Generation and Characterization of In Vitro Transcribed mRNA.

Here we describe the in vitro preparation of mRNA from DNA templates, including setting up the transcription reaction, mRNA capping, and mRNA labeling. We then describe methods used for mRNA characterization, including UV and fluorescence spectrophotometry, as well as gel electrophoresis. Moreover, characterization of the in vitro transcribed RNA using the Bioanalyzer instrument is described, allowing a higher resolution analysis of the target molecules. For the in vitro testing of the mRNA molecules, we include protocols for the transfection of various primary cell cultures and the confirmation of translation by intracellular staining and western blotting.

Immunofluorescence microscopy of G-quadruplexes and R-loops.

A large variety of non-B secondary structures can be formed between DNA and RNA. In this chapter, we focus on G-quadruplexes (G4) and R-loops, which can have a close structural interplay. In recent years, increasing evidence pointed to the fact that they can strongly influence each other in vivo, both having physiological and pathological roles in normal and cancer cells. Here, we detail specific and accurate methods for purification of BG4 and S9.6 antibodies, and their subsequent use in immunofluorescence microscopy, enabling single-cell analysis of extent and localization of G4s and R-loops.

The tongue of the red-eared slider (Trachemys scripta elegans): morphological characterization through gross, light, scanning electron, and immunofluorescence microscopic examination.

The red-eared slider (Trachemys scripta elegans) is renowned for its remarkable adaptations, yet much of its complex biology remains unknown. In this pioneering study, we utilized a combination of gross anatomy, scanning electron microscopy (SEM), light microscopy, and immunofluorescence techniques to examine the tongue's omnivorous adaptation in this species. This research bridges a critical knowledge gap, enhancing our understanding of this intriguing reptile. Gross examination revealed a unique arrowhead-shaped tongue with a median lingual fissure and puzzle-piece-shaped tongue papillae. SEM unveiled rectangular filiform, conical, and fungiform papillae, with taste pores predominantly on the dorsal surface and mucous cells on the lateral surface of the papillae. Histologically, the tongue's apex featured short rectangular filiform and fungiform papillae, while the body exhibited varying filiform shapes and multiple taste buds on fungiform papillae. The tongue's root contained lymphatic tissue with numerous lymphocytes surrounding the central crypt, alongside lingual skeletal musculature, blood and lymph vessels, and Raffin corpuscles in the submucosa. The lingual striated muscle bundles had different orientations, and the lingual hyaline cartilage displayed a bluish coloration of the ground substance, along with a characteristic isogenous group of chondrocytes. Our research represents the first comprehensive application of immunofluorescence techniques to investigate the cellular intricacies of the red-eared slider's tongue by employing seven distinct antibodies, revealing a wide array of compelling and significant findings. Vimentin revealed the presence of taste bud cells, while synaptophysin provided insights into taste bud and nerve bundle characteristics. CD34 and PDGFRα illuminated lingual stromal cells, and SOX9 and PDGFRα shed light on chondrocytes within the tongue's cartilage. CD20 mapped B-cell lymphocyte distribution in the lingual tonsil, while alpha smooth actin (α-SMA) exposed the intricate myofibroblast and smooth muscle network surrounding the lingual blood vessels and salivary glands. In conclusion, our comprehensive study advances our knowledge of the red-eared slider's tongue anatomy and physiology, addressing a significant research gap. These findings not only contribute to the field of turtle biology but also deepen our appreciation for the species' remarkable adaptations in their specific ecological niches.

Phenotyping of Macrophages in Human Immune System Mice.

Human immune system mice, also referred to as humanized mice, are a major research tool for the in vivo study of human immune system function. Upon reconstitution with human hematopoietic stem cells, all major human leukocyte populations develop in immunodeficient mice and can be detected in peripheral blood as well as in lymphatic and nonlymphatic tissue. This includes human macrophages that are intrinsically difficult to study from humans due to their organ-resident nature. In the following chapter, we provide a detailed protocol for generation of human immune system mice. We suggest that these mice are a suitable model to study human macrophage function in vivo.

