SCHENGEN receptor module drives localized ROS production and lignification in plant roots.

Production of reactive oxygen species (ROS) by NADPH oxidases (NOXs) impacts many processes in animals and plants, and many plant receptor pathways involve rapid, NOX-dependent increases of ROS. Yet, their general reactivity has made it challenging to pinpoint the precise role and immediate molecular action of ROS. A well-understood ROS action in plants is to provide the co-substrate for lignin peroxidases in the cell wall. Lignin can be deposited with exquisite spatial control, but the underlying mechanisms have remained elusive. Here, we establish a kinase signaling relay that exerts direct, spatial control over ROS production and lignification within the cell wall. We show that polar localization of a single kinase component is crucial for pathway function. Our data indicate that an intersection of more broadly localized components allows for micrometer-scale precision of lignification and that this system is triggered through initiation of ROS production as a critical peroxidase co-substrate.

Active PD-L1 incorporation within HIV virions functionally impairs T follicular helper cells.

The limited development of broadly neutralizing antibodies (BnAbs) during HIV infection is classically attributed to an inadequate B-cell help brought by functionally impaired T follicular helper (Tfh) cells. However, the determinants of Tfh-cell functional impairment and the signals contributing to this condition remain elusive. In the present study, we showed that PD-L1 is incorporated within HIV virions through an active mechanism involving p17 HIV matrix protein. We subsequently showed that in vitro produced PD-L1high but not PD-L1low HIV virions, significantly reduced Tfh-cell proliferation and IL-21 production, ultimately leading to a decreased of IgG1 secretion from GC B cells. Interestingly, Tfh-cell functions were fully restored in presence of anti-PD-L1/2 blocking mAbs treatment, demonstrating that the incorporated PD-L1 proteins were functionally active. Taken together, the present study unveils an immunovirological mechanism by which HIV specifically exploits the regulatory potential of PD-L1 to suppress the immune system during the course of HIV infection.

Correlated cryo-SEM and CryoNanoSIMS imaging of biological tissue.

BACKGROUND: The development of nanoscale secondary ion mass spectrometry (NanoSIMS) has revolutionized the study of biological tissues by enabling, e.g., the visualization and quantification of metabolic processes at subcellular length scales. However, the associated sample preparation methods all result in some degree of tissue morphology distortion and loss of soluble compounds. To overcome these limitations an entirely cryogenic sample preparation and imaging workflow is required. RESULTS: Here, we report the development of a CryoNanoSIMS instrument that can perform isotope imaging of both positive and negative secondary ions from flat block-face surfaces of vitrified biological tissues with a mass- and image resolution comparable to that of a conventional NanoSIMS. This capability is illustrated with nitrogen isotope as well as trace element mapping of freshwater hydrozoan Green Hydra tissue following uptake of 15N-enriched ammonium. CONCLUSION: With a cryo-workflow that includes vitrification by high pressure freezing, cryo-planing of the sample surface, and cryo-SEM imaging, the CryoNanoSIMS enables correlative ultrastructure and isotopic or elemental imaging of biological tissues in their most pristine post-mortem state. This opens new horizons in the study of fundamental processes at the tissue- and (sub)cellular level. TEASER: CryoNanoSIMS: subcellular mapping of chemical and isotopic compositions of biological tissues in their most pristine post-mortem state.

Genome mapping of a LYST mutation in corn snakes indicates that vertebrate chromatophore vesicles are lysosome-related organelles.

Reptiles exhibit a spectacular diversity of skin colors and patterns brought about by the interactions among three chromatophore types: black melanophores with melanin-packed melanosomes, red and yellow xanthophores with pteridine- and/or carotenoid-containing vesicles, and iridophores filled with light-reflecting platelets generating structural colors. Whereas the melanosome, the only color-producing endosome in mammals and birds, has been documented as a lysosome-related organelle, the maturation paths of xanthosomes and iridosomes are unknown. Here, we first use 10x Genomics linked-reads and optical mapping to assemble and annotate a nearly chromosome-quality genome of the corn snake Pantherophis guttatus The assembly is 1.71 Gb long, with an N50 of 16.8 Mb and L50 of 24. Second, we perform mapping-by-sequencing analyses and identify a 3.9-Mb genomic interval where the lavender variant resides. The lavender color morph in corn snakes is characterized by gray, rather than red, blotches on a pink, instead of orange, background. Third, our sequencing analyses reveal a single nucleotide polymorphism introducing a premature stop codon in the lysosomal trafficking regulator gene (LYST) that shortens the corresponding protein by 603 amino acids and removes evolutionary-conserved domains. Fourth, we use light and transmission electron microscopy comparative analyses of wild type versus lavender corn snakes and show that the color-producing endosomes of all chromatophores are substantially affected in the LYST mutant. Our work provides evidence characterizing xanthosomes in xanthophores and iridosomes in iridophores as lysosome-related organelles.

