Optimizing Transmembrane Protein Assemblies in Nanodiscs for Structural Studies: A Comprehensive Manual.

Membrane protein structures offer a more accurate basis for understanding their functional correlates when derived from full-length proteins in their native lipid environment. Producing such samples has been a primary challenge in the field. Here, we present robust, step-by-step biochemical and biophysical protocols for generating monodisperse assemblies of full-length transmembrane proteins within lipidic environments.

High-resolution yeast actin structures indicate the molecular mechanism of actin filament stiffening by cations.

Actin filament assembly and the regulation of its mechanical properties are fundamental processes essential for eukaryotic cell function. Residue E167 in vertebrate actins forms an inter-subunit salt bridge with residue K61 of the adjacent subunit. Saccharomyces cerevisiae actin filaments are more flexible than vertebrate filaments and have an alanine at this position (A167).

CryoEM Workflow Acceleration with Feret Signatures.

Common challenges in cryogenic electron microscopy, such as orientation bias, conformational diversity, and 3D misclassification, complicate single particle analysis and lead to significant resource expenditure. We previously introduced an in silico method using the maximum Feret diameter distribution, the Feret signature, to characterize sample heterogeneity of disc-shaped samples. Here, we expanded the Feret signature methodology to identify preferred orientations of samples containing arbitrary shapes with only about 1000 particles required.

The GoldX Fiducial Eraser.

Gold nanoparticles with sizes in the range of 5-15 nm are a standard method of providing fiducial markers to assist with alignment during reconstruction in cryogenic electron tomography. However, due to their high electron density and resulting contrast when compared to standard cellular or biological samples, they introduce artifacts such as streaking in the reconstructed tomograms. Here, we demonstrate a tool that automatically detects these nanoparticles and suppresses them by replacing them with a local background as a post-processing step, providing a cleaner tomogram without removing any sample relevant information or introducing new artifacts or edge effects from uniform density replacements.

Occurrence of ultrasonographic assessed placental abnormalities, treatments, pregnancy outcome, and subsequent fertility on a large warmblood stud farm: A retrospective field study.

Little is known about the incidence and outcome of high-risk pregnancies in equine practice and clinical studies on spontaneous occurring placentitis cases and treatments are missing. Therefore, the aims of this retrospective field study were to (1) describe the incidence and severity of ultrasonographic assessed placental abnormalities (UPA) in 4,192 pregnancies on a large commercial warmblood stud farm in 2017 - 2019 and (2) characterize these UPA cases and their pregnancy outcome. UPA severity (Placental abnormality score (PSc) 1-3; low to high), nine treatment regimens (TM1-9) used in UPA cases and treatment duration as well as subsequent fertility were analyzed in the group of UPA mares.

Research Note: Differences in the pecking behavior of turkey hens in proximity to the feeding pan depending on their beak condition.

The present study aimed to provide further insight on different pecking behavior of beak-trimmed and untrimmed turkey hens in proximity to the feeding pan. Investigations were carried out in 4 housing compartments with female fattening turkeys where video were recorded and evaluated with regard to 4 different types of (pecking-) behavior (feeding, pecking on the ground, pecking at conspecifics, being pecked) observed in 1 focal animal in proximity to each feeding pan. Concerning the median duration, there were only small differences between the animals with different beak conditions.

Harmonizing the Generation and Pre-publication Stewardship of FAIR bioimage data.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured bioimage data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable bioimage data sharing in the life sciences.

Heat Stress Trends in Regions of Intensive Turkey Production in Germany-A Challenge in Times of Climate Change.

This study analyzed trends of enthalpy and the temperature-humidity index (THI) over a period of 50 years in outer air, which lead to severe heat stress in turkeys. Weather station data from 15 German districts with high densities of turkey production were used to investigate the heat input into the barns. Therefore, the parameters of enthalpy and THI with specified thresholds were used for heat stress assessment.

Morphological control enables nanometer-scale dissection of cell-cell signaling complexes.

Protein micropatterning enables robust control of cell positioning on electron-microscopy substrates for cryogenic electron tomography (cryo-ET). However, the combination of regulated cell boundaries and the underlying electron-microscopy substrate (EM-grids) provides a poorly understood microenvironment for cell biology. Because substrate stiffness and morphology affect cellular behavior, we devised protocols to characterize the nanometer-scale details of the protein micropatterns on EM-grids by combining cryo-ET, atomic force microscopy, and scanning electron microscopy.

Enhancement of Immunosuppressive Activity of Mesenchymal Stromal Cells by Platelet-Derived Factors is Accompanied by Apoptotic Priming.

The pro-inflammatory phase of bone healing, initiated by platelet activation and eventually hematoma formation, impacts bone marrow mesenchymal stromal cells (MSCs) in unknown ways. Here, we created platelet-rich plasma (PRP) hydrogels to study how platelet-derived factors modulate functional properties of encapsulated MSCs in comparison to a non-inflammatory fibrin (FBR) hydrogel environment. MSCs were isolated from human bone marrow, while PRP was collected from pooled apheresis thrombocyte concentrates and used for hydrogel preparation.

MEPSi: A tool for simulating tomograms of membrane-embedded proteins.

