Environmental and Economic Impact of Using a Higher Efficiency Ventilator and Vaporizer During Surgery Under General Anesthesia: A Randomized Controlled Prospective Cohort.

Background Compared to traditional breathing circuits, low-volume anesthesia machines utilize a lower-volume breathing circuit paired with needle injection vaporizers that supply volatile agents into the circuit mainly during inspiration. We aimed to assess whether or not low-volume anesthesia machines, such as the Maquet Flow-i C20 anesthesia workstation (MQ), deliver volatile anesthetics more efficiently than traditional anesthesia machines, such as the GE Aisys CS2 anesthesia machine (GE), and, secondarily, whether this was in a meaningful economic or environmentally conscious way. Methodology Participants enrolled in the study (Institutional Review Board Identifier: 2014-1248) met the following inclusion criteria: 18-65 years old, scheduled for surgery requiring general anesthesia at the University of California Irvine Health, and expected to receive sevoflurane for the duration of the procedure. Exclusion criteria included age <18 years old, a history of chronic obstructive pulmonary disorder, cardiovascular disease, sevoflurane sensitivity, body mass index >30 kg/m2, American Society of Anesthesiologists >2, pregnancy, or surgery scheduled <120 minutes. We calculated the total amount of sevoflurane delivered and consumption rates during induction and maintenance periods and compared the groups using one-sided parametric testing (Student's t-test). There was no suspicion that the low-volume circuit could use more sevoflurane and that the outcome did not answer our research question. One-sided testing allowed for more power to be more certain of smaller differences in our results. Results In total, 103 subjects (MQ: n = 52, GE: n = 51) were analyzed. Seven subjects were lost to attrition of different types. Overall, the MQ group consumed significantly less sevoflurane (95.5 ± 49.3 g) compared to the GE group (118.3 ± 62.4 g) (p = 0.043), corresponding to an approximately 20% efficiency improvement in overall agent delivery. When accounting for the fresh gas flow setting, agent concentration, and length of induction, the MQ delivered the volatile agent at a significantly lower rate compared to the GE (7.4 ± 3.2 L/minute vs. 9.1 ± 4.1 L/minute; p = 0.017). Based on these results, we estimate that the MQ can save an estimated average of $239,440 over the expected 10-year machine lifespan. This 20% decrease in CO2 equivalent emissions corresponds to 201 metric tons less greenhouse gas emissions over a decade compared to the GE, which is equivalent to 491,760 miles driven by an average passenger vehicle or 219,881 pounds of coal burned. Conclusions Overall, our results from this study suggest that the MQ delivers statistically significantly less (~20%) volatile agent during routine elective surgery using a standardized anesthetic protocol and inclusion/exclusion criteria designed to minimize any patient or provider heterogeneity effects on the results. The results demonstrate an opportunity for economic and environmental benefits.

Endometriosis-Associated Mesenchymal Stem Cells Support Ovarian Clear Cell Carcinoma through Iron Regulation.

Ovarian clear cell carcinoma (OCCC) is a deadly and treatment-resistant cancer, which arises within the unique microenvironment of endometriosis. In this study, we identified a subset of endometriosis-derived mesenchymal stem cells (enMSC) characterized by loss of CD10 expression that specifically support OCCC growth. RNA sequencing identified alterations in iron export in CD10-negative enMSCs and reciprocal changes in metal transport in cocultured OCCC cells. CD10-negative enMSCs exhibited elevated expression of iron export proteins hephaestin and ferroportin and donate iron to associated OCCCs, functionally increasing the levels of labile intracellular iron. Iron is necessary for OCCC growth, and CD10-negative enMSCs prevented the growth inhibitory effects of iron chelation. In addition, enMSC-mediated increases in OCCC iron resulted in a unique sensitivity to ferroptosis. In vitro and in vivo, treatment with the ferroptosis inducer erastin resulted in significant death of cancer cells grown with CD10-negative enMSCs. Collectively, this work describes a novel mechanism of stromal-mediated tumor support via iron donation. This work also defines an important role of endometriosis-associated MSCs in supporting OCCC growth and identifies a critical therapeutic vulnerability of OCCC to ferroptosis based on stromal phenotype. SIGNIFICANCE: Endometriosis-derived mesenchymal stem cells support ovarian clear cell carcinoma via iron donation necessary for cancer growth, which also confers sensitivity to ferroptosis-inducing therapy.

