High affinity GDP binding sites on brown adipose tissue mitochondria of genetically obese ‘fa/fa’ rats

The number of high affinity [³H]GDP binding sites in brown adipose tissue mitochondria is normal in obese ( f a / f a ) rats in contrast to the reduced number of low affinity GDP binding sites. Adrenalectomy corrected the loss of low affinity binding sites in fa/fa rats but had no effect on the number of high affinity sites in either lean or obese rats. Equilibrium dialysis was used to show the presence of both high and low affinity binding sites on the purified 32 kdalton protein.     

Effects of physical training on several glycolytic enzymes in rat heart

In response to a program of daily swimming for 16 weeks, the activities of pyruvate kinase and lactate dehydrogenase increased significantly in the hearts of young male rats. The isozyme composition in M of cardiac lactate dehydrogenase increased from 28.5 to 32.7% in the trained animals. Phosphofructokinase activity and glycogen content were unchanged. The hearts of the exercising animals were 28% heavier than those of sedentary paired weight controls.     

A calcium‐mediated actin redistribution at egg activation in Drosophila

Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.     

Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

An assessment of the seascape genetic structure and hydrodynamic connectivity for subtropical seagrass restoration

Seagrass ecosystems have suffered significant declines globally and focus is shifting to restoration efforts. A key component to successful restoration is an understanding of the genetic factors potentially influencing restoration success. This includes understanding levels of connectivity between restoration locations and neighboring seagrass populations that promote natural recovery (source and sink populations), the identification of potential donor populations, and assessment of genetic diversity of restored meadows and material used for restoration. In this study, we carry out genetic surveys of 352 individuals from 13 populations using 11 polymorphic microsatellite loci to inform seagrass restoration activities by: (1) understanding levels of genetic and genotypic diversity within meadows; and (2) understanding genetic structure and patterns of connectivity among these meadows to determine which source sites may be most appropriate to assist recovery of three restoration sites. The study identified high genotypic diversity within the locations analyzed from the Port of Gladstone and Rodd's Bay region, indicating sexual reproduction is important in maintaining populations. Overall, we detected significant genetic structuring among sites with the Bayesian structure analysis identifying genetic clusters that largely conformed to a northern, central, and southern region. This suggests limited gene flow between regions, although there does appear to be some connectivity within regions. The hydrodynamic models showed that seeds were largely locally retained, while fragments were more widely dispersed. Limited gene flow between regions suggests donor material for restoration should be sourced locally where possible.     

Endometrial Exosomes/Microvesicles in the Uterine Microenvironment: A New Paradigm for Embryo-Endometrial Cross Talk at Implantation

Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100–300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50–150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.     

The response of cerambycid beetles (Coleoptera: Cerambycidae) to long‐term fire frequency regimes in subtropical eucalypt forest

Fire has a varied influence on plant and animal species through direct (e.g. fire‐induced mortality) and indirect (e.g. modification of habitat) effects. Our understanding of the influence of fire regime on invertebrates and their response to fire‐induced modifications to habitat is poor. We aimed to determine the response of a beetle family (Coleoptera: Cerambycidae) to varying fire treatments and hypothesised that the abundance of cerambycid beetles is influenced by fire frequency due to modifications in habitat associated with the fire treatments. Arthropods were sampled across 3 months in annually and triennially burnt areas (treatments starting in 1952 and 1973 respectively), an area unburnt since 1946, and a former unburnt treatment, burnt by wildfire in 2006. Eleven different cerambycid taxa were collected using flight intercept panel traps, dominated by three species (Ipomoria tillides, Adrium sp. and Bethelium signiferum) which made up 99% of individuals collected. Over the sampling period the long unburnt treatment had significantly lower species richness than the triennial and wildfire treatments. Cerambycid abundance was significantly higher in the triennially burnt treatment than in all other fire treatments. Ipomoria tillides was more abundant in both frequently burnt treatments, Adrium sp. was more common in triennially burnt areas, whereas B. signiferum, was more common in the wildfire affected treatment. Some, but not all, cerambycid beetles were more common in areas with a more open understorey (i.e. resulting from frequent burning), and lower tree basal area, as this likely influences their ability to fly easily between food sources. Cerambycid abundance was positively related to the volume of coarse woody debris and healthy tree crowns. Cerambycid beetles were clearly influenced by historic fire regime, suggesting that changes in fire regime can potentially have a profound influence on arthropod assemblages, and subsequent influences on ecosystem processes, which are currently poorly understood.