Bacteria-targeted fluorescence imaging of extracted osteosynthesis devices for rapid visualization of fracture-related infections.

Purpose: Fracture-related infection (FRI) is a serious complication in orthopedic trauma surgery worldwide. Especially, the distinction of infection from sterile inflammation and the detection of low-grade infection are highly challenging. The objective of the present study was to obtain proof-of-principle for the use of bacteria-targeted fluorescence imaging to detect FRI on extracted osteosynthesis devices as a step-up towards real-time image-guided trauma surgery.

Transient von Willebrand factor-mediated platelet influx stimulates liver regeneration after partial hepatectomy in mice.

Background & Aims: In addition to their function in thrombosis and haemostasis, platelets play an important role in the stimulation of liver regeneration. It has been suggested that platelets deliver mitogenic cargo to the regenerating liver, and accumulation of platelets in the regenerating liver has been demonstrated. We studied kinetics of platelet influx in the regenerating liver and investigated the signal that initiates platelet influx.

Particle Bombardment of Ex Vivo Skin to Deliver DNA and Express Proteins.

Particle bombardment of gold microparticles coated with plasmids, which are accelerated to high velocity, is used for transfection of cells within tissue. Using this method, cDNA encoding proteins of interest introduced into ex vivo living human skin enables studying of proteins of interest in real time. Here, technical aspects of particle bombardment of ex vivo skin are described using green fluorescent protein (GFP) as readout for efficiency.

How antibodies alter the cell entry pathway of dengue virus particles in macrophages.

Antibody-dependent enhancement of dengue virus (DENV) infection plays an important role in the exacerbation of DENV-induced disease. To understand how antibodies influence the fate of DENV particles, we explored the cell entry pathway of DENV in the absence and presence of antibodies in macrophage-like P388D1 cells. Recent studies unraveled that both mature and immature DENV particles contribute to ADE, hence, both particles were studied.

Entry of PIP3-containing polyplexes into MDCK epithelial cells by local apical-basal polarity reversal.

The polarized architecture of epithelium presents a barrier to therapeutic drug/gene carriers, which is mainly due to a limited (apical) internalization of the carrier systems. The bacterium Pseudomonas aeruginosa invades epithelial cells by inducing production of apical phosphatidylinositol-3, 4, 5-triphosphate (PIP3), which results in the recruitment of basolateral receptors to the apical membrane. Since basolateral receptors are known receptors for gene delivery vectors, apical PIP3 may improve the internalization of such vectors into epithelial cells.

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge.

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation.

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish.

Destruction of tissue, cells and organelles in type 1 diabetic rats presented at macromolecular resolution.

Finding alternatives for insulin therapy and making advances in etiology of type 1 diabetes benefits from a full structural and functional insight into Islets of Langerhans. Electron microscopy (EM) can visualize Islet morphology at the highest possible resolution, however, conventional EM only provides biased snapshots and lacks context. We developed and employed large scale EM and compiled a resource of complete cross sections of rat Islets during immuno-destruction to provide unbiased structural insight of thousands of cells at macromolecular resolution.

Proteinuria triggers renal lymphangiogenesis prior to the development of interstitial fibrosis.

Proteinuria is an important cause of progressive tubulo-interstitial damage. Whether proteinuria could trigger a renal lymphangiogenic response has not been established. Moreover, the temporal relationship between development of fibrosis, inflammation and lymphangiogenesis in chronic progressive kidney disease is not clear yet.

Correlated light microscopy and electron microscopy.

Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals selected biomolecules or organelles but not the (ultra)structural context, as can be examined by electron microscopy (EM).

Nonviral gene delivery vectors use syndecan-dependent transport mechanisms in filopodia to reach the cell surface.

Lipoplexes and polyplexes, that is, assemblies of cationic lipids and polymers with nucleic acids, respectively, are popular nanocarriers for delivery of genes or siRNA into cells for therapeutic or cell biological purposes. Although endocytosis represents a major mechanism for their cellular entry, very little is known about parameters that govern early events in the initial interaction of such delivery devices with the cell surface. Here, we demonstrate that prior to entry, poly- and lipoplexes are captured by thin, actin-rich filopodial extensions, protruding from the cell surface.

Immunolabeling artifacts and the need for live-cell imaging.

Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation.

Differential effects of TNF (TNFSF2) and IFN-γ on intestinal epithelial cell morphogenesis and barrier function in three-dimensional culture.

Background: The cytokines TNF (TNFSF2) and IFNγ are important mediators of inflammatory bowel diseases and contribute to enhanced intestinal epithelial permeability by stimulating apoptosis and/or disrupting tight junctions. Apoptosis and tight junctions are also important for epithelial tissue morphogenesis, but the effect of TNF and IFNγ on the process of intestinal epithelial morphogenesis is unknown.

