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Since Nicolle, Manceaux and Splendore first described Toxoplasma gondii as a parasite of rodents and rabbits in the early 20th century, a diverse and vigorous research community has been built around studying this fascinating intracellular parasite. In addition to its importance as a pathogen of humans, livestock and wildlife, modern researchers are attracted to T. gondii as a facile experimental system to study many aspects of evolutionary biology, cellular biology, host-microbe interactions, and host immunity. For new researchers entering the field, the extensive literature describing the biology of the parasite, and the interactions with its host, can be daunting. In this review, we examine four foundational studies that describe various aspects of T. gondii biology, presenting a ‘journal club'-style analysis of each. We have chosen a paper that established the beguiling life cycle of the parasite (Hutchison et al., 1971), a paper that described key features of its cellular biology that the parasite shares with related organisms (Gustafson et al., 1954), a paper that characterised the origin of the unique compartment in which the parasite resides within host cells (Jones and Hirsch, 1972), and a paper that established a key mechanism in the host immune response to parasite infection (Pfefferkorn, 1984). These interesting and far-reaching studies set the stage for subsequent research into numerous facets of parasite biology. As well as providing new researchers with an entry point into the literature surrounding the parasite, revisiting these studies can remind us of the roots of our discipline, how far we have come, and the new directions in which we might head.

Review on the paper Wuzong Zhou, Reversed Crystal Growth. Crystals. 2019; 9(1): 7 (16 pp). https://doi.org/10.3390/cryst9010007

Review on the paper Wuzong Zhou, Reversed Crystal Growth. Crystals. 2019; 9(1): 7 (16 pp). https://doi.org/10.3390/cryst9010007

The cost of specialized scientific equipment can be high and with limited funding resources, researchers and students are often unable to access or purchase the ideal equipment for their projects. In the fields of materials science and mechanical engineering, fundamental equipment such as tensile testing devices can cost tens to hundreds of thousands of dollars. While a research lab often has access to a large-scale testing machine suitable for conventional samples, loading devices for meso- and micro-scale samples for in-situ testing with the myriad of microscopy tools are often hard to source and cost prohibitive. Open-source software has allowed for great strides in the reduction of costs associated with software development and open-source hardware and additive manufacturing have the potential to similarly reduce the costs of scientific equipment and increase the accessibility of scientific research. To investigate the feasibility of open-source hardware, a micro-tensile tester was designed with a freely accessible computer-aided design package and manufactured with a desktop 3D-printer and off-the-shelf components. To our knowledge this is one of the first demonstrations of a tensile tester with additively manufactured components for scientific research. The capabilities of the tensile tester were demonstrated by investigating the mechanical properties of Graphene Oxide (GO) paper and thin films. A 3D printed tensile tester was successfully used in conjunction with an atomic force microscope to provide one of the first quantitative measurements of GO thin film buckling under compression. The tensile tester was also used in conjunction with an atomic force microscope to observe the change in surface topology of a GO paper in response to increasing tensile strain. No significant change in surface topology was observed in contrast to prior hypotheses from the literature. Based on this result obtained with the new open source tensile stage we propose an alternative hypothesis we term 'superlamellae consolidation' to explain the initial deformation of GO paper. The additively manufactured tensile tester tested represents cost savings of >99% compared to commercial solutions in its class and offers simple customization. However, continued development is needed for the tensile tester presented here to approach the technical specifications achievable with commercial solutions.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) O Brasil é o segundo maior produtor de celulose do mundo, tornando o setor de árvores plantadas de grande importância para a economia nacional. Porém essas indústrias ocupam o sexto lugar em termos de poluição ambiental, sendo responsáveis por descartar efluentes com alto potencial poluidor se não tratados adequadamente. A maioria das indústrias utilizam tratamentos biológicos. O reator biológico de leito móvel (MBBR) vem ganhando popularidade devido sua estabilidade, tamanho compacto, eficiência na remoção de matéria orgânica, além da baixa produção de lodo. Esse sistema permite o crescimento de biomassa aderida (biofilme) em suportes que, com auxílio da aeração, movimentam-se por todo volume do reator. Nesse estudo foi avaliado o desempenho do meio suporte esponjoso Aquaporousgel (APG), desenvolvido pela empresa japonesa Nisshinbo Chemical Inc., adicionado em MBBR empregado no tratamento de efluente de indústria de celulose. O conjunto MBBR-APG foi construído em escala de bancada com volume útil de 1 L e operado por 240 dias com cargas orgânicas volumétricas (COV) de 0,6 e 1,2 kgDQO m-3 d-1, resultando em tempos de detenção hidráulica (TDH) variando de 1,0 a 2,7 dias. A eficiência do tratamento foi determinada pela remoção de demanda química de oxigênio (DQO), demanda bioquímica de oxigênio (DBO5), carbono orgânico total (COT), cor, turbidez, compostos fenólicos totais (CFT), compostos específicos derivados de lignina e também foi avaliada a matriz de excitação e emissão de fluorescência (MEEF). O desenvolvimento do biofilme foi avaliado com a quantificação dos sólidos aderidos, obtenção de imagens em microscopia eletrônica de varredura (MEV) e análises microbiológicas. O MBBR-APG apresentou melhor desempenho na COV 1,2 kgDQO m-3 d-1, com remoções médias de matéria orgânica de 62,7, 48,4 e 55,7% para DBO5, DQO e COT, respectivamente. Mas o tratamento biológico gerou incrementos de outros compostos fluorogênicos. Discute-se a hipótese desse fenômeno ser resultante da ação de microrganismos anaeróbios presentes em microzonas anóxicas formadas no interior do meio suporte esponjoso devido à dificuldade na difusão de oxigênio. As análises em MEV indicaram que o material esponjoso estudado apresenta estrutura funcional superior às tradicionais esponjas de poliuretano, sendo possível observar o desenvolvimento de uma matriz de biofilme composta por resíduos remanescentes do efluente, substâncias poliméricas extracelulares (EPS) e a presença de fungos e bactérias. As análises microbiológicas confirmaram a presença de fungos do gênero Aspergillus e Trichoderma e bactérias Bacillus sp., Serratia sp. E Lysibacillus sp. O trabalho apresentou importantes contribuições sobre o uso de meios suportes esponjosos, biotransformação de compostos em tratamentos biológicos de efluentes de celulose e o desenvolvimento da matriz de biofilme nesses sistemas. Brazil is the second-largest pulp producer in the world and, therefore, the tree sector is of great importance to the national economy. However, these industries are in sixth place in terms of environmental pollution, being responsible for disposing of effluents with high polluting potential if not properly treated. Most pulp mill industries use biological treatments. The moving bed biofilm reactor (MBBR) has been gaining popularity due to its stability, compact size, removal efficiencies, and low sludge production. This system allows the growth of adhered biomass (biofilm) in supports. The supports, also called carriers, move throughout the reactor volume with the aid of aeration. This study evaluated the performance of the Aquaporousgel (APG), a functional sponge developed by the Japanese company Nisshinbo Chemical Inc., used in an MBBR to treat pulp mill effluent. An MBBR system in a laboratory-scale with utile volume of 1 L was filled by this innovating sponge carrier to treat effluent from a pulp industry. The MBBR-APG operated continuously for 240 d with an organic load rate (OLR) of 0.6 and 1.2 kgDQO m-3 d-1, resulting in hydraulic retention times (HRT) ranging from 1.0 to 2.7 days. The treatment efficiency was determined by the removal of chemical oxygen demand (COD), biochemical oxygen demand (BOD5), total organic carbon (TOC), color, turbidity, total phenolic compounds, lignin derivatives, and fluorescence excitation-emission matrix. Biofilm development was evaluated by quantifying adhered solids, scanning electron microscopy (SEM), and microbiological analyses. The MBBR-APG presented a better performance at OLR of 1.2 kgDQO m-3 d1, achieving average organic matter removals of 62.7, 48.4 and 55.7% for BOD5, COD and TOC, respectively. Despite that, the biological treatment generated increments of other fluorogenic compounds. The hypothesis of this phenomenon is the result of the action of anaerobic microorganisms present in anoxic microzones formed in the inner of the sponge carrier due to the difficulty in oxygen diffusion. The SEM analysis indicated that the APG sponge has a functional structure better than the traditional polyurethane sponges. It is possible to observe the development of a biofilm matrix composed of extracellular polymeric substances (EPS) and the presence of fungi and bacteria. Microbiological analyses confirmed the presence of fungi genus Aspergillus and Trichoderma, and bacterial genus Bacillus sp., Serratia sp. And Lysibacillus sp. This study presented important contributions to the use of sponge carrier, compounds biotransformation in biological treatments of pulp effluents, and the development of biofilm matrix in these systems.

