Interfacing poly(p-anisidine) with photosystem I for the fabrication of photoactive composite films.

Photosystem I (PSI) is an intrinsically photoactive multi-subunit protein that is found in higher order photosynthetic organisms. PSI is a promising candidate for renewable biohybrid energy applications due to its abundance in nature and its high quantum yield. To utilize PSI's light-responsive properties and to overcome its innate electrically insulating nature, the protein can be paired with a biologically compatible conducting polymer that carries charge at appropriate energy levels, allowing excited PSI electrons to travel within a composite network upon light excitation. Here, a substituted aniline, 4-methoxy-aniline (para-anisidine), is chemically oxidized to synthesize poly(p-anisidine) (PPA) and is interfaced with PSI for the fabrication of PSI-PPA composite films by drop casting. The resulting PPA polymer is characterized in terms of its structure, composition, thermal decomposition, spectroscopic response, morphology, and conductivity. Combining PPA with PSI yields composite films that exhibit photocurrent densities on the order of several µA cm-2 when tested with appropriate mediators in a 3-electrode setup. The composite films also display increased photocurrent output when compared to single-component films of the protein or PPA alone to reveal a synergistic combination of the film components. Tuning film thickness and PSI loading within the PSI-PPA films yields optimal photocurrents for the described system, with ∼2 wt% PSI and intermediate film thicknesses generating the highest photocurrents. More broadly, dilute PSI concentrations show significant importance in achieving high photocurrents in PSI-polymer films.

Cultivation, chemistry, and genome of Psilocybe zapotecorum.

Psilocybe zapotecorum is a strongly blue-bruising psilocybin mushroom used by indigenous groups in southeastern Mexico and beyond. While this species has a rich history of ceremonial use, research into its chemistry and genetics have been limited. Herein, we detail mushroom morphology and report on cultivation parameters, chemical profile, and the full genome sequence of P. zapotecorum . First, growth and cloning methods are detailed that are simple, and reproducible. In combination with high resolution microscopic analysis, the strain was barcoded, confirming species-level identification. Full genome sequencing reveals the architecture of the psilocybin gene cluster in P. zapotecorum, and can serve as a reference genome for Psilocybe Clade I. Characterization of the tryptamine profile revealed a psilocybin concentration of 17.9±1.7 mg/g, with a range of 10.6-25.7 mg/g (n=7), and similar tryptamines (psilocin, baeocystin, norbaeocystin, norpsilocin, aeruginascin, 4-HO-tryptamine, and tryptamine) in lesser concentrations for a combined tryptamine concentration of 22.5±3.2 mg/g. These results show P. zapotecorum to be a potent - and variable - Psilocybe mushroom. Chemical profiling, genetic analysis, and cultivation assist in demystifying these mushrooms. As clinical studies with psilocybin gain traction, understanding the diversity of psilocybin mushrooms will assure that psilocybin therapy does not become synonymous with psilocybin mushrooms.

Photoactive and conductive biohybrid films by polymerization of pyrrole through voids in photosystem I multilayer films.

The combination of conducting polymers with electro- and photoactive proteins into thin films holds promise for advanced energy conversion materials and devices. The emerging field of protein electronics requires conductive soft materials in a composite with electrically insulating proteins. The electropolymerization of pyrrole through voids in a drop-casted photosystem I (PSI) multilayer film enables the straightforward fabrication of photoactive and conductive biohybrid films. The rate of polypyrrole (PPy) growth is reduced by the presence of the PSI film but is insensitive to its thickness, suggesting that rapid diffusion of pyrrole through the voids within the PSI film enables initiation at vacant areas on the gold surface. The base thickness of the composite tends to increase with time, as PPy chains propagate through and beyond the PSI film, coalescing to exhibit a tubule-like morphology as observed by scanning electron microscopy. Increasing amounts of PPy greatly increase the capacitance of the composite films in a manner almost identical to that of pure PPy films grown from unmodified gold, consistent with a high polymer/aqueous interfacial area and a conductive composite film. While PPy is not photoactive here, all composite films, including those with large amounts of PPy, exhibit photocurrents when irradiated by white light in the presence of redox mediator species. Optimization of the Py electropolymerization time is necessary, as increasing amounts of PPy lead to decreased photocurrent density due to a combination of light absorbance by the polymer and reduced accessibility of redox species to active PSI sites.

Drop-casted Photosystem I/cytochrome c multilayer films for biohybrid solar energy conversion.

