Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays.

Targeted proteomics strategies present a streamlined hypothesis-driven approach to analyze specific sets of pathways or disease related proteins. goDig is a quantitative, targeted tandem mass tag (TMT)-based assay that can measure the relative abundance differences for hundreds of proteins directly from unfractionated mixtures. Specific protein groups or entire pathways of up to 200 proteins can be selected for quantitative profiling, while leveraging sample multiplexing permits the simultaneous analysis of up to 18 samples. Despite these benefits, implementing goDig is not without challenges, as it requires access to an instrument application programming interface (iAPI), an elution order and spectral library, a web-based method builder, and dedicated companion software. In addition, the absence of an example test assay may dissuade researchers from testing or implementing goDig. Here, we repurpose the TKO11 standard─which is commercially available but may also be assembled in-lab─and establish it as a de facto test assay for goDig. We build a proteome-wide goDig yeast library, quantify protein expression across several gene ontology (GO) categories, and compare these results to a fully fractionated yeast gold-standard data set. Essentially, we provide a guide detailing the goDig-based quantification of TKO11, which can also be used as a template for user-defined assays in other species.

Intravenous Autologous Bone Marrow-derived Mesenchymal Stromal Cells Delay Acute Respiratory Distress Syndrome in Swine.

Rationale: Early post injury mitigation strategies in ARDS are in short supply. Treatments with allogeneic stromal cells are administered after ARDS develops, require specialized expertise and equipment, and to date have shown limited benefit. Objectives: Assess the efficacy of immediate post injury intravenous administration of autologous or allogeneic bone marrow-derived mesenchymal stromal cells (MSCs) for the treatment of acute respiratory distress syndrome (ARDS) due to smoke inhalation and burns. Methods: Yorkshire swine (n = 32, 44.3 ± 0.5 kg) underwent intravenous anesthesia, placement of lines, severe smoke inhalation, and 40% total body surface area flame burns, followed by 72 hours of around-the-clock ICU care. Mechanical ventilation, fluids, pressors, bronchoscopic cast removal, daily lung computed tomography scans, and arterial blood assays were performed. After injury and 24 and 48 hours later, animals were randomized to receive autologous concentrated bone marrow aspirate (n = 10; 3 × 106 white blood cells and a mean of 56.6 × 106 platelets per dose), allogeneic MSCs (n = 10; 6.1 × 106 MSCs per dose) harvested from healthy donor swine, or no treatment in injured control animals (n = 12). Measurements and Main Results: The intravenous administration of MSCs after injury and at 24 and 48 hours delayed the onset of ARDS in swine treated with autologous MSCs (48 ± 10 h) versus control animals (14 ± 2 h) (P = 0.004), reduced ARDS severity at 24 (P < 0.001) and 48 (P = 0.003) hours, and demonstrated visibly diminished consolidation on computed tomography (not significant). Mortality at 72 hours was 1 in 10 (10%) in the autologous group, 5 in 10 (50%) in the allogeneic group, and 6 in 12 (50%) in injured control animals (not significant). Both autologous and allogeneic MSCs suppressed systemic concentrations of TNF-α (tumor necrosis factor-α). Conclusions: The intravenous administration of three doses of freshly processed autologous bone marrow-derived MSCs delays ARDS development and reduces its severity in swine. Bedside retrieval and administration of autologous MSCs in swine is feasible and may be a viable injury mitigation strategy for ARDS.

Quantitative Proteomics and Phosphoproteomics Support a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis.

Protein phosphorylation is one of the most prevalent posttranslational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to environmental stimuli. The Mut9-like kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs.

Outcomes of percutaneous temporary biventricular mechanical support: a systematic review.

Percutaneous biventricular assist devices (BiVAD) are a recently developed treatment option for severe cardiogenic shock. This systematic review sought to identify indications and outcomes of patients placed on percutaneous BiVAD support. An electronic search was performed to identify all appropriate studies utilizing a percutaneous BiVAD configuration. Fifteen studies comprising of 20 patients were identified. Individual patient survival and outcomes data were combined for statistical analysis. All 20 patients were supported with a microaxial LVAD, 12/20 (60%) of those patients were supported with a microaxial (RMA) right ventricular assist device (RVAD), and the remaining 8/20 (40%) patients were supported with a centrifugal extracorporeal RVAD (RCF). All patients presented with cardiogenic shock, and of these, 12/20 (60%) presented with a non-ischemic etiology vs 8/20 (40%) with ischemic disease. For the RMA group, RVAD support was significantly longer [RMA 5 (IQR 4-7) days vs RCF 1 (IQR 1-2) days, p = 0.03]. Intravascular hemolysis post-BiVAD occurred in three patients (27.3%) [RMA 3 (33.3%) vs RCF 0 (0%), p = 0.94]. Five patients received a durable left ventricular assist device, one patient received a total artificial heart, and one patient underwent a heart transplantation. Estimated 30-day mortality was 15.0%, and 78.6% were discharged alive. Both strategies for percutaneous BiVAD support appear to be viable options for severe cardiogenic shock.

