Fluorescent pH Indicator Research Articles

Computationally Optimized Graphene-Based Electrochemical Sensor with Enhanced Signal Stability for the Determination of the Antimicrobial Agent 9-aminoacridine

A hydrophobic aryl diazonium salt has been synthesized from 3,5-bis(trifluoromethyl)aniline and utilized to covalently modify graphene nanoplatelets and carbon nanotubes. The modified nanomaterials were applied on a screen-printed electrode/ion sensing membrane interface resulting in reduced potential drift to 100 μV h−1 compared to control sensors. Characterization was achieved through X-ray photoelectron spectroscopy. The electrode’s response was optimized using response surface methodology and then utilized for determination of 9-Aminoacridine (9-AA) in pharmaceutical gel dosage form and spiked human plasma without prior extraction steps. 9-AA is a fluorescent dye with antimicrobial activity that eradicates a range of microorganisms that can cause oral sores or broken skin and it has been recently used as anticancer among other uses as fluorescent dye and pH indicator. Accurate determination of 9-AA could help in adjusting dosages for each application. The optimized sensor was validated per IUPAC guidelines and obtained a wide linearity range from 1.0 × 10–7 M to 1.0 × 10–2 M, correlation coefficient of 0.9997, improved Nernstian slope 59.72, long term stability, and lower limit of detection (9.0 × 10–8 M). Furthermore, Analytical Eco-scale and AGREE methods were utilized to evaluate the presented method’s greenness.

Intermittent proton bursts of single lactic acid bacteria.

Lactic acid bacteria are a kind of probiotic microorganisms that efficiently convert carbohydrates to lactic acids, thus playing essential roles in fermentation and food industry. While conventional wisdom often suggests continuous release of protons from bacteria during acidification, here we developed a methodology to measure the dynamics of proton release at the single bacteria level, and report on the discovery of a proton burst phenomenon, i.e., the intermittent efflux of protons, of single Lactobacillus plantarum bacteria. When placing an individual bacterium in an oil-sealed microwell, efflux and accumulation of protons consequently reduced the pH in the confined extracellular medium, which was monitored with fluorescent pH indicators in a high-throughput and real-time manner. In addition to the slow and continuous proton release behavior (as expected), stochastic and intermittent proton burst events were surprisingly observed with a typical timescale of several seconds. It was attributed to the regulatory response of bacteria by activating H+-ATPase to compensate the stochastic and transient depolarizations of membrane potential. These findings not only revealed an unprecedented proton burst phenomenon in lactic acid bacteria, but also shed new lights on the intrinsic roles of H+-ATPase in membrane potential homeostasis, with implications for both fermentation industry and bacterial electrophysiology.

Effect of Polypeptide Complex Coacervate Microenvironment on Protonation of a Guest Molecule.

Complex coacervate droplets formed by the liquid-liquid phase separation of polyelectrolyte solutions capture several important features of membraneless organelles including their ability to accumulate guest molecules and to provide distinct microenvironments. Here, we examine how polyions in complex coacervates can influence localized guest molecules, leading to a shifted protonation state of the guest molecule in response to its electrostatic environment. A fluorescent ratiometric pH indicator dye was used as a model guest molecule able to report its protonation state in the coacervate phase. Experimentally observed differences in dye-reported local apparent pH inside versus outside of coacervate droplets were largest for polyion pairs having lower salt stabilities and/or larger polyion length mismatch, which we attribute to the relative concentration of open sites on polyions within the coacervates based on theoretical calculations. Using the transfer matrix method, we confirmed that theoretical phase diagrams and critical salt stabilities generated for each polyion pair were consistent with experimental turbidity measurements and estimated the amount of available binding sites on polyions for guest molecules. We conclude that dye molecules likely experience an effective pKa shift due to interactions with coacervate polyions rather than reporting directly on local proton concentrations. Such a local pKa shift can also be anticipated for other guest molecules having protonatable groups, including, for example, many metabolites, ligands, and/or drug molecules that partition into coacervates or membraneless organelles based on ion pairing interactions.

