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IntroductionHigh intensity exercise is an increasingly popular mode of exercise to elicit similar or greater adaptive responses compared to traditional moderate intensity continuous exercise. However, the molecular mechanisms underlying these adaptive responses are still unclear. The purpose of this pilot study was to compare high and low intensity contractile stimulus on the Nrf2-mediated redox stress response in mouse skeletal muscle.MethodsAn intra-animal design was used to control for variations in individual responses to muscle stimulation by using a stimulated limb (STIM) and comparing to the contralateral unstimulated control limb (CON). High Intensity (HI – 100Hz), Low Intensity (LI – 50Hz), and Naïve Control (NC – Mock stimulation vs CON) groups were used to compare these effects on Nrf2-ARE binding, Keap1 protein content, and downstream gene and protein expression of Nrf2 target genes.ResultsMuscle stimulation significantly increased Nrf2-ARE binding in LI-STIM compared to LI-CON (p = 0.0098), while Nrf2-ARE binding was elevated in both HI-CON and HI-STIM compared to NC (p = 0.0007). The Nrf2-ARE results were mirrored in the downregulation of Keap1, where Keap1 expression in HI-CON and HI-STIM were both significantly lower than NC (p = 0.008) and decreased in LI-STIM compared to LI-CON (p = 0.015). In addition, stimulation increased NQO1 protein compared to contralateral control regardless of stimulation intensity (p = 0.019).ConclusionsTaken together, these data suggest a systemic redox signaling exerkine is activating Nrf2-ARE binding and is intensity gated, where Nrf2-ARE activation in contralateral control limbs were only seen in the HI group. Other research in exercise induced Nrf2 signaling support the general finding that Nrf2 is activated in peripheral tissues in response to exercise, however the specific exerkine responsible for the systemic signaling effects is not known. Future work should aim to delineate these redox sensitive systemic signaling mechanisms.

T-cell–recruiting bispecific molecule therapy has yielded promising results in patients with hematologic malignancies; however, resistance and subsequent relapse remains a major challenge. T-cell exhaustion induced by persistent antigen stimulation or tonic receptor signaling has been reported to compromise outcomes of T-cell–based immunotherapies. The impact of continuous exposure to bispecifics on T-cell function, however, remains poorly understood. In relapsed/refractory B-cell precursor acute lymphoblastic leukemia patients, 28-day continuous infusion with the CD19xCD3 bispecific molecule blinatumomab led to declining T-cell function. In an in vitro model system, mimicking 28-day continuous infusion with the half-life–extended CD19xCD3 bispecific AMG 562, we identified hallmark features of exhaustion arising over time. Continuous AMG 562 exposure induced progressive loss of T-cell function (day 7 vs day 28 mean specific lysis: 88.4% vs 8.6%; n = 6; P = .0003). Treatment-free intervals (TFIs), achieved by AMG 562 withdrawal, were identified as a powerful strategy for counteracting exhaustion. TFIs induced strong functional reinvigoration of T cells (continuous vs TFI-specific lysis on day 14: 34.9% vs 93.4%; n = 6; P < .0001) and transcriptional reprogramming. Furthermore, use of a TFI led to improved T-cell expansion and tumor control in vivo. Our data demonstrate the relevance of T-cell exhaustion in bispecific antibody therapy and highlight that T cells can be functionally and transcriptionally rejuvenated with TFIs. In view of the growing number of bispecific molecules being evaluated in clinical trials, our findings emphasize the need to consider and evaluate TFIs in application schedules to improve clinical outcomes.

Study description Flow Mediated Skin Fluorescence (FMSF) is a novel technique for non-invasive evaluation of the microcirculation and metabolic regulation. This study describes the diagnostic potential of FMSF for type 1 diabetes (DM1). Study population All study participants, in both the control (n = 31) and DM1 (n = 40) groups, were between the ages of 30–49 y. The patients in the DM1 group had all been suffering from diabetes for at least 10 y. Results The parameters HRindex, HRmax and MR inversely correlate with age and BMI. An unidentified compensatory effect was observed among the younger members of the DM1 group. The majority of DM1 patients with HRindex Conclusion FMSF appears to be an extremely useful technique for monitoring diabetic patients over time, enabling early diagnosis of potentially dysfunctional microcirculation and metabolic regulation.

Molecular recognition is a fundamental principle in biological systems. The olfactory detection of both food and predators via ecological relevant odorant cues are abilities of eminent evolutionary significance for many species. Pyrazines are such volatile cues, some of which act as both human-centered key food odorants (KFOs) and semiochemicals. A pyrazine-selective odorant receptor has been elusive. Here we screened 2,3,5-trimethylpyrazine, a KFO and semiochemical, and 2,5-dihydro-2,4,5-trimethylthiazoline, an innate fear-associated non-KFO, against 616 human odorant receptor variants, in a cell-based luminescence assay. OR5K1 emerged as sole responding receptor. Tested against a comprehensive collection of 178 KFOs, we newly identified 18 pyrazines and (2R/2S)-4-methoxy-2,5-dimethylfuran-3(2H)-one as agonists. Notably, OR5K1 orthologs in mouse and domesticated species displayed a human-like, potency-ranked activation pattern of pyrazines, suggesting a domestication-led co-evolution of OR5K1 and its orthologs. In summary, OR5K1 is a specialized olfactory receptor across mammals for the detection of pyrazine-based key food odors and semiochemicals.

The authors wish to make the following erratum to this paper [...]

The recent interest in the clinical applications of Piloty's acid derivatives as HNO donors for the treatment of cardiovascular system dysfunction has led us to the examination of factors controlling HNO release from selected ortho-substituted N-hydroxysulfonamides. Here we present the kinetic and quantum mechanical studies on the mechanism of HNO release from selected ortho-substituted N-hydroxysulfonamides and in vivo examination of the antiaggregatory properties of N-hydroxy-(2-bromobenzene)sulfonamide complex with sodium salt of β-cyclodextrin sulfobutyl ethers-ethyl ethers as compared with Angeli's salt.

A dual-frequency all-in-one Global Navigation Satellite System (GNSS) receiver with a multi-core 32-bit RISC (reduced instruction set computing) application processor was integrated and manufactured as a System-on-Chip (SoC) in a 110 nm CMOS (complementary metal-oxide semiconductor) process. The GNSS RF (radio frequency) front-end with baseband navigation engine is able to receive, simultaneously, Galileo (European Global Satellite Navigation System) E1/E5ab, GPS (US Global Positioning System) L1/L1C/L5, BeiDou (Chinese Navigation Satellite System) B1/B2, GLONASS (GLObal NAvigation Satellite System of Russian Government) L1/L3/L5, QZSS (Quasi-Zenith Satellite System development by the Japanese government) L1/L5 and IRNSS (Indian Regional Navigation Satellite System) L5, as well as all SBAS (Satellite Based Augmentation System) signals. The ability of the GNSS to detect such a broad range of signals allows for high-accuracy positioning. The whole SoC (system-on-chip), which is connected to a small passive antenna, provides precise position, velocity and time or raw GNSS data for hybridization with the IMU (inertial measurement unit) without the need for an external application processor. Additionally, user application can be executed directly in the SoC. It works in the &minus, 40 to +105 &deg, C temperature range with a 1.5 V supply. The assembled test-chip takes 100 pins in a QFN (quad-flat no-leads) package and needs only a quartz crystal for the on-chip reference clock driver and optional SAW (surface acoustic wave) filters. The radio performance for both wideband (52 MHz) channels centered at L1/E1 and L5/E5 is NF = 2.3 dB, G = 131 dB, with 121 dBc/Hz of phase noise @ 1 MHz offset from the carrier, consumes 35 mW and occupies a 4.5 mm2 silicon area. The SoC reported in the paper is the first ever dual-frequency single-chip GNSS receiver equipped with a multi-core application microcontroller integrated with embedded flash memory for the user application program.

