Form Of Nicotinamide Adenine Dinucleotide Research Articles

Enzyme-Mediated Bioorthogonal Cascade Catalytic Reaction for Metabolism Intervention and Enhanced Ferroptosis on Neuroblastoma.

It remains a tremendous challenge to explore effective therapeutic modalities against neuroblastoma, a lethal cancer of the sympathetic nervous system with poor prognosis and disappointing treatment outcomes. Considering the limitations of conventional treatment modalities and the intrinsic vulnerability of neuroblastoma, we herein develop a pioneering sequential catalytic therapeutic system that utilizes lactate oxidase (LOx)/horseradish peroxidase (HRP)-loaded amorphous zinc metal-organic framework, named LOx/HRP-aZIF, in combination with a 3-indole-acetic acid (IAA) prodrug. On the basis of abnormal lactate accumulation that occurs in the tumor microenvironment, the cascade reaction of LOx and HRP consumes endogenous glutathione and a reduced form of nicotinamide adenine dinucleotide to achieve the first stage of killing cancer cells via antioxidative incapacitation and electron transport chain interference. Furthermore, the generation of reactive oxygen species induced by HRP and IAA through bioorthogonal catalysis promotes ferritin degradation and lipid peroxidation, ultimately provoking self-enhanced ferroptosis with positive feedback by initiating an endogenous Fenton reaction. This work highlights the superiority of the natural enzyme-dependent cascade and bioorthogonal catalytic reaction, offering a paradigm for synergistically enzyme-based metabolism-ferroptosis anticancer therapy.

Molecular mechanism of NAD+ and NMN binding to the Nudix homology domains of DBC1

Deleted in breast cancer 1 (DBC1) is a human nuclear protein that modulates the activities of various proteins involved in cell survival and cancer progression. Oxidized form of nicotinamide adenine dinucleotide (NAD+) is suggested to bind to the Nudix homology domains (NHDs) of DBC1, thereby regulating DBC1-Poly (ADP-ribose) polymerase 1 (PARP1) interactions, resulting in the restoration of DNA repair. Using Nuclear Magnetic Resonance (NMR) and Isothermal Titration Calorimetry (ITC), we confirmed NAD+ and its precursor nicotinamide mononucleotide (NMN) both bind the NHD domain of DBC1 (DBC1354–396). NAD+ likely interacts with DBC1354–396 through hydrogen bonding, with a binding affinity (8.99 μM) nearly twice that of NMN (17.0 μM), and the key binding sites are primarily residues E363 and D372, in the agreement with Molecular Docking experiments. Molecular Dynamics (MD) simulation further demonstrated E363 and D372's anchoring role in the binding process. Additional mutagenesis experiments of E363 and D372 confirmed their critical involvement of ligand-protein interactions. These findings lead to a better understanding of how NAD+ and NMN regulate DBC1, thereby offering insights for the development of targeted therapies and drug research focused on DBC1-associated tumors.

Pickering emulsion stabilized with charge separation and transfer modulated photocatalyst for enzyme-photo-coupled catalysis

Biocatalytic reduction is extensively employed in chemical and pharmaceutical industries due to its notable advantages including high activity, selectivity and mild reaction conditions, however, the stoichiometric use of the expensive NADH (reduced form of nicotinamide adenine dinucleotide) hinders its widespread application. Despite the persisting challenges of low charge separation efficiency and limited solubility of organic substrates in aqueous phase, in situ photocatalytic NADH regeneration remains a promising solution. Herein, we report the construction of an enzyme-photo-coupled catalytic system in Pickering emulsion droplet, which was stabilized by a charge separation and transfer modulated photocatalyst. The photocatalyst integrated with visible light driven conjugated polymer, TiO2 and electron mediator greatly enhanced the cascade electron transfer from photocatalyst to NAD+. Consequently, NADH production rate over the integrated photocatalyst reaches as high as 2.4 mmol·g−1·h−1 under visible light irradiation, 12 folds higher than the corresponding physical mixture. Furthermore, the enzyme-photo-coupled catalytic system was constructed with alcohol dehydrogenase/NAD+ confined in the emulsion droplet and photocatalyst assembled at the oil-water interface. The photoactive emulsion continuously catalyzed the n-butyaldehyde reduction to accumulate 16.1 mmol·L−1 butanol, equivalent to 14 cycles for NADH regeneration under visible light irradiation. The primary result demonstrates the potential application prospect of Pickering emulsion in enzyme-photo-coupled catalysis due to the facilitated charge transfer from photocatalyst to NAD+ and fast mass diffusion attributed to the large interfacial area of water and oil.

