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Effect of curcuminoids on lipid mediator profiles of activated M1 and M2 polarized macrophages
No full text<p>Human macrophages of the M1 and M2 phenotype were pre-treated with <strong>3b</strong> or <strong>2f</strong> for 15 min and then stimulated with <i>Staphylococcus aureus</i>-conditioned medium for 180 min, and lipid mediator profiles were determined by UPLC-MS/MS. </p><p>Raw data including the absolute values (ng) and normalized data (% of control) of all individual experiments was uploaded.</p><p>The methods and results were published in Rao et al., Biochem Pharmacol. 2022 Sep:203:115202. doi: 10.1016/j.bcp.2022.115202 </p><p>Human M1 or M2 macrophages were treated with vehicle (0.1 % DMSO), compound 3b or 2f in PBS pH 7.4 plus CaCl2 (1 mM) for 15 min and then stimulated with Staphylococcus aureus-conditioned medium (SACM, 1 %) for 180 min at 37 °C. SACM was prepared. In brief, Staphylococcus aureus (strain 6850) was grown for 18 h with orbital shaking (150 rpm) at 37 °C in brain heart infusion medium (Carl Roth, Karlsruhe, Germany). The conditioned medium was harvested after centrifugation (3350 × g, 10 min) and sterile filtered using a Millex-GP Syringe Filter Unit (0.22 μm; Merck).</p><p>Cell supernatants (1 mL) were collected and mixed with ice-cold methanol (2 mL). d8-5S-HETE, d4-LTB4, d5-LXA4, d5-RvD2, d4-PGE2 (200 nM, 10 µL each) and d8-AA (10 µM, 10 µL) were added as internal standards. Lipid mediators were extracted by solid phase extraction using Sep-Pak C18 6 cc Vac Cartridges (500 mg; Waters). Briefly, samples were stored at −20 °C for at least 45 min to allow protein precipitation. After centrifugation (1200 × g, 4 °C, 10 min), the supernatant was combined with acidified water (pH 3.5, 7 mL) and loaded onto pre-equilibrated solid phase cartridge columns. Washing steps with water and n-hexane (6 mL, each) followed. Lipid mediators were eluted with methyl formate (6 mL), brought to dryness using an evaporation system (TurboVap LV, Biotage, Uppsala, Sweden) and resuspended in methanol/water (50/50, v/v, 100 µL) for UPLC-MS/MS analysis.</p><p>For metabololipidomics analysis, lipid mediators were separated at 50 °C on an Acquity UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 100 mm, Waters). The Acquity Ultraperformance LC system (Waters) was operated at a flow rate of 0.3 mL/min using a mobile phase consisting of methanol, water, and acetic acid (42:58:0.01, v/v/v), which was ramped to 86:14:0.01 (v/v/v) over 12.5 min followed by isocratic elution at 98:2:0.01 (v/v/v) for 3 min. Eluted lipid mediators were detected by (scheduled) multiple reaction monitoring using a QTRAP 5500 mass spectrometer (Sciex, Framingham, MA), which was equipped with an electrospray ionization source that was operated in negative mode. Acquired mass spectra were processed using Analyst 1.6.2 (Sciex).</p>
Analysis of the triglyceride fatty acid composition in α-T-13′-COOH-treated macrophages
No full text<p>RAW264.7 cells were incubated with vehicle (DMSO, 'w/o') or 0.5 or 5.0 µM α-T-13′-COOH for 24 h. The fatty acid distribution of triglyceride was then analyzed by UPLC-MS/MS.</p><p>Raw analyst files (.wiff and .wiff.scan) of the UPLC-MS/MS results were uploaded, together with an Excel file for the sample list.</p><p>The methods and results were published in Liao et al., Int J Mol Sci, 2023 May 25;24(11):9229. doi: 10.3390/ijms24119229 </p><p>Lipids were extracted from RAW264.7 cell pellets by the successive addition of methanol, PBS (pH 7.4), chloroform, and saline (final ratio: 34:14:35:17). After the evaporation of the organic solvent, the remaining lipid fraction was dissolved in methanol, stored at −20 °C, and analyzed by UPLC-MS/MS. Internal standards: 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphatidylcholine (DMPC), 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphatidyl-ethanolamine (DMPE).