Impact of Concomitant Mitral Valve Surgery With LVAD Placement: Systematic Review and Meta-Analysis.

The aim of this systematic review and meta-analysis was to evaluate the outcomes of concomitant mitral valve surgery for significant preexisting mitral regurgitation (MR) in patients undergoing continuous-flow left ventricular assist device (CF-LVAD) implantation. Electronic search was performed to identify all studies in the English literature examining concurrent mitral valve surgery in patients with CF-LVAD implantation. Identified articles were systematically assessed for inclusion and exclusion criteria. Of 2319 studies identified, 8 studies were included. Among 445 patients with moderate to severe or severe MR, 113 (25.4%) patients received concurrent mitral valvular intervention during CF-LVAD implantation. There were no significant differences in cardiopulmonary bypass time (MR Surgery 154 min vs. no MR Surgery 119 min, P = 0.64) or hospital length of stay (MR Surgery 21 days vs. no MR Surgery 18 days, P = 0.93). On follow-up, there were no significant differences in freedom from greater than moderate MR (MR Surgery 100% vs. no MR Surgery 74%, P = 0.12) or left ventricular end-diastolic diameter (MR Surgery: 60 mm vs. no MR Surgery 65 mm, P = 0.51). Survival was comparable at 6-months (MR Surgery 77% vs. no MR Surgery 81%, P = 0.75), 1-year (MR Surgery 72% vs. no MR Surgery 80%, P = 0.36), and 2-years of follow-up (MR Surgery 65% vs. no MR Surgery 70%, P = 0.56). The results of our systematic review and meta-analysis of 8 studies consisting of 445 patients demonstrates that the addition of mitral valve intervention to CF-LVAD implantation appears to be safe with comparable survival to those undergoing CF-LVAD implantation alone. Large prospective randomized clinical trials are needed to elucidate whether concomitant mitral valve intervention during CF-LVAD implantation in patients with severe MR is necessary.

Identification of a functional nuclear translocation sequence in hPPIP5K2.

BACKGROUND: Cells contain several inositol pyrophosphates (PP-InsPs; also known as diphosphoinositol polyphosphates), which play pivotal roles in cellular and organismic homeostasis. It has been proposed that determining mechanisms of compartmentation of the synthesis of a particular PP-InsP is key to understanding how each of them may exert a specific function. Human PPIP5K2 (hPPIP5K2), one of the key enzymes that synthesizes PP-InsPs, contains a putative consensus sequence for a nuclear localization signal (NLS). However, such in silico analysis has limited predictive power, and may be complicated by phosphorylation events that can dynamically modulate NLS function. We investigated if this candidate NLS is functional and regulated, using the techniques of cell biology, mutagenesis and mass spectrometry. RESULTS: Multiple sequence alignments revealed that the metazoan PPIP5K2 family contains a candidate NLS within a strikingly well-conserved 63 amino-acid domain. By analyzing the distribution of hPPIP5K2-GFP in HEK293T cells with the techniques of confocal microscopy and imaging flow cytometry, we found that a distinct pool of hPPIP5K2 is present in the nucleus. Imaging flow cytometry yielded particular insight into the characteristics of the nuclear hPPIP5K2 sub-pool, through a high-throughput, statistically-robust analysis of many hundreds of cells. Mutagenic disruption of the candidate NLS in hPPIP5K2 reduced its degree of nuclear localization. Proximal to the NLS is a Ser residue (S1006) that mass spectrometry data indicate is phosphorylated inside cells. The degree of nuclear localization of hPPIP5K2 was increased when S1006 was rendered non-phosphorylatable by its mutation to Ala. Conversely, a S1006D phosphomimetic mutant of hPPIP5K2 exhibited a lower degree of nuclear localization. CONCLUSIONS: The current study describes for the first time the functional significance of an NLS in the conserved PPIP5K2 family. We have further demonstrated that there is phosphorylation of a Ser residue that is proximal to the NLS of hPPIP5K2. These conclusions draw attention to nuclear compartmentation of PPIP5K2 as being a physiologically relevant and covalently-regulated event. Our study also increases general insight into the consensus sequences of other NLSs, the functions of which might be similarly regulated.