The CD33-targeting bispecific T-cell engager (BiTE) AMG 330 proved to be highly efficient in mediating cytolysis of acute myeloid leukemia (AML) cells in vitro and in mouse models. Yet, T-cell activation is correlated with upregulation of programmed cell death-ligand 1 (PD-L1) and other inhibitory checkpoints on AML cells that confer adaptive immune resistance. PD-1 and PD-L1 blocking agents may counteract T-cell dysfunction, however, at the expense of broadly distributed immune-related adverse events (irAEs). We developed a bifunctional checkpoint inhibitory T cell–engaging (CiTE) antibody that combines T-cell redirection to CD33 on AML cells with locally restricted immune checkpoint blockade. This is accomplished by fusing the extracellular domain of PD-1 (PD-1ex), which naturally holds a low affinity to PD-L1, to an αCD3.αCD33 BiTE-like scaffold. By a synergistic effect of checkpoint blockade and avidity-dependent binding, the PD-1ex attachment increases T-cell activation (3.3-fold elevation of interferon-γ) and leads to efficient and highly selective cytotoxicity against CD33+PD-L1+ cell lines (50% effective concentration = 2.3-26.9 pM) as well as patient-derived AML cells (n = 8). In a murine xenograft model, the CiTE induces complete AML eradication without initial signs of irAEs as measured by body weight loss. We conclude that our molecule preferentially targets AML cells, whereas high-affinity blockers, such as clinically approved anticancer agents, also address PD-L1+ non-AML cells. By combining the high efficacy of T-cell engagers with immune checkpoint blockade in a single molecule, we expect to minimize irAEs associated with the systemic application of immune checkpoint inhibitors and suggest high therapeutic potential, particularly for patients with relapsed/ refractory AML.

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 min after the last stimulation and processed for redox proteomics assay of S-glutathionylation. Using selective reduction with a glutaredoxin enzyme cocktail and resin-assisted enrichment technique, we quantified the levels of site-specific protein S-glutathionylation at rest and following fatiguing contractions. Redox proteomics revealed over 2200 sites of S-glutathionylation modifications, of which 1290 were significantly increased after fatiguing contractions. Muscle contraction leads to the greatest increase in S-glutathionylation in the mitochondria (1.03%) and the smallest increase in the nucleus (0.47%). Regulatory cysteines were significantly S-glutathionylated on mitochondrial complex I and II, GAPDH, MDH1, ACO2, and mitochondrial complex V among others. Similarly, S-glutathionylation of RYR1, SERCA1, titin, and troponin I2 are known to regulate muscle contractility and were significantly S-glutathionylated after just 15 min of fatiguing contractions. The largest fold changes (> 1.6) in the S-glutathionylated proteome after fatigue occurred on signaling proteins such as 14-3-3 protein gamma and MAP2K4, as well as proteins like SERCA1, and NDUV2 of mitochondrial complex I, at previously unknown glutathionylation sites. These findings highlight the important role of redox control over muscle physiology, metabolism, and the exercise adaptive response. This study lays the groundwork for future investigation into the altered exercise adaptation associated with chronic conditions, such as sarcopenia. Keywords: Muscle contraction, Post-translational modification, S-glutathionylation, Redox signaling, Skeletal muscle, Thiol redox proteomics

Ultra-widefield fluorescein angiography (UWFA) is an emerging imaging modality used to characterise pathologies in the retinal vasculature, such as microaneurysms (MAs) and vascular leakages. Despite its potential value for diagnosis and disease screening, objective quantitative assessment of retinal pathologies by UWFA is currently limited because laborious manual processing is required. In this report, we describe a geometrical method for uneven brightness compensation inherent to UWFA imaging technique. The correction function is based on the geometrical eyeball shape, therefore it is fully automated and depends only on pixel distance from the center of the imaged retina. The method’s performance was assessed on a database containing 256 UWFA images with the use of several image quality measures that show the correction method improves image quality. The method is also compared to the commonly used CLAHE approach and was also employed in a pilot study for vascular segmentation, giving a noticeable improvement in segmentation results. Therefore, the method can be used as an image preprocessing step in retinal UWFA image analysis.

Bispecific antibodies represent a promising treatment option for acute myeloid leukemia (AML). We have recently described a novel T-cell bispecific antibody (TCB) targeting the intracellular tumor antigen Wilms tumor 1 (WT1) in the context of HLA-A*02 (Augsberger et al. Blood 2021). Based on these findings a multicenter first-in-human clinical trial was initiated in relapse/refractory AML (NCT04580121). Possible immune escape mechanisms against T-cell based immunotherapy are provided by the tumor microenvironment (TME) of the bone marrow by co-inhibition of T cells or stromal cells shielding leukemic cells from immune effector cells. To overcome the immunosuppressive effect of the TME and to enhance T-cell responses, we evaluated the combination of the WT1-TCB with an antibody fusion protein that targets a stromal antigen (Fibroblast-activation protein; FAP) and provides a positive costimulatory signal (4-1BBL) to T cells. FAP is upregulated on cancer-associated fibroblasts after remodulation of the bone marrow niche by leukemic cells, and the FAP specificity of the molecule therefore provides T-cell co-stimulation tightly restricted to the tumor niche. Efficacy of the combination (WT1-TCB + FAP-4-1BBL antibody fusion protein) was evaluated in co-culture assays over 4 days with primary HLA-A*02 + AML cells, healthy donor (HD) T cells and three NIH-3T3 fibroblast cell lines. NIH-3T3 cell lines were genetically modified to express low and high levels of FAP, respectively. Wild-type NIH-3T3 cells were included as control. Additionally, a control (Ctrl)-TCB and a Ctrl-4-1BBL antibody fusion protein recognizing a non-tumor target derived from the human germline repertoire were included. Enhancement of T-cell mediated cytotoxicity by the FAP-4-1BBL antibody fusion protein was evaluated by (1) specific lysis of primary AML cells, (2) upregulation of the T-cell activation markers CD25 and 4-1BB, (3) T-cell expansion calculated as fold change compared to day 0, and (4) Granzyme B-expression which was evaluated by intracellular staining. After 4 days of co-culture, with an E:T ratio of 1:2, we observed a mean specific lysis of 55.1±8.2% (±SEM; n=4) of primary AML cells mediated by HD T cells and WT1-TCB. Notably, this was reduced to 19.4±5.9% (±SEM; n=4) in the presence of NIH-3T3 cells. However, AML cell lysis was restored by the addition of the FAP-4-1BBL antibody fusion protein in the presence of high FAP expressing NIH-3T3 cells (mean specific lysis: 62.8±7.3%; ±SEM; n=4). Concomitantly, the FAP-4-1BBL antibody fusion protein led to increased expression of the activation molecules CD25 (MFI ratio: 22.1±5.3 vs. 10.4±1.3; ±SEM; n=4) and 4-1BB (MFI ratio: 10.4±6.0 vs. 2.1±0.3; ±SEM; n=4) on CD3 + T cells. Furthermore, lysis was accompanied by increased frequencies of granzyme B expressing T cells (45.0±2.5% vs. 16.1±5.3%; n=3). Importantly, the FAP-4-1BBL antibody fusion protein led to improved T-cell proliferation, especially of CD8 + T cells (fold change on day 4 vs day 0: 5.7±2.2 vs. 1.0±0.3; ±SEM; n=4). Overall similar observations were made in the presence of low FAP expressing NIH-3T3 cells. Taken together, we have established an in vitro model system mimicking the immunoprotective bone marrow TME using NIH-3T3 cells resulting in impaired AML cell lysis. Providing additional T-cell co-stimulation by a tumor-stroma targeted 4-1BB agonist, however, restored WT1-TCB-mediated cytotoxicity of primary AML cells in the presence of FAP expressing cell lines. Importantly, the combination overcame the immunosuppressive effect of the NIH-3T3 cells on T cells as further demonstrated by improved T-cell activation and expansion. The tumor-stroma targeted 4-1BB agonist therefore represents a promising combinatorial approach to enhance T-cell activity at the local tumor site and warrants further investigations in an in vivo model system. Disclosures Pulko: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Claus: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Buecklein: Pfizer: Consultancy, Honoraria, Speakers Bureau; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Miltenyi: Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Umana: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Klein: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; Klinikum der Universität München: Current Employment; Roche: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pfizer: Consultancy, Speakers Bureau; Janssen: Consultancy; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

