, 2003), signal envelope correlations

, 2003), signal envelope correlations buy Ixazomib were particularly robust for high-frequency activity. Gamma-band power envelope correlations have also been reported for sensorimotor networks (He et al., 2008), which were low for slow-wave sleep but high for REM sleep and awake state. Task-related decreases in gamma-band power have been demonstrated in the default-mode network (Jerbi et al., 2010). In addition, anticorrelated gamma-band power fluctuations for different

networks have been observed in invasive human recordings (Keller et al., 2013). Several EEG studies have suggested that the dynamics of the slow fluctuations giving rise to envelope ICMs may be scale-free, that is, not characterized by defined peaks in the power spectrum (Linkenkaer-Hansen et al., 2001, He et al., 2010 and Palva and Palva, 2011). Only recently, a number of studies have aimed to investigate the neurophysiology of ICMs by combining noninvasive MEG recordings with source space analyses. Several of these studies used amplitude or see more power envelope correlations (de Pasquale et al., 2010, Brookes et al., 2011, Brookes et al., 2012, Hipp et al., 2012 and de

Pasquale et al., 2012), while others employed phase coherence (Hipp et al., 2011 and Bardouille and Boe, 2012), phase lag index (Hillebrand et al., 2012), or imaginary coherence (Marzetti et al., 2013). An interesting result is that plain correlation of signal envelopes yields spatially unspecific correlation patterns characterized by high correlation of the seed with neighboring voxels and a monotonic drop off to more distant sites (Hipp et al., 2012). While also comprising true interactions, such patterns are likely to reflect, to a substantial amount, spurious correlations arising from volume spread (Nolte et al., 2004 and Hipp

et al., 2012). However, ICM dynamics can be recovered if correlation patterns resulting from volume conduction are suppressed before analyzing functional connectivity (Hipp et al., 2012, Brookes else et al., 2012, Hillebrand et al., 2012 and Marzetti et al., 2013). A recent study that successfully employed this approach for investigation of envelope ICMs has used phase orthogonalization (Figure 2B) to remove zero-phase coupling (Hipp et al., 2012). Analysis of correlations among power envelopes revealed spatially specific coupling patterns. For instance, signal power was correlated between homologous sensory areas of the two hemispheres (Figure 2D), which matches similar patterns observed in BOLD signals (Figure 1A). Overall, ICMs were most prominent in the alpha and beta band. The power envelope fluctuations were coupled at very slow frequencies below 0.1 Hz (Figure 2C), suggesting a close correspondence to correlated BOLD activity fluctuations (Fox and Raichle, 2007, Deco and Corbetta, 2011 and Raichle, 2010). The data indicate that this approach can reveal a rich set of spectral signatures for functional networks.

Nymphs and adults were dissected, the salivary glands collected a

Nymphs and adults were dissected, the salivary glands collected and immersed in a cell lysis solution for subsequent DNA extraction. DNA was extracted from cervid blood samples (300 μL) with the aid of a Wizard® Genomic DNA Purification Kit (Promega, Madison, BI 6727 ic50 WI, USA) employed according to the manufacturer’s instructions. In order to extract DNA from tick salivary glands, the same commercial kit was used following the manufacturer’s instructions designated for the extraction

of DNA from tissue cultures. The nPCR assay of genomic DNA involved two separate amplification reactions. The first reaction was carried out using the primers RIB-19 (5′CGGGATCCAACCTGGTTGATCCTGC3′) and RIB-20 (5′CCGAATTCCTTGTTACGACTTCTC3′) that are specific

for a 1700 bp segment of the 18S rRNA gene from find more Babesia and Theileria spp. ( Zahler et al., 2000). The reaction mixture comprised 1.2 μL of dNTPs (0.2 mM), 0.15 μL of Taq polymerase (0.05 U), 1.5 μL which buffer (1×), 0.6 μL of a solution containing the mixed primers (10 μM) and sufficient sterile ultra-pure water to give a final volume of 15 μL. A 1.5 μL aliquot of the DNA template was added to the reaction mixture, and amplification was performed using an Eppendorf (São Paulo, SP, Brazil) Mastercycler® thermocycler programmed as follows: 94 °C for 5 min (initial denaturation step), 30 cycles each comprising 92 °C for 1 min (denaturation), 54 °C for 1 min (annealing) and 72 °C for 2 min (extension), and a final extension step at 72 °C for 8 min. Following amplification, reaction mixtures were maintained at 12 °C. PCR amplicons were separated by electrophoresis on 1% agarose gel (40 min, 100 V), stained with ethidium bromide and visualised under ultraviolet light. The second reaction was carried out using primers BabRumF (5′ACCTCACCAGGTCCAGACAG3′) and BabRumR (5′GTACAAAGGGCAGGGACGTA3′) that were designed to amplify a common 420 bp Babesia 18S rRNA fragment identified by aligning sequences from Babesia spp. available at GenBank (http://www.ncbi.nlm.nih.gov), Cytidine deaminase namely, B. bigemina (X59607), B. odocoilei (U16369), Babesia