Role of Protein Kinase C in Metabolic Regulation of Coronary Endothelial Small Conductance Calcium-Activated Potassium Channels.

Small conductance calcium-activated potassium (SK) channels are largely responsible for endothelium-dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes-induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12-myristate 13-acetate, PKC inhibitors, or endothelial PKCα/PKCβ knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKCβ inhibitor LY333531. NADH (30-300 μmol/L) or PKC activator phorbol 12-myristate 13-acetate (30-300 nmol/L) reduced endothelial SK current density, whereas the selective PKCᵦ inhibitor LY333531 significantly reversed the NADH-induced SK channel inhibition. PKCβ small interfering RNA, but not PKCα small interfering RNA, significantly prevented the NADH- and phorbol 12-myristate 13-acetate-induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKCβ expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKCβ inhibitor LY333531. NADH-induced inhibition of endothelial SK channel function is mediated via PKCβ. These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.

An in silico approach to investigate the theranostic potential of coumarin-derived self-immolative luminescent probes.

Till date the challenge exists in the treatments of cancer for various reasons. Most importantly, the available diagnostics are expensive with research gap for enhancing the cancer detection sensitivity. Herein, a series of coumarin-derived fluorescent theranostic probes are reported that can serve as potent anticancer agents as well as in the detection of cancer cells. The potential of these probes to efficiently block one of the well-known cancer drug targets NADPH quinone oxidoreductase-1 (NQO1) is evaluated through various pharmacokinetic methods including absorption, distribution, metabolism and excretion (ADME) properties evaluation, PASS (prediction of activity spectra for substance) algorithm along with molecular docking and dynamic simulations. Further the luminescent properties of these molecules were evaluated by investigating their electronic properties in the ground and excited states with the help of density functional theory methods. Results indicate that the proposed molecules can potentially block the NADPH (reduced form of nicotinamide adenine dinucleotide) binding site of NQO1, thereby inhibiting the activity of the enzyme to ultimately disrupt the metabolism of cancer cells.

Direct Electrochemical Regeneration of NADH and Its Biomimetics

Enzyme-catalyzed redox transformations are an attractive approach for conversion of unactivated alkanes into value-added products under mild reaction conditions. This is especially true since advancements in the area of enzyme engineering has led to a dramatic expansion in the use of modified oxidoreductases for selective functionalization of commodity petrochemicals in non-aqueous media. Despite their appeal, many redox enzymes rely on stoichiometric quantities of expensive coenzymes, such as nicotinamide adenine dinucleotide (NADH), that are not easily regenerated. The use of direct electrochemistry to catalytically regenerate NADH has long been an aspiration of chemists as a convenient and straightforward method that would enable use of the expensive cofactor in enzyme-catalyzed synthesis on an industrial scale. Unfortunately, direct electrochemical reduction of the oxidized form of nicotinamide adenine dinucleotide (NAD+) results in unstable radical intermediates that rapidly form biologically inactive byproducts. Recent progress has been made towards the development of inexpensive artificial NADH biomimetics as replacements for the natural coenzyme, but the strategies to regenerate these biomimetic coenzymes is equally limiting. A primary focus of our research is to design synthetic analogues of NAD+ and NADH that are both enzymatically active and electrochemically regenerable. We will present our recent progress in understanding the structure-reactivity relationships that lead to dimerization over the preferred formation of 1,4-dihydronicotinamide in NAD+ and its biomimetics, and we will describe strategies to prevent dimerization of electrochemically reduced NAD+.

Mask R-CNN provides efficient and accurate measurement of chondrocyte viability in the label-free assessment of articular cartilage