Blinatumomab is a bispecific T-cell engager (BiTE ®) construct approved for treatment of relapsed/refractory (r/r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL). It is applied as continuous infusion over 28 days and induces remissions in 43 % of r/r patients. Responses correlated to T-cell expansion (Topp et al. 2011, Zugmaier et al. 2015). Mimicking the clinical application in an in vitro model system, we showed previously that continuous stimulation (CONT) with AMG 562, a half-life extended CD19xCD3 BiTE ® construct, induces T-cell exhaustion, as seen in chronic infections. Also, we could enhance T-cell function in vitro by treatment-free intervals (TFI) (Zieger et al. ASH 2020). To identify genetic drivers of enhanced T-cell function that could provide anti-exhaustion targets for clinical use, we aimed to characterize the transcriptome of exhausted vs rested T cells by bulk RNA sequencing of CONT and TFI T cells. To simulate CONT vs TFI AMG 562 stimulation, cocultures of healthy donor T cells and CD19 + OCI-Ly1 cells were set up for 28 days under CONT or TFI (7 days on/7 days off) AMG 562 exposure. On day 0, 7, 14 and 21, we sorted 5x10 5 CD3 + T cells for transcriptome assessment (n=3). In parallel, function of TFI vs CONT T cells was analyzed in vitro: (1) AMG 562-mediated killing was evaluated as specific lysis of CD19 + Ba/F3 cells after 72h, (2) T-cell expansion during the killing assay was calculated as fold change (FC) of CD2 + counts, (3) AMG 562-mediated cytokine secretion was evaluated via intracellular staining. We could confirm that function of Day 14 TFI vs CONT T cells was significantly enhanced (% specific lysis: TFI=99±2.2, CONT=34±4.2, p After restimulation of TFI T cells with AMG 562 (Day 21 TFI) we observed higher effector function in TFI vs CONT T cells (% specific lysis, TFI=51±8, CONT=23±7, p Together, our data suggest that TFI functionally and transcriptionally rejuvenates T cells. Upon restimulation (Day 21 TFI), T cells reengage an effector program and are less exhausted compared to CONT T cells. In future analyses we will correlate RNA expression levels to functional traits using whole genome co-expression network analysis (WGCNA). Thereby we aim to identify gene clusters critical for persistent T-cell function that might serve as targets to improve efficacy of T-cell based immunotherapies. Figure 1 Figure 1. Disclosures Lacher: Roche: Research Funding. Brauchle: Adivo: Current Employment. von Bergwelt: Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Theurich: Amgen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Buecklein: Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding. Kischel: Amgen GmbH Munich: Current Employment. Subklewe: Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p < 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p < 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p < 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p < 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p < 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p < 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p < 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p < 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p < 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC > 1 or < -1; p < 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj < 0.0001) and protein secretion (NES=-1.49; p adj < 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

This work presents the results of research toward designing an instruction set extension dedicated to Global Navigation Satellite System (GNSS) baseband processing. The paper describes the state-of-the-art techniques of GNSS receiver implementation. Their advantages and disadvantages are discussed. Against this background, a new versatile instruction set extension for GNSS baseband processing is presented. The authors introduce improved mechanisms for instruction set generation focused on multi-channel processing. The analytical approach used by the authors leads to the introduction of a GNSS-instruction set extension (ISE) for GNSS baseband processing. The developed GNSS-ISE is simulated extensively using PC software and field-programmable gate array (FPGA) emulation. Finally, the developed GNSS-ISE is incorporated into the first-in-the-world, according to the authors’ best knowledge, integrated, multi-frequency, and multi-constellation microcontroller with embedded flash memory. Additionally, this microcontroller may serve as an application processor, which is a unique feature. The presented results show the feasibility of implementing the GNSS-ISE into an embedded microprocessor system and its capability of performing baseband processing. The developed GNSS-ISE can be implemented in a wide range of applications including smart IoT (internet of things) devices or remote sensors, fostering the adaptation of multi-frequency and multi-constellation GNSS receivers to the low-cost consumer mass-market.

International audience; Chromium is a toxic element naturally present in natural waters whose chemical speciation regulates its cycling, mobility and bioavailability. We present here: 1-an improved analytical 16 method for chromium speciation (Cr(VI) vs Cr(III)) in estuarine samples by catalytic adsorptive cathodic stripping voltammetric (cat-AdCSV) and 2-a study highlighting a significant change of redox speciation during summer and winter. Initial measurements first revealed that surface-active substances (SAS) present in estuarine samples strongly influenced the analytical determination of Cr by partially masking the Cr peak through an increase of the background current. We found that the application of a low negative accumulation potential (- 1.65 V) resulted in much better voltammograms compared to those obtained using the usual accumulation potential of-1.0 V. Using humic acid (HA) as a model SAS of natural origin, we show that this negative potential clearly prevents adsorption of SAS on the Hg-electrode surface, which in turns benefits the adsorption of the in-situ formed Cr(III)-DTPA complex and the resulting signal. The optimised method was applied to determine chromium redox speciation and distribution along the 23 km long salinity gradient, well oxygenated, Krka River estuary (Croatia). Cr(VI) was found to be the dominant redox species in both summer and winter, with Cr(III) contribution being lower in summer (up to ~30%, average of ~5%) than in winter (up to ~50%, average of ~30%). In summer, lower concentrations of Cr(VI) were found in the freshwater end-member (2.5 nM) than in the seawater end-member (4-5 nM), while the opposite trend was found in winter. Hexavalent chromium exhibited a non-conservative behaviour along the salinity gradient for both seasons. Chromium predominantlyexists in dissolved phase, and contribution of particles reactive Cr(III) was minor.

