Alcohol dehydrogenase 1 is a tubular mitophagy-dependent apoptosis inhibitor against septic acute kidney injury.

Alcohol dehydrogenase 1 (ADH1) is an alcohol-oxidizing enzyme with poorlydefined biology. Here we report that ADH1 is highly expressed in kidneys of mice with lethal endotoxemia and is transcriptionally upregulated in tubular cells by lipopolysaccharide (LPS) stimuli through TLR4/NF-κB cascade. The Adh1 knockout (Adh1KO) mice with lethal endotoxemia displayed increased susceptibility to acute kidney injury (AKI) but not systemic inflammatory response. Adh1KO mice develop more severe tubular cell apoptosis in comparison to Adh1 wild-type (Adh1WT) mice during course of lethal endotoxemia. ADH1 deficiency facilitates the LPS-induced tubular cell apoptosis in a caspase-dependent manner. Mechanistically, ADH1 deficiency dampens tubular mitophagy that relies on PINK1-Parkin pathway characterized by the reduced membrane potential, reactive oxygen species (ROS) and release of fragmented mtDNA to cytosol. Kidney-specific overexpression of PINK1 and Parkin by adeno-associated viral vector 9 (AAV9) delivery ameliorates AKI exacerbation in Adh1KO mice with lethal endotoxemia. Our study supports the notion that ADH1 is critical for blockade of tubular apoptosis mediated by mitophagy, allowing the rapid identification and targeting of alcohol-metabolic route applicable to septic AKI.

Auto- and paracrine rewiring of NIX-mediated mitophagy by insulin-like growth factor-binding protein 7 in septic AKI escalates inflammation-coupling tubular damage.

AIMS: Inflammation-coupling tubular damage (ICTD) contributes to pathogenesis of septic acute kidney injury (AKI), in which insulin-like growth factor-binding protein 7 (IGFBP-7) serves as a biomarker for risk stratification. The current study aims to discern how IGFBP-7 signalling influences ICTD, the mechanisms that underlie this process and whether blockade of the IGFBP-7-dependent ICTD might have therapeutic value for septic AKI. MATERIALS AND METHODS: In vivo characterization was carried out in B6/JGpt-Igfbp7em1Cd1165/Gpt mice subjected to cecal ligation and puncture (CLP). Transmission electron microscopy, immunofluorescence, flow cytometry, immunoblotting, ELISA, RT-qPCR and dual-luciferase reporter assays were used to determine mitochondrial functions, cell apoptosis, cytokine secretion and gene transcription. KEY FINDINGS: ICTD augments the transcriptional activity and protein secretion of tubular IGFBP-7, which enables an auto- and paracrine signalling via deactivation of IGF-1 receptor (IGF-1R). Genetic knockout (KO) of IGFBP-7 provides renal protection, improves survival and resolves inflammation in murine models of cecal ligation and puncture (CLP), while administering recombinant IGFBP-7 aggravates ICTD and inflammatory invasion. IGFBP-7 perpetuates ICTD in a NIX/BNIP3-indispensable fashion through dampening mitophagy that restricts redox robustness and preserves mitochondrial clearance programs. Adeno-associated viral vector 9 (AAV9)-NIX short hairpin RNA (shRNA) delivery ameliorates the anti-septic AKI phenotypes of IGFBP-7 KO. Activation of BNIP3-mediated mitophagy by mitochonic acid-5 (MA-5) effectively attenuates the IGFBP-7-dependent ICTD and septic AKI in CLP mice. SIGNIFICANCE: Our findings identify IGFBP-7 is an auto- and paracrine manipulator of NIX-mediated mitophagy for ICTD escalation and propose that targeting the IGFBP-7-dependent ICTD represents a novel therapeutic strategy against septic AKI.

Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI.