ObjectiveChondrocyte viability (CV) can be measured with the label-free method using second harmonic generation (SHG) and two-photon excitation autofluorescence (TPAF) imaging. To automate the image processing for the label-free CV measurement, we previously demonstrated a two-step deep-learning method: Step 1 used a U-Net to segment the lacuna area on SHG images; Step 2 used dual CNN networks to count live cells and the total number of cells in extracted cell clusters from TPAF images. This study aims to develop one-step deep learning methods to improve the efficiency of CV measurement. MethodTPAF/SHG images were acquired simultaneously on cartilage samples from rats and pigs using two-photon microscopes and were merged to form RGB color images with red, green, and blue channels assigned to emission bands of oxidized flavoproteins, reduced forms of nicotinamide adenine dinucleotide, and SHG signals, respectively. Based on the Mask R-CNN, we designed a deep learning network and its denoising version using Wiener deconvolution for CV measurement. ResultsUsing training and test datasets from rat and porcine cartilage, we have demonstrated that Mask R-CNN-based networks can segment and classify individual cells with a single-step processing flow. The absolute error (difference between the measured and the ground-truth CV) of the CV measurement using the Mask R-CNN with or without Wiener deconvolution denoising reaches 0.01 or 0.08, respectively; the error of the previous CV networks is 0.18, significantly larger than that of the Mask R-CNN methods. ConclusionsMask R-CNN-based deep-learning networks improve efficiency and accuracy of the label-free CV measurement.

Metalated carbon nitride with facilitated electron transfer pathway for selective NADH regeneration and photoenzyme-coupled CO2 reduction

Photoenzyme-coupled catalysis, featuring the integration of photocatalysis with enzymes, is very promising for next-generation green biomanufacturing. The presence of an Rh complex is a prerequisite for the efficient photocatalytic regeneration of the reduced form of nicotinamide adenine dinucleotide (NADH), which poses the issue of immobilizing homogeneous complexes. In this study, a novel immobilization method based on the thermal polymerization of 2,2‘-bipyridine-5,5‘-diamine (DABP) onto a polymeric carbon nitride (PCN) framework is proposed. PCNbpy4 is metalated by immobilizing Rh on the terminal bipyridine structure. Notably, partial DABP has the ability to undergo high-temperature thermal polymerization, resulting in the formation of N-doped graphene. This N-doped graphene can be grafted onto the terminal amino group, forming a potential electron transfer pathway. Additionally, N-doped graphene, because of its good electrical conductivity, guides the photogenerated electrons toward the anchored Rh sites. The catalyst achieves exclusive regeneration of 1,4-NADH with only a 0.12% Rh atomic ratio and realizes 80% NADH regeneration in 20 min. The competitive relationship between hydrogen production and NADH regeneration is also elucidated. Combined with formate dehydrogenase immobilized on a hydrophobic membrane, CO2 reduction to formate is accomplished efficiently, and the formate concentration can accumulate to 7 mmol L−1 within 48 h.

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.

Protective Effects of Curcumin Against Alcoholic Fatty Liver.

Alcoholism is a global health concern. Due to its role as the principal site of ethanol metabolism, the liver endures the most significant amount of tissue damage from heavy drinking. Numerous liver lesions can result from chronic and heavy alcohol use, including steatosis, hepatitis, and fibrosis/cirrhosis. Fatty liver is caused by a redox shift from the oxidized to the reduced form of nicotinamide adenine dinucleotide (NAD+) caused by the ethanol oxidation reaction. The other molecular mechanisms related to the progression of alcohol-induced liver injury are increasing sterol regulatory element-binding protein-1 (SREBP-1) and decreasing PPAR-α activity, cell signaling pathway impairment, reactive oxygen species (ROS) accumulation, and lipid peroxidation. Curcuma longa L. rhizomes contain a substance called curcumin, which is naturally yellow in color and is also known as turmeric yellow. Curcumin has powerful biological and pharmacological properties, including antioxidant, anti-inflammatory, antifungal, antibacterial, antitumor, and anticancer effects. It's been employed as a hepatoprotective substance. Current studies have demonstrated the ability of curcumin to prevent the activation of NF-κB in Kupffer cells via endotoxins, to suppress the expression of various cytokines, chemokines, cyclooxygenase-2 (COX-2), and iNOS, as well as to modulate immune responses. The present study has shown the vital role of curcumin in a variety of hepatotoxic procedures, and summarizes those effects, focusing on the molecular insights they provide.

Nicotinamide Riboside, a Promising Vitamin B3 Derivative for Healthy Aging and Longevity: Current Research and Perspectives.

