NADH Fluorescence Research Articles

Nadh fluorescence in the skin is a novel biomarker of hypothyroidism-associated impairment of energy metabolism

Nadh fluorescence in the skin is a novel biomarker of hypothyroidism-associated impairment of energy metabolism

Metabolic profiling of single cells by exploiting NADH and FAD fluorescence via flow cytometry

ObjectiveThe metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free. MethodsThe study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry. ResultsOxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes. ConclusionsTaken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.

Image Sensing of Gaseous Acetone Using Secondary Alcohol Dehydrogenase-Immobilized Mesh for Exhaled Air.

Human-borne acetone is a potent marker of lipid metabolism. Here, an enzyme immobilization method for secondary alcohol dehydrogenase (S-ADH), which is suitable for highly sensitive and selective biosensing of acetone, was developed, and then its applicability was demonstrated for spatiotemporal imaging of concentration distribution. After various investigations, S-ADH-immobilized meshes could be prepared with less than 5% variation by cross-linking S-ADH with glutaraldehyde on a cotton mesh at 40 °C for 15 min. Furthermore, high activity was obtained by adjusting the concentration of the coenzyme nicotinamide adenine dinucleotide (NADH) solution added to the S-ADH-immobilized mesh to 500 μM and the solvent to a potassium phosphate buffer solution at pH 6.5. The gas imaging system using the S-ADH-immobilized mesh was able to image the decrease in NADH fluorescence (ex 340 nm, fl 490 nm) caused by the catalytic reaction of S-ADH and the acetone distribution in the concentration range of 0.1-10 ppm-v, including the breath concentration of healthy people at rest. The exhaled breath of two healthy subjects at 6 h of fasting was quantified as 377 and 673 ppb-v, which were consistent with the values quantified by gas chromatography-mass spectrometry.

ПРЕДПОЛАГАЕМЫЙ МЕХАНИЗМ ВЛИЯНИЯ КРАТКОВРЕМЕННОЙ ГИПОКСИИ НА ОБОНЯТЕЛЬНУЮ ЧУВСТВИТЕЛЬНОСТЬ

The article is focused on the effect of short-term hypoxia on the olfactory sensitivity. The effect of short-term hypoxia on the olfactory sensitivity in human and rats were studied by determination thresholds for n-butanol. We have shown that reduced oxygen partial pressure leads to reduction of olfactory sensitivity. As known, odorant detection thresholds characterize the functional state of receptor cells. It can be assumed that the weakening of their sensitivity may be caused by a lack of energy supply to the olfactory perception process in hypoxia. To test this hypothesis, we stimulated rat olfactory sensory neurons with n-butanol in rotenone model. The reaction of cellular respiration of olfactory sensory neurons was evaluated by changing the intensity of intrinsic fluorescence of reduced pyridine nucleotides (NADH) according to the B. Chance method. Analysis of the results showed that under normoxia, stimulation of olfactory neurons by odorant caused an increase in the intensity of NADH fluorescence, which indicates the accumulation of the reduced form of pyridine nucleotides in mitochondria, and, consequently, the activation of cellular respiration due to exposure to odorant. In hypoxia caused by rotenone, an increase in the induced change of fluorescence of NADH was either not registered or the amplitude of the reaction was halved. This means that the recovery of NADH due to the odorant either did not occur at all, or this reaction to the smell was significantly lower than with normoxia, which indicates a weakening of cellular respiration during hypoxia. This is the reason for the decrease in ATP synthesis, which affects the efficiency of the olfactory transduction process and the sensitivity of olfactory sensory neurons. Therefore, based on our results, it can be concluded that the weakening of olfactory sensitivity in rats and humans with hypoxia may be due to a weakening of cellular respiration in olfactory sensory neurons.

Highly-selective optical filter for NADH fluorescence detection in multiphoton microscopy

Colorectal cancer (CRC) is a pressing global health concern, emphasizing the need for early detection tools. In this study an optical filter for precise detection of nicotinamide adenine dinucleotide (NADH) fluorescence via two-photon excitation fluorescence (TPEF) was developed. Fabricated with silicon dioxide and titanium dioxide thin films in a Fabry-Perot structure, the filter achieved a peak transmittance of about 95% at 483 nm, with a 12 nm full-width at half maximum. TPEF measurements using a tailored setup and NADH liquid phantoms underscored the filter's significance in selectively capturing NADH fluorescence while mitigating interference from other fluorophores. This work marks a substantial stride towards integrating multiphoton microscopy into conventional colonoscopy, enabling non-invasive, objective optical biopsy for colorectal tissue analysis. Further refinements of the experimental setup are imperative to advance tissue differentiation and enhance CRC diagnosis.

