Dexamethasone is a synthetic glucocorticoid with potent anti-inflammatory properties. However, its administration causes significant side effects, specially in long-term therapy. A new approach for ...limiting adverse effects consists in the slow and constant deliver of this drug, using dexamethasone-21-phosphate-loaded erythrocytes (RBC) as circulating bioreactors converting the non-diffusible dexamethasone-21-phosphate into the diffusible dexamethasone. In order to evaluate the real possibility to use this new method of administration, a simple, cheap and rapid assay was set to manage a large number of samples originating from clinical studies. Due to the sample complexity and analite polarity, electrospray mass spectrometry (MS) is the most powerful technique to achieve qualitative and quantitative data. In order to overcome the complex, time-consuming and expensive LC–MS/MS methods reported in the literature in the present work a standard fluxes HPLC-ESI-MS method was set up for quantitative evaluation of dexamethasone. Thanks to the extraction ion chromatogram (XIC) feature of the software, it was possible to obtain sharp profiles for dexamethasone (DXM) and for the employed internal standard (IS) flumethasone (FM), in spite of the extremely complicated chromatogram obtained after HPLC separation of acetonitrile extracted plasma sample, thus avoiding the use of the expensive deuterated internal standard. This enabled us to obtain a linear response curve, allowing the quantification of DXM from blood samples at the picomoles level.
ObjectivesTo analyse the prevalence of CECR1 mutations in patients diagnosed with early onset livedo reticularis and/or haemorrhagic/ischaemic strokes in the context of inflammation or polyarteritis ...nodosa (PAN). Forty-eight patients from 43 families were included in the study.MethodsDirect sequencing of CECR1 was performed by Sanger analysis. Adenosine deaminase 2 (ADA2) enzymatic activity was analysed in monocyte isolated from patients and healthy controls incubated with adenosine and with or without an ADA1 inhibitor.ResultsBiallelic homozygous or compound heterozygous CECR1 mutations were detected in 15/48 patients. A heterozygous disease-associated mutation (p.G47V) was observed in two affected brothers. The mean age of onset of the genetically positive patients was 24 months (6 months to 7 years). Ten patients displayed one or more cerebral strokes during their disease course. Low immunoglobulin levels were detected in six patients. Thalidomide and anti-TNF (tumour necrosis factor) blockers were the most effective drugs. Patients without CECR1 mutations had a later age at disease onset, a lower prevalence of neurological and skin manifestations; one of these patients displayed all the clinical features of adenosine deaminase 2deficiency (DADA2) and a defective enzymatic activity suggesting the presence of a missed mutation or a synthesis defect.ConclusionsDADA2 accounts for paediatric patients diagnosed with PAN-like disease and strokes and might explain an unrecognised condition in patients followed by adult rheumatologist. Timely diagnosis and treatment with anti-TNF agents are crucial for the prevention of severe complications of the disease. Functional assay to measure ADA2 activity should complement genetic testing in patients with non-confirming genotypes.
The folded almanac Western MS.8932 (Wellcome Collection, London), produced in England between 1387 and 1405, is a calendar with astrological tables and diagrams used by medical practitioners to ...harness astrological information relating to health. Apart from the great interest for its use in medicine, this object has a unique feature in its exquisite, unique embroidered binding that indicates a prestigious artefact. Considering the scarceness of information on such items, a diagnostic study on the materials used for the outstanding embroidery of MS.8932 is a unique occasion of having information on the methods of production of medieval embroideries. A diagnostic campaign has been carried out to identify the dyes used for this task and the colourants used for the illuminations, and to evaluate them with reference to their commercial value. Preliminary information on the dyes was yielded by means of UV–visible diffuse reflectance spectrophotometry with fibre optics (FORS) and spectrofluorimetry with fibre optics (FOMF). Then a final identification was yielded by micro-invasive analysis involving Surface Enhanced Raman Spectroscopy (SERS) and High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry (HPLC-DAD-MS) 1. Analysis on the micro samples (5–10 mm of very thin threads) taken from the embroidery revealed the presence of orchil for pink hues, indigo/madder double dyeing for purple hues, red safflower for salmon pink hues and indigo/weld double dyeing for green hues. Particularly remarkable was the additional identification of aloe in the purple threads, possibly used as mordant and/or as antibacterial agent, which evidence is the first ever recorded in such materials. This information is very important to understand the significance of the artefact and its historical-artistic location, and will allow to make comparison with similar artworks.
∙The folded almanac Western MS.8932 is famous for its outstanding embroidery∙Orchil, indigo/madder, red safflower and indigo/weld were identified as dyes used for the embroidery∙Aloe was identified, perhaps used as an antiseptic agent∙The palette used for the illuminations, typical of 15th-century Northern Europe, was characterised.
Ephedra foeminea is a member of the Ephedraceae family which is widespread in the eastern Mediterranean area. In Lebanon, Ephedra is a popular remedy in traditional medicine to prevent and/or ...counteract many stress oxidative-related diseases like inflammation and bacterial infections.
Oxidative stress leads to endothelial cell dysfunction, and is a major factor contributing to etiology of atherosclerosis and related diseases. This study aims to investigate the antioxidant and cytoprotective potential of extracts from E. foeminea fruits on human endothelial cells exposed to hydrogen peroxide (H2O2) to mimic in vitro vascular endothelium dysfunction.
Different extracts of E. foeminea fruits were prepared using pure ethanol (EE), methanol/water (EMW), pure hexane (Ehex) or ethyl acetate/water (Epoly) as extraction solvents. The phenolome profile of each extract was characterized using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Total phenolic and flavonoid content, and radical scavenging properties of the extracts were assessed spectrophotometrically. Then, the effects on human endothelial cells HECV were evaluated.
Epoly extract showed the highest phenol and flavonoid content, and the highest radical scavenging capacity. On H2O2-insulted HECV cells Epoly was able: (i) to counteract the ROS/RNS production and lipid peroxidation; (ii) to rescue the ROS-dependent decrease in the mitochondrial membrane potential; (iii) to counteract the apoptosis induction; (iv) to restore endothelial cell viability and migration.
The findings indicated that the polyphenol-enriched extract Epoly from E. foeminea fruits is endowed with in vitro anti-oxidant and anti-apoptotic effects and might be used as nutraceutical for treating ROS-related endothelium dysfunction and inflammation.
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•Fruits from Ephedra foeminea, a popular remedy in Lebanon, are rich in polyphenols, especially flavonoids.•Different extracts of E. foeminea fruits were prepared and their phenolic composition was characterized by HPLC-MS/MS.•Polyphenolic extract showed the highest radical scavenging capacity and protective effect on H2O2-insulted endothelial cells.
There is a growing interest in using brown algal extracts thanks to the bioactive substances they produce for adaptation to the marine benthic environment. We evaluated the anti-aging and ...photoprotective properties of two types of extracts (50%-ethanol and DMSO) obtained from different portions, i.e., apices and thalli, of the brown seaweed, Ericaria amentacea. The apices of this alga, which grow and develop reproductive structures during summer when solar radiation is at its peak, were postulated to be rich in antioxidant compounds. We determined the chemical composition and pharmacological effects of their extracts and compared them to the thallus-derived extracts. All the extracts contained polyphenols, flavonoids and antioxidants and showed significant biological activities. The hydroalcoholic apices extracts demonstrated the highest pharmacological potential, likely due to the higher content of meroditerpene molecular species. They blocked toxicity in UV-exposed HaCaT keratinocytes and L929 fibroblasts and abated the oxidative stress and the production of pro-inflammatory cytokines, typically released after sunburns. Furthermore, the extracts showed anti-tyrosinase and anti-hydrolytic skin enzyme activity, counteracting the collagenase and hyaluronidase degrading activities and possibly slowing down the formation of uneven pigmentation and wrinkles in aging skin. In conclusion, the E. amentacea apices derivatives constitute ideal components for counteracting sunburn symptoms and for cosmetic anti-aging lotions.
ADP-ribosyl cyclases are ubiquitous enzymes responsible for synthesis from NAD+of the intracellular calcium-releasing signal molecules cyclic ADP-ribose (cADPR) and nicotinic acid adenine ...dinucleotide phosphate ($NAADP^+$). Here, we show that cyclases from lower and higher Metazoa also synthesize three adenylic dinucleotides from cADPR and adenine: diadenosine diphosphate and two isomers thereof. These dinucleotides are present and metabolized in mammalian cells and affect intracellular calcium and cell proliferation. The diadenosine diphosphate isomers are naturally occurring nucleotides containing an N-glycosidic bond different from the usual C1'-N9. The identification of these members of the family of$NAD^+-derived$, calcium-active nucleotides opens new areas of investigation into their functional cooperation with cADPR and$NAADP^+$and into their involvement in the physiology and pathology of calcium-controlled cell functions.
Hepatic steatosis is the hallmark of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome and insulin resistance with potential evolution towards ...non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. Key roles of autophagy and oxidative stress in hepatic lipid accumulation and NAFLD progression are recognized. Here, we employed a rat hepatoma cell model of NAFLD progression made of FaO cells exposed to oleate/palmitate followed or not by TNFα treatment to investigate the molecular mechanisms through which silybin, a lipid-lowering nutraceutical, may improve hepatic lipid dyshomeostasis. The beneficial effect of silybin was found to involve amelioration of the fatty acids profile of lipid droplets, stimulation of the mitochondrial oxidation and upregulation of a microRNA of pivotal relevance in hepatic fat metabolism, miR-122. Silybin was also found to restore the levels of Aquaporin-9 (AQP9) and glycerol permeability while reducing the activation of the oxidative stress-dependent transcription factor NF-κB, and autophagy turnover. In conclusion, silybin was shown to have molecular effects on signaling pathways that were previously unknown and potentially protect the hepatocyte. These actions intersect TG metabolism, fat-induced autophagy and AQP9-mediated glycerol transport in hepatocytes.
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•Silybin ameliorates the profile of FA stored in LD in steatotic hepatocytes.•Silybin increases VLCAD and UCP2 and COX activity.•Silybin increases miR-122, a master regulator of hepatic lipid metabolism.•NAFLD amelioration by silybin involves hepatocyte AQP9 and glycerol uptake.•Silybin diminishes fat-stimulated autophagy.
The interactions of Vibrio aestuarianus 01/032 with haemolymph of the bivalves Mytilus galloprovincialis and Crassostrea gigas were investigated to understand if haemolymph components (haemocytes and ...soluble factors) could be involved in the higher resistance to microbial infection shown by mussels in comparison with oysters. Although 01/032 bacteria adhered to haemocytes of both bivalves, they were sensitive to the bactericidal activity of whole haemolymph from mussel, but not from oyster; in addition, adhesion to mussel (but not oyster) haemocytes was affected by D‐mannose. Mussel serum opsonins directed towards D‐mannose‐binding bacterial ligands were purified by affinity chromatography and were shown to mediate 01/032 interactions with M. galloprovincialis haemocytes. Nano‐High Performance Liquid Chromatography‐Electrospray Ionization‐Tandem Mass Spectrometry (HPLC‐ESI‐MS/MS) analysis showed that the purified opsonin matched the protein precursor of Mytilus edulis extrapallial protein (EP). In the presence of M. galloprovincialis EP protein (MgEP), C. gigas haemocytes killed V. aestuarianus 01/032 almost as efficiently as mussel phagocytes. These findings suggest that the different sensitivity of 01/032 strain to the antibacterial activity of oyster and mussel haemolymph might partly depend on the fact that C. gigas serum lacks MgEP‐like opsonins. These results represent the basis for understanding the different sensitivity to microbial infections shown by the two bivalve species.
At least three structural proteins in Paramecium bursaria Chlorella virus (PBCV-1) are glycosylated, including the major capsid protein Vp54. However, unlike other glycoprotein-containing viruses ...that use host-encoded enzymes in the endoplasmic reticulum-Golgi to glycosylate their proteins, PBCV-1 encodes at least many, if not all, of the glycosyltransferases used to glycosylate its structural proteins. As described here, PBCV-1 also encodes two open reading frames that resemble bacterial and mammalian enzymes involved in de novo GDP-L-fucose biosynthesis. This pathway, starting from GDP-D-mannose, consists of two sequential steps catalyzed by GDP-D-mannose 4,6 dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, respectively. The two PBCV-1-encoded genes were expressed in Escherichia coli, and the recombinant proteins had the predicted enzyme activity. However, in addition to the dehydratase activity, PBCV-1 GMD also had a reductase activity, producing GDP-D-rhamnose. In vivo studies established that PBCV-1 GMD and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are expressed after virus infection and that both GDP-L-fucose and GDP-D-rhamnose are produced in virus-infected cells. Thus, PBCV-1 is the first virus known to encode enzymes involved in nucleotide sugar metabolism. Because fucose and rhamnose are components of the glycans attached to Vp54, the pathway could circumvent a limited supply of GDP sugars by the algal host.
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