One of the main barriers to making efficient Photosystem I-based biohybrid solar cells is the need for an electrochemical pathway to facilitate electron transfer between the P700 reaction center of Photosystem I and an electrode. To this end, nature provides inspiration in the form of cytochrome c6, a natural electron donor to the P700 site. Its natural ability to access the P700 binding pocket and reduce the reaction center can be mimicked by employing cytochrome c, which has a similar protein structure and redox chemistry while also being compatible with a variety of electrode surfaces. Previous research has incorporated cytochrome c to improve the photocurrent generation of Photosystem I using time consuming and/or specialized electrode preparation. While those methods lead to high protein areal density, in this work we use the quick and facile vacuum-assisted drop-casting technique to construct a Photosystem I/cytochrome c photoactive composite film with micron-scale thickness. We demonstrate that this simple fabrication technique can result in high cytochrome c loading and improvement in cathodic photocurrent over a drop-casted Photosystem I film without cytochrome c. In addition, we analyze the behavior of the cytochrome c/Photosystem I system at varying applied potentials to show that the improvement in performance can be attributed to enhancement of the electron transfer rate to P700 sites and therefore the PSI turnover rate within the composite film.

Ultraviolet light oxidation of fresh hemoglobin eliminates aggregate formation seen in commercially sourced hemoglobin.

Elevated levels of circulating cell-free hemoglobin (CFH) are an integral feature of several clinical conditions including sickle cell anemia, sepsis, hemodialysis and cardiopulmonary bypass. Oxidized (Fe3+, ferric) hemoglobin contributes to the pathophysiology of these disease states and is therefore widely studied in experimental models, many of which use commercially sourced CFH. In this study, we treated human endothelial cells with commercially sourced ferric hemoglobin and observed the appearance of dense cytoplasmic aggregates (CAgg) over time. These CAgg were intensely autofluorescent, altered intracellular structures (such as mitochondria), formed in multiple cell types and with different media composition, and formed regardless of the presence or absence of cells. An in-depth chemical analysis of these CAgg revealed that they contain inorganic components and are not pure hemoglobin. To oxidize freshly isolated hemoglobin without addition of an oxidizing agent, we developed a novel method to convert ferrous CFH to ferric CFH using ultraviolet light without the need for additional redox agents. Unlike commercial ferric hemoglobin, treatment of cells with the fresh ferric hemoglobin did not lead to CAgg formation. These studies suggest that commercially sourced CFH may contain stabilizers and additives which contribute to CAgg formation.

Adsorption and Electropolymerization of p-Aminophenol Reduces Reproducibility of Electrochemical Immunoassays.

This paper investigates the electrochemical behavior of p-aminophenol (PAP) on commercially available carbon screen-printed electrodes (CSPEs) and gold screen-printed electrodes (GSPEs) at neutral and basic pHs for the development of inexpensive immunoassays. The electrochemical oxidative signal from PAP results from its adsorption to the electrode. The formation of self-assembled monolayers on gold electrodes prevented PAP adsorption but also reduced its oxidative current, confirming that adsorption increases signal production. On bare electrodes, PAP adsorption results in oxidative current variability depending on the electroactive surface area of the screen-printed electrode. This variability could not be remedied by cleaning and reusing the same GSPE. Decreasing the PAP concentration to 3.8 µM greatly improved the consistency of the measurements, suggesting that the adsorption of PAP is concentration-dependent. Multiple PAP oxidations on the same electrode caused polymerization, limiting PAP in continuous monitoring applications. Infrared and Raman spectroscopy allow the distinction between adsorbed PAP and electropolymerized PAP on the surface of a gold wafer. The results from this study suggest that the use of PAP production in immunoassays with SPEs must be fine-tuned, and electrodes must be cleaned or disposed of between measurements.

Trace Oxygen Affects Osmium Redox Polymer Synthesis for Wired Enzymatic Biosensors.

Electrochemical sensors that utilize enzymes are a sensitive, inexpensive means of detecting biologically relevant analytes. These sensors are categorized based on their construction and method of signal transport. Type I sensors consist of a crosslinked enzyme on an electrode surface and are potentially subject to interference from byproducts and other biological analytes. However, type II sensors help alleviate this problem with the addition of a redox polymer layer that assists in signal transduction, thus minimizing interferences. An osmium-loaded poly(vinylimidazole) polymer (Os-PVI) is commonly used with successful results, and when combined with an enzyme yields a type II sensor. Our initial attempts at the synthesis of this polymer resulted in an unexpected osmium precursor, which had fluorescent and redox properties that did not match with the desired Os-PVI polymer. Careful exclusion of oxygen during the Os complex precursor synthesis was necessary to avoid this unexpected oxygen containing Os-precursor, which had been seen previously in mass spectrometry studies. All precursors and osmium polymers were characterized with 1H NMR, fluorescence, mass spectrometry, and cyclic voltammetry to provide a better understanding of these compounds and assist in the building of new sensors.

