Flux Analysis Research Articles

Mathematical reconstruction of the metabolic network in an in-vitro multiple myeloma model.

It is increasingly apparent that cancer cells, in addition to remodelling their metabolism to survive and proliferate, adapt and manipulate the metabolism of other cells. This property may be a telling sign that pre-clinical tumour metabolism studies exclusively utilising in-vitro mono-culture models could prove to be limited for uncovering novel metabolic targets able to translate into clinical therapies. Although this is increasingly recognised, and work towards addressing the issue is becoming routinary much remains poorly understood. For instance, knowledge regarding the biochemical mechanisms through which cancer cells manipulate non-cancerous cell metabolism, and the subsequent impact on their survival and proliferation remains limited. Additionally, the variations in these processes across different cancer types and progression stages, and their implications for therapy, also remain largely unexplored. This study employs an interdisciplinary approach that leverages the predictive power of mathematical modelling to enrich experimental findings. We develop a functional multicellular in-silico model that facilitates the qualitative and quantitative analysis of the metabolic network spawned by an in-vitro co-culture model of bone marrow mesenchymal stem- and myeloma cell lines. To procure this model, we devised a bespoke human genome constraint-based reconstruction workflow that combines aspects from the legacy mCADRE & Metabotools algorithms, the novel redHuman algorithm, along with 13C-metabolic flux analysis. Our workflow transforms the latest human metabolic network matrix (Recon3D) into two cell-specific models coupled with a metabolic network spanning a shared growth medium. When cross-validating our in-silico model against the in-vitro model, we found that the in-silico model successfully reproduces vital metabolic behaviours of its in-vitro counterpart; results include cell growth predictions, respiration rates, as well as support for observations which suggest cross-shuttling of redox-active metabolites between cells.

13C-metabolic flux analysis of lipid accumulation in the green microalgae Tetradesmus obliquus under nitrogen deficiency stress

Metabolic fluxes (MF) serve as the functional phenotypes of biochemical processes and are crucial to describe the distribution of precursors within metabolic networks. There is a lack of experimental observations for carbon flux towards lipids, which is important for biodiesel generation. Here, the accumulation of lipid, and MF in Tetradesmus obliquus under nitrogen deficiency stress (NF) using a 13C isotope tracer at different time intervals was investigated. The 13C based MF showed enhanced de novo synthesis of G3P and PEP, indicating increased carbon flux from CO2 into lipid synthesis. An increase in palmitic acid (3500 μmol/mg), linoleic acid (2100 μmol/mg), and oleic acid (2000 μmol/mg) was observed. The accumulation of C16:0 under NF was mainly related to de novo synthesis while C18:3 was accumulated through a non de novo pathway. Under NF stress, T. obliquus had higher flux in PPP and glycolysis pathway, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis.

Opposite regulation of glycogen metabolism by cAMP produced in the cytosol and at the plasma membrane

Cyclic AMP is produced in cells by two different types of adenylyl cyclases: at the plasma membrane by the transmembrane adenylyl cyclases (tmACs, ADCY1~ADCY9) and in the cytosol by the evolutionarily more conserved soluble adenylyl cyclase (sAC, ADCY10). By employing high-resolution extracellular flux analysis in HepG2 cells to study glycogen breakdown in real time, we showed that cAMP regulates glycogen metabolism in opposite directions depending on its location of synthesis within cells and the downstream cAMP effectors. While the canonical tmAC-cAMP-PKA signaling promotes glycogenolysis, we demonstrate here that the non-canonical sAC-cAMP-Epac1 signaling suppresses glycogenolysis. Mechanistically, suppression of sAC-cAMP-Epac1 leads to Ser-15 phosphorylation and thereby activation of the liver-form glycogen phosphorylase to promote glycogenolysis. Our findings highlight the importance of cAMP microdomain organization for distinct metabolic regulation and establish sAC as a novel regulator of glycogen metabolism.

Measurements of laminar burning velocities and an improved kinetic model of methyl isopropyl ketone

Methyl isopropyl ketone (MIPK) is the simplest branched ketone and a promising biofuel. In this work, laminar burning velocities (SL) of MIPK + air flames were measured using the heat flux method at atmospheric pressure, over initial mixture temperatures of 298–358 K and equivalence ratios of 0.7–1.4. With the help of the temperature dependence of the SL, data inconsistency between the present measurements and the experimental data reported by Li et al. (Proc. Combust. Inst. 38 (2021) 2135) was demonstrated. Moreover, existing kinetic models for MIPK combustion notably deviate from the present SL measurements. Therefore, the MIPK model suggested by Lin et al. (Proc. Combust. Inst. 39 (2023) 315) was updated by revisiting the MIPK H-abstraction reactions and methyl isopropenyl ketone sub-model. Furthermore, a new di-methyl ketene (critical intermediate during MIPK oxidation) sub-model was constructed and integrated into the MIPK model. Flux and sensitivity analyses revealed that integration of the new di-methyl ketene model improves predictions of the laminar burning velocities as well as shock tube ignition delay times over the pressures of 1–40 bar due to converting di-methyl ketene into C3H5-T (CH2 = C˙CH3) rather than C3H6 or C3H5-S (C˙H = CHCH3) predicted by other MIPK models from the literature. Updates of the MIPK H-abstraction reactions yield more reasonable products branching ratios of formation of the primary fuel radicals, and improve prediction of the SL. It was also found that the rate constants of the MIPK decomposition reaction (MIPK (+M) = CH3CO + IC3H7 (+M)) in the model proposed by Li et al. (Proc. Combust. Inst. 38 (2021) 2135) are significantly underestimated, resulting in underestimation of the present SL measurements and significant overprediction of the ignition delay times.

Heat Flux Analysis and Assessment of Drying Kinetics during Lyophilization of Fruits in a Pilot-Scale Freeze Dryer.

Vacuum freeze-drying as a process for achieving high product quality has attracted increasing attention in the last decade. Particularly in the pharmaceutical field and food processing industries, lyophilization can produce high-quality products compared to samples dried by conventional methods. Despite its benefits, lyophilization is a time-consuming and costly process that requires optimization of a number of process parameters, including shelf temperature, chamber pressure, freezing rate, and process time. This paper reports on the implementation of heat flux measurements that allow noninvasive real-time determination of the endpoint of the primary drying stage as an essential parameter for the effective optimization of the overall drying time. Quantitative analysis of the drying kinetics of five fruits (kiwifruit, avocado, Asian pear, persimmon, and passion fruit) was assessed by comparing the heat flux and temperature profiles of samples during the lyophilization process. For a 24 h lyophilization cycle, average heat fluxes in the primary drying phase ranged from 250 to 570 W/m2. A significant correlation was found between the temperature and heat flux distributions at the estimated endpoint of the sublimation process and the corresponding transition into the secondary drying stage. Furthermore, good agreement was also found for the freezing phase. The use of real-time heat flux measurements proved to be a cost-effective experimental method to better understand the process variables in order to reduce the lyophilization cycle time and overall energy consumption.

Bubble behaviour and critical heat flux investigations on electrodeposited metallic tubes during pool boiling

There is colossal research scope in achieving greater functionality and durability in microelectronic devices as conventional air cooling systems cannot handle these devices' cooling requirements which may be due to their low heat transfer performance. In such instances, phase change heat transfer mechanisms are appropriate for heat extraction since they utilise both sensible and latent heat. The ability to remove huge amount of heat at low wall superheats and the lack of moving parts make pool boiling appealing. This experimental study emphasises the analysis of bubble behaviour and critical heat flux (CHF) on electrodeposited tubes developed via in-house built set-up. Copper has been deposited over SS 304 tubes of a length of 100 mm and diameter of 2.5–4.5 mm. Their surface morphologies have been investigated using various characterization techniques such as SEM, EDS, AFM, and XRD. The surface roughness measurement confirms that electrodeposition increases the roughness of the tube. The contact angle analysis confirms that the coated surfaces are hydrophobic in nature. The CHF visualization is carried out under uniform heat flux conditions. It is observed that with an increase in diameter, the magnitude of CHF reduces irrespective of the coated and uncoated surface. The hydrophobic surface exhibits a lesser CHF magnitude than the uncoated surface. The reduction in CHF magnitude is within a range of 4.27%–8.93%. At lower heat flux magnitude, the bubble nucleation is rigorous in coated tubes, making it efficient for phase change heat transfer applications.