The throughput and fidelity of cryogenic cellular electron tomography (cryo-ET) is constantly increasing through advances in cryogenic electron microscope hardware, direct electron detection devices, and powerful image processing algorithms. However, the need for careful optimization of sample preparations and for access to expensive, high-end equipment, make cryo-ET a costly and time-consuming technique. Generally, only after the last step of the cryo-ET workflow, when reconstructed tomograms are available, it becomes clear whether the chosen imaging parameters were suitable for a specific type of sample in order to answer a specific biological question.

Characterization of heterogeneity in nanodisc samples using Feret signatures.

Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process.

Novel cryo-tomography workflow reveals nanometer-scale responses of epithelial cells to matrix stiffness and dimensionality.

Matrix stiffness and dimensionality have been shown to be major determinants of cell behavior. However, a workflow for examining nanometer-scale responses of the associated molecular machinery is not available. Here, we describe a comprehensive, quantitative workflow that permits the analysis of cells responding to mechanical and dimensionality cues in their native state at nanometer scale by cryogenic electron tomography.

Occurrence of claw asymmetries in fattening pigs and potential impact on the development of sole ulcerations.

Background: Claw abnormalities, particularly claw asymmetries, are associated with lameness in pigs and can be a welfare issue. However, the prevalence and development of claw asymmetries in pigs of different age is unknown. Therefore, the aim of this study was to analyze the claw symmetry over the pig lifetime from birth to slaughter as well as the occurrence of sole ulcerations in fattening pigs possibly caused by such asymmetric claws.

Keypoint Detection for Injury Identification during Turkey Husbandry Using Neural Networks.

Injurious pecking against conspecifics is a serious problem in turkey husbandry. Bloody injuries act as a trigger mechanism to induce further pecking, and timely detection and intervention can prevent massive animal welfare impairments and costly losses. Thus, the overarching aim is to develop a camera-based system to monitor the flock and detect injuries using neural networks.

ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells.

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion.

Footpad Monitoring: Reliability of an Automated System to Assess Footpad Dermatitis in Turkeys () During Slaughter.

Footpad dermatitis (FPD) is an indicator of animal welfare in turkeys, giving evidence of the animals' physical integrity and providing information on husbandry management. Automated systems for assessing FPD at slaughter can present a useful tool for objective data collection. However, using automated systems requires that they reliably assess the incidence.

The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.

Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles.

Rapid tool for cell nanoarchitecture integrity assessment.

With the rapid increase and accessibility of high-resolution imaging technologies of cells, the interpretation of results relies more and more on the assumption that the three-dimensional integrity of the surrounding cellular landscape is not compromised by the experimental setup. However, the only available technology for directly probing the structural integrity of whole-cell preparations at the nanoscale is electron cryo-tomography, which is time-consuming, costly, and complex. We devised an accessible, inexpensive and reliable screening assay to quickly report on the compatibility of experimental protocols with preserving the structural integrity of whole-cell preparations at the nanoscale.

Learn to Train: Improving Training Data for a Neural Network to Detect Pecking Injuries in Turkeys.

This study aimed to develop a camera-based system using artificial intelligence for automated detection of pecking injuries in turkeys. Videos were recorded and split into individual images for further processing. Using specifically developed software, the injuries visible on these images were marked by humans, and a neural network was trained with these annotations.

The actomyosin interface contains an evolutionary conserved core and an ancillary interface involved in specificity.

Plasmodium falciparum, the causative agent of malaria, moves by an atypical process called gliding motility. Actomyosin interactions are central to gliding motility. However, the details of these interactions remained elusive until now.

On-farm detection of claw lesions in dairy cows based on acoustic analyses and machine learning.

Claw lesions are a serious problem on dairy farms, affecting both the health and welfare of the cow. Automated detection of lameness with a practical, on-farm application would support the early detection and treatment of lame cows, potentially reducing the number and severity of claw lesions. Therefore, in this study, a method was proposed for the detection of claw lesions based on the acoustic analysis of a cow's gait.

Walking on Tiptoes: Digital Pads Deserve Increased Attention When Scoring Footpad Dermatitis as an Animal Welfare Indicator in Turkeys.

Animal welfare is one of the most challenging issues in modern farm animal husbandry. Animal welfare indicators can be used to monitor welfare on farms or at slaughterhouses, with footpad dermatitis (FPD) being one of the most important indicators used in turkeys. Up to now, the severity of FPD has been measured by evaluating the size of altered lesions on the metatarsal pad of birds.

Large Group Housing Systems in Fattening Bulls-Comparison of Behavior and Performance.

According to international housing recommendations, fattening bulls should not be housed in groups of more than 12-20 animals. However, there are no scientific studies supporting these recommendations as most studies on fattening cattle refer to smaller groups. Therefore, the aim of this study was to analyze and compare behavior and performance of 187 fattening bulls housed in different group sizes of 16, 22, and 33 animals.

[Lying synchronization in fattening cattle at different space allowances].

Objective: Lying synchronization is an important characteristic of the natural behavior in cattle. The guideline for the housing of fattening cattle published in 2018 in Lower Saxony, Germany, requests for the first time that the lying area should be large enough to enable all animals to lie down simultaneously. However, up to now, lying synchrony in fattening cattle has only been scientifically analyzed to a small extent and detailed data on lying space requirements of fattening bulls are lacking altogether.