Polyacrylamide gel technique for the histochemical demonstration of soluble enzymes

Summary Soluble enzymes were immobilized and visualized by polyacrylamide gel slabs, impregnated with the incubation medium including auxiliairy enzymes. The method has several advantages over existing techniques which make use of gel films or a semipermeable membrane. The diffusion of tissue compounds is effectively limited, while auxiliary enzymes may be operative. Moreover the viscosity of the medium is temperature-independent so that the incubation temperature can be varied.To demonstrate the suitability of the method glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, phosphoglucomutase and aldolase were visualized in human or rat skeletal muscle. Cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase were both visualized in the absence of added NAD⁺ and menadione.For the visualization of ATP producing enzymes, like creatine kinase and pyruvate kinase, the method is not suitable.     

Developmental structuring of phenotypic variation: A case study with a cellular automata model of ontogeny

Developmental mechanisms not only produce an organismal phenotype, but they also structure the way genetic variation maps to phenotypic variation. Here, we revisit a computational model for the evolution of ontogeny based on cellular automata, in which evolution regularly discovered two alternative mechanisms for achieving a selected phenotype, one showing high modularity, the other showing morphological integration. We measure a primary variational property of the systems, their distribution of fitness effects of mutation. We find that the modular ontogeny shows the evolution of mutational robustness and ontogenic simplification, while the integrated ontogeny does not. We discuss the wider use of this methodology on other computational models of development as well as real organisms.     

Soil fauna diversity increases CO2 but suppresses N2O emissions from soil

Soil faunal activity can be a major control of greenhouse gas (GHG) emissions from soil. Effects of single faunal species, genera or families have been investigated, but it is unknown how soil fauna diversity may influence emissions of both carbon dioxide (CO₂, end product of decomposition of organic matter) and nitrous oxide (N₂O, an intermediate product of N transformation processes, in particular denitrification). Here, we studied how CO₂ and N₂O emissions are affected by species and species mixtures of up to eight species of detritivorous/fungivorous soil fauna from four different taxonomic groups (earthworms, potworms, mites, springtails) using a microcosm set‐up. We found that higher species richness and increased functional dissimilarity of species mixtures led to increased faunal‐induced CO₂ emission (up to 10%), but decreased N₂O emission (up to 62%). Large ecosystem engineers such as earthworms were key drivers of both CO₂ and N₂O emissions. Interestingly, increased biodiversity of other soil fauna in the presence of earthworms decreased faunal‐induced N₂O emission despite enhanced C cycling. We conclude that higher soil fauna functional diversity enhanced the intensity of belowground processes, leading to more complete litter decomposition and increased CO₂ emission, but concurrently also resulting in more complete denitrification and reduced N₂O emission. Our results suggest that increased soil fauna species diversity has the potential to mitigate emissions of N₂O from soil ecosystems. Given the loss of soil biodiversity in managed soils, our findings call for adoption of management practices that enhance soil biodiversity and stimulate a functionally diverse faunal community to reduce N₂O emissions from managed soils.     

Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1

Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.