Methods/principal Findings: We have employed a three-dimensional cell culture system, reproducing in vivo-like multicellular organization of intestinal epithelial cells, to study the effect of TNF on intestinal epithelial morphogenesis and permeability.

CLSM as quantitative method to determine the size of drug crystals in a solid dispersion.

Purpose: To test whether confocal laser scanning microscopy (CLSM) can be used as an analytical tool to determine the drug crystal size in a powder mixture or a crystalline solid dispersion.

Methods: Crystals of the autofluorescent drug dipyridamole were incorporated in a matrix of crystalline mannitol by physical mixing or freeze-drying. Laser diffraction analysis and dissolution testing were used to validate the particle size that was found by CLSM.

Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4.

Formation and maturation of parallel fiber-Purkinje cell synapses in the Staggerer cerebellum ex vivo.

In vivo, homozygous staggerer (Rora(sg/sg)) Purkinje cells (PCs) remain in an early stage of development with rudimentary spineless dendrites, associated with a lack of parallel fiber (PF) input and the persistence of multiple climbing fibers (CFs). In this immunocytochemical study we used cerebellar organotypic cultures to monitor the development of Rora(sg/sg) PF-PC synapses in the absence of CF innervation. Ex vivo the vesicular glutamate transporters VGluT1 and VGluT2 reactivity was preferentially localized around the Rora(sg/sg) PC soma and proximal dendrites, which are typically CF domains.

Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles.

Neurons are highly polarized cells, and neuron-neuron communication is based on directed transport and release of neurotransmitters, neuropeptides, and neurotrophins. Directed communication may also be attributed to neuron-microglia signaling, since neuronal damage can induce a microglia reaction at specific sites only. However, the mechanism underlying this site-specific microglia reaction is not yet understood.

The unprocessed preprotein form IATC103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing.

Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT(C103S)) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein.

Dynamin-related proteins Vps1p and Dnm1p control peroxisome abundance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains three dynamin-related-proteins, Vps1p, Dnm1p and Mgm1p. Previous data from glucose-grown VPS1 and DNM1 null mutants suggested that Vps1p, but not Dnm1p, plays a role in regulating peroxisome abundance. Here we show that deletion of DNM1 also results in reduction of peroxisome numbers.

Nitrate-dependent [Fe(II)EDTA]2- oxidation by Paracoccus ferrooxidans sp. nov., isolated from a denitrifying bioreactor.

Enrichments with [Fe(II)EDTA]2- as electron donor and nitrate or nitrite as electron acceptor were established using an inoculum from a bioreactor performing denitrification. A nitrate-reducing, [Fe(II)EDTA]2- oxidizing strain was isolated and named strain BDN-1. The G + C content of strain BDN-1 was 67%, and the organism was closely affiliated to Paracoccus denitrificans, P.

Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells.

Background: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E.

Microautophagy and macropexophagy may occur simultaneously in Hansenula polymorpha.

We subjected methanol-grown cells of wild type Hansenula polymorpha simultaneously to nitrogen depletion and excess glucose conditions. Both treatments induce the degradation of peroxisomes, either selective (via excess glucose) or non-selective (via nitrogen limitation). Our combined data strongly suggest that both processes occur simultaneously under these conditions.

Collimonas fungivorans gen. nov., sp. nov., a chitinolytic soil bacterium with the ability to grow on living fungal hyphae.

A polyphasic approach was used to describe the phylogenetic position of 22 chitinolytic bacterial isolates that were able to grow at the expense of intact, living hyphae of several soil fungi. These isolates, which were found in slightly acidic dune soils in the Netherlands, were strictly aerobic, Gram-negative rods. Cells grown in liquid cultures were flagellated and possessed pili.

Thialkalivibrio nitratireducens sp. nov., a nitrate-reducing member of an autotrophic denitrifying consortium from a soda lake.

Strain ALEN 2(T) was isolated from a mixed culture capable of complete autotrophic denitrification with thiosulfate as electron donor at pH 10; the mixed culture was enriched from sediment from Lake Fazda (Wadi Natrun, Egypt), a hypersaline alkaline lake. The isolate had large, non-motile, coccoid or barrel-shaped cells with intracellular sulfur globules. The bacterium was obligately chemolithoautotrophic.

Thioalkalispira microaerophila gen. nov., sp. nov., a novel lithoautotrophic, sulfur-oxidizing bacterium from a soda lake.

An anaerobic enrichment medium (pH 10) with thiosulfate as electron donor and nitrate as electron acceptor was inoculated with sediment from soda lake Fazda (Wadi Natrun, Egypt); a novel strain, ALEN 1(T), was isolated from the subsequent enrichment culture. Cells of strain ALEN 1(T) had a spiral morphology (0.3-0.