Co-expression of TNF receptors 1 and 2 on melanomas facilitates soluble TNF-induced resistance to MAPK pathway inhibitors.

BACKGROUND: The effectiveness of MAPK pathway inhibitors (MAPKi) used to treat patients with BRAF-mutant melanoma is limited by a range of resistance mechanisms, including soluble TNF (solTNF)-mediated NF-kB signaling. solTNF preferentially signals through type-1 TNF receptor (TNFR1), however, it can also bind to TNFR2, a receptor that is primarily expressed on leukocytes. Here, we investigate the TNFR2 expression pattern on human BRAFV600E+ melanomas and its role in solTNF-driven resistance reprogramming to MAPKi. METHODS: Flow cytometry was used to test TNFR1, TNFR2 and CD271 expression on, as well as NF-kB phosphorylation in human BRAF-mutant melanoma. The ability of melanoma cell lines to acquire MAPKi resistance in response to recombinant or macrophage-derived TNF was evaluated using the MTT cytotoxicity assay. Gene editing was implemented to knock out or knock in TNF receptors in melanoma cell lines. Knockout and knock-in cell line variants were employed to assess the intrinsic roles of these receptors in TNF-induced resistance to MAPKi. Multicolor immunofluorescence microscopy was utilized to test TNFR2 expression by melanoma in patients receiving MAPKi therapy. RESULTS: TNFR1 and TNFR2 are co-expressed at various levels on 4/7 BRAFV600E+ melanoma cell lines evaluated in this study. In vitro treatments with solTNF induce MAPKi resistance solely in TNFR2-expressing BRAFV600E+ melanoma cell lines. TNFR1 and TNFR2 knockout and knock-in studies indicate that solTNF-mediated MAPKi resistance in BRAFV600E+ melanomas is predicated on TNFR1 and TNFR2 co-expression, where TNFR1 is the central mediator of NF-kB signaling, while TNFR2 plays an auxiliary role. solTNF-mediated effects are transient and can be abrogated with biologics. Evaluation of patient specimens indicates that TNFR2 is expressed on 50% of primary BRAFV600E+ melanoma cells and that MAPKi therapy may lead to the enrichment of TNFR2-expressing tumor cells. CONCLUSIONS: Our data suggest that TNFR2 is essential to solTNF-induced MAPKi resistance and a possible biomarker to identify melanoma patients that can benefit from solTNF-targeting therapies.

Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells.

Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

Attenuated cell-cycle division protein 2 and elevated mitotic roles of polo-like kinase 1 characterize deficient myoblast fusion in peripheral arterial disease.

INTRODUCTION: We propose that MuSC-derived myoblasts in PAD have transcriptomic differences that can highlight underlying causes of ischemia-induced myopathy. METHODS: Differentiation capacity among perfused and ischemic human myoblasts was compared. Following next generation sequencing of mRNA, Ingenuity Pathway Analysis (IPA) was performed for canonical pathway enrichment. Live cell imaging and immunofluorescence were performed to determine myocyte fusion index and protein expression based on insights from IPA, specifically concerning cell cycle regulators including cell-division cycle protein 2 (CDC2) and polo-like kinase 1 (PLK1). RESULTS: Ischemic myoblasts formed attenuated myotubes indicative of reduced fusion. Additionally, myoblasts from ischemic segments showed significant differences in canonical pathways associated with PLK1 (upregulated) and G2/M DNA damage checkpoint regulation (downregulated). PLK1 inhibition with BI2536 did not affect cell viability in any group over 24 h but deterred fusion more significantly in PAD myoblasts. Furthermore, PLK1 inhibition reduced the expression of checkpoint protein CDC2 in perfused but not ischemic cells. CONCLUSION: Differentiating myoblasts derived from ischemic muscle have significant differences in gene expression including those essential to DNA-damage checkpoint regulation and cell cycle progress. DNA-damage checkpoint dysregulation may contribute to myopathy in PAD.

Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins.

UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions.

Detection of arterial pressure waveform error using machine learning trained algorithms.

In critically ill and high-risk surgical room patients, an invasive arterial catheter is often inserted to continuously measure arterial pressure (AP). The arterial waveform pressure measurement, however, may be compromised by damping or inappropriate reference placement of the pressure transducer. Clinicians, decision support systems, or closed-loop applications that rely on such information would benefit from the ability to detect error from the waveform alone. In the present study we hypothesized that machine-learning trained algorithms could discriminate three types of transducer error from accurate monitoring with receiver operator characteristic (ROC) curve areas greater than 0.9. After obtaining written consent, patient arterial line waveform data was collected in the operating room in real-time during routine surgery requiring arterial pressure monitoring. Three deliberate error conditions were introduced during monitoring: Damping, Transducer High, and Transducer Low. The waveforms were split up into 10 s clips that were featurized. The data was also either calibrated against the patient's own baseline or left uncalibrated. The data was then split into training and validation sets, and machine-learning algorithms were run in a Monte-Carlo fashion on the training data with variable sized training sets and hyperparameters. The algorithms with the highest balanced accuracy were pruned, then the highest performing algorithm in the training set for each error state (High, Low, Damped) for both calibrated and uncalibrated data was finally tested against the validation set and the ROC and precision-recall curve area-under the curve (AUC) calculated. 38 patients were enrolled in the study with a mean age of 52 ± 15 years. A total of 40 h of monitoring time was recorded with approximately 120,000 heart beats featurized. For all error states, ROC AUCs for algorithm performance on classification of the state were greater than 0.9; when using patient-specific calibrated data AUCs were 0.94, 0.95, and 0.99 for the transducer low, transducer high, and damped conditions respectively. Machine-learning trained algorithms were able to discriminate arterial line transducer error states from the waveform alone with a high degree of accuracy.

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

BNIP3L-mediated mitophagy is required for mitochondrial remodeling during the differentiation of optic nerve oligodendrocytes.

Retinal ganglion cell axons are heavily myelinated (98%) and myelin damage in the optic nerve (ON) severely affects vision. Understanding the molecular mechanism of oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes will be essential for developing new therapeutic approaches for ON demyelinating diseases. To this end, we developed a new method for isolation and culture of ON-derived oligodendrocyte lineage cells and used it to study OPC differentiation. A critical aspect of cellular differentiation is macroautophagy/autophagy, a catabolic process that allows for cell remodeling by degradation of excess or damaged cellular molecules and organelles. Knockdown of ATG9A and BECN1 (pro-autophagic proteins involved in the early stages of autophagosome formation) led to a significant reduction in proliferation and survival of OPCs. We also found that autophagy flux (a measure of autophagic degradation activity) is significantly increased during progression of oligodendrocyte differentiation. Additionally, we demonstrate a significant change in mitochondrial dynamics during oligodendrocyte differentiation, which is associated with a significant increase in programmed mitophagy (selective autophagic clearance of mitochondria). This process is mediated by the mitophagy receptor BNIP3L (BCL2/adenovirus E1B interacting protein 3-like). BNIP3L-mediated mitophagy plays a crucial role in the regulation of mitochondrial network formation, mitochondrial function and the viability of newly differentiated oligodendrocytes. Our studies provide novel evidence that proper mitochondrial dynamics is required for establishment of functional mitochondria in mature oligodendrocytes. These findings are significant because targeting BNIP3L-mediated programmed mitophagy may provide a novel therapeutic approach for stimulating myelin repair in ON demyelinating diseases.Abbreviations: A2B5: a surface antigen of oligodendrocytes precursor cells, A2B5 clone 105; ACTB: actin, beta; APC: an antibody to label mature oligodendrocytes, anti-adenomatous polyposis coli clone CC1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9A: autophagy related 9A; AU: arbitrary units; BafA1: bafilomycin A1; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CNP: 2',3'-cyclic nucleotide 3'-phosphodiesterase; Ctl: control; COX8: cytochrome c oxidase subunit; CSPG4/NG2: chondroitin sulfate proteoglycan 4; DAPI: 4'6-diamino-2-phenylindole; DNM1L: dynamin 1-like; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; GFP: green fluorescent protein; HsESC: human embryonic stem cell; IEM: immunoelectron microscopy; LAMP1: lysosomal-associated membrane protein 1; LC3B: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MFN2: mitofusin 2; Mito-Keima: mitochondria-targeted monomeric keima-red; Mito-GFP: mitochondria-green fluorescent protein; Mito-RFP: mitochondria-red fluorescent protein; MitoSOX: red mitochondrial superoxide probe; MKI67: antigen identified by monoclonal antibody Ki 67; MMP: mitochondrial membrane potential; O4: oligodendrocyte marker O4; OLIG2: oligodendrocyte transcription factor 2; ON: optic nerve; OPA1: OPA1, mitochondrial dynamin like GTPase; OPC: oligodendrocyte progenitor cell; PDL: poly-D-lysine; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; RGC: retinal ganglion cell; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SEM: standard error of the mean; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; TUBB3: tubulin, beta 3 class III.

