Adenosine Triphosphate Generation Research Articles

Bioelectricity in Plants: From So Simple a Beginning

Bioelectricity in Plants: From So Simple a Beginning

Methionine adenosyltransferase2A inhibition restores metabolism to improve regenerative capacity and strength of aged skeletal muscle

We investigate the age-related metabolic changes that occur in aged and rejuvenated myoblasts using in vitro and in vivo models of aging. Metabolic and signaling experiments reveal that human senescent myoblasts and myoblasts from a mouse model of premature aging suffer from impaired glycolysis, insulin resistance, and generate Adenosine triphosphate by catabolizing methionine via a methionine adenosyl-transferase 2A-dependant mechanism, producing significant levels of ammonium that may further contribute to cellular senescence. Expression of the pluripotency factor NANOG downregulates methionine adenosyltransferase 2 A, decreases ammonium, restores insulin sensitivity, increases glucose uptake, and enhances muscle regeneration post-injury. Similarly, selective inhibition of methionine adenosyltransferase 2 A activates Akt2 signaling, repairs pyruvate kinase, restores glycolysis, and enhances regeneration, which leads to significant enhancement of muscle strength in a mouse model of premature aging. Collectively, our investigation indicates that inhibiting methionine metabolism may restore age-associated impairments with significant gain in muscle function.

PHPB Attenuated Cognitive Impairment in Type 2 Diabetic KK-Ay Mice by Modulating SIRT1/Insulin Signaling Pathway and Inhibiting Generation of AGEs.

Diabetes mellitus (DM) has been recognized as an increased risk factor for cognitive impairment, known as diabetic encephalopathy (DE). Hyperglycemia and insulin resistance are the main initiators of DE, which is related to the accumulation of advanced glycation end products (AGEs). Potassium 2-(1-hydroxypentyl)-benzoate (PHPB), a derivative of 3-n-butylphthalide (dl-NBP), has emerged various properties including improved mitochondrial function, antioxidant, anti-neuroinflammation, and neuroprotective effects. The present study aimed to investigate the neuroprotective effect of PHPB against AGEs accumulation in type 2 diabetic KK-Ay mice model with DE and further explore the underlying mechanisms. The results showed that PHPB markedly ameliorated the spatial learning ability of KK-Ay mice in the Morris water maze and decreased AD-like pathologic changes (Tau hyperphosphorylation) in the cortex. Furthermore, we found that PHPB treatment significantly reduced AGEs generation via up-regulation of glyoxalase-1 (GLO1) protein and enhancement of methylglyoxal (MG) trapping, while there was no obvious difference in levels of glucose in plasma or brain, contents of total cholesterol (TC), triglycerides (TG), and plasma insulin. Also, PHPB treatment improved the insulin signaling pathway by increasing sirtuin1 (SIRT1) deacetylase activity and attenuated oxidative stress evidenced by elevating glucose-6-phosphate dehydrogenase (G-6-PD) protein expression, promoting the production of reduced glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), restoring mitochondrial membrane potential, increasing adenosine triphosphate (ATP) generation, and reducing malondialdehyde (MDA) levels in the brain. Taken together, PHPB exhibited a beneficial effect on DE, which involved modulating the SIRT1/insulin signaling pathway and reducing oxidative stress by inhibiting the generation of AGEs.

Ketone Bodies and Cardiovascular Disease: An Alternate Fuel Source to the Rescue.

The increased metabolic activity of the heart as a pump involves a high demand of mitochondrial adenosine triphosphate (ATP) production for its mechanical and electrical activities accomplished mainly via oxidative phosphorylation, supplying up to 95% of the necessary ATP production, with the rest attained by substrate-level phosphorylation in glycolysis. In the normal human heart, fatty acids provide the principal fuel (40-70%) for ATP generation, followed mainly by glucose (20-30%), and to a lesser degree (<5%) by other substrates (lactate, ketones, pyruvate and amino acids). Although ketones contribute 4-15% under normal situations, the rate of glucose use is drastically diminished in the hypertrophied and failing heart which switches to ketone bodies as an alternate fuel which are oxidized in lieu of glucose, and if adequately abundant, they reduce myocardial fat delivery and usage. Increasing cardiac ketone body oxidation appears beneficial in the context of heart failure (HF) and other pathological cardiovascular (CV) conditions. Also, an enhanced expression of genes crucial for ketone break down facilitates fat or ketone usage which averts or slows down HF, potentially by avoiding the use of glucose-derived carbon needed for anabolic processes. These issues of ketone body utilization in HF and other CV diseases are herein reviewed and pictorially illustrated.

