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Inhalation exposure to environmental and occupational aerosol contaminants is associated with many respiratory health problems. To realistically mimic long-term inhalation exposure for toxicity testing, lung epithelial cells need to maintained and exposed under air-liquid interface (ALI) conditions for a prolonged period of time. In addition, to study cellular responses to aerosol particles, lung epithelial cells have to be co-cultured with macrophages. To that aim, we evaluated human bronchial epithelial Calu-3, 16HBE14o- (16HBE), H292, and BEAS-2B cell lines with respect to epithelial morphology, barrier function and cell viability under prolonged ALI culture conditions. Only the Calu-3 cells can retain the monolayer structure and maintain a strong tight junction under long-term ALI culture at least up to 2 weeks. As such, Calu-3 cells were applied as the structural barrier to create co-culture models with human monocyte-derived macrophages (MDMs) and THP-1 derived macrophages (TDMs). Adhesion of macrophages onto the epithelial monolayer was allowed for 4 h with a density of 5 × 104 macrophages/cm2. In comparison to the Calu-3 mono-culture model, Calu-3 + TDM and Calu-3 + MDM co-culture models showed an increased sensitivity in inflammatory responses to lipopolysaccharide (LPS) aerosol at Day 1 of co-culture, with the Calu-3 + MDM model giving a stronger response than Calu-3 + TDM. Therefore, the epithelial monolayer integrity and increased sensitivity make the Calu-3 + MDM co-culture model a preferred option for ALI exposure to inhaled aerosols for toxicity testing.

Purpose The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. Procedures CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated in a xenograft breast cancer mouse model using ductal carcinoma in situ cells (DCIS). Results Specific anti-CAIX nanobodies were obtained. Administration of a CAIX-specific nanobody into mice with DCIS xenografts overexpressing CAIX showed after 2 h a mean tumor-to-normal tissue ratio (TNR) of 4.3 ± 0.6, compared to a TNR of 1.4 ± 0.2 in mice injected with the negative control nanobody R2-IR. In DCIS mice, a TNR of 1.8 ± 0.1 was obtained. Biodistribution studies demonstrated an uptake of 14.0 ± 1.1 %I.D./g in DCIS + CAIX tumors, 4.6 ± 0.8 %I.D./g in DCIS tumors, while 2.0 ± 0.2 %I.D./g was obtained with R2-IR. Conclusions These results demonstrate the successful generation of a CAIX-specific nanobody-IRDye800CW conjugate that can be used for rapid imaging of (pre-)invasive breast cancer. Electronic supplementary material The online version of this article (doi:10.1007/s11307-015-0909-6) contains supplementary material, which is available to authorized users.

The rapidly expanding manufacturing, production and use of nanomaterials have raised concerns for both worker and consumer safety. Various studies have been published in which induction of pulmonary inflammation after inhalation exposure to nanomaterials has been described. Nanomaterials can vary in aspects such as size, shape, charge, crystallinity, chemical composition, and dissolution rate. Currently, efforts are made to increase the knowledge on the characteristics of nanomaterials that can be used to categorise them into hazard groups according to these characteristics. Grouping helps to gather information on nanomaterials in an efficient way with the aim to aid risk assessment. Here, we discuss different ways of grouping nanomaterials for their risk assessment after inhalation. Since the relation between single intrinsic particle characteristics and the severity of pulmonary inflammation is unknown, grouping of nanomaterials by their intrinsic characteristics alone is not sufficient to predict their risk after inhalation. The biokinetics of nanomaterials should be taken into account as that affects the dose present at a target site over time. The parameters determining the kinetic behaviour are not the same as the hazard-determining parameters. Furthermore, characteristics of nanomaterials change in the life-cycle, resulting in human exposure to different forms and doses of these nanomaterials. As information on the biokinetics and in situ characteristics of nanomaterials is essential but often lacking, efforts should be made to include these in testing strategies. Grouping nanomaterials will probably be of the most value to risk assessors when information on intrinsic characteristics, life-cycle, biokinetics and effects are all combined.

