Exploring the antibiofilm and toxicity of tin oxide nanoparticles: Insights from in vitro and in vivo investigations.

BACKGROUND INFORMATION: The advancement of biological-mediated nanoscience towards higher levels and novel benchmarks is readily apparent, owing to the use of non-toxic synthesis processes and the incorporation of various additional benefits. This study aimed to synthesize stable tin oxide nanoparticles (SnO2-NPs) using S. rhizophila as a mediator. METHODS: The nanoparticles that were created by biosynthesis was examined using several analytical techniques, including Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), UV-visible (UV-vis) spectroscopy, and energy dispersive X-ray spectroscopy (EDS). RESULTS: The results obtained from the characterization techniques suggest that S. rhizophila effectively catalyzed the reduction of SnCl2 to SnO2-NPs duration of 90 min at ambient temperature with the ƛmax of 328 nm. The size of the nano crystallite formations was measured to be 23 nm. The present study investigates nanoscale applications' antibacterial efficacy against four bacterial strains, including Klebsiella Sp, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The observed zone of inhibition for the nanoparticles (NPs) varied from 10 to 25 mm. The research findings demonstrate that the nanoparticles (NPs) are effective as antibacterial, phytotoxic, and cytotoxic agents.

Malformation of the endoplasmic reticulum system evolving into giant inclusions and Auer bodies in acute promyelocytic leukemia: an ultrastructural study of 6 cases.

The endoplasmic reticulum（ER）is the largest membranous network serving as a region for protein, lipid and steroid synthesis, transport and storage. Detailed information about ER-cisternae, ER-tubules and rough endoplasmic reticulum (rER) is scarce in human blood cells. This study describes a series of giant inclusions and Auer bodies in promyeloblasts in six patients with acute promyelocytic leukemia (APL), by light microscopy, transmission electron microscopy (TEM) and cytochemical stains. TEM revealed that giant inclusions and pro-Auer bodies were associated with rER and surrounded by tubular structures composed of degenerated or redundant membrane in promyeloblasts, which corresponded with elements of the ER system. This paper reveals that in the promyeloblasts of APL, ER is the source of and transforms progressively into giant inclusions and Auer bodies.

[Automatic classification of immune-mediated glomerular diseases based on multi-modal multi-instance learning].

OBJECTIVE: To develop a multi-modal deep learning method for automatic classification of immune-mediated glomerular diseases based on images of optical microscopy (OM), immunofluorescence microscopy (IM), and transmission electron microscopy (TEM). METHODS: We retrospectively collected the pathological images from 273 patients and constructed a multi-modal multi- instance model for classification of 3 immune-mediated glomerular diseases, namely immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN), and lupus nephritis (LN). This model adopts an instance-level multi-instance learning (I-MIL) method to select the TEM images for multi-modal feature fusion with the OM images and IM images of the same patient. By comparing this model with unimodal and bimodal models, we explored different combinations of the 3 modalities and the optimal methods for modal feature fusion. RESULTS: The multi-modal multi-instance model combining OM, IM, and TEM images had a disease classification accuracy of (88.34±2.12)%, superior to that of the optimal unimodal model [(87.08±4.25)%] and that of the optimal bimodal model [(87.92±3.06)%]. CONCLUSION: This multi- modal multi- instance model based on OM, IM, and TEM images can achieve automatic classification of immune-mediated glomerular diseases with a good classification accuracy.

Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

Morphological and biochemical characteristics associated with autophagy in gastrointestinal diseases.

Autophagy is a cellular catabolic process characterized by the formation of double-membrane autophagosomes. Transmission electron microscopy is the most rigorous method to clearly visualize autophagic engulfment and degradation. A large number of studies have shown that autophagy is closely related to the digestion, secretion, and regeneration of gastrointestinal (GI) cells. However, the role of autophagy in GI diseases remains controversial. This article focuses on the morphological and biochemical characteristics of autophagy in GI diseases, in order to provide new ideas for their diagnosis and treatment.

Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

Genetically Fused Resilin-like Polypeptide-Coiled Coil Bundlemer Conjugates Exhibit Tunable Multistimuli-Responsiveness and Undergo Nanofibrillar Assembly.