Detection of G-Quadruplex DNA Structures in Macrophages.

In addition to the canonical B-DNA conformation, DNA can fold into different secondary structures. Among them are G-quadruplex structures (G4s). G4 structures are very stable and can fold in specific guanine-rich regions in DNA and RNA. Different in silico, in vitro, and in cellulo experiments have shown that G4 structures form so far in all tested organisms. There are over 700,000 predicted G4s in higher eukaryotes, but it is so far assumed that not all will form at the same time. Their formation is dynamically regulated by proteins and is cell type-specific and even changes during the cell cycle or during different exogenous or endogenous stimuli (e.g., infection or developmental stages) can alter the G4 level. G4s have been shown to accumulate in cancer cells where they contribute to gene expression changes and the mutagenic burden of the tumor. Specific targeting of G4 structures to impact the expression of oncogenes is currently discussed as an anti-cancer treatment. In a tumor microenvironment, not only the tumor cells will be targeted by G4 stabilization but also immune cells such as macrophages. Although G4s were detected in multiple organisms and different cell types, only little is known about their role in immune cells. Here, we provide a detailed protocol to detect G4 formation in the nucleus of macrophages of vertebrates and invertebrates by microscopic imaging.

Immunoelectron microscopy findings in a patient with C3 glomerulonephritis.

We performed a kidney biopsy in a 36-year-old man to evaluate microscopic hematuria and proteinuria. Light microscopy showed increased mesangial matrix and partial swelling of the glomerular basement membrane (GBM), and immunofluorescence showed positive staining only for C3. Immunoelectron microscopy showed that gold particle-labeled C3 was localized in the electron-dense and moderately electron-dense deposits shown by electron microscopy in the mesangium, the thickened GBM near the paramesangium, and the thickened distal portion of the GBM but was not localized in the non-thickened GBM. Gold-labeled immunoglobulin G, κ, and λ were not seen. C3 glomerulonephritis was more evident in gold-labeled electron microscopy, which further clarified the localization of C3 deposition.

Comparing and Correcting Spectral Sensitivities between Multispectral Microscopes: A Prerequisite to Clinical Implementation.

Multispectral, multiplex immunofluorescence (mIF) microscopy has been used to great effect in research to identify cellular co-expression profiles and spatial relationships within tissue, providing a myriad of diagnostic advantages. As these technologies mature, it is essential that image data from mIF microscopes is reproducible and standardizable across devices. We sought to characterize and correct differences in illumination intensity and spectral sensitivity between three multispectral microscopes. We scanned eight melanoma tissue samples twice on each microscope and calculated their average tissue region flux intensities. We found a baseline average standard deviation of 29.9% across all microscopes, scans, and samples, which was reduced to 13.9% after applying sample-specific corrections accounting for differences in the tissue shown on each slide. We used a basic calibration model to correct sample- and microscope-specific effects on overall brightness and relative brightness as a function of the image layer. We tested the generalizability of the calibration procedure and found that applying corrections to independent validation subsets of the samples reduced the variation to 2.9 ± 0.03%. Variations in the unmixed marker expressions were reduced from 15.8% to 4.4% by correcting the raw images to a single reference microscope. Our findings show that mIF microscopes can be standardized for use in clinical pathology laboratories using a relatively simple correction model.

Interactions of Tissue-Resident T Cells.

Resident memory T cells (TRM) are non-circulating cells that play a critical role in protection from local infections and cancers. Flow cytometric and transcriptional analyses of these cells have defined their distinct phenotypes; imaging allows study of their morphology, localization, and interactions within tissues. Here, we describe commonly used methods to generate cutaneous CD8+ TRM and to prepare skin samples for analysis, including staining of cryostat sections, epidermal sheets, and tissue whole mounts.

Liquid-Liquid Phase Separation of the DEAD-Box Cyanobacterial RNA Helicase Redox (CrhR) into Dynamic Membraneless Organelles in Synechocystis sp. Strain PCC 6803.