The immunosuppressive, inflammatory microenvironment orchestrated by neutrophil extracellular traps (NETs) plays a principal role in pathogenesis of sepsis. Fibroblast growth factor-inducible molecule 14 (Fn14) has been established as a potential target for septic acute kidney injury (AKI), making further therapeutic benefits from combined NETs and Fn14 blockade possible. Methods: The concurrence of NETs and Fn14 in mice and patients with septic AKI were assessed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and in silico studies. Survival, histopathological and biochemical analyses of wild-type and PAD4-deficient CMV-Cre; PAD4 fl/fl mice with septic AKI were applied to evaluate the efficacy of either pharmacological or genetic NETs interruption in combination with Fn14 blockade. Molecular mechanisms underlying such effects were determined by CRISPR technology, fluorescence-activated cell sorter analysis (FACS), cycloheximide (CHX) pulse-chase, luciferase reporter and chromatin immunoprecipitation (ChIP) assay. Results: NETs formation is concurred with Fn14 upregulation in murine AKI models of abdominal, endotoxemic, multidrug-resistant sepsis as well as in serum samples of patients with septic AKI. Pharmacological or genetic interruption of NETs formation synergizes with ITEM-2, a monoclonal antibody (mAb) of Fn14, to prolong mice survival and provide renal protection against abdominal sepsis, the effects that could be abrogated by elimination of macrophages. Interrupting NETs formation predominantly perpetuates infiltration and survival of efferocytic growth arrest-specific protein 6+ (GAS6+) macrophages in combination with ITEM-2 therapy and enhances transcription of tubular cell-intrinsic Fn14 in a DNA methyltransferase 3a (DNMT3a)-independent manner through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5) upon abdominal sepsis challenge or LPS stimuli. Pharmacological NETs interruption potentiates the anti-septic AKI efficacy of ITEM-2 in murine models of endotoxemic and multidrug-resistant sepsis. Conclusion: Our preclinical data propose that interrupting NETs formation in combination with Fn14 mAb might be a feasible therapeutic strategy for septic AKI.

Ascorbate uptake enables tubular mitophagy to prevent septic AKI by PINK1-PARK2 axis.

Ascorbate (Vitamin C) has been proposed as a promising therapeutic agent against sepsis in clinical trials, but there is little experimental evidence on its anti-septic efficacy. We report that Toll-like receptor 4 (TLR4) activation by LPS stimuli augments ascorbate uptake in murine and human tubular cells through upregulation of two ascorbate transporters SVCT-1 and -2 mediated by Fn14/SCFFbxw7α cascade. Ascorbate restriction, or knockout of SVCT-1 and -2, the circumstance reminiscent to blockade of ascorbate uptake, endows tubular cells more vulnerable to the LPS-inducible apoptosis, whereas exogenous administration of ascorbate overrides the ruin execution, for which the PINK1-PARK2, rather than BNIP3-NIX axis is required. Ascorbate increases, while SVCT-1 and -2 knockout or ascorbate restriction dampens tubular mitophagy upon LPS stimuli. Treatment of endotoxemic mice with high-dose ascorbate confers mitophagy and substantial protection against mortality and septic acute kidney injury (AKI). Our work provides a rationale for clinical management of septic AKI with high doses of ascorbate.

Redox DAPK1 destabilizes Pellino1 to govern inflammation-coupling tubular damage during septic AKI.

Tubular damage initiated by inflammatory response and ischemic/hypoxic stress is a hallmark of septic acute kidney injury (AKI), albeit the molecular mechanism coupling the two events remains unclear. We investigated the intrinsic nature of tubular damage with respect to inflammatory/hypoxic stress during septic AKI. Methods: The apoptotic response of tubular cells to LPS stimuli was analyzed before and after hypoxia exposure. Cellular ubiquitination, co-immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and CRISPR technology were adopted to determine the molecular mechanism underlying this process. In vivo characterization was performed in wild-type and DAPK1-/- mice models of cecal ligation and puncture (CLP). Results: We found that the MyD88-dependent inflammatory response couples to tubular damage during LPS stimuli under hypoxia in a Fn14/SCFFbxw7α-dispensable manner via recruitment of caspase-8 with TRIF-RIP1 signalosome mediated by DAPK1, which directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and turnover. Either pharmacological deactivation or genetic ablation of DAPK1 makes tubular cells refractory to the LPS-induced damage in the context of hypoxia, while kinase activity of DAPK1 is essential for ruin execution. Targeting DAPK1 effectively protects mice against septic AKI and potentiates the efficacy of a MyD88 homodimerization inhibitor, ST2825. Conclusion: Our findings provide a rationale for the mechanism whereby inflammation intersects with hypoxic tubular damage during septic AKI through a previously unappreciated role of DAPK1-inducible Ser39 phosphorylation in Pellino1 turnover and underscore that combined targeting DAPK1 and MyD88 might be a feasible strategy for septic AKI management.