Extracellularly Detectable Electrochemical Signals of Living Cells Originate from Metabolic Reactions.

Direct detection of cellular redox signals has shown immense potential as a novel living cell analysis tool. However, the origin of such signals remains unknown, which hinders the widespread use of electrochemical methods for cellular research. In this study, the authors found that intracellular metabolic pathways that generate adenosine triphosphate (ATP) are the main contributors to extracellularly detectable electrochemical signals. This is achieved through the detection of living cells (4,706 cells/chip, linearity: 0.985) at a linear range of 7,466-48,866. Based on this discovery, the authors demonstrated that the cellular signals detected by differential pulse voltammetry (DPV) can be rapidly amplified with a developed medium containing metabolic activator cocktails (MACs). The DPV approach combined with MAC treatment shows a remarkable performance to detect the effects of the anticancer drug CPI-613 on cervical cancer both at a low drug concentration (2µm) and an extremely short treatment time (1hour). Furthermore, the senescence of mesenchymal stem cells could also be sensitively quantified using the DPV+MAC method even at a low passage number (P6). Collectively, their findings unveiled the origin of redox signals in living cells, which has important implications for the characterization of various cellular functions and behaviors using electrochemical approaches.

The physiological polyphosphate as a healing biomaterial for chronic wounds: Crucial roles of its antibacterial and unique metabolic energy supplying properties

• Wound healing, especially the healing of chronic wounds, requires an increased demand for metabolic energy (ATP). • The physiological polymer polyphosphate (polyP) is an efficient source for the generation of ATP during wound regeneration. • In addition, polyP undergoes coacervation, a process during which bacteria are killed. • These two pillars are the basis for the successful regeneration of chronic wounds in humans. Insufficient metabolic energy, in the form of adenosine triphosphate (ATP), and bacterial infections are among the main causes for the development of chronic wounds. Previously we showed that the physiological inorganic polymer polyphosphate (polyP) massively accelerates wound healing both in animals (diabetic mice) and, when incorporated into mats, in patients with chronic wounds. Here, we focused on a hydrogel-based gel formulation, supplemented with both soluble sodium polyP (Na-polyP) and amorphous calcium polyP nanoparticles (Ca-polyP-NP). Exposure of human epidermal keratinocytes to the gel caused a significant increase in extracellular ATP level, an effect that was even enhanced when Na-polyP was combined with Ca-polyP-NP. Furthermore, it is shown that the added polyP in the gel is converted into a coacervate, leading to encapsulation and killing of bacteria. The data on human chronic wounds showed that the administration of hydrogel leads to the complete closure of these wounds. Histological analysis of biopsies showed an increased granulation of the wounds and an enhanced microvessel formation. The results indicate that the polyP hydrogel, due to its properties to entrap bacteria and generate metabolic energy, is a very promising formulation for a new therapy for chronic wounds.

A cellular overview of immunometabolism in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease, characterized by a breakdown of immune tolerance and the development of autoantibodies against nucleic self-antigens. Immunometabolism is a rapidly expanding scientific field investigating the metabolic programming of cells of the immune system. During the normal immune response, extensive reprogramming of cellular metabolism occurs, both to generate adenosine triphosphate and facilitate protein synthesis, and also to manage cellular stress. Major pathways upregulated include glycolysis, oxidative phosphorylation, the tricarboxylic acid cycle and the pentose phosphate pathway, among others. Metabolic reprogramming also occurs to aid resolution of inflammation. Immune cells of both patients with SLE and lupus-prone mice are characterized by metabolic abnormalities resulting in an altered functional and inflammatory state. Recent studies have described how metabolic reprogramming occurs in many cell populations in SLE, particularly CD4+ T cells, e.g. favouring a glycolytic profile by overactivation of the mechanistic target of rapamycin pathway. These advances have led to an increased understanding of the metabolic changes affecting the inflammatory profile of T and B cells, monocytes, dendritic cells and neutrophils, and how they contribute to autoimmunity and SLE pathogenesis. In the current review, we aim to summarize recent advances in the field of immunometabolism involved in SLE and how these could potentially lead to new therapeutic strategies in the future.