The aim of this study was to understand the influence of seasons on the variability of paper mechanical properties, whose influence is normally overcome by important additions of starch with unfavourable economical and environmental repercussions. Several ways of research were explored such as storage conditions of old papers after collecting, influence of circuit water temperatures and influence of the quantity and type of fine elements.To this end, several approaches were developed and adapted (coloration, immunolabeling, PATAg, fluorescent staining, PARA-Gold...) together with conventional techniques of microscopy (optical, transmission, scanning, confocal...). This provided a better understanding of the modifications undergone by fibres in their micromorphology and ultrastructure depending on the different processes in relation with the variations of the mechanical properties of papers. Technical analyses of fibres wood and their components were applied to study fibres morphology (MorFi analyser) and to evaluate quantitatively their different constituents (carbohydrate analysis, gas-liquid chromatography, 13C NMR). In the first part of the project the methodological approach was validated and implemented for correlating observations of fibres at ultrastructural and morphological level with the mechanical properties of papers. The second part has consisted in the study of the influence of old papers storage conditions in fonction of duration, temperature and moisture and pointed out the adverse effects of winter conditions. Also we concluded to the necessity of controlling the circuit water temperature in summer. In the third part of the work devoted to the influence of cellulosic fine elements and their behaviour with fibres in the papermaking, we developed a new method for the specific labelling of fines allowing whereby it was possible to follow their fate when they are added to the fibres. Data on the nature of their interaction at the surface and within the fibres are given., L'objectif de cette étude était de comprendre l'influence des saisons sur la variabilité des propriétés mécaniques des papiers recyclés impliquant l'adjonction de charges en amidon importantes, phénomène ayant des répercussions économiques et environnementales défavorables. Plusieurs voies de recherche ont été explorées comme les conditions de stockage des papiers récupérés après collecte, l'influence des températures des eaux de circuit et l'influence du taux et du type d'éléments fins.Pour atteindre ces objectifs, plusieurs outils permettant une observation à l'échelle de la microfibrille de cellulose (10nm) ont été développés ou adaptés (coloration, immunomarquage, PATAg, marquage fluorescent...) couplés à des techniques de microscopie conventionnelle (optique, transmission, balayage, confocal...) afin de mieux comprendre les modifications de la micromorphologie et de l'ultrastructure des fibres en fonction des différents traitements appliqués et d'établir des corrélations avec les variations des propriétés mécaniques des papiers correspondants. Des techniques d'analyse des fibres de bois et de ses composants ont également été utilisées afin d'étudier la morphologie des fibres (analyseur MorFi) ainsi que d'évaluer de manière plus quantitative leurs différents composants (analyse des sucres, chromatographie en phase gazeuse, 13C RMN). La première partie du projet a permis de mettre en place une démarche méthodologique permettant de corréler les observations au niveau ultrastructural et morphologique des fibres et les propriétés mécaniques des papiers fabriqués à partir de celles-ci. L'application de cette méthode nous a permis dans un second temps de montrer que les papiers récupérés par la collecte devaient être stockés de manière plus protégée en hiver par les centres de tri. De même, nous avons pu mettre en évidence la nécessité de réfrigérer les eaux de circuit dans la machine à papier lorsque la fabrication a lieu en été. Enfin, le dernier chapitre a souligné, par la mise en place d'une nouvelle méthode de marquage (PARA-Gold), le fait que le recyclage entraîne la production d'éléments fins aux propriétés de liaison aux fibres diminuées et des solutions pour réactiver ces derniers ont été proposées aux papetiers.

Supercalendered and coated papers (SC and LWC) were examined using an environmental scanning electron microscope (ESEM). Moderate structural surface changes were observed as water condensed on the surface in a high moisture environment. The changes were fully or partially reversible depending on the sample origin. A wide range of contact angles could be observed when condensing water on uncoated wood fibers. While there was no visible indication of irradiation damage on the commercial paper samples examined nor on mechanical pulp fibers, attempts to look at chemical pulp fibers during wetting to examine fiber swelling were unsuccessful because of very rapid irradiation damage.

Single‐crystalline domains in intergrown microcrystalline material of the new compounds Ba22.5+x La55−x [Si129N240−x O x ]O3:Ce3+ and Ba25.5+x La77−x [Si170N312−x O9+x ]O4:Ce3+ were identified by transmission electron microscopy (TEM). Precise diffraction data from these domains were collected with microfocused synchrotron radiation so that crystal structure elucidation of the complex disordered networks became possible. They are composed of two different interconnected slabs of which one is similar in both compounds, which explains their notorious intergrowth. The distribution of Ba and La is indicated by the analysis of bond‐valence sums and by comparison with isostructural Sr28.5+x La75−x [Si170N312−x O9+x ]O4. Ce3+ doping leads to yellow luminescence. This is a showcase that highlights the discovery and accurate characterization of new compounds relevant for luminescence applications from heterogeneous microcrystalline samples by exploiting the capability of the combination of TEM and diffraction using the latest focusing techniques for synchrotron radiation., Single‐crystalline domains of microcrystalline intergrown oxonitridosilicate oxides Ba22.5+x La55−x [Si129N240−x O x ]O3 and Ba25.5+x La77−x [Si170N312−x O9+x ]O4 and related Sr analogues were identified by TEM. Diffraction data collected with microfocused synchrotron radiation reveal extraordinarily complex tetrahedra networks. This combination of analytical methods enables the characterization of new luminescent materials in heterogeneous powder samples.

The authors examined the ultrastructure of mitochondrial apparatus of skeletal muscles of naked mole rats (Heterocephalus glaber) from the age of 6 months to 11 years. The obtained results have demonstrated that the mitochondria in skeletal muscles of naked mole rats aged below 5 years is not well-developed and represented by few separate small mitochondria. Mitochondrial reticulum is absent. Starting from the age of 5 years, a powerful mitochondrial structure is developed. By the age of 11 years, it become obvious that the mitochondrial apparatus formed differs from that in the skeletal muscle of adult rats and mice, but resembles that of cardiomyocytes of rats or naked mole rats cardiomyocytes. From the age of 6 months to 11 years, percentage area of mitochondria in the skeletal muscle of naked mole rat is increasing by five times. The growth of mitochondria is mainly driven by increased number of organelles. Such significant growth of mitochondria is not associated with any abnormal changes in mitochondrial ultrastructure. We suppose that specific structure of mitochondrial apparatus developed in the skeletal muscle of naked mole rats by the age of 11 years is necessary for continual skeletal muscle activity of these small mammals burrowing very long holes in stony earth, resembling continual activity of heart muscle. In any case, ontogenesis of naked mole rat skeletal muscles is much slower than of rats and mice (one more example of neoteny).

Clear cell renal cell carcinoma (ccRCC) is the most common form of renal cancer. Due to inactivation of the von Hippel–Lindau tumour suppressor, the hypoxia‐inducible transcription factors (HIFs) are constitutively activated in these tumours, resulting in a pseudo‐hypoxic phenotype. The HIFs induce the expression of genes involved in angiogenesis and cell survival, but they also reset the cellular metabolism to protect cells from oxygen and nutrient deprivation. ccRCC tumours are highly vascularized and the cytoplasm of the cancer cells is filled with lipid droplets and glycogen, resulting in the histologically distinctive pale (clear) cytoplasm. Intratumoural heterogeneity may occur, and in some tumours, areas with granular, eosinophilic cytoplasm are found. Little is known regarding these traits and how they relate to the coexistent clear cell component, yet eosinophilic ccRCC is associated with higher grade and clinically more aggressive tumours. In this study, we have for the first time performed RNA sequencing comparing histologically verified clear cell and eosinophilic areas from ccRCC tissue, aiming to analyse the characteristics of these cell types. Findings from RNA sequencing were confirmed by immunohistochemical staining of biphasic ccRCC. We found that the eosinophilic phenotype displayed a higher proliferative drive and lower differentiation, and we confirmed a correlation to tumours of higher stage. We further identified mutations of the tumour suppressor p53 (TP53) exclusively in the eosinophilic ccRCC component, where mTORC1 activity was also elevated. Also, eosinophilic areas were less vascularized, yet harboured more abundant infiltrating immune cells. The cytoplasm of clear cell ccRCC cells was filled with lipids but had very low mitochondrial content, while the reverse was found in eosinophilic tissue. We herein suggest possible transcriptional mechanisms behind these phenomena. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Amyloid‐β peptide (Aβ) oligomers are pathogenic species of amyloid aggregates in Alzheimer's disease. Like certain protein toxins, Aβ oligomers permeabilize cellular membranes, presumably through a pore formation mechanism. Owing to their structural and stoichiometric heterogeneity, the structure of these pores remains to be characterized. We studied a functional Aβ42‐pore equivalent, created by fusing Aβ42 to the oligomerizing, soluble domain of the α‐hemolysin (αHL) toxin. Our data reveal Aβ42‐αHL oligomers to share major structural, functional, and biological properties with wild‐type Aβ42‐pores. Single‐particle cryo‐EM analysis of Aβ42‐αHL oligomers (with an overall 3.3 Å resolution) reveals the Aβ42‐pore region to be intrinsically flexible. The Aβ42‐αHL oligomers will allow many of the features of the wild‐type amyloid oligomers to be studied that cannot be otherwise, and may be a highly specific antigen for the development of immuno‐base diagnostics and therapies., A functional and structural Aβ42‐oligomer equivalent, created by fusing Aβ42 to the oligomerizing, soluble domain of the α‐hemolysin (αHL) toxin, shares major structural, functional, and biological properties with Alzheimer's wild‐type Aβ42 oligomers. Cryo‐EM studies reveal that the αHL‐displayed oligomers structurally remain in a well‐defined stoichiometry, providing a stable mimetic antigen for the development of conformation‐specific antibodies.

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab., タンパク質の抗体ラベリング技術を改良し、構造解析をアシスト --電子顕微鏡やX線結晶解析による構造決定を加速化--. 京都大学プレスリリース. 2021-04-20.

Electron microscopy (EM) continues to provide near-atomic resolution structures for well-behaved proteins and protein complexes. Unfortunately, structures of some complexes are limited to low- to medium-resolution due to biochemical or conformational heterogeneity. Thus, the application of unbiased systematic methods for fitting individual structures into EM maps is important. A method that employs co-evolutionary information obtained solely from sequence data could prove invaluable for quick, confident localization of subunits within these structures. Here, we incorporate the co-evolution of intermolecular amino acids as a new type of distance restraint in the Integrative Modeling Platform (IMP) in order to build three-dimensional models of atomic structures into EM maps ranging from 10-14 a in resolution. We validate this method using four complexes of known structure, where we highlight the conservation of intermolecular couplings despite dynamic conformational changes using the BAM complex. Finally, we use this method to assemble the subunits of the bacterial holo-translocon into a model that agrees with previous biochemical data. The use of evolutionary couplings in integrative modeling improves systematic, unbiased fitting of atomic models into medium- to low-resolution EM maps, providing additional information to integrative models lacking in spatial data. This article is protected by copyright. All rights reserved.

Preparing catalysts with highly dispersed metal nanoparticles and narrow particle size distribution has been in the focus of numerous studies. Besides size and size distribution, the location of metal nanoparticles within and local metal loading of the support can have significant impact on catalytic performance. This study revealed that great variations in Pt loading between individual Pt/zeolite Y crystals occurred irrespective of the metal deposition method, namely ion‐exchange (IE) or incipient wetness impregnation (IWI). The variation in Pt loading was found to be directly related to different Si/Al ratios of individual zeolite crystals. Results indicate that this Si/Al variation was likely induced by post‐synthesis treatments, commonly performed to introduce mesoporosity. In view of the great importance of zeolite‐based catalysts for oil refining, understanding the origin of such metal loading heterogeneities may lead to further improvement of zeolite‐based catalytic performance.

A new certified reference material (CRM) for size and shape analysis of elongated nanoparticles has been developed by the European Commission’s Joint Research Centre. The CRM consists of titanium dioxide nanorods dispersed in 1-butanol, was coded ERM-FD103 and has been certified for different electron microscopy–based operationally defined measurands such as the modal and median values of the particle number-weighted distributions of the minimum and maximum Feret diameter, the maximum inscribed circle diameter, the area-equivalent circular diameter and the aspect ratio. The nanorods have nominal dimensions of 15 nm in width and 55 nm in length. Homogeneity and stability measurements were performed using transmission electron microscopy. The relative standard uncertainty for homogeneity ranged from 0.3 to 1.7%. No significant instability was detected for a shelf life of 18 months and a storage temperature of 18 °C. The certified values have been determined from the results of an interlaboratory comparison in which qualified expert laboratories participated with scanning and transmission electron microscopy. The certified values are traceable to the unit of length in the International System of Units, the metre, and the relative expanded uncertainties (confidence level of approximately 95%) range from 4 to 6%. These properties allow the CRM to be used for quality assurance and calibration of electron microscopy methods for nanoparticle size and shape analysis in ranges relevant for the implementation of EU legislation related to nanomaterials. The presented study discusses the purpose and results of the different steps that were followed to turn an industrially relevant raw titanium dioxide nanorod material into a fit-for-purpose CRM. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02984-z) contains supplementary material, which is available to authorized users.

Zeolites are becoming more versatile in their chemical functions through rational design of their frameworks. Therefore, direct imaging of all atoms at the atomic scale, basic units (Si, Al, and O), heteroatoms in the framework, and extra‐framework cations, is needed. TEM provides local information at the atomic level, but the serious problem of electron‐beam damage needs to be overcome. Herein, all framework atoms, including oxygen and most of the extra‐framework Na cations, are successfully observed in one of the most electron‐beam‐sensitive and lowest framework density zeolites, Na‐LTA. Zeolite performance, for instance in catalysis, is highly dependent on the location of incorporated heteroatoms. Fe single atomic sites in the MFI framework have been imaged for the first time. The approach presented here, combining image analysis, electron diffraction, and DFT calculations, can provide essential structural keys for tuning catalytically active sites at the atomic level., Financial support is acknowledged from CħEM, SPST, ShanghaiTech in the form of grant no. EM02161943 (A.M., Q.Z., Y.M., and O.T.), NFSC‐21850410448 (A.M.), NSFC‐21835002 (A.M., Y.M., J.Y., and O.T.), basic funding of the MPG (P.L., W.S., and F.S.), Foreign 1000 Talents plan, China (O.T.), CityU‐9610369 (H.H.), 111 project of China B17020 (A.M., J.Y., O.T.), and the Alexander von Humboldt foundation (P.L.). A.M. acknowledges the Spanish Ministry of Science and Innovation (Ramon y Cajal Program RYC2018‐024561‐I).

The comprehensive targets of innervation in the intestinal mucosa are unknown, partly because of the diversity of cell types and the complexity of the neural circuits. Herein, we investigated the comprehensive targets of neural connectivity and analyzed the precise characteristics of their contact structures in the mucosa of rat ileum. We examined target cells of neural connections and the characteristics of their contact structures by serial block-face scanning electron microscopy at four portions of the rat ileal mucosa: the apical and basal portions in the villi, and the lateral and basal portions around/in the crypts. Nerve fibers were in contact with several types of fibroblast-like cells (FBLCs), macrophage-like cells, eosinophils, lymphocyte-like cells, and other types of cells. The nerve fibers almost always ran more inside of lamina propria than subepithelial FBLC, and thus contacts with epithelial cells were very scarce. The contact structures of the nerve fibers were usually contained synaptic vesicle-like structures, and we classified them into patterns based on the number of nerve fiber contacting the target cells at one site, the maximum diameter of the contact structures, and the relationship between nerve fibers and nerve bundles. The contact structures for each type of cells occasionally dug into the cellular bodies of the target cells. We revealed the comprehensive targets of neural connectivity based on the characteristics of contact structures, and identified FBLCs, immunocompetent cells, and eosinophils as the candidate targets for innervation in the rat ileal mucosa.

We observed a correlation between acid growth and crossed-polylamellate cell walls which was dependent on CSI1 and microtubules. This is significant for re-evaluating current models of cell morphogenesis., Auxin-induced cell elongation relies in part on the acidification of the cell wall, a process known as acid growth that presumably triggers expansin-mediated wall loosening via altered interactions between cellulose microfibrils. Cellulose microfibrils are a major determinant for anisotropic growth and they provide the scaffold for cell wall assembly. Little is known about how acid growth depends on cell wall architecture. To explore the relationship between acid growth-mediated cell elongation and plant cell wall architecture, two mutants (jia1-1 and csi1-3) that are defective in cellulose biosynthesis and cellulose microfibril organization were analyzed. The study revealed that cell elongation is dependent on CSI1-mediated cell wall architecture but not on the overall crystalline cellulose content. We observed a correlation between loss of crossed-polylamellate walls and loss of auxin- and fusicoccin-induced cell growth in csi1-3. Furthermore, induced loss of crossed-polylamellate walls via disruption of cortical microtubules mimics the effect of csi1 in acid growth. We hypothesize that CSI1- and microtubule-dependent crossed-polylamellate walls are required for acid growth in Arabidopsis hypocotyls.

Precomputed multipolar electron scattering factors are employed for electron crystallography. Because this takes into account the fact that atoms are partially charged and aspherical, model fitting statistics and atomic thermal parameters are visibly improved., A revolution in resolution is occurring now in electron microscopy arising from the development of methods for imaging single particles at cryogenic temperatures and obtaining electron diffraction data from nanocrystals of small organic molecules or macromolecules. Near-atomic or even atomic resolution of molecular structures can be achieved. The basis of these methods is the scattering of an electron beam due to the electrostatic potential of the sample. To analyse these high-quality experimental data, it is necessary to use appropriate atomic scattering factors. The independent atom model (IAM) is commonly used although various more advanced models, already known from X-ray diffraction, can also be applied to enhance the analysis. In this study a comparison is presented of IAM and TAAM (transferable aspherical atom model), the latter with the parameters of the Hansen–Coppens multipole model transferred from the University at Buffalo Databank (UBDB). By this method, TAAM takes into account the fact that atoms in molecules are partially charged and are not spherical. Structure refinements were performed on a carbamazepine crystal using electron structure-factor amplitudes determined experimentally [Jones et al. (2018 ▸). ACS Cent. Sci. 4, 1587–1592] or modelled with theoretical quantum-mechanical methods. The results show the possibilities and limitations of the TAAM method when applied to electron diffraction. Among others, the method clearly improves model fitting statistics, when compared with IAM, and allows for reliable refinement of atomic thermal parameters. The improvements are more pronounced with poorer-resolution diffraction data.

Background: Nanocrystallization is a promising field for the development of new drugs. This study aims to present the use of nanocrystallization via intraperitoneal nanoaerosol therapy (INAT) for the treatment of peritoneal metastases. Methods: A continuous aerosol generation device was used to aerosolize a highly concentrated doxorubicin solution within a dry CO2 environment. The produced nanoaerosol was directed into an ex vivo abdominal model and collision of aerosol particles with placed samples was subject to further analysis via scanning-electron microscopy (SEM). SEM detected structural changes of particles caused by migration to different locations. Results: It was possible to visualize the contact of doxorubicin aerosol particles with the surface. Larger particles as well as particles closer to the aerosol generation chamber collided with the glass sample creating liquid drops, while smaller particles with more distance to the aerosol chamber collided as highly concentrated nanocrystals. The amount of nanocrystal particles outweighed the amount of fluid aerosol particles by far. Conclusions: Under optimal conditions, the formation of nanocrystals via aerosol creation device is possible. While a wide range of possible applications of nanocrystals is conceivable, surface coating with drug particles is especially interesting as it may serve as an alternative to conventional liquid intraperitoneal chemotherapy. Further studies are required to investigate nanocrystallization of chemotherapeutic solutions as well as its physical and pharmacological properties and side effects.

Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction. This study aimed to analyze whether early HFpEF is already associated with ultrastructural alterations and whether they differ quantitatively among the layers of the left ventricular wall. HFpEF was induced in pigs by deoxy‐corticosterone acetate (DOCA) treatment along with a high‐salt/high lipid diet over 3 months and compared with weight‐matched normal pigs (n = 5 each). Samples of the left ventricle were taken and processed for light and electron microscopy. Interstitial fibrosis, subcellular composition of cardiomyocytes and mean cardiomyocyte diameter were evaluated by stereology in subendocardial, midmyocardial and subepicardial regions. DOCA enhanced the mean cardiomyocyte diameter in all locations of the ventricle wall to the same degree. The subcellular composition did not differ between the locations and was not altered by DOCA. The volume fraction of interstitium was smaller in the subendocardium of DOCA group than of control group. Within the interstitium, the volume fraction of collagen fibrils (between cardiomyocytes) was increased in the subendocardial and midmyocardial wall layers of the DOCA group but not in the subepicardial layer. Although the capillary length density and average supply area were not altered in response to DOCA in any of the wall layers, the volume fraction of blood vessels related to the interstitial space was enhanced in the subendocardium of the DOCA group but not in the other wall layers. In conclusion, cardiomyocyte changes due to DOCA were similar in subepicardial, midmyocardial and subendocardial regions but DOCA‐induced changes in the interstitium appeared to be more pronounced in the subendocardial ventricular wall layers. This suggests a pivotal role of the subendocardial interstitium in the pathogenesis of HFpEF., Heart failure with preserved ejection fraction is a relatively new entity in cardiac research. Here, we used a large animal model to analyze differential morphological changes across the left ventricular wall. Our data support the concept that the subendocardial interstitium plays a pivotal role in the pathophysiology of heart failure with preserved ejection fraction.

Structural changes of two patient-derived glioblastoma cell lines after Zika virus infection were investigated using scanning transmission electron tomography on high-pressure-frozen, freeze-substituted samples. In Zika-virus-infected cells, Golgi structures were barely visible under an electron microscope, and viral factories appeared. The cytosol outside of the viral factories resembled the cytosol of uninfected cells. The viral factories contained largely deranged endoplasmic reticulum (ER), filled with many so-called replication organelles consisting of a luminal vesicle surrounded by the ER membrane. Viral capsids were observed in the vicinity of the replication organelles (cell line #12537 GB) or in ER cisternae at large distance from the replication organelles (cell line #15747 GB). Near the replication organelles, we observed many about 100-nm-long filaments that may represent viral ribonucleoprotein complexes (RNPs), which consist of the RNA genome and N protein oligomers. In addition, we compared Zika-virus-infected cells with cells infected with a phlebovirus (sandfly fever Turkey virus). Zika virions are formed in the ER, whereas phlebovirus virions are assembled in the Golgi apparatus. Our findings will help to understand the replication cycle in the virus factories and the building of the replication organelles in glioblastoma cells. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-021-02028-2.

We report the working of a novel detector design based on a Bessel Box (BB) electron energy analyser in a scanning electron microscope (SEM). We demonstrate the application of our detector for elemental identification through Auger electron detection in an SEM environment and its potential as a complementary technique to energy dispersive X‐ray (EDX) spectroscopy. We also demonstrate energy‐filtered secondary electron imaging of a copper‐on‐silicon sample using an electron pass energy of 12 eV., Journal of Microscopy, 279 (3), ISSN:0022-2720, ISSN:1365-2818

The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM‐CLEM approach thus presents a new approach to understand these pathogens during infection.

In this article, a method is presented to estimate a new local quality measure for 3D cryoEM maps that adopts the form of a `local resolution' type of information. The algorithm (DeepRes) is based on deep-learning 3D feature detection. DeepRes is fully automatic and parameter-free, and avoids the issues of most current methods, such as their insensitivity to enhancements owing to B-factor sharpening (unless the 3D mask is changed), among others, which is an issue that has been virtually neglected in the cryoEM field until now. In this way, DeepRes can be applied to any map, detecting subtle changes in local quality after applying enhancement processes such as isotropic filters or substantially more complex procedures, such as model-based local sharpening, non-model-based methods or denoising, that may be very difficult to follow using current methods. It performs as a human observer expects. The comparison with traditional local resolution indicators is also addressed., The authors would like to acknowledge financial support from: the Comunidad de Madrid through grant CAM (S2017/BMD-3817), the Spanish Ministry of Economy and Competitiveness (BIO2016-76400-R) and the European Union and Horizon 2020 through INSTRUCT-ULTRA (INFRADEV-03-016-2017, Proposal 731005), iNEXT (INFRAIA-1-2014-2015, Proposal 653706) and West-Life (EINFRA-2015-1, Proposal 675858).

Journal of synchrotron radiation 26(5), 1769 - 1781 (2019). doi:10.1107/S160057751900660X, Two in situ `nanoreactors' for high-resolution imaging of catalysts have been designed and applied at the hard X-ray nanoprobe endstation at beamline P06 of the PETRA III synchrotron radiation source. The reactors house samples supported on commercial MEMS chips, and were applied for complementary hard X-ray ptychography (23 nm spatial resolution) and transmission electron microscopy, with additional X-ray fluorescence measurements. The reactors allow pressures of 100 kPa and temperatures of up to 1573 K, offering a wide range of conditions relevant for catalysis. Ptychographic tomography was demonstrated at limited tilting angles of at least ±35° within the reactors and ±65° on the naked sample holders. Two case studies were selected to demonstrate the functionality of the reactors: (i) annealing of hierarchical nanoporous gold up to 923 K under inert He environment and (ii) acquisition of a ptychographic projection series at ±35° of a hierarchically structured macroporous zeolite sample under ambient conditions. The reactors are shown to be a flexible and modular platform for in situ studies in catalysis and materials science which may be adapted for a range of sample and experiment types, opening new characterization pathways in correlative multimodal in situ analysis of functional materials at work. The cells will presently be made available for all interested users of beamline P06 at PETRA III., Published by IUCr, Chester

Background Pseudomonas aeruginosa (PA) is the most important opportunistic pathogen in causing nosocomial infections and, furthermore, poses a permanent threat for severe chronic infections in patients with cystic fibrosis or COPD. The transmembrane protein CD26 with dipeptidyl peptidase-4 (DPP4) activity shows an increased expression in inflamed tissue. We tested whether CD26/DPP4 deficiency leads to reduced inflammation and decreased structural damage when infected with PA. Methods CD26/DPP4+ and CD26/DPP4− rats were instilled intratracheally with NaCl (controls) or with PA. Six hours later, bacterial distribution was detected with the in vivo imaging system 200 (IVIS). Lungs were then processed for molecular biology, light and electron microscopy and analyzed qualitatively, quantitatively and stereologically. Bacterial numbers were determined in homogenized lungs. Results Compared to saline treated controls, in both infected groups (1) the acinar airspace was significantly increased, (2) the volume density of the alveolar epithelium was significantly decreased, (3) the septal thickness was significantly reduced, (4) more than 40% of the alveolar epithelial surface was damaged, and up to 36% of the epithelial surface was covered with edema. In infected CD26− rats, the increase in lung weight was significantly less pronounced, the portion of edematous alveolar airspace was significantly lower and the part of edema interspersed with PA was decreased significantly. Conclusions CD26/DPP4 deficiency resulted in reduced pulmonary edema under sublethal PA infection, implicating a role for CD26 in infection progression. The partly pronounced structural damage may mask further possible influences of CD26 on the inflammatory response.

The objective of this study is to verify the feasibility of using biochar made from crop straw as a bitumen additive to improve some properties of bitumen. The differences between crop straw biochar prepared in a laboratory and commercial charcoal were investigated through scanning electron microscopy and laser particle size analyses. Furthermore, biochar-modified asphalt was prepared using the high-speed shear method, and the penetration, softening point, ductility at 15°C, and apparent viscosity of the asphalt binder with 6% biochar were measured at 120, 135, 150, 160, and 175°C. It was found that both the crop straw biochar and the commercial charcoal consist mainly of C, O, Si, and K, but the C content of crop straw biochar is slightly higher than that of commercial charcoal. The particle size of biochar is smaller than that of commercial charcoal, while the specific surface area is larger. It was determined that the addition of crop straw biochar significantly improved the high-temperature performance of asphalt, and that biochar and commercial charcoal have a similar influence on the high temperature performance of asphalt.

BACKGROUND: This study aimed to investigate the morphology and chemical composition of dental caries related to ionizing radiation (DCIR), an aggressive and progressive disease that affects dental hard tissues. MATERIALS AND METHODS: Eight human teeth with DCIR were paired with sixteen control teeth (8 teeth with conventional caries and 8 without caries) and included in this study. An analysis of the morphology of the lesions was performed using the following techniques: periapical radiography, cone beam computed tomography, computed microtomography, and scanning electron microscopy. The chemical composition was assessed using X-ray dispersive spectroscopy. RESULTS: There was more demineralization in DCIR lesions when compared to conventional dental caries, even though there was no cavitation in the cervical region of the teeth. The superficial roughness and topography of DCIR lesions were similar to those of healthy teeth. On the other hand, lesions of conventional dental caries showed greater surface and topographic irregularity when compared to DCIR and healthy teeth (p = 0.001). Calcium (Ca) and phosphorus (P) levels were lower in DCIR lesions when compared to controls. However, higher levels of carbon (C) have been observed in DCIR lesions. There was a greater loss of the mineral matrix in DCIR followed by conventional caries. The reduction in the mineral matrix (Ca and P) was compatible with the imaging patterns observed in teeth with DCIR and conventional caries. CONCLUSION: Despite their rapid evolution, DCIR presents an irregular, apparently intact surface with significant changes in the amount of Ca, P, and C.

In recent years, three‐dimensional density maps reconstructed from single particle images obtained by electron cryo‐microscopy (cryo‐EM) have reached unprecedented resolution. However, map interpretation can be challenging, in particular if the constituting structures require de‐novo model building or are very mobile. Herein, we demonstrate the potential of convolutional neural networks for the annotation of cryo‐EM maps: our network Haruspex has been trained on a carefully curated set of 293 experimentally derived reconstruction maps to automatically annotate RNA/DNA as well as protein secondary structure elements. It can be straightforwardly applied to newly reconstructed maps in order to support domain placement or as a starting point for main‐chain placement. Due to its high recall and precision rates of 95.1 % and 80.3 %, respectively, on an independent test set of 122 maps, it can also be used for validation during model building. The trained network will be available as part of the CCP‐EM suite., Haruspex is a new tool to identify secondary structure and RNA/DNA in cryo‐EM reconstruction maps. It uses a neural network which has been trained on a carefully curated set of 293 experimentally derived reconstructions and can be straightforwardly applied to newly reconstructed maps to support model building and validation.