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Antibacterial formulations based on zinc oxide nanoparticles (ZnO NPs) are widely used for antibiotic replacement in veterinary medicine and animal nutrition. However, the undesired environmental impact of ZnO NPs triggers a search for alternative, environmentally safer solutions. Here, we show that Zn2+in its ionic form is a more eco-friendly antibacterial, and its biocidal action rivals that of ZnO NPs (<100 nm size), with a minimal biocidal concentration being 41(82) μg mL–1vs 5 μg mL–1of ZnO NPs, as determined for 103(106) CFU mL–1E. coli. We demonstrate that the biocidal activity of Zn2+ions is primarily associated with their uptake by E. coliand spontaneous in vivo transformation into insoluble ZnO nanocomposites at an internal bacterial pH of 7.7. Formed in vivo nanocomposite then damages E. colimembrane and intracellular components from the inside, by forming insoluble biocomposites, whose formation can also trigger ZnO characteristic reactions damaging the cells (e.g., by generation of high-potential reactive oxygen species). Our study defines a special route in which Zn2+metal ions induce the death of bacterial cells, which might be common to other metal ions capable of forming semiconductor oxides and insoluble hydroxides at a slightly alkaline intracellular pH of some bacteria.

The merger and acquisition trend among US businesses is changing the insurance intermediary marketplace and may have a major impact on the industry. The reinsurance market community is one sector of the industry that has not been spared by this trend. To protect their corporate objectives, small and large reinsurers must address certain issues regarding the operational impact of consolidation. These include volume, business mix, geographic expansion and insurance acquisitions.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the “phosphate tweezer” CLR01, a “carboxylate tweezer” CLR05, and a “phosphate clip” PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

ABSTRACTThe respiratory tract is a major entry site for microbial pathogens. To combat bacterial infections, the immune system has various defense mechanisms at its disposal, including antimicrobial peptides (AMPs). To search for novel AMPs from the respiratory tract, a peptide library from human broncho-alveolar-lavage (BAL) fluid was screened for antimicrobial activity by radial diffusion assays allowing the efficient detection of antibacterial activity within a small sample size. After repeated testing-cycles and subsequent purification, we identified ß-2-microglobulin (B2M) in antibacterially active fractions. B2M belongs to the MHC-1 receptor complex present at the surface of nucleated cells. It is known to inhibit the growth of Listeria monocytogenesand Escherichia coliand to facilitate phagocytosis of Staphylococcus aureus. Using commercially available B2M we confirmed a dose-dependent inhibition of Pseudomonas aeruginosaand L. monocytogenes. To characterize AMP activity within the B2M sequence, peptide fragments of the molecule were tested for antimicrobial activity. Activity could be localized to the C-terminal part of B2M. Investigating pH dependency of the antimicrobial activity of B2M demonstrated an increased activity at pH values of 5.5 and below, a hallmark of infection and inflammation. Sytox green uptake into bacterial cells following the exposure to B2M was determined and revealed a pH-dependent loss of bacterial membrane integrity. TEM analysis showed areas of disrupted bacterial membranes in L. monocytogenesincubated with B2M and high amounts of lysed bacterial cells. In conclusion, B2M as part of a ubiquitous cell surface complex may represent a potent antimicrobial agent by interfering with bacterial membrane integrity.

Businesses should reanalyze their reinsurance needs so that when a problem arises, operations can continue. The reinsurance industry has not been well regulated, and some policy holders may find it difficult to collect on their policies. Reading a contract closely is recommended, along with reassessing the firm's insurance needs. Inadequate reinsurance coverage is a problem when a natural disaster strikes and a firm does not have enough coverage to repair a facility., Historically, the placement of reinsurance contracts, as well as the financial quality of reinsurers, received little attention from the regulatory community, and even less notice from policyholders. Within the last [...]

Byline: Paul Walther In the parlance of the insurance-buying community, it may not be too uncommon to hear the term 'chief risk officer,'' or 'CRO,'' being applied to the executive [...]

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The authors’ recent concept of bioinorganic filtration devices made up of solid-state membranes (SSMs) accommodating ring-shaped, ribonucleic acid (RNA)-stabilized tobacco mosaic virus (TMV) coat protein (CP) assemblies with central 4 nm holes as genetically encoded ‘pore-in-pore’ fittings, to convey size and charge specificity to the membranes’ permeability, has been elaborated. Key developments for simplifying and finishing the unique combination apply to both soft- and hard-matter components: previous SSMs with millions of conical pores demanded sophisticated lithography to achieve a taper trapping the bionanopores upon insertion in a flow. Now focused helium (He) ion beam technology has enabled efficient, fast preparation of silicon nitride templates adapted to the nanorings. Proof-of-concept experiments reveal that negative charges imparted by nucleic acids exposed on the bionanopores might improve electrophoretic implantation further. Suitable peptides installed on the outer nanoring rim had been shown to nucleate spatially confined silica deposition from liquid precursors, which have been optimized in order to seal the annular gaps between bio and inorganic SSM pores by ‘bionic glue’. Finally, two engineered CP variants and a modified scaffold RNA were established for novel TMV nanoring types with altered pore charges, which also allow installing accessory molecules for advanced filtration and conversion tasks.

Molecular self-assembly of short peptide building blocks leads to the formation of various material architectures that may possess unique physical properties. Recent studies had confirmed the key role of biaromaticity in peptide self-assembly, with the diphenylalanine (FF) structural family as an archetypal model. Another significant direction in the molecular engineering of peptide building blocks is the use of fluorenylmethoxycarbonyl (Fmoc) modification, which promotes the assembly process and may result in nanostructures with distinctive features and macroscopic hydrogel with supramolecular features and nanoscale order. Here, we explored the self-assembly of the protected, noncoded fluorenylmethoxycarbonyl-β,β-diphenyl-Ala-OH (Fmoc-Dip) amino acid. This process results in the formation of elongated needle-like crystals with notable aromatic continuity. By altering the assembly conditions, arrays of spherical particles were formed that exhibit strong light scattering. These arrays display vivid coloration, strongly resembling the appearance of opal gemstones. However, unlike the Rayleigh scattering effect produced by the arrangement of opal, the described optical phenomenon is attributed to Mie scattering. Moreover, by controlling the solution evaporation rate, i.e., the assembly kinetics, we were able to manipulate the resulting coloration. This work demonstrates a bottom-up approach, utilizing self-assembly of a protected amino acid minimal building block, to create arrays of organic, light-scattering colorful surfaces.

ABSTRACTMucormycoses are life-threatening infections in immunocompromised patients. This study characterizes the response of human mononuclear cells to different Mucorales and Ascomycota. PBMC, monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors were stimulated with resting and germinated stages of Mucorales and Ascomycota. Cytokine response and expression of activation markers were studied. Both inactivated germ tubes and resting spores of Rhizopus arrhizusand other human pathogenic Mucorales species significantly stimulated mRNA synthesis and secretion of proinflammatory cytokines. Moreover, R. arrhizusspores induced the upregulation of co-stimulatory molecules on moDCs and a specific T-helper cell response. Removal of rodlet hydrophobins by hydrofluoric acid treatment of A. fumigatusconidia resulted in enhanced immunogenicity, whereas the cytokine response of PBMCs to dormant R. arrhizusspores was not influenced by hydrofluoric acid. Scanning electron micrographs of Mucorales spores did not exhibit any morphological correlates of rodlet hydrophobins. Taken together, this study revealed striking differences in the response of human mononuclear cells to resting stages of Ascomycota and Mucorales, which may be explained by absence of an immunoprotective hydrophobin layer in Mucorales spores.

Insurers with sudden financial obligations to meet due to an 'old portfolio of assumed reinsurance' may take three steps to mitigate their losses. These pertain to the identification of the sources and circumstances of their reinsurance exposure, the administration of its portfolio and the commutation of their liabilities. These requires a review of contracts with a reinsurer's ceding companies and businesses covered by the contracts as well as procedures for claims processing.

Aggregation of misfolded disease–related proteins is a hallmark of neurodegenerative diseases. Aggregate propagation accompanying disease progression has been demonstrated for different proteins (eg, for α-synuclein). Additional evidence supports aggregate cross-seeding activity for α-synuclein. For mutated superoxide dismutase 1 (SOD1), which causes familial amyotrophic lateral sclerosis (ALS), self-propagation of aggregation and cell-to-cell transmission have been demonstrated in vitro. However, there is a prominent lack of in vivodata concerning aggregation and cross-aggregation processes of SOD1. We analyzed the effect of α-synuclein and SOD1 seeds in cell culture using protein fragment complementation assay and intracerebral injection of α-synuclein and SOD1 seeds into SOD1G93Atransgenic ALS mice. Survival of injected mice was determined, and SOD1 aggregates in the facial nuclei were quantified during disease course. We found that α-synuclein preformed fibrils increased the oligomerization rate of SOD1 in vivoand in vitro, whereas aggregated SOD1 did not exert any effect in both experimental setups. Notably, survival of ALS mice was not changed after inoculation of preformed fibrils. We conclude that misfolded α-synuclein can increase SOD1 aggregation and suppose that α-synuclein seeds are transported from the temporal cortex to the facial nuclei. However, unlike other proteins, the further enhancement of a self-aggregation process by additional SOD1 could not be confirmed in our models.

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmaniaparasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3+, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmaniainduced proliferation of CD4+T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmaniahijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.

abstractOxygen partial pressures (O2pp) inside rhizomes of Phragmites australiswere measured to monitor diurnal dynamics. Root-associated biofilms were characterised regarding methane-oxidising bacteria (MOB) by immuno labelling. Runs of O2pp showed distinct diurnal patterns repeating day-to-day. Soon after sunrise O2pp increased steeply to around 185 hPa, remained on this level over mid-day and decreased exponentially over night to about 80 hPa. Root surfaces were densely packed with bacteria of which 34–37% accounted for potential MOB. This emphasises the importance of the oxic rhizosphere in the degradation of organic matter and methane oxidation and diurnal fluctuation of O2pp may implicate for variation of biogeochemical processes involved in wastewater treatment.

Damage of cartilage structures in the head and neck region as well as in orthopedic sites are frequently caused by trauma, tumor resection, or congenital defects. Despite a high demand in many clinical fields, until today, no adequate cartilage replacement matrix is available for these fields of application. Materials that are clinically applied for joint cartilage repair still need optimization due to difficult intraoperative handling and risk of early mechanical damage. We have developed and applied a novel chemical process to completely decellularize and sterilize human and porcine cartilage tissues (meniscus cartilage and nasal septum) to generate a new type of bioimplant matrix. To characterize this matrix and to determine the effect of the decellularization process, the content of denatured collagen (wD) and the content of glycosaminoglycans (GAGs) (wG) were determined. Possible cytotoxic effects and cellular compatibility of the matrix in vitrohave been examined by seeding processed cartilage biomatrices with human primary chondrocytes as well as murine fibroblasts (L929). Vitality and state of metabolism of cells were measured using MTS assays. Both cell types adhered to scaffold surfaces and proliferated. No areas of growth inhibition or cytotoxic effects were detected. New synthesis of cartilage-specific extracellular matrix was observed. By histological staining, electron microscopy, and μCT analysis, an increase of matrix porosity, complete cell elimination, and high GAG removal were demonstrated. Being from natural-origin, processed xenogenic and allogeneic cartilage biomatrices are highly versatile with regard to shape, size, and biomechanics, making them promising candidates for various biomedical applications.

The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg2+). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg2+at physiological concentrations (<1 mM) resulted in networks whose structure was similar to that of keratin networks in epithelial cells. Moreover, the elastic moduli of networks assembled in vitro were found to be within the same magnitude as those measured in keratin networks of detergent-extracted epithelial cells. These findings suggest that Mg2+-induced filament cross-linking represents a valid model for studying the cytoskeletal mechanics of keratin networks.

The primary diseases of the pancreas include diabetes mellitus, acute and chronic pancreatitis, as well as pancreatic carcinoma. This review presents findings and emerging questions on the diseases of the pancreas obtained by the consortium of the Collaborative Research Center 518 (SFB 518), "Inflammation, Regeneration, and Transformation in the Pancreas" at the University of Ulm. During the last 12 years, the SFB 518 contributed considerably to the understanding of the cellular and molecular basis of pancreatic diseases and established the basis for the development of new strategies for prevention and causal therapy for diabetes, pancreatitis, and pancreatic cancer.

Ribosomal RNA molecules are synthesized as precursors that have to undergo several processing steps to generate the functional rRNA. The 5S rRNA in the archaeon Haloferax volcanii is transcribed as part of a multicistronic transcript containing both large rRNAs and one or two tRNAs. Release of the 5S rRNA from the precursor requires two endonucleolytic cleavages by enzymes as yet not identified. Here we report the first identification of an archaeal 5S rRNA processing endonuclease. The enzyme tRNase Z, which was initially identified as tRNA processing enzyme, generates not only tRNA 3' ends but also mature 5S rRNA 5' ends in vitro. Interestingly, the sequence upstream of the 5S rRNA can be folded into a mini-tRNA, which might explain the processing of this RNA by tRNase Z. The endonuclease is active only at low salt concentrations in vitro, which is in contrast to the 2-4 M KCl concentration present inside the cell in vivo. Electron microscopy studies show that there are no compartments inside the Haloferax cell that could provide lower salt environments. Processing of the 5S rRNA 5' end is not restricted to the haloarchaeal tRNase Z since tRNase Z enzymes from a thermophilic archaeon, a lower and a higher eukaryote, are as well able to cleave the tRNA-like structure 5' of the 5S rRNA. Knock out of the tRNase Z gene in Haloferax volcanii is lethal, showing that the protein is essential for the cell.

The solid and thermally instable azoinitiators V-65 and VR110 were embedded within a polymer particle by using the miniemulsion process and afterwards quickly decomposed by thermal treatment below the glass temperature of the polymer. The resulting nitrogen gas overpressure inside the particles leads to a disruption of the polymer particle and a possible sudden release of encapsulated substances. It is shown, by electron microscopic measurements, that the number of burst particles correlates with the applied temperatures as well as the heating time. The surface deformation could be verified by scanning electron microscope analyses.

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37°C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.

High-pressure frozen rat pancreas tissue samples were cryo-fractured and cryo-sectioned with a diamond knife in the microtome of a freeze-etching device. The bulk fracture faces and block faces were investigated in the frozen-hydrated state by use of a cryo-stage in an in-lens SEM. With this combination, relevant biological structures can be investigated with a few nanometers resolution in a near lifelike state, preventing the artifacts of conventional fixation techniques. Compared to TEM replica techniques, the presented method is more versatile since no replica cleaning is necessary. Additional structures can be made visible by controlled sublimation of ice, leading to a better understanding of the three-dimensional organization of organelles, such as the endoplasmic reticulum.

Abstract. The gastropods Cratena peregrina and Flabellina affinis (Nudibranchia, Aeolidacea) feed on Eudendrium racemosum (Hydrozoa). The question is addressed how the snails are protected against discharging nematocysts. When nematocysts of the hydrozoan discharged against the skin of the gastropods, the impact caused the epidermis to fold, and packets of epidermal vesicles were sloughed off. As vesicles opened, characteristic filamentous spindle-like structures were released. The discharging small microbasic eurytele nematocysts of the hydroid tentacles were blocked at the surface of the skin by mucus and/or by a layer of microvilli. Many secreted spindles from the epidermis of the snails were found among the everted nematocyst components. Discharging holotrichous isorhiza nematocysts of the hydroid cnidophores, however, caused complete lysis of epidermal cells of the gastropods, presumably due to the action of the hydrozoan toxins, which resulted in release of masses of spindles. Spindles then coated the nematocyst threads. It is proposed that the spindles in the epidermal cells of the snails serve a protective role, which ensures detachment of the hydroid tentacles and subsequent regeneration of the injured region of epidermis.

The problem of interaction of human serum albumin (HSA), unconjugated bilirubin (UB) and high porosity activated HSGD carbons is investigated in this study. The decrease of UB to HSA molecular ratio by more than 300 times was demonstrated while the batch experiments in HSA–UB admixtures after contact with HSGD. HSGD carbons express extremely high activity for the removal of UB from HSA containing solutions (more than 100 mg of UB per 1 g of activated carbon). Ex-tempore albumin-coating of carbon surface decreases adsorbent capacity by bilirubin on 21. At the same time ex-tempore albumin-coating of HSGD carbon surface as well as blood citratization prevent platelet and leukocytes loss and clotting inside of the column. Pharmacopoeia solution of HSA containing acetyl-tryptophan or octanoate used for albumin-coating of HSGD adsorbents, becomes ligand-free and rather more active in complexing with protein-bound substances. Combination of albumin-coated HSGD carbon as haemosorbent with HSA ligand-free solution as a transfusion media seems a new prospective modality of the extracorporeal removal of protein-bound toxins.

Hyaluronan (HA), an extracellular matrix component, is involved mainly in the control of cell proliferation, neural crest and tumor cell migration, and wound repair. We investigated the effect of hyaluronan on neural crest (NC) cell migration and its ultrastructural localization in dark (wild-type) and white mutant embryos of the Mexican axolotl (Ambystoma mexicanum,Amphibia). The axolotl system is an accepted model for studying mechanims of NC cell migration. Using a biotinylated hyaluronan binding protein (HABP), major extracellular matrix (ECM) spaces, including those of NC cell migration, reacted equally positive on cryosections through dark and white embryos. Since neural crest-derived pigment cells migrate only in subepidermal spaces of dark embryos, HA does not seem to influence crest cell migration in vivo.However, when tested on different alternating substrates in vitro,migrating NC cells in dark and white embryos prefer HA to fibronectin. In vivo,such an HA migration stimulating effect might exist as well, but be counteracted to differing degrees in dark and white embryos. The ultrastructural localization of HA was studied by means of transmission electron microscopic immunohistochemistry using HABP and different protocols of standard chemical fixation, cryofixation, embedding, and immunolabeling. The binding reaction of HA to HABP was strong and showed an equal distribution throughout ECM spaces after both standard chemical fixation/freeze substitution and cryofixation. A preference for the somite or subepidermal side was not observed. Following standard fixation/freeze substitution HABP-labeled “honeycomb”-like networks reminiscent of fixation artifacts were more prominent than labeled fibrillar or irregular net-like structures. The latter predominated in adequately frozen specimens following high-pressure freezing/freeze substitution. For this reason fibrillar or irregular net-like structures very likely represent hyaluronan in the complex subepidermal matrix of the axolotl embryo in its native arrangement.

In article number 2100453by Steffen Stenger, Mika Lindén, and co‐workers, the activity of the antimicrobial peptide NapFab against intracellular Mycobacterium tuberculosis (Mtb) is significantly increased by loading onto dendritic mesoporous silica nanoparticles (DMSN). This enhanced activity is based on a 30‐fold increased, DMSN‐mediated peptide uptake. Livecell fluorescence microscopy and electron microscopy reveal fast DMSN uptake in endosomal vesicles, intracellular degradation of the carrier, and a continuous peptide release.

Tuberculosis remains a serious global health problem causing 1.3 million deaths annually. The causative pathogen Mycobacterium tuberculosis(Mtb) has developed several mechanisms to evade the immune system and resistances to many conventional antibiotics, so that alternative treatment strategies are urgently needed. By isolation from bronchoalveolar lavage and peptide optimization, a new antimicrobial peptide named NapFab is discovered. While showing robust activity against extracellular Mtb, the activity of NapFab against intracellular bacteria is limited due to low intracellular availability. By loading NapFab onto dendritic mesoporous silica nanoparticles (DMSN) as a carrier system, cellular uptake, and consequently antimycobacterial activity against intracellular Mtbis significantly enhanced. Furthermore, using lattice light‐sheet fluorescence microscopy, it can be shown that the peptide is gradually released from the DMSN inside living macrophages over time. By electron microscopy and tomography, it is demonstrated that peptide loaded DMSN are stored in vesicular structures in proximity to mycobacterial phagosomes inside the cells, but the nanoparticles are typically not in direct contact with the bacteria. Based on the combination of functional and live‐cell imaging analyses, it is hypothesized that after being released from the DMSN NapFab is able to enter the bacterial phagosome and gain access to the bacilli. The activity of the antimicrobial peptide NapFab against intracellular Mycobacterium tuberculosis(Mtb) is significantly increased by loading onto dendritic mesoporous silica nanoparticles (DMSN). This enhanced activity is based on a 30‐fold increased, DMSN‐mediated peptide uptake. Live‐cell fluorescence microscopy and electron microscopy reveal fast DMSN uptake in endosomal vesicles, intracellular degradation of the carrier, and a continuous peptide release.

A surface layer (thickness 1–10 μm) containing colloidal TiO2or ZnO particles was prepared in EVA (a copolymer of ethylene and vinyl acetate). The inorganic particles were formed in situ by hydrolysis of incorporated titanium tetrachloride or diethyl zinc. The resulting materials were analyzed with UV spectroscopy, electron microscopy, X‐ray diffraction, thermogravimetric analysis and atomic emission spectroscopy. The average diameter of the embedded TiO2particles was 70 nm; these particles absorb UV radiation but also induce opacity in the polymer sheets in the visible wavelengths range. The ZnO particles were smaller (average diameter 15 nm); with a surface layer of embedded ZnO, transparent polymer sheets can be obtained that absorb UV radiation. © 1997 John Wiley & Sons, Ltd.

PurposeIn this electron microscopical study, we compared effects of chemical fixation versus cryofixation on the ultrastructure of acute rod outer segment alterations in the rat retina.MethodsThe alterations were induced by toxic levels of diffuse white light. Albino rats were exposed to 2000 lux for 30 min. Samples from one eye of each animal were fixed by high pressure freezing and samples from the other eye were fixed by standard glutaraldehyde procedures.ResultsLight exposed retina showed major differences in their rod outer segments, inner segments and photoreceptor synaptic regions in chemical fixation. In particular gross vesiculations of outer segment membranes were produced in light exposed experiments. In contrast, in cryo-fixed samples such prominent changes were not observed in outer segment membranes. There was, however, occasional formation of small vesicles and a reduction of the cilium diameter in response to light damage. In the dark adapted control group the morphology of chemically fixed and cryo-fixed photoreceptors was similar.ConclusionsWe conclude, that cryo-fixed samples better represent the living state of the retina, because high pressure freezing is a purely physical method and acts much faster than chemical fixation. Curr. Eye Res. 15: 807-814, 1996.

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.

In the present study, we compare expression, storage and secretion of the atrial natriuretic factor (ANF) in atrial and ventricular adult rat cardiomyocytes (aARC and vARC) in long-term culture. The influence of insulin-like growth factor-I (IGF-I) and of basic fibroblast growth factor (bFGF) on ANF production and secretion, as well as on the expression of a structural component,α-smooth muscle actin (α-sm actin), was studied in the two cell types. Antibodies againstα-ANF were used for immunocytochemical localization of ANF. aARC contained more ANF-granules than vARC, and they were distributed throughout the cell bodies. Quantitative determination of ANF storage and secretion was done by radioimmunoassay (RIA;125I), and it was demonstrated that aARC stored and secreted ANF 18- and 16-times more, respectively, when compared to vARC. Immuno-electron microscopy confirmed that ANF storing secretory granules were present in both types of cardiomyocytes. Expression of ANF andα-sm actin in aARC and vARC responded differently to treatment with either IGF-I or bFGF. In aARC, neither IGF-I nor bFGF had an influence on expression of ANF. In vARC, expression of ANF was downregulated by IGF-I and upregulated by bFGF with regard to both immunoreactivity and message. In contrast to vARC, expression ofα-sm actin was not affected by IGF-I in aARC, whereas bFGF produced a strong upregulation similar to that found in vARC. Mitogen-activated protein kinases (MAPK) 42 and 44, though, were equally activated by bFGF and IGF-I in both aARC and vARC.

Intracellular accumulation of α-synuclein (α-syn) and formation of Lewy bodies are neuropathological characteristics of Parkinson’s disease (PD) and related α-synucleinopathies. Oligomerization and spreading of α-syn from neuron to neuron have been suggested as key events contributing to the progression of PD. To directly visualize and characterize α-syn oligomerization and spreading in vivo, we generated two independent conditional transgenic mouse models based on α-syn protein complementation assays using neuron-specifically expressed split Gaussia luciferase or split Venus yellow fluorescent protein (YFP). These transgenic mice allow direct assessment of the quantity and subcellular distribution of α-syn oligomers in vivo. Using these mouse models, we demonstrate an age-dependent accumulation of a specific subtype of α-syn oligomers. We provide in vivoevidence that, although α-syn is found throughout neurons, α-syn oligomerization takes place at the presynapse. Furthermore, our mouse models provide strong evidence for a transsynaptic cell-to-cell transfer of de novogenerated α-syn oligomers in vivo.

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.The figures show high pressure frozen bakers yeast saccharomyces cerevisiaein the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

Joy and Pawley (1992) defined the limitations of spatial resolution in the SEM as follows. “The spatial resolution of the scanning electron microscope is limited by at least three factors: the diameter of the electron probe, the size and shape of the beam/specimen interaction volume with the solid for the mode of imaging employed and the Poisson statistics of the detected signal.” The interaction volume of the primary beam in the sample increases with increasing accelerating voltage (Vo), because electrons with higher energy penetrate deeper into the sample. The interaction volume affects the spatial resolution, because the electrons that finally form the image do not only originate from the impact point of the primary beam, but from the much larger interaction volume. It is not simple to understand how this affects spatial resolution, since resolution is also heavily influenced by the properties of the sample surface (the surface coating) and it is the issue of ongoing debates.

High-pressure facilitates freezing of biological specimens: pressurizing to 2000 bar before cooling retards growth and reduces size of ice crystals in the specimen. Although the lung is 80% air and is generally difficult to freeze, we report here good cellular ultrastructure in high-pressure frozen lung when: 1) the lung is allowed to collapse (empty of air) prior to sampling, or 2) air-filled lung is allowed to compress during pressurization in the high-pressure freezer prior to freezing or 3) airspaces are filled with relatively incompressible fluorocarbon liquid.Small Balb mice (28-30g) were anaesthetized and ventilated via endotracheal tube. The chest was opened and a 2mm diameter disk of lung at the thin anterior margin of the right lung was clamped between two aluminum tophats (200μm wells), sealing in air. In other experiments, the lung was allowed to collapse to 0 cm water transpulmonary pressure prior to clamping. In the third series of experiments, air was gradually replaced with fluorocarbon liquid (FC5312, 3M, Minneapolis MN) and the lung inflated with liquid to functional residual capacity. The top hat assembly containing the sealed lung was transferred to the high-pressure freezer (HPM 010, Bal-Tec, Balzers, Lichtenstein) where they were pressurized and frozen.

Sample preparation techniques for electron-microscopy (EM) dictate our perception of the microworld: any structural information which is lost or distorted during preparation can not be regenerated later and might lead to wrong interpretation of the observed micrograph.Cryofixation based procedures for specimen preparation can avoid most of the structural alterations associated with standard techniques based on chemical fixation. The ultrastructure can be represented in a near “native state” thanks to the high time resolution for dynamic cellular events (1).High pressure freezing (2) permits to cryoimmobilize biological samples up to approx. 200μm thick, in contrast to rapid freezing procedures at ambient pressure that are useful to cryoimmobilize samples up to 10-20 μm thick. The actual samplethickness that can be adequately frozen (=without visible damage due to ice crystal formation in freeze-substituted or freeze-fractured specimens) depends on the concentration of naturally occuring substances that exhibit cryoprotective activities.

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High-pressure freezing–freeze substitution (HPF-FS) methods were applied to germinating pea leaves. Good ultrastructural preservation without visible freezing damage was obtained up to 200 μm in thickness. Compared to conventional chemical fixation (CF), cellular membranes were smoother without undulation, and organelles appeared more turgid. The matrices of cytoplasm and organelles were denser and more homogeneous. These features imply that HPF-FS samples retain more substances and ultrastructure closer to the living state. There were differences in membrane stainability among organelles in HPF-FS specimens, which were not seen after CF. Bundles of microfilaments were observed frequently after HPF-FS.

The impact of the apple rust mite, Aculus schlechtendali (Nalepa), on net CO2 exchange, transpiration rate, and leaf color of field-grown Jonagold and Golden Delicious apples was investigated. Apple rust mite feeding causes leaf browning. Changes in leaf color were measured to assess the cumulative leaf damage. Significant negative relationships were found between cumulative leaf damage and single-leaf net CO2 exchange as well as transpiration rate. The same trends were observed on both varieties, but the effect of apple rust mite feeding was more severe on Jonagold than on Golden Delicious. Leaf tissue injury was analyzed by cryoscanning electron microscopy and light microscopy. The pictures show that apple rust mites penetrate epidermal cells with their stylets, causing multiple puncture wounds. On heavily infested leaves, apple rust mite feeding causes desiccation of the epidermis and the spongy parenchyma. The resulting malfunction of the stomata and problems in gas exchange within the spongy parenchyma are likely to be the main reason for the reduction of gas exchange.