Chlorpyrifos Disrupts Acetylcholine Metabolism Across Model Blood-Brain Barrier.

Despite the significant progress in both scientific understanding and regulations, the safety of agricultural pesticides continues to be called into question. The need for complementary analytics to identify dysregulation events associated with chemical exposure and leverage this information to predict biological responses remains. Here, we present a platform that combines a model organ-on-chip neurovascular unit (NVU) with targeted mass spectrometry (MS) and electrochemical analysis to assess the impact of organophosphate (OP) exposure on blood-brain barrier (BBB) function. Using the NVU to simulate exposure, an escalating dose of the organophosphate chlorpyrifos (CPF) was administered. With up to 10 µM, neither CPF nor its metabolites were detected across the BBB (limit of quantitation 0.1 µM). At 30 µM CPF and above, targeted MS detected the main urinary metabolite, trichloropyridinol (TCP), across the BBB (0.025 µM) and no other metabolites. In the vascular chamber where CPF was directly applied, two primary metabolites of CPF, TCP and diethylthiophosphate (DETP), were both detected (0.1-5.7 µM). In a second experiment, a constant dose of 10 µM CPF was administered to the NVU, and though neither CPF nor its metabolites were detected across the BBB after 24 h, electrochemical analysis detected increases in acetylcholine levels on both sides of the BBB (up to 24.8 ± 3.4 µM) and these levels remained high over the course of treatment. In the vascular chamber where CPF was directly applied, only TCP was detected (ranging from 0.06 µM at 2 h to 0.19 µM at 24 h). These results provide chemical evidence of the substantial disruption induced by this widely used commercial pesticide. This work reinforces previously observed OP metabolism and mechanisms of impact, validates the use of the NVU for OP toxicology testing, and provides a model platform for analyzing these organotypic systems.

Layer-by-Layer Assembly of Photosystem I and PEDOT:PSS Biohybrid Films for Photocurrent Generation.

The design of electrode interfaces to achieve efficient electron transfer to biomolecules is important in many bioelectrochemical processes. Within the field of biohybrid solar energy conversion, constructing multilayered Photosystem I (PSI) protein films that maintain good electronic connection to an underlying electrode has been a major challenge. Previous shortcomings include low loadings, long deposition times, and poor connection between PSI and conducting polymers within composite films. Here, we show that PSI protein complexes can be deposited into monolayers within a 30 min timespan by leveraging the electrostatic interactions between the protein complex and the poly(3,4-ethylenedioxythiophene)-polystyrenesulfonate (PEDOT:PSS) polymer. Further, we follow a layer-by-layer (LBL) deposition procedure to produce up to 9-layer pairs of PSI and PEDOT:PSS with highly reproducible layer thicknesses as well as distinct changes in surface composition. When tested in an electrochemical cell employing ubiquinone-0 as a mediator, the photocurrent performance of the LBL films increased linearly by 83 ± 6 nA/cm2 per PSI layer up to 6-layer pairs. The 6-layer pair samples yielded a photocurrent of 414 ± 13 nA/cm2, after which the achieved photocurrent diminished with additional layer pairs. The turnover number (TN) of the PSI-PEDOT:PSS LBL assemblies also greatly exceeds that of drop-casted PSI multilayer films, highlighting that the rate of electron collection is improved through the systematic deposition of the protein complexes and conducting polymer. The capability to deposit high loadings of PSI between PEDOT:PSS layers, while retaining connection to the underlying electrode, shows the value in using LBL assembly to produce PSI and PEDOT:PSS bioelectrodes for photoelectrochemical energy harvesting applications.

A bistable, multiport valve enables microformulators creating microclinical analyzers that reveal aberrant glutamate metabolism in astrocytes derived from a tuberous sclerosis patient.

There is a need for valves and pumps that operate at the microscale with precision and accuracy, are versatile in their application, and are easily fabricated. To that end, we developed a new rotary planar multiport valve to faithfully select solutions (contamination = 5.22 ± 0.06 ppb) and a rotary planar peristaltic pump to precisely control fluid delivery (flow rate = 2.4 ± 1.7 to 890 ± 77 µL/min). Both the valve and pump were implemented in a planar format amenable to single-layer soft lithographic fabrication. These planar microfluidics were actuated by a rotary motor controlled remotely by custom software. Together, these two devices constitute an innovative microformulator that was used to prepare precise, high-fidelity mixtures of up to five solutions (deviation from prescribed mixture = ±|0.02 ± 0.02| %). This system weighed less than a kilogram, occupied around 500 cm3, and generated pressures of 255 ± 47 kPa. This microformulator was then combined with an electrochemical sensor creating a microclinical analyzer (µCA) for detecting glutamate in real time. Using the chamber of the µCA as an in-line bioreactor, we compared glutamate homeostasis in human astrocytes differentiated from human-induced pluripotent stem cells (hiPSCs) from a control subject (CC-3) and a Tuberous Sclerosis Complex (TSC) patient carrying a pathogenic TSC2 mutation. When challenged with glutamate, TSC astrocytes took up less glutamate than control cells. These data validate the analytical power of the µCA and the utility of the microformulator by leveraging it to assess disease-related alterations in cellular homeostasis.

Reversing the Thermodynamics of Galvanic Replacement Reactions by Decreasing the Size of Gold Nanoparticles.

Here, we describe the surprising reactivity between surface-attached (a) 0.9, 1.6, and 4.1 nm diameter weakly stabilized Au nanoparticles (NPs) and aqueous 1.0 × 10-4 M Ag+ solution, and (b) 1.6 and 4.1 nm diameter weakly stabilized Au NPs and aqueous 1.0 × 10-5 M PtCl42-, which are considered to be antigalvanic replacement (AGR) reactions because they are not thermodynamically favorable for bulk-sized Au under these conditions. Anodic Stripping Voltammetry (ASV) and Scanning Transmission Electron Microscopy with Energy-Dispersive X-ray Spectroscopy (STEM-EDS) mapping provide quantitation of the extent of Ag and Pt replacement as a function of Au NP diameter. The extent of the reaction increases as the Au NP size decreases. The percentage of Ag in the AuAg alloy following AGR based on ASV is 17.8 ± 0.6% for 4.1 nm diameter Au NPs, 87.2 ± 2.9% for 1.6 nm Au NPs, and an unprecedented full 100% Ag for 0.9 nm diameter Au NPs. STEM-EDS mapping shows very close agreement with the ASV-determined compositions. In the case of PtCl42-, STEM-EDS mapping shows AuPt alloy NPs with 3.9 ± 1.3% and 41.1 ± 8.7% Pt following replacement with 4.1 and 1.6 nm diameter Au NPs, respectively, consistent with qualitative changes to the ASV. The size-dependent AGR correlates well with the negative shift in the standard potential (E0) for Au oxidation with decreasing NP size.

A low-interference, high-resolution multianalyte electrochemical biosensor.

Electrochemical sensors are used by millions of patients and health care providers every year, yet these measurements are hindered by compounds that also exhibit inherent redox activity. Acetaminophen (APAP) is one such interferent that falls into this extensive class. In this work, an osmium-based redox polymer was used for electrochemical detection in a sensor that was operated at a decreased voltage, allowing for decreased interference. These sensors demonstrated better selectivity (40-fold for glucose and 200-fold for lactate) for their respective analyte over APAP, possessed higher sensitivity (0.350 ± 0.006 µA mM-1 for glucose and 2.00 ± 0.05 µA mM-1 for lactate) over a broad range of analyte concentrations (50 µM-10 mM for glucose and 2-324 µM for lactate), and displayed similar operational stability (26% decrease for glucose and 29% decrease for lactate) over 7 days compared to first-generation sensors. To test this platform under biologically-relevant conditions, glucose metabolism was monitored in a model liver cell line, Alpha Mouse Liver 12 (AML12) after treatment with APAP and/or insulin. This work represents a high-resolution electrochemical biosensor for microphysiological monitoring of glucose and lactate in the presence of APAP.

Optical and electrochemical tuning of hydrothermally synthesized nitrogen-doped carbon dots.

Carbon dots (CDs) are a rapidly progressing class of nanomaterial which show promise towards applications in solar energy conversion due to their low toxicity, favorable electrochemical properties, and tunability. In recent years there have been a number of reported CD syntheses, both top-down and bottom-up methods, producing a diverse range of CDs with intrinsic properties dependent on the starting materials and utilized dopants. This work presents a citrate buffer-facilitated synthesis of nitrogen-doped carbon dots (NCD) and explores the impact of urea concentration on observed electrochemical and optical properties. Optical absorbance and quantum yield of NCDs were found to increase with the dopant concentrations present in the hydrothermal reaction mixture. Electrochemical analysis demonstrates that increased nitrogen content results in the shifting of carbon dot oxidation potentials without the need of post-synthesis surface modifications. Over the range of molar ratios of dopant-to-citrate tested, the oxidation potentials of NCDs shifted up to 150 mV towards more negative potentials. X-ray photoelectron spectroscopy confirms the addition of pyrrolic and pyridinic nitrogen at different levels in different batches of NCDs, which are likely the source of the observed changes.

Photosystem I Enhances the Efficiency of a Natural, Gel-Based Dye-Sensitized Solar Cell.

The photosystem I (PSI) protein complex is known to enhance bioelectrode performance for many liquid-based photoelectrochemical cells. A hydrogel as electrolyte media allows for simpler fabrication of more robust and practical solar cells in comparison to liquid-based devices. This paper reports a natural, gel-based dye-sensitized solar cell that integrates PSI to improve device efficiency. TiO2-coated FTO slides, dyed by blackberry anthocyanin, act as a photoanode, while a film of PSI deposited onto copper comprises the photocathode. Ascorbic acid (AscH) and 2,6-dichlorophenolindophenol (DCPIP) are the redox mediator couple inside an agarose hydrogel, enabling PSI to produce excess oxidized species near the cathode to improve device performance. A comparison of performance at low pH and neutral pH was performed to test the pH-dependent properties of the AscH/DCPIP couple. Devices at neutral pH performed better than those at lower pH. The PSI film enhanced photovoltage by 75 mV to a total photovoltage of 0.45 V per device and provided a mediator concentration-dependent photocurrent enhancement over non-PSI devices, reaching an instantaneous power conversion efficiency of 0.30% compared to 0.18% without PSI, a 1.67-fold increase. At steady state, power conversion efficiencies for devices with and without PSI were 0.042 and 0.028%, respectively.

Fibrotic Encapsulation Is the Dominant Source of Continuous Glucose Monitor Delays.

Continuous glucose monitor (CGM) readings are delayed relative to blood glucose, and this delay is usually attributed to the latency of interstitial glucose levels. However, CGM-independent data suggest rapid equilibration of interstitial glucose. This study sought to determine the loci of CGM delays. Electrical current was measured directly from CGM electrodes to define sensor kinetics in the absence of smoothing algorithms. CGMs were implanted in mice, and sensor versus blood glucose responses were measured after an intravenous glucose challenge. Dispersion of a fluorescent glucose analog (2-NBDG) into the CGM microenvironment was observed in vivo using intravital microscopy. Tissue deposited on the sensor and nonimplanted subcutaneous adipose tissue was then collected for histological analysis. The time to half-maximum CGM response in vitro was 35 ± 2 s. In vivo, CGMs took 24 ± 7 min to reach maximum current versus 2 ± 1 min to maximum blood glucose (P = 0.0017). 2-NBDG took 21 ± 7 min to reach maximum fluorescence at the sensor versus 6 ± 6 min in adipose tissue (P = 0.0011). Collagen content was closely correlated with 2-NBDG latency (R = 0.96, P = 0.0004). Diffusion of glucose into the tissue deposited on a CGM is substantially delayed relative to interstitial fluid. A CGM that resists fibrous encapsulation would better approximate real-time deviations in blood glucose.

Polyviologen as Electron Transport Material in Photosystem I-Based Biophotovoltaic Cells.

The photosynthetic protein complex, photosystem I (PSI), can be photoexcited with a quantum efficiency approaching unity and can be integrated into solar energy conversion devices as the photoactive electrode. The incorporation of PSI into conducting polymer frameworks allows for improved conductivity and orientational control in the photoactive layer. Polyviologens are a unique class of organic polycationic polymers that can rapidly accept electrons from a primary donor such as photoexcited PSI and subsequently can donate them to a secondary acceptor. Monomeric viologens, such as methyl viologen, have been widely used as diffusible mediators in wet PSI-based photoelectrochemical cells on the basis of their suitable redox potentials for accepting electrons. Polyviologens possess similar electronic properties to their monomers with the added advantage that they can shuttle electrons in the solid state. Depositing polyviologen directly onto a film of PSI protein results in significant photocurrent enhancement, which confirms its role as an electron-transport material. The polymer film not only improves the photocurrent by aiding the electron transfer but also helps preserve the protein film underneath. The composite polymer-PSI assembly enhances the charge-shuttling processes from individual protein molecules within the PSI multilayer, greatly reducing charge-transfer resistances. The resulting PSI-based solid-state platform demonstrates a much higher photocurrent than the corresponding photoelectrochemical cell built using a similar architecture.

An Electrochemical Reaction-Diffusion Model of the Photocatalytic Effect of Photosystem I Multilayer Films.

The photosynthetic protein, photosystem I (PSI), has been used as a photoactive species within a host of biohybrid photoelectrochemical systems. PSI multilayer films at electrode surfaces provide greatly improved solar energy conversion relative to homologous monolayer films. While the photocatalytic effect of PSI multilayers has been theorized as an electrolyte-mediated mechanism, no comprehensive, first-principles modeling study has been presented. In this work, we develop and optimize an electrochemical reaction-diffusion model to replicate the significant electrochemical, physicochemical, and transport processes that underpin photocurrent development of a PSI multilayer film. We use this model to provide strong evidence that PSI's terminal cofactors rapidly exchange electrons with diffusible mediators and stimulate photocurrent principally due to alteration of mediator concentrations at a solution-electrode interface as governed by Butler-Volmer kinetics. Our fitted model accurately replicates photocurrent trends under a variety of conditions, including variable applied bias and PSI multilayer film thickness.

Multianalyte Physiological Microanalytical Devices.

Advances in scientific instrumentation have allowed experimentalists to evaluate well-known systems in new ways and to gain insight into previously unexplored or poorly understood phenomena. Within the growing field of multianalyte physiometry (MAP), microphysiometers are being developed that are capable of electrochemically measuring changes in the concentration of various metabolites in real time. By simultaneously quantifying multiple analytes, these devices have begun to unravel the complex pathways that govern biological responses to ischemia and oxidative stress while contributing to basic scientific discoveries in bioenergetics and neurology. Patients and clinicians have also benefited from the highly translational nature of MAP, and the continued expansion of the repertoire of analytes that can be measured with multianalyte microphysiometers will undoubtedly play a role in the automation and personalization of medicine. This is perhaps most evident with the recent advent of fully integrated noninvasive sensor arrays that can continuously monitor changes in analytes linked to specific disease states and deliver a therapeutic agent as required without the need for patient action.

Elucidation of the Role of Lectin-Like oxLDL Receptor-1 in the Metabolic Responses of Macrophages to Human oxLDL.

Atherogenesis is the narrowing of arteries due to plaque build-up that results in cardiovascular disease that can lead to death. The macrophage lectin-like oxidized LDL receptor-1 (LOX-1), also called the oxidized low-density lipoprotein receptor 1 (OLR1), is currently thought to aid in atherosclerotic disease progression; therefore metabolic studies have potential to both provide mechanistic validation for the role of LOX-1 in disease progression and provide valuable information regarding biomarker strategies and clinical imaging. One such mechanistic study is the upregulation of LOX-1 by methylated bacterial DNA and deoxy-cytidylate-phosphate-deoxy-guanylate-DNA (CpG)-DNA exposure. CpG-DNA is known to promote oxidative burst responses in macrophages, due to its direct binding to toll-like receptor 9 (TLR9) leading to the initiation of an NF-κB mediated immune response. In addition to the upregulation of macrophage LOX-1 expression, these studies have also examined the macrophage metabolic response to murine LOX-1/OLR1 antibody exposure. Our data suggests the antibody exposure effectively blocks LOX-1 dependent oxLDL metabolic activation of the macrophage, which was quantified using the multianalyte microphysiometer (MAMP). Using the MAMP to examine metabolic fluctuations during various types of oxLDL exposure, LOX-1 upregulation and inhibition provide valuable information regarding the role of LOX-1 in macrophage activation of oxidative burst.

Analysis of a Nitroreductase-Based Hypoxia Sensor in Primary Neuronal Cultures.

The ability to assess oxygenation within living cells is much sought after to more deeply understand normal and pathological cell biology. Hypoxia Red manufactured by Enzo Life Sciences is advertised as a novel hypoxia detector dependent on nitroreducatase activity. We sought to use Hypoxia Red in primary neuronal cultures to test cell-to-cell metabolic variability in response to hypoxic stress. Neurons treated with 90 min of hypoxia were labeled with Hypoxia Red. We observed that, even under normoxic conditions neurons expressed fluorescence robustly. Analysis of the chemical reactions and biological underpinnings of this method revealed that the high uptake and reduction of the dye is due to active nitroreductases in normoxic cells that are independent of oxygen availability.