Synergistic glycolysis disturbance for cancer therapy by a MOF-based nanospoiler.

Increased glycolysis for promoting adenosine triphosphate (ATP) generation is one of the hallmarks of cancer. Although reducing glucose intake or depriving cellular glucose can delay the growth of tumors to some extent, their therapeutic efficacy is a highly needed improvement for clinical translation. Herein, we found that mannose synergistic with glucose oxidase (GOx) can induce cell death by ATP inhibition, autophagy activation, and apoptosis protein upgradation. By using biodegradable zeolitic imidazolate frameworks (ZIF-8) as a nanocarrier (denoted as ZIF-8/M&G), the mannose and GOx can accumulate at the tumor site while having no obvious long-term toxicity. At the tumor site, GOx inhibits glycolysis by converting glucose and oxygen to H 2O 2 and gluconic acid, realizing oxidation therapy and expediting the degradation of the pH-responsive ZIF-8 nanoparticles, respectively. Simultaneously, mannose disturbs sugar metabolism and reduces oxygen consumption, which in turn promotes the GOx oxidation process. The concerted glycolysis inhibition through interactions between mannose and GOx endows ZIF-8/M&G nanospolier with excellent therapeutic efficacy both in vitro and in vivo. Synergistic glycolysis disturbance by the designed nanospoiler in this work proposes a versatile approach for metabolism disturbance to tumor treatment.

Glycolysis inhibition for synergistic phototherapy of triple-negative breast cancer.

Phototherapy is a local and precise therapeutic technique for tumor treatment. However, the therapeutic effects of photothermal and photodynamic therapies are inevitably encountered by hypoxia of the tumor microenvironment and heat shock protein induced by hyperthermia, respectively. Herein, we found that mannose, a glucose analog, could reverse tumor hypoxia by inhibiting glycolysis of cancer cells and suppressing the expression of heat shock protein through inhibiting cellular adenosine triphosphate (ATP) generation. Next, we used lipid nanoparticles simultaneously loaded with indocyanine green (ICG) and mannose molecules, named imLipo, for tumor therapy. Both in vitro and in vivo experiments evidenced that the imLipo nanoplatform has significant therapeutic efficacy through synergistic phototherapy under single near-infrared laser irradiation. This work shows that glycolysis inhibition can overcome the challenges of phototherapy. In addition, all three parts (mannose, ICG, and lipid) of imLipo are clinically approved and our designed nanoplatforms have great potential for future tumor treatment.

Physiological level of LDL cholesterol and lipoprotein metabolism with apoprotein B100. An update for the clinician

Teleologically (physiological end or purpose), LP-apoB100 are macromolecules whose biological objective is to package in the liver within very-low-density lipoproteins (VLDL) (density 0.96-1.006 g/L), essential hydrophobic lipids, especially triglycerides (TG) and esterified cholesterol. Once assembled, the hepatocyte secretes VLDL into the systemic circulation; this way, essential hydrophobic lipids (insoluble in water) can be transported from the liver to peripheral tissues in an aqueous medium such as plasma. In the systemic circulation and influenced by the energy balance, VLDL (by hydrolysis of its triglycerides), fulfill its primary physiological purpose, releasing glycerol and especially fatty acids (FA) towards the cardiac and skeletal muscle tissues. Which, in turn, will be used as an energy substrate for beta-oxidation and adenosine triphosphate (ATP) generation or towards the adipose tissue to be used as a substrate for the resynthesis of triglycerides (adipocyte lipogenesis) and its eventual rehydrolysis with the release of fatty acids (adipocyte lipolysis).

