cytoskeleton rearrangement modulates proton-induced uptake.

Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process.

Electroendocytosis is driven by the binding of electrochemically produced protons to the cell's surface.

Electroendocytosis involves the exposure of cells to pulsed low electric field and is emerging as a complementary method to electroporation for the incorporation of macromolecules into cells. The present study explores the underlying mechanism of electroendocytosis and its dependence on electrochemical byproducts formed at the electrode interface. Cell suspensions were exposed to pulsed low electric field in a partitioned device where cells are spatially restricted relative to the electrodes. The cellular uptake of dextran-FITC was analyzed by flow cytometery and visualized by confocal microscopy. We first show that uptake occurs only in cells adjacent to the anode. The enhanced uptake near the anode is found to depend on electric current density rather than on electric field strength, in the range of 5 to 65 V/cm. Electrochemically produced oxidative species that impose intracellular oxidative stress, do not play any role in the stimulated uptake. An inverse dependence is found between electrically induced uptake and the solution's buffer capacity. Electroendocytosis can be mimicked by chemically acidifying the extracellular solution which promotes the enhanced uptake of dextran polymers and the uptake of plasmid DNA. Electrochemical production of protons at the anode interface is responsible for inducing uptake of macromolecules into cells exposed to a pulsed low electric field. Expanding the understanding of the mechanism involved in electric fields induced drug-delivery into cells, is expected to contribute to clinical therapy applications in the future.

Enhancement of cell membrane invaginations, vesiculation and uptake of macromolecules by protonation of the cell surface.

The different pathways of endocytosis share an initial step involving local inward curvature of the cell's lipid bilayer. It has been shown that to generate membrane curvature, proteins or lipids enforce transversal asymmetry of the plasma membrane. Thus it emerges as a general phenomenon that transversal membrane asymmetry is the common required element for the formation of membrane curvature. The present study demonstrates that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesiculation accompanied by efficient uptake of macromolecules (Dextran-FITC, 70 kD), relative to the constitutive one. The insensitivity of proton induced uptake to inhibiting treatments and agents of the known endocytic pathways suggests the entry of macromolecules to proceeds via a yet undefined route. This is in line with the fact that neither ATP depletion, nor the lowering of temperature, abolishes the uptake process. In addition, fusion mechanism such as associated with low pH uptake of toxins and viral proteins can be disregarded by employing the polysaccharide dextran as the uptake molecule. The proton induced uptake increases linearly in the extracellular pH range of 6.5 to 4.5, and possesses a steep increase at the range of 4> pH>3, reaching a plateau at pH ≤ 3. The kinetics of the uptake implies that the induced vesicles release their content to the cytosol and undergo rapid recycling to the plasma membrane. We suggest that protonation of the cell's surface induces local charge asymmetries across the cell membrane bilayer, inducing inward curvature of the cell membrane and consequent vesiculation and uptake.

Cell-based detection of electrochemical oxidative stress by a fluorescent tryptophan intermediate.

The present study suggests a sensitive and rapid cell analysis method to evaluate the oxidative stress produced in a physiological culture medium, by anodic electrochemical products. The detection of these oxidizing agents, probably involving hypochlorite, is carried out by measuring the presence of an oxidized tryptophan intermediate, entrapped and stabilized in the cell cytoplasm. The formation of this tryptophan intermediate depends solely on the presence of a free tryptophan in the extracellular medium near the anode. This intermediate possesses a characteristic emission maximum at λ~560 nm, which can be abolished by the presence of anti-oxidants in the media during the cells' exposure to electric current. However, this intermediate's emission is unaffected by increased concentrations of intracellular anti-oxidants. This suggests that the anodic produced unstable tryptophan intermediate permeates the cell plasma membrane and becomes stabilized by cytoplasmic proteins. Tryptophan oxidative intermediates with similar spectra could also be formed by the chemical reaction of hypochlorite with tryptophan in solution. The analysis of the intracellularly stabilized tryptophan intermediate by flow cytometry can be used for measuring external oxidation stress without the disturbance of intracellular anti-oxidative capacity.

Primary myogenic cells see the light: improved survival of transplanted myogenic cells following low energy laser irradiation.

BACKGROUND AND OBJECTIVES: There is a substantial need for finding new avenues to promote muscle recovery when acute skeletal muscle loss extends beyond the natural capacity of the muscle to recover. Maintenance and regeneration of skeletal muscles depend mainly on resident stem cells known as satellite cells. Nevertheless, there are situations in which a significant loss of muscle tissue exhausts the satellite cell pool. For such cases, cell therapy and tissue engineering are becoming promising alternatives. Thus far, attempts to supplement damaged host muscles with donor satellite cells by means of myoblast transplantation therapy were mostly unsuccessful due to massive and rapid loss of donor cells within few hours after transplantation. This study aims at following the effects of low-energy-laser irradiation on the fate of implanted myoblasts. STUDY DESIGN: Primary myogenic cells, harvested from male rat skeletal muscles, were irradiated with low energy laser, seeded on a biodegradable scaffold and expanded in vitro. The scaffold containing cells was transplanted into partially excised muscles of host female rats. Donor cells were identified in the host muscle tissue, using Y-chromosome in situ hybridization. RESULTS: In this study, we show that laser irradiated donor primary myogenic cells not only survive, but also fuse with host myoblasts to form a host-donor syncytium. CONCLUSIONS: Our data show that the use of low energy laser irradiation (LELI), a non-surgical tool, is a promising means to enhance both the survival and functionality of transplanted primary myogenic cells.