New fluorescent pH indicators for characterizing cement carbonation

Fluorescent indicators of a wider labeling pH range of 7.0–12.5 were prepared to characterize the pH evolution of the accelerated/natural carbonation of cement and the corrosion of steel bar. Compared with conventional indicators (phenolphthalein, pH range of 8.2–8.5), these indicators indicate multiple pH-induced switches of different fluorescence emissions for the fluorescence resonance energy transfer between donor–acceptor. The results showed that the newly-developed indicators are sensitive and applicable for labeling both natural and accelerated carbonation of cement. Investigations by Fourier Transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and thermogravimetric analysis demonstrated the reliability of these indicators, that is, in the “uncarbonated area” shown by phenolphthalein, carbonization actually occurs as exhibited by fluorescent indicators. Furthermore, electrochemical impedance spectroscopy, as well as dynamic potential polarization studies showed that rusting/corrosion of reinforcing steel started when the pH dropped to 11.5 in the simulation study. This work promoted the contribution of fluorescent indicators in cement carbonation characterization, with a wide pH response.

Quantifying pH in Malaria Using pHluorin and Flow Cytometry.

Intracellular pH (pHi) plays a critical role in the regulation of numerous biological functions where specific pH ranges are required for optimal operation within cells. Slight pH changes can impact the regulation of diverse molecular processes, including enzymatic activities, ion channels, and transporters, which all play a role in cell functions. Methods for quantifying pHi continue to evolve and include various optical methods using fluorescent pH indicators. Here, we provide a protocol to measure pHi in the cytosol of Plasmodium falciparum blood stage parasites by means of flow cytometry and using pHluorin2, a pH-sensitive fluorescent protein that has been introduced into the genome of the parasite.

Developing a fluorometric urease activity microplate assay suitable for automated microbioreactor experiments.

Quantifying urease activity is an important task for Microbial Induced Calcite Precipitation research. A new urease activity microplate assay using a fluorescent pH indicator is presented. The method is also suitable for automated measurements during microbioreactor experiments. The assay reagent consists of the green fluorescent pH-indicator fluorescein, urea and a phosphate buffer. After sample addition, the microbial urease hydrolyses urea, which results in a pH and hence fluorescence increase. The fluorescence signal can be measured with a microplate reader or with the microbioreactor system BioLector, allowing for automated urease activity measurements during cultivation experiments. In both measurement systems, the fluorescence signal slope highly correlates with the urease activity measured offline with standard methods. Automated measurement is possible, as no sample preparation such as centrifugation or adjusting of the optical density is required. The assay was developed so that the culture samples turbidity, salinity or buffer concentration does not have a negative impact on the fluorescence signal. The assay allows for straightforward, non-hazardous, parallelized, cheap and reliable measurements, making research on ureolytic bacteria for Microbial Induced Calcite Precipitation more efficient. The assay could be adapted to other enzymes, which have a strong impact on the pH value.

The Dual Use of the Pyranine (HPTS) Fluorescent Probe: A Ground-State pH Indicator and an Excited-State Proton Transfer Probe.