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

Prospective Study Evaluating a Pain Assessment Tool in a Postoperative Environment: Protocol for Algorithm Testing and Enhancement.

BACKGROUND: Assessment of pain is critical to its optimal treatment. There is a high demand for accurate objective pain assessment for effectively optimizing pain management interventions. However, pain is a multivalent, dynamic, and ambiguous phenomenon that is difficult to quantify, particularly when the patient's ability to communicate is limited. The criterion standard of pain intensity assessment is self-reporting. However, this unidimensional model is disparaged for its oversimplification and limited applicability in several vulnerable patient populations. Researchers have attempted to develop objective pain assessment tools through analysis of physiological pain indicators, such as electrocardiography, electromyography, photoplethysmography, and electrodermal activity. However, pain assessment by using only these signals can be unreliable, as various other factors alter these vital signs and the adaptation of vital signs to pain stimulation varies from person to person. Objective pain assessment using behavioral signs such as facial expressions has recently gained attention. OBJECTIVE: Our objective is to further the development and research of a pain assessment tool for use with patients who are likely experiencing mild to moderate pain. We will collect observational data through wearable technologies, measuring facial electromyography, electrocardiography, photoplethysmography, and electrodermal activity. METHODS: This protocol focuses on the second phase of a larger study of multimodal signal acquisition through facial muscle electrical activity, cardiac electrical activity, and electrodermal activity as indicators of pain and for building predictive models. We used state-of-the-art standard sensors to measure bioelectrical electromyographic signals and changes in heart rate, respiratory rate, and oxygen saturation. Based on the results, we further developed the pain assessment tool and reconstituted it with modern wearable sensors, devices, and algorithms. In this second phase, we will test the smart pain assessment tool in communicative patients after elective surgery in the recovery room. RESULTS: Our human research protections application for institutional review board review was approved for this part of the study. We expect to have the pain assessment tool developed and available for further research in early 2021. Preliminary results will be ready for publication during fall 2020. CONCLUSIONS: This study will help to further the development of and research on an objective pain assessment tool for monitoring patients likely experiencing mild to moderate pain. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/17783.

Structural and functional divergence of GDAP1 from the glutathione S-transferase superfamily.

Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) alter mitochondrial morphology and result in several subtypes of the inherited peripheral neuropathy Charcot-Marie-Tooth disease; however, the mechanism by which GDAP1 functions has remained elusive. GDAP1 contains primary sequence homology to the GST superfamily; however, the question of whether GDAP1 is an active GST has not been clearly resolved. Here, we present biochemical evidence, suggesting that GDAP1 has lost the ability to bind glutathione without a loss of substrate binding activity. We have revealed that the α-loop, located within the H-site motif is the primary determinant for substrate binding. Using structural data of GDAP1, we have found that critical residues and configurations in the G-site which canonically interact with glutathione are altered in GDAP1, rendering it incapable of binding glutathione. Last, we have found that the overexpression of GDAP1 in HeLa cells results in a mitochondrial phenotype which is distinct from oxidative stress-induced mitochondrial fragmentation. This phenotype is dependent on the presence of the transmembrane domain, as well as a unique hydrophobic domain that is not found in canonical GSTs. Together, we data point toward a non-enzymatic role for GDAP1, such as a sensor or receptor.

Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity.

Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases, but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific mitochondria in defined neuronal groups. We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo. MG2I, a chemical fluorogen, produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light. Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits, caused by neuronal bioenergetic crisis and acute neuronal depolarization. These abnormalities resulted from loss of neuronal respiration, associated with mitochondrial fragmentation, swelling and elimination of cristae. Remaining cellular ultrastructure was preserved initially, but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death. Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function, neuronal biogenetics and whole-animal behavior.

Most life processes require the energy produced by small cellular compartments called mitochondria. Many internal and external factors can harm these miniature powerhouses, potentially leading to cell death. For instance, in patients with Parkinson's or Alzheimer's disease, dying neurons often show mitochondrial damage. However, it is unclear exactly how injured mitochondria trigger the demise of these cells. Gaining a better understanding of this process requires studying the impact of mitochondrial damage in live neurons, something that is still difficult to do. As a response to this challenge, Xie, Jiao, Bai, Ilin et al. designed a new tool that can specifically injure mitochondria in the neurons of live zebrafish larvae at will, and fine-tune the amount of damage inflicted. The zebrafish are genetically engineered so that the mitochondria in their neurons carry a protein which can bind to a chemical compound called MG2I. When attached to each other, MG2I and the protein respond to far-red light by locally creating highly damaging chemicals. This means that whenever far-red light is shone onto the larvae, mitochondria in their neurons are harmed ­ the brighter the light, the stronger the damage. Zebrafish larvae exposed to these conditions immediately stopped swimming: mitochondria in their neurons could not produce enough energy and these cells could therefore no longer communicate properly. The neurons then started to die about 24 hours after exposure to the light, suggesting that the mitochondrial damage triggered other downstream processes that culminated in cell death. This new light-controlled tool could help to understand the consequences of mitochondrial damage, potentially revealing new ways to rescue impaired neurons in patients with Parkinson's or Alzheimer's disease. In the future, the method could be adapted to work in any type of cell and deactivate other cell compartments, so that it can be used to study many types of diseases.

Blood pressure variability in surgical and intensive care patients: Is there a potential for closed-loop vasopressor administration?

Blood pressure management in the operating rooms (OR) and intensive care units (ICU) frequently involves manually titrated vasopressor therapy to an optimal range of mean arterial pressure (MAP). Ideally, changes in vasopressor infusion rates have to quickly follow variations in blood pressure measurements. However, such a tightly controlled feedback loop is difficult to achieve. Few studies have examined blood pressure control when vasopressor therapy is administered manually in OR and ICU patients. We extracted MAP data from 3623 patients (2530 from the ORs and 1093 from the ICU) on vasopressors from our electronic medical records. Coefficient of variation (= standard deviation/mean value) *100) was calculated and the values were additionally categorized into different MAP ranges (MAP < 60 mmHg, 60 < MAP < 80 and MAP > 80 mmHg). There was no statistically significant difference between both centres for MAP across all time points (80 ± 12 vs. 80 ± 16, P = 0.996, 95% CI -6 to 6). The coefficients of variation of MAP were 13.7 ± 5.4% and 18.4 ± 9.8% in the OR and in ICU respectively. Patients on vasopressors spent 48.8% treatment time with a MAP between 60 and 80 mmHg (11.2% time with MAP < 60 mmHg, and 40% with MAP > 80 mmHg). These results provide a reasonable baseline from which to establish whether 'reduced variability' may be achieved with a closed-loop vasopressor administration system.

Synthesis and Evaluation of a Mitochondria-Targeting Poly(ADP-ribose) Polymerase-1 Inhibitor.