divergens (U07885) and B. bovis (L31922). The reaction mixture comprised 2.0 μL of dNTPs (0.2 mM), 0.25 μL of Taq polymerase (0.05 U), 2.5 μL which buffer (1×), 1.0 μL of a solution containing the mixed primers (10 μM) and sufficient sterile ultra-pure water to give a final volume of 25 μL. An aliquot (2.5 μL) of amplicon obtained in the first reaction were added to the reaction mixture and amplification was carried out under the conditions described above. Products were separated by electrophoresis and visualised as described above, and subsequently purified with the aid of QIAquick PCR Purification Kit (Qiagen Biotecnologia Brasil, São Paulo, SP, Brazil) used according to the recommendations of the manufacturer.

The RV144 vaccine trial demonstrated modest success, leading to a

The RV144 vaccine trial demonstrated modest success, leading to a 31% lowered rate of HIV-1 infection in a specific www.selleckchem.com/products/pci-32765.html subset of vaccinees versus placebo groups [14]. While the correlates of immunity of that trial remain to be understood, viral diversity is likely to be at least partially responsible for the limited coverage. HIV-1-specific CD4+ T helper cells and CD8+ cytotoxic T cells have been

shown to play a central role in control of the virus following infection [15], [16], [17], [18], [19], [20] and [21]. CD4+ T helper cells are essential for the generation of both humoral and cellular responses against the virus [22] and [23], while cytotoxic T cells play an important role in the resolution of acute viremia and in control of persistent

HIV-1 viral replication [17] and [24]. Recent longitudinal studies following first CD8+ CTL responses to founder virus in early infection have defined a narrow window of opportunity for the CTL response to control infection and revealed multiple evolutionary pathways utilized by the virus during acute infection to retain replicative fitness [25], [26], [27] and [28]. Moreover, roles for both cytolytic function of CD8+ T cells during nonproductive infection and noncytolytic functions (e.g., MIP-1β, MIP-1α, IFNγ, TNFα, and IL-1) in resolution of peak viremia have been identified [29] and [30]. Therefore, vaccines that stimulate

virus-specific T-cell responses may be selleck inhibitor able to boost humoral immune responses and may also delay the progression of HIV-1 to AIDS in infected individuals. A robust T-cell response will be a necessary component of any successful HIV vaccine; however, the ability of a vaccine to account for the extraordinary viral diversity of HIV-1 continues to be a challenge. This diversity extends not only to T-cell epitope differences across clades, but also to isolates from a number of diverse clades that occupy a single geographic area [31]. One approach because to address the problem of HIV-1 diversity is to develop multiple vaccines. These vaccines could be developed on a clade-by-clade basis, whereby a single vaccine represents isolates from a single clade, or on a geographically specific basis, whereby vaccines are derived from isolates commonly circulating in a particular country or region. However, this multiple vaccine approach raises the question of how many vaccines would be needed to protect against each of the many clades of HIV. In a time of increasing global connectedness and mobility, the notion of controlling a particular viral population and keeping it geographically sequestered is unlikely to bear fruit. In contrast to region-specific vaccine efforts, our approach is to develop a globally effective vaccine.

