Transmembrane potential induced on the internal organelle by a time-varying magnetic field: a model study

<p>Abstract</p> <p>Background</p> <p>When a cell is exposed to a time-varying magnetic field, this leads to an induced voltage on the cytoplasmic membrane, as well as on the membranes of the internal organelles, such as mitochondria. These potential changes in the organelles could have a significant impact on their functionality. However, a quantitative analysis on the magnetically-induced membrane potential on the internal organelles has not been performed.</p> <p>Methods</p> <p>Using a two-shell model, we provided the first analytical solution for the transmembrane potential in the organelle membrane induced by a time-varying magnetic field. We then analyzed factors that impact on the polarization of the organelle, including the frequency of the magnetic field, the presence of the outer cytoplasmic membrane, and electrical and geometrical parameters of the cytoplasmic membrane and the organelle membrane.</p> <p>Results</p> <p>The amount of polarization in the organelle was less than its counterpart in the cytoplasmic membrane. This was largely due to the presence of the cell membrane, which "shielded" the internal organelle from excessive polarization by the field. Organelle polarization was largely dependent on the frequency of the magnetic field, and its polarization was not significant under the low frequency band used for transcranial magnetic stimulation (TMS). Both the properties of the cytoplasmic and the organelle membranes affect the polarization of the internal organelle in a frequency-dependent manner.</p> <p>Conclusions</p> <p>The work provided a theoretical framework and insights into factors affecting mitochondrial function under time-varying magnetic stimulation, and provided evidence that TMS does not affect normal mitochondrial functionality by altering its membrane potential.</p

Microwell Array Integrating Ring Nanoelectrodes for The Monitoring of Metabolic Responses at Isolated Mitochondria

International audienceMitochondria are major cell organelles since they are the main source of ATP owing to the oxidative phosphorylation pathway. They also play an important role into several other metabolic pathways (Krebs cycle, lipid synthesis…) and when defective, they are involved into severe pathologies (myopathies, neurological diseases, cancers...). Consequently, new methodological approaches [1, 2] are required in order to decipher mitochondria metabolisms and to provide efficient tools for diagnosis. In this context, we have developed microsystems, namely ElecWell platforms, which combine electrochemical [1] and optical [2] sensing abilities. These are based on the integration of platinum ring nanoelectrodes (RNE, surface: 10-15 µm 2) into SiO2-based microwell arrays (well radius: 3 to 4.5 µm, depth: 5 µm, individual volume < 1 pL), as shown on figure 1. Similarly to ultramicroelectrodes, RNE exhibit high current density, fast response time, reduced charging current and high signal-to-noise ratio. These electrochemical devices were characterized by cyclic voltammetry using single well or array configurations, and optimised according to COMSOL™ simulations. In addition, the glass substrate of the microsystems allows the observation by microscopy of the content and reactivity within each well. Then, a suspension of isolated mitochondria (yeast origin) was deposited on the ElecWell array and allowed to sediment within wells. We monitored by fluorescence the presence of individual mitochondria within wells (Fig. 1b) owing to their NADH content and variations [2]. Simultaneously, we monitored by cyclic voltammetry the variations of their oxygen consumption rate in response to specific activators and inhibitors of respiratory chain activity (ethanol, ADP, antimycine A). The resolution offered by the ElecWell platform is nearly a few thousands of mitochondria (Fig. 1c), corresponding to the mitochondrial content of a single cell. a) b) c) Figure 1. a) Integration of a platinum ring nanoelectrode into a SiO2-based microwell (radius: 3 µm) within the array (10 6 wells); b) Monitoring by fluorescence (NADH content) of isolated mitochondria deposited within wells. c) Detection by cyclic voltammetry (reduction current sampling) of the oxygen variations during mitochondrial activation (EtOH/ADP) and inhibition (Antimycin A)

Toward the Analysis of Mitochondria Isolated from Leukemic Cells with Electrochemically Instrumented Microwell Arrays

International audienceThis work deals with the development of electrochemical transducers for the analysis of the metabolic status of mitochondria isolated from leukemic cells. It proposes the use of ring nanoelectrodes (RNE) integrated into microwell arrays for the simultaneous monitoring of the oxygen (O2) consumption and the hydrogen peroxide (H2O2) production. The sensor enabled the real-time recording of the oxygen consumption of approximately 10,000 isolated mitochondria. Solutions are now proposed to detect H2O2 production and to reduce the number of mitochondria under test, targeting the single mitochondrion analysis