</p><p>TGs were separated on an Acquity UPLC BEH C8 column (130 Å, 1.7 μm, 2.1 × 100 mm; Waters) using either an Acquity UPLC system (Waters), which was coupled to a QTRAP 5500 mass spectrometer (Sciex) equipped with a Turbo V Ion Source and an electrospray ionization probe, or an ExionLC AD UHPLC system (Sciex), which was coupled to a QTRAP 6500+ mass spectrometer (Sciex) equipped with an IonDrive Turbo V Ion Source and an electrospray ionization probe. In brief, both LC systems were operated at a flow rate of 0.75 mL/min and a column temperature of 45 °C. The mobile phase consisted of eluent A (acetonitrile/water, 95/5, with 2 mM ammonium acetate) and eluent B (isopropanol). For the separation of TGs, eluent A was reduced from 90% to 70% within 6 min followed by isocratic elution for 4 min. The fragmentation of [M + NH4]+ adduct ions to [M-fatty acid anion]+ ions was measured by multiple reaction monitoring. The ion spray voltage was set to 5500 V, the curtain gas to 30 psi (QTRAP 5500) or 40 psi (QTRAP 6500+), the collision gas to low, and the heated capillary temperature to 400 °C. The sheath gas pressure was set to 60 psi and the auxiliary gas pressure was set to 70 psi. The declustering potential was set to 120 V, the entrance potential to 10 V, the collision energy to 35 eV, and the collision cell exit potential to 26 V.</p><p>The instruments were either operated with Analyst 1.6.2 (QTRAP 5500, Sciex) or Analyst 1.7.1 (QTRAP 6500+, Sciex). </p>
Effect of α-amplexichromanol (27a) on lipid mediator formation in zymosan induced murine peritonitis
No full text<p>Neukirch, K. et al. Exploration of Long-Chain Vitamin E Metabolites for the Discovery of a Highly Potent, Orally Effective, and Metabolically Stable 5-LOX Inhibitor that Limits Inflammation. J Med Chem 64, 11496-11526 (2021). https://doi.org/10.1021/acs.jmedchem.1c00806</p><p>CD-1 mice received <strong>27a</strong> or zileuton, with DMSO (2%) in saline (0.5 mL) as a vehicle for i.p. administration and carboxymethylcellulose (0.5%) in 10% Tween 20 (0.5 mL) as a vehicle for p.o. administration. Zymosan (2 mg/mL insaline, i.p., 0.5 mL, Sigma-Aldrich) was injected at 30 min (i.p.) or 60min (p.o.) post compound administration. Mice were sacrificed by inhalation of CO2 after another 30 min to determine LTC4 levels, lipid mediator profiles, metabolites, and vascular permeability and after 4 h to analyze LTB4 levels and cell infiltration. Plasma and peritoneal exudates were collected, and cells were counted in exudates after trypan blue staining. Compound <strong>27a</strong>, its metabolites, and lipid mediators were extracted and analyzed by UPLC-MS/MS as described in Neukirch et al., 2021.</p>
Active Brownian particles in a circular disk with an absorbing boundary
No full text<p>We solve the time-dependent Fokker-Planck equation for a two-dimensional active Brownian particle exploring a circular region with an absorbing boundary. Using the passive Brownian particle as basis states and dealing with the activity as a perturbation, we provide a matrix representation of the Fokker-Planck operator and we express the propagator in terms of the perturbed eigenvalues and eigenfunctions. Alternatively, we show that the propagator can be expressed as a combination of the equilibrium eigenstates with weights depending only on time and on the initial conditions, and obeying exact iterative relations. Our solution allows also obtaining the survival probability and the first-passage time distribution. These latter quantities exhibit peculiarities induced by the non-equilibrium character of the dynamics,; in particular, they display a strong dependence on the activity of the particle and, to a less extent, also on its rotational diffusivity.</p>
