Identification of alkaline-induced thiolyl-chlorogenic acid conjugates with cysteine and glutathione.

Alkaline reactions of chlorogenic acid (CGA) yield undesirable development of brown or green pigments, limiting the utilization of alkalized CGA-rich foods. Thiols such as cysteine and glutathione mitigate pigment formation through several mechanisms, including redox coupling to reduce CGA quinones, and thiol conjugation, which forms colorless thiolyl-CGA compounds that do not readily participate in color-generating reactions. This work provided evidence of the formation of both aromatic and benzylic thiolyl-CGA conjugate species formed with cysteine and glutathione under alkaline conditions in addition to hydroxylated conjugate species hypothesized to arise from reactions with hydroxyl radicals. Formation of these conjugates proceeds more quickly than CGA dimerization and amine addition reactions mitigating pigment development. Differentiation between aromatic and benzylic conjugates is enabled by characteristic fragmentation of CS bonds. Acyl migration and hydrolysis of the quinic acid moiety of thiolyl-CGA conjugates yielded a variety of isomeric species also identified through untargeted LC-MS methods.

Intravital assessment of precapillary pulmonary arterioles of type 1 diabetic mice shows oxidative damage and increased tone in response to NOS inhibition.

Microvascular dilation, important for peripheral tissue glucose distribution, also modulates alveolar perfusion and is inhibited by loss of bioavailable nitric oxide (NO) in diabetes mellitus (DM). We hypothesized that DM-induced oxidative stress decreases bioavailable NO and pulmonary precapillary arteriolar diameter, causing endothelial injury. We examined subpleural pulmonary arterioles after acute NO synthase (NOS) inhibition with NG-nitro-l-arginine methyl ester (l-NAME) in streptozotocin (STZ)- and saline (CTRL)-treated C57BL/6J mice. Microvascular changes were assessed by intravital microscopy in the right lung of anesthetized mice with open chest and ventilated lungs. Arteriolar tone in pulmonary arterioles (27.2-48.7 µm diameter) increased in CTRL mice (18.0 ± 11% constriction, P = 0.034, n = 5) but decreased in STZ mice (13.6 ± 7.5% dilation, P = 0.009, n = 5) after l-NAME. Lung tissue dihydroethidium (DHE) fluorescence (superoxide), inducible NOS expression, and protein nitrosylation (3-nitrotyrosine) increased in STZ mice and correlated with increased glucose levels (103.8 ± 8.8 mg/dL). Fluorescently labeled fibrinogen administration and fibrinogen immunostaining showed fibrinogen adhesion, indicating endothelial injury in STZ mice. In CTRL mice, vasoconstriction to l-NAME was likely due to the loss of bioavailable NO. Vasodilation in STZ mice may be due to decreased formation of a vasoconstrictor or emergence of a vasodilator. These findings provide novel evidence that DM targets the pulmonary microcirculation and that decreased NO bioavailability and increased precapillary arteriolar tone could potentially lead to ventilation-perfusion abnormalities, exacerbating systemic DM complications.NEW & NOTEWORTHY Diabetes pulmonary and microvascular consequences are well recognized but have not been characterized. We assessed lung microvascular changes in a live anesthetized mouse model of type 1 diabetes, using a novel intravital microscopy technique. Our results show new evidence that a diabetes-induced decrease in lung nitric oxide bioavailability underlies oxidative damage, enhanced platelet activation, and endothelial injury causing pulmonary microvascular dysfunction and altered vasoreactivity. These findings could provide novel strategies to prevent or reverse diabetes systemic consequences.

A consideration of factors affecting palliative oral appliance effectiveness for obstructive sleep apnea: a scoping review.