A combination of atomic force microscopy (AFM), high-resolution scanning electron microscopy (HR-SEM), focused-ion-beam scanning electron microscopy (FIB-SEM), X-ray photoelectron spectroscopy (XPS), confocal fluorescence microscopy (CFM), and UV/Vis and synchrotronbased IR microspectroscopy was used to investigate the dealumination processes of zeolite ZSM-5 at the individual crystal level. It was shown that steaming has a significant impact on the porosity, acidity, and reactivity of the zeolite materials. The catalytic performance, tested by the styrene oligomerization and methanol-to-olefin reactions, led to the conclusion that mild steaming conditions resulted in greatly enhanced acidity and reactivity of dealuminated zeolite ZSM-5. Interestingly, only residual surface mesoporosity was generated in the mildly steamed ZSM- 5 zeolite, leading to rapid crystal coloration and coking upon catalytic testing and indicating an enhanced deactivation of the zeolites. In contrast, harsh steaming conditions generated 5–50 nm mesopores, extensively improving the accessibility of the zeolites. However, severe dealumination decreased the strength of the Brønsted acid sites, causing a depletion of the overall acidity, which resulted in a major drop in catalytic activity.

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs. © 2011 Informa UK, Ltd.

Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h. The methods were validated in immuno-TEM experiments on THP-1 monocytes, human umbilical vein endothelial cells (HUVECs) and Madin–Darby canine kidney (MDCK-II) cells. The fixation and retention of neutral lipids is demonstrated, offering unique prospects for the application of immuno-TEM in the lipidomics field. Furthermore, the VIS2FIX methods were successfully employed in correlative fluorescence and electron microscopy.

Break on through: Steaming-induced mesopores of individual ZSM-5 crystals were studied by a combination of focused ion beam (FIB) and scanning electron microscopy (SEM) tomography (see picture). In this manner, quantitative insight into the width, length, morphology, and distribution of mesopores generated within zeolite crystals has been obtained. Keywords:crystal intergrowth;scanning probe microscopy;mesoporosity;tomography;zeolites

Understanding the internal mechanisms controlling fault friction is crucial for understanding seismogenic slip on active faults. Displacement in such fault zones is frequently localized on highly reflective (mirrorlike) slip surfaces, coated with thin films of nanogranular fault rock. We show that mirror-slip surfaces developed in experimentally simulated calcite faults consist of aligned nanogranular chains or fibers that are ductile at room conditions. These microstructures and associated frictional data suggest a fault-slip mechanism resembling classical Ashby-Verrall superplasticity, capable of producing unstable fault slip. Diffusive mass transfer in nanocrystalline calcite gouge is shown to be fast enough for this mechanism to control seismogenesis in limestone terrains. With nanogranular fault surfaces becoming increasingly recognized in crustal faults, the proposed mechanism may be generally relevant to crustal seismogenesis.

BACKGROUND: Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50% and 90% of the cases, respectively. Interestingly, only 24 and 37% of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature. CONCLUSIONS/SIGNIFICANCE: Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of a higher fungus is not continuous per se.

Coronaviruses are enveloped viruses containing the largest reported RNA genomes. As a result of their pleomorphic nature, our structural insight into the coronavirion is still rudimentary, and it is based mainly on 2D electron microscopy. Here we report the 3D virion structure of coronaviruses obtained by cryo-electron tomography. Our study focused primarily on the coronavirus prototype murine hepatitis virus (MHV). MHV particles have a distinctly spherical shape and a relatively homogenous size (approximate to 85 nm envelope diameter). The viral envelope exhibits an unusual thickness (7.8 +/- 0.7 nm), almost twice that of a typical biological membrane. Focal pairs revealed the existence of an extra internal layer, most likely formed by the C-terminal domains of the major envelope protein M. In the interior of the particles, coiled structures and tubular shapes are observed, consistent with a helical nucleocapsid model. Our reconstructions provide no evidence of a shelled core. Instead, the ribonucleoprotein seems to be extensively folded onto itself, assuming a compact structure that tends to closely follow the envelope at a distance of approximate to 4 nm. Focal contact points and thread-like densities connecting the envelope and the ribonucleoprotein are revealed in the tomograms. Transmissible gastroenteritis coronavirion tomograms confirm all the general features and global architecture observed for MHV. We propose a general model for the structure of the coronavirion in which our own and published observations are combined.

