Efferocytosis and extrusion of leukocytes determine the progression of early mycobacterial pathogenesis.

Macrophages and neutrophils are the first responders to invading pathogens and contribute strongly to the host defense against intracellular pathogens. The collective interplay and dynamic interactions between these leukocytes are to a large extent not understood. In the present study, we have investigated their role using a combination of confocal laser-scanning and electron microscopy in a zebrafish model for tuberculosis, a local Mycobacterium marinum infection in the tissue of the larval tail fin.

Innexin7a forms junctions that stabilize the basal membrane during cellularization of the blastoderm in Tribolium castaneum.

In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophila melanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects.

Elucidation of the serosal cuticle machinery in the beetle Tribolium by RNA sequencing and functional analysis of Knickkopf1, Retroactive and Laccase2.

Insects have been extraordinary successful in colonizing terrestrial habitats and this success is partly due to a protective cuticle that mainly contains chitin and proteins. The cuticle has been well studied in larvae and adults, but little attention has been paid to the cuticle of the egg. This cuticle is secreted by the serosa, an extraembryonic epithelium that surrounds the yolk and embryo in all insect eggs, but was lost in the Schizophoran flies to which Drosophila belongs.

Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model.

High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques.

The DNA damage-regulated autophagy modulator DRAM1 links mycobacterial recognition via TLR-MYD88 to autophagic defense [corrected].

Autophagy is an important defense mechanism against mycobacteria, the causative agents of tuberculosis. The molecular mechanisms that link mycobacterial recognition to autophagy remain unclear. Our analysis in zebrafish and human macrophage models of mycobacterial infection reveals that the DNA damage-regulated autophagy modulator DRAM1 functions downstream of pathogen recognition by the Toll-like receptor (TLR)/interleukin-1 receptor (IL1R)-MYD88-NF-κB innate immune sensing pathway to activate selective autophagy.

Interaction between the GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX families of transcription factors.

KNOTTED1-LIKE HOMEOBOX (KNOX) genes are important regulators of meristem function, and a complex network of transcription factors ensures tight control of their expression. Here, we show that members of the GROWTH-REGULATING FACTOR (GRF) family act as players in this network. A yeast (Saccharomyces cerevisiae) one-hybrid screen with the upstream sequence of the KNOX gene Oskn2 from rice (Oryza sativa) resulted in isolation of OsGRF3 and OsGRF10.

The extraembryonic serosa protects the insect egg against desiccation.

Insects have been extraordinarily successful in occupying terrestrial habitats, in contrast to their mostly aquatic sister group, the crustaceans. This success is typically attributed to adult traits such as flight, whereas little attention has been paid to adaptation of the egg. An evolutionary novelty of insect eggs is the serosa, an extraembryonic membrane that enfolds the embryo and secretes a cuticle.

Single-walled carbon nanotubes as scaffolds to concentrate DNA for the study of DNA-protein interactions.

Genomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single-walled carbon nanotubes (Strep-SWNTs) through biotin-streptavidin interactions. We reasoned that confining DNA within a defined space through mechanical constraints, rather than by manipulating buffer conditions, would more closely resemble physiological conditions.

Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model.

Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within 1 week.

Peptide modified mesoporous silica nanocontainers.

Here we report a new peptide modified mesoporous silica nanocontainer (PMSN) as a novel controlled release system. The peptides are part of a stimuli responsive nanovalve and ensure an efficient cellular uptake.

Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2.

From a pool of transgenic Arabidopsis (Arabidopsis thaliana) plants harboring an activator T-DNA construct, one mutant was identified that developed spontaneous necrotic spots (sns-D) on the rosette leaves under aseptic conditions. The sns-D mutation is dominant and homozygous plants are embryo lethal. The mutant produced smaller rosettes with a different number of stomata than the wild-type.

Detection of bacterial biofilm on cochlear implants removed because of device failure, without evidence of infection.

Objective: To investigate the formation of bacterial biofilms on the surface of the electrode array of cochlear implants (CI) explanted because of device failure, without evidence of infection, by use of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).

Study Design: Prospective study.

Setting: Patients from 2 tertiary-care referral centers.

Transcriptomic insights into the physiology of Aspergillus niger approaching a specific growth rate of zero.

The physiology of filamentous fungi at growth rates approaching zero has been subject to limited study and exploitation. With the aim of uncoupling product formation from growth, we have revisited and improved the retentostat cultivation method for Aspergillus niger. A new retention device was designed allowing reliable and nearly complete cell retention even at high flow rates.

