Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

A portfolio is presented documenting economic, high-resolution correlative focused ion beam scanning electron microscopy (FIB/SEM) in routine, comprising: (i) the use of custom-labeled slides and coverslips, (ii) embedding of cells in thin, or ultra-thin resin layers for correlative light and electron microscopy (CLEM) and (iii) the claim to reach the highest resolution possible with FIB/SEM in xyz. Regions of interest (ROIs) defined in light microscope (LM), can be relocated quickly and precisely in SEM. As proof of principle, HeLa cells were investigated in 3D context at all stages of the cell cycle, documenting ultrastructural changes during mitosis: nuclear envelope breakdown and reassembly, Golgi degradation and reconstitution and the formation of the midzone and midbody.     

Polyploid speciation across a suture zone: phylogeography and species delimitation in S French <Emphasis Type="Italic">Leucanthemum</Emphasis> Mill. representatives (Compositae–Anthemideae)

Chromosome counts suggest that the three varieties of the SE French and NW Italian Leucanthemum vulgare subsp. glaucophyllum (var. glaucophyllum: decaploid; var. esterellense: octoploid; var. subglaucum: hexaploid) deserve acknowledgement as independent species. Here we use AFLP fingerprinting and sequence information from two chloroplast regions (psbA-trnH, rpl16) to gain insight into the evolutionary relationships among those species, along with those with the co-occurring and ecologically similar L. pallens (hexaploid) and the closely related L. ircutianum subsp. leucolepis (tetraploid). Both genetic marker systems reveal a geographical differentiation pattern in L. pallens with the Rhône valley forming a suture zone between the western (SW France, Spain) and eastern (SE France, NW Italy) populations, arguing for the existence of two lineages in the species that may have survived the Last Glacial Maximum (LGM) in refugia on the Iberian and Apennine Peninsulas. Transgressions of this suture zone are observable in the case of the hexaploid L. subglaucum from the Massif Central, which shows genetic relationships to the more eastern decaploid L. glaucophyllum from the Alps Maritimes, and the octoploid L. esterellense from the Esterel Massif (Côte d´Azur), for which relationships to the more western populations of the hexaploids L. pallens and L. subglaucum are observable. The genetic and cytological survey of a mixed stand of L. pallens and L. glaucophyllum on Monte Bignone (Liguria, NW Italy) reveals patterns of recent hybridisation of these two species with intermediate octoploid and nonaploid cytotypes that are, however, genetically distinct from L. esterellense.     

The giant mycoheterotrophic orchid Erythrorchis altissima is associated mainly with a divergent set of wood‐decaying fungi

The climbing orchid Erythrorchis altissima is the largest mycoheterotroph in the world. Although previous in vitro work suggests that E. altissima has a unique symbiosis with wood‐decaying fungi, little is known about how this giant orchid meets its carbon and nutrient demands exclusively via mycorrhizal fungi. In this study, the mycorrhizal fungi of E. altissima were molecularly identified using root samples from 26 individuals. Furthermore, in vitro symbiotic germination with five fungi and stable isotope compositions in five E. altissima at one site were examined. In total, 37 fungal operational taxonomic units (OTUs) belonging to nine orders in Basidiomycota were identified from the orchid roots. Most of the fungal OTUs were wood‐decaying fungi, but underground roots had ectomycorrhizal Russula. Two fungal isolates from mycorrhizal roots induced seed germination and subsequent seedling development in vitro. Measurement of carbon and nitrogen stable isotope abundances revealed that E. altissima is a full mycoheterotroph whose carbon originates mainly from wood‐decaying fungi. All of the results show that E. altissima is associated with a wide range of wood‐ and soil‐inhabiting fungi, the majority of which are wood‐decaying taxa. This generalist association enables E. altissima to access a large carbon pool in woody debris and has been key to the evolution of such a large mycoheterotroph.     

Male reproductive health statement (XIIIth international symposium on Spermatology,may 9th–12th 2018, Stockholm,Sweden

On the occasion of the XIIIth International Symposium on Spermatology held from 9 to 13 May 2018 in Stockholm (Sweden), participants (guest speakers and audience) collectively felt the need to make a public statement on the general issue of male reproductive health. Our intention is to raise awareness of what we believe is a neglected area of research despite alarming situations around the world. The disclosure strategy desired by the co-authors is to bring it to the attention of the greatest number partly by considering co-publication in the various periodicals dealing with Reproductive Biology and Andrology. BaCA’s editorial office accepted this mission and found it natural that our periodical, the official journal of the French Andrology Society (SALF), should carry this message.     