Antibiotic Exposure Leads to Reduced Phage Susceptibility in Vancomycin Intermediate Staphylococcus aureus (VISA).

In the time of antimicrobial resistance, phage therapy is frequently suggested as a possible solution for such difficult-to-treat infections. Vancomycin-intermediate Staphylococcus aureus (VISA) remains a relatively rare yet increasing occurrence in the clinic for which phage therapy may be an option. However, the data presented herein suggest a potential cross-resistance mechanism to phage following vancomycin exposure in VISA strains. When comparing genetically similar strains differing in their susceptibility to vancomycin, those with intermediate levels of vancomycin resistance displayed decreased sensitivity to phage in solid and liquid assays. Serial passaging with vancomycin induced both reduced vancomycin susceptibility and phage sensitivity. As a consequence, the process of phage infection was shown to be interrupted after DNA ejection from adsorbed phage but prior to phage DNA replication, as demonstrated through adsorption assays, lysostaphin sensitivity assays, electron microscopy, and quantitative PCR (qPCR). At a time when phage products are being used for experimental treatments and tested in clinical trials, it is important to understand possible interference between mechanisms underlying antibiotic and phage resistance in order to design effective therapeutic regimens.

Hidden cell diversity in Placozoa: ultrastructural insights from Hoilungia hongkongensis.

From a morphological point of view, placozoans are among the most simple free-living animals. This enigmatic phylum is critical for our understanding of the evolution of animals and their cell types. Their millimeter-sized, disc-like bodies consist of only three cell layers that are shaped by roughly seven major cell types. Placozoans lack muscle cells and neurons but are able to move using their ciliated lower surface and take up food in a highly coordinated manner. Intriguingly, the genome of Trichoplax adhaerens, the founding member of the enigmatic phylum, has disclosed a surprising level of genetic complexity. Moreover, recent molecular and functional investigations have uncovered a much larger, so-far hidden cell-type diversity. Here, we have extended the microanatomical characterization of a recently described placozoan species-Hoilungia hongkongensis. In H. hongkongensis, we recognized the established canonical three-layered placozoan body plan but also came across several morphologically distinct and potentially novel cell types, among them novel gland cells and "shiny spheres"-bearing cells at the upper epithelium. Thus, the diversity of cell types in placozoans is indeed higher than anticipated.

Tracking the cargo of extracellular symbionts into host tissues with correlated electron microscopy and nanoscale secondary ion mass spectrometry imaging.

Extracellular bacterial symbionts communicate biochemically with their hosts to establish niches that foster the partnership. Using quantitative ion microprobe isotopic imaging (nanoscale secondary ion mass spectrometry [NanoSIMS]), we surveyed localization of 15 N-labelled molecules produced by the bacterium Vibrio fischeri within the cells of the symbiotic organ of its host, the Hawaiian bobtail squid, and compared that with either labelled non-specific species or amino acids. In all cases, two areas of the organ's epithelia were significantly more 15 N enriched: (a) surface ciliated cells, where environmental symbionts are recruited, and (b) the organ's crypts, where the symbiont population resides in the host. Label enrichment in all cases was strongest inside host cell nuclei, preferentially in the euchromatin regions and the nucleoli. This permissiveness demonstrated that uptake of biomolecules is a general mechanism of the epithelia, but the specific responses to V. fischeri cells recruited to the organ's surface are due to some property exclusive to this species. Similarly, in the organ's deeper crypts, the host responds to common bacterial products that only the specific symbiont can present in that location. The application of NanoSIMS allows the discovery of such distinct modes of downstream signalling dependent on location within the host and provides a unique opportunity to study the microbiogeographical patterns of symbiotic dialogue.

Correction: Comparative genomics and the nature of placozoan species.

[This corrects the article DOI: 10.1371/journal.pbio.2005359.].

Comparative genomics and the nature of placozoan species.

Placozoans are a phylum of nonbilaterian marine animals currently represented by a single described species, Trichoplax adhaerens, Schulze 1883. Placozoans arguably show the simplest animal morphology, which is identical among isolates collected worldwide, despite an apparently sizeable genetic diversity within the phylum. Here, we use a comparative genomics approach for a deeper appreciation of the structure and causes of the deeply diverging lineages in the Placozoa. We generated a high-quality draft genome of the genetic lineage H13 isolated from Hong Kong and compared it to the distantly related T. adhaerens. We uncovered substantial structural differences between the two genomes that point to a deep genomic separation and provide support that adaptation by gene duplication is likely a crucial mechanism in placozoan speciation. We further provide genetic evidence for reproductively isolated species and suggest a genus-level difference of H13 to T. adhaerens, justifying the designation of H13 as a new species, Hoilungia hongkongensis nov. gen., nov. spec., now the second described placozoan species and the first in a new genus. Our multilevel comparative genomics approach is, therefore, likely to prove valuable for species distinctions in other cryptic microscopic animal groups that lack diagnostic morphological characters, such as some nematodes, copepods, rotifers, or mites.

Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis.

The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development.

Cuticular Defects in Oryza sativa ATP-binding Cassette Transporter G31 Mutant Plants Cause Dwarfism, Elevated Defense Responses and Pathogen Resistance.

The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense.

The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle in Arabidopsis.

The cuticle is an essential diffusion barrier on aerial surfaces of land plants whose structural component is the polyester cutin. The PERMEABLE CUTICLE1/ABCG32 (PEC1) transporter is involved in plant cuticle formation in Arabidopsis. The gpat6 pec1 and gpat4 gapt8 pec1 double and triple mutants are characterized. Their PEC1-specific contributions to aliphatic cutin composition and cuticle formation during plant development are revealed by gas chromatography/mass spectrometry and Fourier-transform infrared spectroscopy. The composition of cutin changes during rosette leaf expansion in Arabidopsis. C16:0 monomers are in higher abundance in expanding than in fully expanded leaves. The atypical cutin monomer C18:2 dicarboxylic acid is more prominent in fully expanded leaves. Findings point to differences in the regulation of several pathways of cutin precursor synthesis. PEC1 plays an essential role during expansion of the rosette leaf cuticle. The reduction of C16 monomers in the pec1 mutant during leaf expansion is unlikely to cause permeability of the leaf cuticle because the gpat6 mutant with even fewer C16:0 monomers forms a functional rosette leaf cuticle at all stages of development. PEC1/ABCG32 transport activity affects cutin composition and cuticle structure in a specific and non-redundant fashion.

Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.

In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼ 500 nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB-SEM tomography, became available to image much larger volumes. During TEM tomography and FIB-SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples.

Host-derived organic acids enable gut colonization of the honey bee symbiont Snodgrassella alvi.

Diverse bacteria can colonize the animal gut using dietary nutrients or by engaging in microbial crossfeeding interactions. Less is known about the role of host-derived nutrients in enabling gut bacterial colonization. Here we examined metabolic interactions within the evolutionary ancient symbiosis between the honey bee (Apis mellifera) and the core gut microbiota member Snodgrassella alvi. This betaproteobacterium is incapable of metabolizing saccharides, yet colonizes the honey bee gut in the presence of a sugar-only diet. Using comparative metabolomics, 13C-tracers and nanoscale secondary ion mass spectrometry (NanoSIMS), we show in vivo that S. alvi grows on host-derived organic acids, including citrate, glycerate and 3-hydroxy-3-methylglutarate, which are actively secreted by the host into the gut lumen. S. alvi also modulates tryptophan metabolism in the gut by converting kynurenine to anthranilate. These results suggest that S. alvi is adapted to a specific metabolic niche in the honey bee gut that depends on host-derived nutritional resources.

In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with ß-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.

Uptake without inactivation of human adenovirus type 2 by Tetrahymena pyriformis ciliates.

Human adenoviruses are ubiquitous contaminants of surface water. Indigenous protists may interact with adenoviruses and contribute to their removal from the water column, though the associated kinetics and mechanisms differ between protist species. In this work, we investigated the interaction of human adenovirus type 2 (HAdV2) with the ciliate Tetrahymena pyriformis. In co-incubation experiments in a freshwater matrix, T. pyriformis was found to efficiently remove HAdV2 from the aqueous phase, with ≥4 log10 removal over 72 hours. Neither sorption onto the ciliate nor secreted compounds contributed to the observed loss of infectious HAdV2. Instead, internalization was shown to be the dominant removal mechanism, resulting in the presence of viral particles inside food vacuoles of T. pyriformis, as visualized by transmission electron microscopy. The fate of HAdV2 once ingested was scrutinized and no evidence of virus digestion was found over the course of 48 hours. This work shows that T. pyriformis can exert a dual role in microbial water quality: while they remove infectious adenovirus from the water column, they can also accumulate infectious viruses.

SEM3De: image restoration for FIB-SEM.

Motivation: FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is a technique to generate 3D images of samples up to several microns in depth. The principle is based on the alternate use of SEM to image the surface of the sample (a few nanometers thickness) and of FIB to mill the surface of the sample a few nanometers at the time. In this way, huge stacks of images can thus be acquired.Although this technique has proven useful in imaging biological systems, the presence of some visual artifacts (stripes due to sample milling, detector saturation, charge effects, focus or sample drift, etc.) still raises some challenges for image interpretation and analyses. Results: With the aim of meeting these challenges, we developed a freeware (SEM3De) that either corrects artifacts with state-of-the-art approaches or, when artifacts are impossible to correct, enables the replacement of artifactual slices by an in-painted image created from adjacent non-artifactual slices. Thus, SEM3De improves the overall usability of FIB-SEM acquisitions. Availability and implementation: SEM3De can be downloaded from https://sourceforge.net/projects/sem3de/ as a plugin for ImageJ.

