pH-sensitive Dye Research Articles

In vivo targeted-imaging of mitochondrial acidification in an aristolochic acid I-induced nephrotoxicity mouse model by a fluorescent/photoacoustic bimodal probe

Aristolochic acid I (AAI), a natural compound in aristolochia type Chinese medicinal herb, is generally acknowledged to have nephrotoxicity, which may be associated with mitophagy. Mitophagy is a cellular process with important functions that drive AAI-induced renal injury. Mitochondrial pH is currently measured by fluorescent probes in cell culture, but existing probes do not allow for in situ imaging of AAI-induced mitophagy in vivo. We developed a ratiometric fluorescent/PA dual-modal probe with a silicon rhodamine fluorophore and a pH-sensitive hemicyanine dye covalently linked via a short chain to obtain a FRET type probe. The probe was used to measure AAI-mediated mitochondrial acidification in live cells and in vivo. The Förster resonance energy transfer (FRET)-mediated ratiometric and bimodal method can efficiently eliminate signal variability associated with the commonly used one-emission and single detection mode by ratiometric two channels of the donor and acceptor. The probe has good water-solubility and low molecular weight with two positively charged, facilitating its precise targeting into renal mitochondria, where the fluorescent/PA changes in response to mitochondrial acidification, enabling dynamic and semi-quantitative mapping of subtle changes in mitochondrial pH in AAI-induced nephrotoxicity mouse model for the first time. Also, the joint use of L-carnitine could mitigate the mitophagy in AAI-induced nephrotoxicity.

Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies.

Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones.

Alkaline range pH sensor based on chitosan hydrogel: A novel approach to pH sensing

Monitoring of pH under extreme alkaline range is still a challenge due to the lack of accuracy and validity. This research developed a novel pH sensor (hydrogel/BTB) based on the transition of bromothymol blue from the hydrogel matrix into the pH-examining sample solution. The hydrogel/BTB sensor was synthesized through the solvent casting of chitosan, citric acid as the crosslinker, and bromothymol blue as a pH-sensitive dye. The structure of hydrogel/BTB was characterized using Fourier-transform infrared spectroscopy (FT-IR), Energy-dispersive X-ray spectroscopy (EDS), Field emission scanning electron microscopy (FE-SEM), Brunauer-Emmett-Teller (BET) analysis, and thermogravimetric analysis (TGA). The effect of various parameters on pH determination was investigated. The developed pH sensor demonstrated a linear detection range validated from pH 10 to 14 using the gravimetric method, and from pH 11 to 14 using the colorimetric method. The sensor successfully detected pH in alkaline tap water, carbonate buffer, and ethanol amine buffer. The transition of bromothymol blue is described by the Peppas-Korsmeyer kinetic model. The activation and Gibbs free energy were obtained as 357.1 J/mol and 260 J/mol, respectively. This work furnished a new mechanism for pH detection, and it has excellent potential for developing novel sensors in this field.

A versatile pH-sensitive hydrogel based on a high-performance dye: Monitoring the freshness of milk and chicken meat

There is a growing interest in soft-matter sensors at the research and application level. Colorimetric hydrogel-based sensors can work as smart tools for the fast, simple, and low-cost monitoring of food freshness and spoilage. Herein we developed a hydrogel pH indicator working from pH 2–12 with a specific colorimetric pattern. A fast-responsive, easily formed and mouldable hydrogel was obtained by adding a minimal amount of a single pH-sensitive dye to an agar matrix. Optical, mechanical, and morphologic characteristic of the hydrogel were examined in detail. An easy-to-use smartphone app ensures color tone capture and correlation of color changes to pH levels. Practical applications of the versatile gel sensor were tested in solution and in the gaseous phase. The sensor was employed to assess the freshness of milk samples and to monitor the volatile basic nitrogen compounds resulting from packaged chicken spoilage.

Abstract We123: Cardiomyocyte Specific Sustained Atg7 Overexpression Activates Mitochondrial Autophagy in the Heart

Introduction: Accumulating evidence indicates that autophagy-related protein 7 (Atg7) is essential in cellular autophagy. Our earlier studies showed that Atg7 is required to promote macro-autophagy in the heart. However, it is unknown if the activation of Atg7 has a role in the heart to regulate selective mitochondrial autophagy (mitophagy). Objectives: We employed in vitro and in vivo approaches to delineate the molecular function and mechanisms of Atg7-mediated activation of mitophagy in the heart. Methods and Results: Subcellular fractionation and quantum dot-labeled electron microscopy revealed Atg7’s localization on the mitochondrial fraction in the heart. Atg7 co-localization to Mitotracker-labeled mitochondria was confirmed using isolated heart mitochondria. To quantitate the mitophagy, we overexpressed Atg7 in cultured primary cardiomyocytes, treated with cyanide 3-chlorophenylhydrazone (CCCP) to induce mitophagy, and stained with pH-sensitive fluorescent mitophagy dye to enumerate mitophagy dots by microscopy. We found significantly increased mitophagy dots in Atg7 overexpressed cardiomyocytes than in β-gal expressing control. To assess the activation of mitophagy in vivo , we treated the cardiac-specific Atg7 overexpressing transgenic (αMHC-Atg7 Tg) and their littermate non-transgenic (Ntg) mice with CCCP. We found an increased accumulation of mature autophagolysosomal marker protein LC3-II in isolated mitochondria from Atg7 Tg mice hearts post-CCCP treatment. To assess the extent of mitochondrial lysosomal delivery in Atg7 overexpressing hearts, we crossed αMHC-Atg7 Tg mice with cardiac-specific Mito-Keima transgenic (Mt-Keima Tg) mice. Mt-Keima Tg mice bear mitochondria-targeted pH-dependent fluorescent lysosomal protease-resistance Keima protein, enabling the measurement of mitophagy activation in vivo . We observed increased Mt-Keima high red-to-green areas in αMHC-Atg7xMt-Keima Tg mice hearts compared to Mt-Keima mice. Conclusions: Our biochemical and genetic approaches revealed that Atg7 localizes to the mitochondria, leading to the recruitment and initiation of mitophagy machinery. Our data further confirm that genetic activation of Atg7 leads to increased mitophagy activity in the heart.

Assessing the mechanism of facilitated proton transport across GUVs trapped in a microfluidic device

Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H+ permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin.

Characterization of PLGA versus PEG-PLGA intracochlear drug delivery implants: Degradation kinetics, morphological changes, and pH alterations

Drug delivery to the inner ear presents a unique challenge due to the complex inner ear anatomy and its tight physiological barriers. This study investigates the degradation behavior of intracochlear drug delivery implants (IDDI) composed of dexamethasone and poly(lactic-co-glycolic acid) (PLGA) or polyethylene glycol–poly(lactic-co-glycolic acid) (PEG-PLGA), respectively. IDDI were incubated in artificial perilymph and implants' degradation kinetics, morphological changes, water uptake behavior, and pH alterations were assessed. Microscopy revealed significant changes in appearance, with PLGA IDDI exhibiting rapid expansion, reaching up to 183 % in diameter and 185 % in length. PEG-PLGA implants showed gradual expansion, reaching a maximum of 178 % in diameter and 144 % in length. Despite these morphological changes, the IDDIs could still be applicable in terms of cochlear dimensions in combination with cochlear implants (CI) in humans or in a domestic pig animal model. Scanning electron microscopy analysis demonstrated surface alterations of PLGA implants, while PEG-PLGA implants remained shape-stable. Gravimetric analysis and gel permeation chromatography revealed distinct degradation profiles, with PLGA implants displaying rapid water uptake and mass loss, while PEG-PLGA implants showed delayed water uptake and minimal mass reduction. pH measurements using the pH-sensitive fluorescent dye SNARF™-1 showed initial pH reduction in artificial perilymph for PLGA implants while PEG-PLGA implants maintained pH stability.

Point-of-Care Molecular Testing with tinto rangTM: A Food Grade Safe Fluorophore for Colorimetric LAMP Assays at Low Resource Settings

LAMP (loop-mediated isothermal amplification) has proven to be a highly robust, sensitive, and cost-effective method for detecting specific target nucleic acid sequences in a single-tube reaction without the need for sophisticated instruments. Traditionally, pH-sensitive dyes, DNA-binding dyes like Ethidium Bromide(EtBr), and SYBR have been employed to detect amplified products. However, these dyes present several drawbacks, limiting the widespread adoption of LAMP in diagnostic applications. To address this limitation, a novel dye called tinto rangTM has been introduced with promising applications in nucleic acid analysis. In this study, we present an evaluation of tinto rangTM's performance in comparison to commercially available dyes commonly used in LAMP assays. One of the unique advantages of tinto rangTM is its capability to facilitate the analysis of amplified nucleic acid products through three distinct methods: visual color change, changes in relative fluorescence, and turbidity. In addition to assessing tinto rangTM's performance, we also investigated various approaches to tackle the issue of non-specific amplification, which has been a common challenge in LAMP reactions. The experimental results unequivocally demonstrate that tinto rangTM outperforms pH-indicating and other DNA-binding dyes used in LAMP assays, making it a superior alternative. This study paves the way for the broader adoption of tinto rangTM in various applications, including point-of-care diagnostics, disease monitoring, and molecular biology research.

In situ encapsulation of anthocyanin within soybean protein isolate chains driven by pH for detecting the freshness of food

Intelligent indicator labels have been widely demonstrated as functional material to real-time monitor the freshness of food, whereas the migration problem of natural pH-sensitive dyes from the matrix into surface of packaging film restricted the practical application. Herein, a facile in situ encapsulation strategy was proposed to design and fabricate soybean protein isolate (SPI)/carboxymethyl cellulose sodium (CMC)/anthocyanin (Anth) (SCA) composite film as smart labels to monitor the freshness of protein-based foods. Inspired by the stimulation-response behaviors of Mimosa, Anth was in situ wrapped by the spatial structural mutation of SPI chains driven by different pH conditions, in which tannic acid (TA) served as pH modulator. Compared to CMC/Anth composite film free of pH modulation, the cumulative release of Anth from SCA-7.0 composite film was decreased from 90.97%, 82.45% and 70.52% to 64.14%, 53.88% and 39.45% in distilled water, 10% ethanol and 90% ethanol, respectively. Furthermore, the prepared SCA-7.0 composite film possessed reinforced UV-shielding, gas barrier and water resistance performances. When employed as intelligent label, this composite film can be an intelligent indicator label for real-time meat freshness monitoring. A facile strategy was carried out to in situ encapsulate the Anth within matrix, boosting the reliability and lifespan of intelligent packaging in practical application.

Recent advancements in chitosan-based intelligent food freshness indicators: Categorization, advantages, and applications

With an increasing emphasis on food safety and public health, there is an ongoing effort to develop reliable, non-invasive methods to assess the freshness of diverse food products. Chitosan-based food freshness indicators, leveraging properties such as biocompatibility, biodegradability, non-toxicity, and high stability, offer an innovative approach for real-time monitoring of food quality during storage and transportation. This review introduces intelligent food freshness indicators, specifically those utilizing pH-sensitive dyes like anthocyanins, curcumin, alizarin, shikonin, and betacyanin. It highlights the benefits of chitosan-based intelligent food freshness indicators, emphasizing improvements in barrier and mechanical properties, antibacterial activity, and composite film solubility. The application of these indicators in the food industry is then explored, alongside a concise overview of chitosan's limitations. The paper concludes by discussing the challenges and potential areas for future research in the development of intelligent food freshness indicators using chitosan. Thus, chitosan-based smart food preservation indicators represent an innovative approach to providing real-time data for monitoring food quality, offering valuable insights to both customers and retailers, and playing a pivotal role in advancing the food industry.

A real-time antibody-dependent cellular phagocytosis assay by live cell imaging

Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated in vitro. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.

Differentially Charged Liposomes Stimulate Dendritic Cells with Varying Effects on Uptake and Processing When Used Alone or in Combination with an Adjuvant.

Liposomes carrying differential charges have been extensively studied for their role in stimulating dendritic cells (DCs), major antigen-presenting cells, known to serve as a pivotal bridge between innate and adaptive immunity. However, the impact of the differentially charged liposomes on activating DCs remains to be understood. In this study, we have investigated the impact of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based neutral, anionic, and cationic liposomes on the uptake, immunostimulation, and intracellular fate in mouse bone-marrow-derived DCs. We observed that liposomes could induce phenotypic maturation of DCs by inducing the expression of costimulatory molecules (CD40 and CD86) and production of cytokines tumor necrosis factor-α, interleukin-12,and nitric oxide. Interestingly, admixing monophosphoryl lipid A with charged liposomes further enhances the expression of the costimulatory molecules and production of cytokines, with preferential activation by positively charged liposomes. Fluorometric analysis using a pH-sensitive dye and flow-cytometry-based pathway inhibition assays revealed that cationic liposomes were taken up more efficiently by DCs through endocytosis and transported to neutral compartments for further processing, whereas anionic and neutral liposomes were inclined to accumulate in acidic compartments. These findings therefore endorse the use of cationic DSPC liposomes as a preferred option for vaccine delivery vehicles over neutral and negatively charged liposomes, particularly for the preferential activation of DCs.

Development of a Sensitive Colorimetric Indicator for Detecting Beef Spoilage in Smart Packaging.

This study aimed to fabricate and characterize a novel colorimetric indicator designed to detect ammonia (NH3) and monitor meat freshness. The sensing platform was constructed using electrospun nanofibers made from polylactic acid (PLA), which were then impregnated with anthocyanins as a natural pH-sensitive dye, extracted from red cabbage. This research involved investigating the relationship between the various concentrations of anthocyanins and the colorimetric platform's efficiency when exposed to ammonia vapor. Scanning electron microscope (SEM) results were used to examine the morphology and structure of the nanofiber mats before and after the dip-coating process. The study also delved into the selectivity of the indicator when exposed to various volatile organic compounds (VOCs) and their stability under extreme humidity levels. Furthermore, the platform's sensitivity was evaluated as it encountered ammonia (NH3) in concentrations ranging from 1 to 100 ppm, with varying dye concentrations. The developed indicator demonstrated an exceptional detection limit of 1 ppm of MH3 within just 30 min, making it highly sensitive to subtle changes in gas concentration. The indicator proved effective in assessing meat freshness by detecting spoilage levels in beef over time. It reliably identified spoilage after 10 h and 7 days, corresponding to bacterial growth thresholds (107 CFU/mL), both at room temperature and in refrigerated environments, respectively. With its simple visual detection mechanism, the platform offered a straightforward and user-friendly solution for consumers and industry professionals alike to monitor packaged beef freshness, enhancing food safety and quality assurance.

Acidochromic Free-Standing Multilayered Chitosan-Pyranoflavylium/Alginate Membranes toward Food Smart Packaging Applications.

Food smart packaging has emerged as a promising technology to address consumer concerns regarding food conservation and food safety. In this context, we report the rational design of azide-containing pyranoflavylium-based pH-sensitive dye for subsequent click chemistry conjugation toward a chitosan-modified alkyne. The chitosan-pyranoflavylium conjugate was characterized by infrared (ATR-FTIR), ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) spectroscopies, and dynamic light scattering (DLS), as well as its thermodynamic parameters related to their pH-dependent chromatic features. The fabrication of thin-films through electrostatic-driven layer-by-layer (LbL) assembly technology was first screened by quartz crystal microbalance with dissipation monitoring (QCM-D) onto gold substrates, and then free-standing (FS) multilayered membranes from polypropylene substrate were obtained using a homemade automatic dipping robot. The membranes' characterization included morphology analysis and thickness evaluation, assessed by scanning electron microscopy (SEM), pH-responsive color change performance tests using buffer solutions at different pH levels, and biogenic amines-enriched model solutions, demonstrating the feasibility and effectiveness of the chitosan-pyranoflavylium/alginate biomembranes for food spoilage monitoring. This work provides insights toward the development of innovative pH-responsive smart biomaterials for advanced and sustainable technological packaging solutions, which could significantly contribute to ensuring food safety and quality, while reducing food waste.

Reusable, Green, Portable Ionogels Based on Terpyridine-Imidazole Salt for Visual Monitoring of Pork Spoilage.

Ionogels have emerged as a promising approach because they combine the advantageous properties of ionic liquids and gels. Herein, a novel gelator bearing terpyridine and imidazolium salt units was designed and synthesized, which assembled into ionogels in three ionic liquids by a heating-cooling procedure. The properties of ionogels were characterized by FT-IR, UV-vis spectroscopy, POM, XRD, and rheology, and resonance light scattering and opacity measurements were conducted to investigate the gelation kinetics. Furthermore, the ionogels incorporating pH-sensitive dyes (BTB and MR) were exploited as colorimetric sensor to monitor total volatile basic nitrogen (TVB-N) of meat at -4 °C, which can easily and reliably estimate the quality of meat by naked eye recognition, and the results demonstrated a positive correlation between the color variation and TVB-N levels. Notably, the hydrophobic ionogel indicators are more suitable for potential application at high humidity thanks to their antiswelling advantage, which could prevent the inaccurate information produced by hydrogel indicators. In addition, the ionogels could be reused up to three times as colorimetric indicators, suggesting potential applications and competitiveness. Our research sheds new light on the novel application of ionogels in the food industry.

Acidification of α-granules in megakaryocytes by vacuolar-type adenosine triphosphatase is essential for organelle biogenesis

BackgroundPlatelets coordinate blood coagulation at sites of vascular injury and play fundamental roles in a wide variety of (patho)physiological processes. Key to many platelet functions is the transport and secretion of proteins packaged within α-granules, organelles produced by platelet precursor megakaryocytes. Prominent among α-granule cargo are fibrinogen endocytosed from plasma and endogenously synthesized von Willebrand factor. These and other proteins are known to require acidic pH for stable packaging. Luminal acidity has been confirmed for mature α-granules isolated from platelets, but direct measurement of megakaryocyte granule acidity has not been reported. ObjectivesTo determine the luminal pH of α-granules and their precursors in megakaryocytes and assess the requirement of vacuolar-type adenosine triphosphatase (V-ATPase) activity to establish and maintain the luminal acidity and integrity of these organelles. MethodsCresyl violet staining was used to detect acidic granules in megakaryocytes. Endocytosis of fibrinogen tagged with the pH-sensitive fluorescent dye fluorescein isothiocyanate was used to load a subset of these organelles. Ratiometric fluorescence analysis was used to determine their luminal pH. ResultsWe show that most of the acidic granules detected in megakaryocytes appear to be α-granules/precursors, for which we established a median luminal pH of 5.2 (IQR, 5.0-5.5). Inhibition of megakaryocyte V-ATPase activity led to enlargement of cargo-containing compartments detected by fluorescence microscopy and electron microscopy. ConclusionThese observations reveal that V-ATPase activity is required to establish and maintain a luminal acidic pH in megakaryocyte α-granules/precursors, confirming its importance for stable packaging of cargo proteins such as von Willebrand factor.

Extrusion of Sodium Hypochlorite in Oval-Shaped Canals: A Comparative Study of the Potential of Four Final Agitation Approaches Employing Agarose-Embedded Mandibular First Premolars.

Background: The aim of this investigation was to assess the apical extrusion potential of sodium hypochlorite (NaOCl) in agarose-embedded mandibular first premolars employing four final agitation procedures. Methods: Based on CBCT confirmation of single oval-shaped canals, one hundred extracted mandibular first premolars were chosen. Using 5.25% NaOCl, the teeth were prepared using the XP Endo Shaper and divided into experimental and control groups. The following were the experimental groups: Group 1 comprised the XP-Endo Finisher, Group 2 the Ultrasonic Activation, Group 3 the Gentle File Finisher Brush, and Group 4 the 27-gauge side-vented needle. To test extrusion, the teeth were placed in a 0.2% agarose gel that contained the pH-sensitive dye m-cresol purple, allowing pixel quantification via ImageJ software (version 1.54i). Results: The XP Endo Finisher featured the most pixels, depicting higher apical extrusion (p < 0.01), followed by the side-vented needle, Gentle File Finisher Brush, and PUI, while the Control Group endured no extrusion. Conclusions: The effective irrigation method for root canal therapy is critical, especially in situations of open apices, resorption, or perforation. According to in vitro experiments, the XP-Endo Finisher has the maximum sodium hypochlorite extrusion, which is determined by parameters such as apical preparation size and irrigation system.

Innovative PNA-LB mediated allele-specific LAMP for KRAS mutation profiling on a compact lab-on-a-disc device

Tailored healthcare, an approach focused on individual patients, requires integrating emerging interdisciplinary technologies to develop accurate and user-friendly diagnostic tools. KRAS mutations, prevalent in various common cancers, are crucial determinants in selecting patients for novel KRAS inhibitor therapies. This study presents a novel state-of-the-art Lab-on-a-Disc system utilizing peptide nucleic acids-loop backward (PNA-LB) mediated allele-specific loop-mediated isothermal amplification (LAMP) for detecting the frequent G12D KRAS mutation, signifying its superiority over alternative mutation detection approaches. The designed Lab-on-a-Disc system demonstrated exceptional preclinical and technical precision, accuracy, and versatility. By applying varying cutoff values to PNA- LB LAMP reactions, the assay's sensitivity and specificity were increased by 80 % and 90 %, respectively. The device's key advantages include a robust microfluidic Lab-on-a-Disc design, precise rotary control, and a cutting-edge induction heating module. These features enable multiplexing of LAMP reactions with high reproducibility and repeatability, with CV% values less than 3.5 % and 5.5 %, respectively. The device offers several methods for accurate endpoint result detection, including naked-eye observation, RGB image analysis using Python code, and time of fluorescence (Tf) values. Preclinical specificity and sensitivity, assessed using different cutoffs for Eva-Green fluorescence Tf values and pH-sensitive dyes, demonstrated comparable performance to the best standard methods. Overall, this study represents a significant step towards tailoring treatment strategies for cancer patients through precise and efficient mutation detection technologies.

