Glycated walnut meal peptide­calcium chelates (COS-MMGGED-Ca): Preparation, characterization, and calcium absorption-promoting.

This study isolated a novel peptide MMGGED with strong calcium-binding capacity from defatted walnut meal and synthesized a novel peptide­calcium chelate COS-MMGGED-Ca with high stability via glycation. Structural characterization and computer simulation identified binding sites, while in vitro digestion stability and calcium transport experiments explored the chelate's properties. Results showed that after glycation, COS-MMGGED bound Ca2+ with 88.75 ± 1.75 %, mainly via aspartic and glutamic acids. COS-MMGGED-Ca released Ca2+ steadily (60.27 %), with thermal denaturation temperature increased by 18 °C and 37 °C compared to MMGGED-Ca, indicating good processing performance. Furthermore, COS-MMGGED significantly enhanced Ca2+ transport across Caco-2 monolayers, 1.13-fold and 1.62-fold higher than CaCl2 and MMGGED, respectively, at 240 h. These findings prove glycation enhances structural properties, stability, calcium loading, and transport of peptide­calcium chelates, providing a scientific basis for developing novel efficient calcium supplements and high-value utilization of walnut meal.

Measuring Calcium Signaling at the Primary Cilia.

Cellular signaling is nature's ingenious way for cells to perceive their surroundings and transmit external cues to internal compartments. Due to its critical role in cellular functions, the intricate machinery of molecular signaling has been intensively studied. A diverse arsenal of techniques exists to quantify the molecules involved in these processes. Among them, calcium stands out as a ubiquitous signaling molecule with roles in countless biological pathways. To elucidate its function as a second messenger, methods for measuring intracellular calcium have steadily evolved. This chapter introduces various methods for investigating calcium signaling cascades in cells as well as in cilia (thin hairlike projections) specifically, where calcium signaling is triggered by different cilial manipulation techniques.

Photothermal switch by gallic acid-calcium grafts synthesized by coordination chemistry for sequential treatment of bone tumor and regeneration.

The residual bone tumor and defects which is caused by surgical therapy of bone tumor is a major and important problem in clinicals. And the sequential treatment for irradiating residual tumor and repairing bone defects has wildly prospects. In this study, we developed a general modification strategy by gallic acid (GA)-assisted coordination chemistry to prepare black calcium-based materials, which combines the sequential photothermal therapy of bone tumor and bone defects. The GA modification endows the materials remarkable photothermal properties. Under the near-infrared (NIR) irradiation with different power densities, the black GA-modified bone matrix (GBM) did not merely display an excellent performance in eliminating bone tumor with high temperature, but showed a facile effect of the mild-heat stimulation to accelerate bone regeneration. GBM can efficiently regulate the microenvironments of bone regeneration in a spatial-temporal manner, including inflammation/immune response, vascularization and osteogenic differentiation. Meanwhile, the integrin/PI3K/Akt signaling pathway of bone marrow mesenchymal stem cells (BMSCs) was revealed to be involved in the effect of osteogenesis induced by the mild-heat stimulation. The outcome of this study not only provides a serial of new multifunctional biomaterials, but also demonstrates a general strategy for designing novel blacked calcium-based biomaterials with great potential for clinical use.

Interaction of Ca2+ and Fe3+ in co-precipitation process induced by Virgibacillus dokdonensis and its application.

Biomineralization has garnered significant attention in the field of wastewater treatment due to its notable cost reduction compared to conventional methods. The reinjection water from oilfields containing an exceedingly high concentration of calcium and ferric ions will pose a major hazard in production. However, the utilization of biomineralization for precipitating these ions has been scarcely investigated due to limited tolerance among halophiles towards such extreme conditions. In this study, free and immobilized halophiles Virgibacillus dokdonensis were used to precipitate these ions and the effects were compared, at the same time, biomineralization mechanisms and mineral characteristics were further explored. The results show that bacterial concentration and carbonic anhydrase activity were higher when additionally adding ferric ion based on calcium ion; the content of protein, polysaccharides, deoxyribonucleic acid and humic substances in the extracellular polymers also increased compared to control. Calcium ions were biomineralized into calcite and vaterite with multiple morphology. Due to iron doping, the crystallinity and thermal stability of calcium carbonate decreased, the content of OC = O, NC = O and CO-PO3 increased, the stable carbon isotope values became much more negative, and ß-sheet in minerals disappeared. Higher calcium concentrations facilitated ferric ion precipitation, while ferric ions hindered calcium precipitation. The immobilized bacteria performed better in ferric ion removal, with a precipitation ratio exceeding 90%. Free bacteria performed better in calcium removal, and the precipitation ratio reached a maximum of 56%. This research maybe provides some reference for the co-removal of calcium and ferric ions from the oilfield wastewater.