MicroRNA-19a Targets Fibroblast Growth Factor-Inducible Molecule 14 and Prevents Tubular Damage in Septic AKI.

Fibroblast growth factor-inducible molecule 14 (Fn14) plays a principal role in triggering tubular damage during septic acute kidney injury (AKI). Here, we explore the mechanism underlying Fn14 deregulation in septic AKI. We identify Fn14 as a bona fide target of miR-19a, which directly binds to 3' UTR of Fn14 for repression independent of cylindromatosis (CYLD), the deubiquitinase (DUB) downstream of miR-19a, and thereby antagonizes the LPS-induced tubular cell apoptosis. Genetic ablation of Fn14, but not of CYLD, abolishes the ability of miR-19a to antagonize the tubular apoptosis by lipopolysaccharide (LPS). In mice, systemic delivery of miR-19a confers protection against septic AKI. Our findings implicate that miR-19a may serve as a promising therapeutic candidate in the prevention of septic AKI.

Expert recommendations on blood purification treatment protocol for patients with severe COVID-19.

Coronavirus disease (COVID-19) was first diagnosed in Wuhan in December 2019. The World Health Organization defined the subsequent outbreak of COVID-19 worldwide as a public health emergency of international concern. Epidemiological data indicate that at least 20% of COVID-19 patients have severe disease. In addition to impairment of the respiratory system, acute kidney injury (AKI) is a major complication. Immune damage mediated by cytokine storms and concomitant AKI is a key factor for poor prognosis. Based on previous experience of blood purification for patients with severe acute respiratory syndrome and Middle East respiratory syndrome combined with clinical front-line practice, we developed a blood purification protocol for patients with severe COVID-19. This protocol is divided into four major steps. The first step is to assess whether patients with severe COVID-19 require blood purification. The second step is to prescribe a blood purification treatment for patients with COVID-19. The third step is to monitor and adjust parameters of blood purification. The fourth step is to evaluate the timing of discontinuation of blood purification. It is expected that blood purification will play a key role in effectively reducing the mortality of patients with severe COVID-19 through the standardized implementation of the present protocol.

miR-133b Downregulation Reduces Vulnerable Plaque Formation in Mice with AS through Inhibiting Macrophage Immune Responses.

Atherosclerosis (AS) is a chronic inflammatory disease characterized by accumulating deposition of lipids in the arterial intima. Notably, macrophages participate centrally in the pathogenesis of this deadly disease. In this study, we established AS mouse models in order to investigate the effect of microRNA-133b (miR-133b) on vulnerable plaque formation and vascular remodeling in AS and explore the potential functional mechanisms. The expression of miR-133b was altered or the Notch-signaling pathway was blocked in the AS mouse models in order to evaluate the proliferation, migration, and apoptosis of macrophages. It was observed that miR-133b was upregulated in AS, which might target MAML1 to regulate the Notch-signaling pathway. AS mice with downregulated miR-133b or inhibited Notch-signaling pathway presented with a reduced AS plaque area, a decreased positive rate of macrophages, and an increased positive rate of vascular smooth muscle cells. Moreover, Notch-signaling pathway blockade or miR-133b downregulation inhibited the macrophage viability and migration and accelerated the apoptosis. This study provides evidence that downregulated miR-133b expression may inhibit the immune responses of macrophages and attenuate the vulnerable plaque formation and vascular remodeling in AS mice through the MAML1-mediated Notch-signaling pathway, highlighting miR-133b as a novel therapeutic target for AS.