STUDY OBJECTIVES: This scoping review allows physicians, researchers, and others interested in obstructive sleep apnea to consider effectiveness of oral appliances (OAs). The intent is to improve understanding of OA effectiveness by considering morphologic interaction in patients with obstructive sleep apnea. METHODS: Morphologic and biomechanical criteria for positional alterations of the mandible assessed success rates of OA appliances. Searches of databases (Medline, PubMed, The Cochrane Library, EBSCO) using terms: OA treatment effectiveness and positive and/or negative outcome predictors. Craniofacial predictors of OAs and obstructive sleep apnea biomechanical factors of anatomical traits associated with OA effectiveness were included. Databases searched radiographic cephalometric imaging for morphology/phenotypes and apnea-hypopnea index responses. Articles were excluded if title or abstract was not relevant or a case report. If the analysis did not report mean or standard deviation for apnea-hypoxia index, it was excluded. No language, age, or sex restrictions were applied. RESULTS: Analysis of 135 articles included in searched literature indicated alterations in musculature and pharyngeal airway structure through OA use. These alterations were individually unpredictable with wide variability 61.81% ± 12.29 (apnea-hypoxia index mean ± standard deviation). Morphologic variations as predictors were typically weak and idiosyncratic. Biomechanical factors and wide variations in the metrics of appliance application were unclear, identifying gaps in knowledge and practice of OAs. CONCLUSIONS: An integrated basis to identify morphologic and biomechanical elements of phenotypic expressions of sleep-disordered breathing in the design and application of OAs is needed. Current knowledge is heterogeneous and shows high variability. Identification of subgroups of patients with obstructive sleep apnea responding to OAs is needed.

Acute lung injury and persistent small airway disease in a rabbit model of chlorine inhalation.

Chlorine is a pulmonary toxicant to which humans can be exposed through accidents or intentional releases. Acute effects of chlorine inhalation in humans and animal models have been well characterized, but less is known about persistent effects of acute, high-level chlorine exposures. In particular, animal models that reproduce the long-term effects suggested to occur in humans are lacking. Here, we report the development of a rabbit model in which both acute and persistent effects of chlorine inhalation can be assessed. Male New Zealand White rabbits were exposed to chlorine while the lungs were mechanically ventilated. After chlorine exposure, the rabbits were extubated and were allowed to survive for up to 24h after exposure to 800ppm chlorine for 4min to study acute effects or up to 7days after exposure to 400ppm for 8min to study longer term effects. Acute effects observed 6 or 24h after inhalation of 800ppm chlorine for 4min included hypoxemia, pulmonary edema, airway epithelial injury, inflammation, altered baseline lung mechanics, and airway hyperreactivity to inhaled methacholine. Seven days after recovery from inhalation of 400ppm chlorine for 8min, rabbits exhibited mild hypoxemia, increased area of pressure-volume loops, and airway hyperreactivity. Lung histology 7days after chlorine exposure revealed abnormalities in the small airways, including inflammation and sporadic bronchiolitis obliterans lesions. Immunostaining showed a paucity of club and ciliated cells in the epithelium at these sites. These results suggest that small airway disease may be an important component of persistent respiratory abnormalities that occur following acute chlorine exposure. This non-rodent chlorine exposure model should prove useful for studying persistent effects of acute chlorine exposure and for assessing efficacy of countermeasures for chlorine-induced lung injury.

Diabetic Microvascular Disease and Pulmonary Fibrosis: The Contribution of Platelets and Systemic Inflammation.

Diabetes is strongly associated with systemic inflammation and oxidative stress, but its effect on pulmonary vascular disease and lung function has often been disregarded. Several studies identified restrictive lung disease and fibrotic changes in diabetic patients and in animal models of diabetes. While microvascular dysfunction is a well-known complication of diabetes, the mechanisms leading to diabetes-induced lung injury have largely been disregarded. We described the potential involvement of diabetes-induced platelet-endothelial interactions in perpetuating vascular inflammation and oxidative injury leading to fibrotic changes in the lung. Changes in nitric oxide synthase (NOS) activation and decreased NO bioavailability in the diabetic lung increase platelet activation and vascular injury and may account for platelet hyperreactivity reported in diabetic patients. Additionally, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has been reported to mediate pancreatic islet damage, and is implicated in the onset of diabetes, inflammation and vascular injury. Many growth factors and diabetes-induced agonists act via the JAK/STAT pathway. Other studies reported the contribution of the JAK/STAT pathway to the regulation of the pulmonary fibrotic process but the role of this pathway in the development of diabetic lung fibrosis has not been considered. These observations may open new therapeutic perspectives for modulating multiple pathways to mitigate diabetes onset or its pulmonary consequences.