The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a crucial role in cellular and molecular processes including DNA damage detection and repair and transcription; indeed, PARP inhibitors are under clinical evaluation as chemotherapeutic adjuncts given their capacity to impede genomic DNA repair in tumor cells. Conversely, overactivation of PARP can lead to NAD+ depletion, mitochondrial energy failure, and cell death. Since PARP activation facilitates genomic but impedes mitochondrial DNA repair, nonselective PARP inhibitors are likely to have opposing effects in these cellular compartments. Herein, we describe the synthesis and evaluation of the mitochondria-targeting PARP inhibitor, XJB-veliparib. Attachment of the hemigramicidin S pentapeptide isostere for mitochondrial targeting using a flexible linker at the primary amide site of veliparib did not disrupt PARP affinity or inhibition. XJB-veliparib was effective at low nanomolar concentrations (10-100 nM) and more potent than veliparib in protection from oxygen-glucose deprivation (OGD) in primary cortical neurons. Both XJB-veliparib and veliparib (10 nM) preserved mitochondrial NAD+ after OGD; however, only XJB-veliparib prevented release of NAD+ into cytosol. XJB-veliparib (10 nM) appeared to inhibit poly(ADP-ribose) polymer formation in mitochondria and preserve mitochondrial cytoarchitecture after OGD in primary cortical neurons. After 10 nM exposure, XJB-veliparib was detected by LC-MS in mitochondria but not nuclear-enriched fractions in neurons and was observed in mitoplasts stripped of the outer mitochondrial membrane obtained from HT22 cells. XJB-veliparib was also effective at preventing glutamate-induced HT22 cell death at micromolar concentrations. Importantly, in HT22 cells exposed to H2O2 to produce DNA damage, XJB-veliparib (10 µM) had no effect on nuclear DNA repair, in contrast to veliparib (10 µM) where DNA repair was retarded. XJB-veliparib and analogous mitochondria-targeting PARP inhibitors warrant further evaluation in vitro and in vivo, particularly in conditions where PARP overactivation leads to mitochondrial energy failure and maintenance of genomic DNA integrity is desirable, e.g., ischemia, oxidative stress, and radiation exposure.

Vertebrate myosin 1d regulates left-right organizer morphogenesis and laterality.

Establishing left-right asymmetry is a fundamental process essential for arrangement of visceral organs during development. In vertebrates, motile cilia-driven fluid flow in the left-right organizer (LRO) is essential for initiating symmetry breaking event. Here, we report that myosin 1d (myo1d) is essential for establishing left-right asymmetry in zebrafish. Using super-resolution microscopy, we show that the zebrafish LRO, Kupffer's vesicle (KV), fails to form a spherical lumen and establish proper unidirectional flow in the absence of myo1d. This process requires directed vacuolar trafficking in KV epithelial cells. Interestingly, the vacuole transporting function of zebrafish Myo1d can be substituted by myosin1C derived from an ancient eukaryote, Acanthamoeba castellanii, where it regulates the transport of contractile vacuoles. Our findings reveal an evolutionary conserved role for an unconventional myosin in vacuole trafficking, lumen formation, and determining laterality.

4-1BB costimulation induces T cell mitochondrial function and biogenesis enabling cancer immunotherapeutic responses.

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to these therapies. The tumor microenvironment can impose metabolic restrictions on T cell function, creating a resistance mechanism to immunotherapy. We have previously shown tumor-infiltrating T cells succumb to progressive loss of metabolic sufficiency, characterized by repression of mitochondrial activity that cannot be rescued by PD-1 blockade. 4-1BB, a costimulatory molecule highly expressed on exhausted T cells, has been shown to influence metabolic function. We hypothesized that 4-1BB signaling might provide metabolic support to tumor-infiltrating T cells. 4-1BB costimulation of CD8+ T cells results in enhanced mitochondrial capacity (suggestive of fusion) and engages PGC1α-mediated pathways via activation of p38-MAPK. 4-1BB treatment of mice improves metabolic sufficiency in endogenous and adoptive therapeutic CD8+ T cells. 4-1BB stimulation combined with PD-1 blockade results in robust antitumor immunity. Sequenced studies revealed the metabolic support afforded by 4-1BB agonism need not be continuous and that a short course of anti-4-1BB pretreatment was sufficient to provide a synergistic response. Our studies highlight metabolic reprogramming as the dominant effect of 4-1BB therapy and suggest that combinatorial strategies using 4-1BB agonism may help overcome the immunosuppressive metabolic landscape of the tumor microenvironment.

