Brachiaria brizantha Grass as a Feedstock for Ethanol Production

HIGHLIGHTS Brachiaria brizantha proved to be a promising biomass for ethanol production. Fermentation was not impaired by the inhibitors furfural and hydroxymethylfurfural.

Abstract Different lignocellulosic biomasses are found worldwide and each country has its own important industrial crop that can be converted into high-value products, such as ethanol. Therefore, evaluation of new biomasses to be used in biorefineries is important to decrease the dependence on non-renewable resources and to guarantee sustainable development. This work evaluated Brachiaria brizantha, a grass commonly used as animal forage, and the standard biomass for 2G-ethanol, sugarcane bagasse. The chemical compositions of both biomasses were determined and different times and temperature of acid pretreatment were tested. Morphological analysis via scanning electron microscopy showed more deconstructed fibers after harsher biomass pretreatments. Simultaneous saccharification and fermentation of pretreated Brachiaria brizantha presented higher efficiency than when using sugarcane bagasse as the carbon source. A biomass conversion of 46 % was achieved when Brachiaria brizantha grass was pretreated with 2% sulfuric acid for 60 minutes. Moreover, fermentation was not impaired by the inhibitors furfural and hydroxymethylfurfural. It was concluded that Brachiaria brizantha is a promising biomass for ethanol production.

Diagnostic Electron Microscopy of Viruses With Low-voltage Electron Microscopes.

Diagnostic electron microscopy is a useful technique for the identification of viruses associated with human, animal, or plant diseases. The size of virus structures requires a high optical resolution (i.e., about 1 nm), which, for a long time, was only provided by transmission electron microscopes operated at 60 kV and above. During the last decade, low-voltage electron microscopy has been improved and potentially provides an alternative to the use of high-voltage electron microscopy for diagnostic electron microscopy of viruses. Therefore, we have compared the imaging capabilities of three low-voltage electron microscopes, a scanning electron microscope equipped with a scanning transmission detector and two low-voltage transmission electron microscopes, operated at 25 kV, with the imaging capabilities of a high-voltage transmission electron microscope using different viruses in samples prepared by negative staining and ultrathin sectioning. All of the microscopes provided sufficient optical resolution for a recognition of the viruses tested. In ultrathin sections, ultrastructural details of virus genesis could be revealed. Speed of imaging was fast enough to allow rapid screening of diagnostic samples at a reasonable throughput. In summary, the results suggest that low-voltage microscopes are a suitable alternative to high-voltage transmission electron microscopes for diagnostic electron microscopy of viruses.

Microbialite-like structures in Cladophora sp. (Ulvophyceae) mats from a subtropical Andean basin: ecological implications / Estructuras de tipo microbialito en Cladophora sp. (Ulvophyceae) de una cuenca andina subtropical: implicaciones ecológicas

Abstract The solubility equilibrium of calcite is influenced by physicochemical, climatic and biological factors. Annual cycles of exceptionally prolonged drought, in conjunction with naturally occurring diffuse organic pollution, generate the unique conditions for the precipitation of lithified carbonate structures (microbialites). The aim of this article is to analyze the possible implications of calcite precipitation produced in mats of Cladophora sp. in an Andean subtropical basin, considering it is the first time this phenomenon is described for the region. We collected samples from selected sites at the Lules River Basin, in four sampling dates between the years 2003 and 2004, within a monitoring work of 15 years. Samples were analyzed using an electron microscope and X-ray diffraction analysis. We found that Gomphonema sp. attached to Cladophora sp. contributes to precipitation of calcite and formation of microbialite like structures, in the studied area. This work presents an initial discussion of the discovery of microbialites-like structures attached to Cladophora sp. mats in a subtropical Andean stream and the environmental conditions that lead to their production, as well as the possible ecological implications of these microbialites.(AU)

Resumen La fase sólida de las sales de los cuerpos de agua juega un papel importante en la concentración de los elementos mayoritarios. El equilibrio de solubilidad de la calcita está influenciado por factores fisicoquímicos, climáticos y biológicos. Los ciclos anuales de sequía excepcionalmente prolongada, junto con la contaminación orgánica difusa de origen natural, generan condiciones únicas para la precipitación de estructuras de carbonato litificado (microbialitos). El objetivo de este artículo es analizar las posibles implicaciones de la precipitación de calcita producida en Cladophora sp. en una cuenca subtropical andina, considerando que es la primera vez que se describe este fenómeno para la región. Recolectamos muestras en sitios seleccionados en la Cuenca del Río Lules, en cuatro fechas de muestreo entre 2003 y 2004, en el marco de un trabajo de monitoreo de quince años. Usamos red Surber para realizar los muestreos. Las muestras de algas, una vez libres de macroinvertebrados, se secaron y pesaron. Analizamos utilizando microscopía electrónica y difracción de rayos X. Encontramos que Gomphonema sp. unido a Cladophora contribuye a la precipitación de calcita y la formación de estructuras similares a microbialitos, en el área estudiada. Este trabajo presenta una discusión inicial sobre el descubrimiento de estructuras similares a microbialitos unidas a filamentos de Cladophora sp. en una cuenca andina subtropical y las condiciones ambientales que conducen a su producción, así como las posibles implicaciones ecológicas de lo mencionado anteriormente.(AU)

Correlative light and electron microscopy is a powerful tool to study interactions of extracellular vesicles with recipient cells.

Extracellular vesicles (EVs) and their interactions with recipient cells constitute a rapidly growing research area. However, due to the limitations in current methodologies, the mechanisms of these interactions are still unclear. Microscopic studies of EVs are challenging, because their typical diameter is near the resolution limit of light microscopy, and electron microscopy has restricted possibilities for protein labelling. The objective of this study was to combine these two techniques to demonstrate in detail the interactions of EVs by recipient cells. Hyaluronan synthase 3 (HAS3) is an integral transmembrane protein that is enriched in EVs. In this work, GFP-HAS3 was utilized to study the interactions of EVs with the recipient cells. Surprisingly, confocal analysis correlation with scanning electron microscopy (SEM) revealed that most of the EVs were indeed lying on the recipient cell's plasma membrane, while the level of EV-derived intracellular signal was low. Immunoelectron microscopy supported this finding. Furthermore, hyaluronan oligosaccharides decreased the numbers of bound EVs, suggesting that CD44 participates in the regulation of their binding. This study indicates that correlative light and electron microscopy is a reliable method to analyze EV interactions with recipient cells. Detailed 3D confocal imaging of EV carrying a GFP-label on their plasma membrane combined with high-resolution electron microscopy provides significantly more information than either of the techniques alone. In the future studies it is crucial to utilize these techniques and their combinations to solve in detail the ambiguous fate of EV in target cells. Furthermore, live cell imaging at high resolution will be required to obtain definite answers on the detailed mechanisms of binding, fusion and endocytosis of EVs.

Porous Silicon Nanoneedles Modulate Endocytosis to Deliver Biological Payloads.

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane-nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state-of-the-art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin-coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae- and clathrin-mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle-mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell-manipulation technologies.

Imaging extracellular vesicles: current and emerging methods.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells. They range from 30 nm to several micrometers in diameter, and ferry biological cargos such as proteins, lipids, RNAs and DNAs for local and distant intercellular communications. EVs have since been found to play a role in development, as well as in diseases including cancers. To elucidate the roles of EVs, researchers have established different methods to visualize and study their spatiotemporal properties. However, since EV are nanometer-sized, imaging them demands a full understanding of each labeling strategy to ensure accurate monitoring. This review covers current and emerging strategies for EV imaging for prospective studies.

Sequential Immunofluorescent Light Microscopy and Electron Microscopy of Recombination Nodules During Meiotic Prophase I.

Immunolocalization using either fluorescence for light microscopy (LM) or gold particles for electron microscopy (EM) has become a common tool to pinpoint proteins involved in recombination during meiotic prophase. Each method has its advantages and disadvantages. For example, LM immunofluorescence is comparatively easier and higher throughput compared to immunogold EM localization. In addition, immunofluorescence has the advantages that a faint signal can often be enhanced by longer exposure times and colocalization using two (or more) probes with different absorbance and emission spectra is straightforward. However, immunofluorescence is not useful if the object of interest does not label with an antibody probe and is below the resolution of the LM. In comparison, immunogold EM localization is higher resolution than immunofluorescent LM localization, and individual nuclear structures, such as recombination nodules, can be identified by EM regardless of whether they are labeled or not. However, immunogold localization has other disadvantages including comparatively low signal-to-noise ratios, more difficult colocalization using gold particles of different sizes, and the inability to evaluate labeling efficiency before examining the sample using EM (a more expensive and time-consuming technique than LM). Here we describe a method that takes advantage of the good points of both immunofluorescent LM and EM to analyze two classes of late recombination nodules (RNs), only one of which labels with antibodies to MLH1 protein, a marker of crossovers. The method can be used readily with other antibodies to analyze early recombination nodules or other prophase I structures.

Ultrasensitive Detection of α-Fetoprotein by Total Internal Reflection Scattering-Based Super-Resolution Microscopy for Superlocalization of Nano-Immunoplasmonics.

Superlocalization of immunoplasmonic nanotags on antibody-bound gold-nanoislands (GNIs) along the x and y coordinates was determined using total internal reflection scattering-based super-resolution microscopy (TIRS-SRM) at subdiffraction limit resolution. Individual immunoplasmonic nanotags (20 nm silver nanoparticles) and 100 nm GNIs were selectively acquired in the evanescent field layer by wavelength-dependent plasmonic scattering using two illumination lasers (405 and 635 nm, respectively). α-Fetoprotein (AFP), a liver cancer-related model protein, was immobilized as a target molecule on the GNI arrays. The centroid position of a localized immunoplasmonic nanotag on the GNI was resolved at less than 10 nm of spatial resolution by applying 2D Gaussian fitting to its point spread function. This method showed enhanced sensitive quantification with a limit of detection (LOD) of 7.04 zM (1-2 molecules of AFP/GNI), which was 100-5000000000 times lower than detection limits obtained with previous AFP detection methods. Furthermore, the method was also successfully applied to quantify AFP molecules at the single-molecule level in human serum samples. The wavelength-dependent TIRS-SRM method was demonstrated to be an effective tool for superlocalizing individual protein molecules and interactions in nanoscale regions and was a reliable method for the ultrasensitive quantitative detection of disease-related protein molecules as a nanosensor and for diagnosis at the single-molecule level.

Correlative stochastic optical reconstruction microscopy and electron microscopy.

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.

Dificuldades no diagnóstico diferencial entre mesotelioma pleural e adenocarcinoma metastático para pleura patológica / Difficulties in the differential diagnosis between pleural mesothelioma and metastatic adenocarcinoma for pathological pleura

As dificuldades diagnósticas entre mesotelioma pleural e adenocarcinoma metastático na pleura exigem estudo amplo. Os autores ilustram um caso clínico cujo diagnóstico só foi estabelecido após realização de toracotomia, com retirada de material para análise imuno-histoquímica. A diferenciação diagnóstica é de fundamental importância, uma vez que envolvem conduta terapêutica e prognóstico distintos. O estudo adequado deve utilizar material obtido através de toracoscopia ou toracotomia e empregar coloração imuno-histoquímica, estudos com anticorpos monoclonais, microscopia eletrônica e pesquisa de marcadores tumorais.

The diagnosis difficulties distinguishing mesothelioma from pleural metastatic adenocarcinoma request wide study. The authors illustrate a clinical case whose diagnosis was only established after thoracotomy removing material for immune-histochemical analysis. The diagnosis differentiating is very important, because involves distinct treatment and prognosis. The appropriate study should use material obtained through thoracoscopy or thoracotomy and submitted to immune-histochemical coloration, studies with monoclonal antibodies, electronic microscopy and research of tumor makers.

Imaging of protease functions--current guide to spotting cysteine cathepsins in classical and novel scenes of action in mammalian epithelial cells and tissues.