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms.

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis.

Genetic tools for tagging Gram-negative bacteria with mCherry for visualization in vitro and in natural habitats, biofilm and pathogenicity studies.

Live-cell imaging techniques are essential to gain a better understanding of microbial functioning in natural systems, for example in biofilms. Autofluorescent proteins, such as the green fluorescent protein (GFP) and the red fluorescent protein (DsRed), are valuable tools for studying microbial communities in their natural environment. Because of the functional limitations of DsRed such as slow maturation and low photostability, new and improved variants were created such as mCherry.

Watching the native supramolecular architecture of photosynthetic membrane in red algae: topography of phycobilisomes and their crowding, diverse distribution patterns.

The architecture of the entire photosynthetic membrane network determines, at the supramolecular level, the physiological roles of the photosynthetic protein complexes involved. So far, a precise picture of the native configuration of red algal thylakoids is still lacking. In this work, we investigated the supramolecular architectures of phycobilisomes (PBsomes) and native thylakoid membranes from the unicellular red alga Porphyridium cruentum using atomic force microscopy (AFM) and transmission electron microscopy.

Light-induced energetic decoupling as a mechanism for phycobilisome-related energy dissipation in red algae: a single molecule study.

Background: Photosynthetic organisms have developed multiple protective mechanisms to prevent photodamage in vivo under high-light conditions. Cyanobacteria and red algae use phycobilisomes (PBsomes) as their major light-harvesting antennae complexes. The orange carotenoid protein in some cyanobacteria has been demonstrated to play roles in the photoprotective mechanism.

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation.

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA.

A spatially restricted increase in receptor mobility is involved in directional sensing during Dictyostelium discoideum chemotaxis.

The directed cell migration towards a chemotactic source, chemotaxis, involves three complex and interrelated processes: directional sensing, cell polarization and motility. Directional sensing allows migrating eukaryotic cells to chemotax in extremely shallow gradients (<2% across the cell body) of the chemoattractant. Although directional sensing has been observed as spatially restricted responses along the plasma membrane, our understanding of the ;compass' of the cell that controls the gradient-induced translocation of proteins during chemotactic movements is still largely lacking.

ZebraFISH: fluorescent in situ hybridization protocol and three-dimensional imaging of gene expression patterns.

We present a method and protocol for fluorescent in situ hybridization (FISH) in zebrafish embryos to enable three-dimensional imaging of patterns of gene expression using confocal laser scanning microscopy. We describe the development of our protocol and the processing workflow of the three-dimensional images from the confocal microscope. We refer to this protocol as zebraFISH.

Characterization of mucosal biofilms on human adenoid tissues.

Objectives: To demonstrate the presence of mucosal biofilm in adenoid tissue using double staining for visualization of both the bacterial matrix and the bacterial cells. To identify bacterial species present on the surface of the studied adenoids.

Study Design: Prospective study.

Identification and real-time imaging of a myc-expressing neutrophil population involved in inflammation and mycobacterial granuloma formation in zebrafish.

By enhancer trap screening we identified a transgenic zebrafish line showing leukocyte-specific YFP expression during late embryo and early larval development. Its enhancer detection insertion was mapped near a novel member of the myc proto-oncogene family, encoding transcription factors known to be important for regulating human myelopoiesis. Characterization of the zebrafish myc family showed that only this particular myc gene is strongly expressed in leukocytes.

Demonstration of bacterial cells and glycocalyx in biofilms on human tonsils.

Objectives: To demonstrate mucosal biofilms in human tissue by direct visualization of bacteria and glycocalyx using confocal laser scanning microscopy with double fluorescent staining on tonsils and to compare the findings with the results of scanning electron microscopy analysis.

Design: Prospective study.

Setting: Tertiary university-based referral center.

A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin.

Rhizobium bacteria produce different surface polysaccharides which are either secreted in the growth medium or contribute to a capsule surrounding the cell. Here, we describe isolation and partial characterization of a novel high molecular weight surface polysaccharide from a strain of Rhizobium leguminosarum that nodulates Pisum sativum (pea) and Vicia sativa (vetch) roots. Carbohydrate analysis showed that the polysaccharide consists for 95% of mannose and glucose, with minor amounts of galactose and rhamnose.