Improving ATP availability by sod1 deletion with a strategy of precursor feeding enhanced S-adenosyl-L-methionine accumulation in Saccharomyces cerevisiae

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15–33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g−1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L−1L-met and 0.60 g L−1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L−1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L−1 to 10.1 g L−1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

Drugless nanoparticles tune-up an array of intertwined pathways contributing to immune checkpoint signaling and metabolic reprogramming in triple-negative breast cancer

Metabolic reprogramming ‘Warburg effect’ and immune checkpoint signaling are immunosuppressive hallmarks of triple-negative breast cancer (TNBC) contributing to the limited clinical applicability of immunotherapy. Biomaterials arise as novel tools for immunomodulation of the tumor microenvironment that can be used alongside conventional immunotherapeutics. Chitosan and lecithin are examples of versatile biomaterials with interesting immunomodulatory properties. In this study, we aimed at investigation of the role of carefully designed hybrid nanoparticles (NPs) on common mediators of both programmed death ligand 1 (PD-L1) expression and glycolytic metabolism. Hybrid lecithin–chitosan NPs were prepared and characterized. Their intracellular concentration, localization and effect on the viability of MDA-MB-231 cells were assessed. Glycolytic metabolism was quantified by measuring glucose consumption, adenosine triphosphate (ATP) generation, lactate production and extracellular acidification. Nitric oxide production was quantified using Greiss reagent. Gene expression of inducible nitric oxide synthase (iNOS), phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB or Akt), mammalian target of rapamycin (mTOR), hypoxia-inducible factor 1α (HIF-1α) and PD-L1 was quantified by quantitative reverse transcription polymerase chain reaction (q-RT-PCR). Chitosan, lecithin and the NPs-formulated forms have been shown to influence the ‘Warburg effect’ and immune checkpoint signaling of TNBC cells differently. The composition of the hybrid systems dictated their subcellular localization and hence the positive or negative impact on the immunosuppressive characteristics of TNBC cells. Carefully engineered hybrid lecithin–chitosan NPs could convert the immune-suppressive microenvironment of TNBC to an immune-active microenvironment via reduction of PD-L1 expression and reversal of the Warburg effect.

Label-free and non-destructive identification of naïve and primed embryonic stem cells based on differences in cellular metabolism

Pluripotent stem cells (PSCs) exist in naïve or primed states based on their origin. For in vitro culture, these PSCs require different supplements and growth factors. However, owing to their similar phenotypic features, identifying both cell types without harming cellular functions is challenging. This study reports an electrochemical method that enables simple, label-free, and non-destructive detection of naïve embryonic stem cells (ESCs) derived from mouse ESCs, based on the differences in cellular metabolism. Two major metabolic pathways to generate adenosine triphosphate (ATP)—glycolysis and oxidative phosphorylation (OXPHOS)—were blocked, and it was found that mitochondrial energy generation is the origin of the strong electrochemical signals of naïve ESCs. The number of ESCs is quantified when mixed with primed ESCs or converted from naïve–primed switchable metastable ESCs. The mouse PSCs derived from doxycycline-inducible mouse embryonic fibroblasts (MEFs) are also sensitively identified among other cell types such as unconverted MEFs and primed PSCs. The developed sensing platform operates in a non-invasive and label-free manner. Thus, it can be useful in the development of stem cell-derived therapeutics.

Effects of colonization-associated gene yqiC on global transcriptome, cellular respiration, and oxidative stress in Salmonella Typhimurium

BackgroundyqiC is required for colonizing the Salmonella enterica serovar Typhimurium (S. Typhimurium) in human cells; however, how yqiC regulates nontyphoidal Salmonella (NTS) genes to influence bacteria–host interactions remains unclear.MethodsThe global transcriptomes of S. Typhimurium yqiC-deleted mutant (ΔyqiC) and its wild-type strain SL1344 after 2 h of in vitro infection with Caco-2 cells were obtained through RNA sequencing to conduct comparisons and identify major yqiC-regulated genes, particularly those involved in Salmonella pathogenicity islands (SPIs), ubiquinone and menaquinone biosynthesis, electron transportation chains (ETCs), and carbohydrate/energy metabolism. A Seahorse XFp Analyzer and assays of NADH/NAD+ and H2O2 were used to compare oxygen consumption and extracellular acidification, glycolysis parameters, adenosine triphosphate (ATP) generation, NADH/NAD+ ratios, and H2O2 production between ΔyqiC and SL1344.ResultsAfter S. Typhimurium interacts with Caco-2 cells, yqiC represses gene upregulation in aspartate carbamoyl transferase, type 1 fimbriae, and iron–sulfur assembly, and it is required for expressing ilvB operon, flagellin, tdcABCD, and dmsAB. Furthermore, yqiC is required for expressing mainly SPI-1 genes and specific SPI-4, SPI-5, and SPI-6 genes; however, it diversely regulates SPI-2 and SPI-3 gene expression. yqiC significantly contributes to menD expression in menaquinone biosynthesis. A Kyoto Encyclopedia of Genes and Genomes analysis revealed the extensive association of yqiC with carbohydrate and energy metabolism. yqiC contributes to ATP generation, and the analyzer results demonstrate that yqiC is required for maintaining cellular respiration and metabolic potential under energy stress and for achieving glycolysis, glycolytic capacity, and glycolytic reserve. yqiC is also required for expressing ndh, cydA, nuoE, and sdhB but suppresses cyoC upregulation in the ETC of aerobically and anaerobically grown S. Typhimurium; priming with Caco-2 cells caused a reversed regulation of yiqC toward upregulation in these ETC complex genes. Furthermore, yqiC is required for maintaining NADH/NAD+ redox status and H2O2 production.ConclusionsSpecific unreported genes that were considerably regulated by the colonization-associated gene yqiC in NTS were identified, and the key role and tentative mechanisms of yqiC in the extensive modulation of virulence factors, SPIs, ubiquinone and menaquinone biosynthesis, ETCs, glycolysis, and oxidative stress were discovered.

