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Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.

Nitroxyl (HNO/NO(-)), the reduced form of nitric oxide, has gained attention based on its separate chemistry and biology from nitric oxide. The inherent reactivity of HNO requires new and mechanistically unique donors for the detailed study of HNO chemistry and biology. Oxidation of cyclohexanone oxime with lead tetraacetate yields 1-nitrosocyclohexyl acetate, whereas oxidation of oximes in the presence of excess carboxylic acid gives various acyloxy nitroso compounds. These bright blue compounds exist as monomers as indicated by their infrared, proton, and carbon NMR spectra, and X-ray crystallographic analysis reveals the nitroso groups possess a "nitroxyl-like" bent configuration. Hydrolysis of these compounds produces nitrous oxide, the dimerization and dehydration product of HNO, and provides evidence for the intermediacy of HNO. Both thiols and oxidative metal complexes inhibit nitrous oxide formation. Hydrolysis of these compounds in the presence of ferric heme complexes forms ferrous nitrosyl complexes providing further evidence for the intermediacy of HNO. Kinetic analysis shows that the rate of hydrolysis depends on pH and the structure of the acyl group of the acyloxy nitroso compound. These compounds relax pre-constricted rat aortic rings similar to known HNO donors. Together, these results identify acyloxy nitroso compounds as a new class of HNO donors.

Introductory ParagraphThe coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this O 2 sensing/NO bioactivity linkage postulates an essential role for the conserved hemoglobin β93Cys residue and, specifically, for S-nitrosation of β93Cys to form S-nitrosohemoglobin (SNO-Hb) 1 . The SNO-Hb hypothesis, which conceptually linked hemoglobin and NO biology, has been debated intensely in recent years 2,3 . This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe novel mouse models that express exclusively either human wild type hemoglobin or human hemoglobin in which the β93cys residue is replaced with alanine to assess the role of SNO-Hb in red cell mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red cell S-nitrosothiol levels but shifted S-nitrosothiol distribution to lower MWt species, consistent with the loss of SNO-Hb. Loss of β93cys resulted in no deficits in systemic nor pulmonary hemodynamics under basal conditions and, importantly, did not affect isolated red cell dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO-bioactivity in vivo. *Co corresponding Authors: Rakesh P Patel, PhD, Department of Pathology, University of Alabama at Birmingham, 901 19 th street south, BMR 2, room 302, Birmingham, AL 35294, E mail: E-mail: rakeshp@uab.edu. Tim M Townes, PhD, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Kaul Human Genetics Bldg, room 502, 720 20 th street south, Birmingham, AL 35294, E mail: E-mail: ttownes@uab.edu. # these authors contributed equally to this work Author Contributions TSI, CWS, LCW, XT, DAV and KMP were responsible for performing experiments. TSI, CWS, DAV, RPP and TMT were responsible for planning all experiments, analyzing data and writing manuscript. MBR contributed to mass spectrometry assays, LS was responsible for exercise related studies, CGK and BGB for capillary density measurements, NP and JW contributed to blood pressure measurements and NA for assessment of pulmonary hemodynamics. JR did the ES cell injections to generate the chimeras. In addition to hemoglobin oxygen affinity, blood flow is a key component of the processes that match oxygen delivery to demand. Increased blood flow in response to hypoxia is a critical physiological response which does not correlate with dissolved oxygen tensions but does correlate with hemoglobin oxygen fractional saturation 4 . These observations have led to the concept that the red blood cell (RBC) itself is a regulator of flow and to the general paradigm that RBC/hemoglobin deoxygenation is coupled to the stimulation of vasodilation 1,5,6 . Three mechanisms for this coupling have been proposed (...

Breast cancer is a major public health problem worldwide in women and current therapeutic strategies are not adequately effective for this deadly disease. We have previously shown the anti-proliferative activity of bitter melon extract (BME) in breast cancer cells. In this study, we observed that BME treatment induces autophagosome-bound Long chain 3 (LC3)-B and accumulates protein p62/SQSTM1 (p62) in breast cancer cells. Additionally, we observed that BME treatment in breast cancer cells increases phospho-AMPK expression and inhibits the mTOR/Akt signaling pathway. Subsequently, we demonstrated that BME feeding effectively inhibited breast cancer growth in syngeneic and xenograft mouse models. Further, we observed the increased p62 accumulation, induction of autophagy and apoptotic cell death in tumors from BME-fed animals. Taken together, our results demonstrate that BME treatment inhibits breast tumor growth, and this anti-tumor activity in breast cancer is, in part, mediated by induction of autophagy and modulation of the AMPK/mTOR pathway. The antitumor activity of BME by oral feeding in breast cancer models suggested the high potential for a clinical application.

Dietary supplements such as grape seed extract (GSE) enriched in proanthocyanidins (PA) (oligomeric polyphenols) have been suggested to have multiple health benefits, due to antioxidant and other beneficial activities of the PA. However, a systematic analysis of the molecular basis of these benefits has not been demonstrated. Because the brain is vulnerable to age-related oxidative damage and other insults including inflammation, it was hypothesized that rats ingesting GSE would experience changes in expression or modifications of specific brain proteins that might protect against pathologic events. Normal adult female rats were fed diets supplemented with 5% GSE for 6 weeks. Proteomics analysis (2D electrophoresis and mass spectrometry) of brain homogenates from these animals identified 13 proteins that were altered in amount and/or charge. Because many of these changes were quantitatively in the opposite direction from previous findings for the same proteins in either Alzheimer disease or mouse models of neurodegeneration, the data suggest that these identified proteins may mediate the neuroprotective actions of GSE. This is the first identification and quantitation of specific proteins in mammalian tissues modulated by a dietary supplement, as well as the first to demonstrate links of such proteins with any disease.

Sepsis is a major cause of mortality among hospitalized patients worldwide. Shorter time to administration of broad-spectrum antibiotics is associated with improved outcomes, but early recognition of sepsis remains a major challenge. In a two-center cohort study with prospective sample collection from 1400 adult patients in emergency departments suspected of sepsis, we sought to determine the diagnostic and prognostic capabilities of a machine-learning algorithm based on clinical data and a set of uncommonly measured biomarkers. Specifically, we demonstrate that a machine-learning model developed using this dataset outputs a score with not only diagnostic capability but also prognostic power with respect to hospital length of stay (LOS), thirty-day mortality, and thirty-day inpatient readmission both in our entire testing cohort and various subpopulations. The area under the Receiver Operating Curve (AUROC) for diagnosis of sepsis was 0.83. Predicted risk scores for patients with septic shock were higher compared to patients with sepsis but without shock (p < 0.0001). Scores for patients with infection and organ dysfunction were higher compared to those without either condition (p < 0.0001). Stratification based on predicted scores of the patients into low, medium and high-risk groups showed significant differences in length of stay (p < 0.0001), thirty-day mortality (p < 0.0001), and thirty-day inpatient readmission (p < 0.0001). In conclusion, a machine-learning algorithm based on EMR data and three non-routinely measured biomarkers demonstrated good diagnostic and prognostic capability at the time of initial blood culture.