Semantic segmentation-based detection algorithm for challenging cryo-electron microscopy RNP samples.

In this study, we present a novel and robust methodology for the automatic detection of influenza A virus ribonucleoproteins (RNPs) in single-particle cryo-electron microscopy (cryo-EM) images. Utilizing a U-net architecture-a type of convolutional neural network renowned for its efficiency in biomedical image segmentation-our approach is based on a pretraining phase with a dataset annotated through visual inspection. This dataset facilitates the precise identification of filamentous RNPs, including the localization of the filaments and their terminal coordinates. A key feature of our method is the application of semantic segmentation techniques, enabling the automated categorization of micrograph pixels into distinct classifications of particle and background. This deep learning strategy allows to robustly detect these intricate particles, a crucial step in achieving high-resolution reconstructions in cryo-EM studies. To encourage collaborative advancements in the field, we have made our routines, the pretrained U-net model, and the training dataset publicly accessible. The reproducibility and accessibility of these resources aim to facilitate further research and validation in the realm of cryo-EM image analysis.

Understanding and Mitigating Lattice Collapse Degradation in Layered Oxide Materials for Sodium-Ion Battery Anode.

Na2Ti3O7 has attracted significant attention due to its ecofriendliness and cost-effectiveness for sodium-ion batteries. However, their limited cycling stability hampers their practical applications. Herein, we elucidate a mechanism of structural degradation caused by the heterogeneous phase transition in the Na2Ti3O7 anode using aberration-corrected (scanning) transmission electron microscopy (S)TEM and in situ TEM. It is found that the unevenly distributed phase transition results in the accumulation of strain, which promotes the growth of microcracks and eventually leads to structural decomposition and electrochemical failure. Motivated by this degradation mechanism, nanowires were proposed, and the structural stability is thus improved with the lattice strain effectively released. These findings deepen our understanding of ion transport and degradation mechanisms in intercalated layered electrode materials while emphasizing the significance of the material structure engineered for improving electrode performance.

Sorption-oxidation mechanism for the removal of arsenic (III) using Cu-doped ZnO in an alkaline medium.

1-D oxides Zn1-xCuxO and spherical composites Zn1-xCuxO/CuO were obtained by thermolysis of formate-glycolate complexes Zn1-xCux (HCOO)(OCH2CH2O)1/2 (0 ≤ x ≤ 0.15). The structural and property characteristics showed that Cu was introduced into the Zn site of the ZnO lattice to form the Zn0.95Cu0.05O solid solution. The concentration of copper in the precursors regulates the topological and structural features of the formation of Zn1-xCuxO oxides, which determine their sorption and photocatalytic properties. The materials were tested in As3+ photooxidation reaction under UV and visible radiation. It has been established that Cu+ is an effective dopant in the composition of 1-D oxide Zn1-xCuxO (0 ≤ x < 0.1). The presence of Cu2+ in the shell of Zn1-xCuxO/CuO composite reduces the photoactivity of the material. The maximum efficiency of arsenic extraction (up to 80% for Zn0.95Cu0.05O) was achieved from dilute arsenic-containing solutions (3.8 mg/L As) and an adsorbent concentration of 0.8 g/L for 24 h. In saturated solutions (380 mg/L As) this value is reduced by a factor of 100. According to XPS data, the primary process is As3+ sorption on the catalyst surface followed by its oxidation to As5+. Using the EPR method it was found that singly charged oxygen vacancies V O + $$ {V}_O^{+} $$ associated with Cu in Zn1-xCuxO are directly involved in the photostimulated oxidation of As3+. PRACTITIONER POINTS: Two types of Zn1-x Cux O photocatalysts were obtained by thermolysis of the Zn1-x Сux (HCOO)(OCH2 CH2 O)1/2 complex (0 ≤ x ≤ 0.15) in air. Sorption of arsenic from dilute solutions reaches 80% on 1-D oxide Zn0.95 Cu0.05 O. Sorption of As3+ on the catalyst surface is at primary process followed by its oxidation to As5+ . Removal of As3+ from alkaline solutions occurs due to successful combination of sorption and photocatalytic properties of the 1-D oxides Zn1-x Cux O.

Chemical Composition and Thermogravimetric Behaviors of Glanded and Glandless Cottonseed Kernels.

Common "glanded" (Gd) cottonseeds contain the toxic compound gossypol that restricts human consumption of the derived products. The "glandless" (Gl) cottonseeds of a new cotton variety, in contrast, show a trace gossypol content, indicating the great potential of cottonseed for agro-food applications. This work comparatively evaluated the chemical composition and thermogravimetric behaviors of the two types of cottonseed kernels. In contrast to the high gossypol content (3.75 g kg-1) observed in Gd kernels, the gossypol level detected in Gl kernels was only 0.06 g kg-1, meeting the FDA's criteria as human food. While the gossypol gland dots in Gd kernels were visually observed, scanning electron microcopy was not able to distinguish the microstructural difference between ground Gd and Gl samples. Chemical analysis and Fourier transform infrared (FTIR) spectroscopy showed that Gl kernels and Gd kernels had similar chemical components and mineral contents, but the former was slightly higher in protein, starch, and phosphorus contents. Thermogravimetric (TG) processes of both kernels and their residues after hexane and ethanol extraction were based on three stages of drying, de-volatilization, and char formation. TG-FTIR analysis revealed apparent spectral differences between Gd and Gl samples, as well as between raw and extracted cottonseed kernel samples, indicating that some components in Gd kernels were more susceptible to thermal decomposition than Gl kernels. The TG and TG-FTIR observations suggested that the Gl kernels could be heat treated (e.g., frying and roasting) at an optimal temperature of 140-150 °C for food applications. On the other hand, optimal pyrolysis temperatures would be much higher (350-500 °C) for Gd cottonseed and its defatted residues for non-food bio-oil and biochar production. The findings from this research enhance the potential utilization of Gd and Gl cottonseed kernels for food applications.

Insights into neurodegeneration from electron microscopy studies.

Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, pose an increasingly severe burden for individuals and society in an ageing population. The causes and mechanisms of the diseases are poorly understood and as yet there are no effective treatments. Some of the molecular complexes involved in degeneration have been identified and electron microscopy has provided an essential tool in the investigations. The focus of this review is to show how electron microscopy has contributed historically to the understanding of disease and to summarize the most striking current advances. It does not seek to cover in detail the recent technical developments in microscopy, involving better microscopes, better electron detectors and more powerful image processing techniques, which have made possible the new insights. In many instances pathological filament assemblies are associated with brain cells that die in the disease, causing the observed symptoms such as dementia or movement disorders. Using electron microscopy it is now possible to go beyond morphological descriptions to produce atomic structures of many of the filaments. This information may help to understand the seeding and assembly of the filaments, with the aim of finding small molecule inhibitors that could potentially provide a form of treatment for the diseases.

Virus Assembly Pathways: Straying Away but Not Too Far.

Non-enveloped RNA viruses pervade all domains of life. In a cell, they co-assemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native one. However, they contain large native structure fragments that can be recycled to form BMV virions, should a viral genome become available. The existence of such structures suggest the possibility of a previously unknown regulatory pathway for the packaging process inside cells.

Spectroscopic studies on free radical coalescing antioxidants and brain protein cystatin.

Cystatins are the inhibitors of thiol proteinases and are ubiquitously present in mammalian system. In brain, they put off unwanted proteolysis and are also involved in several neurodegenerative diseases. In the present study, it was demonstrated that photo-activated HOCl-induced modifications in brain cystatin leading to its inactivation and degradation due to hydroxyl radicals. It has been shown that oxidation of cystatin by ROS in vivo leads to oxidative modification which may direct the damage of this significant protein, as it is so well pronounced in vitro. The interplay between free radicals, antioxidants and co-factors is important in maintaining health, aging and age-related diseases. Body's endogenous antioxidant systems stabilize free radical-induced oxidative stress by the ingestion of exogenous antioxidants. If the generation of free radicals goes beyond the protective effect of antioxidants, this can cause oxidative damage which accumulates during the life cycle and has been implicated in aging and age-related diseases such as cardiovascular disease, cancer, neurodegenerative disorders and other chronic conditions. Activation of neutrophils in certain diseases (e.g., inflammatory conditions and atherosclerosis) results in the production of highly reactive species, such as OH• and the release of the enzyme myeloperoxidase. Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. Hypochlorous acid (HOCl) is a potent oxidant formed by myeloperoxidase that causes aggregation of many proteins and damage of proteins by reaction with amino-acid side-chains or backbone cleavage. Communicated by Ramaswamy H. Sarma.

First haplosporidan parasite reported infecting a member of the Superfamily Pinnoidea (Pinna nobilis) during a mortality event in Alicante (Spain, Western Mediterranean).

Several stages of a haplosporidan parasite, including spores, were detected infecting three out of four specimens of the Pen Shell Pinna nobilis from the coast of Alicante (Western Mediterranean). A mortality event initiated few weeks before the sampling. The infection was systemic in the connective tissue, with free uni-nucleate stages and early plasmodia, whereas sporulation process took place in the digestive tubules disrupting them. Morphological details, by light and transmission electron microscopy, and PCR amplification confirmed that the parasite belongs to the haplosporidan group. Spores were pleomorphic, usually elongated ovoid, with round to elongated haplosporosomes-like in the sporoplasma. The operculum was situated in the apical zone of the wall, with an external lid, and the nucleus tended to be eccentric in the basal zone. Spore ornamentation was not observed. The single uninfected specimen appeared to be healthy. This is the first report of a haplosporidan parasite infecting a member of the Superfamily Pinnoidea and this is the first histopathological study of a mortality event in the endangered and protected P. nobilis.

Assessing Vacuolar Escape of Listeria Monocytogenes.

Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L. monocytogenes.

Effects of long-term exposure to 900 megahertz electromagnetic field on heart morphology and biochemistry of male adolescent rats.

The pathological effects of exposure to an electromagnetic field (EMF) during adolescence may be greater than those in adulthood. We investigated the effects of exposure to 900 MHz EMF during adolescence on male adult rats. Twenty-four 21-day-old male rats were divided into three equal groups: control (Cont-Gr), sham (Shm-Gr) and EMF-exposed (EMF-Gr). EMF-Gr rats were placed in an EMF exposure cage (Plexiglas cage) for 1 h/day between postnatal days 21 and 59 and exposed to 900 MHz EMF. Shm-Gr rats were placed inside the Plexiglas cage under the same conditions and for the same duration, but were not exposed to EMF. All animals were sacrificed on postnatal day 60 and the hearts were extracted for microscopic and biochemical analyses. Biochemical analysis showed increased levels of malondialdehyde and superoxide dismutase, and reduced glutathione and catalase levels in EMF-Gr compared to Cont-Gr animals. Hematoxylin and eosin stained sections from EMF-Gr animals exhibited structural changes and capillary congestion in the myocardium. The percentage of apoptotic myocardial cells in EMF-Gr was higher than in either Shm-Gr or Cont-Gr animals. Transmission electron microscopy of myocardial cells of EMF-Gr animals showed altered structure of Z bands, decreased myofilaments and pronounced vacuolization. We found that exposure of male rats to 900 MHz EMF for 1 h/day during adolescence caused oxidative stress, which caused structural alteration of male adolescent rat heart tissue.

Comparison between magnetic and non magnetic multi-walled carbon nanotubes-dispersive solid-phase extraction combined with ultra-high performance liquid chromatography for the determination of sulfonamide antibiotics in water samples.

In this manuscript, a new method based on the use of off-line dispersive solid-phase extraction (dSPE) combined with ultra-high performance liquid chromatography with diode-array detection was developed to determine 11 sulfonamide antibiotics (sulfanilamide, sulfacetamide, sulfadiazine, sulfathiazole, sulfamerazine, sulfadimidin, sulfamethoxypyridazine, sulfadoxine, sulfamethoxazole, sulfisoxazole and sulfadimethoxine) in mineral waters with different mineral content. For this purpose, pristine multi-walled carbon nanotubes (MWCNTs) and magnetic-MWCNTs (m-MWCNTs) were used as sorbents. Magnetic nanoparticles were synthesized by means of a solvothermal process, assembled onto CNTs through an "aggregation wrap" mechanism and characterized by scanning electron microscopy. Parameters affecting the extraction such as volume and pH of the sample, amount of sorbent and type and volume of eluent were optimized. Once optimum extraction conditions (250 mL of water at pH 6.0 and elution with 25 mL of MeOH) were obtained, the extraction efficiency of the different carbon nanomaterials was compared. Results demonstrated the higher extraction capacity of pristine MWCNTs with recoveries between 61 and 110% (except for sulfacetamide which ranged between 40 and 53%) and between 22 and 77% for m-MWCNTs. Limits of detection lower than 32 ng/L were achieved for all of the analyzed samples.

Skeletal muscle mitochondrial and lipid droplet content assessed with standardized grid sizes for stereology.

UNLABELLED: Skeletal muscle mitochondrial (Mito) and lipid droplet (Lipid) content are often measured in human translational studies. Stereological point counting allows computing Mito and Lipid volume density (Vd) from micrographs taken with transmission electron microscopes. Former studies are not specific as to the size of individual squares that make up the grids, making reproducibility difficult, particularly when different magnifications are used. Our objective was to determine which size grid would be best at predicting fractional volume efficiently without sacrificing reliability and to test a novel method to reduce sampling bias. METHODS: ten subjects underwent vastus lateralis biopsies. Samples were fixed, embedded, and cut longitudinally in ultrathin sections of 60 nm. Twenty micrographs from the intramyofibrillar region were taken per subject at ×33,000 magnification. Different grid sizes were superimposed on each micrograph: 1,000 × 1,000 nm, 500 × 500 nm, and 250 × 250 nm. RESULTS: mean Mito and Lipid Vd were not statistically different across grids. Variability was greater when going from 1,000 × 1,000 to 500 × 500 nm grid than from 500 × 500 to 250 × 250 nm grid. DISCUSSION: this study is the first to attempt to standardize grid size while keeping with the conventional stereology principles. This is all in hopes of producing replicable assessments that can be obtained universally across different studies looking at human skeletal muscle mitochondrial and lipid droplet content.

Insights into cell entry and intracellular trafficking of peptide and protein drugs provided by electron microscopy.

For widening the arsenal of protein and peptide therapeutics that act within cells, their cell-entry mechanisms, intracellular trafficking and distribution need to be characterized in detail. Immunofluorescence microscopy has been a prevalent tool for these studies. However, due to the limited resolution, it is often complemented with other methods. This article focuses on the perspectives of electron microscopy in tracking the intracellular delivery and trafficking of proteins, peptides and their carriers. This review introduces the electron microscopy techniques and labeling methods currently used for studying the cellular whereabouts of peptides and proteins with a focus on their intracellular trafficking. Since cell-penetrating peptides have widely been harnessed as carriers for proteins and peptides, and their usage is rapidly expanding, a particular emphasis has been placed on their applications and cell-entry mechanisms.

Influência de variáveis metodológicas no valor da resistência de união material adesivo/substrato dentinário e, no padrão de fratura: ensaio de micro cisalhamento / Methodological variables influence on bonding of adhesive material/dentin substrate and fracture pattern micro shear test

A influência de variáveis metodológicas no valor da resistência de união e no padrão de fratura, por ensaio de microcisalhamento, foi verificada em dentina planificada de molares humanos e estudada em quatro diferentes experimentos, nos quais dois sistemas adesivos foram utilizados (Adper Single Bond 2 e XP Bond) e uma resina composta (Filtek Z250). Foram eles: Experimento 1- momento de fotoativação do adesivo: antes ou após o posicionamento das cânulas sobre o substrato dentinário; Experimento 2- tempo de armazenagem: 10min, 24 horas, 48 horas e 7 dias em água destilada a 37oC; Experimento 3- área de secção transversal dos corpos de prova: 0,44mm2, 0,82mm2, 1,98mm2 e 4,44mm2; Experimento 4- momento de fotoativação da resina / remoção da cânula para o ensaio. Os dados obtidos de resistência de união, em MPa, foram tratados por métodos de ANOVA. Os corpos de prova resultantes de todos os experimentos foram observados em microscopia eletrônica de varredura e analisados de forma qualitativa quanto ao modo de fratura. Houve diferença significativa no Experimento 1, sendo que os valores médios de resistência de união foram mais altos quando os adesivos foram fotoativados antes de posicionar as cânulas. Não houve diferença estatisticamente significante para as variáveis tempo de armazenagem e sistema adesivo (Experimento 2), e nem para as variáveis área de secção transversal e sistema adesivo estudadas (Experimento 3). Para o Experimento 4, houve diferença estatisticamente significante para a condição experimental resina fotoativada e remoção da cânula quando previamente realizada à adesão ao substrato e entre os adesivos. Em todos os experimentos o modo de fratura foi predominantemente adesivo/misto. Concluiu-se que algumas variáveis do método são fundamentais para sua padronização, pois com base nos resultados deste estudo elas influenciam os valores médios da resistência de união.

The effect of methodological variables on the microshear bond strength and fracture pattern was assessed in flat human molars dentin in four different experiments using two adhesive systems (Adper Single Bond 2 and XP Bond) and a composite resin (Filtek Z250). The experiments were: 1. Adhesive light activation moment prior or after mold positioning on dentin; 2. Storage period 10 min, 24h, 48h or 7 days in distilled water at 37oC; 3. Mold transversal section area 0.44mm2, 0.82mm2, 1.98mm2 and 4.44mm2; 4. Resin light activation moment with or without mold removal prior to testing. Mean data values (MPa) were subjected to ANOVAs methods. All fractured specimens were qualitatively analyzed in SEM to verify the fracture mode. Regarding Experiment 1 a significative difference was shown for the main factor Adhesive Light Activation Moment; higher bond strength values were achieved with adhesive system ligth activated before mold positioning. No significant differences were found for storage period or section area analyzed on Experiments 2 and 3, respectively. The main factor Resin Light Activation Moment presented differences when the resin composite was previously light activated and mold was removed prior to bonding the resin with the adhesive systems. In all experiments, mode of failure was predominantly adhesive/mixed. The conclusion is that some variables of the method are essential for its standardization, given that they influence the bond strength values.