Compartmentalization of macromolecules into discrete non-lipid-bound bodies by liquid-liquid phase separation (LLPS) is a well-characterized regulatory mechanism frequently associated with the cellular stress response in eukaryotes. In contrast, the formation and importance of similar complexes is just becoming evident in bacteria. Here, we identify LLPS as the mechanism by which the DEAD-box RNA helicase, cyanobacterial RNA helicase redox (CrhR), compartmentalizes into dynamic membraneless organelles in a temporal and spatial manner in response to abiotic stress in the cyanobacterium Synechocystis sp. strain PCC 6803. Stress conditions induced CrhR to form a single crescent localized exterior to the thylakoid membrane, indicating that this region is a crucial domain in the cyanobacterial stress response. These crescents rapidly dissipate upon alleviation of the stress conditions. Furthermore, CrhR aggregation was mediated by LLPS in an RNA-dependent reaction. We propose that dynamic CrhR condensation performs crucial roles in RNA metabolism, enabling rapid adaptation of the photosynthetic apparatus to environmental stresses. These results expand our understanding of the role that functional compartmentalization of RNA helicases and thus RNA processing in membraneless organelles by LLPS-mediated protein condensation performs in the bacterial response to environmental stress. IMPORTANCE Oxygen-evolving photosynthetic cyanobacteria evolved ~3 billion years ago, performing fundamental roles in the biogeochemical evolution of the early Earth and continue to perform fundamental roles in nutrient cycling and primary productivity today. The phylum consists of diverse species that flourish in heterogeneous environments. A prime driver for survival is the ability to alter photosynthetic performance in response to the shifting environmental conditions these organisms continuously encounter. This study demonstrated that diverse abiotic stresses elicit dramatic changes in localization and structural organization of the RNA helicase CrhR associated with the photosynthetic thylakoid membrane. These dynamic changes, mediated by a liquid-liquid phase separation (LLPS)-mediated mechanism, reveal a novel mechanism by which cyanobacteria can compartmentalize the activity of ribonucleoprotein complexes in membraneless organelles. The results have significant consequences for understanding bacterial adaptation and survival in response to changing environmental conditions.

Peptidic Inhibitors and a Fluorescent Probe for the Selective Inhibition and Labelling of Factor XIIIa Transglutaminase.

Factor XIIIa (FXIIIa) is a transglutaminase of major therapeutic interest for the development of anticoagulants due to its essential role in the blood coagulation cascade. While numerous FXIIIa inhibitors have been reported, they failed to reach clinical evaluation due to their lack of metabolic stability and low selectivity over transglutaminase 2 (TG2). Furthermore, the chemical tools available for the study of FXIIIa activity and localization are extremely limited. To combat these shortcomings, we designed, synthesised, and evaluated a library of 21 novel FXIIIa inhibitors. Electrophilic warheads, linker lengths, and hydrophobic units were varied on small molecule and peptidic scaffolds to optimize isozyme selectivity and potency. A previously reported FXIIIa inhibitor was then adapted for the design of a probe bearing a rhodamine B moiety, producing the innovative KM93 as the first known fluorescent probe designed to selectively label active FXIIIa with high efficiency (kinact/KI = 127,300 M-1 min-1) and 6.5-fold selectivity over TG2. The probe KM93 facilitated fluorescent microscopy studies within bone marrow macrophages, labelling FXIIIa with high efficiency and selectivity in cell culture. The structure-activity trends with these novel inhibitors and probes will help in the future study of the activity, inhibition, and localization of FXIIIa.

Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA-MurG interaction.

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

Methods to Evaluate the Effects of HAT/KAT Inhibition on SIAH2-Driven Reactive Oxygen Species Generation in Helicobacter pylori-Infected Gastric Epithelial Cells.

Helicobacter pylori infection is one of the leading factors that promotes, among other diseases, gastric cancer (GC). Infection of gastric epithelial cells (GECs) by H. pylori enhances the expression as well as acetylation of the E3 ubiquitin ligase SIAH2 which promotes GC progression. The histone acetyltransferase (HAT) activity of p300 catalyzes SIAH2 acetylation following H. pylori infection. Since reactive oxygen species (ROS) generation in H. pylori-infected GECs accelerates GC progression, acetylation-mediated SIAH2 regulation might be a crucial modifier of ROS generation in the infected GECs. Here, we describe a compendium of methods to evaluate the effects of HAT/lysine acetyl transferase (KAT) inhibitors (HAT/KATi) on SIAH2-mediated ROS regulation in H. pylori-infected GECs.