Dataset for Stegner et al. 2023: Frozen mountain pine needles: The endodermis discriminates between the ice-containing central tissue and the ice-free fully functional mesophyll. Physiologia Plantarum
No full textMetabolism of α-amplexichromanol (27a) in a human liver-on-chip model
No full text<p>Neukirch, K. et al. Exploration of Long-Chain Vitamin E Metabolites for the Discovery of a Highly Potent, Orally Effective, and Metabolically Stable 5-LOX Inhibitor that Limits Inflammation. J Med Chem 64, 11496-11526 (2021). https://doi.org/10.1021/acs.jmedchem.1c00806</p><p>Biochips were made from polystyrene by injection molding and were equipped with a poly(ethylene terephthalate) (PET) membrane (TRAKETCH, thickness: 12μm; pore diameter: 8μm; pore density: 1×10^5 pores/cm2; Sabeu, Radeberg, Germany) that was integrated in the upper and lower parts of the biochip by heat-sealing with the bulk material. The top and bottom sides of the biochips and channels were sealed with an extruded PS bonding foil (thickness: 125 μm) by a low-temperature bonding method. The upper and lower parts of the biochips were assembled using a double-sided adhesive film, and the chip surface was hydrophilized by oxygen plasma treatment to facilitate cell adhesion and prevent air bubble formation within the chambers and channels. HUVECs were isolated from human umbilical cord veins and cultivated in an endothelial cell growth medium MV (Promocell, Heidelberg, Germany) with penicillin (100 U/mL) / streptomycin (100 μg/mL, GE Healthcare) up to passage 4. On reaching 95% confluence, cells were sub-cultured at a density of 1.5×10^4 cells/cm2. HepaRG cells (Biopredic International, Rennes, France) were grown in William's medium E (Biochrom, Berlin, Germany) with 10% FCS, 5 μg/mL insulin (Sigma-Aldrich), 2 mM glutamine (Thermofisher Scientific), 50 μM hydrocortisone-hemisuccinate (Sigma-Aldrich), and penicillin (100 U/mL) / streptomycin (100μg/mL, GE Healthcare) at 37°C and 5% CO2 for 14 days prior to differentiation with 2% DMSO for another 14 days. The medium was changed every 3−4 days, and differentiated cells were used up to 4weeks. The liver-on-chip was prepared by seeding HUVECs (top: 3×10^5, bottom: 1.5×10^5) in an endothelial cell growth medium MV on top of the membrane in the upper chamber and on the bottom of the membrane in the lower chamber, giving the cells 3−4 h to adhere before flipping the chip upside and seeding the bottom layer. After 2 days, differentiated HepaRG cells (3×10^5) were seeded on top of the lower and on the bottom of the upper membrane in a hepatocyte culture medium with hydrocortisone-hemisuccinate adjusted to 5 μM for 24 h at 37°C and 5% CO2. The medium between the two membranes was renewed, and the vehicle (DMSO), <strong>12a</strong>, or <strong>27a</strong> were added. After incubation for 48 h at 37°C and 5% CO2, the medium was collected and the system was washed with 500 μL of methanol. <strong>12a, 27a</strong>, and their metabolites were extracted from the combined fractions and analyzed by UPLC-MS/MS as described in Neukirch et al., 2021.</p>
UIBK Avalanche dataset
No full text<p>Folder annotations: Avalanche segmentation by type</p><p>Folder images: Avalanche images by type</p><p>File train.txt: Files used for training</p><p>File test.txt: Files used for evaluation (test)</p>
Analysis of the phosphatidylethanolamine fatty acid composition in α-T-13′-COOH-treated macrophages
No full text<p>RAW264.7 cells were incubated with vehicle (DMSO, 'w/o') or 0.5 or 5.0 µM α-T-13′-COOH for 24 h. The fatty acid distribution of phosphatidylethanolamine was then analyzed by UPLC-MS/MS.</p><p>Raw analyst files (.wiff and .wiff.scan) of the UPLC-MS/MS results were uploaded, together with an Excel file for the sample list.</p><p>The methods and results were published in Liao et al., Int J Mol Sci, 2023 May 25;24(11):9229. doi: 10.3390/ijms24119229.</p><p>Lipids were extracted from RAW264.7 cell pellets by the successive addition of methanol, PBS (pH 7.4), chloroform, and saline (final ratio: 34:14:35:17). After the evaporation of the organic solvent, the remaining lipid fraction was dissolved in methanol, stored at −20 °C, and analyzed by UPLC-MS/MS. Internal standards: 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphatidylcholine (DMPC), 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphatidyl-ethanolamine (DMPE).</p><p>Phospholipids (PC, PE, PI, PS, PG) were separated on an Acquity UPLC BEH C8 column (130 Å, 1.7 μm, 2.1 × 100 mm; Waters, Milford, MA, USA) using an Acquity UPLC system (Waters), which was coupled to a QTRAP 5500 mass spectrometer (Sciex, Framingham, MA, USA) equipped with a Turbo V Ion Source and an electrospray ionization probe. Chromatographic separation was performed at a flow rate of 0.75 mL/min and at a column temperature of 45 °C. The mobile phase was composed of eluent A (acetonitrile/water, 95/5, with 2 mM ammonium acetate) and eluent B (water/acetonitrile, 90/10, with 2 mM ammonium acetate). The gradient was ramped from 70% to 80% A within 5 min and to 100% A within 2 min, followed by isocratic elution for another 2 min. Eluted phospholipids were detected upon the fragmentation of parental ions (PC: [M+OAc]−, all other phospholipids: [M-H]−) to fatty acid anions derived from <i>sn</i>-1 and <i>sn</i>-2 positions by multiple reaction monitoring using a QTRAP 5500 mass spectrometer. The ion spray voltage was set to −4500 V, the curtain gas to 30 psi, the collision gas to medium, and the heated capillary temperature to either 350 °C (PC), 500 °C (PI), 550 °C (PS, PG), or 650 °C (PE). The sheath gas pressure was set to 45 (PS) or 55 psi (PC, PE, PI, PG) and the auxiliary gas pressure was set to either to 75 psi (PC, PE, PI, PG) or 80 psi (PS). The declustering potential was set to −40 V (PS), −44 V (PC), −45 V (PG), or −50 V (PE, PI), the entrance potential to −10 eV (PC; PE, PI, PS, PG), the collision energy to −38 eV (PE), −46 eV (PC), −52 eV (PG), −56 eV (PS), or −62 eV (PI), and the collision cell exit potential to −11 V (PC, PI), −12 V (PE), −18 V (PG), or −20 V (PS).</p><p>The instruments were either operated with Analyst 1.6.2 (QTRAP 5500, Sciex) or Analyst 1.7.1 (QTRAP 6500+, Sciex).</p>
Effect of curcuminoids on cell-free microsomal prostaglandin E synthase-1 activity
No full text<p>Human mPGES-1 was pre-treated with DMSO or curcuminoids, the formation of PGE2 was initiated by the addition of PGH2 and the mPGES-1 product PGE2 was analyzed by RP-UV-HPLC.</p><p>Raw data including the absolute values (ng) and normalized data (% of control) of all individual experiments was uploaded.</p><p>The methods and results were published in Rao et al., Biochem Pharmacol. 