A human macrophage – hepatocyte co-culture model for comparative studies of infection and replication of Francisella tularensis LVS strain and subspecies holarctica and mediasiatica

Detection of intracellular LPS in macrophage / hepatocyte co-cultures infected with LVS (open bars), spp. holarctica (grey filled bars) or spp. mediasiatica (black filled bars) and untreated control (hatched bars). A) Different amounts of macrophages in the co-culture were tested (6, 12 and 22 % of macrophages on total cell count). Flow cytometric detection of intracellular LPS in macrophages (MFI mean fluorescence intensity); B-D) percentage of remaining detectable macrophages after infection of the co-cultures with B) 6 % macrophages/94 % hepatocytes, C) 12 % macrophages/ 88 % hepatocytes and D) 22 % macrophages/ 88 % hepatocytes 72 h post infection. (TIF 32735 kb

High‐Saturated‐Fat Diet Increases Circulating Angiotensin‐Converting Enzyme, Which Is Enhanced by the rs4343 Polymorphism Defining Persons at Risk of Nutrient‐Dependent Increases of Blood Pressure

Background Angiotensin‐converting enzyme (ACE) plays a major role in blood pressure regulation and cardiovascular homeostasis. Contrary to the assumption that ACE levels are stable, circulating ACE has been shown to be altered in obesity and weight loss. We sought to examine effects of a high‐saturated‐fat (HF) diet on ACE within the NUtriGenomic Analysis in Twins (NUGAT) study. Methods and Results Forty‐six healthy and nonobese twin pairs initially consumed a carbohydrate‐rich, low‐fat diet over a period of 6 weeks to standardize for nutritional behavior prior to the study, followed by 6 weeks of HF diet under isocaloric conditions. After 6 weeks of HF diet, circulating ACE concentrations increased by 15% (P=1.6×10−30), accompanied by an increased ACE gene expression in adipose tissue (P=3.8×10−6). Stratification by ACE rs4343, a proxy for the ACE insertion/deletion polymorphism (I/D), revealed that homozygous carriers (GG) of the variant had higher baseline ACE concentrations (P=7.5×10−8) and additionally showed a 2‐fold increase in ACE concentrations in response to the HF diet as compared to non‐ or heterozygous carriers (AA/AG, P=2×10−6). GG carriers also responded with higher systolic blood pressure as compared to AA/AG carriers (P=0.008). The strong gene‐diet interaction was confirmed in a second independent, cross‐sectional cohort, the Metabolic Syndrome Berlin Potsdam (MeSyBePo) study. Conclusions The HF‐diet‐induced increase of ACE serum concentrations reveals ACE to be a potential molecular link between dietary fat intake and hypertension and cardiovascular disease (CVD). The GG genotype of the ACE rs4343 polymorphism represents a robust nutrigenetic marker for an unfavorable response to high‐saturated‐fat diets. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT01631123

Functional integration of natural killer cells in a microfluidically perfused liver on-a-chip model.

OBJECTIVE The liver acts as an innate immunity-dominant organ and natural killer (NK) cells, are the main lymphocyte population in the human liver. NK cells are in close interaction with other immune cells, acting as the first line of defense against pathogens, infections, and injury. A previously developed, three-dimensional, perfused liver-on-a-chip comprised of human cells was used to integrate NK cells, representing pivotal immune cells during liver injury and regeneration. The objective of this study was to integrate functional NK cells in an in vitro model of the human liver and assess utilization of the model for NK cell-dependent studies of liver inflammation. RESULTS NK cells from human blood and liver specimen were isolated by Percoll separation with subsequent magnetic cell separation (MACS), yielding highly purified blood and liver derived NK cells. After stimulation with toll-like-receptor (TLR) agonists (lipopolysaccharides, Pam3CSK4), isolated NK cells showed increased interferon (IFN)-gamma secretion. To study the role of NK cells in a complex hepatic environment, these cells were integrated in the vascular compartment of a microfluidically supported liver-on-a-chip model in close interaction with endothelial and resident macrophages. Successful, functional integration of NK cells was verified by immunofluorescence staining (NKp46), flow cytometry analysis and TLR agonist-dependent secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. Lastly, we observed that inflammatory activation of NK cells in the liver-on-a-chip led to a loss of vascular barrier integrity. Overall, our data shows the first successful, functional integration of NK cells in a liver-on-a-chip model that can be utilized to investigate NK cell-dependent effects on liver inflammation in vitro

Tailoring the Degradation Time of Polycationic PEG-Based Hydrogels toward Dynamic Cell Culture Matrices