Background Loss of mitochondrial function contributes to fatigue, exercise intolerance and muscle weakness, and is a key factor in the disability that develops with age and a wide variety of chronic disorders. Here, we describe the impact of a first-in-class cardiolipin-binding compound that is targeted to mitochondria and improves oxidative phosphorylation capacity (Elamipretide, ELAM) in a randomized, double-blind, placebo-controlled clinical trial. Methods Non-invasive magnetic resonance and optical spectroscopy provided measures of mitochondrial capacity (ATPmax) with exercise and mitochondrial coupling (ATP supply per O2 uptake; P/O) at rest. The first dorsal interosseous (FDI) muscle was studied in 39 healthy older adult subjects (60 to 85 yrs of age; 46% female) who were enrolled based on the presence of poorly functioning mitochondria. We measured volitional fatigue resistance by force-time integral over repetitive muscle contractions. Results A single ELAM dose elevated mitochondrial energetic capacity in vivo relative to placebo (ΔATPmax; P = 0.055, %ΔATPmax; P = 0.045) immediately after a 2-hour infusion. No difference was found on day 7 after treatment, which is consistent with the half-life of ELAM in human blood. No significant changes were found in resting muscle mitochondrial coupling. Despite the increase in ATPmax there was no significant effect of treatment on fatigue resistance in the FDI. Conclusions These results highlight that ELAM rapidly and reversibly elevates mitochondrial capacity after a single dose. This response represents the first demonstration of a pharmacological intervention that can reverse mitochondrial dysfunction in vivo immediately after treatment in aging human muscle.

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(•), CO3(•-),(•)NO2 and GS(•)). The rate constants for the Amplex® Red oxidation by N3(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.

The synthesis of new analogues of cyclolinopeptide A (CLA) and their linear precursors modified with (R)- and (S)-4-methylpseudoproline in the Pro3-Pro4 fragment are presented. The peptides were tested in comparison with cyclosporine A (CsA) in concanavalin A (Con A) and pokeweed mitogen (PWM)-induced mouse splenocyte proliferation and in secondary humoral immune response in vitro to sheep erythrocytes (SRBC). Their effects on expression of selected signaling molecules in the Jurkat T cell line were also determined. In addition, the structural features of the peptides, applying nuclear magnetic resonance and circular dichroism, were analyzed. The results showed that only peptides 7 and 8 modified with (R)-4-methylpseudoproline residue (c(Leu1-Val2-(R)-(αMe)Ser(ΨPro)3-Pro4-Phe5-Phe6-Leu7-Ile8-Ile9) and c(Leu1-Val2-Pro3-(R)-(αMe)Ser(ΨPro)4-Phe5-Phe6-Leu7-Ile8-Ile9), respectively) strongly suppressed mitogen-induced splenocyte proliferation and the humoral immune response, with peptide 8 being more potent. Likewise, peptide 8 more strongly elevated expression of Fas, a proapoptotic signaling molecule in Jurkat cells. We postulate that the increased biological activity of peptide 8, compared to the parent molecule and other studied peptides, resulted from its more flexible structure, found on the basis of both CD and NMR studies. CD and NMR spectra showed that replacement of Pro3 by (R)-(αMe)Ser(¬Pro) caused much greater conformational changes than the same replacement of the Pro4 residue. Such a modification could lead to increased conformational freedom of peptide 8, resulting in a greater ability to adopt a more compact structure, better suited to its putative receptor. In conclusion, peptide 8 is a potent immune suppressor which may find application in controlling immune disorders.

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day sS-31. Mitochondrial function was assessed in vivo using (31)P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.

Summary Changes in mitochondrial function with age vary between different muscle types, and mechanisms underlying this variation remain poorly defined. We examined whether the rate of mitochondrial protein turnover contributes to this variation. Using heavy label proteomics, we measured mitochondrial protein turnover and abundance in slow‐twitch soleus (SOL) and fast‐twitch extensor digitorum longus (EDL) from young and aged mice. We found that mitochondrial proteins were longer lived in EDL than SOL at both ages. Proteomic analyses revealed that age‐induced changes in protein abundance differed between EDL and SOL with the largest change being increased mitochondrial respiratory protein content in EDL. To determine how altered mitochondrial proteomics affect function, we measured respiratory capacity in permeabilized SOL and EDL. The increased mitochondrial protein content in aged EDL resulted in reduced complex I respiratory efficiency in addition to increased complex I‐derived H2O2 production. In contrast, SOL maintained mitochondrial quality, but demonstrated reduced respiratory capacity with age. Thus, the decline in mitochondrial quality with age in EDL was associated with slower protein turnover throughout life that may contribute to the greater decline in mitochondrial dysfunction in this muscle. Furthermore, mitochondrial‐targeted catalase protected respiratory function with age suggesting a causal role of oxidative stress. Our data clearly indicate divergent effects of age between different skeletal muscles on mitochondrial protein homeostasis and function with the greatest differences related to complex I. These results show the importance of tissue‐specific changes in the interaction between dysregulation of respiratory protein expression, oxidative stress, and mitochondrial function with age.

High levels of the flame retardant 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) have been detected in Pacific salmon sampled near urban areas, raising concern over the safety of salmon consumption. However, salmon fillets also contain the antioxidants eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), whose oxidation products induce cellular antioxidant responses. Because oxidative stress is a mechanism of BDE 47 toxicity, we hypothesized that oxidized EPA and DHA can ameliorate the cellular and mitochondrial toxicity of BDE 47. HepG2 cells were treated with a mixture of oxidized EPA and DHA (oxEPA/oxDHA) at a ratio relevant to salmon consumption (1.5/1 oxEPA/oxDHA) followed by exposure to 100 μM BDE 47. Pretreatment with oxEPA/oxDHA for 12 h prior to BDE 47 exposure prevented BDE 47-mediated depletion of glutathione, and increased expression of antioxidant response genes. oxEPA/oxDHA also reduced the level of reactive oxygen species production by BDE 47. The oxEPA/oxDHA antioxidant responses were associated with partial protection against BDE 47-induced loss of viability and also mitochondrial membrane potential. Mitochondrial electron transport system functional analysis revealed extensive inhibition of State 3 respiration and maximum respiratory capacity by BDE 47 were partially reversed by oxEPA/oxDHA. Our findings indicate that the antioxidant effects of oxEPA/oxDHA protect against short exposures to BDE 47, including a protective role of these compounds on maintaining cellular and mitochondrial function.

Nitroxyl (HNO), which according to the IUPAC recommended nomenclature should be named azanone, is the protonated one-electron reduction product of nitric oxide. Recently, it has gained a considerable attention due to the interesting pharmacological effects of its donors. Although there has been great progress in the understanding of HNO chemistry and chemical biology, it still remains the most elusive reactive nitrogen species, and its selective detection is a real challenge. The development of reliable methodologies for the direct detection of azanone is essential for the understanding of important signaling properties of this reactive intermediate and its pharmacological potential. Over the last decade, there has been considerable progress in the development of low-molecular-weight fluorogenic probes for the detection of HNO, and therefore, in this review, we have focused on the challenges and limitations of and perspectives on nitroxyl detection based on the use of such probes.

Fatigue is the symptom most commonly reported by long-term cancer survivors and is increasingly recognized as related to skeletal muscle dysfunction. Traditional chemotherapeutic agents can cause acute toxicities including cardiac and skeletal myopathies. To investigate the mechanism by which chemotherapy may lead to persistent skeletal muscle dysfunction, mature adult mice were injected with a single cyclophosphamide dose and evaluated for 6 weeks. We found that exposed mice developed a persistent decrease in treadmill running time compared to baseline (25.7±10.6 vs. 49.0±16.8 min, P = 0.0012). Further, 6 weeks after drug exposure, in vivo parameters of mitochondrial function remained below baseline including maximum ATP production (482.1 ± 48.6 vs. 696.2 ± 76.6, P = 0.029) and phosphocreatine to ATP ratio (3.243 ± 0.1 vs. 3.878 ± 0.1, P = 0.004). Immunoblotting of homogenized muscles from treated animals demonstrated a transient increase in HNE adducts 1 week after exposure that resolved by 6 weeks. However, there was no evidence of an oxidative stress response as measured by quantitation of SOD1, SOD2, and catalase protein levels. Examination of mtDNA demonstrated that the mutation frequency remained comparable between control and treated groups. Interestingly, there was evidence of a transient increase in NF-ĸB p65 protein 1 day after drug exposure as compared to saline controls (0.091±0.017 vs. 0.053±0.022, P = 0.033). These data suggest that continued impairment in muscle and mitochondria function in cyclophosphamide-treated animals is not linked to persistent oxidative stress and that alternative mechanisms need to be considered.

Nitroxyl (HNO), the protonated one-electron reduction product of NO, remains an enigmatic reactive nitrogen species. Its chemical reactivity and biological activity are still not completely understood. HNO donors show biological effects different from NO donors. Although HNO reactivity with molecular oxygen is described in the literature, the product of this reaction has not yet been unambiguously identified. Here we report that the decomposition of HNO donors under aerobic conditions in aqueous solutions at physiological pH leads to the formation of peroxynitrite (ONOO−) as a major intermediate. We have specifically detected and quantified ONOO− with the aid of boronate probes, e.g. coumarin-7-boronic acid or 4-boronobenzyl derivative of fluorescein methyl ester. In addition to the major phenolic products, peroxynitrite-specific minor products of oxidation of boronate probes were detected under these conditions. Using the competition kinetics method and a set of HNO scavengers, the value of the second order rate constant of the HNO reaction with oxygen (k = 1.8 × 104 m−1 s−1) was determined. The rate constant (k = 2 × 104 m−1 s−1) was also determined using kinetic simulations. The kinetic parameters of the reactions of HNO with selected thiols, including cysteine, dithiothreitol, N-acetylcysteine, captopril, bovine and human serum albumins, and hydrogen sulfide, are reported. Biological and cardiovascular implications of nitroxyl reactions are discussed.

Background Genomic loss of mismatched HLAs ("HLA loss") represents a frequent modality by which acute myeloid leukemia (AML) evades immune recognition from donor T cells after partially HLA-incompatible allogeneic hematopoietic cell transplantation (allo-HCT). One important consequence of this post-transplantation relapse mechanism is that infusions of lymphocytes from the original donor become ineffectual, prompting the search for alternative therapeutic options. Here, to circumvent the loss of physiological T cell receptor-HLA interactions in these patients, we tested the ability of an anti-CD3/CD33 bispecific antibody (BsAb) to re-target donor T cells towards HLA loss relapses. Methods For short-term in vitro experiments, T cells were co-cultured with the MOLM-13 AML cell line or with primary patient blasts for 96 hours in presence or absence of an anti-CD3/CD33 BsAb. As readouts, we measured T cell activation (as surface expression of CD25 and CD69) and the absolute counts and relative proportion of effectors and targets. For long-term in vitro experiments, we established mixed lymphocyte cultures (MLCs) of T cells purified from two patients after haploidentical HCT and primary AML blasts obtained from the same patients at the time of diagnosis. After sequential stimulations, the co-cultures were tested against targets of interest, with or without addition of the BsAb. Functional readouts were T cell degranulation (measured as CD107a expression), antigen-specific activation (as CD137/41-BB expression) and target-specific cytotoxicity (measured by time-lapse live cell imaging over a 48 hour time span). For in vivo experiments, human leukemic cells were infused intravenously into non-irradiated NSG mice, followed by intraperitoneal infusion of T cells and daily administration of the BiTE compound. Results First, we retrospectively analyzed immunophenotypic data of 36 AML patients who experienced HLA loss relapses at our Institution, documenting robust expression of CD33 on the surface of the relapsed leukemia in 35 of them (97%; Figure 1A). By short-term co-culture experiments we titrated the BsAb concentration to be used for subsequent in vitro assays to 100 ng/ml, and the most informative effector:target ratio to 1:3. Then, we established MLCs by stimulating T cells collected from two patients after partially HLA-incompatible allo-HCT with AML blasts collected from the same patients at the time of diagnosis. In both cases, donor-derived T cells robustly responded against the patient blasts both in term of degranulation (Figure 1B) and of antigen-specific activation (Figure 1C). As expected, when we tested the same T cells against the patient leukemia at time of HLA loss relapse, we detected no T cell-mediated responses. Noticeably, when the BsAb was added, in both cases we detected a strong response not only against the diagnosis but also against the HLA loss variants, indicating that T cells were effectively re-targeted towards leukemic cells. Similar results were obtained also by live cell imaging, measuring target cell apoptosis over 48 hours of recording: also in this assay, in fact, donor T cells recognized and killed leukemia at diagnosis (45% of detection area positive for apoptosis dye) and failed to recognize its HLA loss relapse counterpart (32% of area positive for apoptosis dye). Addition of the BsAb to the co-cultures had a minor effect on recognition of the original disease (45% of area positive for apoptosis dye) but drove dramatic cell death of HLA loss blasts (80% of area positive for apoptosis dye), demonstrating that the BsAb induced not only T cell activation but also and most importantly target cell killing (Figure 1D). Finally, we modeled the BsAb activity in vivo, showing that, whereas the sole infusion of human T cells is not able to prevent the outgrowth of leukemia in the bone marrow of NSG mice, addition of the bispecific antibody leads to effective disease clearance (Figure 1E). Conclusions Our results demonstrate that anti-CD3/CD33 BsAbs can effectively redirect donor T cells against HLA loss leukemia variants, resulting in their rapid and effective killing. Taken together, these promising findings strongly support translation of this approach to ad hoc designed early-phase clinical trials, to provide a rational therapy for this increasingly recognized but still treatment-orphan modality of post-transplantation relapse. Figure 1 Disclosures Subklewe: Janssen: Consultancy; Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Roche: Consultancy, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding. Vago:Moderna Therapeutics: Research Funding; GenDx: Research Funding.

The free-radical theory of aging was proposed more than 50 years ago. As one of the most popular mechanisms explaining the aging process, it has been extensively studied in several model organisms. However, the results remain controversial. The mitochondrial version of free-radical theory of aging proposes that mitochondria are both the primary sources of reactive oxygen species (ROS) and the primary targets of ROS-induced damage. One critical ROS is hydrogen peroxide, which is naturally degraded by catalase in peroxisomes or glutathione peroxidase within mitochondria. Our laboratory developed mice-overexpressing catalase targeted to mitochondria (mCAT), peroxisomes (pCAT), or the nucleus (nCAT) in order to investigate the role of hydrogen peroxide in different subcellular compartments in aging and age-related diseases. The mCAT mice have demonstrated the largest effects on life span and healthspan extension. This chapter will discuss the mCAT phenotype and review studies using mCAT to investigate the roles of mitochondrial oxidative stresses in various disease models, including metabolic syndrome and atherosclerosis, cardiac aging, heart failure, skeletal muscle pathology, sensory defect, neurodegenerative diseases, and cancer. As ROS has been increasingly recognized as essential signaling molecules that may be beneficial in hormesis, stress response and immunity, the potential pleiotropic, or adverse effects of mCAT are also discussed. Finally, the development of small-molecule mitochondrial-targeted therapeutic approaches is reviewed.

Periods of elevated reactive oxygen species (ROS) production are a normal part of mitochondrial physiology. However, little is known about age-related changes in the mitochondrial response to elevated ROS in vivo. Significantly, ROS-induced uncoupling of oxidative phosphorylation has received attention as a negative feedback mechanism to reduce mitochondrial superoxide production. Here we use a novel in vivo spectroscopy system to test the hypothesis that ROS-induced uncoupling is diminished in aged mitochondria. This system simultaneously acquires (31)P magnetic resonance and near-infrared optical spectra to non-invasively measure phosphometabolite and O(2) concentrations in mouse skeletal muscle. Using low dose paraquat to elevate intracellular ROS we assess in vivo mitochondrial function in young, middle aged, and old mice. Oxidative phosphorylation was uncoupled to the same degree in response to ROS at each age, but this uncoupling was associated with loss of phosphorylation capacity and total ATP in old mice only. Using mice lacking UCP3 we demonstrate that this in vivo uncoupling is independent of this putative uncoupler of skeletal muscle mitochondria. These data indicate that ROS-induced uncoupling persists throughout life, but that oxidative stress leads to mitochondrial deficits and loss of ATP in aged organisms that may contribute to impaired function and degeneration.

Protein S-glutathionylation (P-SSG) is a reversible redox modification known to alter the activity of transcription factors, kinases, and contractile proteins as well as protect critical cysteine residues from irreversible oxidation. Exercise elicits an acute oxidative stress that is reported to result in beneficial adaptive responses, a process known as redox signaling. We hypothesized that redox signaling will occur through P-SSG of regulatory cysteines following fatiguing muscle contractions. To test this hypothesis, we performed acute fatiguing contractions using an in vivo stimulation protocol on anesthetized CB6F1 (BALB/cBy x C57BL/6) mice. Muscle force production was monitored by a force transducer throughout the 15 minutes of fatiguing stimulations. The right (stimulated) and left (unstimulated) gastrocnemius were collected 60 minutes after the last stimulation and processed for redox proteomics using resin-assisted thiol enrichment and selective reduction. Redox proteomics revealed over 1,500 P-SSG modifications that were significantly increased by fatiguing contractions. Mitochondrial proteins were among the most sensitive to glutathionylation. Ingenuity Pathway Analysis revealed mitochondrial dysfunction, calcium signaling, cytoskeleton signaling, and oxidative stress response in the top ten affected pathways. Critical cysteines on beta-actin, troponin I, ryanodine receptor 1, SERCA1, ATP synthase subunit alpha and NADH dehydrogenase flavoprotein 1 were among those significantly glutathionylated that have previously been reported to alter protein and muscle function. These modifications of redox sensitive proteins implicate rapid regulation of metabolism, excitation/contraction coupling, kinase activity, and contractile protein function, and protection from irreversible oxidation. Together, these data suggest a direct role of ROS in acute exercise signaling and map the redox sensitive proteome in mouse muscle. This study lays the groundwork for future investigation into the redox signaling of altered exercise adaptation associated with chronic conditions, such as sarcopenia, the age-related loss of muscle mass and function.

Bispecific T-cell engagers (BiTE® antibody constructs) represent a novel immunotherapeutic strategy relying on the recruitment of T cells against tumor cells independent of TCR specificity. In Acute Myeloid Leukemia (AML), CD33 represents a suitable target antigen with high expression levels in >90 % of primary AML samples (Krupka et al, 2014). A CD33-BiTE® antibody construct (AMG 330) was developed mediating cytotoxicity against primary AML in vitro although to a variable degree (Krupka et al, 2016). Several parameters have been identified which modulate AMG 330-mediated cytotoxicity, including CD33 expression level as well as effector to target cell (E:T) ratio. However, the exact mechanism of T-cell activation through BiTE® antibody constructs is only partly understood. Physiological T-cell activation is based on engagement of the T-cell receptor complex together with costimulatory molecules whereas the absence of positive costimulation leads to T-cell anergy. In line with this concept, we hypothesized that BiTE®-mediated cytotoxicity requires positive costimulatory signals on the target cells for T-cell activation. We hypothesize that the ratio of costimulatory and coinhibitory molecules on AML cells determines the susceptibility to AMG 330-mediated cytotoxicity independent of target antigen expression level. A stable expression system was established utilizing murine Ba/F3 cells expressing human CD33 ± CD80 ± CD86 ± PD-L1. Co-cultures of Ba/F3 constructs and T cells were performed in presence of AMG 330 or a control BiTE® (cBiTE®) (5 ng/ml). For some experiments, T cells were separated into naive (CD45RA+/CCR7+) vs memory (CD45RADIM) cells using fluorescence-activated cell sorting. After 3 days, specific lysis was determined by flow cytometry and calculated as % specific lysis = 100 × (1 - live CD33+ cellsAMG 330 / live CD33+ cellscBiTE). T-cell proliferation was defined as number of CD2+ cells on day 3 compared to day 0. The expression pattern of CD33, CD80, CD86 and PD-L1 on primary AML cells was evaluated by specific fluorescence intensity (SFI) using multiparameter flow cytometry. A sample was considered positive at an SFI of > 1.5. Characterized primary AML patient samples were used in a long-term culture assay to determine the influence of the checkpoint molecule expression profile on AMG 330-mediated cytotoxicity. CD33 single positive Ba/F3 cells were not lysed upon the addition of AMG 330 and allogeneic T cells. Cytotoxicity could be restored by expression of CD80, CD86 and CD80+CD86 with following tendency: CD80+CD86 >> CD80 > CD86 (see table 1). There was a direct correlation of T-cell proliferation to AMG 330 mediated cytotoxicity. Memory T cells showed increased cytotoxicity compared to naive T cells against the different Ba/F3 cell lines. The influence of co-inhibition was investigated by additionally transducing PD-L1 into the different Ba/F3 cells. This led to a reduced AMG 330-mediated cytotoxicity in all PD-L1 expressing Ba/F3 cells (Table 1). This was accompanied by a reduction in T-cell proliferation. Looking at the expression profile of CD80 and CD86 in primary AML samples, we observed expression of CD80 in 7/123 and of CD86 in 188/226 of cases (respectively 5.7 % and 83.2 %). When comparing AMG 330-mediated cytotoxicity against primary AML cells for patient pairs with similar CD33 expression levels, a higher CD86/PD-L1 ratio led to an increased AMG 330-mediated cytotoxicity compared to patient samples with a lower CD86/PD-L1 ratio (exemplary data: SFI CD33+: 81.7; SFI-ratio CD86/PD-L1: 4; specific cytotoxicity: 64.2 % vs. SFI CD33+: 89.5; SFI-ratio CD86/PD-L1: 15.9; specific cytotoxicity: 96.4 %). In summary, this data supports the hypothesis that AMG 330-mediated cytotoxicity and T-cell proliferation are influenced by the ratio of costimulatory and coinhibitory molecules on AML cells. Our data supports the notion that the checkpoint profile on AML, rather than one molecule by itself, determines T-cell response to AMG 330. Prospective analyses in clinical trials are needed to validate the relevance of checkpoint molecules on target cells as a predictive biomarker for response. Disclosures Marcinek: AMGEN Research Munich: Research Funding. Brauchle:AMGEN Inc.: Research Funding. Kischel:AMGEN: Employment. Kufer:AMGEN Research Munich: Employment. Subklewe:Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche AG: Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Muscle redox status and post-translational modifications (PTMs) regulate diverse aspects of skeletal muscle function, including mitochondrial energetics and contractile performance. In aging skeletal muscle mitochondrial oxidative stress contributes to altered redox homeostasis. We have previously demonstrated that reducing mitochondrial oxidant production with acute (1 hr) and chronic (8 week) treatment with the peptide elamipretide (SS-31) can reverse skeletal muscle and mitochondrial dysfunction and reduce redox stress in aging mice. These improvements were also associated with increased exercise tolerance and greater fatigue resistance. However, the mechanisms by which improving mitochondrial function with SS-31 reverses age-related decline in skeletal muscle function remain unknown. To investigate the effect of SS-31 on the skeletal muscle proteome we assessed global changes in protein abundance, and phosphorylation and S-glutathionylation PTMs in young vs. aged and aged+SS-31 vs. aged mice. Osmotically regulated pumps were surgically implanted into 26-month old female C57Bl/6 mice to deliver saline or SS-31 (3mg/kg/day) for 8 weeks. At the end of 8-week treatment, animals were dissected and gastrocnemius muscle was separated and flash-frozen for analysis via mass spectrometry. Seven-month old female C57Bl/mice were sacrificed as healthy young controls. Relatively few significant protein abundance changes occur with age in skeletal muscle (43) using a false discovery rate of

Hydroethidine (HE) is a fluorogenic probe widely used for the detection of superoxide radical anion in cellular studies. Recently, we have synthesized cell-impermeant analogue of HE called hydropropidine (HPr +). HPr+ is a water-soluble molecule, possessing highly localized positive charge on the nitrogen atom of the alkyl group, that prevents its cellular uptake. Analogously to its precursor, HPr+ reacts with superoxide forming a specific fluorescent product 2-hydroxypropidine (2-OH-Pr++). According to the proposed mechanism of superoxide-mediated oxidation of HE and HPr+, the probe undergoes one-electron oxidation to the radical cation, and next, in the presence of superoxide, is converted to the appropriate hydroxylation product. Here, we show the spectroscopic characetrization of radical cation of HPr+ and HE by pulse radiolysis technique. The reactivity of both probes toward biologically relevant radicals, e.g. nitrogen dioxide (•NO2) and carbonate (CO3•−) radicals (products of peroxynitrite decomposition), as well as glutathionyl radicals (GS•), will be presented. These results will be discussed in relation to the mechanism of oxidation of hydroethidine based probes in biological milieu.

The aim of this study was to examine changes in activities of cytochrome c oxidase (CCO), glucose-6-phosphate dehydrogenase (G6PDH), Cu-Zn superoxide dismutase (Cu-Zn SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) levels and copper (Cu), zinc (Zn) and selenium (Se) concentrations, and to assess the possible differences between preterm placentas, placentas from term pregnancies complicated by intrauterine growth restriction (IUGR) and full-term control placentas. The enzyme activities and the level of GSH decreased in IUGR and preterm placentas in comparison with the control group. CCO activity and GSH level in preterm placentas were markedly lower compared with the IUGR (P0.01; P0.05) and control (P0.01; P0.05) placentas, respectively. In IUGR placentas the level of Cu was reduced by 23% (P0.05) and Zn by 37%. In preterm placentas the level of Cu was reduced by 19% and Zn by 42%. Se level in IUGR and preterm placentas was higher (P0.05) by 28% and 32% than in control group, respectively. The strong relation was observed between birth weight and CCO activity, birth weight and Cu-Zn SOD activity, and a low level of Zn and Cu influenced the birth weight especially in IUGR cases. Moreover, the strong inverse correlation between Se level and birth weight, Se level and placental weight and Se level and CCO activity are new findings.

Mitochondria integrate the key metabolic fluxes in the cell. This role places this organelle at the center of cellular energetics and, hence, mitochondrial dysfunction underlies a growing number of human disorders and age-related degenerative diseases. Here we present novel analytical and technical methods for evaluating mitochondrial metabolism and (dys)function in human muscle in vivo. Three innovations involving advances in optical spectroscopy (OS) and magnetic resonance spectroscopy (MRS) permit quantifying key compounds in energy metabolism to yield mitochondrial oxidation and phosphorylation fluxes. The first of these uses analytical methods applied to optical spectra to measure hemoglobin (Hb) and myoglobin (Mb) oxygenation states and relative contents ([Hb]/[Mb]) to determine mitochondrial respiration (O2 uptake) in vivo. The second uses MRS methods to quantify key high-energy compounds (creatine phosphate, PCr, and adenosine triphosphate, ATP) to determine mitochondrial phosphorylation (ATP flux) in vivo. The third involves a functional test that combines these spectroscopic approaches to determine mitochondrial energy coupling (ATP/O2), phosphorylation capacity (ATP(max)) and oxidative capacity (O2max) of muscle. These new developments in optical and MR tools allow us to determine the function and capacity of mitochondria noninvasively in order to identify specific defects in vivo that are associated with disease in human and animal muscle. The clinical implication of this unique diagnostic probe is the insight into the nature and extent of dysfunction in metabolic and degenerative disorders, as well as the ability to follow the impact of interventions designed to reverse these disorders.

To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until approximately 5 weeks of age, when ataxic signs began, progressing to death at approximately 7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters.

Aim: Skeletal muscle nitric oxide–cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSμ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. Results: GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSμ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1−/− muscle. Functional analyses of GC1−/− muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA–IIX fiber balance. Force deficits in GC1−/− muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. Innovation: GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. Conclusions: These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency.

