Zebrafish Larvae Microinjection and Automated Fluorescence Microscopy for Studying Klebsiella pneumoniae Infection and the Host Immune Response.

Studying host-pathogen interactions is essential for understanding infectious diseases and developing possible treatments, especially for priority pathogens with increased virulence and antibiotic resistance, such as Klebsiella pneumoniae. Over time, this subject has been approached from different perspectives, often using mammal host models and invasive endpoint measurements (e.g., sacrifice and organ extraction). However, taking advantage of technological advances, it is now possible to follow the infective process by noninvasive visualization in real time, using optically amenable surrogate hosts. In this line, this chapter describes a live-cell imaging approach to monitor the interaction of K. pneumoniae and potentially other bacterial pathogens with zebrafish larvae in vivo. This methodology is based on the microinjection of fluorescent bacteria into the otic vesicle, followed by time-lapse observation by automated fluorescence microscopy with environmental control, monitoring the dynamics of immune cell recruitment, bacterial load, and larvae survival.

Bioactive compound from marine seagrass Streptomyces argenteolus TMA13: combatting fish pathogens with time-kill kinetics and live-dead cell imaging.

Actinobacteria, pervasive in aquatic and terrestrial environments, exhibit a filamentous morphology, possess DNA with a specific G + C content and production of numerous secondary metabolites. This study, focused on actinobacteria isolated from marine seagrass, investigating their antibacterial activity against fish pathogens. Among 28 isolates, Streptomyces argenteolus TMA13 displayed the maximum zone of inhibition against fish pathogens-Aeromonas hydrophila (10 mm), Aeromonas caviae (22 mm), Edwardsiella tarda (17 mm), Vibrio harveyi (22 mm) and Vibrio anguillarum (12 mm) using the agar plug method. Optimization of this potent strain involved with various factors, including pH, temperature, carbon source and salt condition to enhance both yield production and antibacterial efficacy. In anti-biofilm assay shows the maximum percentage of inhibition while increasing concentration of TMA13 extract. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) assays with TMA13 crude extract demonstrated potent activity against fish pathogens at remarkably low concentrations. Time-kill kinetics assay showcased growth curve variations over different time intervals for all fish pathogens treated with a 100 µg/ml concentration of crude extract, indicating a decline in cells viability and progression into the death phase. Additionally, fluorescence microscopic visualization of bacterial cells exposed to the extracts emitting green and red fluorescence, enabling live-dead cell differentiation was also studied. Further characterization of the crude extract through GC-MS and FT-IR analyses performed and identified secondary metabolites with functional groups exhibiting significant antibacterial activity. This study elucidates the capacity of Streptomyces argenteolus TMA13 to enhance the production of antibiotic compounds effective against fish pathogens.

Chromatin structure and dynamics: one nucleosome at a time.

Eukaryotic genomes store information on many levels, including their linear DNA sequence, the posttranslational modifications of its constituents (epigenetic modifications), and its three-dimensional folding. Understanding how this information is stored and read requires multidisciplinary collaborations from many branches of science beyond biology, including physics, chemistry, and computer science. Concurrent recent developments in all these areas have enabled researchers to image the genome with unprecedented spatial and temporal resolution. In this review, we focus on what single-molecule imaging and tracking of individual proteins in live cells have taught us about chromatin structure and dynamics. Starting with the basics of single-molecule tracking (SMT), we describe some advantages over in situ imaging techniques and its current limitations. Next, we focus on single-nucleosome studies and what they have added to our current understanding of the relationship between chromatin dynamics and transcription. In celebration of Robert Feulgen's ground-breaking discovery that allowed us to start seeing the genome, we discuss current models of chromatin structure and future challenges ahead.

Human neutrophil extracellular traps do not impair in vitro Toxoplasma gondii infection.

Introduction: Toxoplasma gondii, responsible for causing toxoplasmosis, is a prevalent food and waterborne pathogen worldwide. It commonly infects warm-blooded animals and affects more than a third of the global human population. Once ingested, the parasite enters the host's small intestine and rapidly disseminates throughout the body via the bloodstream, infiltrating various tissues. Leukocyte-driven responses are vital against T. gondii, with neutrophils playing a dual role: swiftly recruited to infection sites, releasing inflammatory mediators, and serving as a replication hub and Trojan horses, aiding parasite spread. Neutrophils from various hosts release extracellular traps (NETs) against the protozoan. However, gaps persist regarding the mechanisms of NETs production to parasite and their significance in infection control. This study investigates the interplay between human neutrophils and T. gondii, exploring dynamics, key molecules, and signaling pathways involved in NETs production upon protozoan challenge. Methods and Results: Using confocal and electron microscopy, live cell imaging, pharmacological inhibitors, and DNA quantification assays, we find that human neutrophils promptly release both classical and rapid NETs upon pathogen stimulation. The NETs structure exhibits diverse phenotypes over time and is consistently associated with microorganisms. Mechanisms involve neutrophil elastase and peptidylarginine deiminase, along with intracellular calcium signaling and the PI3K pathway. Unexpectedly, human traps do not diminish viability or infectivity, but potentially aid in capturing parasites for subsequent neutrophil phagocytosis and elimination. Discussion: By revealing NETs formation mechanisms and their nuanced impact on T. gondii infection dynamics, our findings contribute to broader insights into host-pathogen relationships.

The Virus-Induced Cytopathic Effect.

The cytopathic effect comprises the set of cellular alterations produced by a viral infection. It is of great relevance since it constitutes a direct marker of infection. Likewise, these alterations are often virus-specific which makes them a phenotypic marker for many viral species. All these characteristics have been used to complement the study of the dynamics of virus-cell interactions through the kinetic study of the progression of damage produced by the infection. Various approaches have been used to monitor the cytopathic effect, ranging from light microscopy, immunofluorescence assays, and direct labeling with fluorescent dyes, to plaque assay for the characterization of the infection over time. Here we address the relevance of the study of cytopathic effect and describe different experimental alternatives for its application.

pH-Dependence Cytotoxicity Evaluation of Artepillin C against Tumor Cells.

Brazilian green propolis is a well-known product that is consumed globally. Its major component, Artepillin C, showed potential as an antitumor product. This study explored the impact of Artepillin C on fibroblast and glioblastoma cell lines, used as healthy and very aggressive tumor cell lines, respectively. The focus of the study was to evaluate the pH-dependence of Artepillin C cytotoxicity, since tumor cells are known to have a more acidic extracellular microenvironment compared to healthy cells, and Artepillin C was shown to become more lipophilic at lower pH values. Investigations into the pH-dependency of Artepillin C (6.0-7.4), through viability assays and live cell imaging, revealed compelling insights. At pH 6.0, MTT assays showed the pronounced cytotoxic effects of Artepillin C, yielding a notable reduction in cell viability to less than 12% among glioblastoma cells following a 24 h exposure to 100 µM of Artepillin C. Concurrently, LDH assays indicated significant membrane damage, affecting approximately 50% of the total cells under the same conditions. Our Laurdan GP analysis suggests that Artepillin C induces autophagy, and notably, provokes a lipid membrane packing effect, contributing to cell death. These combined results affirm the selective cytotoxicity of Artepillin C within the acidic tumor microenvironment, emphasizing its potential as an effective antitumor agent. Furthermore, our findings suggest that Artepillin C holds promise for potential applications in the realm of anticancer therapies given its pH-dependence cytotoxicity.

A pH-Sensitive Fluorescent Chemosensor Turn-On Based in a Salen Iron (III) Complex: Synthesis, Photophysical Properties, and Live-Cell Imaging Application.

pH regulation is essential to allow normal cell function, and their imbalance is associated with different pathologic situations, including cancer. In this study, we present the synthesis of 2-(((2-aminoethyl)imino)methyl)phenol (HL1) and the iron (III) complex (Fe(L1)2Br, (C1)), confirmed by X-ray diffraction analysis. The absorption and emission properties of complex C1 were assessed in the presence and absence of different physiologically relevant analytes, finding a fluorescent turn-on when OH- was added. So, we determined the limit of detection (LOD = 3.97 × 10-9 M), stoichiometry (1:1), and association constant (Kas = 5.86 × 103 M-1). Using DFT calculations, we proposed a spontaneous decomposition mechanism for C1. After characterization, complex C1 was evaluated as an intracellular pH chemosensor on the human primary gastric adenocarcinoma (AGS) and non-tumoral gastric epithelia (GES-1) cell lines, finding fluorescent signal activation in the latter when compared to AGS cells due to the lower intracellular pH of AGS cells caused by the increased metabolic rate. However, when complex C1 was used on metastatic cancer cell lines (MKN-45 and MKN-74), a fluorescent turn-on was observed in both cell lines because the intracellular lactate amount increased. Our results could provide insights about the application of complex C1 as a metabolic probe to be used in cancer cell imaging.

The Contribution of Cell Imaging to the Study of Anterior Pituitary Function and Its Regulation.

Advances in the knowledge of the neuroendocrine system are closely related to the development of cellular imaging and labeling techniques. This synergy ranges from the staining techniques that allowed the first characterizations of the anterior pituitary gland, its relationship with the hypothalamus, and the birth of neuroendocrinology; through the development of fluorescence microscopy applications, specific labeling strategies, transgenic systems, and intracellular calcium sensors that enabled the study of processes and dynamics at the cellular and tissue level; until the advent of super-resolution microscopy, miniscopes, optogenetics, fiber photometry, and other imaging methods that allowed high spatiotemporal resolution and long-term three-dimensional cellular activity recordings in living systems in a conscious and freely moving condition. In this review, we briefly summarize the main contributions of cellular imaging techniques that have allowed relevant advances in the field of neuroendocrinology and paradigm shifts that have improved our understanding of the function of the hypothalamic-pituitary axes. The development of these methods and equipment is the result of the integration of knowledge achieved by the integration of several disciplines and effort to solve scientific questions and problems of high impact on health and society that this system entails.

Assessment of Intracellular Amyloid Formation in Fixed and Live Bacteria Using Fluorescence Microscopy.

Although amyloid aggregation has been generally associated with protein misfolding and neurodegenerative diseases in mammals, bacteria and other organisms have harnessed amyloidogenesis to perform diverse biological processes. These functional amyloids, some of them secreted and others intracellular, require that the producing cells keep aggregation under control in the cytoplasm upon protein translation, preventing their inherent toxicity. Thus, it is highly relevant to understand how intracellular amyloid formation occurs and is regulated, its metabolic consequences, and the formation dynamics and fate of the amyloid inclusions upon cell division. This chapter describes methods leveraging fluorescence microscopy and fixed- or live-cell imaging to monitor intracellular amyloid formation in bacterial cells.

Targeting chalcone binding sites in living Leishmania using a reversible fluorogenic benzochalcone probe.

Chalcones (1,3-diphenyl-2-propen-1-ones) either natural or synthetic have a plethora of biological properties including antileishmanial activities, but their development as drugs is hampered by their largely unknown mechanisms of action. We demonstrate herein that our previously described benzochalcone fluorogenic probe (HAB) could be imaged by fluorescence microscopy in live Leishmania amazonensis promastigotes where it targeted the parasite acidocalcisomes, lysosomes and the mitochondrion. As in the live zebrafish model, HAB formed yellow-emitting fluorescent complexes when associated with biological targets in Leishmania. Further, we used HAB as a reversible probe to study the binding of a portfolio of diverse chalcones and analogues in live promastigotes, using a combination of competitive flow cytometry analysis and cell microscopy. This pharmacological evaluation suggested that the binding of HAB in promastigotes was representative of chalcone pharmacology in Leishmania, with certain exogenous chalcones exhibiting competitive inhibition (ca. 20-30%) towards HAB whereas non-chalconic inhibitors showed weak capacity (ca. 3-5%) to block the probe intracellular binding. However, this methodology was restricted by the strong toxicity of several competing chalcones at high concentration, in conjunction with the limited sensitivity of the HAB fluorophore. This advocates for further optimization of this undirect target detection strategy using pharmacophore-derived reversible fluorescent probes.

Tuning the photoluminescence by engineering surface states/size of S, N co-doped carbon dots for cellular imaging applications.

In this research, we have synthesized carbon dots (CDs) co-doped with nitrogen and sulfur by facile hydrothermal method, using citric acid and cysteine as carbon source. The effect of solid-state thermic treatment (STT) at 303-453 K on the size, surface, fluorescence and cellular cytotoxicity of the CDs were systematically investigated. Through a simple STT, it was possible to tune surface states and the average size of the CDs, causing a permanent red shift. Initially, CDs showed a decrease in cell viability with increasing concentration. However, after STT, its viability remained constant with an increase in concentration. Here, we show the possibility to label the cells cytoplasm according to the CDs fluorescence emission before (blue emission) and after STT (red emission). The CDs studied in this paper show selective luminescence properties, which are fundamental for any cell imaging application.

The dendritic ERGIC: Microtubule and actin cytoskeletons participate in stop-and-go movement of mobile carriers between stable structures.

The endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the ER and the Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae, located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized yet. Here, we show that long-distance ERGIC transport occurs via an intermittent mechanism in dendrites, with mobile elements moving between stationary structures. Slow and fast live-cell imaging have captured stable ERGIC structures remaining in place over long periods of time, as well as mobile ERGIC structures advancing very short distances along dendrites. These short distances have been consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Interestingly, this movement apparently depends not only on the integrity of the microtubule cytoskeleton, as previously reported, but on the actin cytoskeleton as well. Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement in which both the microtubule and the actin cytoskeletons participate.

Morphometric analysis of aerobic Eimeria bovis sporogony using live cell 3D holotomographic microscopy imaging.

M onoxenous Eimeria species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by E. bovis-infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent E. bovis sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant Eimeria species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating E. bovis oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different E. bovis sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating E. bovis oocysts accomplished aerobic sporogony in a synchronized manner. E. bovis sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2-3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian Eimeria sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree.

Fluorescent Benzothiadiazole Derivatives as Fluorescence Imaging Dyes: A Decade of New Generation Probes.

The current review describes advances in the use of fluorescent 2,1,3-benzothiadiazole (BTD) derivatives after nearly one decade since the first description of bioimaging experiments using this class of fluorogenic dyes. The review describes the use of BTD-containing fluorophores applied as, inter alia, bioprobes for imaging cell nuclei, mitochondria, lipid droplets, sensors, markers for proteins and related events, biological processes and activities, lysosomes, plasma membranes, multicellular models, and animals. A number of physicochemical and photophysical properties commonly observed for BTD fluorogenic structures are also described.

A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging.

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.

Generation and Implementation of Reporter BHK-21 Cells for Live Imaging of Flavivirus Infection.

The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirus-activatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent protein adopts a conformation promoting fluorescence. Here we provide a detailed protocol for the generation, selection and implementation of stable BHK-21 cells expressing our flavivirus genetically-encoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. We also describe the image analysis procedures and provide the required software pipelines. Our reporter cells allow the implementation of single-cell infection kinetics as well as plaque assays for both reference and native strains of flaviviruses by live-cell imaging. Graphic abstract: Workflow for the generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection.

Unraveling the molecular interactions involved in phase separation of glucocorticoid receptor.

BACKGROUND: Functional compartmentalization has emerged as an important factor modulating the kinetics and specificity of biochemical reactions in the nucleus, including those involved in transcriptional regulation. The glucocorticoid receptor (GR) is a ligand-activated transcription factor that translocates to the nucleus upon hormone stimulation and distributes between the nucleoplasm and membraneless compartments named nuclear foci. While a liquid-liquid phase separation process has been recently proposed to drive the formation of many nuclear compartments, the mechanisms governing the heterogeneous organization of GR in the nucleus and the functional relevance of foci formation remain elusive. RESULTS: We dissected some of the molecular interactions involved in the formation of GR condensates and analyzed the GR structural determinants relevant to this process. We show that GR foci present properties consistent with those expected for biomolecular condensates formed by a liquid-liquid phase separation process in living human cells. Their formation requires an initial interaction of GR with certain chromatin regions at specific locations within the nucleus. Surprisingly, the intrinsically disordered region of GR is not essential for condensate formation, in contrast to many nuclear proteins that require disordered regions to phase separate, while the ligand-binding domain seems essential for that process. We finally show that GR condensates include Mediator, a protein complex involved in transcription regulation. CONCLUSIONS: We show that GR foci have properties of liquid condensates and propose that active GR molecules interact with chromatin and recruit multivalent cofactors whose interactions with additional molecules lead to the formation of a focus. The biological relevance of the interactions occurring in GR condensates supports their involvement in transcription regulation.

Plasma membrane imaging with a fluorescent benzothiadiazole derivative.

This work describes a novel fluorescent 2,1,3-benzothiadiazole derivative designed to act as a water-soluble and selective bioprobe for plasma membrane imaging. The new compound was efficiently synthesized in a two-step procedure with good yields. The photophysical properties were evaluated and the dye proved to have an excellent photostability in several solvents. DFT calculations were found in agreement with the experimental data and helped to understand the stabilizing intramolecular charge-transfer process from the first excited state. The new fluorescent derivative could be applied as selective bioprobe in several cell lines and displayed plasma-membrane affinity during the imaging experiments for all tested models.

Deep Learning Neural Networks Highly Predict Very Early Onset of Pluripotent Stem Cell Differentiation.

Deep learning is a significant step forward for developing autonomous tasks. One of its branches, computer vision, allows image recognition with high accuracy thanks to the use of convolutional neural networks (CNNs). Our goal was to train a CNN with transmitted light microscopy images to distinguish pluripotent stem cells from early differentiating cells. We induced differentiation of mouse embryonic stem cells to epiblast-like cells and took images at several time points from the initial stimulus. We found that the networks can be trained to recognize undifferentiated cells from differentiating cells with an accuracy higher than 99%. Successful prediction started just 20 min after the onset of differentiation. Furthermore, CNNs displayed great performance in several similar pluripotent stem cell (PSC) settings, including mesoderm differentiation in human induced PSCs. Accurate cellular morphology recognition in a simple microscopic set up may have a significant impact on how cell assays are performed in the near future.

Assessing real-time signaling and agonist-induced CRHR1 internalization by optical methods.

The development of live-cell sensors for real-time measurement of signaling responses, with improved spatial and temporal resolution with respect to classical biochemical methods, has changed our understanding of cellular signaling. Examination of cAMP generation downstream activated GPCRs has shown that signaling responses can be short-lived (generated from the cell surface) or prolonged after receptor internalization. Class B secretin-like Corticotropin-releasing hormone receptor 1 (CRHR1) is a key player in stress pathophysiology. By monitoring real-time signaling in living cells, we uncovered cell context-dependent temporal characteristics of CRHR1-elicited cAMP responses and disclosed a specific link between cAMP generation and receptor signaling from internal compartments. We describe technical aspects and elaborate the protocols for cell line expression of Förster resonance energy transfer (FRET)-based biosensors to study the dynamics of cAMP and calcium signaling responses downstream activated CRHR1, live-cell imaging and analysis, and fluorescence flow cytometry to determine receptor levels at the cell surface.