Poly(ethylene glycol)-based (PEG) hydrogels provide an ideal platform to obtain well-defined and tailor-made cell culture matrices to enhance in vitro cell culture conditions, although cell adhesion is often challenging when the cells are cultivated on the substrate surface. We herein demonstrate two approaches for the synthesis of polycationic PEG-based hydrogels which were modified to enhance cell-matrix interactions, to improve two-dimensional (2D) cell culture, and catalyze hydrolytic degradation. While the utilization of N , N -(bisacryloxyethyl) amine (BAA) as cross-linker for in situ gelation provides degradable scaffolds for dynamic cell culture, the incorporation of short segments of poly( N -(3-(dimethylamino)propyl)acrylamide) (PDMAPAam) provides high local cationic charge density leading to PEG-based hydrogels with high selectivity for fibroblastic cell lines. The adsorption of transforming growth factor (TGF-β) into the hydrogels induced stimulation of fibrosis and thus the formation of collagen as a natural ECM compound. With this, these dynamic hydrogels enhance in vitro cell culture by providing a well-defined, artificial, and degradable matrix that stimulates cells to produce their own natural scaffold within a defined time frame

A versatile and customizable low-cost 3D-printed open standard for microscopic imaging

Modern microscopes used for biological imaging often present themselves as black boxes whose precise operating principle remains unknown, and whose optical resolution and price seem to be in inverse proportion to each other. With UC2 (You. See. Too.) we present a low-cost, 3D-printed, open-source, modular microscopy toolbox and demonstrate its versatility by realizing a complete microscope development cycle from concept to experimental phase. The self-contained incubator-enclosed brightfield microscope monitors monocyte to macrophage cell differentiation for seven days at cellular resolution level (e.g. 2 μm). Furthermore, by including very few additional components, the geometry is transferred into a 400 Euro light sheet fluorescence microscope for volumetric observations of a transgenic Zebrafish expressing green fluorescent protein (GFP). With this, we aim to establish an open standard in optics to facilitate interfacing with various complementary platforms. By making the content and comprehensive documentation publicly available, the systems presented here lend themselves to easy and straightforward replications, modifications, and extensions

Facilitating advanced Sentinel-2 analysis through a simplified computation of Nadir BRDF Adjusted Reflectance

The Sentinel-2 (S2) mission from the European Space Agency’s Copernicus program provides essential data for Earth surface analysis. Its Level-2A products deliver high-to-medium resolution (10–60 m) surface reflectance (SR) data through the MultiSpectral Instrument (MSI). To enhance the accuracy and comparability of SR data, adjustments simulating a nadir viewing perspective are essential. These corrections address the anisotropic nature of SR and the variability in sun and observation angles, ensuring consistent image comparisons over time and under different conditions. The c-factor method, a simple yet effective algorithm, adjusts observed S2 SR by using the MODIS BRDF model to achieve Nadir BRDF Adjusted Reflectance (NBAR). Despite the straightforward application of the c-factor to individual images, a cohesive Python framework for its application across multiple S2 images and Earth System Data Cubes (ESDCs) from cloud-stored data has been lacking. Here we introduce sen2nbar, a Python package crafted to convert S2 SR data to NBAR, supporting both individual images and ESDCs derived from cloud-stored data. This package simplifies the conversion of S2 SR data to NBAR via a single function, organized into modules for efficient process management. By facilitating NBAR conversion for both SAFE files and ESDCs from SpatioTemporal Asset Catalogs (STAC), sen2nbar is developed as a flexible tool that can handle diverse data format requirements. We anticipate that sen2nbar will considerably contribute to the standardization and harmonization of S2 data, offering a robust solution for a diverse range of users across various applications. sen2nbar is an open-source tool available at https://github.com/ESDS-Leipzig/sen2nbar

On the analytic-numeric treatment of weakly singular integrals on arbitrary polygonal domains

An alternative analytical approach to calculate the weakly singular free-space static potential integral associated to uniform sources is presented. Arbitrary oriented flat polygons are considered as integration domains. The technique stands out by its mathematical simplicity and it is based on a novel integral transformation. The presented formula is equivalent to others existing in literature, being also concise and suitable within a singularity subtraction framework. Generalized Cartesian product rules built on the double exponential formula are utilized to integrate numerically the resulting analytical 2D potential integral. As a consequence, drawbacks associated to endpoint singularities in the derivative of the potential are tempered. Numerical examples within a surface integral equation-Method of Moments framework are finally provided

A genome scan for milk production traits in dairy goats reveals two new mutations in <i>Dgat1</i> reducing milk fat content

The quantity of milk and milk fat and proteins are particularly important traits in dairy livestock. However, little is known about the regions of the genome that influence these traits in goats. We conducted a genome wide association study in French goats and identified 109 regions associated with dairy traits. For a major region on chromosome 14 closely associated with fat content, the Diacylglycerol O-Acyltransferase 1 (DGAT1) gene turned out to be a functional and positional candidate gene. The caprine reference sequence of this gene was completed and 29 polymorphisms were found in the gene sequence, including two novel exonic mutations: R251L and R396W, leading to substitutions in the protein sequence. The R251L mutation was found in the Saanen breed at a frequency of 3.5% and the R396W mutation both in the Saanen and Alpine breeds at a frequencies of 13% and 7% respectively. The R396W mutation explained 46% of the genetic variance of the trait, and the R251L mutation 6%. Both mutations were associated with a notable decrease in milk fat content. Their causality was then demonstrated by a functional test. These results provide new knowledge on the genetic basis of milk synthesis and will help improve the management of the French dairy goat breeding program