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community

Although uncultured microorganisms have important roles in ecosystems, their ecophysiology in situ remains elusive owing to the difficulty of obtaining live cells from their natural habitats. In this study, we employed a novel magnetic nanoparticle-mediated isolation (MMI) method to recover metabolically active cells of a group of previously uncultured phenol degraders, Burkholderiales spp., from coking plant wastewater biosludge; five other culturable phenol degraders—Rhodococcus sp., Chryseobacterium sp. and three different Pseudomonas spp.—were also isolated from the same biosludge using traditional methods. The kinetics of phenol degradation by MMI-recovered cells (MRCs) was similar to that of the original sludge. Stable isotope probing (SIP) and pyrosequencing of the 16S rRNA from the ‘heavy’ DNA (13C-DNA) fractions indicated that Burkholderiales spp. were the key phenol degraders in situ in the biosludge, consistent with the results of MRCs. Single-cell Raman micro-spectroscopy was applied to probe individual bacteria in the MRCs obtained from the SIP experiment and showed that 79% of them were fully 13C-labelled. Biolog assays on the MRCs revealed the impact of various carbon and nitrogen substrates on the efficiency of phenol degradation in the wastewater treatment plant biosludge. Specifically, hydroxylamine, a metabolite of ammonia oxidisation, but not nitrite, nitrate or ammonia, inhibited phenol degradation in the biosludge. Our results provided a novel insight into the occasional abrupt failure events that occur in the wastewater treatment plant. This study demonstrated that MMI is a powerful tool to recover live and functional cells in situ from a complex microbial community to enable further characterisation of their physiology

Microsystems for the electrochemical and optical monitoring of bioenergetic activities of isolated mitochondria

International audienceMitochondria are known as central players in many cellular processes including oxidative phosphorylation, oxidative stress and signaling through the production of reactive oxygen species, or the activation of apoptosis by the cytochrome c release. Consequently, they play a key role in the progression of diseases linked to ageing, including cancers and neurodegenerative troubles. Thus, lots of efforts are currently devoted to develop innovative therapies based on the modulation of mitochondrial activity. This implies increasing demand for devices allowing the analysis of metabolic processes at the scale of isolated mitochondria. In this context, we developed the ElecWell (electrochemical microwell), based on the integration of ring nanoelectrodes (RNE) into silica microwell arrays made on glass substrates [1]. The new generation of ElecWell devices was adapted to a temperature-controlled microscopy platform. Two planar electrodes were integrated to obtain a complete electrochemical cell and allow experiments in closed-flow-through configuration (Figure 1A). Methods were developed to enhance the filling rate of microwells by mitochondria and to reduce biofouling (Figure 1B). First results were obtained with mitochondria isolated from rodent cardiomyocytes. Oxygen consumption was measured locally by cyclic voltammetry at the RNEs (Figure 1C) whereas individual variations of mitochondrial membrane potential were monitored by fluorescence microscopy (Figure 1D), [2]. Next steps consist in performing simultaneous measurements and to reach the electrochemical detection of the bioenergetic activity of single mitochondria with individually addressable microwells. [1] Sékli Belaïdi F. et al., Sensors Actuators B-Chemical, 2016, 232, 345 [2] Vajrala V.S. et al, Integrative Biology, 2016, 8, 836. Figure 1: (A) the 2 nd generation of the ElecWell device mounted on its microscopy platform; (B) mitochondria into microwells (TMRM fluorescence); (C) variations of dissolved oxygen concentration versus time; (D) variations of mitochondrial membrane potential versus time, both as function of activator/inhibitor additions

Stress response of a marine ammonia-oxidizing archaeon informs physiological status of environmental populations

High representation by ammonia-oxidizing archaea (AOA) in marine systems is consistent with their high affinity for ammonia, efficient carbon fixation, and copper (Cu)-centric respiratory system. However, little is known about their response to nutrient stress. We therefore used global transcriptional and proteomic analyses to characterize the response of a model AOA, Nitrosopumilus maritimus SCM1, to ammonia starvation, Cu limitation and Cu excess. Most predicted protein-coding genes were transcribed in exponentially growing cells, and of ∼74% detected in the proteome, ∼6% were modified by N-terminal acetylation. The general response to ammonia starvation and Cu stress was downregulation of genes for energy generation and biosynthesis. Cells rapidly depleted transcripts for the A and B subunits of ammonia monooxygenase (AMO) in response to ammonia starvation, yet retained relatively high levels of transcripts for the C subunit. Thus, similar to ammonia-oxidizing bacteria, selective retention of amoC transcripts during starvation appears important for subsequent recovery, and also suggests that AMO subunit transcript ratios could be used to assess the physiological status of marine populations. Unexpectedly, cobalamin biosynthesis was upregulated in response to both ammonia starvation and Cu stress, indicating the importance of this cofactor in retaining functional integrity during times of stress.http://deepblue.lib.umich.edu/bitstream/2027.42/191241/2/ISME Journal_2018.pdfPublished versionDescription of ISME Journal_2018.pdf : Accepted versio