Sections were mounted on 1 mm Superfrost slides (Fisher) and moun

Sections were mounted on 1 mm Superfrost slides (Fisher) and mounted with Vectashield (Vector Labs) mounting medium. Z-stack, tiles, and single images were acquired on a Zeiss LSM Z10 confocal microscope using a 20×, 40×, or 63× objective and analyzed using ZEN 2009 and Image J software. For cell counting, images were marked/counted using digital image editing software. For quantification

of ChR2-eYFP fluorescence Sotrastaurin in vitro intensity, images were acquired using identical pinhole, gain, and laser settings for the NAc, DMS, and DLS sections, and for the VTA and Sn sections. No additional post-processing was performed on any of the collected images. ChR2-eYFP fluorescence intensity was then quantified using a scale from 0–255 in Image J to determine the mean intensity across the entire image. For determination of optical fiber placements, tissue was imaged at 10× and 20× on an upright conventional fluorescent microscope. Optical stimulation sites Palbociclib datasheet were recorded as the location in tissue 0.5 mm more ventral than where visible optical fiber tracks terminated. Behavioral data was analyzed using Neuroexplorer, Microsoft Excel,

and Prism. Electrophysiological data was analyzed in ClampFit and Prism. Voltammetry data was analyzed with TarHeel CV. Imagining data was analyzed with ZEN 2009 and Image J. Between- and within-subjects t tests and ANOVAs followed by Bonferroni post-hoc tests were used when applicable with an α = 0.05. We thank Drs. Bradford Lowell and Linh Vong for providing the VGat-ires-Cre mice and Dr. Karl Deisseroth for AAV-DIO-ChR2-eYFP, and second AAV-DIO-GFP constructs. We also thank Randall Ung and Dr. Vladimir Gukassyan and the UNC Neuroscience Center Microscopy Core Facility for assistance. This study was supported by funds from NARSAD, The Whitehall Foundation, The Foundation

of Hope, NIDA (DA029325 and DA032750), and startup funds provided by the Department of Psychiatry at UNC Chapel Hill (G.D.S.) and DA021634 (E.A.B.). R.v.Z. was supported by the Hendrik Muller Fonds, Fundatie van de Vrijvrouwe van Renswoude, David de Wied Stichting, and Vreedefonds. R.v.Z. and G.D.S. conceived and designed all experiments. R.v.Z., J.P., E.A.B., and G.D.S. performed experiments and analyzed the data. R.v.Z. and G.D.S. wrote the manuscript. “
“Neuroplasticity, the capacity of the nervous system to modify its organization, involves a complex, multistep process that includes numerous time-dependent events occurring at the molecular, synaptic, electrophysiological, and structural organization levels. The scope of neuroplasticity is wide, ranging from short-term weakening and strengthening of existing synapses, through induction of long-term potentiation (LTP), to formation of long-lasting new neuronal connections. These modifications include subtle changes at the synaptic level (e.g.

In PFC and FEF,

previous-trial information persisted into

In PFC and FEF,

previous-trial information persisted into the current trial to affect neuronal activity in various epochs. We recorded from single neurons in the KU-55933 ic50 FEF, PFC, and SEF while monkeys performed a visual oculomotor task in which they monitored their own decisions. Neuronal activity correlated with decisions and bets was found in all three areas, but joint activity that linked decisions to appropriate bets was found exclusively in the SEF. This putative metacognitive activity began swiftly in the SEF during the decision stage and continued into the bet stage. Monkey behavior was independent of previous trial outcome, as was SEF activity (but not PFC or FEF activity). We had predicted that both the SEF and PFC would participate in metacognitive monitoring, but our data supported a role only for the SEF. The putative metacognitive activity in SEF arose early in trials (Figures 5A and 5C), beginning soon after the start of the decision-related signal (Figure 2F) and before the monkey reported its decision with a saccade. The time course suggests that monitoring a decision occurs in near simultaneity with making the decision. This seems analogous to the time course of monitoring motor operations (“corollary

http://www.selleckchem.com/products/erastin.html discharge”); when motor areas finalize a movement command, upstream areas monitor it within milliseconds (Sommer and Wurtz, 2004). It should be noted that most (15/20) of our individual SEF neurons with a metacognitive signal also exhibited a decision-related Oxalosuccinic acid signal. This close relationship between metacognitive and decision-related signals may be no coincidence: in the SEF, decision-related signals may evolve into metacognitive signals. A decision-related signal that outlasts the decisive act (the saccade to the target) provides information that could be monitored for later behavior (the bet). Although decision-related signals occurred in all three areas, our data suggest differences

in how the signals are used. In SEF, the prolonged decision-related signal seems to be maintained for internal use (e.g., determining the bet to make). In PFC and FEF, the briefer signal may guide only immediate acts (e.g., planning the decision saccade). Metacognition-related activity in SEF had not been reported previously. No fMRI studies reported human SEF signals during metacognition tasks, although many fMRI results have implicated regions interconnected with SEF, such as anterior cingulate and medial prefrontal regions (Chua et al., 2006; Kikyo et al., 2002). Our recording strategy was to study every neuron encountered, so our population data may be considered a representative sample of SEF neurons (leaving aside issues of sampling biases related to neuron size, e.g., Sommer and Wurtz, 2000).