Unhatched bovine blastocysts express all transcripts of the estrogen biosynthetic pathway, but steroid hormone synthesis could not yet be demonstrated.

Early embryos of rodent species and rabbits but also farm animals such as pigs, horses and cattle produce estrogens, which are considered important regulators of the implantation process. In cattle, the exact stage at which embryonic estrogen synthesis commences is yet unknown. However, this information is regarded as important to consider a possible role of embryonic estrogens in preimplantation development. Therefore, in this study, we first used quantitative reverse transcription PCR to examine the mRNA expression of the enzymes required for the conversion of cholesterol into free and sulfonated estrogens (CYP11A1, CYP17A1, HSD3B, CYP19A1, and SULT1E1), the cholesterol carrier protein STAR, and the estrogen receptors ESR1 and ESR2 in in vitro produced morulae and unhatched blastocysts (d 6-9). Only in the blastocysts, were the mRNAs of the entire estrogen biosynthesis chain and of both estrogen receptors clearly present, whereas mRNA specific to ESRs was already detectable in the morulae. We also examined the expression of the corresponding enzymes in blastocysts at the protein level. None of the enzymes were detectable by capillary-based western analysis. Immunofluorescence methods were established for the detection of CYP17A1, CYP19A1, and SULT1E1. CYP17A1 was observed in the inner cell mass and trophectoderm, whereas CYP19A1 and SULT1E1 were present only in trophectoderm. An attempt to detect estrogen sulfotransferase activity was unsuccessful. Despite clear evidence that some elements of the estrogen biosynthetic pathway are also present at the protein level, it remains to be clarified whether the enzyme cascade underlying estrogen production is already functional in unhatched blastocysts.

Quantum Dot-Antibody Conjugates for Immunofluorescence Studies of Biomolecules and Subcellular Structures.

Quantum dots, or nanoscale semiconductors, are one of the most important materials for various research and development purposes. Due to their advantageous photoluminescence and electronic properties, namely, their unique photostability, high brightness, narrow emission spectra from visible to near-infrared wavelengths, convey them significant advantages over widely used fluorochromes, including organic dyes, fluorescent probes. Quantum dots are a unique instrument for a wide range of immunoassays with antibodies. The paper provides an overview of the developed and already applied methods of quantum dot surface modification, quantum dots conjugation to different antibodies (non-covalent, direct covalent linkage or with the use of special adapter molecules), as well as practical examples of recent quantum dot-antibody applications in the immunofluorescence microscopy for cell and cell structure imaging, fluorescent assays for biomolecules detection and in diagnostics of various diseases. The review presents advantages of quantum dot-antibody conjugation technology over the existing methods of immunofluorescence studies and a forward look into its potential prospects in biological and biomedical research.

Detection of Extracellular Traps in Canine Steroid-Responsive Meningitis-Arteritis.

Extracellular traps (ETs) are DNA networks formed by immune cells to fight infectious diseases by catching and attacking pathogenic microorganisms. Uncontrolled ET formation or impaired ET clearance can cause tissue and organ damage. Steroid-responsive meningitis-arteritis (SRMA) represents an immune-mediated, presumably non-infectious, purulent leptomeningitis and fibrinoid-necrotizing arteritis and periarteritis of young-adult dogs. Chronic and recurrent cases of SRMA are characterized by lymphohistiocytic inflammatory cell infiltration in the meninges and perivascular tissue. This study aimed to identify extracellular traps in dogs with SRMA, a model for immune-mediated diseases in the central nervous system (CNS). Hematoxylin and eosin-stained samples of two young dogs with chronic, recurrent SRMA were examined by light microscopy for characteristic lesions and consecutive slices of affected tissues were stained for detection of ETs by immunofluorescence microscopy using antibodies against DNA-histone-1 complexes, myeloperoxidase, and citrullinated histone H3. Histology revealed purulent and lymphohistiocytic leptomeningitis (n = 2/2) with meningeal periarteritis (n = 2/2) and periadrenal located lymphohistiocytic periarteritis (n = 1). Extracellular DNA networks and inflammatory cell infiltrates of macrophages, neutrophil granulocytes, and lymphocytes were detected in the subarachnoid space of the leptomeninx (n = 2/2) and perivascularly in meningeal (n = 2/2) as well as periadrenal vessels (n = 1/1). In summary, extracellular DNA fibers and attached ET markers are detectable in affected perivascular and meningeal tissues of dogs suffering from SRMA. The proof of principle could be confirmed that ETs are present in canine, inflammatory, and non-infectious CNS diseases and possibly play a role in the pathogenesis of SRMA.

In Vitro and In Vivo Biocompatibility Studies on Engineered Fabric with Graphene Nanoplatelets.

To produce clothes made with engineered fabrics to monitor the physiological parameters of workers, strain sensors were produced by depositing two different types of water-based inks (P1 and P2) suitably mixed with graphene nanoplatelets (GNPs) on a fabric. We evaluated the biocompatibility of fabrics with GNPs (GNP fabric) through in vitro and in vivo assays. We investigated the effects induced on human keratinocytes by the eluates extracted from GNP fabrics by the contact of GNP fabrics with cells and by seeding keratinocytes directly onto the GNP fabrics using a cell viability test and morphological analysis. Moreover, we evaluated in vivo possible adverse effects of the GNPs using the model system Caenorhabditis elegans. Cell viability assay, morphological analysis and Caenorhabditis elegans tests performed on smart fabric treated with P2 (P2GNP fabric) did not show significant differences when compared with their respective control samples. Instead, a reduction in cell viability and changes in the membrane microvilli structure were found in cells incubated with smart fabric treated with P1. The results were helpful in determining the non-toxic properties of the P2GNP fabric. In the future, therefore, graphene-based ink integrated into elastic fabric will be developed for piezoresistive sensors.

Visualization of Cytosolic Galectin Accumulation Around Damaged Vesicles and Organelles.

Galectins are animal lectins that recognize ß-galactoside and bind glycans. Recent studies have indicated that cytosolic galectins recognize cytosolically exposed glycans and accumulate around endocytic vesicles or organelles damaged by various disruptive substances. Accumulated galectins engage other cytosolic proteins toward damaged vesicles, leading to cellular responses, such as autophagy. Disruptive substances include bacteria, viruses, particulate matters, and protein aggregates; thus, this process is implicated in the pathogenesis of various diseases. In this chapter, we describe methods for studying three disruptive substances: photosensitizers, Listeria monocytogenes, and Helicobacter pylori. We summarize the tools used for the detection of cytosolic galectin accumulation around damaged vesicles.

Immunofluorescence Detection of Callose in Plant Tissue Sections.

The accumulation of the cell wall component callose at plasmodesmata (PD) is crucial for the regulation of symplastic intercellular transport in plants. Here we describe protocols to fluorescently image callose in sectioned plant tissue using monoclonal antibodies. This protocol achieves high-resolution images by the fixation, embedding, and sectioning of plant material to expose internal cell walls. By using this protocol in combination with high-resolution confocal microscopy, we can detect PD callose in a variety of plant tissues and species.

Analyzing Lymphatic Vessel Patterning in Adult Tissue.

Whole-mount immunostaining allows intact tissue to be surveyed in three dimensions, avoiding the more restricted fields of view provided by visualizing thin sections. This technique is particularly useful for imaging lymphatic and blood networks by high-resolution confocal microscopy, revealing how such vessels are spatially positioned, the subcellular arrangements of individual antigens, and interactions with individual cells within the interstitium or vessel lumen. The purpose of this chapter is to provide a practical guide for obtaining images of lymphatic vessels following immunofluorescence staining, primarily in mouse skin.