Regulation of Fn14 stability by SCFFbxw7α during septic acute kidney injury.

Acute kidney injury (AKI) initiated by sepsis remains a thorny problem despite recent advancements in its clinical management. Having been found to be activated during AKI, fibroblast growth factor-inducible molecule 14 (Fn14) may be a potential therapeutic target because of its involvement in the molecular basis of injury. Here, we report that LPS induces apoptosis of mouse cortical tubule cells mediated by Fn14, for which simultaneous Toll-like receptor (TLR)4 activation is required. Mechanistically, TLR4 activation by lipopolysaccharide, through disassociating E3 ligase SCFFbxw7α from Fn14, dismantles Lys48-linked polyubiquitination of Fn14 and stabilizes it. Pharmacological deactivation of Fn14 with monoclonal antibody ITEM-2 provides effective protection against lethal sepsis and AKI in mice. Our study underscores an adaptive mechanism whereby TLR4 regulates SCFFbxw7α-dependent Fn14 stabilization during inflammatory tubular damage and further supports investigation of targeting Fn14 in clinical trials of patients with septic AKI.

Productive transcription of miR-124-3p by RelA and RNA polymerase II directs RIP1 ubiquitination-dependent apoptosis resistance during hypoxia.

The K63-linked ubiquitination of RIP1 coordinates survival/death homeostasis by driving transcription of genes downstream of RelA. Previously, we demonstrated that EGF-dependent RelA transactivation overcomes hypoxia-initiated apoptosis, yet the underlying mechanisms remain mysterious. We report here that UBXN1 deficiency empowers apoptosis resistance against hypoxia through triggering IκBα degradation, for which K63-linked ubiquitination of RIP1 is required. MiR-124-3p is a bona fide inhibitor upstream of UBXN1, thereby antagonizing the hypoxia-initiated apoptosis. UBXN1 repression by miR-124-3p restores the K63-linked ubiquitination of RIP1, IKKß phosphorylation, IκBα-RelA disassembly, RelA nuclear localization and transactivation of EGF gene as well as EGF secretion under hypoxia. Reconstitution of wild-type UBXN1, but not a truncated UBXN1ΔUBA mutant, or pharmacological inhibition of RelA transactivation in miR-124-3p-replete cells compromises the apoptosis-resistant phenotypes of miR-124-3p. Hypoxia transcriptionally downregulates miR-124-3p by disassociating RelA and RNAP II from its promoter. EGFR activation renders the K63-linked ubiquitination of RIP1 and hypoxic tolerance in conjunction with miR-124-3p. Our findings identify a pivotal role of miR-124-3p in ubiquitin conjugation of RIP1 against hypoxic damage and underscore that productive transcription of miR-124-3p by RelA and RNAP II might be a switching mechanism for this process.

Pharmacological Inhibition of PTEN Restores Remote Ischemic Postconditioning Cardioprotection in Hypercholesterolemic Mice: Potential Role of PTEN/AKT/GSK3ß SIGNALS.

Although remote ischemic postconditioning (RIPC) was shown to confer cardioprotection against myocardial ischemia/reperfusion (I/R) injury in normal animals, whether RIPC-induced cardioprotection is altered in the presence of hypercholesterolemia, a comorbidity with acute myocardial infarction (AMI) patients has yet to be determined. Normal or 2% cholesterol chow was fed to male C57BL/6J mice for 12 weeks to induce hypercholesterolemia, then normal or hypercholesterolemic murine hearts were exposed to AMI by coronary artery ligation. RIPC was induced by four episodes of 5 min femoral artery occlusion followed by 5 min reperfusion immediately after myocardial reperfusion in mice. Following I/R, RIPC significantly attenuated postischemic infarct size, hindered cardiomyocyte apoptosis, improved cardiac systolic function, decreased phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression, and further increased Akt and GSK-3ß phosphorylation in non-hypercholesterolemic, but not in hypercholesterolemic mice. Application of the PTEN inhibitor bisperoxovanadium (BpV) (1.0 mg/kg) reduced postischemic infarct size, attenuated cardiomyocyte apoptosis, and improved cardiac dysfunction in normal, but not in hypercholesterolemic mice. Further, increased dose of BpV (2 mg/kg or 10 mg/kg) failed to rescue the detrimental effects of hypercholesterolemia on I/R in mice following I/R. Especially important, we demonstrated that the combination BpV and RIPC exerted marked cardioprotective effects both in normal and hypercholesterolemic mice with I/R, indicating that PTEN inhibition restores RIPC-elicited myocardial protection in the presence of hypercholesterolemia. Our results demonstrated that hypercholesterolemia attenuated RIPC-induced cardioprotection against I/R injury by alteration of PTEN/Akt/GSK3ß signals, and inhibition of PTEN rescued RIPC-induced cardioprotection in the presence of hypercholesterolemia.

Prevalence and phenotypic characterization of carbapenem-resistant Klebsiella pneumoniae strains recovered from sputum and fecal samples of ICU patients in Zhejiang Province, China.

OBJECTIVE: To understand the prevalence and transmission of carbapenem-resistant Klebsiella pneumoniae (CRKP) in ICU patients in Zhejiang Province, China, and determined the genetic and phenotypic characteristics of these CRKP strains. MATERIALS AND METHODS: A total of 202 ICU patients from eight tertiary hospitals were recruited and 55 non-duplicate CRKP strains were collected during July and August in 2017. These strains were subjected to determination of MICs, carriage of carbapenemase genes and tet(A) variants, PFGE, MLST and virulence potential using G. mellonella larvae infection model. RESULTS: A total of 55 CRKP strains were recovered from 42 patients, representing a carriage rate of 20.8%. CRKP strains were recovered from both the intestinal and respiratory tract of 13 patients. Importantly, strains isolated from sputum and fecal samples often displayed identical PFGE profiles, suggesting that CRKP may also colonize the respiratory tract. The most dominant ST type of these CRKP strains was ST11, accounting for 78% (43/55) of the test strains. The majority of CRKP strains were resistant to multiple antibiotics, with the exception of tigecycline and ceftazidime/avibactam. Interestingly, 32 strains were found to harbor the tet(A) variant, which is known to confer reduced tigecycline susceptibility. Assessment of the virulence potential of these CRKP strains by string test showed that results were negative for 53 of the 55 test strains. However, further assessment of virulence potential using a G. mellonella larvae infection model showed that CRKP isolated from sputum consistently exhibited a higher virulence level than strains recovered from fecal samples. CONCLUSION: CRKP is highly prevalent in ICU patients in Zhejiang Province with strains isolated from respiratory exhibiting higher virulence potential than those from GI tract. These data provide essential insight into development of new infection control measures to halt the transmission of CRKP in clinical settings.

Prognostic value of the biomarkers serum amyloid A and nitric oxide in patients with sepsis.

OBJECTIVE: Sepsis is a major cause of mortality among critically ill patients in the intensive care unit (ICU). Alterations in serum amyloid A (SAA) and nitric oxide (NO) levels have been associated with mortality in critically ill patients. In the present study, we investigated the predictive value of SAA and/or NO compared to traditional predictive markers such as C-reactive protein (CRP) and Acute Physiology and Chronic Health Evaluation II (APACHE II) score. METHODS: 100 adult patients with sepsis and 25 without sepsis were enrolled in a prospective, randomized study in our ICU. The APACHE II score was calculated, and their peripheral venous blood SAA, NO and CRP levels were evaluated on days 1, 3, and 7 after sepsis was diagnosed. The patients were sorted based on incidence of septic shock into septic shock (A) and non-septic shock (B) groups. Comparative analyses of altered levels of these indicators between the two groups were performed, and correlations between SAA, NO, and the more traditional APACHE II score were probed. Patients were sorted based on survival status into death (D) and survival (S) groups based on death endpoint within 28 days after admission. RESULTS: We observed that the difference in APACHE II score, SAA and CRP levels were statistically significantly (p < 0.05) between groups A and B on days 1, 3 and 7 post-diagnosis, while inter-group NO level significantly differed (p < 0.05) on days 1 and 3 post-diagnosis, no apparent difference was observed on day 7 post-diagnosis. For groups D and S, SAA, CRP and NO levels significantly differed (p < 0.05) on days 3 and 7 post-diagnosis, with no apparent difference on day 1. APACHE II score was significantly different on day 7 (p < 0.05), however the difference on days 1 and 3 were non-significant. We also demonstrated a positive correlation between APACHE II scores, SAA levels on days 1, 3, and 7, as well as NO levels on days 1 and 3. In addition, for the D and S groups, SAA at all time points, NO on day 3 and CRP on day 7 positively correlated with increased death events. CONCLUSION: The dynamic monitoring of SAA and NO serum levels with APACHE II scores better reflect the severity of sepsis than traditional indicators like CRP and may serve as independent prognosticators of sepsis in critically ill patients, shorten time to diagnosis confirmation and improve therapeutic decision-making.

Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis.

Cell apoptosis is one of the main pathological alterations during oxidative stress (OS) injury. Previously, we corroborated that nuclear factor-κB (NF-κB) transactivation confers apoptosis resistance against OS in mammalian cells, yet the underlying mechanisms remain enigmatic. Here we report that microRNA-19a (miR-19a) transcriptionally regulated by reactive oxygen species (ROS) production and NF-κB deactivation prevents OS-initiated cell apoptosis through cylindromatosis (CYLD) repression. CYLD contributes to OS-initiated cell apoptosis, for which NF-κB deactivation is essential. MiR-19a directly represses CYLD via targeting 3' UTR of CYLD, thereby antagonizing OS-initiated apoptosis. CYLD repression by miR-19a restores the IKKß phosphorylation, RelA disassociation from IκBα, IκBα polyubiquitination and degradation, RelA recruitment at VEGF gene promoter as well as VEGF secretion in the context of OS. Either pharmacological deactivation of NF-κB or genetic upregulation of CYLD compromises the apoptosis-resistant phenotypes of miR-19a. Furthermore, miR-19a is transcriptionally downregulated upon OS in two distinct processes that require ROS production and NF-κB deactivation. VEGF potentiates the ability of miR-19a to activate NF-κB and render apoptosis resistance. Our findings underscore a putative mechanism whereby CYLD repression-mediated and NF-κB transactivation-dependent miR-19a regulatory feedback loop prevents cell apoptosis in response to OS microenvironment.

MicroRNA-130b transcriptionally regulated by histone H3 deacetylation renders Akt ubiquitination and apoptosis resistance to 6-OHDA.

Apoptosis of DA neurons is a contributing cause of disability and death for Parkinson's disease (PD). Akt may become a potential therapeutic target for PD since Akt has been deactivated during DA neuron apoptosis. We previously demonstrated that Akt confers apoptosis resistance against 6-OHDA in DA neuron-like PC12 cells, yet the underlying mechanisms accounted for this are not fully understood. Here we report that microRNA-130b (miR-130b)-dependent and cylindromatosis (CYLD) repression-mediated Akt ubiquitination renders apoptosis resistance of PC12 cells to 6-OHDA, which elicits histone H3 deacetylation-induced transcriptional downregulation of miR-130b vice versa. CYLD deficiency ubiquitinates Akt at Lys63, thereby phosphorylating Akt and antagonizing 6-OHDA-initiated apoptosis. MiR-130b targetedly represses CYLD and increases apoptosis resistance to 6-OHDA. CYLD repression by miR-130b restores Akt ubiquitination and activation, GSK3ß and FoxO3a phosphorylation, FoxO3a removal from Bim promoter as well as Bim downregulation during 6-OHDA administration. CYLD deficiency-mediated Akt activation is instrumental for the apoptosis-resistant phenotypes of miR-130b. In addition, 6-OHDA transcriptionally downregulates miR-130b through recruitment of HDAC3 at the promoter. Furthermore, EPO potentiates the ability of miR-130b to activate Akt and augment apoptosis resistance. Our findings identify the apoptosis-resistant function of miR-130b and suggest that histone H3 deacetylation plays a pivotal role in regulating miR-130b transcription in response to 6-OHDA.

Does training improve diagnostic accuracy and inter-rater agreement in applying the Berlin radiographic definition of acute respiratory distress syndrome? A multicenter prospective study.

BACKGROUND: Poor inter-rater reliability in chest radiograph interpretation has been reported in the context of acute respiratory distress syndrome (ARDS), although not for the Berlin definition of ARDS. We sought to examine the effect of training material on the accuracy and consistency of intensivists' chest radiograph interpretations for ARDS diagnosis. METHODS: We conducted a rater agreement study in which 286 intensivists (residents 41.3%, junior attending physicians 35.3%, and senior attending physician 23.4%) independently reviewed the same 12 chest radiographs developed by the ARDS Definition Task Force ("the panel") before and after training. Radiographic diagnoses by the panel were classified into the consistent (n = 4), equivocal (n = 4), and inconsistent (n = 4) categories and were used as a reference. The 1.5-hour training course attended by all 286 intensivists included introduction of the diagnostic rationale, and a subsequent in-depth discussion to reach consensus for all 12 radiographs. RESULTS: Overall diagnostic accuracy, which was defined as the percentage of chest radiographs that were interpreted correctly, improved but remained poor after training (42.0 ± 14.8% before training vs. 55.3 ± 23.4% after training, p < 0.001). Diagnostic sensitivity and specificity improved after training for all diagnostic categories (p < 0.001), with the exception of specificity for the equivocal category (p = 0.883). Diagnostic accuracy was higher for the consistent category than for the inconsistent and equivocal categories (p < 0.001). Comparisons of pre-training and post-training results revealed that inter-rater agreement was poor and did not improve after training, as assessed by overall agreement (0.450 ± 0.406 vs. 0.461 ± 0.575, p = 0.792), Fleiss's kappa (0.133 ± 0.575 vs. 0.178 ± 0.710, p = 0.405), and intraclass correlation coefficient (ICC; 0.219 vs. 0.276, p = 0.470). CONCLUSIONS: The radiographic diagnostic accuracy and inter-rater agreement were poor when the Berlin radiographic definition was used, and were not significantly improved by the training set of chest radiographs developed by the ARDS Definition Task Force. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov (registration number NCT01704066 ) on 6 October 2012.

Hypercholesterolemia Abrogates Remote Ischemic Preconditioning-Induced Cardioprotection: Role of Reperfusion Injury Salvage Kinase Signals.

Remote ischemic preconditioning (RIPC) is one of the most powerful intrinsic cardioprotective strategies discovered so far and experimental data indicate that comorbidity may interfere with the protection by RIPC. Therefore, we investigate whether RIPC-induced cardioprotection was intact in hypercholesterolemic rat hearts exposed to ischemia reperfusion in vivo. Normal or hypercholesterolemic rat hearts were exposed to 30 min of ischemia and 2 h of reperfusion, with or without RIPC, PI3K inhibitor wortmannin, MEK-ERK1/2 inhibitor PD98059, GSK3ß inhibitor SB216763. Infarct size, apoptosis, MG53, PI3K-p85, p-Akt, p-ERK1/2, p-GSK3ß, and cleaved Caspase-3 were determined. RIPC reduced infarct size, limited cardiomyocyte apoptosis following IR that was blocked by wortmannin but not PD98059. RIPC triggered unique cardioprotective signaling including MG53, phosphorylation of Akt, and glycogen synthase kinase-3ß (GSK3ß) in concert with reduced proapoptotic active caspase-3. In contrast, RIPC failed to reduce myocardial necrosis and apoptosis as well as to increase the phosphorylated Akt and GSK3ß in hypercholestorolemic myocardium. Importantly, we found that inhibition of GSK with SB216763 reduced myocardial infarct size in healthy and hypercholesterolemic hearts, but no additional cardioprotective effect was achieved when combined with RIPC. Our results suggest that acute GSK3ß inhibition may provide a novel therapeutic strategy for hypercholesterolemic patients during acute myocardial infarction, whereas RIPC is less effective due to signaling events that adversely affect GSK3ß.