The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development

MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA‐generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain‐derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca²⁺‐dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre‐miR16, was impaired. Decreased production of miR‐16‐5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane‐targeted TRBP. Moreover, miR‐16‐5p or membrane‐targeted TRBP expression blocked BDNF‐induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity‐dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.     

Hepatic Rac1 GTPase contributes to liver-mediated basal immune homeostasis and LPS-induced endotoxemia

Background

The Ras-homologous GTPase Rac1 plays a key role in the regulation of gene expression, cytoskeleton-associated processes and cell death as well as carcinogenesis and inflammation. Here, we investigated the impact of Rac1 signaling on liver-mediated immune homeostasis.

Anaerobic biotransformation of hexachlorocyclohexane isomers by <Emphasis Type="Italic">Dehalococcoides</Emphasis> species and an enrichment culture

The biotransformation of hexachlorocyclohexane isomers (HCH) by two Dehalococcoides mccartyi strains (195 and BTF08) and an enrichment culture was investigated and compared to conversion by the obligate anaerobic strain Clostridium pasteurianum strain DSMZ 525. The D. mccartyi strains preferentially transformed γ-HCH over α-HCH and δ-HCH isomers while β-HCH biotransformation was not significant. In case of the enrichment culture, γ-HCH was preferentially transformed over the δ-HCH, β-HCH and α-HCH isomers. Major observed metabolites in both cases were tetrachlorocyclohexene and as end products monochlorobenzene (MCB) and benzene. Dechlorination of the γ-HCH isomer was linked to an increase in cell numbers for strain 195. γ-HCH transformation was linked to considerable carbon stable isotope fractionation with the enrichment factor ε_c?=???5.5?±?0.8‰ for D. mccartyi strain 195, ε_c?=???3.1?±?0.4‰ for the enrichment culture and ε_c?=???4.1?±?0.6‰ for co-metabolic transformation by C. pasteurianum.     

Density-dependent shift from facilitation to competition in a dwarf <Emphasis Type="Italic">Avicennia germinans</Emphasis> forest

The global effort to rehabilitate and restore destroyed mangrove forests is unable to keep up with the high mangrove deforestation rates, which exceed the average pace of global deforestation. Although facilitation theory presents new possibilities for the restoration of heavily degraded mangrove sites, knowledge of tree–tree interactions in stressed mangrove forest ecosystems is too limited to utilize facilitation appropriately. The aim was to determine the mode of local interaction among stressed mangrove trees by investigating the effect of clustering on tree size and crown morphology under contrasting stand densities. The study was conducted in a dwarf Avicennia germinans forest in Northern Brazil, in which tree growth is limited by infrequent inundation and high pore-water salinity. Autoregressive regression, Voronoi tessellation and spatial point pattern statistics were used to address the spatial processes underlying tree interaction. Under low stand density (1.2 trees m^?2) dwarf trees which grew in clustered cohorts of A. germinans had a less stunted crown morphology revealing the dominance of a positive neighborhood influence among plants. In contrast, dwarf trees in the denser forest stand (2.7 trees m^?2) were interacting competitively as indicated by the more negative effect of neighbors on crown morphology and size. The shift from facilitative to competitive interactions is an important feature of mangrove forest regeneration under harsh environmental conditions. If mangrove trees are unable to regenerate naturally on severely degraded sites, intraspecific facilitation could be used to assist regeneration by planting seedlings in clusters and not evenly spaced.     

Trace Metal Imaging of Sulfate-Reducing Bacteria and Methanogenic Archaea at Single-Cell Resolution by Synchrotron X-Ray Fluorescence Imaging

Metal cofactors are required for many enzymes in anaerobic microbial respiration. This study examined iron, cobalt, nickel, copper, and zinc in cellular and abiotic phases at the single-cell scale for a sulfate-reducing bacterium (Desulfococcus multivorans) and a methanogenic archaeon (Methanosarcina acetivorans) using synchrotron X-ray fluorescence microscopy. Relative abundances of cellular metals were also measured by inductively coupled plasma mass spectrometry. For both species, zinc and iron were consistently the most abundant cellular metals. M. acetivorans contained higher nickel and cobalt content than D. multivorans, likely due to elevated metal requirements for methylotrophic methanogenesis. Cocultures contained spheroid zinc sulfides and cobalt/copper sulfides.