Biological evaluation of levofloxacin and its thionated derivatives: antioxidant activity, aldehyde dehydrogenase enzyme inhibition, and cytotoxicity on A549 cell line.

Levofloxacin (LVX) is among the fluoroquinolones antibiotics that has also been studied in vitro and in vivo for its anticancer effects. In this study, we used LVX and novel LVX thionated derivatives; compounds 2 and 3, to evaluate their antioxidant activity, aldehyde dehydrogenase (ALDH) enzymes activity inhibition, and anticancer activity. Combination treatments with doxorubicin (DOX) were investigated as well. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to determine the antioxidant activity. The NADH fluorescence spectrophotometric activity assay was used to determine the ALDH inhibitory effects. Resazurin dye method was applied for cell viability assays. Molecular Operating Environment software was used for the molecular docking experiments. Compared to ascorbic acid, DPPH assay showed that compound 3 had the highest antioxidant activity among the tested compounds with approximately 35% scavenging activity. On ALDH enzymes, compound 3 showed a significant ALDH activity inhibition compared to compound 2 at 200 µM. The IC50 values for the tested compounds were approximately 100 µM on A549 cell line, a non-small cell lung cancer (NSCLC) cell line. However, significant enhancement of cytotoxicity and reduction of IC50 values were observed by combining DOX and synergism was achieved with LVX with a combination index value of 0.4. The molecular docking test showed a minimum binding energy with a good affinity for compound 3 towards ALDH enzymes. Thionated LVX derivatives, may be repurposed for NSCLC therapy in combination with DOX, taking into account the antioxidant activity, ALDH activity inhibition, and the molecular docking results of compound 3.

Semiconducting polymer dots based l-lactate sensor by enzymatic cascade reaction system

Backgroundl-lactate detection is important for not only assessing exercise intensity, optimizing training regimens, and identifying the lactate threshold in athletes, but also for diagnosing conditions like L-lactateosis, monitoring tissue hypoxia, and guiding critical care decisions. Moreover, l-lactate has been utilized as a biomarker to represent the state of human health. However, the sensitivity of the present l-lactate detection technique is inadequate. ResultsHere, we reported a sensitive ratiometric fluorescent probe for l-lactate detection based on platinum octaethylporphyrin (PtOEP) doped semiconducting polymer dots (Pdots-Pt) with enzymatic cascade reaction. With the help of an enzyme cascade reaction, the l-lactate was continuously oxidized to pyruvic and then reduced back to l-lactate for the next cycle. During this process, oxygen and NADH were continuously consumed, which increased the red fluorescence of Pdots-Pt that responded to the changes of oxygen concentration and decreased the blue fluorescence of NADH at the same time. By comparing the fluorescence intensities at these two different wavelengths, the concentration of l-lactate was accurately measured. With the optimal conditions, the probes showed two linear detection ranges from 0.5 nM to 5.0 μM and 5.0 μM–50.0 μM for l-lactate detection. The limit of detection was calculated to be 0.18 nM by 3σ/slope method. Finally, the method shows good detection performance of l-lactate in both bovine serum and artificial serum samples, indicating its potential usage for the selective analysis of l-lactate for health monitoring and disease diagnosis. SignificanceThe successful application of the sensing system in the complex biological sample (bovine serum and artificial serum samples) demonstrated that this method could be used for sensitive l-lactate detection in practical clinical applications. This detection system provided an extremely low detection limit, which was several orders of magnitude lower than methods proposed in other literatures.

Abstract 5549: Multicellular spheroid structural and metabolic characterization through radial line profiling

Abstract Three-dimensional multicellular tumor spheroids have emerged as a step between in vitro and in vivo models that can offer biological insight in fields such as tumor biology. Spheroids closely resemble in vivo solid tumors regarding their heterogeneous architecture and gradients of nutrients and oxygenation. These characteristics give the spheroid model great potential for studying basic tumor biology. However, traditional characterization methods investigate changes in the discrete regions within the spheroid, limiting the amount of spatial information regarding cellular metabolism. In this study, multicellular spheroids were created through a combination of hanging drops and gentle agitation using murine RAW 264.7 macrophages and colorectal adenocarcinoma CT26 cells. Hanging drops were maintained for three days then transferred to 6-well plates and placed on an orbital shaker set to 70 RPM for seven days. Prior to metabolic imaging, spheroids were moved to a microincubator with controllable temperature and humidified gas delivery (5% CO2). A custom inverted multiphoton imaging system (Bruker) equipped with an Ultrafast Ti:Sapphire via a 60x/1.2NA water immersion objective (Olympus) and four close-proximity, high-efficiency GaAsP detectors. NADH fluorescence was captured with a 460 (± 20) nm band pass filter at 755 nm excitation and FAD fluorescence with a 525 (± 25) nm band pass filter at 855 nm excitation. NADH and FAD fluorescence were normalized by PMT gain and laser power. An integrated fluorescence lifetime imaging microscopy (FLIM) module was used to measure mitochondrial function regarding the different components contributing to NADH autofluorescence. Immunofluorescence was used to characterize macrophage populations (M1 vs M2) and cellular proliferation (Ki67) and apoptosis (CC3). CD206, a M2 marker, showed a lower normalized fluorescent intensity at the spheroid core (0.429) compared to the proliferative edge (0.570). Similar trends are also observed for CD80, a M1 marker and Ki67. CC3 showed a higher normalized fluorescent intensity at the spheroid core (0.823) compared to the proliferative edge (0.004). The optical redox ratio displays a slightly lower value at the core (0.429) compared to the proliferative edge (0.570). Overall, these results indicate that the proliferative edge shows higher numbers of immune cells and more oxidative metabolism compared to the core. Future work includes exposing this spheroid model to low levels of oxygen to investigate how environmental changes in oxygen can affect spheroid structure and metabolism. Citation Format: Shelby N. Bess, Matthew J. Igoe, Gaven K. Smart, Timothy J. Muldoon. Multicellular spheroid structural and metabolic characterization through radial line profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5549.

The use of NADH anisotropy to investigate mitochondrial cristae alignment

Life may be expressed as the flow of electrons, protons, and other ions, resulting in large potential difference. It is also highly photo-sensitive, as a large proportion of the redox capable molecules it relies on are chromophoric. It is thus suggestive that a key organelle in eukaryotes, the mitochondrion, constantly adapt their morphology as part of the homeostatic process. Studying unstained in vivo nano-scale structure in live cells is technically very challenging. One option is to study a central electron carrier in metabolism, reduced nicotinamide adenine dinucleotide (NADH), which is fluorescent and mostly located within mitochondria. Using one and two-photon absorption (340–360 nm and 730 nm, respectively), fluorescence lifetime imaging and anisotropy spectroscopy of NADH in solution and in live cells, we show that mitochondria do indeed appear to be aligned and exhibit high anisotropy (asymmetric directionality). Aqueous solution of NADH showed an anisotropy of ~ 0.20 compared to fluorescein or coumarin of < 0.1 and 0.04 in water respectively and as expected for small organic molecules. The anisotropy of NADH also increased further to 0.30 in the presence of proteins and 0.42 in glycerol (restricted environment) following two-photon excitation, suggesting more ordered structures. Two-photon NADH fluorescence imaging of Michigan Cancer Foundation-7 (MCF7) also showed strong anisotropy of 0.25 to 0.45. NADH has a quantum yield of fluorescence of 2% compared to more than 40% for photoionisation (electron generation), when exposed to light at 360 nm and below. The consequence of such highly ordered and directional NADH patterns with respect to electron ejection upon ultra-violet (UV) excitation could be very informative—especially in relation to ascertaining the extent of quantum effects in biology, including electron and photonic cascade, communication and modulation of effects such as spin and tunnelling.

3D convolutional neural networks predict cellular metabolic pathway use from fluorescence lifetime decay data.

