ATP Consumption Research Articles

The Mycobacterium tuberculosis PE15/PPE20 complex transports calcium across the outer membrane.

The mechanisms by which nutrients traverse the Mycobacterium tuberculosis (Mtb) outer membrane remain mostly unknown and, in the absence of classical porins, likely involve specialized transport systems. Calcium ions (Ca2+) are an important nutrient and serve as a second messenger in eukaryotes, but whether bacteria have similar Ca2+ signaling systems is not well understood. To understand the basis for Ca2+ transport and signaling in Mtb, we determined Mtb's transcriptional response to Ca2+. Overall, only few genes changed expression, suggesting a limited role of Ca2+ as a transcriptional regulator. However, 2 of the most strongly down-regulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localize to the outer membrane and comprise many intrinsically disordered proteins. PE15 and PPE20 formed a complex and PPE20 directly bound Ca2+. Ca2+-associated phenotypes such as increased ATP consumption and biofilm formation were reversed in a pe15/ppe20 knockout (KO) strain, suggesting a direct role in Ca2+ homeostasis. To test whether the PE15/PPE20 complex has a role in Ca2+ transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca2+ reporter strain. A pe15/ppe20 KO in the FRET background showed a specific and selective loss of Ca2+ influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca2+-binding protein complex that selectively imports Ca2+, show a distinct transport function for an intrinsically disordered protein, and support the emerging idea of a general family-wide role of PE/PPE proteins as idiosyncratic transporters across the outer membrane.

High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS.

The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB, a protein that binds to centromeric-like <i>parS</i> sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements of plasmid dynamics. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We develop a unified stochastic model that quantitatively explains this behaviour and predicts that cells with the lowest plasmid concentration transition to oscillatory dynamics. We confirm this prediction for F plasmid as well as a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, which according to our model, minimises ATP consumption. Our work also clarifies how various plasmid dynamics are achievable in a single unified stochastic model. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.

Controlling the Periodicity of a Reaction-Diffusion Wave in Artificial Cells by a Two-Way Energy Supplier.

Reaction-diffusion (RD) waves, which are dynamic self-organization structures generated by nanosize molecules, are a fundamental mechanism from patterning in nano- and micromaterials to spatiotemporal regulations in living cells, such as cell division and motility. Although the periods of RD waves are the critical element for these functions, the development of a system to control their period is challenging because RD waves result from nonlinear physical dynamics under far-from-equilibrium conditions. Here, we developed an artificial cell system with tunable period of an RD-driven wave (Min protein wave), which determines a cell division site plane in living bacterial cells. The developed system is based on our finding that Min waves are generated by energy consumption of either ATP or dATP, and the period of the wave is different between these two energy suppliers. We showed that the Min-wave period was modulated linearly by the mixing ratio of ATP and dATP and that it was also possible to estimate the mixing ratio of ATP and dATP from the period. Our findings illuminated a previously unidentified principle to control the dissipative dynamics of biomolecules and, simultaneously, built an important framework to construct molecular robots with spatiotemporal units.

Abscisic Acid Improves Rice Thermo-Tolerance by Affecting Trehalose Metabolism.

Heat stress that occurs during the flowering stage severely decreases the rice (Oryza sativa L.) seed-setting rate. This damage can be reversed by abscisic acid (ABA), through effects on reactive oxygen species, carbohydrate metabolism, and heat shock proteins, but the exact role of trehalose and ATP in this process remains unclear. Two rice genotypes, namely, Zhefu802 (heat-resistant plant, a recurrent parent) and its near-isogenic line (faded green leaf, Fgl, heat-sensitive plant), were subjected to 38 °C heat stress after being sprayed with ABA or its biosynthetic inhibitor, fluridone (Flu), at the flowering stage. The results showed that exogenous ABA significantly increased the seed-setting rate of rice under heat stress, by 14.31 and 22.40% in Zhefu802 and Fgl, respectively, when compared with the H2O treatment. Similarly, exogenous ABA increased trehalose content, key enzyme activities of trehalose metabolism, ATP content, and F1Fo-ATPase activity. Importantly, the opposite results were observed in plants treated with Flu. Therefore, ABA may improve rice thermo-tolerance by affecting trehalose metabolism and ATP consumption.