The human genome encodes some hundreds of proteases. Many of these are well studied and understood with respect to their biochemistry, molecular mechanisms of proteolytic cleavage, expression patterns, molecular structure, substrate preferences and regulatory mechanisms, including their endogenous inhibitors. Moreover, precise determination of protease localisation within subcellular compartments, peri- and extracellular spaces has been extremely useful in elucidating biological functions of peptidases. This can be achieved by refined methodology as will be demonstrated herein for the cysteine cathepsins. Besides localisation, it is now feasible to study in situ enzymatic activity at the various levels of subcellular compartments, cells, tissues, and even whole organisms including mouse.

3D versus 2D cell culture implications for electron microscopy.

Cell culture systems are indispensable tools for basic research and a wide range of clinical in vitro studies. However, conventional 2D cell cultures poorly mimic the conditions in the living organism. This limitation may seriously compromise the reliability and significance of data obtained from such approaches. Therefore, we present here a comparative study on selected 3D and 2D cell cultures of U87-MG human glioblastoma cells that were processed by means of high-pressure freezing and freeze-substitution as well as by conventional chemical fixation and Tokuyasu cryo-section immuno-labeling. Three-dimensional cultures comprised pseudo-vascularized cultures, fiber and bead scaffold cultures, and spheroid cultures. Cell cultures in dishes and on coverslips were the static 2D culture systems used as reference models. We will discuss morphological and immuno-cytochemical observations with respect to the feasibility of the cell culture systems investigated for the state-of-the-art electron microscopy.

Optimization and validation of FePro cell labeling method.

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 microg/ml of Fe and 3 microg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 microg/ml of Fe and 3 microg/ml of Pro is effective in labeling cells for cellular MRI.

Electron microscopy of whole cells in liquid with nanometer resolution.

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 micros were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 +/- 1 microm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods.

Electron microscopic imaging and analysis of isolated dynein particles.

Despite more than 40 years of investigation since the discovery of dynein [Gibbons, I. R. and Rowe, A. J. (1965). Science149, 424-426] our understanding of how this microtubule-based motor generates force and movement remains frustratingly incomplete at the atomic level. Electron microscopy (EM) has played a major role in establishing dynein's complex architecture and its nucleotide-dependent conformational changes. In this chapter we review recent structural studies and describe in detail negative stain EM and computational single-particle image processing techniques that have been used to investigate dynein. We describe studies of both Chlamydomonas flagellar inner arm dynein-c and recombinant cytoplasmic dynein from Dictyostelium. We also detail methods for locating green fluorescent protein (GFP) and blue fluorescent protein (BFP) tags inserted at specific locations within the dynein motor, which can be used to map subdomains and conformational changes.

Correlated light and electron microscopy of the cytoskeleton.

The cytoskeleton of cultured cells can be most easily visualized in the electron microscope by simultaneous extraction and fixation with Triton-glutaraldehyde mixtures, followed by negative staining. Actin filaments are better preserved by stabilization with phalloidin, either during or after the primary fixation step. A technique is described for the combination of this procedure with live cell microscopy. Optimal conditions for light microscopy are achieved by culturing cells on coverslips coated with formvar film. For cell relocation a gold finder grid pattern is embossed on the film by evaporation through a tailor-made mask. After video microscopy and fixation, the film is floated from the coverslip and an electron microscope grid added to the film with the central hole of the grid over the region of interest. Accurate positioning is achieved under a dissecting microscope, using forceps mounted in a micromanipulator. Examples are shown of the changes in organization of actin filaments in the lamellipodia of migrating melanoma cells resulting from changes in protrusion rate. The technique is applicable to alternative processing procedures after fixation, including cryoelectron tomography.

Oxide formation and alloying elements enrichment on TRIP steel surface during inter-critical annealing.

The gas atmosphere in continuous annealing and galvanizing lines alters both composition and microstructure of the surface and sub-surface of sheet steels. The alloying element enrichments and the oxide morphology on transformation-induced plasticity steel surfaces are strongly influenced by the dew point of the furnace atmosphere and annealing temperature. The formation of a thin oxide film and enrichment of the alloying elements during annealing may result in surface defects on galvanized sheet products. The present contribution reports on the use of microanalysis techniques such as electron backscatter diffraction, glow discharge optical emission spectroscopy and electron probe micro-analysis for the detailed surface analysis of inter-critically annealed transformation-induced plasticity steel such as oxide phase determination, microstructure and microtexture evolutions.