Many studies have suggested that the oxidized form of nicotinamide adenine dinucleotide (NAD+) is involved in an extensive spectrum of human pathologies, including neurodegenerative disorders, cardiomyopathy, obesity, and diabetes. Further, healthy aging and longevity appear to be closely related to NAD+ and its related metabolites, including nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). As a dietary supplement, NR appears to be well tolerated, having better pharmacodynamics and greater potency. Unfortunately, NR is a reactive molecule, often unstable during its manufacturing, transport, and storage. Recently, work related to prebiotic chemistry discovered that NR borate is considerably more stable than NR itself. However, immediately upon consumption, the borate dissociates from the NR borate and is lost in the body through dilution and binding to other species, notably carbohydrates such as fructose and glucose. The NR left behind is expected to behave pharmacologically in ways identical to NR itself. This review provides a comprehensive summary (through Q1 of 2023) of the literature that makes the case for the consumption of NR as a dietary supplement. It then summarizes the challenges of delivering quality NR to consumers using standard synthesis, manufacture, shipping, and storage approaches. It concludes by outlining the advantages of NR borate in these processes.

Unraveling the role of polymeric ferric sulfate in promoting propionic acid in anaerobic sludge hydrogen production fermentation

Polymerized ferric sulfate (PFS) is widely employed in the pretreatment of industrial wastewater and sludge dewatering. Its widespread use results in its accumulation in waste activated sludge (WAS) and may impact anaerobic fermentation (AF). This work fills the gap in the effects and mechanisms of PFS on hydrogen (H2) production from AF under mesophilic and thermophilic conditions. PFS (40 g/kg total solids, TS) reduced cumulative H2 production by 59.2% (mesophilic conditions) and 52.6% (thermophilic conditions) (p < 0.001), whereas propionic acid production increased by 299.2% and 192.0%, respectively. Model-based analysis showed that PFS inhibited solubilization, hydrolysis, and acidification below 10 g/kg TS but promoted them above 10 g/kg TS. Changes in the ratio of the two forms of nicotinamide adenine dinucleotide (NADH/NAD+) and hydrogenase activity led to unfavorable pathways for H2 production. Thermophilic fermentation exhibited strong tolerance to PFS due to the increased release of extracellular polymeric substances (EPSs). By analyzing 16S-rRNA gene operational taxonomic units (OTUs), the effects of environmental factors on microbial communities and microbial interactions under the combined effects of PFS and temperature were identified, revealing the important factors affecting H2 production. Sludge containing PFS was not conducive to anaerobic H2 production; however, mesophilic fermentation with PFS may be a potential way to recover propionic acid with low energy consumption. This study provided insights into the effects of potential contaminants in WAS on AF for H2 production and information for the effective disposal and resource utilization of WAS.

Developmental changes in cerebral NAD and neuroenergetics of an antioxidant compromised mouse model of schizophrenia

Defects in essential metabolic regulation for energy supply, increased oxidative stress promoting excitatory/inhibitory imbalance and phospholipid membrane dysfunction have been implicated in the pathophysiology of schizophrenia (SZ). The knowledge about the developmental trajectory of these key pathophysiological components and their interplay is important to develop new preventive and treatment strategies. However, this assertion is so far limited. To investigate the developmental regulations of these key components in the brain, we assessed, for the first time, in vivo redox state from the oxidized (NAD+) and reduced (NADH) form of Nicotinamide Adenine Dinucleotide (NAD), energy and membrane metabolites, inhibitory and excitatory neurotransmitters by 31P and 1H MRS during the neurodevelopment of an SZ animal model with genetically compromised glutathione synthesis (gclm-KO mice). When compared to age-matched wild type (WT), an increase in NAD+/NADH redox ratio was found in gclm-KO mice until early adulthood, followed by a decrease in full adults as observed in patients. Especially, in early postnatal life (P20, corresponding to childhood), levels of several metabolites were altered in gclm-KO mice, including NAD+, NAD+/NADH, ATP, and glutamine + glutamate, suggesting an interactive compensation for redox dysregulation between NAD, energy metabolism, and neurotransmission. The identified temporal neurometabolic regulations under deficits in redox regulation provide insights into preventive treatment targets for at-risk individuals, and other neurodevelopmental disorders involving oxidative stress and energetic dysfunction.

Aloe vera-derived graphene-coupled phenosafranin photocatalyst for generation and regeneration of ammonia and NADH by mimicking natural photosynthetic route.

Aloe vera-derived graphene (ADG) coupled system photocatalyst, mimicking natural photosynthesis, is one of the most promising ways for converting solar energy into ammonia (NH3 ) and nicotinamide adenine dinucleotide (NADH) that have been widely used to make the numerous chemicals such as fertilizer and fuel. In this study, we report the synthesis of the aloe vera-derived graphene-coupled phenosafranin (ADGCP) acting as a highly efficient photocatalyst for the generation of NH3 and regeneration of NADH from nitrogen (N2 ) and oxidized form of nicotinamide adenine dinucleotide (NAD+ ). The results show a benchmark instance for mimicking natural photosynthesis activity as well as the practical applications for the solar-driven selective formation ofNH3 and the regeneration of NADH by using the newly designed photocatalyst.