Analysis of GFP-expressing neurons at P18 revealed a significant

Analysis of GFP-expressing neurons at P18 revealed a significant increase in the number of dendritic spines on CA1 pyramidal neurons in NgRTKO−/− mice relative to their triple heterozygous littermate controls ( Figures 5A and 5B). These findings are

consistent with the idea that the NgR family members function together in vivo to limit the number of excitatory synapses. To extend this analysis using an independent approach, we performed transmission electron microscopy to visualize the ultrastructural features of excitatory synapses. In micrographs from NgRTKO−/− mice, we observed asymmetric synapses of typical morphology, suggesting that the overall structure and vesicle content of Selleckchem Venetoclax excitatory synapses are normal in the absence of NgRs. However, quantification of the number of excitatory synapses in the apical dendritic regions of CA1 revealed that NgRTKO−/− mice had a significant

increase in the density of excitatory synapses relative to heterozygous littermate controls ( Figures 5C and 5D). Furthermore, this effect was not limited to CA1 neurons, since analysis of CA3 neurons also revealed a clear increase in the number of PSDs in NgRTKO−/− animals ( Figure 5E). Thus, analysis by confocal and electron microscopy suggests that the NgR family functions to limit the number of excitatory synapses in vivo. To address whether the observed increase in synapse number reflects an increase Pictilisib research buy in functional synapses, we performed whole-cell patch-clamp electrophysiology on CA1 pyramidal neurons from acute hippocampal slices obtained from NgRTKO−/− mice and control littermates to quantify the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs). This analysis revealed a significant increase in the frequency of mEPSCs in

NgRTKO−/− mice relative to littermate controls ( Figure 5F and S5C), suggesting that the NgR family restricts the development of functional excitatory synapses. Interestingly, there was a small but significant Oxymatrine decrease in the amplitude of mEPSCs ( Figure 5G and S5D), consistent with the immature spine types observed in NgR1 knockouts ( Lee et al., 2008 and Zagrebelsky et al., 2010). Thus, reducing the expression of the NgR family results in an increase in functional synapses that are slightly reduced in strength. The question remained as to how NgRs work at a mechanistic level to restrict excitatory synapse number. One possibility was that NgRs limit the formation of new synapses in part by inhibiting dendritic growth, thereby reducing the possibility of contact between axons and dendrites. Therefore, we asked whether loss of NgR family members affects dendritic branching.

Depending on the dosage and duration, rapamycin may affect glucos

Depending on the dosage and duration, rapamycin may affect glucose

homeostasis in different ways (Blagosklonny, 2011b). Rapamycin has also been proposed to have a hormetic effect on aging and aging-related diseases (Blagosklonny, 2011a). Short-term rapamycin transiently affects intracellular signaling activated by nutrient overload; it does not affect body weight but abrogates the anorexia induced by nutrients such as leucine (Cota et al., 2006). On the other hand, long-term rapamycin treatment has a profound impact on neuronal morphology and functions. Ion channels typically have slow turnover rates (Crane and Aguilar-Bryan, 2004), and any loss of neurite projection may require a few days to be restored (Keck et al., 2011). Similarly, Mori and colleagues have shown that activating mTOR in POMC neurons by deleting the Tsc1 gene also results in enlarged POMC neuron soma and reduced neurite projections to the PVN, and chronic check details intraperitoneal injection of high doses of rapamycin restores the projection ( Mori et al., 2009). In our study, we have identified that aging is one of the physiological factors to activate mTOR in POMC neurons and the genetic activation Selleck CP-868596 of mTOR in conditional knockout mice recapitulates the physiological consequences of aging by activating mTOR in POMC neurons. Glucose intolerance and diabetes are commonly associated with obesity (Mokdad et al.,