Fluorescence lifetime imaging of the co-enzyme reduced nicotinamide adenine dinucleotide (NADH) offers a label-free approach for detecting cellular metabolic perturbations. However, the relationships between variations in NADH lifetime and metabolic pathway changes are complex, preventing robust interpretation of NADH lifetime data relative to metabolic phenotypes. Here, a three-dimensional convolutional neural network (3D CNN) trained at the cell level with 3D NAD(P)H lifetime decay images (two spatial dimensions and one time dimension) was developed to identify metabolic pathway usage by cancer cells. NADH fluorescence lifetime images of MCF7 breast cancer cells with three isolated metabolic pathways, glycolysis, oxidative phosphorylation, and glutaminolysis were obtained by a multiphoton fluorescence lifetime microscope and then segmented into individual cells as the input data for the classification models. The 3D CNN models achieved over 90% accuracy in identifying cancer cells reliant on glycolysis, oxidative phosphorylation, or glutaminolysis. Furthermore, the model trained with human breast cancer cell data successfully predicted the differences in metabolic phenotypes of macrophages from control and POLG-mutated mice. These results suggest that the integration of autofluorescence lifetime imaging with 3D CNNs enables intracellular spatial patterns of NADH intensity and temporal dynamics of the lifetime decay to discriminate multiple metabolic phenotypes. Furthermore, the use of 3D CNNs to identify metabolic phenotypes from NADH fluorescence lifetime decay images eliminates the need for time- and expertise-demanding exponential decay fitting procedures. In summary, metabolic-prediction CNNs will enable live-cell and in vivo metabolic measurements with single-cell resolution, filling a current gap in metabolic measurement technologies.

Обзор спектрофлуориметрических исследований кожных покровов

Background. Nicotinamide adenine dinucleotide (NADN) is involved in many important biochemical reactions in human metabolism, including participation in mitochondrial energy production. Mitochondrial activity, which affects skin microcirculation and the efficiency of local blood supply, can be indirectly assessed by the fluorescence of NADH in its reduced form. Changes in major skin fluorophores have been studied in both animals and humans, at rest and under different conditions, using spectrofluorimetry. The purpose of the work is, based on scientific literature data, to evaluate the possibility of using a spectrofluorimetric study of NADH levels, as well as the NADH/FAD ratio when assessing the condition of both free skin flaps and pedicled flaps after reconstructive operations, in particular in ophthalmology. Materials and methods. A search of scientific publications in PubMed, Google Scolar and Elibrary was conducted, as a result 61 articles for the period of 1957-2022 were selected for the final scientific review. Results and discussion. A brief historical background of the first spectrofluorimetric studies is presented. A brief overview of spectrofluorimetric methods for studying the skin of both humans and experimental animals, as well as other organs and systems, is given. The disadvantages and advantages of various research methods are listed. A review of changes in spectrofluorimetric parameters of the skin in patients with various somatic diseases of organs and systems was carried out. Conclusion. A review of the abovementioned methods for assessing tissue metabolism is applicable in surgical treatment, plastic surgery and onco-ophthalmology. Of particular value for surgery will be research methods that make it possible to predict the likelihood of rejection of transplanted skin flaps and their cicatricial changes. The presented literature review confirms the effectiveness of spectrofluorimetry for assessing metabolic disorders in the skin. The relevance of the method for predicting cosmetic outcomes during plastic surgery, including on the adnexal apparatus of the eye, requires continued research into the main fluorophores of the skin.

In vivo measurement of NADH fluorescence lifetime in skeletal muscle via fiber-coupled time-correlated single photon counting

Nicotinamide adenine dinucleotide (NADH) is a cofactor that serves to shuttle electrons during metabolic processes such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS). NADH is autofluorescent, and its fluorescence lifetime can be used to infer metabolic dynamics in living cells. Fiber-coupled time-correlated single photon counting (TCSPC) equipped with an implantable needle probe can be used to measure NADH lifetime in vivo, enabling investigation of changing metabolic demand during muscle contraction or tissue regeneration. This study illustrates a proof of concept for point-based, minimally-invasive NADH fluorescence lifetime measurement in vivo. Volumetric muscle loss (VML) injuries were created in the left tibialis anterior (TA) muscle of male Sprague Dawley rats. NADH lifetime measurements were collected before, during, and after a 30[Formula: see text]s tetanic contraction in the injured and uninjured TA muscles, which was subsequently fit to a biexponential decay model to yield a metric of NADH utilization (cytoplasmic vs protein-bound NADH, the A[Formula: see text]/A[Formula: see text] ratio). On average, this ratio was higher during and after contraction in uninjured muscle compared to muscle at rest, suggesting higher levels of free NADH in contracting and recovering muscle, indicating increased rates of glycolysis. In injured muscle, this ratio was higher than uninjured muscle overall but decreased over time, which is consistent with current knowledge of inflammatory response to injury, suggesting tissue regeneration has occurred. These data suggest that fiber-coupled TCSPC has the potential to measure changes in NADH binding in vivo in a minimally invasive manner that requires further investigation.