Extracellular vesiculo-tubular structures associated with suberin deposition in plant cell walls.

Suberin is a fundamental plant biopolymer, found in protective tissues, such as seed coats, exodermis and endodermis of roots. Suberin is deposited in most suberizing cells in the form of lamellae just outside of the plasma membrane, below the primary cell wall. How monomeric suberin precursors, thought to be synthesized at the endoplasmic reticulum, are transported outside of the cell, for polymerization into suberin lamellae has remained obscure. Using electron-microscopy, we observed large numbers of extracellular vesiculo-tubular structures (EVs) to accumulate specifically in suberizing cells, in both chemically and cryo-fixed samples. EV presence correlates perfectly with root suberization and we could block suberin deposition and vesicle accumulation by affecting early, as well as late steps in the secretory pathway. Whereas many previous reports have described EVs in the context of biotic interactions, our results suggest a developmental role for extracellular vesicles in the formation of a major cell wall polymer.

Renal tubular peroxisomes are dispensable for normal kidney function.

Peroxisomes are specialized cellular organelles involved in a variety of metabolic processes. In humans, mutations leading to complete loss of peroxisomes cause multiorgan failure (Zellweger's spectrum disorders, ZSD), including renal impairment. However, the (patho)physiological role of peroxisomes in the kidney remains unknown. We addressed the role of peroxisomes in renal function in mice with conditional ablation of peroxisomal biogenesis in the renal tubule (cKO mice). Functional analyses did not reveal any overt kidney phenotype in cKO mice. However, infant male cKO mice had lower body and kidney weights, and adult male cKO mice exhibited substantial reductions in kidney weight and kidney weight/body weight ratio. Stereological analysis showed an increase in mitochondria density in proximal tubule cells of cKO mice. Integrated transcriptome and metabolome analyses revealed profound reprogramming of a number of metabolic pathways, including metabolism of glutathione and biosynthesis/biotransformation of several major classes of lipids. Although this analysis suggested compensated oxidative stress, challenge with high-fat feeding did not induce significant renal impairments in cKO mice. We demonstrate that renal tubular peroxisomes are dispensable for normal renal function. Our data also suggest that renal impairments in patients with ZSD are of extrarenal origin.

Low number of neurosecretory vesicles in neuroblastoma impairs massive catecholamine release and prevents hypertension.

Introduction: Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system. It produces and releases metanephrines, which are used as biomarkers for diagnosis in plasma and urine. However, plasma catecholamine concentrations remain generally normal in children with NB. Thus, unlike pheochromocytoma and paraganglioma (PHEO/PGL), two other non-epithelial neuroendocrine tumors, hypertension is not part of the usual clinical picture of patients with NB. This suggests that the mode of production and secretion of catecholamines and metanephrines in NB is different from that in PHEO/PGL, but little is known about these discrepancies. Here we aim to provide a detailed comparison of the biosynthesis, metabolism and storage of catecholamines and metanephrines between patients with NB and PHEO. Method: Catecholamines and metanephrines were quantified in NB and PHEO/PGL patients from plasma and tumor tissues by ultra-high pressure liquid chromatography tandem mass spectrometry. Electron microscopy was used to quantify neurosecretory vesicles within cells derived from PHEO tumor biopsies, NB-PDX and NB cell lines. Chromaffin markers were detected by qPCR, IHC and/or immunoblotting. Results: Plasma levels of metanephrines were comparable between NB and PHEO patients, while catecholamines were 3.5-fold lower in NB vs PHEO affected individuals. However, we observed that intratumoral concentrations of metanephrines and catecholamines measured in NB were several orders of magnitude lower than in PHEO. Cellular and molecular analyses revealed that NB cell lines, primary cells dissociated from human tumor biopsies as well as cells from patient-derived xenograft tumors (NB-PDX) stored a very low amount of intracellular catecholamines, and contained only rare neurosecretory vesicles relative to PHEO cells. In addition, primary NB expressed reduced levels of numerous chromaffin markers, as compared to PHEO/PGL, except catechol O-methyltransferase and monoamine oxidase A. Furthermore, functional assays through induction of chromaffin differentiation of the IMR32 NB cell line with Bt2cAMP led to an increase of neurosecretory vesicles able to secrete catecholamines after KCl or nicotine stimulation. Conclusion: The low amount of neurosecretory vesicles in NB cytoplasm prevents catecholamine storage and lead to their rapid transformation by catechol O-methyltransferase into metanephrines that diffuse in blood. Hence, in contrast to PHEO/PGL, catecholamines are not secreted massively in the blood, which explains why systemic hypertension is not observed in most patients with NB.