Monitoring Calcium-Sensing Receptor (CaSR)-Induced Intracellular Calcium Flux Using an Indo-1 Flow Cytometry Assay.

The calcium-sensing receptor (CaSR) has a critical role in maintaining serum calcium concentrations within the normal physiological range, and mutations in the receptor, or components of its signaling and trafficking pathway, cause disorders of calcium homeostasis. Inactivating mutations cause neonatal severe hyperparathyroidism or familial hypocalciuric hypercalcemia (FHH), while gain-of-function mutations cause autosomal dominant hypocalcemia (ADH). Characterizing the functional impact of mutations of the CaSR, and components of the CaSR-signaling pathway, is clinically important to enable correct diagnoses of FHH and ADH, optimize management, and prevent inappropriate parathyroidectomy or vitamin D supplementation. CaSR signals predominantly by activating the G-alpha subunit-11 to mobilize calcium release from intracellular stores. Thus, measurement of CaSR-induced intracellular calcium (Ca2+i) signaling is the gold standard method to investigate the pathogenicity of CaSR genetic variants. This protocol describes a method to assess CaSR-induced Ca2+I signaling using the Indo-1 calcium indicator dye and flow cytometry. This method has been used to assess multiple genetic variants in CaSR and components of its signaling and trafficking pathway in HEK293 cells.

Measuring Calcium-Sensing Receptor Agonist-Driven Insertional Signaling (ADIS) and Trafficking by TIRF Microscopy.

The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor. Deciphering these mechanisms is important to understand how mutations in the CaSR and components of its signaling and trafficking pathways cause human disorders of calcium homeostasis.This chapter describes a protocol to measure CaSR ADIS and endocytosis in parallel using total internal reflection fluorescence (TIRF) microscopy. This utilizes a mammalian expression construct comprising the full-length human CaSR with an N-terminal bungarotoxin minimal-binding site that can be labeled with commercially available fluorescent ligands to measure endocytosis, and a super-ecliptic pHluorin (SEP) to measure total cell surface expression and exocytic events. This protocol could easily be adapted to simultaneously assess forward trafficking and endocytosis of other membrane proteins by TIRF microscopy.

Quantifying Gq Signaling Using the IP1 Homogenous Time-Resolved Fluorescence (HTRF) Assay.

G protein-coupled receptors (GPCRs) play pivotal roles in cellular signaling and can regulate several cellular functions such as proliferation, secretion, protein expression, and cellular metabolism. Coupling of GPCRs to members of the Gq/11 protein family results in activation of inositol trisphosphate (IP3) and accumulation of calcium intracellularly. This protocol chapter outlines a step-by-step guide for utilizing the inositol phosphate-1 (IP1) accumulation assay, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, to investigate Gq-IP3 signaling. The assay serves as a valuable tool for those conducting pharmacological investigations and compound screening targeting this critical cellular pathway. This protocol chapter covers experimental setup, sample preparation, and data analysis, providing researchers with an in-depth guide to explore the pharmacology of Gq-coupled receptors.

Monitoring ER Ca2+ by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).

The endoplasmic reticulum (ER) is the main cellular reservoir of Ca2+, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca2+ dynamics within the ER lumen is best achieved using genetically encoded and targeted reporters. Luminescent probes based on the photoprotein aequorin have provided significant insight to measure subcellular Ca2+. Here we describe a robust and quantitative method based on the Ca2+ indicator of the GFP-Aequorin Protein (GAP) family, targeted to the ER lumen. A low Ca2+ affinity version of GAP, GAP1, carrying mutations in two EF-hands of aequorin, reconstituted with coelenterazine n has a reduced affinity for Ca2+ such that it conforms with the [Ca2+] values found in the ER and it slows the consumption of the probe by Ca2+. This feature is advantageous because it avoids fast aequorin consumption allowing long-term (longer than 1 h) ER Ca2+ measurements. GAP1 targeted to the ER allows monitoring of resting [Ca2+]ER and Ca2+ dynamics in intact cells stimulated with IP3-produced agonists. In addition, GAP1 can record Ca2+ mobilization in permeabilized cells challenged with IP3. We also provide a detailed calibration procedure which allows to accurately convert the luminescence signal into [Ca2+]ER.