ConspectusMolecular fluorescent probes are an essential experimental tool in many fields, ranging from biology to chemistry and materials science, to study the localization and other environmental properties surrounding the fluorescent probe. Thousands of different molecular fluorescent probes can be grouped into different families according to their photophysical properties. This Account focuses on a unique class of fluorescent probes that distinguishes itself from all other probes. This class is termed photoacids, which are molecules exhibiting a change in their acid–base transition between the ground and excited states, resulting in a large change in their pKa values between these two states, which is thermodynamically described using the Förster cycle. While there are many different photoacids, we focus only on pyranine, which is the most used photoacid, with pKa values of ∼7.4 and ∼0.4 for its ground and excited states, respectively. Such a difference between the pKa values is the basis for the dual use of the pyranine fluorescent probe. Furthermore, the protonated and deprotonated states of pyranine absorb and emit at different wavelengths, making it easy to focus on a specific state. Pyranine has been used for decades as a fluorescent pH indicator for physiological pH values, which is based on its acid–base equilibrium in the ground state. While the unique excited-state proton transfer (ESPT) properties of photoacids have been explored for more than a half-century, it is only recently that photoacids and especially pyranine have been used as fluorescent probes for the local environment of the probe, especially the hydration layer surrounding it and related proton diffusion properties. Such use of photoacids is based on their capability for ESPT from the photoacid to a nearby proton acceptor, which is usually, but not necessarily, water. In this Account, we detail the photophysical properties of pyranine, distinguishing between the processes in the ground state and the ones in the excited state. We further review the different utilization of pyranine for probing different properties of the environment. Our main perspective is on the emerging use of the ESPT process for deciphering the hydration layer around the probe and other parameters related to proton diffusion taking place while the molecule is in the excited state, focusing primarily on bio-related materials. Special attention is given to how to perform the experiments and, most importantly, how to interpret their results. We also briefly discuss the breadth of possibilities in making pyranine derivatives and the use of pyranine for controlling dynamic reactions.

Advanced multimodal solid-state optochemical pH and dual pH/O2 sensors for cell analysis

Optochemical sensors are actively used in cell analysis, however existing systems have limitations with respect to their robustness and analytical performance. We have developed advanced multimodal and multi-parametric solid-state pH sensors for cell analysis based on hydrophobic protonable metal-free porphyrins, such as octaethylporphine (OEP), and octaethylporphine-ketone (OEPK), as fluorescent pH indicators. The internally referenced ratiometric intensity and nanosecond lifetime-based versions of the pH sensors were developed and also multiplexed with the O2 sensors based on phosphorescent PtOEP dye. We optimised the key parameters of the pH sensor, including: dye encapsulation matrix, the type and concentration of proton transfer reagent, measurement pH range and pKa, dye concentrations and cross-talk with the O2 sensor. Subsequently, sensor coatings were deposited on common substrates used in cell analysis (96-well plates), fine-tuned for their operational performance, dual O2/pH sensing functionally and the ability to measure Extracellular Acidification (ECAR) and Oxygen Consumption (OCR) rates in biological samples containing cells. The optimised sensors with stable internally referenced calibrations, convenient spectral characteristics and low cytotoxicity, were demonstrated with cultured cells and 3D spheroid structures, measuring their ECAR, OCR and responses to stimulation. These pH and dual pH/O2 sensors are well-suited for detailed metabolic studies of biological samples on widely available laboratory equipment.

Circular Permutants of BrUSLEE Protein as Fluorescent pH Indicators

—Two variants of circularly permuted BrUSLEE, a green fluorescent protein with a short fluorescence lifetime, have been engineered. We characterized the pH-dependence of fluorescence decay kinetics of these fluorophores. It was shown that both permutants (cpBrUS and cpBrUS-145) exhibit three-component fluorescence decay kinetics, with the lifetime of the one component varying within the ~3000–300 ps range upon pH shift from 5.5 to 9.0. At the same time, the original BrUSLEE does not show a significant change in the fluorescence decay kinetics within the physiologically relevant pH-range of 6.0–8.5. The described pH-dependence allows considering the BrUSLEE permutants as pH indicators with the fluorescence lifetime readout.

A Patient-Ready Wearable Transcutaneous CO2 Sensor

Continuously monitoring transcutaneous CO2 partial pressure is of crucial importance in the diagnosis and treatment of respiratory and cardiac diseases. Despite significant progress in the development of CO2 sensors, their implementation as portable or wearable devices for real-time monitoring remains under-explored. Here, we report on the creation of a wearable prototype device for transcutaneous CO2 monitoring based on quantifying the fluorescence of a highly breathable CO2-sensing film. The developed materials are based on a fluorescent pH indicator (8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt or HPTS) embedded into hydrophobic polymer matrices. The film’s fluorescence is highly sensitive to changes in CO2 partial pressure in the physiological range, as well as photostable and insensitive to humidity. The device and medical-grade films are based on our prior work on transcutaneous oxygen-sensing technology, which has been extensively validated clinically.

Pulmonary Arterial Smooth Muscle Cell Monocarboxylate Transporter 1/4 Contributes to Extracellular Acidosis following Chronic Hypoxia‐induced Pulmonary Hypertension

Pulmonary hypertension (pHTN) is defined by mean pulmonary arterial pressure at rest exceeding 20 mmHg with mortality often resulting from right ventricular failure that is secondary to increased afterload. During pHTN, increased reliance on aerobic glycolysis and inhibition of mitochondrial oxidative respiration is observed in pulmonary arterial smooth muscle cells (PASMCs). This metabolic shift is associated with hyperproliferation and resistance to apoptosis contributing to the pathogenesis of pHTN. Along with these phenotypic changes, cells undergoing aerobic respiration exhibit increased glucose uptake and subsequent lactate and H+ efflux, contributing to extracellular acidosis. Acid‐sensing ion channel 1 (ASIC1) is a voltage‐independent, proton‐gated cation channel that contributes to the development of chronic hypoxia (CH)‐induced pHTN. While the function of ASIC1 is well characterized in pHTN, it has not been determined whether the metabolic shift observed during pHTN contributes to the activation of ASIC1. Therefore, we hypothesized that enhanced glucose uptake and subsequent acidification of the extracellular microenvironment in PASMCs lead to the activation of ASIC1 in CH‐induced pHTN. As an initial step in testing this hypothesis, rats were housed in a hypobaric chamber (barometric pressure: ~380 mmHg) to induce pHTN and compared to age‐matched normoxic controls. After 4 weeks, PASMCs were collected and transiently cultured under normoxia for 3‐4 days. As expected, PASMCs from CH rats showed a reduced oxygen consumption rate (p<0.0001) and greater extracellular acidification (p<0.0001) compared to those from control animals, indicating increased glycolytic activity. Utilizing the fluorescent pH indicators SNARF‐5/AM, we found that PASMCs from CH rats exhibit intracellular alkalization (p<0.0001 vs. control) and extracellular acidosis (p<0.001 vs. control). We further examined the H+ transporter(s) involved in mediating extracellular acidosis by inhibiting carbonic anhydrase IX (CA‐IX), the Na+/H+ exchanger (NHE1), the vacuolar‐type H+‐type ATPase (V‐ATPase), or monocarboxylate transporters 1 and ‐4 (MCT1 and MCT4). The dual MCT1/4 inhibitor, syrosingopine, prevented intracellular alkalization and extracellular acidosis following CH (Figure 1A), which corresponded with an increase in MCT4, but not MCT1, expression (Figure 1B). These data suggest MCT plays an important role in regulating PASMC pH homeostasis. Future studies will assess the role of MCT to activate ASIC1 and contribute to pHTN.

Anagliptin promotes apoptosis in mouse colon carcinoma cells via MCT-4/lactate-mediated intracellular acidosis.

Cancer cells frequently exhibit an acidic extracellular microenvironment, where inversion of the transmembrane pH gradient is associated with tumor proliferation and metastasis. To elucidate a new therapeutic target against cancer, the current study aimed to determine the mechanism by which the dipeptidyl peptidase-4 inhibitor anagliptin regulates the cellular pH gradient and concomitant extracellular acidosis during cancer progression. A total of 5x105 CT-26 cells (resuspended in phosphate buffer saline) were injected subcutaneously in the right flank of male BALB/c mice (weighing 25-28 g). The tumor samples were harvested, and lactate was detected using a lactate assay kit. Immunohistochemistry was used to detect the Ki67 and PCNA. MTT assay and flow cytometric were used to detect cell viability. Intracellular pH was detected by fluorescence pH indicator. The results revealed that anagliptin effectively reduced tumor growth, but did not affect the body weight of treated mice. Anagliptin reduced the accumulation of lactate in tumor sample. Treatment with anagliptin stimulated the apoptosis of CT-26 cells. And lactate excretion inhibition is accompanied by an increase in extracellular pH (pHe) after treatment with anagliptin. Furthermore, anagliptin induced intracellular acidification and reversed the low pHe gradient via monocarboxylate transporter-4 (MCT-4)-mediated lactate excretion. Additionally, anagliptin reversed the aberrant transmembrane extracellular/intracellular pH gradient by suppressing MCT-4-mediated lactate excretion, while also reducing mitochondrial membrane potential and inducing apoptosis. These data revealed a novel function of anagliptin in regulating lactate excretion from cancer cells, suggesting that anagliptin may be used as a potential treatment for cancer.

Multi-functional rhodamine-based chitosan hydrogels as colorimetric Hg2+ adsorbents and pH-triggered biosensors

HypothesisWater contamination from heavy metal ions is a major global environmental concern. Adsorbents based on biomaterials have been demonstrated to possess remarkable removal efficiency for metal ions, but the adsorption model of biosorbents is not clear and much efforts should be devoted to study the adsorption behaviors and understand the adsorption mechanism. ExperimentsThe multifunctional rhodamine-modified chitosan (RMC) hydrogel for Hg2+ adsorption with fluorescent turn-ON properties was fabricated through grafting the rhodamine-modified poly (ethylene glycol) benzaldehyde (RM-PEG) onto the hydrogel network serving as the fluorescence/colorimetric sensing receptor. The adsorption behaviors and colorimetric sensing mechanism of RMC hydrogel towards Hg2+ were investigated in detail. FindingsRMC hydrogel can remove more than 96.5% of Hg2+ from aqueous solution with significant fluorescence response and colorimetric change. The high adsorption selectivity and colorimetric sensing mechanism of RMC hydrogel towards Hg2+ can be explained by the hard and soft acid/base (HSAB) theory. The O atom in hydroxyl and carbonyl groups together with the N atom in amine/imine groups of RMC hydrogel play a vital role in the adsorption of Hg2+, while the colorimetric response and fluorescence enhancement of the hydrogel after adsorption are attributed to the specific spiro-lactam structure of rhodamine moieties. The adsorption isotherms and kinetics were investigated and well described by Freundlich isotherm and pseudo-second-order kinetic model. Furthermore, RM-PEG showed low cytotoxicity towards mouse embryonic fibroblast cells and RMC hydrogel can be used as a fluorescent pH indicator from 4.2 to 7.4, demonstrating the potential applications of RMC hydrogel in biological diagnosis.

Improved Plasmodium falciparum dilution cloning through efficient quantification of parasite numbers and c-SNARF detection

BackgroundMolecular and genetic studies of blood-stage Plasmodium falciparum parasites require limiting dilution cloning and prolonged cultivation in microplates. The entire process is laborious and subject to errors due to inaccurate dilutions at the onset and failed detection of parasite growth in individual microplate wells.MethodsTo precisely control the number of parasites dispensed into each microplate well, parasitaemia and total cell counts were determined by flow cytometry using parasite cultures stained with ethidium bromide or SYBR Green I. Microplates were seeded with 0.2 or 0.3 infected cells/well and cultivated with fresh erythrocytes. The c-SNARF fluorescent pH indicator was then used to reliably detect parasite growth.ResultsFlow cytometry required less time than the traditional approach of estimating parasitaemia and cell numbers by microscopic examination. The resulting dilutions matched predictions from Poisson distribution calculations and yielded clonal lines. Addition of c-SNARF to media permitted rapid detection of parasite growth in microplate wells with high confidence.ConclusionThe combined use of flow cytometry for precise dilution and the c-SNARF method for detection of growth improves limiting dilution cloning of P. falciparum. This simple approach saves time, is scalable, and maximizes identification of desired parasite clones. It will facilitate DNA transfection studies and isolation of parasite clones from ex vivo blood samples.

Bioelectrical and cytoskeletal patterns correlate with altered axial polarity in the follicular epithelium of the Drosophila mutant gurken

BackgroundBioelectrical signals are known to be involved in the generation of cell and tissue polarity as well as in cytoskeletal dynamics. The epithelium of Drosophila ovarian follicles is a suitable model system for studying connections between electrochemical gradients, patterns of cytoskeletal elements and axial polarity. By interactions between soma and germline cells, the transforming growth factor-α homolog Gurken (Grk) establishes both the anteroposterior and the dorsoventral axis during oogenesis.ResultsIn the follicular epithelium of the wild-type (wt) and the polarity mutant grk, we analysed stage-specific gradients of membrane potentials (Vmem) and intracellular pH (pHi) using the potentiometric dye DiBAC4(3) and the fluorescent pH-indicator 5-CFDA,AM, respectively. In addition, we compared the cytoskeletal organisation in the follicular epithelium of wt and grk using fluorescent phalloidin and an antibody against acetylated α-tubulin. Corresponding to impaired polarity in grk, the slope of the anteroposterior Vmem-gradient in stage S9 is significantly reduced compared to wt. Even more striking differences in Vmem- and pHi-patterns become obvious during stage S10B, when the respective dorsoventral gradients are established in wt but not in grk. Concurrent with bioelectrical differences, wt and grk exhibit differences concerning cytoskeletal patterns in the follicular epithelium. During all vitellogenic stages, basal microfilaments in grk are characterised by transversal alignment, while wt-typical condensations in centripetal follicle cells (S9) and in dorsal centripetal follicle cells (S10B) are absent. Moreover, in grk, longitudinal alignment of microtubules occurs throughout vitellogenesis in all follicle cells, whereas in wt, microtubules in mainbody and posterior follicle cells exhibit a more cell-autonomous organisation. Therefore, in contrast to wt, the follicular epithelium in grk is characterised by missing or shallower electrochemical gradients and by more coordinated transcellular cytoskeletal patterns.ConclusionsOur results show that bioelectrical polarity and cytoskeletal polarity are closely linked to axial polarity in both wt and grk. When primary polarity signals are altered, both bioelectrical and cytoskeletal patterns in the follicular epithelium change. We propose that not only cell-specific levels of Vmem and pHi, or the polarities of transcellular electrochemical gradients, but also the slopes of these gradients are crucial for cytoskeletal modifications and, thus, for proper development of epithelial polarity.

Auxin Signaling-Mediated Apoplastic pH Modification Functions in Petal Conical Cell Shaping

The flowers of angiosperm species typically contain specialized conical cells. Although substantial progress has been achieved regarding the mechanisms underlying flower development, little is known about how petal cells achieve final conical shape. Here, we use 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as a fluorescent pH indicator for analyzing the apoplastic pH of conical cells in Arabidopsis and show that normal conical cell expansion requires auxin signaling and apoplastic pH changes. By combining imaging analysis and genetic and pharmacological experiments, we demonstrate that apoplastic acidification and alkalization correlate with an increase and decrease in tip sharpening of conical cells, respectively. Initial expansion of conical cells is accompanied by decreased apoplastic pH, which is associated with increased auxin signaling. Decreased auxin levels, transport, or signaling abolishes cell wall acidification and causes reduced tip sharpening and heights of conical cells. These findings provide an insight into apoplastic pH regulation of conical cell expansion.

Neuronal Glutamatergic Synaptic Clefts Alkalinize Rather Than Acidify during Neurotransmission.

The dogma that the synaptic cleft acidifies during neurotransmission is based on the corelease of neurotransmitters and protons from synaptic vesicles, and is supported by direct data from sensory ribbon-type synapses. However, it is unclear whether acidification occurs at non-ribbon-type synapses. Here we used genetically encoded fluorescent pH indicators to examine cleft pH at conventional neuronal synapses. At the neuromuscular junction of female Drosophila larvae, we observed alkaline spikes of over 1 log unit during fictive locomotion in vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only ∼0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca2+ movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca2+/H+ antiporting activity of the plasma membrane Ca2+-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage gated Ca2+ channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extrasynaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non-ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses.SIGNIFICANCE STATEMENT Neurotransmission is highly sensitive to the pH of the extracellular milieu. This is readily evident in the neurological symptoms that accompany systemic acid/base imbalances. Imaging data from sensory ribbon-type synapses show that neurotransmission itself can acidify the synaptic cleft, likely due to the corelease of protons and glutamate. It is not clear whether the same phenomenon occurs at conventional neuronal synapses due to the difficulties in collecting such data. If it does occur, it would provide for an additional layer of activity-dependent modulation of neurotransmission. Our findings of alkalinization, rather than acidification, within the cleft of two different neuronal synapses encourages a reassessment of the scope of activity-dependent pH influences on neurotransmission and short-term synaptic plasticity.