MultipleXed Population Selection and Enrichment single nucleus RNA sequencing (XPoSE-seq) enables sample identity retention during transcriptional profiling of rare populations.

Single nucleus RNA-sequencing is critical in deciphering tissue heterogeneity and identifying rare populations. However, current high throughput techniques are not optimized for rare target populations and require tradeoffs in design due to feasibility. We provide a novel snRNA pipeline, MulipleXed Population Selection and Enrichment snRNA-sequencing (XPoSE-seq), to enable targeted snRNA-seq experiments and in-depth transcriptomic characterization of rare target populations while retaining individual sample identity.

Comparison of Synthetic Computed Tomography Generation Methods, Incorporating Male and Female Anatomical Differences, for Magnetic Resonance Imaging-Only Definitive Pelvic Radiotherapy.

PURPOSE: There are several means of synthetic computed tomography (sCT) generation for magnetic resonance imaging (MRI)-only planning; however, much of the research omits large pelvic treatment regions and female anatomical specific methods. This research aimed to apply four of the most popular methods of sCT creation to facilitate MRI-only radiotherapy treatment planning for male and female anorectal and gynecological neoplasms. sCT methods were validated against conventional computed tomography (CT), with regard to Hounsfield unit (HU) estimation and plan dosimetry. METHODS AND MATERIALS: Paired MRI and CT scans of 40 patients were used for sCT generation and validation. Bulk density assignment, tissue class density assignment, hybrid atlas, and deep learning sCT generation methods were applied to all 40 patients. Dosimetric accuracy was assessed by dose difference at reference point, dose volume histogram (DVH) parameters, and 3D gamma dose comparison. HU estimation was assessed by mean error and mean absolute error in HU value between each sCT and CT. RESULTS: The median percentage dose difference between the CT and sCT was <1.0% for all sCT methods. The deep learning method resulted in the lowest median percentage dose difference to CT at -0.03% (IQR 0.13, -0.31) and bulk density assignment resulted in the greatest difference at -0.73% (IQR -0.10, -1.01). The mean 3D gamma dose agreement at 3%/2 mm among all sCT methods was 99.8%. The highest agreement at 1%/1 mm was 97.3% for the deep learning method and the lowest was 93.6% for the bulk density method. Deep learning and hybrid atlas techniques gave the lowest difference to CT in mean error and mean absolute error in HU estimation. CONCLUSIONS: All methods of sCT generation used in this study resulted in similarly high dosimetric agreement for MRI-only planning of male and female cancer pelvic regions. The choice of the sCT generation technique can be guided by department resources available and image guidance considerations, with minimal impact on dosimetric accuracy.

A mutation in SCARB2 is a modifier in Gaucher disease.

Lysosomal integral membrane protein type 2 (LIMP-2) is responsible for proper sorting and lysosomal targeting of glucocerebrosidase, the enzyme deficient in Gaucher disease (GD). Mutations in the gene for LIMP-2, SCARB2, are implicated in inherited forms of myoclonic epilepsy, and myoclonic epilepsy is part of the phenotypic spectrum associated with GD. We investigated whether SCARB2 mutations impact the Gaucher phenotype focusing on patients with myoclonic epilepsy, including a pair of siblings with GD who were discordant for myoclonic seizures. Sequencing of SCARB2 genomic and cDNA identified a heterozygous, maternally inherited novel mutation, c.1412A>G (p.Glu471Gly), in the brother with GD and myoclonic epilepsy, absent from his sibling and controls. Glucocerebrosidase activity, Western blots, real-time PCR, and immunofluorescence studies demonstrated markedly decreased LIMP-2 and glucocerebrosidase in cells from the sibling with (p.Glu471Gly) LIMP-2, and diminished glucocerebrosidase in lysosomes. The cells secreted highly glycosylated enzyme and showed mistrafficking of glucocerebrosidase. Sequencing of SCARB2 in 13 other subjects with GD and myoclonic epilepsy and 40 controls failed to identify additional mutations. The study provides further evidence for the association of LIMP-2 and myoclonic epilepsy, explains the drastically different phenotypes encountered in the siblings, and demonstrates that LIMP-2 can serve as a modifier in GD.

Unified immune modulation by 4-1BB triggering leads to diverse effects on disease progression in vivo.

4-1BB (CD137) is a powerful T-cell costimulatory molecule in the treatment of virus infections and tumors, but recent studies have also uncovered regulatory functions of 4-1BB signaling. Since 4-1BB triggering suppresses autoimmunity by accumulating indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) in an interferon (IFN)-γ-dependent manner, we asked whether similar molecular and cellular changes were induced by 4-1BB triggering in virus-infected mice. 4-1BB triggering increased IFN-γ and IDO, and suppressed CD4(+) T cells, in C57BL/6 mice infected with the type 1 KOS strain of Herpes simplex virus (HSV-1), as it does in an autoimmune disease model. Detailed analysis of the CD4(+) T suppression showed that freshly activated CD62L(high) T cells underwent apoptosis in the early phase of suppression, and CD62L(low) effector/memory T cells in the later phase. Although 4-1BB triggering resulted in similar cellular changes - increased CD8(+) T and decreased CD4(+) T cells, it had different effects on mortality in mice infected with HSV-1 RE, influenza, and Japanese encephalitis virus (JEV); it increased mortality in influenza-infected mice but decreased it in JEV-infected mice. Since the dominant type of immune cell generated to protect the host was different for each virus - CD4(+) T cells and neutrophils in HSV-1 RE infection, both CD4(+) T and CD8(+) T cells in influenza infection, and a crucial role for B cells in JEV infection, 4-1BB triggering resulted in different therapeutic outcomes. We conclude that the therapeutic outcome of 4-1BB triggering is determined by whether the protective immunity generated against the virus was beneficially altered by the 4-1BB triggering.

4-1BB-mediated immunotherapy of rheumatoid arthritis.

Collagen type II-induced arthritis is a CD4(+) T-cell-dependent chronic inflammation in susceptible DBA/1 mice and represents an animal model of human rheumatoid arthritis. We found that development of this condition, and even established disease, are inhibited by an agonistic anti-4-1BB monoclonal antibody. Anti-4-1BB suppressed serum antibodies to collagen type II and CD4(+) T-cell recall responses to collagen type II. Crosslinking of 4-1BB evoked an antigen-specific, active suppression mechanism that differed from the results of blocking the interaction between 4-1BB and its ligand, 4-1BBL. Anti-4-1BB monoclonal antibodies induced massive, antigen-dependent clonal expansion of CD11c(+)CD8(+) T cells and accumulation of indoleamine 2,3-dioxygenase in CD11b(+) monocytes and CD11c(+) dendritic cells. Both anti-interferon-gamma and 1-methyltryptophan, a pharmacological inhibitor of indoleamine 2,3-dioxygenase, reversed the anti-4-1BB effect. We conclude that the suppression of collagen-induced arthritis was caused by an expansion of new CD11c(+)CD8(+) T cells, and that interferon-gamma produced by these cells suppresses antigen-specific CD4(+) T cells through an indoleamine 2,3-dioxygenase-dependent mechanism.

A System for Continuous Pre- to Post-reperfusion Intra-carotid Cold Infusion for Selective Brain Hypothermia in Rodent StrokeModels.

Intra-carotid cold infusion (ICCI) appears as a promising method for hypothermia-mediated brain protection from ischemic stroke. Recent clinical pilot studies indicate easy implementation of ICCI into endovascular acute ischemic stroke treatment. Current rodent ICCI-in-stroke models limit ICCI to the post-reperfusion phase. To establish a method for continuous ICCI over the duration of intra-ischemia to post-reperfusion in rodent stroke models, a novel system was developed. Eighteen male Sprague-Dawley rats were included. Intraluminal filament method was used for transient middle cerebral artery occlusion (MCAO). Normal saline (~ 0 °C) was delivered (≤ 2.0 mL/min) into the internal carotid artery via a customized infusion system without interruption during MCAO (intra-ischemia) to after filament withdrawal (post-reperfusion). Bilateral cortical and striatal temperatures were monitored. Hypothermia goals were a temperature reduction in the ischemic hemisphere by 2 °C prior to reperfusion and thereafter maintenance of regional brain hypothermia at ~ 32 °C limiting the administered ICCI volume to ½ of each rat's total blood volume. During ischemia, maximum brain cooling rate was achieved with ICCI at 0.5 mL/min. It took 2 min to reduce ischemic striatal temperature by 2.3 ± 0.3 °C. After reperfusion, brain cooling was continued at 2 mL/min ICCI first (over 42 s) and maintained at 32.1 ± 0.3 °C at 0.7 mL/min ICCI over a duration of 15 ± 0.8 min. ICCI (total 12.6 ± 0.6 mL) was uninterrupted over the duration of the studied phases. First system that allows continuous ICCI during the phases of intra-ischemia to post-reperfusion in small animals for selective brain cooling and for investigations of other neuroprotective infusions.

Optimisation and validation of an integrated magnetic resonance imaging-only radiotherapy planning solution.

BACKGROUND AND PURPOSE: Magnetic resonance imaging (MRI)-only treatment planning is gaining in popularity in radiation oncology, with various methods available to generate a synthetic computed tomography (sCT) for this purpose. The aim of this study was to validate a sCT generation software for MRI-only radiotherapy planning of male and female pelvic cancers. The secondary aim of this study was to improve dose agreement by applying a derived relative electron and mass density (RED) curve to the sCT. METHOD AND MATERIALS: Computed tomography (CT) and MRI scans of forty patients with pelvic neoplasms were used in the study. Treatment plans were copied from the CT scan to the sCT scan for dose comparison. Dose difference at reference point, 3D gamma comparison and dose volume histogram analysis was used to validate the dose impact of the sCT. The RED values were optimised to improve dose agreement by using a linear plot. RESULTS: The average percentage dose difference at isocentre was 1.2% and the mean 3D gamma comparison with a criteria of 1%/1 mm was 84.0% ± 9.7%. The results indicate an inherent systematic difference in the dosimetry of the sCT plans, deriving from the tissue densities. With the adapted REDmod table, the average percentage dose difference was reduced to -0.1% and the mean 3D gamma analysis improved to 92.9% ± 5.7% at 1%/1 mm. CONCLUSIONS: CT generation software is a viable solution for MRI-only radiotherapy planning. The option makes it relatively easy for departments to implement a MRI-only planning workflow for cancers of male and female pelvic anatomy.

Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA): update and insights into current practices and future directions for research and implementation.

BACKGROUND: In this review, we assess the state of Resuscitative Endovascular Occlusion of the Aorta (REBOA) today with respect to out-of-hospital (OOH) vs. inhospital (H) use in blunt and penetrating trauma, as well as discuss areas of promising research that may be key in further advancement of REBOA applications. METHODS: To analyze the trends in REBOA use, we conducted a review of the literature and identified articles with human or animal data that fit the respective inclusion and exclusion criteria. In separate tables, we compiled data extracted from selected articles in categories including injury type, zone and duration of REBOA, setting in which REBOA was performed, sample size, age, sex and outcome. Based on these tables as well as more detailed review of some key cases of REBOA usage, we assessed the current state of REBOA as well as coagulation and histological disturbances associated with its usage. All statistical tests were 2-sided using an alpha=0.05 for significance. Analysis was done using SAS 9.5 (Cary, NC). Tests for significance was done with a t-test for continuous data and a Chi Square Test for categorical data. RESULTS: In a total of 44 cases performed outside of a hospital in both military and civilian settings, the overall survival was found to be 88.6%, significantly higher than the 50.4% survival calculated from 1,807 cases of REBOA performed within a hospital (p<.0001). We observe from human data a propensity to use Zone I in penetrating trauma and Zone III in blunt injuries. We observe lower final metabolic markers in animal studies with shorter REBOA time and longer follow-up times. CONCLUSIONS: Further research related to human use of REBOA must be focused on earlier initiation of REBOA after injury which may depend on development of rapid vascular access devices and techniques more so than on any new improvements in REBOA. Future animal studies should provide detailed multisystem organ assessment to accurately define organ injury and metabolic burden associated with REBOA application. Overall, animal studies must involve realistic models of injury with severe clinical scenarios approximating human trauma and exsanguination, especially with long-term follow-up after injury.

Mitigating Ischemia-Reperfusion Injury Using a Bilobed Partial REBOA Catheter: Controlled Lower-Body Hypotension.

BACKGROUND: Non-compressible torso hemorrhage (NCTH) is the leading cause of potentially preventable death on the battlefield. Resuscitative endovascular balloon occlusion of the aorta (REBOA) aims to restore central blood pressure and control NCTH below the balloon, but risks ischemia-reperfusion injury to distal organs when prolonged. We tested a bilobed partial REBOA catheter (pREBOA), which permits some of the blood to flow past the balloon. METHODS: Female swine (n = 37, 6 groups, n = 5-8/group), anesthetized and instrumented, were exponentially hemorrhaged 50% of estimated blood volume (all except time controls [TC]). Negative controls (NC) did not receive REBOA or resuscitation. Positive controls (PC) received retransfusion after 120 min. REBOA groups received REBOA for 120 min, then retransfusion. Balloon was fully inflated in the full REBOA group (FR), and was partially inflated in partial REBOA groups (P45 and P60) to achieve a distal systolic blood pressure of 45 mm Hg or 60 mm Hg. RESULTS: Aortic occlusion restored baseline values of proximal mean arterial pressure, cardiac output, and carotid flow in pREBOA groups. Lactate reached high values during occlusion in all REBOA groups (9.9 ±â€Š4.2, 8.0 ±â€Š4.1, and 10.7 ±â€Š2.9 for P45, P60, and FR), but normalized by 6 to 12 h post-deflation in the partial groups. All TC and P60 animals survived 24 h. The NC, PC, and P45 groups survived 18.2 ±â€Š9.5, 19.3 ±â€Š10.6, and 21.0 ±â€Š8.4 h. For FR animals mean survival was 6.2 ±â€Š5.8 h, significantly worse than all other animals (P < 0.01, logrank test). CONCLUSIONS: In this porcine model of hemorrhagic shock, animals undergoing partial REBOA for 120 min survived longer than those undergoing full occlusion.

Role of casein kinase 1 in the glucose sensor-mediated signaling pathway in yeast.

BACKGROUND: In yeast, glucose-dependent degradation of the Mth1 protein, a corepressor of the glucose transporter gene (HXT) repressor Rgt1, is a crucial event enabling expression of several HXT. This event occurs through a signaling pathway that involves the Rgt2 and Snf3 glucose sensors and yeast casein kinase 1 and 2 (Yck1/2). In this study, we examined whether the glucose sensors directly couple with Yck1/2 to convert glucose binding into an intracellular signal that leads to the degradation of Mth1. RESULTS: High levels of glucose induce degradation of Mth1 through the Rgt2/Snf3 glucose signaling pathway. Fluorescence microscopy analysis indicates that, under glucose-limited conditions, GFP-Mth1 is localized in the nucleus and does not shuttle between the nucleus and cytoplasm. If glucose-induced degradation is prevented due to disruption of the Rgt2/Snf3 pathway, GFP-Mth1 accumulates in the nucleus. When engineered to be localized to the cytoplasm, GFP-Mth1 is degraded regardless of the presence of glucose or the glucose sensors. In addition, removal of Grr1 from the nucleus prevents degradation of GFP-Mth1. These results suggest that glucose-induced, glucose sensor-dependent Mth1 degradation occurs in the nucleus. We also show that, like Yck2, Yck1 is localized to the plasma membrane via C-terminal palmitoylation mediated by the palmitoyl transferase Akr1. However, glucose-dependent degradation of Mth1 is not impaired in the absence of Akr1, suggesting that a direct interaction between the glucose sensors and Yck1/2 is not required for Mth1 degradation. CONCLUSION: Glucose-induced, glucose sensor-regulated degradation of Mth1 occurs in the nucleus and does not require direct interaction of the glucose sensors with Yck1/2.

Diagnostic correlates of Alzheimer dementia in a U.S. Nationwide inpatient sample.

INTRODUCTION: Alzheimer dementia (ALZ-D) is among the most frequent diseases in the elderly. Several somatic and psychiatric disorders have been suggested to be related to this diagnosis. The aim of this analysis of a large and representative U.S. nationwide inpatient sample (NIS) was to identify diagnostic correlates of ALZ-D in subjects aged 60 years and older. METHODS: Of the total sample of 800,457 inpatient subjects ( approximately 2% of all inpatients in 2004), 315,244 individuals were 60 years or older. Of these, 9,572 (3.03%) received a diagnosis of ALZ-D, whereas 33,367 (10.59%) were diagnosed with osteoarthritis (OA) and served as a comparison group. Comparisons of potential somatic and psychiatric diagnostic correlates were conducted. RESULTS: As determined by both univariate comparison and multivariate logistic regression analysis, after controlling for age and gender, subjects with ALZ-D (versus OA) had an overall higher rate of diagnoses of diseases of the vascular system (stroke: odds ratio 1.69; 95% confidence interval: 1.25-2.30) and psychotic and affective disorders (bipolar: 2.78 [1.26-6.12]; schizoaffective: 3.06 [2.10-4.47]). Increasing age and male gender were positively associated with the diagnosis of ALZ-D. DISCUSSION: Many somatic diagnoses related to ALZ-D were confirmed by these analyses of the NIS. However, psychotic and affective disorders were identified to be equally significant correlates of ALZ-D, even in the presence of all other disorders. Prospective and longitudinal data are needed to investigate potential causal and temporal relationships between ALZ-D with somatic and psychiatric disorders.

Selective Brain Hypothermia in Acute Ischemic Stroke: Reperfusion Without Reperfusion Injury.

In acute ischemic stroke, early recanalization of the occluded artery is crucial for best outcome to be achieved. Recanalization aims at restoring blood flow to the ischemic tissue (reperfusion) and is achieved with pharmacological thrombolytic drugs, endovascular thrombectomy (EVT) devices, or both. The introduction of modern endovascular devices has led to tremendous anatomical and clinical success with rates of substantial reperfusion exceeding 80% and proven clinical benefit in patients with anterior circulation large vessel occlusions (LVOs). However, not every successful reperfusion procedure leads to the desired clinical outcome. In fact, the rate of non-disabled outcome at 3 months with current EVT treatment is ~1 out of 4. A constraint upon better outcomes is that reperfusion, though resolving ischemic stress, may not restore the anatomic structures and metabolic functions of ischemic tissue to their baseline states. In fact, ischemia triggers a complex cascade of destructive mechanisms that can sometimes be exacerbated rather than alleviated by reperfusion therapy. Such reperfusion injury may cause infarct progression, intracranial hemorrhage, and unfavorable outcome. Therapeutic hypothermia has been shown to have a favorable impact on the molecular elaboration of ischemic injury, but systemic hypothermia is limited by slow speed of attaining target temperatures and clinical complications. A novel approach is endovascular delivery of hypothermia to cool the affected brain tissue selectively and rapidly with tight local temperature control, features not available with systemic hypothermia devices. In this perspective article, we discuss the possible benefits of adjunctive selective endovascular brain hypothermia during interventional stroke treatment.

Cellular energetic status supervises the synthesis of bis-diphosphoinositol tetrakisphosphate independently of AMP-activated protein kinase.

Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP](2)-InsP(4)), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP](2)-InsP(4) synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP](2)-InsP(4) synthesis without affecting other inositol phosphates. In addition, we report that the shortterm increases in [PP](2)-InsP(4) levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP](2)-InsP(4) synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2'-amino-3'-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP](2)-InsP(4) is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks.

Brain cooling maintenance with cooling cap following induction with intracarotid cold saline infusion: a quantitative model.

Intracarotid cold saline infusion (ICSI) is potentially much faster than whole-body cooling and more effective than cooling caps in inducing therapeutic brain cooling. One drawback of ICSI is hemodilution and volume loading. We hypothesized that cooling caps could enhance brain cooling with ICSI and minimize hemodilution and volume loading. Six-hour-long simulations were performed in a 3D mathematical brain model. The Pennes bioheat equation was used to propagate brain temperature. Convective heat transfer through jugular venous return and the circle of Willis was simulated. Hemodilution and volume loading were modeled using a two-compartment saline infusion model. A feedback method of local brain temperature control was developed where ICSI flow rate was varied based on the rate of temperature change and the deviation of temperature to a target (32 degrees C) within a voxel in the treated region of brain. The simulations confirmed the inability of cooling caps alone to induce hypothermia. In the ICSI and the combination models (ICSI and cap), the control algorithm guided ICSI to quickly achieve and maintain the target temperature. The combination model had lower ICSI flow rates than the ICSI model resulting in a 55% reduction of infusion volume over a 6h period and higher hematocrit values compared to the ICSI model. Moreover, in the combination model, the ICSI flow rate decreased to zero after 4h, and hypothermia was subsequently maintained solely by the cooling cap. This is the first study supporting a role of cooling caps in therapeutic hypothermia in adults.