Colonies of Aspergillus niger are characterized by zonal heterogeneity in growth, sporulation, gene expression and secretion. For instance, the glucoamylase gene glaA is more highly expressed at the periphery of colonies when compared to the center. As a consequence, its encoded protein GlaA is mainly secreted at the outer part of the colony. Here, multiple copies of amyR were introduced in A. niger. Most transformants over-expressing this regulatory gene of amylolytic genes still displayed heterogeneous glaA expression and GlaA secretion. However, heterogeneity was abolished in transformant UU-A001.13 by expressing glaA and secreting GlaA throughout the mycelium. Sequencing the genome of UU-A001.13 revealed that transformation had been accompanied by deletion of part of the fluG gene and disrupting its 3' end by integration of a transformation vector. Inactivation of fluG in the wild-type background of A. niger also resulted in breakdown of starch under the whole colony. Asexual development of the ∆fluG strain was not affected, unlike what was previously shown in Aspergillus nidulans. Genes encoding proteins with a signal sequence for secretion, including part of the amylolytic genes, were more often downregulated in the central zone of maltose-grown ∆fluG colonies and upregulated in the intermediate part and periphery when compared to the wild-type. Together, these data indicate that FluG of A. niger is a repressor of secretion.

Insight into processes leading to rupture of intracranial aneurysms (IAs) may identify biomarkers for rupture or lead to management strategies reducing the risk of rupture. We characterized and quantified (ultra)structural differences between unruptured and ruptured aneurysmal walls. Six unruptured and 6 ruptured IA fundi were resected after microsurgical clipping and analyzed by correlative light microscopy for quantitative analysis (proportion of the vessel wall area) and transmission electron microscopy for qualitative ultrastructural analysis. Quantitative analysis revealed extensive internal elastic lamina (IEL) thickening in ruptured IA (36.3% ± 15%), while thin and fragmented IEL were common in unruptured IA (5.6% ± 7.1%). Macrophages were increased in ruptured IA (28.3 ± 24%) versus unruptured IA (2.7% ± 5.5%), as were leukocytes (12.85% ± 10% vs 0%). Vasa vasorum in ruptured but not in unruptured IA contained vast numbers of inflammatory cells and extravasation of these cells into the vessel wall. In conclusion, detection of thickened IEL, leaky vasa vasorum, and heavy inflammation as seen in ruptured IA in comparison to unruptured IA may identify aneurysms at risk of rupture, and management strategies preventing development of vasa vasorum or inflammation may reduce the risk of aneurysmal rupture.

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture. © 2006 Elsevier Inc. All rights reserved.

The Focused Ion Beam – Scanning Electron Microscope (FIB-SEM) is a versatile instrument originating from the semiconductor industry. The FIB is used to produce cross sections of pre-defined locations of interest, which are imaged and analyzed with the SEM. Repeated FIB cross sectioning and subsequent SEM imaging –a process called FIB-SEM tomography– results in a full three-dimensional analysis of the structure of interest, allowing for the 3D reconstruction of cellular materials or porous media. In recent years, the capabilities of the FIB-SEM have attracted a considerable amount of attention from various scientific disciplines. This PhD Thesis presents a survey across samples from Life Sciences, Earth Sciences and Material Sciences, exploring and validating the application of the FIB-SEM instrument to scientific questions from the three disciplines. The common denominator of the various samples under investigation is their electrically insulating nature, requiring additional care in dealing with the samples in the microscope. Life Sciences Electron Microscopy (EM) in Life Sciences can be split in room temperature (cells are dehydrated, optionally embedded) and cryo applications. A contribution is made in both fields. Room temperature FIB-SEM tomography is demonstrated on cells embedded in a resin. The cellular membranes are stained with heavy metals, providing sufficient contrast in Back Scattered Electron (BSE) mode. It is shown that the distribution of endoplasmic reticulum and the mitochondria can be reconstructed in three dimensions. As for cryo-EM, a major quest in Life Sciences is the preparation of artifact-free thick cryo-sections or lamellas (>200 nm) for cryo-Transmission Electron Microscopy (TEM) tomography. A dedicated workflow has been developed to produce such thick cryo-sections with the FIB, while is SEM is used to check the quality of the sections in-situ. Earth Sciences The importance of 3D reconstructions over 2D imaging is demonstrated with an olivine sample. The permeability of olivine is linked to the wetting angle –the dihedral angle– of the melt and the rock. FIB-SEM tomography data of melt pockets indicates that measuring the dihedral angle based on 2D data may not be valid because of the presence of facets in the third dimension. Material Sciences Two different Material Sciences samples are investigated: 1) Large steamed zeolite ZSM-5 crystals and 2) Fluid Catalytic Cracking (FCC) particles. The steaming process of large ZSM-5 crystals creates mesoporosity, which is characterized by FIB-SEM tomography. To prevent charging artifacts, low-kV BSE imaging is used successfully. FIB-SEM tomography through the FCC particles revealed two major challenges considering the post-processing of the 3D data. The first challenge considers automated segmentation of the pore space from FIB-SEM tomography data. A combination of several techniques is found to be sufficiently accurate. The second challenge involves upscaling of transport properties from the dimensions analyzed by the FIB-SEM. A workflow is presented which employs experimentally based statistical means to extrapolate high resolution data to much larger length scales.

Human Immunodeficiency Virus type 1 (HIV-1) is one of the major health problems worldwide and has been for over thirty years. Most (67%) of the people infected with HIV-1 are living in sub-Saharan Africa. Here, the access to treatments is limited and most women are not in a position to protect themselves from getting infected. The aim of this study was to identify VHH that are both broad and potent HIV-1 neutralizers, which can be used in the fight against HIV-1. A VHH is an antibody fragment containing the antigen binding domain derived from a heavy chain only antibody. This special type of antibody can be found in members of the camel family. The advantage of VHH is that they are relatively small, stable, cheap to produce and easy to work with. In order to raise VHH against HIV-1, two llamas were immunized with a mix of its envelope proteins. After cloning of the llama’s VHH repertoire into a phagemid expression system, multiple different strategies were followed to identify neutralizing VHH. In total, twelve families of neutralizing VHH were found and characterized in more detail. These VHH target four independent areas on the HIV-1 envelope protein. The best VHH of them being J3, which neutralized 96 out of a 100 tested viruses with a median IC50 of 0.9 µg/ml and is targeting the CD4 binding site. The CD4 binding site on the envelope protein is considered an important area as the first interaction between virus and its target cell occurs via CD4. Previous studies have shown that linking two VHH together into so-called biheads may increase their breadth and potency. For this reason we have linked two different VHH, targeting independent epitopes, together to form bi-specific biheads. Several combinations of VHH have been tried, the best (J3 linked to 2E7) yielded upto 1400 times increase in potency compared to a mix of the individual VHH. Another bihead (J3 linked to 1F10) was capable of neutralizing a viral strain J3 alone could not neutralize, possibly improving the breadth of this molecule above 96%.

Alzheimer's Disease (AD) is the most common form of dementia among elderly in the Western world. AD is a devastating neurodegenerative disease where patients starting with episodic memory problems end up completely bedridden and care dependent. At present there is no real therapy stopping or reversing AD progression. Roughly 100 years ago Alois Alzheimer described the deposition of amyloid-beta (Aβ) in post-mortem brains, one of the hallmarks of AD. Since this description AD has had great attention. However, the exact mechanism of AD remains unclear, a general consensus is reached over Aβ as a peptide involved in AD progression. Aβ is derived from amyloid-beta precursor protein (APP) by the proteolytic cleavage activity of beta-secretase BACE1. Interesting, next to AD patients, healthy individuals also produce Aβ in their brain. So next to the production and presence of Aβ, an extra mechanism is in place to exert toxicity and therefore AD progression. The hydrophobic nature of Aβ makes this peptide prone to aggregate. Overwhelming evidence point to an aggregated form of Aβ (oligomer) as the culprit of toxicity. This makes aggregation of Aβ the mechanism responsible for the generation of toxic Aβ species. Members from the Camelidae family possess, next to conventional IgGs, a heavy chain only antibody. The antigen-binding domain of this antibody (VHH) is fully function while being about ten times smaller compared to a conventional antibody. Next to size, VHH have several other benefits like stability, production costs and genetic modulation. Next to that, VHH are found that inhibit protein aggregation and enzyme activity. We exploited the capability of VHH to inhibit protein aggregation by selecting and generation VHH against Aβ. Llamas were immunized with patient material containing severe Aβ deposition. The subsequently generated VHH library yielded one VHH that inhibits aggregation, and reduced Aβ toxicity. Mutation studies and an additional crystal structure led to the hypothesis that this VHH binds to a small aggregated form of Aβ. A secondary strategy to prevent Aβ toxicity is to inhibit Aβ production by targeting BACE1. We selected a VHH that inhibits BACE1 activity in vitro and in vivo. The in vivo pilot experiment showed a reduced concentration of Aβ and therefore might be of therapeutical value. Problematic is the delivery of compounds to the brain due to the blood brain barrier (BBB). We provided a proof of principle to deliver compounds over cell layers that were BBB models. Transferrin Receptor (TfR) was used to piggy bag an anti-TfR covalently coupled to an anti-Aβ over the cell layer. The aim of this thesis was to find VHH against Ab targets and characterize these VHH. We made great progress in characterizing VHH G7 in vitro and explain characteristics needed for aggregation and toxicity inhibition. Besides this we found a VHH inhibiting BACE1 activity in vitro and in vivo providing a secondary therapeutic strategy for AD. Furthermore, proof of principle is provided to deliver compounds over cellular monolayers modeling the blood brain barrier.

Delivery of drugs to sites of disease can be hampered by the presence of cellular barriers in the human body. The presence of such barriers reduces the therapeutic activity of drugs on the disease target. The barrier present between the bloodstream and the brain for instance, stringently controls what goes into the brain and what not. Diseases that take place in such enclosed systems may be difficult to reach for pharmaceutical compounds because of these barriers and this can reduce their therapeutic effectivity. In this thesis we have described the development of molecules that interact with specific barriers in the human body and that mediate translocation across these barriers. As platform we used antibodies from the llama. Llama antibodies have several favourable properties as compared with other mammalian antibodies. The llama antibody platform was used in the described studies to make entities capable of translocating across cellular barriers. Part of the described studies has looked at the removal of compounds from the body by efflux via the intestinal wall (a cellular barrier). We developed molecules which can interact specifically with that barrier and mediate the passage of unwanted molecules. These molecules can be used for example for the removal of viruses that have infected the body. Fusion of the translocation molecules with compounds that bind viruses would enable the resulting compounds to both bind the virus and mediate its translocation across the intestinal wall. Such therapeutic strategies could tackle the infections of persistent viruses such as HIV. The other part of the described studies has looked at the delivery of blood borne compounds across the blood brain barrier to the central nervous system. Efficient platforms that mediate brain delivery from the bloodstream are scarce/ non-existent. We found molecules which have favourable properties in artificial model systems mimicking the blood-brain barrier. Application of these molecules to mice showed that there was a significant proportion of the injected dose able to accumulate in the brain, which indicates that our molecules indeed can target the brain. Further genetic adaptation of the antibodies might improve the translocation process to the brain environment and deliver a bigger pool of therapeutics to the site of disease. Development of these molecules might yield entities capable of delivering therapeutically relevant compounds to the brain. At the moment, brain delivery of pharmaceuticals is still a feature that is hardly possible without invasive procedures. Due to this aberrant delivery, there is no proper treatment possible of diseases like Alzheimer’s, Parkinson’s and brain cancer. We hope that with further development, our molecules will be able to give a solution to the brain delivery problem and thereby aid in the treatment of brain afflictions. This thesis has given examples of how antibodies from the llama can be used as retargeting agents. We have shown that they can be designed to translocate across cellular barriers and to take along cargo in this process. Development of the antibodies may yield molecules which can help in the diagnosis and treatment of several diseases.