The present in vivo studies demonstrated that diallyl disulfide (DADS), occurring in garlic, elevated hepatic sulfane sulfur level and activities of gamma-cystathionase and 3-mercaptopyruvate sulfotransferase in healthy mice but did not affect the hepatic glutathione level. DADS efficiently corrected the concentrations of glutathione and sulfane sulfur, and ameliorated gamma-cystathionase activity that had been lowered in the livers of Ehrlich ascites tumor-bearing mice. In Ehrlich ascites tumor cells, diallyl disulfide did not alter bound sulfane sulfur level, sulfotransferases activity or glutathione level. These data indicate that this compound is capable of acting efficiently and selectively only in the liver and can be used for hepatoprotection during chemotherapy.

Mitochondrial changes are at the centre of a wide range of maladies, including diabetes, neurodegeneration and ageing-related dysfunctions. Here we describe innovative optical and magnetic resonance spectroscopic methods that non-invasively measure key mitochondrial fluxes, ATP synthesis and O(2) uptake, to permit the determination of mitochondrial coupling efficiency in vivo (P/O: half the ratio of ATP flux to O(2) uptake). Three new insights result. First, mitochondrial coupling can be measured in vivo with the rigor of a biochemical determination and provides a gold standard to define well-coupled mitochondria (P/O approximately 2.5). Second, mitochondrial coupling differs substantially among muscles in healthy adults, from values reflective of well-coupled oxidative phosphorylation in a hand muscle (P/O = 2.7) to mild uncoupling in a leg muscle (P/O = 2.0). Third, these coupling differences have an important impact on cell ageing. We found substantial uncoupling and loss of cellular [ATP] in a hand muscle indicative of mitochondrial dysfunction with age. In contrast, stable mitochondrial function was found in a leg muscle, which supports the notion that mild uncoupling is protective against mitochondrial damage with age. Thus, greater mitochondrial dysfunction is evident in muscles with higher type II muscle fibre content, which may be at the root of the preferential loss of type II fibres found in the elderly. Our results demonstrate that mitochondrial function and the tempo of ageing varies among human muscles in the same individual. These technical advances, in combination with the range of mitochondrial properties available in human muscles, provide an ideal system for studying mitochondrial function in normal tissue and the link between mitochondrial defects and cell pathology in disease.

Faster aging is predicted in more active tissues and animals because of greater reactive oxygen species generation. Yet age-related cell loss is greater in less active cell types, such as type II muscle fibers. Mitochondrial uncoupling has been proposed as a mechanism that reduces reactive oxygen species production and could account for this paradox between longevity and activity. We distinguished these hypotheses by using innovative optical and magnetic resonance spectroscopic methods applied to noninvasively measured ATP synthesis and O 2 uptake in vivo in human muscle. Here we show that mitochondrial function is unchanged with age in mildly uncoupled tibialis anterior muscle (75% type I) despite a high respiratory rate in adults. In contrast, substantial uncoupling and loss of cellular [ATP] indicative of mitochondrial dysfunction with age was found in the lower respiring and well coupled first dorsal interosseus (43–50% type II) of the same subjects. These results reject respiration rate as the sole factor impacting the tempo of cellular aging. Instead, they support mild uncoupling as a mechanism protecting mitochondrial function and contributing to the paradoxical longevity of the most active muscle fibers.

The mitochondrial respiratory chain (RC) produces most of the cellular ATP and requires strict quality-control mechanisms. To examine RC subunit proteostasis in vivo, we measured RC protein half-lives (HLs) in mice by liquid chromatography-tandem mass spectrometry with metabolic [2H3]-leucine heavy isotope labeling under divergent conditions. We studied 7 tissues/fractions of young and old mice on control diet or one of 2 diet regimens (caloric restriction or rapamycin) that altered protein turnover (42 conditions in total). We observed a 6.5-fold difference in mean HL across tissues and an 11.5-fold difference across all conditions. Normalization to the mean HL of each condition showed that relative HLs were conserved across conditions (Spearman’s ρ = 0.57; P < 10–4), but were highly heterogeneous between subunits, with a 7.3-fold mean range overall, and a 2.2- to 4.6-fold range within each complex. To identify factors regulating this conserved distribution, we performed statistical analyses to study the correlation of HLs to the properties of the subunits. HLs significantly correlated with localization within the mitochondria, evolutionary origin, location of protein-encoding, and ubiquitination levels. These findings challenge the notion that all subunits in a complex turnover at comparable rates and suggest that there are common rules governing the differential proteolysis of RC protein subunits under divergent cellular conditions.—Karunadharma, P. P., Basisty, N., Chiao, Y. A., Dai, D.-F., Drake, R., Levy, N., Koh, W. J., Emond, M. J., Kruse, S., Marcinek, D., Maccoss, M. J., Rabinovitch, P. S. Respiratory chain protein turnover rates in mice are highly heterogeneous but strikingly conserved across tissues, ages, and treatments.

It is claimed that novel beta-adrenolytic drugs possess superior antioxidant properties as compared to classical selective or non-selective beta-adrenoceptor antagonists. Here we tested this notion by analyzing radical scavenging properties of selected beta-adrenolytic drugs and their ability to release nitric oxide in biological preparations. Selective beta1-adrenolytics such as nebivolol, atenolol, metoprolol and non-selective beta-adrenolytics with alpha1-receptor blocking properties such as carvedilol and labetalol were chosen for analysis. NO-releasing properties of nebivolol and carvedilol distinguished third generation beta-adrenolytics from their older counterparts while the reactivity towards hydroxyl and peroxyl radicals discerns only carvedilol but not nebivolol. Thus, superior clinical efficacy of third generation beta-adrenolytics may be related to their ability to release NO rather then to their direct antioxidant properties.

The mitochondrial theory of ageing proposes that the accumulation of oxidative damage to mitochondria leads to mitochondrial dysfunction and tissue degeneration with age. However, no consensus has emerged regarding the effects of ageing on mitochondrial function, particularly for mitochondrial coupling (P/O). One of the main barriers to a better understanding of the effects of ageing on coupling has been the lack of in vivo approaches to measure P/O. We use optical and magnetic resonance spectroscopy to independently quantify mitochondrial ATP synthesis and O2 uptake to determine in vivo P/O. Resting ATP demand (equal to ATP synthesis) was lower in the skeletal muscle of 30-month-old C57Bl/6 mice compared to 7-month-old controls (21.9 +/- 1.5 versus 13.6 +/- 1.7 nmol ATP (g tissue)(-1) s(-1), P = 0.01). In contrast, there was no difference in the resting rates of O2 uptake between the groups (5.4 +/- 0.6 versus 8.4 +/- 1.6 nmol O2 (g tissue)(-1) s(-1)). These results indicate a nearly 50% reduction in the mitochondrial P/O in the aged animals (2.05 +/- 0.07 versus 1.05 +/- 0.36, P = 0.02). The higher resting ADP (30.8 +/- 6.8 versus 58.0 +/- 9.5 micromol g(-1), P = 0.05) and decreased energy charge (ATP/ADP) (274 +/- 70 versus 84 +/- 16, P = 0.03) in the aged mice is consistent with an impairment of oxidative ATP synthesis. Despite the reduced P/O, uncoupling protein 3 protein levels were not different in the muscles of the two groups. These results demonstrate reduced mitochondrial coupling in aged skeletal muscle that alters cellular metabolism and energetics.