Energy metabolism and intracellular pH regulation reveal different physiological acclimation mechanisms of Chlorella strains to high concentrations of CO2

The intolerance of high CO2 in the exhaust gas is the “bottleneck” limiting the wide application of microalgae for CO2 biosequestration. Around this topic, we selected high-CO2-tolerant (LAMB 33 and 31) and nontolerant (LAMB 122) Chlorella strains to study their different energy metabolisms and cytoplasmic pH regulations in response to high CO2. Under 40 % CO2, LAMB 33 and 31 both showed elevated ATP synthesis, accelerated ATP consumption and fast cytoplasmic pH regulation while exhibiting different acclimating strategies therein: chloroplast acclimations were reflected by high chlorophyll contents in 33 but photosystem transitions in 31; faster mitochondrial acclimations occurred in 33 than in 31; cellular organic carbon mainly flowed to monosaccharide synthesis for 33 but to monosaccharide and protein synthesis for 31; and cytoplasmic pH regulation was attributed to V-ATPase in 31 but not in 33. All the above metabolic processes gradually collapsed in 122, leading to growth inhibition. Our study identified different metabolic acclimation strategies among Chlorella strains to high CO2 and provided new traits for breeding microalgae for CO2 biosequestration.

Myosin Heavy Chain as a Novel Key Modulator of Striated Muscle Resting State.

After years of intense research using structural, biological, and biochemical experimental procedures, it is clear that myosin molecules are essential for striated muscle contraction. However, this is just the tip of the iceberg of their function. Interestingly, it has been shown recently that these molecules (especially myosin heavy chains) are also crucial for cardiac and skeletal muscle resting state. In the present review, we first overview myosin heavy chain biochemical states and how they influence the consumption of ATP. We then detail how neighboring partner proteins including myosin light chains and myosin binding protein C intervene in such processes, modulating the ATP demand in health and disease. Finally, we present current experimental drugs targeting myosin ATP consumption and how they can treat muscle diseases.

Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function

AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single molecule dwell time using total internal reflection fluorescence microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX. Substrates comprising a stable domain resistant to unfolding and a C-terminal unstructured tail, tagged with a degron for initiating translocase insertion, were used to determine dwell time in relation to tail length and composition. We found greater tail length promoted substrate retention during futile unfolding. Additionally, we tested two tail compositions known to frustrate unfolding. A poly-glycine tract (polyG) promoted release, but only when adjacent to the folded domain, whereas glycine-alanine repeats (GAr) did not promote release. A high complexity motif containing polar and charged residues also promoted release. We further investigated the impact of these and related motifs on substrate degradation rates and ATP consumption, using the unfoldase–protease complex ClpXP. Here, substrate domain stability modulates the effects of substrate tail sequences. polyG and GAr are both inhibitory for unfolding, but act in different ways. GAr motifs only negatively affected degradation of highly stable substrates, which is accompanied by reduced ClpXP ATPase activity. Together, our results specify substrate characteristics that affect unfolding and degradation by ClpXP.

Inhibitors targeting the autophosphorylation of serine/threonine kinase of Streptococcus suis show potent antimicrobial activity.

