Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino ...acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.
Vaccination protects against COVID-19 via the spike protein receptor-binding domain (RBD)-specific antibody formation, but it also affects the innate immunity. The effects of specific antibody ...induction on neutrophils that can cause severe respiratory inflammation are important, though not completely investigated. In the present study, using a mouse model mimicking SARS-CoV-2 virus particle inhalation, we investigated neutrophil phenotype and activity alterations in the presence of RBD-specific antibodies. Mice were immunized with RBD and a week after a strong antibody response establishment received 100 nm particles in the RBD solution. Control mice received injections of a phosphate buffer instead of RBD. We show that the application of 100 nm particles in the RBD solution elevates neutrophil recruitment to the blood and the airways of RBD-immunized mice rather than in control mice. Analysis of bone marrow cells of mice with induced RBD-specific antibodies revealed the increased population of CXCR2+CD101+ neutrophils. These neutrophils did not demonstrate an enhanced ability of neutrophil extracellular traps (NETs) formation compared to the neutrophils from control mice. Thus, the induction of RBD-specific antibodies stimulates the activation of mature neutrophils that react to RBD-coated particles without triggering excessive inflammation.
Microbial rhodopsin (MRs) ion channels and pumps have become invaluable optogenetic tools for neuroscience as well as biomedical applications. Recently, MR-optogenetics expanded towards subcellular ...organelles opening principally new opportunities in optogenetic control of intracellular metabolism and signaling
precise manipulations of organelle ion gradients using light. This new optogenetic field expands the opportunities for basic and medical studies of cancer, cardiovascular, and metabolic disorders, providing more detailed and accurate control of cell physiology. This review summarizes recent advances in studies of the cellular metabolic processes and signaling mediated by optogenetic tools targeting mitochondria, endoplasmic reticulum (ER), lysosomes, and synaptic vesicles. Finally, we discuss perspectives of such an optogenetic approach in both fundamental and applied research.
This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, ...drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins—drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed.
People are constantly exposed to airborne fungal spores, including
conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, ...immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense
conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the
conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII
antigen-presenting cells, including IE-DCs, and CD11b
phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of
conidia increased the number of CD11b
phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b
phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b
phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b
phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b
phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.
Susceptibility to fungal infection is commonly associated with impaired neutrophil responses. To study the mechanisms underlying this association, we investigated neutrophil recruitment to the ...conducting airway wall after Aspergillus fumigatus conidium inhalation in mouse models of drug-induced immunosuppression and antibody-mediated neutrophil depletion (neutropenia) by performing three-dimensional confocal laser-scanning microscopy of whole-mount primary bronchus specimens. Actin staining enabled visualization of the epithelial and smooth muscle layers that mark the airway wall. Gr-1+ or Ly6G+ neutrophils located between the epithelium and smooth muscles were considered airway wall neutrophils. The number of airway wall neutrophils for immunocompetent, immunosuppressed, and neutropenic mice before and 6 h after A. fumigatus infection were analyzed and compared. Our results show that the number of conducting airway wall neutrophils in immunocompetent mice significantly increased upon inflammation, while a dramatic reduction in this number was observed following immunosuppression and neutropenia. Interestingly, a slight increase in the infiltration of neutrophils into the airway wall was detected as a result of infection, even in immunosuppressed and neutropenic mice. Taken together, these data indicate that neutrophils are present in intact conducting airway walls and the number elevates upon A. fumigatus infection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.
Mirror proteorhodopsins Okhrimenko, Ivan S; Kovalev, Kirill; Petrovskaya, Lada E ...
Communications chemistry,
05/2023, Letnik:
6, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining ...mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name "mirror proteorhodopsins", from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest.
BACKGROUND: Airborne pathogens such as virus particles undergo elimination from the respiratory tract by mucociliary clearance and phagocytosis by immune cells. The data about phagocytic cell type ...infiltration and stimuli that attract phagocytic cells to conducting airway are required for the anti-virus immune response mechanism understanding and the treatment strategy development.
AIM: To detect the role of the receptor-binding domain of SARS-CoV-2 in neutrophil immune response activation in conducting airway mucosa after 100 nm particles application.
MATERIALS AND METHODS: C57BL/6 mice received an oropharyngeal application of fluorescent 100 nm particles suspended in the receptor-binding domain of SARS-CoV-2 solution. 24 hours after, conducting airways of mice were dissected and subjected for immunohistochemistry as whole-mounts. Three-dimensional images of conducting airway regions were obtained using confocal microscopy. Quantitative image analysis was performed to estimate the ingestion activity of neutrophils in conducting airway mucosa.
RESULTS: Neutrophil migration to conducting airway mucosa was detected in case of the application of particles in receptor-binding domain solution, but not in phosphate buffer or bovine serum albumin solution. Receptor-binding domain solution alone also induced neutrophil migration to conducting airway mucosa. Infiltrating conducting airway wall mucosa neutrophils contributed to particles internalization.
CONCLUSIONS: The receptor-binding domain of SARS-CoV-2 can activate the neutrophil-mediated response in conducting airway mucosa.
BACKGROUND: Daily, people inhale airborne viral particles, some of which have a size of about 100 nm, such as particles of SARS-CoV-2. Kinetics of such 100 nm particle distribution in the respiratory ...tract is important, however, not a properly investigated question.
AIM: To estimate the dissemination of inert viral particles based on the analysis of the spatial distribution of fluorescent 100 nm particles in the mouse lungs at different time points after the application.
MATHERIALS AND METHODS: Fluorescent particles of 100 nm size were applied to C57BL/6 mice. 6, 24, 48 and 72 hours after, lungs were excised and fixed. Lung lobes were stained with immunohistochemistry as whole-mounts and then underwent optical clearance. Three-dimensional images of whole-mount mouse lung lobes were acquired using confocal laser scanning microscopy.
RESULTS: 6 hours after the particle application particles were detected in lungs both as single particles and as particle agglomerates. Particles were both free and internalized by phagocytic cells. 24 hours after the application particles were detected both in bronchial lumen and in the alveolar space. Particles were detected in the mouse lungs up to 72 hours after the application.
CONCLUSIONS: Reaching the respiratory tract of mammalian, inert particles which size equal to SARS-CoV-2 particle size distribute both in bronchi and in alveoli and undergoes internalization of phagocytic cells.
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