Measuring GPCR-Induced Intracellular Calcium Signaling Using a Quantitative High-Throughput Assay.

Alterations in intracellular calcium are integral to signal transduction pathways for many G-protein-coupled receptors, but this signaling is not well studied. This is mostly due to a lack of reliable, robust, high-throughput, quantitative methods to monitor intracellular calcium concentrations in live cells. Recently, we developed a reliable, robust, quantitative method to measure intracellular calcium levels in which HEK293 cell suspensions loaded with Fura-2/AM are placed in 96-well plates. Minimum and maximum intracellular calcium levels, which are required for converting fluorescence into calcium concentrations, are calibrated using EGTA to chelate calcium and ionomycin to load calcium into cells, respectively. Fluorescence is monitored with a PHERAstar FS plate reader. We provide a detailed method for this high-throughput assay that can be used to quantitate intracellular calcium in endogenous and exogenously (stable or transient) expressed GPCRs in HEK293 cells.

Analysis of Calcium-Sensing Receptor Signaling Using Dual Luciferase Assays.

Luciferases catalyze a reaction that involves the emission of light, a phenomenon referred to as "bioluminescence". The calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), induces characteristic signaling pathways that stimulate extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ mobilization from the endoplasmic reticulum. ERK1/2 causes an activation of the serum response element (SRE), whereas Ca2+ causes an activation of the nuclear factor of activated T-cells response element (NFAT-RE). Transfection of cells with a vector containing a firefly luciferase reporter gene under the control of the SRE or NFAT-RE allows the monitoring of ERK1/2 activation and Ca2+ mobilization, respectively, by measuring luminescence. In a dual luciferase assay, firefly luminescence is normalized by co-transfecting an internal control vector, which contains a constitutively active promoter driving the expression of a second luciferase, namely, Renilla luciferase, whose activity can be quantified within the same sample. Here, a protocol for the analysis of CaSR signaling using dual luciferase assays in HEK293 cells is provided. The assays can, for example, be used to investigate functional consequences of mutations in the CaSR gene.

Calcium Imaging in Brain Tissue Slices.

Calcium imaging is a method that was first developed in the mid-1970s yet kept developing until current days to allow accurate measurement of free calcium ions in tissues. This widely used method has provided significant advances to our understanding of cellular signal transduction, including the discovery of subcellular compartmentalization of neurons and astrocytes, the identification of multiple signaling pathways, and mapping the functional connectivity between astrocytes and neuronal networks. Here we describe a method for the loading and imaging of cell-permeable AM ester calcium-sensitive dyes for the in vitro measurement of free intracellular Ca2+ ions in acute brain slices.

Analyzing Mitochondrial Calcium Influx in Isolated Mitochondria.

Mitochondria play a crucial role in Ca2+ signaling and homeostasis and can contribute to shaping the cytosolic Ca2+ landscape as well as regulate a variety of pathways including energy production and cell death. Dysregulation of mitochondrial Ca2+ homeostasis promotes pathologies including neurodegenerative diseases, cardiovascular disorders, and metabolic syndromes. The significance of mitochondria to Ca2+ signaling and regulation underscores the value of methods to assess mitochondrial Ca2+ import. Here we present a plate reader-based method using the Ca2+-sensitive fluorescent probe calcium green-5 N to measure mitochondrial Ca2+ import in isolated cardiac mitochondria. This technique can be expanded to measure Ca2+ uptake in mitochondria isolated from other tissue types and from cultured cells.

Measuring Calcium Levels in Bone-Resorbing Osteoclasts and Bone-Forming Osteoblasts.

Bone remodeling is a crucial, dynamic process that renews bone and maintains mineral homeostasis. It consists of several steps, including osteoclastic bone resorption and osteoblastic bone formation and mineralization. Intracellular calcium signaling is essential for osteoclast and osteoblast differentiation and activity. Here, we describe the differentiation of human osteoclasts and osteoblasts in vitro and provide common methods to determine cell differentiation and activity. We then describe protocols for measuring intracellular calcium in these cells using Fura2-AM.

Two-Photon Microscopy to Measure Calcium Signaling in the Living Brain.

Two-photon microscopy enables imaging of calcium signaling at cellular or subcellular resolution up to hundreds of microns deep in the living brain. Changes in the brightness of fluorescent calcium indicators provide a readout of calcium levels over time, affording information about neuronal activity and/or calcium-dependent subcellular signaling. Here, we describe a protocol for repeated two-photon imaging of calcium signals in mice expressing a genetically encoded calcium indicator that have been implanted with a chronic cranial window.

Methods for Imaging Intracellular Calcium Signals in the Mouse Mammary Epithelium in Two and Three Dimensions.

The mammary gland has a central role in optimal mammalian development and survival. Contractions of smooth muscle-like basal (or myoepithelial) cells in the functionally mature mammary gland in response to oxytocin are essential for milk ejection and are tightly regulated by intracellular calcium (Ca2+). Using mice expressing a genetically encoded Ca2+ indicator (GCaMP6f), we present in this chapter a method to visualize at high spatiotemporal resolution changes in intracellular Ca2+ in mammary epithelial cells, both in vitro (2D) and ex vivo (3D). The procedure to optimally prepare mammary tissue and primary cells is presented in detail.

Live-Cell Calcium Imaging in 3D Intestinal Organoids.

Live-cell Ca2+ imaging is an important tool to detect activation of receptors by a putative ligand/drug and complements studies on transport processes, as intracellular Ca2+ changes provide direct evidence for substrate fluxes. Organoid-based systems offer numerous advantages over other in vitro systems such as cell lines, primary cells, or tissue explants, and in particular, intestinal organoid culture has revolutionized research on functional gastrointestinal processes. Calcium imaging using the fluorescent Ca2+ indicator Fura-2-AM can be applied to 3D intestinal organoids, which show an excellent dye-loading efficiency. Here we describe live-cell Ca2+ imaging in intestinal organoids, an important technique to improve research on malabsorption syndromes, secretory diarrhea, and metabolic disorders.

Quantitative Intravital Calcium Imaging in Mouse Kidney.

Intracellular calcium is an important regulator of solute transport in renal epithelial cells, and disordered calcium signaling may underlie the pathogenesis of certain kidney diseases. Intravital multiphoton imaging of the kidney in transgenic mice expressing highly sensitive fluorescent reporters allows detailed study of calcium signals within different specialized segments of the renal tubule and how these are integrated with other cellular processes. Moreover, changes in activity can be observed in real time in response to physiological interventions or disease-causing insults. In this chapter, we will provide a detailed protocol for performing this powerful research technique.

Real-Time Imaging of Calcium Dynamics in Human Sperm After Precise Single-Cell Stimulation.

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.

Computational Analysis of Calcium Flux Data Using R.

Calcium imaging has emerged as a powerful tool for studying cellular dynamics, with applications spanning neuroscience, cell biology, and beyond. In this chapter, we present a comprehensive guide to the computational analysis of calcium flux data using the R programming language. Using an example of in vivo live imaging of GCaMP signal in zebrafish hepatocytes, we demonstrate techniques for segmentation, normalization, and quantification of calcium transients. We provide a step-by-step code example showcasing extraction of meaningful information from calcium imaging datasets. The code allows insights into the number of oscillating cells, number of oscillations per cell within a time frame, and generation of publication-ready plots for showcasing calcium dynamics. This chapter serves as a valuable resource for researchers seeking to leverage freely available computational tools for analyzing calcium flux data at cellular resolution and uncovering novel insights into cellular physiology.

Calcium Imaging and Analysis in Beta Cells in Acute Mouse Pancreas Tissue Slices.

Ca2+ ions play a central role in the stimulus-secretion coupling cascade of pancreatic beta cells. The use of confocal microscopy in conjunction with the acute pancreas tissue slice technique offers valuable insights into changes in the intracellular calcium concentration following stimulation by secretagogues. This allows the study of beta cells on a single cell level, as well as their behavior on a multicellular scale within an intact environment. With the use of advanced analytical tools, this approach offers insight into how single cells contribute to the functional unit of islets of Langerhans and processes underlying insulin secretion. