Role of Autophagy in Haematococcus lacustris Cell Growth under Salinity

The microalga Haematococcus lacustris (formerly H. pluvialis) is able to accumulate high amounts of the carotenoid astaxanthin in the course of adaptation to stresses like salinity. Technologies aimed at production of natural astaxanthin for commercial purposes often involve salinity stress; however, after a switch to stressful conditions, H. lacustris experiences massive cell death which negatively influences astaxanthin yield. This study addressed the possibility to improve cell survival in H. lacustris subjected to salinity via manipulation of the levels of autophagy using AZD8055, a known inhibitor of TOR kinase previously shown to accelerate autophagy in several microalgae. Addition of NaCl in concentrations of 0.2% or 0.8% to the growth medium induced formation of autophagosomes in H. lacustris, while simultaneous addition of AZD8055 up to a final concentration of 0.2 µM further stimulated this process. AZD8055 significantly improved the yield of H. lacustris cells after 5 days of exposure to 0.2% NaCl. Strikingly, this occurred by acceleration of cell growth, and not by acceleration of aplanospore formation. The level of astaxanthin synthesis was not affected by AZD8055. However, cytological data suggested a role of autophagosomes, lysosomes and Golgi cisternae in cell remodeling during high salt stress.

Optimization of pineapple juice amount used as a negative oral contrast agent in magnetic resonance cholangiopancreatography

To evaluate the potential variability of Manganese (Mn2+) in commercial pineapple juice (PJ) produced in different years and to identify the optimal Mn2+ concentration in the correct amount of PJ to be administered prior to Magnetic Resonance Cholangiopancreatography (MRCP) in order to suppress the gastroduodenal (GD) liquid signal. The Mn2+ concentration in PJ produced in different years was defined using Atomic Absorption Spectrometry. The optimal Mn2+ concentration and the amount of PJ, were estimated in an in-vitro analysis, and were then prospectively tested in a population of patients who underwent MRCP. The results were compared with those achieved with the previous standard amount of PJ used in a similar population. The concentrations of Mn2+ in commercial PJ produced in different years did not differ. A total amount of 150 ml (one glass) of PJ having a high Mn2+ content (2.37 mg/dl) was sufficient for the suppression of the GD liquid signal, despite the additional dilution caused by GD liquids since it led to a final concentration of Mn2+ of 0.5–1.00 mg/dl. The optimized single-dose oral administration of 150 ml (approximately one glass) of PJ having a high Mn2+ concentration prior to MRCP was adequate to guarantee the correct amount of Mn2+ to suppress the GD signal.

Function of oxygen transport in the blood of patients in a state of alcohol deprivation in blood in vitro incubation with ethanol solution

Objective. To estimate the in vitro effect of ethanol on the function of oxygen transport in the blood of patients in a state of alcohol deprivation and in healthy donors.Materials and methods. We analyzed blood samples from 13 male patients aged 22–56 in a state of alcohol deprivation and 11 males aged 20-45 in whom this diagnosis was excluded. The blood was incubated with ethanol solution at a final concentration of 50 μmol/L. The parameters of the function of oxygen transport (р50, рО2, рСО2, рН, АВЕ, НСО3-, ТСО2,SВЕ, SBC) of the blood taken from the cubital vein using an ABL-330 “Radiometr” microgas analyzer were determined.Results. The patients in a state of alcohol deprivation reveal increased affinity of hemoglobin for oxygen, and their acid-base status of the blood becomes alkaline. When ethanol is added in vitro to the blood of patients, the affinity of hemoglobin for oxygen decreases.Conclusion. Decreased affinity of hemoglobin for oxygen upon the in vitro addition of ethanol in the examined patients indicates compensatory and adaptive changes in the oxygen transport of the blood in response to chronic ethanol consumption.

Oenothein B in Eucalyptus Leaf Extract Suppresses Fructose Absorption in Caco-2 Cells

Inhibition of fructose absorption may suppress adiposity and adiposity-related diseases caused by fructose ingestion. Eucalyptus leaf extract (ELE) inhibits intestinal fructose absorption (but not glucose absorption); however, its active compound has not yet been identified. Therefore, we evaluated the inhibitory activity of ELE obtained from Eucalyptus globulus using an intestinal fructose permeation assay with the human intestinal epithelial cell line Caco-2. The luminal sides of a cell monolayer model cultured on membrane filters were exposed to fructose with or without the ELE. Cellular fructose permeation was evaluated by measuring the fructose concentration in the medium on the basolateral side. ELE inhibited 65% of fructose absorption at a final concentration of 1 mg/mL. Oenothein B isolated from the ELE strongly inhibited fructose absorption; the inhibition rate was 63% at a final concentration of 5 μg/mL. Oenothein B did not affect glucose absorption. In contrast, the other major constituents (i.e., gallic acid and ellagic acid) showed little fructose-inhibitory activity. To our knowledge, this is the first report that oenothein B in ELE strongly inhibits fructose absorption in vitro. ELE containing oenothein B can prevent and ameliorate obesity and other diseases caused by dietary fructose consumption.

The Application of Ultrasound and Enzymes Could Be Promising Tools for Recovering Polyphenols during the Aging on Lees Process in Red Winemaking

The final concentration of phenolic compounds in wines is usually lower than what might be expected, given their concentration in grapes. This is in part due to the interactions between cell walls from grapes and yeast with phenolics during red winemaking. Most of these aggregates are insoluble and end up precipitating, forming part of the lees. The objective of this study is to determine the capacity of ultrasounds and/or enzymes treatments (β-glucanase and a pectolytic enzyme) to release the anthocyanins and tannins adsorbed in the lees. The ultrasound (US) applied for 120 min slightly favored the extraction of anthocyanins and doubled tannin extraction. Shorter sonication times did not show any positive effect. The combination of β-glucanase and pectolytic enzyme was always more effective in the liberation of anthocyanins (both no-acylated and acylated anthocyanins) and tannins than the enzymes acting separately. The combination of US (120 min), β-glucanase and pectolytic enzyme showed an additive effect, increasing the extraction of phenolic compounds with respect to the individual treatments and also releasing a large quantity of low molecular weight polysaccharides, compounds of enological importance. These results of this study could be of enological interest, facilitating and accelerating the aging on lees process, through the liberation of polysaccharides and the recovery of the phenolic compounds lost during vinification.

Determination of Terpenoid Profile in Dry Cannabis Flowers and Extracts Obtained from Different Cannabis Varietes

Aim: The aim of this study was to determine the terpenoid profile in dried cannabis flowers obtained from different varieties of cannabis plant and in cannabis extracts in order to investigate quantity of terpenes lost during extraction and purification process. Methods: GC/MS method for determination of terpenes was verified. The concentration of terpenes was determined in dry flowers as raw material and in decarboxylated and distillated cannabis extracts, using the same GC/MS analytical method. The extraction was performed using 96% ethanol as a solvent. Results: The obtained results indicate that dry cannabis flowers from different cannabis plant can be distinguished only by their terpenoid profile. The use of standardized cannabis-based extracts can be confirmed by determination of terpenoid profile. The purification process of the cannabis extracts removes terpenes. The percentage of major terpen beta-Myrcene decreased from 68% in dry flower to 15% in decarboxylated and, 1.9% in distillated cannabis oil after purification. The percentage of second major terpene alpha-Pinene decreased from 15% in dry flower to 5% in decarboxylated and, 0.7% in distillated cannabis oil after purification. Conclusion: Terpenes act synergistically with cannabinoids. Following the monograph for quality testing of cannabis extracts in the German Pharmacopoeia, the purification process is necessary to achieve a final concentration of cannabinoids (Tetrahydrocannabinol) of more than 95% in the final active pharmaceutical ingredient. The purification process removes terpenes that have proven synergistically pharmacological effects with cannabinoids.

COVID-19 Is Associated with Endothelial Dysfunction and Enhanced Plasma Thrombin Generation Despite Pharmacological Thromboprophylaxis

Background Hospitalised patients with severe COVID-19 (requiring critical care level support) appear to be at increased risk of thrombosis despite standard pharmacological thromboprophylaxis. The magnitude of thrombotic risk in patients with COVID-19 of moderate severity (not requiring critical care) is less clear. The optimal approach to thromboprophylaxis (and the role of intensified thromboprophylaxis) remains to be determined. Evidence of endothelial dysfunction has been widely reported in COVID-19 (particularly in severe COVID) and this may contribute to hypercoagulability. Aim To assess differences in patterns of hypercoagulability and endothelial dysfunction between a group of patients with moderate COVID-19 and a group of age-matched hospitalized patients (SARS-CoV-2 PCR negative) receiving low molecular weight heparin (LMWH) thromboprophylaxis. Methods Blood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care level support) and a group of age-matched patients admitted with infective/inflammatory illness (SARS-CoV-2 PCR negative). All subjects received standard-dose LMWH thromboprophylaxis, with blood drawn at 12 hours post-dose (and with measurement of anti-FXa activity levels). Circulating levels of endothelial & fibrinolytic markers including ICAM, PAI-1, VCAM, soluble thrombomodulin (sTM), and tissue plasminogen activator (tPA) were determined by ELISA. Thrombin generation (TG) in platelet-poor plasma was assessed by calibrated automated thrombography in the presence of tissue factor (Final concentration, 1pM & 5pM), thrombomodulin (TM) (Final concentration, 6.25nM), and an inhibitory anti-tissue factor pathway inhibitor antibody (anti-TFPI; Final concentration 100μg/mL). Results 14 COVID-19 positive subjects and 11 hospitalized controls were recruited. There were no differences in mean age (69.7±4.5 vs 61.6±4.7 years; p= 0.2) or mean Body mass index (25.7±1.1 vs 22.7±1.2 Kg/m2; p=0.1) between groups. No COVID-19 patient or control required critical care support. In the COVID group, radiological evidence of pneumonitis [diffuse (n=3) or peripheral infiltrates (n=7)] was present in the majority of cases. None of the COVID-19 cases were requiring supplemental oxygen at the time of recruitment. All controls were admitted with either respiratory or urinary infection [radiological evidence of pneumonia in 4/11; supplemental oxygen requirement in 2/11, (28-36% FiO2 via nasal cannula)]. Plasma levels of sTM, ICAM, PAI-1 & VCAM were similar in both groups. Levels of t-PA were significantly higher in the COVID group (8.31±4.35 vs 4.91±2.37 ng/mL; p= 0.005). Despite similar plasma anti-Xa activity in both groups (0.06 vs 0.04 IU/mL; p=0.2), mean endogenous thrombin potential (ETP) was significantly higher in the COVID group (1929±119.7 vs 1528±138.9 nM*min; p=0.02), although peak thrombin was similar (173.6±26 vs 161.5±31nM). ETP-TM ratio was similar between groups (0.3±0.1 vs 0.2±0.1; p=0.3). Despite increased ETP, the lag time to thrombin generation was significantly prolonged in the COVID group (8.3±0.6 vs 5.8±0.5 mins, p= 0.006). This pattern has previously been observed in vascular diseases associated with altered plasma tissue factor pathway inhibitor (TFPI) activity. In the presence of an anti-TFPI antibody, the difference in lagtime between groups was attenuated (4.7±0.2 vs 3.5±0.1 mins; p= 0.002) and the difference in overall thrombin generation (delta TG) between both groups became significantly increased (Fig.1). Conclusion Plasma thrombin generation is enhanced in patients with non-severe COVID-19 despite pharmacological thromboprophylaxis. Endothelial dysfunction is also observed in this group and appears to modulate parameters of plasma thrombin generation. The clinical implications of these observations are not known although clinical studies of intensified thromboprophylaxis in attenuating thrombotic risk and other complications are ongoing. Fig 1. Inhibition of TFPI activity enhances thrombin generation in COVID-19. In the presence of an inhibitory anti-TFPI antibody, peak plasma thrombin generation was enhanced in COVID-19 in contrast to that observed among SARS-CoV-2 PCR negative hospitalised patients (339.6+25.2 vs 247.4+10.1, p=0.01). Figure 1 Figure 1. Disclosures Maguire: Actelion: Research Funding; Bayer Pharma: Research Funding. Ni Ainle: Daiichi-Sankyo: Research Funding; Actelion: Research Funding; Leo Pharma: Research Funding; Bayer Pharma: Research Funding. Kevane: Leo Pharma: Research Funding.

Recipe for standard BG-11 media v1

Stanier RY, Deruelles J, Rippka R, Herdman M, Waterbury JB: Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology 1979, 111:1–61. Recipes for standard and alternative BG11 for culturing freshwater cyanobacteria, such as Synechocystis sp. PCC 6803, as described. Media is usually not suitable for marine cyanobacteria. Final Concentration of Medium. CaCl2*2 H2O 0.036 g/L Citric acid 0.006 g/L NaNO3 1.4958 g/L MgSO4* 7 H2O 0.0749 g/L 0.25M Na2EDTA (pH 8) 0.0056 mL/L Na2CO3 20 µg/ml Fe(III) Ammonium citrate 6 µg/ml K2HPO4 * 3H2O 30 µg/ml TES Buffer (pH 8) 10 mM H3BO3 2.86 mg/L MnCl2 * 4 H2O 1.81 mg/L ZnSO4 * 7 H2O 0.222 mg/L Na2MoO4 * 2 H2O 0.390 mg/L Co(NO3)2 *6 H2O 0.049 mg/L (CuSO4 * 5 H2O 0.079 mg/L if required)

The Effect of A2E on the Uptake and Release of Calcium in the Lysosomes and Mitochondria of Human RPE Cells Exposed to Blue Light

We aimed to explore the effect of N-retinylidene-N-retinylethanolamine (A2E) on the uptake and release of calcium in lysosomes and mitochondria by establishing a model of human retinal pigment epithelial (RPE) cell injury induced by exposure to blue light. Primary human RPE cells were cultured from passages 4 to 6 and exposed to blue light at an intensity of 2000 ± 500 lux for 6 hours. After blue light exposure, the culture was maintained for 24 hours. A2E at a final concentration of 25 μM was added to the culture 2 hours before light exposure, and nifedipine at a final concentration of 10−4 M was added 1 hour before light exposure. The levels of Ca2+ in the cytosol (CaTM/2AM), mitochondria (Rhod/2AM), and lysosomes (LysoTracker Red and Fluo-3/AM) were determined. In order to measure the calcium levels in the different organelles, RPE were imaged using a laser scanning confocal microscope. Moreover, changes in the mitochondrial membrane potential were detected by flow cytometry analysis of JC-1-stained cells. The obtained results revealed that blue light illumination increased the calcium fluorescence intensity in the cytoplasm, mitochondria, and lysosomes of human RPE cells when compared with the control cells ( P < 0.05 ). After A2E treatment, the fluorescence intensity of the calcium in the cytoplasm was further increased ( P < 0.05 ), while that in the mitochondria and lysosomes decreased ( P < 0.05 ). In addition, we observed that nifedipine reduced the fluorescence intensity of calcium in the RPE cells. Our results also showed that the mitochondrial membrane potential in the RPE treated with blue light and A2E was lower than that in the control, blue light, and A2E-treated cells ( P < 0.05 ). Blue light increased calcium levels in the cytoplasm, lysosomes, and mitochondria of RPE cells. A2E damages the lysosomal and mitochondrial membranes, resulting in calcium release into the cytoplasm. Finally, our results demonstrated that both blue light and A2E treatments reduced mitochondrial membrane potential, increasing cytosolic Ca2+ levels, which can contribute to the activation of RPE death.

Alpha-Ketoglutarate: A Potential Inner Mitochondrial and Cytosolic Protector against Peroxynitrite and Peroxynitrite-Induced Nitration?

The generation of peroxynitrite (ONOO−) is associated with several diseases, including atherosclerosis, hypertension, neurodegeneration, cancer, inflammation, and sepsis. Alpha-ketoglutarate (αKG) is a known potential highly antioxidative agent for radical oxidative species such as peroxides. The question arises as to whether αKG is also a potential scavenger of ONOO− and a potential protector against ONOO−-mediated nitration of proteins. NMR studies of 1 mM αKG in 100 mM phosphate-buffered saline at pH 7.4 and pH 6.0 were carried out in the presence or absence of a final concentration of 2 mM ONOO−. An ONOO−–luminol-induced chemiluminescence reaction was used to measure the scavenging function of several concentrations of αKG; quantification of αKG was performed via spectrophotometric enzymatic assay of αKG in the absence or presence of 0, 1, or 2 mM ONOO−. The nitration of tyrosine residues on proteins was measured on ONOO−-treated bovine serum albumin (BSA) in the presence or absence of 0–24 mM αKG by an ELISA technique using a specific anti-IgG against nitro-tyrosine. The addition of ONOO− to αKG led to the formation of succinic acid and nitrite at pH 7.0, but not at pH 6.0, as αKG was stable against ONOO−. The absorbance of enzymatically estimated αKG at the time point of 30 min was significantly lower in favour of ONOO− (1 mM: 0.21 ± 0.03, 2 mM: 0.12 ± 0.05 vs. 0 mM: 0.32 ± 0.02; p < 0.001). The luminol technique showed an inverse logarithmic correlation of the ONOO− and αKG concentrations (y = −2 × 105 ln(x) + 1 × 106; r2 = 0.99). The usage of 4 mM αKG showed a significant reduction by nearly half in the chemiluminescence signal (284,456 ± 29,293 cps, p < 0.001) compared to the control (474,401 ± 18,259); for 20 and 200 mM αKG, there were further reductions to 163,546 ± 26,196 cps (p < 0.001) and 12,658 ± 1928 cps (p < 0.001). Nitrated tyrosine residues were estimated using the ELISA technique. A negative linear correlation was obtained by estimating nitrated tyrosine residues in the presence of αKG (r2 = 0.94): a reduction by half of nitrated tyrosine was estimated using 12 mM αKG compared to the control (326.1 ± 39.6 nmol vs. 844.5 ± 128.4 nmol; p < 0.001).