Peptide-based materials are diverse candidates for self-assembly into modularly designed and stimuli-responsive nanostructures with precisely tunable compositions. Here, we genetically fused computationally designed coiled coil-forming peptides to the N- and C-termini of compositionally distinct multistimuli-responsive resilin-like polypeptides (RLPs) of various lengths. The successful expression of these hybrid polypeptides in bacterial hosts was confirmed through techniques such as gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism spectroscopy and ultraviolet-visible turbidimetry demonstrated that despite the fusion of disparate structural and responsive units, the coiled coils remained stable in the hybrid polypeptides, and the sequence-encoded differences in thermoresponsive phase separation of the RLPs were preserved. Cryogenic transmission electron microscopy and coarse-grained modeling showed that after thermal annealing in solution, the hybrid polypeptides adopted a closed loop conformation and assembled into nanofibrils capable of further hierarchically organizing into cluster structures and ribbon-like structures mediated by the self-association tendency of the RLPs.

Square condenser apertures for square cameras in low-dose transmission electron microscopy.

In transmission electron microscopy (TEM), cameras are square or rectangular but beams are round so the circular lobes irradiate adjacent areas, precluding further neighboring acquisition for beam-sensitive samples. We present condenser aperture plates with square and rectangular shapes that improve the efficiency of area usage by 70% and enhance montage imaging for beam-sensitive specimens. We demonstrate the compatibility of these condenser aperture plates with high-resolution cryogenic TEM by reconstructing a 1.8-Å map of equine apo-ferritin.

Sedimentation and Laser Light Scattering Methods for Quantifying Synthetic Tau Aggregation Propensity.

Tau aggregation assays detect and quantify the conversion of soluble tau monomers into species having filamentous or oligomeric structure. Assays for filamentous aggregates in cross-ß-sheet conformation leverage optical, biochemical, or biophysical methods, each with their own advantages and throughput capacity. Here we provide protocols for two medium-throughput assays based on sedimentation and laser light scattering and compare their performance, their utility for characterizing tau aggregation dynamics, and their limitations relative to other approaches. Additionally, a protocol for transmission electron microscopy analysis is updated so as to be compatible with the truncated tau variants that have emerged as powerful tools for interrogating the structural basis of tau polymorphism. Together these methods contribute to a rich tool kit for interrogating tau aggregation kinetics and propensity over a wide range of experimental conditions.

Ultrastructure of human brain tissue vitrified from autopsy revealed by cryo-ET with cryo-plasma FIB milling.

Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following fixation, staining, and sectioning, which limit resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) allows higher resolution imaging of unfixed cellular samples while preserving architecture, but it requires samples to be vitreous and thin enough for transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue via plunge-freezing and use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid at variable depth inside the tissue. Lamellae generated in Alzheimer's disease brain tissue reveal intact subcellular structures including components of autophagy and potential pathologic tau fibrils. Furthermore, we reveal intact compact myelin and functional cytoplasmic expansions. These images indicate that plasma FIB milling with cryo-ET may be used to elucidate nanoscale structures within the human brain.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants.

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Visualization of Engineered M13 Phages Bound to Bacterial Targets by Transmission Electron Microscopy.

The filamentous phage M13 is one of the most well-studied and characterized phages, particularly since it was introduced as a scaffold for phage display, a technique to express and evolve fusion proteins on the M13 phage's coat to study protein or peptide binding interactions. Since phages can be engineered or evolved to specifically bind to a variety of targets, engineered M13 phages have been explored for applications such as drug delivery, biosensing, and cancer therapy, among others. Specifically, with the rising challenge of antimicrobial resistance among bacteria, chimeric M13 phages have been explored both as detection and therapeutic agents due to the flexibility in tuning target specificity. Transmission electron microscopy (TEM) is a powerful tool enabling researchers to directly visualize and characterize binding of phages to bacterial surfaces. However, the filamentous phage structure poses a challenge for this technique, as the phages have similar morphology to bacterial structures such as pili. In order to differentiate between bacterial structures and the filamentous phages, here we describe a protocol to prepare TEM samples of engineered M13 phages bound to bacterial cells, in which the phage virions have been specifically labeled by decoration of the major capsid proteins with gold nanoparticles. This protocol enables clear visualization and unambiguous identification of attached filamentous phages within the context of bacterial cells expressing numerous pili.

Construction Strategy and Mechanism of a Novel Wood Preservative with Excellent Antifungal Effects.