2022 Sep:203:115202. doi: 10.1016/j.bcp.2022.115202 </p><p>mPGES-1 activity was measured in microsomal membranes of IL-1β-treated human lung adenocarcinoma epithelial A549 cells. A549 cells were incubated with IL-1β (2 ng/mL, Peprotech, Hamburg, Germany) in DMEM/high glucose (4.5 g/L) medium plus FCS (10 %) and penicillin/streptomycin (100 U/mL and 100 μg/mL; GE Healthcare, Freiburg, Germany) at 37 °C and 5 % CO2 for 48 h. Cell pellets were harvested and snap-frozen in liquid nitrogen. After resuspension and incubation in ice-cold homogenization buffer [potassium phosphate buffer (0.1 M, pH 7.4), phenylmethylsulphonyl fluoride (1 mM, Cayman Chemical), soybean trypsin inhibitor (60 μg/mL, Cayman Chemical), leupeptin (1 μg/mL, Cayman Chemical), glutathione (2.5 mM, Cayman Chemical) and sucrose (250 mM, Cayman Chemical)] for 15 min on ice, cells were sonicated (3 times, 20 s, each, at 4 °C). Differential centrifugation (first 10,000 × g, 10 min; then 174,000 × g, 60 min; at 4 °C) yielded the microsomal membrane fraction, which was resuspended in homogenization buffer and diluted in potassium phosphate buffer (0.1 M, pH 7.4) plus glutathione (2.5 mM, Cayman Chemical). </p><p>To determine mPGES-1 activity, membranes (containing 2.5–5 μg total protein in 50 μL homogenization buffer) were pre-treated with vehicle (DMSO), test compounds or the mPGES-1 inhibitor MK-886 (10 μM, Cayman Chemical) for 15 min at 4 °C. The formation of PGE2 was initiated by the addition of PGH2 [20 μM in 50 µL potassium phosphate buffer (0.1 M, pH 7.4) containing phenylmethylsulphonyl fluoride (1 mM, Cayman Chemical), soybean trypsin inhibitor (60 μg/mL, Cayman Chemical), leupeptin (1 μg/mL, Cayman Chemical), glutathione (2.5 mM, Cayman Chemical)]. After 1 min incubation at 4 °C, the reaction was terminated with an equal volume of stop solution containing FeCl2 (40 mM) and citric acid (80 mM) as well as the internal standard 11β-PGE2 (1 nmol, Cayman Chemical). The mPGES-1 product PGE2 was extracted by solid phase extraction using Sep-Pak C18 35 cc Vac Cartridges (Waters), separated on Nova-Pak C18 Radial-Pak Column (4 μm, 5 × 100 mm, Waters) under isocratic conditions (30 % acetonitrile, 70 % water, 0.007 % trifluoroacetic acid) at a flow rate of 1 mL/min and detected at 195 nm. The mPGES-1 reference inhibitor MK-886 (10 μM, Cayman Chemical) inhibited PGE2 formation by 86.4 ± 0.5 %.</p>
UIBK Avalanche Dataset
No full text<h2>UIBK Avalanche Dataset</h2><p>Dataset of 4090 labelled avalanche photographs. Each photograph in the dataset was assigned to one of four categories: glide, loose, slab, or none. These labels refer to the different avalanche release mechanisms: glide, loose-snow, and slab, with none indicating the absence of avalanches. In addition, avalanche experts outlined each visible avalanche with a polygonal bounding box and assigned it a single label of glide, loose, or slab. </p><p>The photographs were taken in the field in the winters of 2000/2001 - 2021/2022 and each image has the date and approximate location at which it was taken in the image name. Most photographs in the dataset were taken from the ground: additional images from low-flying helicopters were included only if the annotators deemed that they could feasibly have been taken by webcams.</p><p>Accompanying code for training ResNet, VGG, and YOLOv3 models to detect avalanches is available at <a href="https://github.com/j-f-ox/avalanche-detection">github.com/j-f-ox/avalanche-detection</a></p><h3>Folders</h3><ul><li>annotations # Polygonal and rectangular bounding boxes for each avalanche image<ul><li>glide</li><li>loose</li><li>slab</li></ul></li><li>images # The .jpg images sorted by overall image label<ul><li>glide</li><li>loose</li><li>none</li><li>slab</li></ul></li><li>test.txt # Test images</li><li>train.txt # Training images</li></ul><p> </p><h3>Label Distribution </h3><ul><li>The images folder contains 716 glide, 416 loose, and 1887 slab avalanche photographs and a further 1071 "none" images without visible avalanche regions</li><li>The dataset contains a total of 2489 annotated glide avalanches, 1827 loose-snow avalanches, and 2912 slab avalanches</li></ul>