Exposure to the Functional Bacterial Amyloid Protein Curli Enhances Alpha-Synuclein Aggregation in Aged Fischer 344 Rats and Caenorhabditis elegans.

Misfolded alpha-synuclein (AS) and other neurodegenerative disorder proteins display prion-like transmission of protein aggregation. Factors responsible for the initiation of AS aggregation are unknown. To evaluate the role of amyloid proteins made by the microbiota we exposed aged rats and transgenic C. elegans to E. coli producing the extracellular bacterial amyloid protein curli. Rats exposed to curli-producing bacteria displayed increased neuronal AS deposition in both gut and brain and enhanced microgliosis and astrogliosis compared to rats exposed to either mutant bacteria unable to synthesize curli, or to vehicle alone. Animals exposed to curli producing bacteria also had more expression of TLR2, IL-6 and TNF in the brain than the other two groups. There were no differences among the rat groups in survival, body weight, inflammation in the mouth, retina, kidneys or gut epithelia, and circulating cytokine levels. AS-expressing C. elegans fed on curli-producing bacteria also had enhanced AS aggregation. These results suggest that bacterial amyloid functions as a trigger to initiate AS aggregation through cross-seeding and also primes responses of the innate immune system.

Increased pulmonary arteriolar tone associated with lung oxidative stress and nitric oxide in a mouse model of Alzheimer's disease.

Vascular dysfunction and decreased cerebral blood flow are linked to Alzheimer's disease (AD). Loss of endothelial nitric oxide (NO) and oxidative stress in human cerebrovascular endothelium increase expression of amyloid precursor protein (APP) and enhance production of the Aß peptide, suggesting that loss of endothelial NO contributes to AD pathology. We hypothesize that decreased systemic NO bioavailability in AD may also impact lung microcirculation and induce pulmonary endothelial dysfunction. The acute effect of NO synthase (NOS) inhibition on pulmonary arteriolar tone was assessed in a transgenic mouse model (TgAD) of AD (C57BL/6-Tg(Thy1-APPSwDutIowa)BWevn/Mmjax) and age-matched wild-type controls (C57BL/6J). Arteriolar diameters were measured before and after the administration of the NOS inhibitor, L-NAME Lung superoxide formation (DHE) and formation of nitrotyrosine (3-NT) were assessed as indicators of oxidative stress, inducible NOS (iNOS) and tumor necrosis factor alpha (TNF-α) expression as indicators of inflammation. Administration of L-NAME caused either significant pulmonary arteriolar constriction or no change from baseline tone in wild-type (WT) mice, and significant arteriolar dilation in TgAD mice. DHE, 3-NT, TNF-α, and iNOS expression were higher in TgAD lung tissue, compared to WT mice. These data suggest L-NAME could induce increased pulmonary arteriolar tone in WT mice from loss of bioavailable NO In contrast, NOS inhibition with L-NAME had a vasodilator effect in TgAD mice, potentially caused by decreased reactive nitrogen species formation, while significant oxidative stress and inflammation were present. We conclude that AD may increase pulmonary microvascular tone as a result of loss of bioavailable NO and increased oxidative stress. Our findings suggest that AD may have systemic microvascular implications beyond central neural control mechanisms.

GIANT API: an application programming interface for functional genomics.

