Application of Advanced Microscopic Methods to Study the Interaction of Carboxylated Fluorescent Nanodiamonds with Membrane Structures in THP-1 Cells: Activation of Inflammasome NLRP3 as the Result of Lysosome Destabilization.

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.

What keeps polyhydroxyalkanoates in bacterial cells amorphous? A derivation from stress exposure experiments.

Polyhydroxyalkanoates (PHA) are storage polymers accumulated by numerous prokaryotes in form of intracellular granules. Native PHA granules are formed by amorphous polymer which reveals considerably higher elasticity and flexibility as compared to crystalline pure PHA polymers. The fact that bacteria store PHA in amorphous state has great biological consequences. It is not clear which mechanisms protect amorphous polymer in native granules from transition into thermodynamically favorable crystalline state. Here, we demonstrate that exposition of bacterial cells to particular stressors induces granules aggregation, which is the first but not sufficient condition for PHA crystallization. Crystallization of the polymer occurs only when the stressed bacterial cells are subsequently dried. The fact that both granules aggregation and cell drying must occur to induce crystallization of PHA indicates that both previously suggested hypotheses about mechanisms of stabilization of amorphous state of native PHA are valid and, in fact, both effects participate synergistically. It seems that the amorphous state of the polymer is stabilized kinetically by the low rate of crystallization in limited volume in small PHA granules and, moreover, water present in PHA granules seems to function as plasticizer protecting the polymer from crystallization, as confirmed experimentally for the first time by the present work.

Monitoring Candida parapsilosis and Staphylococcus epidermidis Biofilms by a Combination of Scanning Electron Microscopy and Raman Spectroscopy.

The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

A Versatile High-Vacuum Cryo-transfer System for Cryo-microscopy and Analytics.

Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented.

Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.

The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.

Candida parapsilosis biofilm identification by Raman spectroscopy.

Colonies of Candida parapsilosis on culture plates were probed directly in situ using Raman spectroscopy for rapid identification of specific strains separated by a given time intervals (up to months apart). To classify the Raman spectra, data analysis was performed using the approach of principal component analysis (PCA). The analysis of the data sets generated during the scans of individual colonies reveals that despite the inhomogeneity of the biological samples unambiguous associations to individual strains (two biofilm-positive and two biofilm-negative) could be made.

A Comparison of the Effects of Continuous Illumination and Day/Night Regimes on PHB Accumulation in Synechocystis Cells.

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable polymer with properties comparable to polypropylene and therefore has the potential to replace conventional plastics. PHB is intracellularly accumulated by prokaryotic organisms. For the cells PHB functions manly as carbon and energy source, but all possible functions of PHB are still not known. Synechocystis (cyanobacteria) accumulates PHB using light as energy and CO2 as carbon source. The main trigger for PHB accumulation in cyanobacteria is nitrogen and phosphorous depletion with simultaneous surplus of carbon and energy. For the above reasons, obtaining knowledge about external factors influencing PHB accumulation is of highest interest. This study compares the effect of continuous light exposure and day/night (16/8 h) cycles on selected physiology parameters of three Synechocystis strains. We show that continuous illumination at moderate light intensities leads to an increased PHB accumulation in Synechocystis salina CCALA 192 (max. 14.2% CDW - cell dry weight) compared to day/night cycles (3.7% CDW). In addition to PHB content, glycogen and cell size increased, while cell density and cell viability decreased. The results offer new approaches for further studies to gain deeper insights into the role of PHB in cyanobacteria to obtain bioplastics in a more sustainable and environmentally friendly way.

Drosophila GGA model: an ultimate gateway to GGA analysis.

Golgi-localized, γ-ear-containing, ADP ribosylation factor-binding (GGA) proteins are monomeric adaptors implicated in clathrin-mediated vesicular transport between the trans Golgi network and endosomes, characterized mainly from cell culture analysis of lysosomal sorting. To provide the first demonstration of GGA's role in vivo, we used Drosophila which has a single GGA and a single lysosomal sorting receptor, lysosomal enzyme receptor protein (LERP). Using RNAi knockdowns, we show that the Drosophila GGA is required for lysosomal sorting. We further identified authentic components of the Drosophila lysosomal sorting system--the sorting receptor LERP, the sorting adaptor GGA and the lysosomal cargo cathepsins B1, D and L--to show that GGA depletion results in lysosomal dysfunction. Abnormal lysosomal morphology, missorting of lysosomal cathepsins and impaired lysosomal proteolysis show disturbed LERP trafficking after GGA depletion. GGA is highly expressed in the mushroom bodies and the pigment cells of the retina, and increasing or decreasing the levels of GGA in the eyes leads to retinal defects. Reduced GGA levels also enhance an eye defect caused by overexpression of the autophagy-associated protein Blue cheese (Bchs), implicating GGA in autophagic processes. This shows that Drosophila provides an excellent whole-animal model to gain new insights into the function of GGA in the physiological environment of a multicellular organism.

Following the mechanisms of bacteriostatic versus bactericidal action using Raman spectroscopy.

Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations-MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms' "fingerprint" (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.

Urany-Less Low Voltage Transmission Electron Microscopy: A Powerful Tool for Ultrastructural Studying of Cyanobacterial Cells.

Sample preparation protocols for conventional high voltage transmission electron microscopy (TEM) heavily rely on the usage of staining agents containing various heavy metals, most commonly uranyl acetate and lead citrate. However high toxicity, rising legal regulations, and problematic waste disposal of uranyl acetate have increased calls for the reduction or even complete replacement of this staining agent. One of the strategies for uranyless imaging is the employment of low-voltage transmission electron microscopy. To investigate the influence of different imaging and staining strategies on the final image of cyanobacterial cells, samples stained by uranyl acetate with lead citrate, as well as unstained samples, were observed using TEM and accelerating voltages of 200 kV or 25 kV. Moreover, to examine the possibilities of reducing chromatic aberration, which often causes issues when imaging using electrons of lower energies, samples were also imaged using a scanning transmission electron microscopy at 15 kV accelerating voltages. The results of this study demonstrate that low-voltage electron microscopy offers great potential for uranyless electron microscopy.

Liposomal form of erlotinib for local inhalation administration and efficiency of its transport to the lungs.

This contribution is focused on the preparation of a liposomal drug delivery system of erlotinib resisting the nebulization process that could be used for local treatment of non-small-cell lung cancer. Liposomes with different compositions were formulated to reveal their influence on the encapsulation efficiency of erlotinib. An encapsulation efficiency higher than 98 % was achieved for all vesicles containing phosphatidic acid (d ≈ 100 nm, ζ = - 43 mV) even in the presence of polyethylene glycol (d ≈ 150 nm, ζ = - 17 mV) which decreased this value in all other formulas. The three most promising formulations were nebulized by two air-jet and two vibrating mesh nebulizers, and the aerosol deposition in lungs was calculated by tools of computational fluid and particle mechanics. According to the numerical simulations and measurements of liposomal stability, air-jet nebulizers generated larger portion of the aerosol able to penetrate deeper into the lungs, but the delivery is likely to be more efficient when the formulation is administered by Aerogen Solo vibrating mesh nebulizer because of a higher portion of intact vesicles after the nebulization. The leakage of encapsulated drug from liposomes nebulized by this nebulizer was lower than 2 % for all chosen vesicles.

Robust Local Thickness Estimation of Sub-Micrometer Specimen by 4D-STEM.

A quantitative four-dimensional scanning transmission electron microscopy (4D-STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub-micrometer amorphous specimen using both direct detection and light conversion 2D-STEM detectors in a coincident FIB-SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam-sensitive materials.

Artificial forisomes are ideal models of forisome assembly and activity that allow the development of technical devices.

Forisomes are protein polymers found in leguminous plants that have the remarkable ability to undergo reversible "muscle-like" contractions in the presence of divalent cations and in extreme pH environments. To gain insight into the molecular basis of forisome structure and assembly, we used confocal laser scanning microscopy to monitor the assembly of fluorescence-labeled artificial forisomes in real time, revealing two distinct assembly processes involving either fiber elongation or fiber alignment. We also used scanning and transmission electron microscopy and X-ray diffraction to investigate the ultrastructure of forisomes, finding that individual fibers are arranged into compact fibril bundles that disentangle with minimal residual order in the presence of calcium ions. To demonstrate the potential applications of artificial forisomes, we created hybrid protein bodies from forisome subunits fused to the B-domain of staphylococcal protein A. This allowed the functionalization of the artificial forisomes with antibodies that were then used to target forisomes to specific regions on a substrate, providing a straightforward approach to develop forisome-based technical devices with precise configurations. The functional contractile properties of forisomes are also better preserved when they are immobilized via affinity reagents rather than by direct contact to the substrate. Artificial forisomes produced in plants and yeast therefore provide an ideal model for the investigation of forisome structure and assembly and for the design and testing of tailored artificial forisomes for technical applications.

Cathodoluminescence imaging of cellular structures labeled with luminescent iridium or rhenium complexes at cryogenic temperatures.

We report for the first time the use of two live-cell imaging agents from the group of luminescent transition metal complexes (IRAZOLVE-MITO and REZOLVE-ER) as cathodoluminescent probes. This first experimental demonstration shows the application of both probes for the identification of cellular structures at the nanoscale and near the native state directly in the cryo-scanning electron microscope. This approach can potentially be applied to correlative and multimodal approaches and used to target specific regions within vitrified samples at low electron beam energies.

Combination of Hypotonic Lysis and Application of Detergent for Isolation of Polyhydroxyalkanoates from Extremophiles.

Production of polyhydroxyalkanoates (PHA), microbial biopolyesters, employing extremophilic microorganisms is a very promising concept relying on robustness of such organisms against microbial contamination, which provides numerous economic and technological benefits. In this work, we took advantage of the natural susceptibility of halophilic and thermophilic PHA producers to hypotonic lysis and we developed a simple and robust approach enabling effective isolation of PHA materials from microbial cells. The method is based on the exposition of microbial cells to hypotonic conditions induced by the diluted solution of sodium dodecyl sulfate (SDS) at elevated temperatures. Such conditions lead to disruption of the cells and release of PHA granules. Moreover, SDS, apart from its cell-disruptive function, also solubilizes hydrophobic components, which would otherwise contaminate PHA materials. The purity of obtained materials, as well as the yields of recovery, reach high values (values of purity higher than 99 wt.%, yields close to 1). Furthermore, we also focused on the removal of SDS from wastewater. The simple, inexpensive, and safe technique is based on the precipitation of SDS in the presence of KCl. The precipitate can be simply removed by decantation or centrifugation. Moreover, there is also the possibility to regenerate the SDS, which would substantially improve the economic feasibility of the process.

The role of polyhydroxyalkanoates in adaptation of Cupriavidus necator to osmotic pressure and high concentration of copper ions.

Polyhydroxyalkanoates (PHA) are abundant microbial polyesters accumulated in the form of intracellular granules by numerous prokaryotes primarily as storage of carbon and energy. Apart from their storage function, the presence of PHA also enhances the robustness of the microbial cells against various stressors. In this work, we investigated the role of PHA in Cupriavidus necator, a model organism concerning PHA metabolism, for adaptation to osmotic pressure and copper ions. In long-term laboratory evolution experiments, the bacterial culture was cultivated in presence of elevated doses of sodium chloride or copper ions (incubations lasted 78 passages for Cu2+ and 68 passages for NaCl) and the evolved strains were compared with the wild-type strain in terms of growth and PHA production capacity, cell morphology (investigated by various electron microscopy techniques), activities of selected enzymes involved in PHA metabolism and other crucial metabolic pathways, the chemical composition of bacterial biomass (determined by infrared and Raman spectroscopy) and also considering robustness against various stressors. The results confirmed the important role of PHA metabolism for adaptation to both tested stressors.

The chlorosome of Chlorobaculum tepidum: size, mass and protein composition revealed by electron microscopy, dynamic light scattering and mass spectrometry-driven proteomics.

Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (±28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.

Powder Nano-Beam Diffraction in Scanning Electron Microscope: Fast and Simple Method for Analysis of Nanoparticle Crystal Structure.

We introduce a novel scanning electron microscopy (SEM) method which yields powder electron diffraction patterns. The only requirement is that the SEM microscope must be equipped with a pixelated detector of transmitted electrons. The pixelated detectors for SEM have been commercialized recently. They can be used routinely to collect a high number of electron diffraction patterns from individual nanocrystals and/or locations (this is called four-dimensional scanning transmission electron microscopy (4D-STEM), as we obtain two-dimensional (2D) information for each pixel of the 2D scanning array). Nevertheless, the individual 4D-STEM diffractograms are difficult to analyze due to the random orientation of nanocrystalline material. In our method, all individual diffractograms (showing randomly oriented diffraction spots from a few nanocrystals) are combined into one composite diffraction pattern (showing diffraction rings typical of polycrystalline/powder materials). The final powder diffraction pattern can be analyzed by means of standard programs for TEM/SAED (Selected-Area Electron Diffraction). We called our new method 4D-STEM/PNBD (Powder NanoBeam Diffraction) and applied it to three different systems: Au nano-islands (well diffracting nanocrystals with size ~20 nm), small TbF3 nanocrystals (size < 5 nm), and large NaYF4 nanocrystals (size > 100 nm). In all three cases, the STEM/PNBD results were comparable to those obtained from TEM/SAED. Therefore, the 4D-STEM/PNBD method enables fast and simple analysis of nanocrystalline materials, which opens quite new possibilities in the field of SEM.

High Resolution Powder Electron Diffraction in Scanning Electron Microscopy.

A modern scanning electron microscope equipped with a pixelated detector of transmitted electrons can record a four-dimensional (4D) dataset containing a two-dimensional (2D) array of 2D nanobeam electron diffraction patterns; this is known as a four-dimensional scanning transmission electron microscopy (4D-STEM). In this work, we introduce a new version of our method called 4D-STEM/PNBD (powder nanobeam diffraction), which yields high-resolution powder diffractograms, whose quality is fully comparable to standard TEM/SAED (selected-area electron diffraction) patterns. Our method converts a complex 4D-STEM dataset measured on a nanocrystalline material to a single 2D powder electron diffractogram, which is easy to process with standard software. The original version of 4D-STEM/PNBD method, which suffered from low resolution, was improved in three important areas: (i) an optimized data collection protocol enables the experimental determination of the point spread function (PSF) of the primary electron beam, (ii) an improved data processing combines an entropy-based filtering of the whole dataset with a PSF-deconvolution of the individual 2D diffractograms and (iii) completely re-written software automates all calculations and requires just a minimal user input. The new method was applied to Au, TbF3 and TiO2 nanocrystals and the resolution of the 4D-STEM/PNBD diffractograms was even slightly better than that of TEM/SAED.