Multiparameter Sensing of Oxygen and pH at Biological Interfaces via Hyperspectral Imaging of Luminescent Sensor Nanoparticles.

Chemical dynamics in biological samples are seldom stand-alone processes but represent the outcome of complicated cascades of interlinked reaction chains. In order to understand these processes and how they correlate, it is important to monitor several parameters simultaneously at high spatial and temporal resolution. Hyperspectral imaging is a promising tool for this, as it provides broad-range spectral information in each pixel, enabling the use of multiple luminescent indicator dyes, while simultaneously providing information on sample structures and optical properties. In this study, we first characterized pH- and O2-sensitive indicator dyes incorporated in different polymer matrices as optical sensor nanoparticles to provide a library for (hyperspectral) chemical imaging. We then demonstrate the successful combination of a pH-sensitive indicator dye (HPTS(DHA)3), an O2-sensitive indicator dye (PtTPTBPF), and two reference dyes (perylene and TFPP), incorporated in polymer nanoparticles for multiparameter chemical imaging of complex natural samples such as green algal biofilms (Chlorella sorokiniana) and seagrass leaves (Zostera marina) with high background fluorescence. We discuss the system-specific challenges and limitations of our approach and further optimization possibilities. Our study illustrates how multiparameter chemical imaging with hyperspectral read-out can now be applied on natural samples, enabling the alignment of several chemical parameters to sample structures.

Abstract LB189: Elucidating a mechanistic link between progranulin and lysosomal function in SW13 cells: Implications for inflammation and lipid metabolism

Abstract The SW13 human adrenocortical carcinoma line is a uniquely powerful model in which to probe the relationship between progranulin processing and cellular activity as it exists in two epigenetically distinct subtypes. The SW13- subtype has an epithelial morphology and is highly proliferative, while the SW13+ subtype has a mesenchymal-like phenotype and a higher metastatic potential. These two subtypes both express progranulin and interconvert by epigenetic mechanisms, as treatment of SW13- cells with histone deacetylase inhibitors (HDACi) adopt a SW13+ phenotype. Progranulin is composed of 7½ highly conserved granulin repeats which are subject to proteolytic processing and may serve as a prognostic biomarker in cancer as progranulin units regulate varied cellular activities including proliferation, migration, inflammation, and lysosomal function. Thus, biological activity of progranulin is dependent on the cellular proteases expressed. Indicative of differential processing, treatment of SW13- cells with exogenous progranulin results in an increase in growth rate, while no effect on SW13+ cell growth was observed. As the SW13+ subtype expresses a higher amount of progranulin than the SW13- subtype, these data are consistent with an inability of SW13+ to process endogenous progranulin to mitogenic granulin unit(s). Immunoblotting with granulin-specific antibodies indicates that progranulin may be processed differently in each subtype. In this work, we focused on lysosomes as a potential site for differential processing of progranulin for two reasons. First, lysosomal proteases have been shown to process progranulin and progranulin itself plays a role in lysosome maturation. Second, differences in lysosomal pH between the two subtypes have been detected using two pH-sensitive dyes which localize to the lysosome. Interestingly, proteomic analysis of lysosomes isolated from each subtype confirms lysosomal dysfunction and suggests upregulation of inflammatory proteins in SW13+ cells. Both proteomics and lipidomics suggest alterations in lipid metabolism. Ongoing experiments will address whether restoration of lysosomal function impacts progranulin production and/or processing in SW13 subtypes. Citation Format: Kathryn J. Leyva, Andrew Z. Yang, Pegah Biparva, Chika Okonya, Chandana K. Uppalapati, Alisha M. Harrison, Agnes S. Pascual, Elizabeth E. Hull. Elucidating a mechanistic link between progranulin and lysosomal function in SW13 cells: Implications for inflammation and lipid metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB189.