Electrochemical Recycling of Adenosine Triphosphate in Biocatalytic Reaction Cascades.

Adenosine triphosphate (ATP) provides the driving force necessary for critical biological functions in all living organisms. In synthetic biocatalytic reactions, this cofactor is recycled in situ using high-energy stoichiometric reagents, an approach that generates waste and poses challenges with enzyme stability. On the other hand, an electrochemical recycling system would use electrons as a convenient source of energy. We report a method that uses electricity to turn over enzymes for ATP generation in a simplified cellular respiration mimic. The method is simple, robust, and scalable, as well as broadly applicable to complex enzymatic processes including a four-enzyme biocatalytic cascade in the synthesis of the antiviral molnupiravir.

Cu Single Atom Nanozyme Based High-Efficiency Mild Photothermal Therapy through Cellular Metabolic Regulation.

Upregulation of heat shock proteins (HSPs) drastically compromises the treatment effect of mild photothermal therapy (PTT). Herein, we designed a polyporous Cu single atom nanozyme (Cu SAzyme) loaded with licogliflozin (LIK066) for HSP-silencing induced mild PTT. On one hand, LIK066 inhibits glucose uptake by shutting sodium-dependent glucose transporter (SGLT) "valve", effectively blocking the energy source for adenosine triphosphate (ATP) generation. Without sufficient energy, cancer cells cannot synthesize HSPs. On the other hand, Cu SAzyme presents extraordinary multienzyme activities to induce reactive oxygen species (ROS) storm formation, which can damage the existing HSPs in cancer cells. Through a two-pronged strategy of SGLT inhibitor and ROS storm, LIK066-loaded Cu SAzyme shows high efficiency for comprehensive removal of HSPs to realize mild PTT.

Cu Single Atom Nanozyme Based High‐Efficiency Mild Photothermal Therapy through Cellular Metabolic Regulation

AbstractUpregulation of heat shock proteins (HSPs) drastically compromises the treatment effect of mild photothermal therapy (PTT). Herein, we designed a polyporous Cu single atom nanozyme (Cu SAzyme) loaded with licogliflozin (LIK066) for HSP‐silencing induced mild PTT. On one hand, LIK066 inhibits glucose uptake by shutting sodium‐dependent glucose transporter (SGLT) “valve”, effectively blocking the energy source for adenosine triphosphate (ATP) generation. Without sufficient energy, cancer cells cannot synthesize HSPs. On the other hand, Cu SAzyme presents extraordinary multienzyme activities to induce reactive oxygen species (ROS) storm formation, which can damage the existing HSPs in cancer cells. Through a two‐pronged strategy of SGLT inhibitor and ROS storm, LIK066‐loaded Cu SAzyme shows high efficiency for comprehensive removal of HSPs to realize mild PTT.

Metabolomic analysis and pharmacological validation of the cerebral protective effect of 3,4‑dihydroxybenzaldehyde on cerebral ischemia‑reperfusion injury.

