Modulation of Endocannabinoids by Caloric Restriction Is Conserved in Mice but Is Not Required for Protection from Acute Kidney Injury

Acute kidney injury (AKI) is a frequent and critical complication in the clinical setting. In rodents, AKI can be effectively prevented through caloric restriction (CR), which has also been shown to increase lifespan in many species. In Caenorhabditis elegans (C. elegans), longevity studies revealed that a marked CR-induced reduction of endocannabinoids may be a key mechanism. Thus, we hypothesized that regulation of endocannabinoids, particularly arachidonoyl ethanolamide (AEA), might also play a role in CR-mediated protection from renal ischemia-reperfusion injury (IRI) in mammals including humans. In male C57Bl6J mice, CR significantly reduced renal IRI and led to a significant decrease of AEA. Supplementation of AEA to near-normal serum concentrations by repetitive intraperitoneal administration in CR mice, however, did not abrogate the protective effect of CR. We also analyzed serum samples taken before and after CR from patients of three different pilot trials of dietary interventions. In contrast to mice and C. elegans, we detected an increase of AEA. We conclude that endocannabinoid levels in mice are modulated by CR, but CR-mediated renal protection does not depend on this effect. Moreover, our results indicate that modulation of endocannabinoids by CR in humans may differ fundamentally from the effects in animal models.     

Immune Cells Invade the Collateral Circulation during Human Stroke: Prospective Replication and Extension

It remains unclear if principal components of the local cerebral stroke immune response can be reliably and reproducibly observed in patients with acute large-vessel-occlusion (LVO) stroke. We prospectively studied a large independent cohort of n = 318 consecutive LVO stroke patients undergoing mechanical thrombectomy during which cerebral blood samples from within the occluded anterior circulation and systemic control samples from the ipsilateral cervical internal carotid artery were obtained. An extensive protocol was applied to homogenize the patient cohort and to standardize the procedural steps of endovascular sample collection, sample processing, and laboratory analyses. N = 58 patients met all inclusion criteria. (1) Mean total leukocyte counts were significantly higher within the occluded ischemic cerebral vasculature (I) vs. intraindividual systemic controls (S): +9.6%, I: 8114/µL ± 529 vs. S: 7406/µL ± 468, p = 0.0125. (2) This increase was driven by neutrophils: +12.1%, I: 7197/µL ± 510 vs. S: 6420/µL ± 438, p = 0.0022. Leukocyte influx was associated with (3) reduced retrograde collateral flow (R² = 0.09696, p = 0.0373) and (4) greater infarct extent (R² = 0.08382, p = 0.032). Despite LVO, leukocytes invade the occluded territory via retrograde collateral pathways early during ischemia, likely compromising cerebral hemodynamics and tissue integrity. This inflammatory response can be reliably observed in human stroke by harvesting immune cells from the occluded cerebral vascular compartment.     

A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

Chemokines guide leukocyte migration in different contexts, including homeostasis, immune surveillance and immunity. The chemokines CCL19 and CCL21 control lymphocyte and dendritic cell migration and homing to lymphoid organs. Thereby they orchestrate adaptive immunity in a chemokine receptor CCR7-dependent manner. Likewise, cancer cells that upregulate CCR7 expression are attracted by these chemokines and metastasize to lymphoid organs. In-depth investigation of CCR7 expression and chemokine-mediated signaling is pivotal to understand their role in health and disease. Appropriate fluorescent probes to track these events are increasingly in demand. Here, we present an approach to cost-effectively produce and fluorescently label CCL19 and CCL21 in a semi-automated process. We established a versatile protocol for the production of recombinant chemokines harboring a small C-terminal S6-tag for efficient and site-specific enzymatic labelling with an inorganic fluorescent dye of choice. We demonstrate that the fluorescently labeled chemokines CCL19-S6^Dy649P1 and CCL21-S6^Dy649P1 retain their full biological function as assessed by their abilities to mobilize intracellular calcium, to recruit β-arrestin to engaged receptors and to attract CCR7-expressing leukocytes. Moreover, we show that CCL19-S6^Dy649P1 serves as powerful reagent to monitor CCR7 internalization by time-lapse confocal video microscopy and to stain CCR7-positive primary human and mouse T cell sub-populations.     

Gastric Serotonin Biosynthesis and Its Functional Role in L-Arginine-Induced Gastric Proton Secretion

Among mammals, serotonin is predominantly found in the gastrointestinal tract, where it has been shown to participate in pathway-regulating satiation. For the stomach, vascular serotonin release induced by gastric distension is thought to chiefly contribute to satiation after food intake. However, little information is available on the capability of gastric cells to synthesize, release and respond to serotonin by functional changes of mechanisms regulating gastric acid secretion. We investigated whether human gastric cells are capable of serotonin synthesis and release. First, HGT-1 cells, derived from a human adenocarcinoma of the stomach, and human stomach specimens were immunostained positive for serotonin. In HGT-1 cells, incubation with the tryptophan hydroxylase inhibitor p-chlorophenylalanine reduced the mean serotonin-induced fluorescence signal intensity by 27%. Serotonin release of 147 ± 18%, compared to control HGT-1 cells (set to 100%) was demonstrated after treatment with 30 mM of the satiating amino acid L-Arg. Granisetron, a 5-HT3 receptor antagonist, reduced this L-Arg-induced serotonin release, as well as L-Arg-induced proton secretion. Similarly to the in vitro experiment, human antrum samples released serotonin upon incubation with 10 mM L-Arg. Overall, our data suggest that human parietal cells in culture, as well as from the gastric antrum, synthesize serotonin and release it after treatment with L-Arg via an HTR3-related mechanism. Moreover, we suggest not only gastric distension but also gastric acid secretion to result in peripheral serotonin release.     