α-Lipoic acid promotes α-tubulin hyperacetylation and blocks the turnover of mitochondria through mitophagy.

Lysine acetylation is tightly coupled to the nutritional status of the cell, as the availability of its cofactor, acetyl-CoA, fluctuates with changing metabolic conditions. Recent studies have demonstrated that acetyl-CoA levels act as an indicator of cellular nourishment, and increased abundance of this metabolite can block the induction of cellular recycling programmes. In the present study we investigated the cross-talk between mitochondrial metabolic pathways, acetylation and autophagy, using chemical inducers of mitochondrial acetyl-CoA production. Treatment of cells with α-lipoic acid (αLA), a cofactor of the pyruvate dehydrogenase complex, led to the unexpected hyperacetylation of α-tubulin in the cytosol. This acetylation was blocked by pharmacological inhibition of mitochondrial citrate export (a source for mitochondria-derived acetyl-CoA in the cytosol), was dependent on the α-tubulin acetyltransferase (αTAT) and was coupled to a loss in function of the cytosolic histone deacetylase, HDAC6. We further demonstrate that αLA slows the flux of substrates through autophagy-related pathways, and severely limits the ability of cells to remove depolarized mitochondria through PTEN-associated kinase 1 (PINK1)-mediated mitophagy.

Loss of Foxm1 Results in Reduced Somatotrope Cell Number during Mouse Embryogenesis.

FOXM1, a member of the forkhead box transcription factor family, plays a key role in cell cycling progression by regulating the expression of critical G1/S and G2/M phase transition genes. In vivo studies reveal that Foxm1 null mice have a 91% lethality rate at e18.5 due to significant cardiovascular and hepatic hypoplasia. Thus, FOXM1 has emerged as a key protein regulating mitotic division and cell proliferation necessary for embryogenesis. In the current study, we assess the requirement for Foxm1 in the developing pituitary gland. We find that Foxm1 is expressed in the pituitary at embryonic days 10.5-e18.5 and localizes with markers for active cell proliferation (BrdU). Interestingly, direct analysis of Foxm1 null mice at various embryonic ages, reveals no difference in gross pituitary morphology or cell proliferation. We do observe a downward trend in overall pituitary cell number and a small reduction in pituitary size in e18.5 embryos suggesting there may be subtle changes in pituitary proliferation not detected with our proliferation makers. Consistent with this, Foxm1 null mice have reductions in both the somatotrope and gonadotrope cell populations.

Impaired fertility and FSH synthesis in gonadotrope-specific Foxl2 knockout mice.

Impairments in pituitary FSH synthesis or action cause infertility. However, causes of FSH dysregulation are poorly described, in part because of our incomplete understanding of mechanisms controlling FSH synthesis. Previously, we discovered a critical role for forkhead protein L2 (FOXL2) in activin-stimulated FSH ß-subunit (Fshb) transcription in immortalized cells in vitro. Here, we tested the hypothesis that FOXL2 is required for FSH synthesis in vivo. Using a Cre/lox approach, we selectively ablated Foxl2 in murine anterior pituitary gonadotrope cells. Conditional knockout (cKO) mice developed overtly normally but were subfertile in adulthood. Testis size and spermatogenesis were significantly impaired in cKO males. cKO females exhibited reduced ovarian weight and ovulated fewer oocytes in natural estrous cycles compared with controls. In contrast, ovaries of juvenile cKO females showed normal responses to exogenous gonadotropin stimulation. Both male and female cKO mice were FSH deficient, secondary to diminished pituitary Fshb mRNA production. Basal and activin-stimulated Fshb expression was similarly impaired in Foxl2 depleted primary pituitary cultures. Collectively, these data definitively establish FOXL2 as the first identified gonadotrope-restricted transcription factor required for selective FSH synthesis in vivo.