NAD+ Precursors and Intestinal Inflammation: Therapeutic Insights Involving Gut Microbiota.

The oxidized form of nicotinamide adenine dinucleotide (NAD+) is a critical metabolite for living cells. NAD+ may act either as a cofactor for many cellular reactions as well as a coenzyme for different NAD+-consuming enzymes involved in the physiological homeostasis of different organs and systems. In mammals, NAD+ is synthesized from either tryptophan or other vitamin B3 intermediates that act as NAD+ precursors. Recent research suggests that NAD+ precursors play a crucial role in maintaining the integrity of the gut barrier. Indeed, its deficiency has been associated with enhanced gut inflammation and leakage, and dysbiosis. Conversely, NAD+-increasing therapies may confer protection against intestinal inflammation in experimental conditions and human patients, with accumulating evidence indicating that such favorable effects could be, at least in part, mediated by concomitant changes in the composition of intestinal microbiota. However, the mechanisms by which NAD+-based treatments affect the microbiota are still poorly understood. In this context, we have focused specifically on the impact of NAD+ deficiency on intestinal inflammation and dysbiosis in animal and human models. We have further explored the relationship between NAD+ and improved host intestinal metabolism and immunity and the composition of microbiota in vivo. Overall, this comprehensive review aims to provide a new perspective on the effect of NAD+-increasing strategies on host intestinal physiology.

Highly Efficient Self-Assembled Activated Carbon Cloth-Templated Photocatalyst for NADH Regeneration and Photocatalytic Reduction of 4-Nitro Benzyl Alcohol

This manuscript emphasizes how structural assembling can facilitate the generation of solar chemicals and the synthesis of fine chemicals under solar light, which is a challenging task via a photocatalytic pathway. Solar energy utilization for pollution prevention through the reduction of organic chemicals is one of the most challenging tasks. In this field, a metal-based photocatalyst is an optional technique but has some drawbacks, such as low efficiency, a toxic nature, poor yield of photocatalytic products, and it is expensive. A metal-free activated carbon cloth (ACC)–templated photocatalyst is an alternative path to minimize these drawbacks. Herein, we design the synthesis and development of a metal-free self-assembled eriochrome cyanine R (EC-R) based ACC photocatalyst (EC-R@ACC), which has a higher molar extinction coefficient and an appropriate optical band gap in the visible region. The EC-R@ACC photocatalyst functions in a highly effective manner for the photocatalytic reduction of 4-nitro benzyl alcohol (4-NBA) into 4-amino benzyl alcohol (4-ABA) with a yield of 96% in 12 h. The synthesized EC-R@ACC photocatalyst also regenerates reduced forms of nicotinamide adenine dinucleotide (NADH) cofactor with a yield of 76.9% in 2 h. The calculated turnover number (TON) of the EC-R@ACC photocatalyst for the reduction of 4-nitrobenzyl alcohol is 1.769 × 1019 molecules. The present research sets a new benchmark example in the area of organic transformation and artificial photocatalysis.

Investigating the Ischaemic Phase of Skin NADH Fluorescence Dynamics in Recently Diagnosed Primary Hypertension: A Time Series Analysis

The reduced form of nicotinamide adenine dinucleotide (NADH) is crucial in cellular metabolism. During hypoxia, NADH accumulation results from anaerobic cytoplasmic glycolysis and impaired mitochondrial function. This study aimed to compare the dynamic changes in the 460-nm forearm skin fluorescence, which reflects cellular NADH content, during transient ischaemia between healthy individuals and patients with newly diagnosed, untreated essential hypertension (HA). Sixteen healthy volunteers and sixty-five patients with HA underwent non-invasive measurement of forearm skin NADH content using the Flow Mediated Skin Fluorescence (FMSF) method at rest and during a 100-s transient ischaemia induced by inflation of the brachial cuff. The fluorescent signal was sampled at 25 Hz. All samples were normalised to the end of the ischaemic phase, which is the most stable phase of the whole recording. Slope values of 1 s linear regressions were determined for every 25-sample neighbouring set. The 1-s slopes in the early phase of skin ischaemia, indicating quicker hypoxia-induced NADH accumulation in skin, were significantly higher in patients with HA than in healthy individuals. These findings suggest that some protecting mechanisms postponing the early consequences of early cellular hypoxia and premature NADH accumulation during skin ischaemia are impaired in patients with untreated HA. Further studies are needed to investigate this phenomenon.