Online Use of Physically Based Plasticity Models for Steady State Cold Rolling Processes

A procedure has been developed to incorporate computationally costly physically based crystal plasticity models to calculate texture and anisotropy for steady state forming processes online. When using these models, at every point in the deformed zone, an average and a nonlinear solution procedure for stresses and/or strains in all these grains is required. The online calculation cost is avoided by offline creating a database with texture and anisotropy data for all possible deformation modes of the process. The case studied is a cold rolling process, but can easily be extended to any type of forming process, when the deformation field is known in advance. Textures and anisotropy data are predicted using a viscoplastic self-consistent model, but the method is suitable for any kind of crystal plasticity model. Single crystal plastic parameters, such as the critical resolved shear stress, the single crystal hardening parameters, and the strain-rate sensitivity, have been calibrated based on mechanical tests by means of a direct search simplex algorithm. The online calculated deformation history is compared to the histories stored in the database and the best match is selected. The deformation history is divided in two zones, the one before the neutral point where forward shearing occurs and the one after the neutral point where backward shearing occurs. One online deformation generation and selection procedure requires 0.005 s of CPU time for a database with a division in deformation gradients fine enough to accurately cover all deformations. The method allows calculating yield surfaces at any point in space based on microstructural effects modeled by crystal plasticity, without incremental material updating and necessity to define a kinematic and isotropic hardening, which makes the method suitable for fast models to calculate rolling forces and torques online.     

Predicting crack growth rate vs. temperature behaviour of Type 304 stainless steel in dilute sulphuric acid solutions

The coupled-environment fracture model for intergranular stress corrosion cracking of Type 304 stainless steel in boiling water reactor primary (water) heat transport circuits containing relatively pure water has been extended to incorporate the effects of sulphuric acid additions to the coolant and to include thermal activation of the crack tip strain rate. These extensions allow comparisons to be made between theoretically estimated and experimentally determined crack growth rates (CGRs) over a considerable temperature range after calibration at a single temperature. The model predicts, in agreement with experiment, that the CGR passes through a maximum with increasing temperature at a temperature of about 180 °C. The temperature dependence of the CGR is attributed to the competing effects of temperature on the thermally activated processes that occur at the crack tip and the properties (including ECP and conductivity) of the external environment.     

Hohlfaser-Membrankontaktoren zur Stickstoffrückgewinnung aus Prozesswasser einer kommunalen Kläranlage

Process water deriving from digested sludge dewatering at municipal wastewater treatment plants (WWTP) represents an additional load for the plant in terms of nitrogen. Münster WWTP is the first German plant to operate an innovative full-scale membrane contactor facility for nitrogen removal and recovery from process water. Within the scope of this study, aspects influencing the membrane performance and operation as well as utilization of the produced fertilizer are investigated. First results reveal nitrogen removal efficiencies around 85 % and a regional usability of the produced fertilizer.     

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.     

Moderate Compared to Low Dietary Intake of trans-Fatty Acids Impairs Strength of Old and Aerobic Capacity of Young SAMP8 Mice in Both Sexes

The senescence accelerated SAMP8 mouse is a model for sarcopenia and provides an opportunity to study the effects of lifelong dietary composition on the loss of physical function with age. We studied the effects of trans-fatty acids (2 % of total energy, TFA diet) on the loss of strength and aerobic exercise capacity (VO₂peak) with age. SAMP8 mice were studied at two ages (young, 25 weeks; old, 60 weeks) and on two diets (control vs TFA). Body composition, grip strength, VO₂peak, blood metabolites, and biochemical parameters were assessed. Body weight, fat mass, and body fat percentage all increased with age (p < 0.05) but were not significantly impacted by diet. There was a significant age-related decline in total grip strength as well as that normalized to fat-free mass (FFM) (p < 0.05) with a further decrease at old age in these metrics of strength on the TFA diet vs control diet (p < 0.05). Total VO₂peak exhibited no change with age or diet, but when normalized to FFM, VO₂peak exhibited age and TFA-related declines (p < 0.05). Intramuscular triacylglycerol (p < 0.05) and collagen content (p < 0.05) significantly increased with age, while blood triacylglycerol was increased by the TFA diet (p < 0.05). These data further characterize the SAMP8 mouse as a model for sarcopenia and indicate that dietary fatty acid composition can impact the degree of this age-related loss of physical function.     

Material Evidence for Multiple Firings of Ancient Athenian Red‐Figure Pottery

The production of Athenian fine ware pottery, produced between the 6th and 4th centuries B.C., required alternating the high‐temperature kiln between oxidative and reductive environments during a single firing to create the iconic red and black decorative scenes. Here, we show that the production of this pottery was even more complex, with vessels subjected to two, or possibly more, firings in the kiln, with applications of slip between each firing. On a representative sherd, we compared three painted black decorative features—relief line, contour line, and background slip. Scanning transmission electron microscopy (STEM) of the slips revealed that the relief line had a more melted microstructure than either the contour line or background slip. By characterizing the chemistry and micromorphology of the slips, we find that the relief line microstructure could only be produced through a separate firing, at a hotter temperature, than the other two decorative features.