3,4‑Dihydroxybenzaldehyde (DBD), one of the active components of Gastrodia elata, has a cerebral protective effect and can effectively combat cerebral ischemia/reperfusion (I/R) injury in rats. However, the metabolite profiles and underlying mechanisms associated with DBD remain unclear. To explore the level of energy metabolism and pharmacological targets in brain tissue following DBD treatment of stroke. The right middle cerebral artery of the rats was occluded for 2h and reperfused for 24h to simulate brain I/R injury. Pharmacological results showed that DBD reduced cerebral infarct volume, improved neurological function and increased adenosine triphosphate (ATP) content. Mitochondria are the primary sites for ATP generation and cellular energy supply and decreasing mitochondrial dysfunction can alleviate the energy expenditure of ischemic stroke. Through further experiments, it was found that mitochondrial damage was recovered following DBD treatment, which was manifested by the improvement of mitochondrial morphology under an electron microscope and the reduction of oxidative stress damage. The metabolomics of middle cerebral artery occlusion/reperfusion (MCAO/R) rat brain tissue was studied by the liquid chromatography‑tandem mass spectrometry metabolomics method. Significantly different metabolites were screened and the pathways involved included amino sugar and nucleotide sugar metabolism and pentose phosphate pathway. Finally, the present study performed targeted metabolic profiling and validated potential therapeutic targets. Uridine diphosphate N‑acetylglucosamine (UDP‑GlcNAc) levels were decreased in the MCAO/R group but significantly increased in the DBD group. TdT‑mediated dUTP nick end labeling (TUNEL) staining, hematoxylin and eosin staining and western blotting showed that brain cell apoptosis was inhibited and neuronal morphology improved. Among them, the regulatory enzyme O‑GlcNAc transferase (OGT) of UDP‑GlcNAc appeared to be significantly increased and neuronal apoptosis was inhibited following DBD treatment, which was verified by western blotting. Therefore, by analyzing mitochondrial dysfunction following I/R and the characterization of potential markers in mitochondrial energy metabolism, it was shown that OGT could inhibit neuronal apoptosis as a potential therapeutic target for brain I/R injury.

Hypoxia-Activatable Nanovesicles as In Situ Bombers for Combined Hydrogen-Sulfide-Mediated Respiration Inhibition and Photothermal Therapy.

Photothermal therapy (PTT) has emerged as a promising alternative or supplement to cancer treatments. While PTT induces the ablation of solid tumors, its efficiency is hampered by self-recovery within impaired cancer cells through glycolysis and respiration metabolism. Based on this, the introduction of hydrogen sulfide (H2S)-mediated respiration inhibition is a good choice to make up for the PTT limitation. Herein, nanovesicles (NP1) are integrated by a hypoxia-responsive conjugated polymer (P1), polymetric H2S donor (P2), and near-infrared (NIR) light-harvesting aza-BODIPY dye (B1) for the delivery of H2S and synergistic H2S gas therapy/PTT. The scaffold of NP1 undergoes disassembly in the hypoxic environments, thus triggering the hydrolysis of P2 to continuously long-term release H2S. Dependent on the superior photothermal ability of B1, NP1 elicits high photothermal conversion efficiency (η = 19.9%) under NIR light irradiation for PTT. Moreover, NP1 serves as in situ H2S bombers in the hypoxic tumor environment and suppresses the mitochondrial respiration through inhibiting expression of cytochrome c oxidase (COX IV) and cutting off the adenosine triphosphate (ATP) generation. Both in vitro and in vivo results demonstrate good antitumor efficacy of H2S gas therapy/PTT, which will be recommended as an advanced strategy for cancer therapeutics.

Neuronal mitochondrial morphology is significantly affected by both fixative and oxygen level during perfusion.

Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs), dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating adenosine triphosphate (ATP) and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stressors such as oxidative stress or in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure, induce dramatic fragmentation of mitochondria both in vitro and in vivo. This disruption was not observed following the use of glutaraldehyde (GA) addition or oxygen supplementation, respectively. Finally, using pre-formed fibril α-synuclein treated neurons, we show fixative choice can alter experimental outcomes. Specifically, α-synuclein-induced mitochondrial remodeling could not be observed with PFA only fixation as fixation itself caused mitochondrial fragmentation. Our study provides optimized methods for examining mitochondrial morphology in neurons and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.