Multispectral Imaging of Metabolic Fluorophores: Comparing In Vivo and Fresh Ex Vivo Tissue.

The enhanced uptake of glucose by cancer cells via aerobic glycolysis occurs when the lactic acid pathway is favored over the citric acid cycle. The lactic acid cycle in cancer cells influences the cytosolic concentration of metabolic fluorophores including NADH (the reduced form of nicotinamide adenine dinucleotide) and flavin adenine dinucleotide (FAD). In particular, the literature has shown that breast cancer influences the relative magnitude of fluorescence from NADH and FAD. A multispectral imaging system has been developed for rapid non-destructive imaging of intrinsic fluorescence in tissue. This paper compares in vivo data to fresh ex vivo data gathered as a function of time in mouse models. The data indicate that, if measured within 30 min of excision, a cancer diagnosis in fresh ex vivo tissue correlates with a cancer diagnosis in in vivo tissue. These results justify a plan to evaluate fresh ex vivo human tissue to quantify the sensitivity and specificity of the multispectral system.

Detection of Changes in Macrophage Polarization as a Result of 5-Aminolevulinic Acid Photodynamic Therapy Using Fluorescence-Lifetime Imaging Microscopy

Non-invasive detection of polarized macrophages in tumors is an urgent task in terms of combined antitumor therapy. By analyzing the fluorescence lifetime of the metabolic cofactors—the reduced form of nicotinamide adenine dinucleotide (NADH) and flavins—differences in cellular metabolism of normal tissue, tumor, inflammatory and anti-inflammatory macrophages were demonstrated. In this work we studied changes in the polarization of macrophages obtained from THP-1 monocytes in response to photodynamic therapy with 5-aminolevulinic acid (ALA–PDT). Moderate ALA–PDT in vitro led to changes in M0 macrophages metabolism towards M1 polarization, wherein M1 and M2 macrophages died and were replaced by non-polarized cells. The interstitial distribution of polarized macrophages after ALA–PDT was studied in a mouse tumor model of grafted Lewis lung carcinoma. In response to ALA–PDT, there was an increase in the inflammatory macrophages fraction in the tumor node. Metabolic fluorescence-lifetime imaging microscopy (FLIM) was performed for macrophages in vitro and for tumor cryosections. It was shown that analysis of phasor diagrams for the NADH, flavins, and 5-ALA-induced protoporphyrin IX (PpIX) fluorescence lifetime helps to determine the change in metabolism in response to different modes of PDT at the cellular and tissue levels. These data can be used for post-surgery tissue inspection.

Crosstalk Between the Mitochondrial Dynamics and Oxidative Stress in Zinc-induced Cytotoxicity.

Zinc is an essential trace element, which plays an important role in multiple biological activities. However, excessive exposure to zinc can cause toxic damage to living organism. Here, we investigated the relationship between oxidative stress and mitochondrial dynamics in the zinc-induced cytotoxicity. Results showed that excess exposure to zinc could significantly reduce cell viability and induce cell vacuolation in PK-15 cells. Additionally, zinc exposure caused mitochondrial dynamics disorder, manifested as mitochondrial fission, and the elevated mRNA level of Drp1 and downregulated mRNA levels of OPA1, Mfn1, and Mfn2. Meanwhile, zinc could induce oxidative damage, evidenced by the increasing levels of hydrogen peroxide, malondialdehyde, lipid peroxidation, oxidized form of nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, oxidized glutathione/glutathione, superoxide dismutase activity, and the mRNA expression of SOD-1 and NOQ1, and decreasing levels of catalase activity, glutathione peroxidase activity, glutathione reductase activity, and the mRNA expression of CAT, and GPX1. Interestingly, N-acetyl-L-cysteine, an inhibitor for oxidative stress, could reduce the mitochondrial fission under zinc treatment. Besides, Mdivi-1, a mitochondrial fission inhibitor, could relieve oxidative stress caused by excess zinc. Ingeneral, these results suggested that mitochondrial fission and oxidative stress induced by zinc were interrelated in PK-15 cells, which is conducive to explore the new mechanism of zinc toxicity and proposes a theoretical foundation for selecting effective drugs to alleviate the toxic effects caused by zinc.