2001). Paradoxically, we did not observe such a deleterious metabolic dysfunction in Pomc-cre;Tsc1-f/f mice, despite the fact that they were obese and hyperphagic. Instead, these mice had an improved glucose tolerance ( Figure 4J). Interestingly, a recent study Mephenoxalone has also revealed that direct leptin action in POMC neurons regulates glucose homeostasis independent of body weight ( Berglund et al., 2012). Other than regulating body weight and appetite, the arcuate nucleus is well documented as a central regulator for glucose homeostasis. To keep a constant glucose supply to the

brain, those hypothalamic neurons that are located outside the blood-brain barrier can monitor the glucose level in the periphery and send feedback commands to visceral organs such as the liver and pancreas through the descending autonomic system to counterbalance any fluctuation of the glucose supply to the brain ( Parton et al., 2007). Moreover, activating KATP channels in hypothalamic neurons has been shown to improve glucose metabolism as infusing diazoxide to the third ventricle suppresses hepatic gluconeogenesis ( Pocai et al., 2005) and central GLP-1 reduces peripheral glucose levels by activating KATP channels in POMC neurons ( Sandoval et al., 2008). As obesity impairs glucose tolerance, we suspect that the improved glucose tolerance in the Pomc-cre;Tsc1-f/f mice might be more significant in pair-feeding experiments that ensure matched body weight.

, 2009, Hasenstaub et al ,

, 2009, Hasenstaub et al., LY294002 2005, Sohal et al., 2009, Traub et al., 1996,

Traub et al., 1997 and Wang and Buzsaki, 1996). These fast oscillations take place under a variety of behavioral states, either spontaneously or in response to sensory stimuli and are thought to play a role in the transmission of information across cortical areas. Specifically, because excitatory input is more efficient in depolarizing target neurons when they are active synchronously rather than distributed in time (Azouz and Gray, 2000 and Pouille and Scanziani, 2001), oscillations enable neurons to cooperate in the depolarization of common downstream targets, and thus in the propagation of neuronal signals. Through this mechanism, gamma oscillations are proposed to contribute to the merging of information processed in distinct cortical regions, for example,

by “binding” neuronal ensembles that oscillate in phase (Engel et al., 2001). Inhibition is not only directly involved in the generation of these fast oscillations, but also in synchronizing participating neurons, in setting the pace of the oscillations and in maintaining their coherence in space. Among the various types of inhibitory neurons, basket cells play a key role in gamma oscillations (Cardin et al., find more 2009, Cobb et al., 1995 and Sohal et al., 2009). Two important properties of interneurons appear crucial to the generation of synchronized oscillations. First, interneurons are electrically coupled via gap junctions allowing large populations of interneurons to be synchronized with millisecond Vasopressin Receptor precision (Beierlein et al., 2000, Galarreta and Hestrin, 1999, Galarreta and Hestrin, 2001, Gibson et al., 1999 and Hestrin and Galarreta, 2005). Second, interneurons make reciprocal synaptic connections onto each other (Bartos et al., 2002, Galarreta and Hestrin, 2002, Gibson et al., 1999 and Tamas et al., 1998), a property that models show is important for the robustness of oscillations (Bartos

et al., 2007 and Vida et al., 2006). Two alternate mechanisms, “PING” (pyramidal-interneuron network gamma oscillations) and “ING” (interneuron network gamma oscillations) have been proposed for the role of inhibitory neurons in the generation of gamma oscillations (Tiesinga and Sejnowski, 2009 and Whittington et al., 2000). PING is based on the reciprocal (feedback) connectivity between pyramidal cells and interneurons. Here, the oscillation is generated by the alternation in the firing of interneurons (excited by pyramidal cells) and pyramidal cells (as they reemerge from the inhibition triggered by interneurons). The fact that individual basket cells contact a very large fraction of neighboring (i.e., within ∼100 um) pyramidal cells, and that individual pyramidal cells in turn contact many local inhibitory neurons leads to the synchronous involvement of large populations of neurons in the oscillation.

The response of cells to fimbria or thalamus single-pulse stimula

The response of cells to fimbria or thalamus single-pulse stimulation 50 ms following single-pulse stimulation of the PFC was also considered in a subgroup of cells (n =

13). In some cases (n = 12), we injected depolarizing current through the recording electrode (between −0.2 and 0.2 nA) to record an F1 or T1 response during a depolarized membrane potential similar to that at which F2 and T2 responses were evoked. A subset of cells (n = 13) was also subjected to a stimulus protocol in which a single-pulse stimulus was delivered to the PFC (1.0 mA; 0.5 ms; PFC1), followed at a 500 ms latency by a train stimulation of the fimbria or thalamus (50 Hz train of ten pulses; 1.0 mA; 0.5 ms), after which a second pulse was delivered to the PFC (1.0 mA; 0.5 ms; PFC2). In all cases, responses to stimulation were averaged over all of the repetitions MDV3100 price delivered to the cell. To calculate the magnitude of EPSP suppression, we first determined the CH5424802 price ratio of the control and test pulses. For instance, in the cases in which we stimulated the fimbria, we calculated F2/F1 using response amplitudes. As this quotient represents the proportion of the response retained following PFC train stimulation, we expressed the difference between 1 and F2/F1 as a percentage to indicate the magnitude of EPSP suppression. After baseline