Metabolic engineering of Synechococcus elongatus 7942 for enhanced sucrose biosynthesis

The capability of cyanobacteria to produce sucrose from CO2 and light has a remarkable societal and biotechnological impact since sucrose can serve as a carbon and energy source for a variety of heterotrophic organisms and can be converted into value-added products. However, most metabolic engineering efforts have focused on understanding local pathway alterations that drive sucrose biosynthesis and secretion in cyanobacteria rather than analyzing the global flux re-routing that occurs following induction of sucrose production by salt stress. Here, we investigated global metabolic flux alterations in a sucrose-secreting (cscB-overexpressing) strain relative to its wild-type Synechococcus elongatus 7942 parental strain. We used targeted metabolomics, 13C metabolic flux analysis (MFA), and genome-scale modeling (GSM) as complementary approaches to elucidate differences in cellular resource allocation by quantifying metabolic profiles of three cyanobacterial cultures – wild-type S. elongatus 7942 without salt stress (WT), wild-type with salt stress (WT/NaCl), and the cscB-overexpressing strain with salt stress (cscB/NaCl) – all under photoautotrophic conditions. We quantified the substantial rewiring of metabolic fluxes in WT/NaCl and cscB/NaCl cultures relative to WT and identified a metabolic bottleneck limiting carbon fixation and sucrose biosynthesis. This bottleneck was subsequently mitigated through heterologous overexpression of glyceraldehyde-3-phosphate dehydrogenase in an engineered sucrose-secreting strain. Our study also demonstrates that combining 13C-MFA and GSM is a useful strategy to both extend the coverage of MFA beyond central metabolism and to improve the accuracy of flux predictions provided by GSM.

Characterization of the effect of pomegranate crude extract, and its post-harvesting preservation procedures, on redox tone, cellular growth and metabolic profile of MDA-MB-231 cell line

BackgroundPomegranate is known for its beneficial properties due to its high content in antioxidants and might constitute a natural option for preventing and treatment of different pathologies including cancer. Since mitochondria are involved in tumorigenesis through ROS production and modulation of oxidative metabolism, we investigated the biological effects of pomegranate on cellular redox state, proliferation and metabolism in the breast cancer cell line MDA-MB-231 (MDA).MethodsMDA were treated for 24 h with graded concentration of filtered Pomegranate juice (PJ) and tested for metabolic Flux Analysis with XFe96 Extracellular Flux Analyzer, for proliferation using the xCELLigence System Real-Time Cell Analyzer and for intracellular ROS content by Confocal Microscopy Imaging.ResultsCells-treatment with freshly prepared pomegranate juice (PJ) resulted in a significant reduction of the intracellular ROS content already at the lower concentration of PJ tested. Additionally, it enhanced mitochondria respiration, and decreased glycolysis at high concentrations, inhibiting at the same time cell proliferation. As pomegranate is a seasonal fruit, assessment of optimum storage conditions preserving its bio-active properties was investigated. Our results indicated that storage conditions under controlled atmosphere for 30 days was able to enhance mitochondrial respiration at the same extent than freshly extracted PJ. Conversely, freezing procedure, though retaining the antioxidant and cell-growth inhibitory property, elicited an opposite effect on the metabolic profile as compared with fresh extract.ConclusionOverall, the results of our study, on the one hand, confirms the preventive/therapeutic potential of PJ, as well as of its post-harvested processing, for cancer management. On the other hand, it highlights the intrinsic difficulties in attaining mechanistic insights when a multiplicity of effects is elicited by a crude mixture of bio-active compounds.

Beta cell primary cilia mediate somatostatin responsiveness via SSTR3

ABSTRACT Somatostatin is a paracrine modulator of insulin secretion and beta cell function with pleotropic effects on glucose homeostasis. The mechanism of somatostatin-mediated communication between delta and beta cells is not well-understood, which we address in this study via the ciliary somatostatin receptor 3 (SSTR3). Primary cilia are membrane organelles that act as signaling hubs in islets by virtue of their subcellular location and enrichment in signaling proteins such as G-protein coupled receptors (GPCRs). We show that SSTR3, a ciliary GPCR, mediates somatostatin suppression of insulin secretion in mouse islets. Quantitative analysis of calcium flux using a mouse model of genetically encoded beta cell-specific GCaMP6f calcium reporter shows that somatostatin signaling alters beta cell calcium flux after physiologic glucose stimulation, an effect that depends on endogenous SSTR3 expression and the presence of intact primary cilia on beta cells. Comparative in vitro studies using SSTR isoform antagonists demonstrate a role for SSTR3 in mediating somatostatin regulation of insulin secretion in mouse islets. Our findings support a model in which ciliary SSTR3 mediates a distinct pathway of delta-to-beta cell regulatory crosstalk and may serve as a target for paracrine modulation.

Amiodarone but not propafenone impairs bioenergetics and autophagy of human myocardial cells

Cardiac and extra-cardiac side effects of common antiarrhythmic agents might be related to drug-induced mitochondrial dysfunction. Supratherapeutic doses of amiodarone have been shown to impair mitochondria in animal studies, whilst influence of propafenone on cellular bioenergetics is unknown. We aimed to assess effects of protracted exposure to pharmacologically relevant doses of amiodarone and propafenone on cellular bioenergetics and mitochondrial biology of human and mouse cardiomyocytes. In this study, HL-1 mouse atrial cardiomyocytes and primary human cardiomyocytes derived from the ventricles of the adult heart were exposed to 2 and 7 μg/mL of either amiodarone or propafenone. After 24 h, extracellular flux analysis and confocal laser scanning microscopy were used to measure mitochondrial functions. Autophagy was assessed by western blots and live-cell imaging of lysosomes. In human cardiomyocytes, amiodarone significantly reduced mitochondrial membrane potential and ATP production, in association with an inhibition of fatty acid oxidation and impaired complex I- and II-linked respiration in the electron transport chain. Expectedly, this led to increased anaerobic glycolysis. Amiodarone increased the production of reactive oxygen species and autophagy was also markedly affected. In contrast, propafenone-exposed cardiomyocytes did not exert any impairment of cellular bioenergetics. Similar changes after amiodarone treatment were observed during identical experiments performed on HL-1 mouse cardiomyocytes, suggesting a comparable pharmacodynamics of amiodarone among mammalian species. In conclusion, amiodarone but not propafenone in near-therapeutic concentrations causes a pattern of mitochondrial dysfunction with affected autophagy and metabolic switch from oxidative metabolism to anaerobic glycolysis in human cardiomyocytes.

Chemical flux analysis of low-temperature plasma-enhanced oxidation of methane and hydrogen in argon

Plasma can be used to enhance the reactivity of combustible mixtures at low temperatures. In this article, the chemical pathways predicted by three different reaction mechanisms are investigated for the low-temperature oxidation of hydrogen and methane. To validate our model and the reaction mechanisms, the numerical results are compared against experimental results in a diluted flow reactor. Our model with all three reaction mechanisms predicts trends similar to those observed in the experiments. Moreover, all predicted quantities show reasonable quantitative agreement with the experiments. Flux analysis is used to identify the main pathways of oxidation at different temperatures. Three different modes, each active in a different temperature range, are identified in the oxidation of hydrogen. When the temperature is increased, these modes become increasingly self-sustained. Similarly, three different pathways are identified in the oxidation of methane. Below 1000K, methane quickly removes hydroxyl radicals from the radical pool, inhibiting self-sustained oxidation. From our analysis, we conclude that plasma provides activation of the low-temperature chemistry by the generation of radicals.

Analysis of the North Atlantic offshore energy flux from different reanalysis and hindcasts

To date there is a wide range of wave reanalysis and hindcasts available to the scientific and engineering community which are commonly used for different applications, including downscaling or the estimation of the wave energy resource (Morim et al., 2022). These long datasets have been created using different combinations of forcing fields, physical parameterizations, and numerical choices (like spatial and spectral resolution). All these elements have a direct effect on the accuracy of the wave models’ output (e.g., Alday et al., 2021) and thus, they are one of the main reasons for the differences between these products. In the present study we analyze the significant wave heights and peak periods characteristics from a selection of global datasets. We additionally include results from a hindcast created using the WAVEWATCH III model, with adjustments specially aimed to reduce uncertainties of the wave energy resource along the Atlantic coasts of Europe. Models’ output is compared with buoys and altimeter data from the latest ESA (European Space Agency) CCI Sea State V3 product. Preliminary validation of the hindcast we have generated for the North Atlantic already show an important bias reduction for wave heights in the 2.5 to 11.5 range compared to ERA5 wave product. Using the relevant wave parameters, we estimate the power density and quantify the differences between databases. Then, based on scatter diagrams obtained from the joint distributions of significant wave height and peak period, the differences in the power captured by wave energy converters (WEC) related to different wave data sources will be quantified (e.g., Babarit et al., 2011; Henriques et al., 2016).

Spectral analysis of heat flux across a nanostructured solid diamond-liquid water interface: A nonequilibrium molecular dynamics study

Spectral analysis of heat flux across a nanostructured solid diamond-liquid water interface: A nonequilibrium molecular dynamics study

Analysis of the transient heat flux on divertor surface during plasma disruption in EAST

Analysis of the transient heat flux on divertor surface during plasma disruption in EAST

Swash-by-swash morphology change on a dynamic cobble berm revetment: High-resolution cross-shore measurements

Dynamic cobble berm revetments are a promising soft engineering technique capable of protecting sandy coastlines by armouring the sand and dissipating wave energy to protect the hinterland against wave attack. They also form composite beaches as they are essentially mimicking natural composite beach structure and behaviour. This type of coastal protections and beaches have recently been investigated, and this led to a better understanding of their overall behaviour under varying water levels and wave conditions. However, the short-term dynamics of the wash zone (where all bed changes occur) has never been studied at high-resolution, and this is needed to fully understand the underlying dynamics of such structures and relate it to observed processes at larger scale. To do so, the revetment at North Cove (WA, USA) was monitored for a nine-day period in January 2019 over a spring tidal cycle and with significant wave heights offshore reaching 6 m. A 2-D lidar was used to survey a cross-shore profile of the revetment, and record all surface changes and interaction with swashes at high spatial (0.1 m) and temporal (swash-by-swash) resolution. The revetment was found to rapidly reshape under these energetic conditions, but reached a relatively stable state during the rising tide. The analysis of bed-level changes and net cross-shore mass fluxes over the revetment showed that revetment changes are mainly driven by very small events, with some rare large bed-level changes of a magnitude comparable to the median cobble diameter. The distribution of event mass fluxes nearly balanced out over the duration of a tide, meaning that positive and negative fluxes tended to be symmetrical. Furthermore, measured net fluxes magnitude were 18 times smaller than the absolute fluxes, which demonstrated the dynamic stability of the revetment as substantial movement occur on a wave-by-wave timescale but these balance out over time. The analysis of swash revealed that the revetment section where the swash reaches a maximum depth between 0.15 and 0.45 m undergoes the more extreme fluxes. Swashes deeper than 0.45 m only occurred in zones inundated more than 50% of the time, and smaller extreme fluxes were measured over the revetment section where these deep swashes were recorded. Bed level change oscillations over the revetment were observed, and the cross shore limit of these was correlated with the mean wave period at the toe of the revetment. Overall, the water depth at the toe of the revetment was identified as the key parameter to describe the energy reaching the revetment. This study enables the morphodynamics of dynamic revetment, observed in previous lab and field studies, to be explained at the swash scale, and brought new information on the sediment dynamics of composite beaches and dynamic revetments. These findings allow to suggest some generic guidance for dynamic cobble berm revetment design. Finally, the results are compared to a similar study on sandy beaches.

The extreme wet and large precipitation size increase carbon uptake in Eurasian meadow steppes: Evidence from natural and manipulated precipitation experiments

The distribution of seasonal precipitation would profoundly affect the dynamics of carbon fluxes in terrestrial ecosystems. However, little is known about the impacts of extreme precipitation and size events on ecosystem carbon cycle when compared to the effects of average precipitation amount. The study involved an analysis of carbon fluxes and water exchange using the eddy covariance and chamber based techniques during the growing seasons of 2015–2017 in Bayan, Mongolia and 2019–2021 in Hulunbuir, Inner Mongolia, respectively. The components of carbon fluxes and water exchange at each site were normalized to evaluate of relative response among carbon fluxes and water exchange. The investigation delved into the relationship between carbon fluxes and extreme precipitation over five gradients (control, dry spring, dry summer, wet spring and wet summer) in Hulunbuir meadow steppe and distinct four precipitation sizes (0.1–2, 2–5, 5–10, and 10–25 mm d−1) in Bayan meadow steppe. The wet spring and summer showed the greatest ecosystem respiration (ER) relative response values, 76.2% and 73.5%, respectively, while the dry spring (−16.7%) and dry summer (14.2%) showed the lowest values. Gross primary production (GPP) relative response improved with wet precipitation gradients, and declined with dry precipitation gradients in Hulunbuir meadow steppe. The least value in net ecosystem CO2 exchange (NEE) was found at 10–25 mm d−1 precipitation size in Bayan meadow steppe. Similarly, the ER and GPP increased with size of precipitation events. The structural equation models (SEM) satisfactorily fitted the data (χ2 = 43.03, d.f. = 11, p = 0.215), with interactive linkages among soil microclimate, water exchange and carbon fluxes components regulating NEE. Overall, this study highlighted the importance of extreme precipitation and event size in influencing ecosystem carbon exchange, which is decisive to further understand the carbon cycle in meadow steppes.

