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Microbiology Spectrum May 2019The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and... (Review)
Review
The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.
Topics: Bacterial Capsules; Bacteriological Techniques; Cell Membrane; Cell Wall; Cryoelectron Microscopy; Electron Microscope Tomography; Extracellular Vesicles; Freezing; Gram-Positive Bacteria; Imaging, Three-Dimensional; Lipopolysaccharides; Microscopy, Electron; Microscopy, Electron, Transmission; Mycobacterium; Peptidoglycan; Teichoic Acids
PubMed: 31124431
DOI: 10.1128/microbiolspec.GPP3-0044-2018 -
Science (New York, N.Y.) Sep 2020The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S)...
The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Topics: Amino Acid Substitution; Betacoronavirus; COVID-19 Vaccines; Coronavirus Infections; Cryoelectron Microscopy; Humans; Proline; Protein Domains; Protein Stability; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Viral Vaccines
PubMed: 32703906
DOI: 10.1126/science.abd0826 -
Histochemistry and Cell Biology Sep 2022Megapinosomes are endocytic organelles found in human macrophage colony-stimulating factor (M-CSF) monocyte-derived M macrophages. They are large (several microns) and... (Review)
Review
Megapinosomes are endocytic organelles found in human macrophage colony-stimulating factor (M-CSF) monocyte-derived M macrophages. They are large (several microns) and have a complex internal structure that is connected with the cytosol and consists of interconnected knots and concave bridges with sizes in the range of 100 nm. We called this structure trabecular meshwork. The luminal part of the megapinosome can be connected with luminal tubules and cisterns that form the megapinosome complex. The structures are especially well visible in scanning electron tomography when macrophages are prepared by high-pressure freezing and freeze substitution. Our research received a new impulse after studying the literature on hematopoietic cells, where very similar, most likely homologous, structures have been published in peritoneal macrophages as well as in megakaryocytes and blood platelets. In platelets, they serve as membrane storage that is used for structural changes of platelets during activation.
Topics: Endocytosis; Humans; Macrophages; Megakaryocytes; Trabecular Meshwork
PubMed: 35829814
DOI: 10.1007/s00418-022-02124-x -
Frontiers in Cell and Developmental... 2022Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a...
Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues (), nematode () and yeast (). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.
PubMed: 36003147
DOI: 10.3389/fcell.2022.933376 -
Transfusion Dec 2022OctaplasLG is a frozen solvent/detergent-treated plasma product used for treating complex coagulation factor deficiencies or as substitution therapy in emergency...
BACKGROUND
OctaplasLG is a frozen solvent/detergent-treated plasma product used for treating complex coagulation factor deficiencies or as substitution therapy in emergency situations where specific factor concentrates are not available. A new freeze-dried (also known as lyophilized) form of OctaplasLG, referred as OctaplasLG Lyo (Octapharma AG, Switzerland) offers rapid reconstitution and more flexible storage conditions, improving logistics and utilization. This study compared the biochemical quality of OctaplasLG Lyo with OctaplasLG and single-donor fresh frozen plasma units.
STUDY DESIGN AND METHODS
Three batches of OctaplasLG Lyo, manufactured for production process qualification, and 12 batches of OctaplasLG were provided by Octapharma AB (Sweden). Twelve units of fresh frozen plasma were collected by the local FDA-licensed blood provider. All plasma samples were assessed for global coagulation parameters, coagulation factors and protease inhibitors, activation markers of coagulation and fibrinolysis, and important plasma proteins. Quality control assays were conducted in accordance with European Pharmacopeia requirements.
RESULTS
Frozen and freeze-dried OctaplasLG demonstrated comparable quality profiles upon thawing or reconstitution. All coagulation factor and protease inhibitor activity parameters were in line with levels mandated by the European Pharmacopeia. Fresh frozen plasma units showed comparable coagulation factor activities, with higher protein S and plasmin inhibitor levels than the OctaplasLG products. Fresh frozen plasma parameters showed high lot-to-lot variations.
DISCUSSION
The two pharmaceutical forms of OctaplasLG (frozen and freeze-dried) have comparable biochemical quality. Key features of OctaplasLG Lyo are rapid reconstitution time and storage flexibility, which may improve logistics and utilization, and have particular advantages in emergency situations and pre-hospital settings.
Topics: Humans; Drug Compounding; Solvents; Plasma; Blood Coagulation Factors; Sweden
PubMed: 36181447
DOI: 10.1111/trf.17139 -
Journal of Genetics and Genomics = Yi... Mar 2021Chromatin interactions functionally affect genome architecture and gene regulation, but to date, only fresh samples must be used in High-through chromosome conformation...
Chromatin interactions functionally affect genome architecture and gene regulation, but to date, only fresh samples must be used in High-through chromosome conformation capture (Hi-C) to keep natural chromatin conformation intact. This requirement has impeded the advancement of 3D genome research by limiting sample collection and storage options for researchers and severely limiting the number of samples that can be processed in a short time. Here, we develop a freeze substitution Hi-C (FS-Hi-C) technique that overcomes the need for fresh samples. FS-Hi-C can be used with samples stored in liquid nitrogen (LN): the water in a vitreous form in the sample cells is replaced with ethanol via automated freeze substitution. After confirming that the FS step preserves the natural chromosome conformation during sample thawing, we tested the performance of FS-Hi-C with Drosophila melanogaster and Gossypium hirsutum. Beyond allowing the use of frozen samples and confirming that FS-Hi-C delivers robust data for generating contact heat maps and delineating A/B compartments and topologically associating domains, we found that FS-Hi-C outperforms the in situ Hi-C in terms of library quality, reproducibility, and valid interactions. Thus, FS-Hi-C will probably extend the application of 3D genome structure analysis to the vast number of experimental contexts in biological and medical research for which Hi-C methods have been unfeasible to date.
Topics: Animals; Cost-Benefit Analysis; Drosophila melanogaster; Freeze Substitution
PubMed: 33573880
DOI: 10.1016/j.jgg.2020.11.002 -
Histochemistry and Cell Biology Oct 2013In 1898, the Golgi apparatus was discovered by light microscopy, and since the 1950s, the ultrastructure composition is known by electron microscopic investigation. The... (Review)
Review
In 1898, the Golgi apparatus was discovered by light microscopy, and since the 1950s, the ultrastructure composition is known by electron microscopic investigation. The complex three-dimensional morphology fascinated researchers and was sometimes even the driving force to develop novel visualization techniques. However, the highly dynamic membrane systems of Golgi apparatus are delicate and prone to fixation artifacts. Therefore, the understanding of Golgi morphology and its function has been improved significantly with the development of better preparation methods. Nowadays, cryo-fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in amorphous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy and tomography. Even though the overall morphology of vitrified Golgi stacks is comparable to well-prepared and resin-embedded samples, previously unknown structural details can be observed solely based on their native density. At this point, any further improvement of sample preparation would gain novel insights, perhaps not in terms of general morphology, but on fine structural details of this dynamic organelle.
Topics: Animals; Cryoelectron Microscopy; Golgi Apparatus; Humans
PubMed: 23954988
DOI: 10.1007/s00418-013-1136-3 -
Materials (Basel, Switzerland) May 2022Lanthanum aluminate-based perovskite (LaAlO) has excellent stability at high temperatures, low toxicity, and high chemical resistance and also offers wide versatility to... (Review)
Review
Lanthanum aluminate-based perovskite (LaAlO) has excellent stability at high temperatures, low toxicity, and high chemical resistance and also offers wide versatility to the substitution of La and Al, thus, allowing it to be applied as a catalyst, nano-adsorbent, sensor, and microwave dielectric resonator, amongst other equally important uses. As such, LaAlO perovskites have gained importance in recent years. This review considers the extensive literature of the past 10 years on the synthesis and catalytic applications of perovskites based on lanthanum and aluminum (LaAlO). The aim is, first, to provide an overview of the structure, properties, and classification of perovskites. Secondly, the most recent advances in synthetic methods, such as solid-state methods, solution-mediated methods (co-precipitation, sol-gel, and Pechini synthesis), thermal treatments (combustion, microwave, and freeze drying), and hydrothermal and solvothermal methods, are also discussed. The most recent energetic catalytic applications (the dry and steam reforming of methane; steam reforming of toluene, glycerol, and ethanol; and oxidative coupling of methane, amongst others) using these functional materials are also addressed. Finally, the synthetic challenges, advantages, and limitations associated with the preparation methods and catalytic applications are discussed.
PubMed: 35591622
DOI: 10.3390/ma15093288 -
Biomolecules Oct 2019Tannins are one of the most natural, non-toxic, and highly reactive aromatic biomolecules classified as polyphenols. The reactive phenolic compounds present in their... (Review)
Review
Tannins are one of the most natural, non-toxic, and highly reactive aromatic biomolecules classified as polyphenols. The reactive phenolic compounds present in their chemical structure can be an alternative precursor for the preparation of several polymeric materials for applications in distinct industries: adhesives and coatings, leather tanning, wood protection, wine manufacture, animal feed industries, and recently also in the production of new porous materials (i.e., foams and gels). Among these new polymeric materials synthesized with tannins, organic and carbon gels have shown remarkable textural and physicochemical properties. Thus, this review presents and discusses the available studies on organic and carbon gels produced from tannin feedstock and how their properties are related to the different operating conditions, hence causing their cross-linking reaction mechanisms. Moreover, the steps during tannin gels preparation, such as the gelation and curing processes (under normal or hydrothermal conditions), solvent extraction, and gel drying approaches (i.e., supercritical, subcritical, and freeze-drying) as well as the methods available for their carbonization (i.e., pyrolysis and activation) are presented and discussed. Findings from organic and carbon tannin gels features demonstrate that their physicochemical and textural properties can vary greatly depending on the synthesis parameters, drying conditions, and carbonization methods. Research is still ongoing on the improvement of tannin gels synthesis and properties, but the review evaluates the application of these highly porous materials in multidisciplinary areas of science and engineering, including thermal insulation, contaminant sorption in drinking water and wastewater, and electrochemistry. Finally, the substitution of phenolic materials (i.e., phenol and resorcinol) by tannin in the production of gels could be beneficial to both the bioeconomy and the environment due to its low-cost, bio-based, non-toxic, and non-carcinogenic characteristics.
Topics: Carbon; Flavonoids; Gels; Molecular Structure; Porosity; Tannins
PubMed: 31597350
DOI: 10.3390/biom9100587 -
Frontiers in Neuroanatomy 2021The post-embedding immunogold (PI) technique for immunolabeling of neuronal tissues utilizing standard thin-section transmission electron microscopy (TEM) continues to... (Review)
Review
The post-embedding immunogold (PI) technique for immunolabeling of neuronal tissues utilizing standard thin-section transmission electron microscopy (TEM) continues to be a prime method for understanding the functional localization of key proteins in neuronal function. Its main advantages over other immunolabeling methods for thin-section TEM are (1) fairly accurate and quantifiable localization of proteins in cells; (2) double-labeling of sections using two gold particle sizes; and (3) the ability to perform multiple labeling for different proteins by using adjacent sections. Here we first review in detail a common method for PI of neuronal tissues. This method has two major parts. First, we describe the freeze-substitution embedding method: cryoprotected tissue is frozen in liquid propane plunge-freezing, and is placed in a freeze-substitution instrument in which the tissue is embedded in Lowicryl at low temperatures. We highlight important aspects of freeze-substitution embedding. Then we outline how thin sections of embedded tissue on grids are labeled with a primary antibody and a secondary gold particle-conjugated antibody, and the particular problems encountered in TEM of PI-labeled sections. In the Discussion, we compare our method both to earlier PI methods and to more recent PI methods used by other laboratories. We also compare TEM immunolabeling using PI vs. various pre-embedding immunolabeling methods, especially relating to neuronal tissue.
PubMed: 34720893
DOI: 10.3389/fnana.2021.763427