GIANT API provides biomedical researchers programmatic access to tissue-specific and global networks in humans and model organisms, and associated tools, which includes functional re-prioritization of existing genome-wide association study (GWAS) data. Using tissue-specific interaction networks, researchers are able to predict relationships between genes specific to a tissue or cell lineage, identify the changing roles of genes across tissues and uncover disease-gene associations. Additionally, GIANT API enables computational tools like NetWAS, which leverages tissue-specific networks for re-prioritization of GWAS results. The web services covered by the API include 144 tissue-specific functional gene networks in human, global functional networks for human and six common model organisms and the NetWAS method. GIANT API conforms to the REST architecture, which makes it stateless, cacheable and highly scalable. It can be used by a diverse range of clients including web browsers, command terminals, programming languages and standalone apps for data analysis and visualization. The API is freely available for use at http://giant-api.princeton.edu.

Microvascular dilation evoked by chemical stimulation of C-fibers in rats.

Activation of pulmonary C-fibers can reflexively decrease heart rate, blood pressure, and peripheral vascular resistance. However, the effects of these afferents on microvascular tone remain incompletely understood. In this study, we examined the effects of these afferents on microvascular tone in a striated muscle vascular bed. The right cremaster muscle in pentobarbital-anesthetized rats with intact circulation and innervation was suspended in a tissue bath, and diameters of small arterioles were measured by intravital video microscopy. Stimulation of pulmonary C-fibers by injecting capsaicin (5 µg/kg) or phenylbiguanide (20 µg/kg) into the right atrium dilated small arterioles and decreased blood pressure and heart rate. The effects persisted when the cervical vagus nerves were cooled to 5 to 7°C (blocking myelinated fibers), but were prevented by cooling to 0°C (blocking C-fibers and myelinated fibers), by cutting the genital femoral nerve (GFN) supplying the cremaster to block the nerve supply to the muscle, or by adding 6-hydroxydopamine to the bathing medium to selectively block sympathetic effects by depleting norepinephrine from adrenergic nerve terminals. Our results show that stimulation of pulmonary C-fibers reflexively dilates small arterioles in striated muscle by a mechanism that could involve withdrawal of sympathetic adrenergic tone. In conclusion, pulmonary C-fibers can exert an inhibitory influence on neural tone of the microcirculation at an important site where microvascular resistance and tissue blood flow are regulated.

Listening to Limericks: a pupillometry investigation of perceivers' expectancy.

What features of a poem make it captivating, and which cognitive mechanisms are sensitive to these features? We addressed these questions experimentally by measuring pupillary responses of 40 participants who listened to a series of Limericks. The Limericks ended with either a semantic, syntactic, rhyme or metric violation. Compared to a control condition without violations, only the rhyme violation condition induced a reliable pupillary response. An anomaly-rating study on the same stimuli showed that all violations were reliably detectable relative to the control condition, but the anomaly induced by rhyme violations was perceived as most severe. Together, our data suggest that rhyme violations in Limericks may induce an emotional response beyond mere anomaly detection.

Aquaporin 4 molecular mimicry and implications for neuromyelitis optica.

Neuromyelitis optica (NMO) is associated with antibodies to aquaporin 4 (AQP4). We hypothesized that antibodies to AQP4 can be triggered by exposure to environmental proteins. We compared human AQP4 to plant and bacterial proteins to investigate the occurrence of significantly similar structures and sequences. High similarity to a known epitope for NMO-IgG, AQP4(207-232), was observed for corn ZmTIP4-1. NMO and non-NMO sera were assessed for reactivity to AQP4(207-232) and the corn peptide. NMO patient serum showed reactivity to both peptides as well as to plant tissue. These findings warrant further investigation into the role of the environment in NMO etiology.

Humans have antibodies against a plant virus: evidence from tobacco mosaic virus.

Tobacco mosaic virus (TMV), a widespread plant pathogen, is found in tobacco (including cigarettes and smokeless tobacco) as well as in many other plants. Plant viruses do not replicate or cause infection in humans or other mammals. This study was done to determine whether exposure to tobacco products induces an immune response to TMV in humans. Using a sandwich ELISA assay, we detected serum anti-TMV antibodies (IgG, IgG1, IgG3, IgG4, IgA, and IgM) in all subjects enrolled in the study (20 healthy smokers, 20 smokeless-tobacco users, and 20 non-smokers). Smokers had a higher level of serum anti-TMV IgG antibodies than non-smokers, while the serum level of anti-TMV IgA from smokeless tobacco users was lower than smokers and non-smokers. Using bioinformatics, we also found that the human protein TOMM40L (an outer mitochondrial membrane 40 homolog--like translocase) contains a strong homology of six contiguous amino acids to the TMV coat protein, and TOMM40L peptide exhibited cross-reactivity with anti-TMV antibodies. People who smoke cigarettes or other tobacco products experience a lower risk of developing Parkinson's disease, but the mechanism by which this occurs is unclear. Our results showing molecular mimicry between TMV and human TOMM40L raise the question as to whether TMV has a potential role in smokers against Parkinson's disease development. The potential mechanisms of molecular mimicry between plant viruses and human disease should be further explored.

Platelet-mediated vascular dysfunction during acute lung injury.

CONTEXT: Platelets have significant roles in initiating and mediating reduced alveolar blood flow, microvascular leak, and ventilation/perfusion mismatch caused by metabolic changes and altered signal transduction caused by ischemia-reperfusion. OBJECTIVE: This review focuses on platelet mechanisms of vascular dysfunction in the lung and presents a hypothesis for interplay between platelet activation, endothelial damage and fibrinogen. The purpose is to discuss current knowledge regarding mechanisms of platelet-mediated endothelial injury and implications for new strategies to treat vascular dysfunction associated with acute lung injury (ALI). METHODS: Literature from a number of fields was searched using Medline and Google Scholar. RESULTS: Activated platelets contribute to redox imbalance through reactive oxygen species production, pro-leak molecules such as PAF and serotonin, and recruitment of inflammatory cytokines and leukocytes to the damaged endothelium. CONCLUSION: Platelets are a critical component of pulmonary ALI, acting in conjunction with fibrinogen to mediate endothelial damage through multiple signal transduction pathways.

Fibrinogen-induced increased pial venular permeability in mice.

Elevated blood level of Fibrinogen (Fg) is commonly associated with vascular dysfunction. We tested the hypothesis that at pathologically high levels, Fg increases cerebrovascular permeability by activating matrix metalloproteinases (MMPs). Fibrinogen (4 mg/mL blood concentration) or equal volume of phosphate-buffered saline (PBS) was infused into male wild-type (WT; C57BL/6J) or MMP-9 gene knockout (MMP9-/-) mice. Pial venular leakage of fluorescein isothiocyanate-bovine serum albumin to Fg or PBS alone and to topically applied histamine (10(-5) mol/L) were assessed. Intravital fluorescence microscopy and image analysis were used to assess cerebrovascular protein leakage. Pial venular macromolecular leakage increased more after Fg infusion than after infusion of PBS in both (WT and MMP9-/-) mice but was more pronounced in WT compared with MMP9-/- mice. Expression of vascular endothelial cadherin (VE-cadherin) was less and plasmalemmal vesicle-associated protein-1 (PV-1) was greater in Fg-infused than in PBS-infused both mice groups. However, in MMP9-/- mice, VE-cadherin expression was greater and PV-1 expression was less than in WT mice. These data indicate that at higher levels, Fg compromises microvascular integrity through activation of MMP-9 and downregulation of VE-cadherin and upregulation of PV-1. Our results suggest that elevated blood level of Fg could have a significant role in cerebrovascular dysfunction and remodeling.

Oncolytic targeting of renal cell carcinoma via encephalomyocarditis virus.

Apoptosis is a fundamental host defence mechanism against invading microbes. Inactivation of NF-kappaB attenuates encephalomyocarditis virus (EMCV) virulence by triggering rapid apoptosis of infected cells, thereby pre-emptively limiting viral replication. Recent evidence has shown that hypoxia-inducible factor (HIF) increases NF-kappaB-mediated anti-apoptotic response in clear-cell renal cell carcinoma (CCRCC) that commonly exhibit hyperactivation of HIF due to the loss of its principal negative regulator, von Hippel-Lindau (VHL) tumour suppressor protein. Here, we show that EMCV challenge induces a strong NF-kappaB-dependent gene expression profile concomitant with a lack of interferon-mediated anti-viral response in VHL-null CCRCC, and that multiple established CCRCC cell lines, as well as early-passage primary CCRCC cultured cells, are acutely susceptible to EMCV replication and virulence. Functional restoration of VHL or molecular suppression of HIF or NF-kappaB dramatically reverses CCRCC cellular susceptibility to EMCV-induced killing. Notably, intratumoural EMCV treatment of CCRCC in a murine xenograft model rapidly regresses tumour growth. These findings provide compelling pre-clinical evidence for the usage of EMCV in the treatment of CCRCC and potentially other tumours with elevated HIF/NF-kappaB-survival signature.

Mitochondrial dysfunction may explain the cardiomyopathy of chronic iron overload.

In patients with hemochromatosis, cardiac dysfunction may appear years after they have reached a state of iron overload. We hypothesized that cumulative iron-catalyzed oxidant damage to mitochondrial DNA (mtDNA) might explain the cardiomyopathy of chronic iron overload. Mice were given repetitive injections of iron dextran for a total of 4weeks after which the iron-loaded mice had elevated cardiac iron, modest cardiac hypertrophy, and cardiac dysfunction. qPCR amplification of near-full-length ( approximately 16kb) mtDNA revealed >50% loss of full-length product, whereas amounts of a qPCR product of a nuclear gene (13kb region of beta globin) were unaffected. Quantitative rtPCR analyses revealed 60-70% loss of mRNA for proteins encoded by mtDNA with no change in mRNA abundance for nuclear-encoded respiratory subunits. These changes coincided with proportionate reductions in complex I and IV activities and decreased respiration of isolated cardiac mitochondria. We conclude that chronic iron overload leads to cumulative iron-mediated damage to mtDNA and impaired synthesis of mitochondrial respiratory chain subunits. The resulting respiratory dysfunction may explain the slow development of cardiomyopathy in chronic iron overload and similar accumulation of damage to mtDNA may also explain the mitochondrial dysfunction observed in slowly progressing diseases such as neurodegenerative disorders.

Suppression of hypoxia-inducible factor 2alpha restores p53 activity via Hdm2 and reverses chemoresistance of renal carcinoma cells.

p53 mutations are rarely detected in clear cell renal cell carcinoma (CCRCC), but, paradoxically, these tumors remain highly resistant to chemotherapy and death receptor-induced death. Here, we show that the accumulation of hypoxia-inducible factor 2alpha (HIF2alpha), a critical oncogenic event in CCRCC following the loss of von Hippel-Lindau (VHL) tumor suppressor protein, leads to Hdm2-mediated suppression of p53. Primary CCRCC specimens exhibiting strong hypoxic signatures show increased levels of activated nuclear phospho-Hdm2(Ser(166)), which is concomitant with low p53 expression. The abrogation of Hdm2-p53 interaction using the small-molecule Hdm2 inhibitor nutlin-3 or the downregulation of HIF2alpha via HIF2alpha-specific short hairpin RNA or wild-type VHL reconstitution restores p53 function and reverses the resistance of CCRCC cells to Fas-mediated and chemotherapy-induced cell death. These findings unveil a mechanistic link between HIF2alpha and p53 and provide a rationale for combining Hdm2 antagonists with chemotherapy for the treatment of CCRCC.