and stimulus-response recordings were collected, cells were filled with Neurobiotin by passing positive current (1 nA, 200 ms pulses, 2 Hz) for at least 10 min through the recording electrode. Upon completion of recording experiments, animals and were euthanized with an overdose of sodium pentobarbital (100 mg/kg) and transcardially perfused with cold saline followed by 4% paraformaldehyde. Brains were then removed and postfixed in 4% paraformaldehyde for at least 24 hr before being transferred to a 30% sucrose solution in 0.1 M phosphate buffer. After at least 48 hr in sucrose, brains were cut into 50 μm sections using a freezing microtome and placed

into phosphate buffer. Sections through PFC and fimbria or thalamus were mounted on gelatin-coated slides and Nissl stained to verify placement of stimulating electrodes. Sections through VS were processed for visualization of Neurobiotin-filled cells and then mounted on gelatin-coated slides and Nissl stained. All stained slides were coverslipped and examined microscopically for cell and electrode location. This work was supported by grants from the National Institutes of Health (R01 MH060131) to P.O.’D. and (R31 MH092043) to G.G.C. “
“Coordinated movement relies on the integration of sensory feedback signals with core motor circuits. In mammals, motor performance is refined by sensory feedback signals that convey information from proprioceptive afferents as well as from mechanoreceptive afferents activated by diverse cutaneous receptors.

The rate of oxygen consumption was thus measured

The rate of oxygen consumption was thus measured GDC-0973 price using a Clark oxygen electrode in three clonal populations of stable VCP KD SH-SY5Y cells. VCP protein expression levels were reduced by approximately 90% in stable VCP KD SH-SY5Y cells compared to stable SCR SH-SY5Y cells ( Figure S1B). The basal rate of oxygen consumption was significantly increased

in VCP KD cells compared to control ( Figure 3A; for numbers see Table S2). Furthermore, addition of oligomycin resulted in an inhibition of oxygen consumption in control cells but not in VCP KD cells, while the uncoupler FCCP increased oxygen consumption to the same maximal values in both cell populations. Calculation of Vbasal/Voligomycin and Vbasal/VFCCP revealed a decrease in the Vbasal/Voligomycin index, suggesting an increase in mitochondrial respiration SNS-032 ( Figure 3B; for numbers see Table S2). No differences in the Vbasal/VFCCP index were observed between VCP KD and SCR cells, showing that ETC complexes are not damaged and they are working at a similar rate under normal conditions ( Figure 3C; for numbers see Table S2). To further analyze ETC and OXPHOS functionality and coupling, we permeabilized stable VCP KD and SCR cell lines using a low concentration of digitonin (40 μM) and basal oxygen consumption rates were measured

in the presence of external substrates for the ETC in the absence of ADP. We then added 50 nmol ADP to establish state 3 (V3) respiration. Upon consumption of this ADP, mitochondria resumed an inhibited state, termed state 4 (V4). Respiratory control ratio (RCR) is the ratio of V3 to V4 and is considered an indicator of coupling of OXPHOS and respiration. RCR values confirmed the uncoupling of mitochondrial respiratory chain from

oxidative phosphorylation in stable VCP KD cells compared to control ( Figure 3D; for numbers see Table S3). The “ADP/O” ratio, expressed as the oxygen consumed per nmol ADP added during V3, indicates the efficiency of oxidative phosphorylation. ADP/O ratios indicated that oxidative phosphorylation efficiency was reduced by more than 30% in VCP KD cells compared to SCR cells ( Figure 3E; for numbers see from Table S3). Taken together, these data show that the respiratory rate, driven by the loss of potential and oxidation of the NADH pool, is increased in VCP-deficient cells and that VCP deficiency increases the mitochondrial proton leak causing uncoupling between respiration and OXPHOS. Mitochondrial uncoupling may occur through a variety of mechanisms including altered lipid peroxidation. Using the fluorescent ratiometric oxidation-sensitive dye C11 BODIPY581/591, we therefore determined the levels of lipid peroxidation in VCP-deficient cells.