Antimicrobial resistance (AMR) is a global concern threatening public health. Developing novel antibiotics is one of the effective strategies to tackle AMR. Serine/threonine kinases (STKs) have been recently shown to play critical roles in the physiology and pathogenesis of several important bacterial pathogens which are regarded as a promising antimicrobial drug target. We previously reported the roles of STK in the regulation of bacterial cell division, metabolism, and pathogenesis in Streptococcus suis, an important zoonotic bacterial pathogen. In this study, we firstly identified the Thr167 and Ser175 residues in the activation loop of S. suis STK (ssSTK) as the kinase autophosphorylation sites. Phenotyping results demonstrated that the autophosphorylation deficient strain resembled the stk deletion strain showing essentiality for bacterial growth in minimal medium, abnormal morphology, and decreased virulence when compared with the wild-type S. suis SC19 strain. Based on these findings, we established an ssSTK inhibitor screening approach by measuring the growth of S. suis in a minimal medium and testing the autophosphorylation inhibition by measuring the consumption of ATP in an enzymatic reaction by ssSTK. A series of inhibitors against ssSTK are identified from a commercial kinase inhibitors library, including Staurosporine, K252a, AT9283, and APY29. These inhibitors showed antimicrobial activity in vitro. Moreover, by using Galleria mellonella larvae infection assay, compound APY29 displayed in vivo efficacy against S. suis infection. Additionally, it was predicted by molecular docking that these inhibitors could interact with ssSTK. Collectively, our data illustrated the essential roles of ssSTK autophosphorylation in the physiology and pathogenicity of S. suis and consider these inhibitors as promising antimicrobial lead compounds.

Proximal tubular epithelia-specific transcriptomics of diabetic mice treated with dapagliflozin

Based on recent clinical trials using sodium-glucose co-transporter 2 inhibitor (SGLT2i) demonstrating the significant improvement of outcomes of diabetic kidney disease (DKD), the paradigm shift from “glomerulocentric” to “tubule centric” pathophysiology in DKD progression has been highlighted. Several responsible mechanisms for renoprotective effects by SGLT2i have been proposed recently, but the changes in proximal tubule-specific gene expression by SGLT2i in diabetic mice have not been elucidated. We report the analysis of the proximal tubular-specific pathway, demonstrating the downregulation of oxidative phosphorylation in dapagliflozin-treated db/db mice, a type 2 diabetic model. After 8-week treatment of dapagliflozin for db/db mice having a proximal tubule-specific tdTomato reporter, tdTomato-positive cells were isolated by FACS. Pathway analysis of RNA sequencing of isolated tubular epithelia revealed that oxidative phosphorylation was downregulated in dapagliflozin-treated mice. However, depletion of renal tissue ATP content in db/db mice was ameliorated by dapagliflozin administration. Pimonidazole staining demonstrated renal cortical tissue hypoxia in db/db mice, which was improved by dapagliflozin administration. This study suggests that dapagliflozin can ameliorate the excessive oxygen and ATP consumption, and subsequent tissue hypoxia in the diabetic kidney, which may explain, in part, the responsible mechanisms of the renoprotective effects of dapagliflozin.

Mitochondrial dysfunction in heart failure and its therapeutic implications.

The ATP consumption in heart is very intensive to support muscle contraction and relaxation. Mitochondrion is the power plant of the cell. Mitochondrial dysfunction has long been believed as the primary mechanism responsible for the inability of energy generation and utilization in heart failure. In addition, emerging evidence has demonstrated that mitochondrial dysfunction also contributes to calcium dysregulation, oxidative stress, proteotoxic insults and cardiomyocyte death. These elements interact with each other to form a vicious circle in failing heart. The role of mitochondrial dysfunction in the pathogenesis of heart failure has attracted increasing attention. The complex signaling of mitochondrial quality control provides multiple targets for maintaining mitochondrial function. Design of therapeutic strategies targeting mitochondrial dysfunction holds promise for the prevention and treatment of heart failure.

Excess intracellular ATP causes neuropathic pain following spinal cord injury.

Intractable neuropathic pain following spinal cord injury (NP-SCI) reduces a patient's quality of life. Excessive release of ATP into the extracellular space evokes neuroinflammation via purinergic receptor. Neuroinflammation plays an important role in the initiation and maintenance of NP. However, little is known about whether or not extracellular ATP cause NP-SCI. We found in the present study that excess of intracellular ATP at the lesion site evokes at-level NP-SCI. No significant differences in the body weight, locomotor function, or motor behaviors were found in groups that were negative and positive for at-level allodynia. The intracellular ATP level at the lesion site was significantly higher in the allodynia-positive mice than in the allodynia-negative mice. A metabolome analysis revealed that there were no significant differences in the ATP production or degradation between allodynia-negative and allodynia-positive mice. Dorsal horn neurons in allodynia mice were found to be inactivated in the resting state, suggesting that decreased ATP consumption due to neural inactivity leads to a build-up of intracellular ATP. In contrast to the findings in the resting state, mechanical stimulation increased the neural activity of dorsal horn and extracellular ATP release at lesion site. The forced production of intracellular ATP at the lesion site in non-allodynia mice induced allodynia. The inhibition of P2X4 receptors in allodynia mice reduced allodynia. These results suggest that an excess buildup of intracellular ATP in the resting state causes at-level NP-SCI as a result of the extracellular release of ATP with mechanical stimulation.

Chlamydia trachomatis development requires both host glycolysis and oxidative phosphorylation but has only minor effects on these pathways

The obligate intracellular bacteria Chlamydia trachomatis obtain all nutrients from the cytoplasm of their epithelial host cells and stimulate glucose uptake by these cells. They even hijack host ATP, exerting a strong metabolic pressure on their host at the peak of the proliferative stage of their developmental cycle. However, it is largely unknown whether infection modulates the metabolism of the host cell. Also, the reliance of the bacteria on host metabolism might change during their progression through their biphasic developmental cycle. Herein, using primary epithelial cells and 2 cell lines of nontumoral origin, we showed that between the 2 main ATP-producing pathways of the host, oxidative phosphorylation (OxPhos) remained stable and glycolysis was slightly increased. Inhibition of either pathway strongly reduced bacterial proliferation, implicating that optimal bacterial growth required both pathways to function at full capacity. While we found C. trachomatis displayed some degree of energetic autonomy in the synthesis of proteins expressed at the onset of infection, functional host glycolysis was necessary for the establishment of early inclusions, whereas OxPhos contributed less. These observations correlated with the relative contributions of the pathways in maintaining ATP levels in epithelial cells, with glycolysis contributing the most. Altogether, this work highlights the dependence of C. trachomatis on both host glycolysis and OxPhos for efficient bacterial replication. However, ATP consumption appears at equilibrium with the normal production capacity of the host and the bacteria, so that no major shift between these pathways is required to meet bacterial needs.

Effect of acute hypoxia on the brain energy metabolism of the scorpionfish Scorpaena porcus Linnaeus, 1758: the pattern of oxidoreductase activity and adenylate system.

The activity of oxidoreductases, malate dehydrogenase and lactate dehydrogenase (MDH, 1.1.1.37; LDH, 1.1.1.27), as well as parameters of adenylate system-[ATP], [ADP], [AMP], total adenylate pool (AP), and adenylate energy charge (AEC) in medulla oblongata (MB) and forebrain, midbrain, and diencephalon (FDMB)-were studied in the scorpionfish under acute hypoxia (0.9-1.2mgO2·L-1, 90min). A higher MDH activity level was observed in MB and FDMB, as compared to LDH (p < 0.05). At the same time, MB showed a higher adenylate content and increased AP (p < 0.05). AEC did not exceed ~ 0.7 (vs. the maximum of this index ~ 0.9-1.0) in the brain of the scorpionfish indicating adaptation of the tissue energy status to hypoxia. A rapid decrease in MDH activity (p < 0.05) was observed in MB under acute hypoxia. These changes were accompanied by insignificant LDH activation. A pronounced LDH activation (p < 0.05), a decrease in MDH activity, and the highest AP raise (p < 0.05) were observed in FDMB, suggesting activation of glycolysis and simultaneous decrease in the rate of ATP consumption. MB and FDMB demonstrated the ability to a relative retention of AEC during hypoxia. The unidirectional metabolic adaptation was based on the intensification of glycolysis, a decrease of ATP consumption, and a subsequent increase in adenylate concentration that allowed the scorpionfish brain structures to maintain the energy status under acute hypoxia.

Antibacterial Mechanism of Linalool against Pseudomonas fragi: A Transcriptomic Study.

Pseudomonas fragi is the dominant spoilage bacterium that causes the deterioration of chilled meat. Our previous study showed that linalool has potent antibacterial activity against P. fragi, but its antibacterial mechanism is unclear. To explore the antibacterial mechanism of linalool against P. fragi, this study used RNA-seq technology to perform transcriptome analysis of P. fragi samples with or without linalool treatment (1.5 mL/L) for 2 h. The results showed that linalool treatment disrupted the extracellular lipopolysaccharide synthesis pathway in P. fragi and activated fatty acid metabolism and ribosomal function to compensate for cell membrane damage. The energy metabolism of P. fragi was severely disturbed by linalool, and multiple ATP synthases and ATP transportases were overexpressed in the cells but could not guarantee the consumption of ATP. The simultaneous overexpression of multiple ribosomal functional proteins and transporters may also place an additional burden on cells and cause them to collapse.

Transgenerational effects and phenotypic plasticity in sperm and larvae of the sea urchin Paracentrotus lividus under ocean acidification.

In marine organisms, differing degree of sensitivity to ocean acidification (OA) is expected for each life stage, and disturbance at one stage can carry over into the following stage or following generation. In this study we investigated phenotypic changes of sperm and larvae of the sea urchin Paracentrotus lividus in response to different pH conditions (8.0, 7.7, 7.4) experienced by the parents during gametogenesis. In sperm from two-months exposed males, sperm motility, velocity, ATP content, ATP consumption and respiration rate were evaluated at three pH values of the activating medium (8.0, 7.7 and 7.4). Moreover, larvae from each parental group were reared at pH 8.0 and 7.7 for 20 days and larval mortality and growth were then assessed. Sperm motility and respiration rate were not affected either by exposure of males to low pH or by the post-activation pH. Sperm velocity did not differ among post-activation pH values in all sperm groups, but it decreased slower in sperm developed under acidified conditions, suggesting the presence of positive carryover effect on sperm longevity. This positive carryover effect of exposure of males to low pH values was highlighted also for the sperm ATP content, which was higher in these groups of sperm. ATP consumption rate was affected by post-activation pH with higher values at pH 8.0 in sperm from males maintained at control condition and pH 7.7 while the energy consumption appeared to be differently modulated at different experimental conditions. A negative carry over effect of OA was observed on survival of larvae from parents acclimated at pH 7.4 and additive negative effects of both parental and larval exposure to low pH can be suggested. In all groups of larvae, decreased somatic growth was observed at low rearing pH, thus larvae from parents maintained at low pH did not show an increased capability to cope with OA.

Transcriptional responses of Aurantiochytrium limacinum under light conditions.

Astaxanthin-producing protist Aurantiochytrium limacinum can accumulate higher amounts of astaxanthin under light conditions; however, little is known about the impact of light exposure on its metabolism. Here, we investigated the transcriptional profile of A. limacinum under light conditions. Transcriptomic analyses revealed that 962 genes of A. limacinum showed a significant change in expression under light conditions, most of which (94.5%) were downregulated. Furthermore, gene ontology enrichment analysis indicated that A. limacinum mainly downregulated genes associated with cell motility, proliferation and gene expression processes, whose activities depend on ATP as an energy source. Additionally, the quantification of carotenoid and its transcripts suggested that β-carotene and astaxanthin biosynthesis pathways were rate-limiting and tightly regulated steps, respectively. In comparison, these processes were enhanced under light conditions. Considering that astaxanthin accumulation was highly correlated with reactive oxygen species (ROS) levels in microalgae, our results suggest that A. limacinum reduces ATP consumption to decrease the occurrence of ROS in mitochondria while accumulating astaxanthin to prevent ROS damage. This study provides novel insights into the impact of light exposure on A. limacinum metabolism, thereby facilitating a complete understanding of this protist for efficient astaxanthin production.

Investigation of the Relation between Temperature and M13 Phage Production via ATP Expenditure

M13 bacteriophage is a promising biomolecule capable of various bionano and material science applications. The biomaterial can self-assemble into matrices to fabricate bioscaffolds using high phage concentration and high phage purity. Previous studies aimed to acquire these conditions in large-scale phage production and have identified the optimal culture temperature range at 28–31 °C. However, explanations as to why this temperature range was optimal for phage production is absent from the work. Therefore, in this study, we identified the relation between culture temperature and M13 phage production using ATP expenditure calculations to comprehend the high yield phage production at the optimal temperature range. We extended a coarse-grained model for the evaluation of phage protein and ribosomal protein synthesis with the premise that phage proteins (a ribosomal protein) are translated by bacterial ribosomes in E. coli through expenditure of ATP energy. By comparing the ATP energy for ribosomal protein synthesis estimated using the coarse-grained model and the experimentally calculated ATP expenditure for phage production, we interpreted the high phage yield at the optimal temperature range and recognized ATP analysis as a reasonable method that can be used to evaluate other parameters for phage production optimization.

Does Inosine Monophosphate Formation Facilitate the Bioenergetic Response to Incremental Contractions in Human Skeletal Muscle In Vivo?

Traditionally, it has been accepted that working muscle will endeavor to maintain [ATP] near its resting level in order to maintain a favorable free energy of ATP hydrolysis (∆GATP). Recent reports of significant declines in [ATP] during maximal muscular contractions in vivo spark new interest in this concept. ATP is hydrolyzed to adenosine diphosphate (ADP) in the creatine kinase reaction to provide energy directly to working ATPases. As needed, additional energy can be provided by the breakdown of ADP to adenosine monophosphate (AMP), which is rapidly cleared by the formation of inosine monophosphate (IMP), with accompanying loss of total adenine nucleotides. IMP formation has been thought to occur only during very intense muscular work, wherein ATP production does not keep pace with ATP consumption. Here, we propose a role for IMP in managing both ∆GATP and flux through a key energetic pathway during contractions. To test our hypothesis that IMP formation mitigates the accumulation of ADP, 8 males (28.4 + 3.5 yr, mean + SD) performed 8 min of incremental, isotonic knee extension contractions (1 every 2 s, starting at 6% peak torque and incrementing by 3% every 2 min) on a magnetic resonance (MR)‐compatible ergometer in a 3‐Tesla MR system. 31Phosphorus MR spectra were obtained at rest (60‐s average) and throughout the contraction protocol (10‐s temporal resolution). Peaks corresponding to ATP, phosphocreatine (PCr), inorganic phosphate (Pi), and phosphomonoesters (PME) were fit in jMRUI, and concentrations for these metabolites were determined assuming resting [ATP] = 8.2 mM. [ADP], [AMP], [IMP], and the rate of ATP (mM∙s‐1) production by non‐oxidative glycolysis (i.e., wherein the fate of pyruvate is lactate; ATPgly) were also calculated. As expected, [PCr] decreased and [Pi] increased approximately linearly throughout the incremental contraction protocol. Surprisingly, [ATP] decreased and [IMP] increased (to 7.3 + 0.4 mM and 0.8 + 0.56 mM, respectively) steadily from the onset of contractions until the final stage, at which point no further changes were observed. In contrast, both [ADP] and [AMP] increased linearly from the start of contractions until the final stage, when they accumulated more rapidly; ultimately to 109.9 + 52.1 uM and 1.0 + 0.91 uM, respectively. The glycolytic contribution to ATP production was consistent for the first 3 stages, after which ATPgly increased rapidly to 0.102 + 0.14 mM·s‐1 at 480 s. Collectively, the timing and magnitude of these metabolic changes in working muscle suggest a sequence of responses designed to maintain cytosolic [ADP] sufficiently low as to mitigate changes in ∆GATP, but at a level sufficient to stimulate ATPgly as contraction intensity increases. A novel observation in this study was the decrease in [ATP] (and thus increase in [IMP]) from the onset of the contraction protocol, which then leveled off at the highest contraction intensity. The utility and consequences of these responses remain to be determined, but this study supports the concept that IMP formation is important in maintaining the cellular energy state as well as in directing the bioenergetic response to a series of incremental muscular contractions in vivo.

TNFα Reduces the Maximum Respiratory Capacity of Mitochondria in Human Airway Smooth Muscle Cells

Previously, we reported that airway inflammation mediated by pro‐inflammatory cytokines such as TNFα induces hypercontractility and increased ATP consumption in human airway smooth muscle (hASM). We also found that TNFα induces an increase in maximum O2 consumption rate as well as mitochondrial biogenesis and an increase in mitochondrial volume density. However, when normalized to mitochondrial volume, maximum O2 consumption rate per mitochondrion is reduced in hASM after TNFα exposure. Unfortunately, standard respirometry (e.g., Seahorse) measures only the averaged maximum O2 consumption rate of a large number of cells. In the present study, we used a quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase reaction (SDHmax) together with mitochondrial volume density in individual hASM cells. SDH is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC), and SDHmax reflects the maximum respiratory capacity of individual mitochondrion. We hypothesized that TNFα decreases SDHmax per mitochondrion in hASM cells. hASM cells were dissociated from bronchial biopsies obtained during thoracic surgeries in patients with no history of asthma or chronic obstructive pulmonary disease. The hASM cells were treated with TNFα (20 ng/ml) or vehicle (DMSO) for 24 h, and SDHmax was measured using a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT) as the reaction indicator. As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil‐immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD), reflecting the accumulation of NBT diformazan reaction product in the hASM cell, was measured every 15 s over a 10 min period. Based on the Beer‐Lambert equation, the slope of the change in OD was used to determine SDHmax expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the same hASM cells were also loaded with MitoTracker Green (500 nM) and imaged in 3D. We found that mitochondria were more fragmented after TNFα exposure although mitochondrial volume density increased. When SDHmax was normalized to mitochondrial volume, we found that SDHmax per mitochondrion decreased in hASM cells, consistent with the reduction in maximum O2 consumption per mitochondrion that we previously observed. Thus, the increased energetic demand induced by inflammation (TNFα) are met by mitochondrial fragmentation leading to biogenesis and an increase in mitochondrial volume density rather than an increase in mitochondrial respiratory capacity.

RETRACTED ARTICLE: MIR600HG sponges miR-125a-5p to regulate glycometabolism and cisplatin resistance of oral squamous cell carcinoma cells via mediating RNF44

There is increasing evidence that dysregulated long non-coding RNA (lncRNA) is implicated in tumorigenesis and progression. We aim to explore the role of lncRNA MIR600HG in glycometabolism and cisplatin (DDP) resistance of oral squamous cell carcinoma (OSCC) cells via regulating microRNA-125a-5p (miR-125a-5p) and RING finger 44 (RNF44). Expression of MIR600HG, miR-125a-5p, and RNF44 in OSCC clinical samples, cell lines, and DDP-resistant OSCC cells (SCC-9/DDP) was determined. In SCC-9 cells, proliferation, IC50 value of DDP, migration, invasion, and apoptosis were detected; in SCC-9/DDP cells, proliferation, IC50 value of DDP, apoptosis, glucose consumption, and production of lactic acid and ATP were evaluated. The interaction of MR600HG, miR-125a-5p, and RNF44 was verified. MIR600HG and RNF44 were upregulated while miR-125a-5p was downregulated in OSCC tissues and cell lines, and also in SCC-9/DDP cells. In SCC-9 cells, MIR600HG overexpression improved cell growth, metastasis, and inhibited cell susceptibility to DDP; in SCC-9/DDP cells, silencing of MIR600HG promoted apoptosis, improved DDP sensitivity, and inhibited cell glycolysis. Downregulation of miR-125a-5p showed the opposite effect to downregulation of MIR600HG. MIR600HG bound to miR-125a-5p and miR-125a-5p targeted RNF44. Downregulation of miR-125a-5p reversed the improvement of DDP sensitivity and the inhibition of cell glycolysis by downregulated MIR600HG on SCC-9/DDP cells. Downregulating RNF44 reversed the promotion of DDP resistance and cell glycolysis of SCC-9/DDP cells mediated by downregulation of miR-125a-5p. Collectively, our study addresses that MIR600HG downregulation elevates miR-125a-5p and reduces RNF44 expression, thereby improving DDP sensitivity and inhibiting glycolysis in DDP-resistant OSCC cells.