WNK kinases sense molecular crowding and rescue cell volume via phase separation.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

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.

Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments.

Dopamine transporter (DAT) controls dopamine neurotransmission by clearing synaptically released dopamine. However, trafficking itineraries of DAT, which determine its cell-surface concentration near synapses, are poorly characterized. It is especially unknown how DAT is transported between spatially distant midbrain somatodendritic and striatal axonal compartments. To examine this "long-range" trafficking, the localization and membrane diffusion of HA-epitope tagged DAT in the medial forebrain bundle (MFB) of a knock-in mouse (both sexes) were analyzed using confocal, super-resolution and EM in intact brain and acute brain slices. HA-DAT was abundant in the plasma membrane of MFB axons, similar to the striatum, although the intracellular fraction of HA-DAT in MFB was more substantial. Intracellular HA-DAT colocalized with VPS35, a subunit of the retromer complex mediating recycling from endosomes, in a subset of axons. Late endosomes, lysosomes, and endoplasmic reticulum were abundant in the soma but minimally present in MFB axons, suggesting that biosynthesis and lysosomal degradation of DAT are confined to soma. Together, the data suggest that membrane diffusion is the main mode of long-range DAT transport through MFB, although the contribution of vesicular traffic can be significant in a population of MFB axons. Based on HA-DAT diffusion rates, plasma membrane DAT in MFB axons turns over with a halftime of ∼20 d, which explains the extremely slow turnover of DAT protein in the brain. Unexpectedly, the mean diameter of DAT-labeled MFB axons was observed to be twice larger than reported for striatum. The implications of this finding for dopamine neuron physiology are discussed.SIGNIFICANCE STATEMENT The dopamine transporter (DAT) is a key regulator of dopamine neurotransmission and a target of abused psychostimulants. In the present study, we examined, for the first time, mechanisms of the long-range traffic of DAT in intact brain and acute brain slices from the knock-in mouse expressing epitope-tagged DAT. Using a combination of confocal, super-resolution and EM, we defined DAT localization and its membrane diffusion parameters in medial forebrain bundle axonal tracts connecting midbrain somatodendritic and striatal axonal compartments of dopaminergic neurons. In contrast to the widely accepted model of long-range axonal transport, our studies suggest that DAT traffics between midbrain and striatum, mainly by lateral diffusion in the plasma membrane with only a limited contribution of vesicular transport in recycling endosomes.

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.

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.

An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication.

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

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.

Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission.

Many neurons influence their targets through co-release of neuropeptides and small-molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small-molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both stimulated emission depletion super-resolution and transmission electron microscopy. Ptp4E also controls the presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content.This article has an associated First Person interview with the first author of the paper.

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.

Pain Recognition With Electrocardiographic Features in Postoperative Patients: Method Validation Study.

BACKGROUND: There is a strong demand for an accurate and objective means of assessing acute pain among hospitalized patients to help clinicians provide pain medications at a proper dosage and in a timely manner. Heart rate variability (HRV) comprises changes in the time intervals between consecutive heartbeats, which can be measured through acquisition and interpretation of electrocardiography (ECG) captured from bedside monitors or wearable devices. As increased sympathetic activity affects the HRV, an index of autonomic regulation of heart rate, ultra-short-term HRV analysis can provide a reliable source of information for acute pain monitoring. In this study, widely used HRV time and frequency domain measurements are used in acute pain assessments among postoperative patients. The existing approaches have only focused on stimulated pain in healthy subjects, whereas, to the best of our knowledge, there is no work in the literature building models using real pain data and on postoperative patients. OBJECTIVE: The objective of our study was to develop and evaluate an automatic and adaptable pain assessment algorithm based on ECG features for assessing acute pain in postoperative patients likely experiencing mild to moderate pain. METHODS: The study used a prospective observational design. The sample consisted of 25 patient participants aged 18 to 65 years. In part 1 of the study, a transcutaneous electrical nerve stimulation unit was employed to obtain baseline discomfort thresholds for the patients. In part 2, a multichannel biosignal acquisition device was used as patients were engaging in non-noxious activities. At all times, pain intensity was measured using patient self-reports based on the Numerical Rating Scale. A weak supervision framework was inherited for rapid training data creation. The collected labels were then transformed from 11 intensity levels to 5 intensity levels. Prediction models were developed using 5 different machine learning methods. Mean prediction accuracy was calculated using leave-one-out cross-validation. We compared the performance of these models with the results from a previously published research study. RESULTS: Five different machine learning algorithms were applied to perform a binary classification of baseline (BL) versus 4 distinct pain levels (PL1 through PL4). The highest validation accuracy using 3 time domain HRV features from a BioVid research paper for baseline versus any other pain level was achieved by support vector machine (SVM) with 62.72% (BL vs PL4) to 84.14% (BL vs PL2). Similar results were achieved for the top 8 features based on the Gini index using the SVM method, with an accuracy ranging from 63.86% (BL vs PL4) to 84.79% (BL vs PL2). CONCLUSIONS: We propose a novel pain assessment method for postoperative patients using ECG signal. Weak supervision applied for labeling and feature extraction improves the robustness of the approach. Our results show the viability of using a machine learning algorithm to accurately and objectively assess acute pain among hospitalized patients. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/17783.

Mitochondrial fatty acid oxidation and the electron transport chain comprise a multifunctional mitochondrial protein complex.

Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: the electron transfer chain (ETC), fatty acid ß-oxidation (FAO), and the tricarboxylic acid cycle. The ETC is organized into inner mitochondrial membrane supercomplexes that promote substrate channeling and catalytic efficiency. Although previous studies have suggested functional interaction between FAO and the ETC, their physical interaction has never been demonstrated. In this study, using blue native gel and two-dimensional electrophoreses, nano-LC-MS/MS, immunogold EM, and stimulated emission depletion microscopy, we show that FAO enzymes physically interact with ETC supercomplexes at two points. We found that the FAO trifunctional protein (TFP) interacts with the NADH-binding domain of complex I of the ETC, whereas the electron transfer enzyme flavoprotein dehydrogenase interacts with ETC complex III. Moreover, the FAO enzyme very-long-chain acyl-CoA dehydrogenase physically interacted with TFP, thereby creating a multifunctional energy protein complex. These findings provide a first view of an integrated molecular architecture for the major energy-generating pathways in mitochondria that ensures the safe transfer of unstable reducing equivalents from FAO to the ETC. They also offer insight into clinical ramifications for individuals with genetic defects in these pathways.

Evidence for Compartmentalized Axonal Mitochondrial Biogenesis: Mitochondrial DNA Replication Increases in Distal Axons As an Early Response to Parkinson's Disease-Relevant Stress.

Dysregulation of mitochondrial biogenesis is implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). However, it is not clear how mitochondrial biogenesis is regulated in neurons, with their unique compartmentalized anatomy and energetic demands. This is particularly relevant in PD because selectively vulnerable neurons feature long, highly arborized axons where degeneration initiates. We previously found that exposure of neurons to chronic, sublethal doses of rotenone, a complex I inhibitor linked to PD, causes early increases in mitochondrial density specifically in distal axons, suggesting possible upregulation of mitochondrial biogenesis within axons. Here, we directly evaluated for evidence of mitochondrial biogenesis in distal axons and examined whether PD-relevant stress causes compartmentalized alterations. Using BrdU labeling and imaging to quantify replicating mitochondrial DNA (mtDNA) in primary rat neurons (pooled from both sexes), we provide evidence of mtDNA replication in axons along with cell bodies and proximal dendrites. We found that exposure to chronic, sublethal rotenone increases mtDNA replication first in neurites and later extending to cell bodies, complementing our mitochondrial density data. Further, isolating axons from cell bodies and dendrites, we discovered that rotenone exposure upregulates mtDNA replication in distal axons. Utilizing superresolution stimulated emission depletion (STED) imaging, we identified mtDNA replication at sites of mitochondrial-endoplasmic reticulum contacts in axons. Our evidence suggests that mitochondrial biogenesis occurs not only in cell bodies, but also in distal axons, and is altered under PD-relevant stress conditions in an anatomically compartmentalized manner. We hypothesize that this contributes to vulnerability in neurodegenerative diseases.SIGNIFICANCE STATEMENT Mitochondrial biogenesis is crucial for maintaining mitochondrial and cellular health and has been linked to neurodegenerative disease pathogenesis. However, regulation of this process is poorly understood in CNS neurons, which rely on mitochondrial function for survival. Our findings offer fundamental insight into these regulatory mechanisms by demonstrating that replication of mitochondrial DNA, an essential precursor for biogenesis, can occur in distal regions of CNS neuron axons independent of the soma. Further, this process is upregulated specifically in axons as an early response to neurodegeneration-relevant stress. This is the first demonstration of the compartmentalized regulation of CNS neuronal mitochondrial biogenesis in response to stress and may prove a useful target in development of therapeutic strategies for neurodegenerative disease.

Lack of impact of nil-per-os (NPO) time on goal-directed fluid delivery in first case versus afternoon case starts: a retrospective cohort study.

BACKGROUND: Goal Directed Fluid Therapy (GDFT) represents an objective fluid replacement algorithm. The effect of provider variability remains a confounder. Overhydration worsens perioperative morbidity and mortality; therefore, the impact of the calculated NPO deficit prior to the operating room may reach harm. METHODS: A retrospective single-institution study analyzed patients at UC Irvine Medical Center main operating rooms from September 1, 2013 through September 1, 2015 receiving GDFT. The primary study question asked if GDFT suggested different fluid delivery after different NPO periods, while reducing inter-provider variability. We created two patient groups distinguished by 0715 surgical start time or start time after 1200. We analyzed fluid administration totals with either a 1:1 crystalloid to colloid ratio or a 3:1 ratio. We performed direct group-wise testing on total administered volume expressed as total ml, total ml/hr., and total ml/kg/hr. between the first case start (AM) and afternoon case (PM) groups. A linear regression model included all baseline covariates that differed between groups as well as plausible confounding factors for differing fluid needs. Finally, we combined all patients from both groups, and created NPO time to total administered fluid scatterplots to assess the effect of patient-reported NPO time on fluid administration. RESULTS: Whether reported by total administered volume or net fluid volume, and whether we expressed the sum as ml, ml/hr., or ml/kg/hr., the AM group received more fluid on average than the PM group in all cases. In the general linear models, for all significant independent variables evaluated, AM vs PM case start did not reach significance in both cases at p = 0.64 and p = 0.19, respectively. In scatterplots of NPO time to fluid volumes, absolute adjusted and unadjusted R2 values are < 0.01 for each plot, indicating virtually non-existent correlations between uncorrected NPO time and fluid volumes measured. CONCLUSIONS: This study showed NPO periods do not influence a patient's volume status just prior to presentation to the operating room for surgical intervention. We hope this data will influence the practice of providers routinely replacing calculated NPO period volume deficit; particularly with those presenting with later surgical case start times.

Closed-loop vasopressor control: in-silico study of robustness against pharmacodynamic variability.

Initial feasibility of a novel closed-loop controller created by our group for closed-loop control of vasopressor infusions has been previously described. In clinical practice, vasopressor potency may be affected by a variety of factors including other pharmacologic agents, organ dysfunction, and vasoplegic states. The purpose of this study was therefore to evaluate the effectiveness of our controller in the face of large variations in drug potency, where 'effective' was defined as convergence on target pressure over time. We hypothesized that the controller would remain effective in the face up to a tenfold variability in drug response. To perform the robustness study, our physiologic simulator was used to create randomized simulated septic patients. 250 simulated patients were managed by the closed-loop in each of 7 norepinephrine responsiveness conditions: 0.1 ×, 0.2 ×, 0.5 ×, 1 ×, 2 ×, 5 ×, and 10 × expected population response to drug dose. Controller performance was evaluated for each level of norepinephrine response using Varvel's criteria as well as time-out-of-target. Median performance error and median absolute performance error were less than 5% in all response levels. Wobble was below 3% and divergence remained negative (i.e. the controller tended to converge towards the target over time) in all norepinephrine response levels, but at the highest response level of 10 × the value approached zero, suggesting the controller may be approaching instability. Response levels of 0.1 × and 0.2 × exhibited significantly higher time-out-of-target in the lower ranges (p < 0.001) compared to the 1 × response level as the controller was slower to correct the initial hypotension. In this simulation study, the closed-loop vasopressor controller remained effective in simulated patients exhibiting 0.1 to 10 × the expected population drug response.

Selective knockout of astrocytic Na+ /H+ exchanger isoform 1 reduces astrogliosis, BBB damage, infarction, and improves neurological function after ischemic stroke.

Stimulation of Na+ /H+ exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1flox/flox (Nhe1f/f ) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1f/f mice with Gfap-CreERT2 Cre-recombinase mice. Gfap-CreERT2+/- ;Nhe1f/f mice at postnatal day 60-90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post-injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared with the oil-vehicle group (control), Tam-treated Gfap-CreERT2+/- ;Nhe1f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1-5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri-lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood-brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP-9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP-9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post-MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.

Feasibility of automated titration of vasopressor infusions using a novel closed-loop controller.

Blood pressure management is a central concern in critical care patients. For a variety of reasons, titration of vasopressor infusions may be an ideal use-case for computer assistance. Using our previous experience gained in the bench-to-bedside development of a computer-assisted fluid management system, we have developed a novel controller for this purpose. The aim of this preliminary study was to assess the feasibility of using this controller in simulated patients to maintain a target blood pressure in both stable and variable blood-pressure scenarios. We tested the controller in two sets of simulation scenarios: one with stable underlying blood pressure and a second with variable underlying blood pressure. In addition, in the variable phase of the study, we tested infusion-line delays of 8-60 s. The primary outcome for both testing conditions (stable and variable) was % case time in target range. We determined a priori that acceptable performance on the first phase of the protocol would require greater than 95% case-time in-target given the simple nature of the protocol, and for the second phase of the study 80% or greater given the erratic nature of the blood pressure changes taking place. 250 distinct cases for each simulation condition, both managed and unmanaged, were run over 4 days. In the stable hemodynamic conditions, the unmanaged group had an MAP of 57.5 ± 4.6 mmHg and spent only 5.6% of case time in-target. The managed group had an MAP of 70.3 ± 2.6 and spent a total of 99.5% of case time in-target (p < 0.00001 for both comparisons between groups). In the variable hemodynamic conditions, the unmanaged group had an MAP of 53.1 ± 5.0 mmHg and spent 0% of case time in-target. The managed group had an MAP of 70.5 ± 3.2 mmHg (p < 0.00001 compared to unmanaged group) and spent 88.6% of case time in-target (p < 0.00001 compared to unmanaged group), with 6.4% of case time over and 5.1% of case time under target. Increasing infusion lag increased coefficient of variation by about 10% per 15 s of lag (p = 0.001). This study demonstrated that this novel controller for vasopressor administration is able to main a target mean arterial pressure in a simulated physiologic model in the face of random disturbances and infusion-line lag.

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.

High-mobility group box 1 increases platelet surface P2Y12 and platelet activation in sickle cell disease.

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.

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.