Wood is a naturally porous material prone to microbial erosion and degradation in outdoor environments. Therefore, the development of an environmentally friendly wood preservative with excellent antibacterial effects and low toxicity is urgently needed. In this study, nitrogen-doped carbon quantum dots (N-CQDs) with excellent antifungal performance and fluorescent properties were synthesized using a one-step hydrothermal method with chitosan quaternary ammonium salt (HACC) as the raw material. The fluorescence characteristics of N-CQD preservatives can help track their position and distribution in wood. The minimum inhibitory concentration (MIC) of N-CQDs is 1.8 mg/mL, which was nearly 22 times lower than that of HACC (40.0 mg/mL) in the PDA medium. The decay resistance test demonstrated that wood treated with N-CQDs showed a considerably reduced decay degree and its mass loss rate decreased from 46 ± 0.5% to 3.8 ± 0.5%. Biological transmission electron microscopy revealed that N-CQDs effectively destroyed fungal cell structures, thereby hindering the growth of Coriolus versicolor. N-CQDs synthesized using the one-step hydrothermal method can be used as an efficient wood preservative that can effectively improve the utilization and service life of wood.

A transmission electron microscopy investigation suggests that telocytes, skeletal muscles, myoblasts, and stem cells in common carp (Cyprinus carpio) respond to salinity challenges.

BACKGROUND: Telocytes are modified interstitial cells that communicate with other types of cells, including stem cells. Stemness properties render them more susceptible to environmental conditions. The current morphological investigation examined the reactions of telocytes to salt stress in relation to stem cells and myoblasts. The common carp are subjected to salinity levels of 0.2, 6, and 10 ppt. The gill samples were preserved and prepared for TEM. RESULTS: The present study observed that telocytes undergo morphological change and exhibit enhanced secretory activities in response to changes in salinity. TEM can identify typical telocytes. This research gives evidence for the communication of telocytes with stem cells, myoblasts, and skeletal muscles. Telocytes surround stem cells. Telopodes made planar contact with the cell membrane of the stem cell. Telocytes and their telopodes surrounded the skeletal myoblast. These findings show that telocytes may act as nurse cells for skeletal stem cells and myoblasts, which undergo fibrillogenesis. Not only telocytes undergo morphological alternations, but also skeletal muscles become hypertrophied, which receive telocyte secretory vesicles in intercellular compartments. CONCLUSION: In conclusion, the activation of telocytes is what causes stress adaptation. They might act as important players in intercellular communication between cells. It is also possible that reciprocal interaction occurs between telocytes and other cells to adapt to changing environmental conditions.

Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles.

Introduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered. Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet-visual spectroscopy, interferometric light microscopy and liquid chromatography-mass spectrometry. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.

Mass Spectrometry-Based Characterization of Protein Aggregates in Tissues and Biofluids.

Protein aggregation is a common mechanism in multiple neurodegenerative and heart diseases and the accumulation of proteins in aggregates is toxic to cells, causing injury and death. The degree of protein aggregation directly correlates with the severity of the disease. Misfolded proteins present thermodynamic barriers that culminate in the loss of structure and function and the exposure of hydrophobic residues. The exposure of hydrophobic residues is the driving force behind protein aggregation, as it reduces surface free energy and increases the propensity for the formation of large insoluble aggregates. Exploring the protein content of aggregates is fundamental to understanding their formation mechanism and pathophysiological effects. We demonstrate here a method for isolating aggregated protein content in human plasma and mouse brain samples. The samples were characterized by mass spectrometry analysis, transmission electron microscopy, and western blotting. We report the identification of proteins associated with neurodegenerative diseases in the isolated pellets. The western blotting analyses of the isolated pellet showed the positivity for CD89 and CD63, consolidated markers of exosomes, confirming the presence of exosomes within the pellet but not in the supernatant in human plasma. Notably, the concomitant isolation of exosomes together with the protein aggregates was feasible starting from 200 µL of human plasma. Moreover, the presented methodology separated albumin from the aggregated pellet, allowing identification of larger diversity of proteins through mass spectrometry analysis.

Preparation and Imaging of Specialized ER Using Super-Resolution and TEM Techniques.

The plant endoplasmic reticulum (ER) forms several specialized structures. These include the sieve element reticulum (SER) and the desmotubule formed as the ER passes through plasmodesmata. Imaging both of these structures has been inhibited by the resolution limits of light microscopy and their relatively inaccessible locations, combined with the fragile nature of the ER. Here we describe methods to view desmotubules in live cells under 3D-structured illumination microscopy (3D-SIM) and methods to fix and prepare phloem tissue for both 3D-SIM and transmission electron microscopy (TEM), which preserve the fragile structure and allow the detailed imaging of the SER.

Evaluating the biological (antidiabetic) potential of TEM, FTIR, XRD, and UV-spectra observed berberis lyceum conjugated silver nanoparticles.

Diabetes is a life-threatening disease that affects different parts of the body including the liver, kidney, and pancreas. The core root of diabetes is mainly linked to oxidative stress produced by reactive oxygen species (ROS). Berberis lyceum Royle (BLR) is the source of natural products. It comprises numerous bioactive compounds having antioxidant activities. In the current investigation, silver nanoparticles from BLR root extract were synthesized, characterized, and assessed for antidiabetic potential. UV spectrophotometry, Transmission electron microscopy (TEM), Fourier transform infra-red spectroscopy (FTIR), and x-ray diffraction (XRD) were applied for the characterization of NPs. It was evident from the morphological studies that the synthesized NPs were spherical and the average size was 11.02 nm. Results revealed that BLR-AgNPs showed higher radical scavenging activity as compared to BLR extract. Moreover, BLR-AgNPs displayed superior in vivo and in vitro antidiabetic activity in comparison to BLR extract. Glucose level (116.5 ± 5.1 mg/dL), liver function test (ALAT: 54.038 ± 6.2 IU/L; ASAT: 104.42 ± 13.9 IU/L; ALP: 192.6 ± 2.4 IU/L; bilirubin: 1.434 ± 0.14 mg/dL; total protein: 5.14 ± 0.24 mg/dL), renal function test (urea: 39.6 ± 0.63 mg/dL; uric acid: 21.4 ± 0.94 mg/dL; creatinine: 0.798 ± 0.03 mg/dL; albumin: 4.14 ± 0.2 mg/dL), lipid profile level (cholesterol: 101.62 ± 3 mg/dL; triglyceride: 110.42 ± 7 mg/dL; HDL-C: 29.7 ± 3 mg/dL; LDL-C: 47.056 ± 1 mg/dL; VLDL-C: 22.0 ± 1.3 mg/dL) and hematology (WBCs: 3.82 ± 0.24 103 /µL; RBCs: 4.78 ± 0.42 106 /µL; Hb: 12.6 ± 1.0 g/dL; Hematocrit: 39.4 ± 3.7%; MCV: 65.8 ± 3 fL; platelets: 312 ± 22.4; neutrophils: 34.8 ± 1.87; eosinophils: 3.08 ± 0.43; monocytes: 3.08 ± 0.28; lymphocytes: 75.6 ± 3.77) confirmed the significant antidiabetic potential of BLR-AgNPs. Histopathological examination authenticated that BLR-AgNPs caused a significant revival in the morphology of the liver, kidney, and pancreas. Hence, findings of the study suggested the BLR-AgNPs as a potent antidiabetic agent and could be an appropriate nanomedicine to prevent diabetes in future. RESEARCH HIGHLIGHTS: Berberis lyceum extract as a reducing, capping, and stabilization agent for the BLR-AgNPs synthesis Evaluation of α-amylase inhibition, antioxidant, and α-glucosidase inhibition potential Thorough characterization using Fourier transform infrared spectroscopy, Transmission electron microscopy, x-ray diffraction, and UV-VIS spectrophotometer, which is 1st of its kind In-vivo antidiabetic activity evaluation through multiple biomarkers.

Lighting lantern above Psalteriomonadidae: Unveiling novel diversity within the genus Psalteriomonas (Discoba: Heterolobosea).

Psalteriomonadidae are a small family of anaerobic free-living protists belonging to Heterolobosea, Discoba. We cultured 74 new strains of mostly amoeboid Psalteriomonadidae obtained from mainly freshwater habitats and sequenced their 18S rRNA gene. Based on the phylogenetic analysis and genetic distances, we report multiple novel species, four of which we formally describe based on the light-microscopic morphology (Psalteriomonas minuta, P. australis, P. fimbriata, and P. parva). We also examined the ultrastructure of two Psalteriomonas species using transmission electron microscopy. We transfer Sawyeria marylandensis into the genus Psalteriomonas and synonymize Sawyeria with Psalteriomonas. In addition, we studied the flagellate stage of P. marylandensis comb. nov. for the first time, using light and scanning electron microscopy.

The application and development of electron microscopy for three-dimensional reconstruction in life science: a review.

Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure-function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.