Spatial–temporal analysis of magnetohydrodynamics flow and energy flux of power-law nanofluid in a confined domain

Energy flux analysis of power-law fluid is a novel contribution to recent developments in computational fluid dynamics. The study of the unsteady two-dimensional flow with double diffusive effect inside a complex enclosure exhibits great potential in optimizing the heat transfer rate due to the wavy nature of the side walls. The enclosure is confined with the flow circulation due to the thermal and solutal gradients acting along the left and right wavy walls. The computational time and accuracy in results are estimated and compared by implementing the finite volume method and element-free Galerkin technique. The results are obtained in terms of streamlines, isotherms, isoconcentrations, average Nusselt number, Sherwood number, and total entropy generation due to the effect of conventional parameters, namely, power-law index, Rayleigh number, buoyancy ratio parameter, thermophoresis parameter, Brownian motion parameter, and Lewis number with a fixed Prandtl number throughout the computation. The optimized double-diffusive natural convection analysis is based on entropy generation and a calculated Bejan number. The novelty of this paper lies in the implementation of a mesh-free approach, which may be useful for the further analysis of elliptical/semi-elliptical structures.

GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5’-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

αβ-T cell receptor transduction gives superior mitochondrial function to γδ-T cells with promising persistence

Adoptive cell therapy using allogeneic γδ-T cells is a promising option for off-the-shelf Tcell products with a low risk of graft-versus-host disease (GVHD). Long-term persistence may boost the clinical development of γδ-T cell products. In this study, we found that genetically modified Vγ9+Vδ2+ Tcells expressing a tumor antigen-specific αβ-TCR and CD8 coreceptor (GMC) showed target-specific killing and excellent persistence. To determine the mechanisms underlying these promising effects, we investigated metabolic characteristics. Cytokine secretion by γδ-TCR-stimulated nongene-modified γδ-T cells (NGMCs) and αβ-TCR-stimulated GMCs was equally suppressed by a glycolysis inhibitor, although the cytokine secretion of αβ-TCR-stimulated GMCs was more strongly inhibited by ATP synthase inhibitors than that of γδ-TCR-stimulated NGMCs. Metabolomic and transcriptomic analyses, flow cytometry analysis using mitochondria-labeling dyes and extracellular flux analysis consistently suggest that αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function. In conclusion, αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function with promising persistence.

Emergence of putative energy parasites within Clostridia revealed by genome analysis of a novel endosymbiotic clade.

The Clostridia is a dominant bacterial class in the guts of various animals and are considered to nutritionally contribute to the animal host. Here, we discovered clostridial endosymbionts of cellulolytic protists in termite guts, which have never been reported with evidence. We obtained (near-)complete genome sequences of three endosymbiotic Clostridia, each associated with a different parabasalid protist species with various infection rates: Trichonympha agilis, Pseudotrichonympha grassii, and Devescovina sp. All these protists are previously known to harbor permanently-associated, mutualistic Endomicrobia or Bacteroidales that supplement nitrogenous compounds. The genomes of the endosymbiotic Clostridia were small in size (1.0-1.3 Mbp) and exhibited signatures of an obligately-intracellular parasite, such as an extremely limited capability to synthesize amino acids, cofactors, and nucleotides and a disrupted glycolytic pathway with no known net ATP-generating system. Instead, the genomes encoded ATP/ADP translocase and, interestingly, regulatory proteins that are unique to eukaryotes in general and are possibly used to interfere with host cellular processes. These three genomes formed a clade with metagenome-assembled genomes (MAGs) derived from the guts of other animals, including human and ruminants, and the MAGs shared the characteristics of parasites. Gene flux analysis suggested that the acquisition of the ATP/ADP translocase gene in a common ancestor was probably key to the emergence of this parasitic clade. Taken together, we provide novel insights into the multilayered symbiotic system in the termite gut by adding the presence of parasitism and present an example of the emergence of putative energy parasites from a dominant gut bacterial clade.