Hyperglycemia-induced cardiomyocyte death is mediated by lysosomal membrane injury and aberrant expression of cathepsin D

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. Previous studies have shown an association between lysosomal dysfunction and diabetic heart injury. The present study examined if mimicking hyperglycemia in cultured cardiomyocytes could induce lysosomal membrane permeabilization (LMP), leading to the release of lysosome enzymes and subsequent cell death. High glucose (HG) reduced the number of lysosomes with acidic pH as shown by a fluorescent pH indicator. Also, HG induced lysosomal membrane injury as shown by an accumulation of Galectin3-RFP puncta, which was accompanied by the leakage of cathepsin D (CTSD), an aspartic protease that normally resides within the lysosomal lumen. Furthermore, CTSD expression was increased in HG-cultured cardiomyocytes and in the hearts of 2 mouse models of type 1 diabetes. Either CTSD knockdown with siRNA or inhibition of CTSD activity by pepstatin A markedly diminished HG-induced cardiomyocyte death, while CTSD overexpression exaggerated HG-induced cell death. Together, these results suggested that HG increased CTSD expression, induced LMP and triggered CTSD release from the lysosomes, which collectively contributed to HG-induced cardiomyocyte injury.

2159. Accurate Carbapenem Susceptibility Testing Within 5–6 Hours

BackgroundPatients infected with multi-drug-resistant (MDR) pathogens may experience long delays to targeted therapies due to the incomplete antimicrobial menus and/or breakpoints tested on current commercial antimicrobial susceptibility testing (AST) systems. Detection of carbapenem resistance poses a challenge to rapid, accurate, minimum inhibitory concentrations (MIC) determinations because some resistant organisms may be inhibited by a carbapenem antibiotic until sufficient carbapenemase production has been achieved and traditional AST platforms must wait to make MIC calls. More accurate carbapenem MICs can be determined by implementing a carbapenemase test alongside rapid AST.MethodsWe demonstrate a novel, proprietary test to detect carbapenemase production that enables rapid MIC testing for carbapenem antibiotics. The test is processed in parallel with the Selux next-generation phenotyping (NGP) AST method, enabling rapid, <6-hour, accurate MIC determinations. The carbapenemase assay utilizes high concentrations of intact bacteria. After 3 hours of incubation, a fluorescent pH indicator is read spectroscopically. The solution pH is lowered by carbapenemase-mediated imipenem degradation and is indicative of enzyme activity.ResultsThis assay accurately identifies carabapenemases across multiple enzyme classes and bacterial species. Figure 1 shows the accuracy and speed of NGP AST at determining MICs for representative isolates from the FDA-CDC antimicrobial resistance bank compared with results from overnight broth microdilution (BMD). To date, over 100 challenge strains of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii have been tested with no very major errors and an average time-to-result of 5.3 hours.ConclusionBy incorporating a rapid, on-board carbapenemase activity assay, the NGP AST platform rapidly delivers accurate carbapenem results. Combined with NGP’s comprehensive antibiotic menus, this platform will therefore ensure prompt delivery of personalized antibiotic therapies for all patients, including those infected with MDROs, and enable streamlined antibiotic stewardship coordination. Disclosures All authors: No reported disclosures.

Fluorescent pH Indicators for Neutral to Near-Alkaline Conditions Based on 9-Iminopyronin Derivatives

Monitoring enzymatic activities at pH ranges compatible with their physiological optimum using fluorescent assays is important for high-throughput screening and engineering of novel biocatalysts as...