One of the major challenges for correlative microscopy is the preparation of the sample; the protocols for transmission electron microscopy (TEM) and fluorescence microscopy (FM) often prove to be incompatible. Here, we introduce 2+Staining: an improved contrasting procedure for Tokuyasu sections that yields both excellent positive membrane contrast in the TEM and bright fluorescence of the probe labeled on the section. 2+Staining involves the contrasting of the immunolabeled sections with 1% osmium tetroxide, 2% uranyl acetate and lead citrate in sequential steps, followed by embedding in 1.8% methyl cellulose. In addition, we demonstrate an amplification of the fluorescent signal by introducing additional antibody incubation steps to the immunolabeling procedure. The methods were validated using the integrated laser and electron microscope (iLEM), a novel tool for correlative microscopy combining FM and TEM in a single setup. The approaches were tested on HL-60 cells labeled for lysosomal-associated membrane protein 2 (LAMP-2) and on sections of muscle from a facioscapulohumeral dystrophy mouse model. Yielding excellent results and greatly expediting the workflow, the methods are of great value for those working in the field of correlative microscopy and indispensible for future users of integrated correlative microscopy.

Binding of tissue-type plasminogen activator (tPA) to amyloid and denatured proteins is reported in a number of studies. The binding site has been mapped previously to the finger domain of tPA. In this study, tPA and truncated tPA constructs, lacking the finger domain, were tested for their ability to bind to Aβ and AIAPP amyloid-like fibrils. Surface plasmon resonance experiments and pull-down assays clearly show that indeed tPA binds, but that the finger domain is not essential. Another possible binding mechanism via the lysine binding site on the kringle 2 domain was also not crucial for the binding. Immuno-electron microscopy studies show that tPA binds to fibril sides. This study shows that, besides the finger domain, other domains in tPA are involved in amyloid binding.

Autotransporters (ATs) are proteins secreted by Gram-negative bacteria that often play a role in virulence. Eight different ATs have been identified in Neisseria meningitidis, but only six of them have been characterized. AutA is one of the remaining ATs. Its expression remains controversial. Here, we show that the autA gene is present in many neisserial species, but its expression is often disrupted by various genetic features; however, it is expressed in certain strains of N. meningitidis. By sequencing the autA gene in large panels of disease isolates and Western blot analysis, we demonstrated that AutA expression is prone to phase variation at AAGC nucleotide repeats located within the DNA encoding the signal sequence. AutA is not secreted into the extracellular medium, but remains associated with the bacterial cell surface. We further demonstrate that AutA expression induces autoaggregation in a process that, dependent on the particular strain, may require extracellular DNA (eDNA). This property influences the organization of bacterial communities like lattices and biofilms. In vitro assays evidenced that AutA is a self-associating AT that binds DNA. We suggest that AutA-mediated autoaggregation might be particularly important for colonization and persistence of the pathogen in the nasopharynx of the host.

A number of studies have shown that induction of pulmonary toxicity by nanoparticles of the same chemical composition depends on particle size, which is likely in part due to differences in lung deposition. Particle size mostly determines whether nanoparticles reach the alveoli, and where they might induce toxicity. For the risk assessment of nanomaterials, there is need for a suitable dose metric that accounts for differences in effects between different sized nanoparticles of the same chemical composition. The aim of the present study is to determine the most suitable dose metric to describe the effects of silver nanoparticles after short-term inhalation. Rats were exposed to different concentrations (ranging from 41 to 1105 µg silver/m3 air) of 18, 34, 60 and 160 nm silver particles for four consecutive days and sacrificed at 24 h and 7 days after exposure. We observed a concentration-dependent increase in pulmonary toxicity parameters like cell counts and pro-inflammatory cytokines in the bronchoalveolar lavage fluid. All results were analysed using the measured exposure concentrations in air, the measured internal dose in the lung and the estimated alveolar dose. In addition, we analysed the results based on mass, particle number and particle surface area. Our study indicates that using the particle surface area as a dose metric in the alveoli, the dose–response effects of the different silver particle sizes overlap for most pulmonary toxicity parameters. We conclude that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation.