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A method for high-content functional imaging of intracellular calcium responses in gelatin-immobilized non-adherent cells.
Ritter, P, Bye, LJ, Finol-Urdaneta, RK, Lesko, C, Adams, DJ, Friedrich, O, Gilbert, DF
Experimental cell research. 2020;(2):112210
Abstract
Functional imaging of the intracellular calcium concentration [Ca2+]i using fluorescent indicators is a powerful and frequently applied method for assessing various biological questions in vitro, including ion channel function and intracellular signaling in homeostasis and disease. In functional [Ca2+]i imaging experiments, the fluorescence intensity of single cells is typically recorded during application of a chemical stimulus, i.e. by exchange of modified extracellular media, exposure to drugs and/or ligands. The concomitant mechanical perturbation caused by the perfusion of different solution during experimentation severely hinders calcium imaging in non-adherent cells, including peripheral immune cells, as cells in suspension are dislocated by turbulent flow during chemical stimulation. The quantitative analysis, involving time-courses of intracellular fluorescence signal changes, necessitates cells to remain at the same position throughout the experiment. To prevent dislocation of cells during solution exchange, and to enable imaging as well as analysis of Ca2+ responses in immune cells, a gelatin-based method for immobilization of non-adherent cells was developed. Gelatin has been a long-serving material for cell immobilization, e.g. in 3D bio-printing of cells and has thus, also been employed in the context of this study. To demonstrate the applicability of the established method for functional Ca2+ imaging in gelatin-immobilized suspension cells, a proof-of-concept study was conducted using human peripheral blood model cell lines (Jurkat/T-lymphocytes and THP-1/monocytes), Ca2+ indicators (Fluo-4 and Fura-2) and two different fluorescence microscopy rigs. The data presented that the established methodology is applicable for studying Ca2+ signaling by in vitro high-content functional imaging of [Ca2+]i in suspension cells, including but not restricted to human immune cells.
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Microglial Store-operated Calcium Signaling in Health and in Alzheimer's Disease.
McLarnon, JG
Current Alzheimer research. 2020;(12):1057-1064
Abstract
The dysregulation of calcium signaling mechanisms in neurons has been considered a contributing factor to the pathogenesis evident in early-onset Alzheimer's Disease (AD). However, considerably less is known concerning the possible impairment of Ca2+ mobilization in resident immune cell microglia. This review considers findings which suggest that a prominent pathway for non-excitable microglial cells, store-operated calcium entry (SOCE), is altered in the sporadic form of AD. The patterns of Ca2+ mobilization are first discussed with platelet-activating factor (PAF) stimulation of SOCE in adult, fetal and immortalized cell-line, human microglia in the healthy brain. In all cases, PAF was found to induce a rapid transient depletion of Ca2+ from endoplasmic reticulum (ER) stores, followed by a sustained entry of Ca2+ (SOCE). A considerably attenuated duration of SOCE is observed with ATP stimulation of human microglia, suggested as due to agonist actions on differential subtype purinergic receptors. Microglia obtained from AD brain tissue, or microglia treated with full-length amyloid-β peptide (Aβ42), show significant reductions in the amplitude of SOCE relative to controls. In addition, AD brain and Aβ42-treated microglia exhibit decreased levels of Ca2+ release from ER stores compared to controls. Changes in properties of SOCE in microglia could lead to altered immune cell response and neurovascular unit dysfunction in the inflamed AD brain.
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3.
TRPM2, linking oxidative stress and Ca2+ permeation to NLRP3 inflammasome activation.
Wang, L, Negro, R, Wu, H
Current opinion in immunology. 2020;:131-135
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Abstract
The NLRP3 inflammasome is an innate immune platform that senses various pathogens and sterile insults. NLRP3 stimulation leads to activation of caspase-1, the secretion of pro-inflammatory cytokines and an inflammatory cell death called pyroptosis. Effectors of the NLRP3 inflammasome efficiently drive an immune response, not only providing protection in physiological settings but also promoting pathology when over activated. Generation of reactive oxygen species (ROS) and intracellular calcium mobilization can activate the NLRP3 inflammasome. Recent studies suggest that TRPM2 is a calcium-permeable cation channel mediating ROS-dependent NLRP3 activation. Here, we review the role of TRPM2 in NLRP3 inflammasome activation and provide an update on new functional and structural discoveries. Understanding the molecular mechanism of TRPM2 dependent NLRP3 inflammasome activation will shed lights on this complex pathway and help the developing of therapeutic strategies.
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Vitamin D in burn-injured patients.
Rech, MA, Colon Hidalgo, D, Larson, J, Zavala, S, Mosier, M
Burns : journal of the International Society for Burn Injuries. 2019;(1):32-41
Abstract
Recently, many studies have demonstrated pleotropic effects of vitamin D, including immune modulation and cardiovascular system activity. Sufficient vitamin D concentrations and supplementation of vitamin D may be of benefit in burn-injured patients. Low 25(OH)D has been observed in nearly all pediatric and most adult burn patients. Vitamin D has primarily been studied in pediatric burn patients, focusing on bone marker measurements and the incidence of fractures. The preferred vitamin D dose, formulation, and route of administration remain unknown, and there is limited data on the impact of vitamin D status on clinical outcomes. Further research should focus on determining optimal monitoring strategies, supplementation regimens and clinical outcomes like mortality, length of stay and incidence of sepsis.
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Calcium Signalling in Plant Biotic Interactions.
Aldon, D, Mbengue, M, Mazars, C, Galaud, JP
International journal of molecular sciences. 2018;(3)
Abstract
Calcium (Ca2+) is a universal second messenger involved in various cellular processes, leading to plant development and to biotic and abiotic stress responses. Intracellular variation in free Ca2+ concentration is among the earliest events following the plant perception of environmental change. These Ca2+ variations differ in their spatio-temporal properties according to the nature, strength and duration of the stimulus. However, their conversion into biological responses requires Ca2+ sensors for decoding and relaying. The occurrence in plants of calmodulin (CaM) but also of other sets of plant-specific Ca2+ sensors such as calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs) and calcineurin B-like proteins (CBLs) indicate that plants possess specific tools and machineries to convert Ca2+ signals into appropriate responses. Here, we focus on recent progress made in monitoring the generation of Ca2+ signals at the whole plant or cell level and their long distance propagation during biotic interactions. The contribution of CaM/CMLs and CDPKs in plant immune responses mounted against bacteria, fungi, viruses and insects are also presented.
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6.
Single-Cell Ca2+ Imaging.
Liu, S
Methods in molecular biology (Clifton, N.J.). 2018;:161-168
Abstract
In rheumatological studies, visualization of Ca2+ dynamics in intact cells as direct experimental evidence of Ca2+-dependent signal pathways is generally used to monitor the function of immune cells at first glance. Ability to monitor Ca2+ signaling in living cells would greatly facilitate advances in the functional dissection of immune cells. In this chapter, we describe a basic technique and methods of data analysis for single-cell real-time Ca2+ monitoring using Fluo-4 labeling, which is a single-wavelength Ca2+ indicator.
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The Biological Activities of Vitamin D and Its Receptor in Relation to Calcium and Bone Homeostasis, Cancer, Immune and Cardiovascular Systems, Skin Biology, and Oral Health.
Khammissa, RAG, Fourie, J, Motswaledi, MH, Ballyram, R, Lemmer, J, Feller, L
BioMed research international. 2018;:9276380
Abstract
Vitamin D plays an important role in calcium homeostasis and bone metabolism, with the capacity to modulate innate and adaptive immune function, cardiovascular function, and proliferation and differentiation of both normal and malignant keratinocytes. 1,25(OH)2D, the biologically active form of vitamin D, exerts most of its functions through the almost universally distributed nuclear vitamin D receptor (VDR). Upon stimulation by 1,25(OH)2D, VDR forms a heterodimer with the retinoid X receptor (RXR). In turn, VDR/RXR binds to DNA sequences termed vitamin D response elements in target genes, regulating gene transcription. In order to exert its biological effects, VDR signalling interacts with other intracellular signalling pathways. In some cases 1,25(OH)2D exerts its biological effects without regulating either gene expression or protein synthesis. Although the regulatory role of vitamin D in many biological processes is well documented, there is not enough evidence to support the therapeutic use of vitamin D supplementation in the prevention or treatment of infectious, immunoinflammatory, or hyperproliferative disorders. In this review we highlight the effects of 1,25(OH)2D on bone and calcium homeostasis, on cancer, and refer to its effects on the cardiovascular and immune systems.
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Mechanistic Understanding of Single-Cell Behavior is Essential for Transformative Advances in Biomedicine.
Francis, EA, Heinrich, V
The Yale journal of biology and medicine. 2018;(3):279-289
Abstract
Most current efforts to advance medical technology proceed along one of two tracks. The first is dedicated to the improvement of clinical tasks through the incremental refinement of medical instruments. The second comprises engineering endeavors to support basic science studies that often only remotely relate to human medicine. Here we survey emerging research approaches that aim to populate the sprawling frontier between these tracks. We focus on interdisciplinary single-live-cell techniques that have overcome limitations of traditional biological methods to successfully address vital questions about medically relevant cellular behavior. Most of the presented case studies are based on the controlled manipulation of nonadherent human immune cells using one or more micropipettes. The included studies have (i) examined one-on-one encounters of immune cells with real or model pathogens, (ii) assessed the physiological role of the expandable surface area of immune cells, and (iii) started to dissect the spatiotemporal organization of signaling processes within these cells. The unique aptitude of such single-live-cell studies to fill conspicuous gaps in our quantitative understanding of medically relevant cause-effect relationships provides a sound basis for new insights that will inform and drive future biomedical innovation.
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9.
Calcium signatures and signaling events orchestrate plant-microbe interactions.
Yuan, P, Jauregui, E, Du, L, Tanaka, K, Poovaiah, BW
Current opinion in plant biology. 2017;:173-183
Abstract
Calcium (Ca2+) acts as an essential second messenger connecting the perception of microbe signals to the establishment of appropriate immune and symbiotic responses in plants. Accumulating evidence suggests that plants distinguish different microorganisms through plasma membrane-localized pattern recognition receptors. The particular recognition events are encoded into Ca2+ signatures, which are sensed by diverse intracellular Ca2+ binding proteins. The Ca2+ signatures are eventually decoded to distinct downstream responses through transcriptional reprogramming of the defense or symbiosis-related genes. Recent observations further reveal that Ca2+-mediated signaling is also involved in negative regulation of plant immunity. This review is intended as an overview of Ca2+ signaling during immunity and symbiosis, including Ca2+ responses in the nucleus and cytosol.
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10.
Calcium in the pathomechanism of amyotrophic lateral sclerosis - Taking center stage?
Patai, R, Nógrádi, B, Engelhardt, JI, Siklós, L
Biochemical and biophysical research communications. 2017;(4):1031-1039
Abstract
Amyotrophic lateral sclerosis is an incurable, relentlessly progressive disease primarily affecting motor neurons. The cause of the disease, except for the mutations identified in a small fraction of patients, is unknown. The major mechanisms contributing to the degeneration of motor neurons have already been disclosed and characterized, including excitotoxicity, oxidative stress, mitochondrial dysfunction, and immune/inflammatory processes. During the progression of the disease these toxic processes are not discrete, but each facilitates the deleterious effect of the other. However, due to their common reciprocal calcium dependence, calcium ions may act as a common denominator and through a positive feedback loop may combine the individual pathological processes into a unified escalating mechanism of neuronal destruction. This mini-review provides an overview of the mutual calcium dependence of the major toxic mechanisms associated with amyotrophic lateral sclerosis.