Sperm induce a secondary increase in ATP levels in mouse eggs that is independent of Ca2+ oscillations.

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred ∼1 h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations.

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

BackgroundHuman mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source.MethodsIn this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality—two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred.ResultsDuring osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, we validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and α-CHC respectively. A reduction in mineralization was found after treatment with BPTES and α-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism.ConclusionIn summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis.Graphical

Modulating Effect of Extremely High-Frequency Low-Intensity Electromagnetic Radiation on Tissue Oxidative Metabolism and Skin Microhemodynamics

We set out to examine the indicators of microhemodynamics and tissue oxidative metabolism in rats after their tenfold exposure to extremely high-frequency low-intensity electromagnetic radiation (EHF EMR). The aim was to elucidate the specifics of skin microcirculation and tissue oxidative metabolism following exposure to tenfold electromagnetic radiation of extremely high frequency. The experiment was carried out on 40 mature male Wistar rats weighing 200–220 g, which were kept in standard vivarium conditions under natural light regimen. The animals were divided into two groups with 20 rats each. The animals in the first group were biological controls and were exposed to false EHF EMR (placebo); the animals in the second group were exposed to mm-exposure in the morning, 10 sessions daily. On the 10th day of EMR exposure, the indicators of skin tissue fluorescence on the tail base were recorded. Tenfold exposure to low-intensity EHF EMR was shown to increase the concentration and intensity of NADH fluorescence, as well as the FAD and redox ratio. This indicates an increased cellular demand for ATP and the predominance of oxidative phosphorylation over other processes, thus demonstrating the activation of the respiratory chain. At the same time, an increase in endothelium-dependent vasodilation, a decrease in peripheral resistance, an increase in blood flow to the nutritive microvascular bed, and an improvement in venular outflow were observed in the microcirculatory bed. The conclusion is made that the modulating effect of EHF EMR is manifested in the rearrangements of correlations. Thus, the coefficient of variation comes to the fore – the final calculated indicator of microcirculation, which has strong negative associations with all indicators of tissue metabolism (FAD, NADH, RR). In addition, the amplitudes of endothelial rhythms associated with periodic releasing of nitric oxide by the endothelium show a strong negative association with FAD.

Near-infrared absorption and emission probes with optimal connection bridges for live monitoring of NAD(P)H dynamics in living systems

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Probe solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 µM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe A in sensing changes in NAD(P)H levels within a whole organism.

Live-Cell Imaging Quantifies Changes in Function and Metabolic NADH Autofluorescence During Macrophage-Mediated Phagocytosis of Tumor Cells

ABSTRACT Background The immune system has evolved to detect foreign antigens and deliver coordinated responses, while minimizing “friendly fire.” Until recently, studies investigating the behavior of immune cells were limited to static in vitro measurements. Although static measurements allow for real-time imaging, results are often difficult to translate to an in vivo setting. Multiphoton microscopy is an emerging method to capture spatial information on subcellular events and characterize the local microenvironment. Previous studies have shown that multiphoton microscopy can monitor changes in single-cell macrophage heterogeneity during differentiation. Therefore, there is a need to use multiphoton microscopy to monitor molecular interactions during immunological activities like phagocytosis. Here we investigate the correlation between phagocytic function and changes in endogenous optical reporters during phagocytosis. Methods In vitro autofluorescence imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) was used to detect metabolic changes in macrophages during phagocytosis. More specifically, optical redox ratio, mean NADH fluorescence lifetime and ratio of free to protein-bound NADH were used to quantify changes in metabolism. Results Results show that IFN-γ (M1) macrophages showed decreased optical redox ratios and mean NADH lifetime while phagocytosing immunogenic cancer cells compared to metastatic cells. To validate phagocytic function, a fluorescence microscopy-based protocol using a pH-sensitive fluorescent probe was used. Results indicate that M0 and M1 macrophages show similar trends in phagocytic potential. Conclusion Overall, this work demonstrates that in vitro multiphoton imaging can be used to longitudinally track changes in phagocytosis and endogenous metabolic cofactors.

EXTH-83. BIRC3 AS A PREDICTOR OF OUTCOME AND A NEW THERAPEUTIC TARGET IN TMZ RESISTANT GLIOBLASTOMA TUMORS

Abstract Glioblastoma (GBM) is an incurable malignant brain tumor, with a highly unfavorable prognosis. Apart from MGMT promoter methylation and TMZ response, molecular biomarkers associated with therapeutic response or survival prognosis are still lacking in GBM. Previously, using NAD(P)H-fluorescence lifetime imaging, a new drug screening precision medicine ex-vivo approach, we perturbed patients-derived vital GBM tumor explants (GB-EXPs) by TMZ treatment and obtained a stratification in TMZ responder (Res) and non-responder (Non-Resp) tumors, identifying several genes differentially expressed between the two groups. To validate the approach we enlarged our glioblastoma case study. Transcriptome analysis was performed to characterize TMZ Res and Non-Resp cases. In-silico and functional cellular investigations were carried on to explore the biomarker's efficiency. The majority of the genes found dysregulated in the previous study, showed the same directional regulation of expression and we confirmed, in TMZ-resistant samples the statistically significant upregulation of BIRC3, a strong inhibitor of apoptosis. The TCGA dataset provided an opportunity to fully assess the BIRC3 mRNA expression complementarity with the MGMT status as a predictor of outcome. BIRC3 was validated as a prognostic factor of survival also in large French and Italian glioblastoma cohorts. Most importantly, AZD5582 drug, which is a specific antagonist of BIRC3, combined with TMZ treatment, is able to reverse the TMZ resistance, in glioblastoma cell lines, by restoring apoptosis, and in GB-EXPs that were resistant to TMZ treatment. BIRC3 represents a potential biomarker of prognosis and a predictor of TMZ treatment response in GBM and complements MGMT status as an outcome predictor. The level of BIRC3 expression could stratify patients who will benefit from AZD5582 or its combination with TMZ. Our study provides a potentially new treatment approach and valuable leads to determine whether functional precision medicine combined with BIRC3 expression assessment become a standard tool in glioblastoma clinical oncology.

Investigating Carvedilol's Repurposing for the Treatment of Non-Small Cell Lung Cancer via Aldehyde Dehydrogenase Activity Modulation in the Presence of β-Adrenergic Agonists.

Repurposing existing drugs appears to be a potential solution for addressing the challenges in the treatment of non-small cell lung cancer (NSCLC). β-adrenoceptor antagonist drugs (β-blockers) have tumor-inhibiting effects, making them promising candidates for potential NSCLC treatment. This study investigates the anticancer potential of a subset of β-blockers in NSCLC cell lines; A549 and H1299. Additionally, it investigates the underlying mechanism behind β-blockers' anticancer effect by influencing a potential novel target named aldehyde dehydrogenase (ALDH). The MTT assay assessed β-blockers' cytotoxicity on both cell lines, while Western blot and NADH fluorescence assays evaluated their influence on ALDH protein expression and activity. Carvedilol (CAR) was the most effective blocker in reducing cell survival of A549 and H1299 with IC50 of 18 µM and 13.7 µM, respectively. Significantly, CAR led to a 50% reduction in ALDH expression and 80% decrease in ALDH activity in A549 cells, especially when combined with β-agonists, in comparison to the control. This effect might be attributed to β-agonist blockade or an alternative pathway. This novel finding adds to our understanding of CAR's multifaceted anticancer properties, implying that combining CAR with β-agonists could be a useful strategy for lung cancer treatment.

Arterial Blood Pressure Features of Hypertensive Patients with Typical and Atypical 460 nm Skin Fluorescence Response to Transient Ischaemia.

Flow-mediated skin fluorescence (FMSF) at 460 nm is a non-invasive method for assessing dynamic changes in the reduced form of nicotinamide adenine dinucleotide (NADH) and microcirculation in forearm skin under varying conditions of tissue perfusion. Typically, fluorescence increases during ischaemia, but atypical cases show a temporary signal decrease instead of a constant increase. This study aimed to explore the clinical implications of atypical FMSF patterns in patients with newly diagnosed untreated hypertension. NADH fluorescence and pulse wave analysis were performed on 65 patients. Differences in peripheral and arterial pulse pressure profiles were examined based on FMSF curve courses. Patients with atypical curve courses had significantly (p < 0.05 or lower for all) higher heart rate, peripheral and central diastolic pressure, tension time index, central rate pressure product, shorter diastole duration, and reservoir pressure-time integral. Hypertensive patients with atypical FMSF signals had less advantageous blood pressure profiles. Although the underlying factors causing these symptoms are unknown, the atypical FMSF pattern may reflect increased sympathetic stimulation and vascular resistance. The visual assessment of the FMSF curve may have important clinical implications that deserve further investigation.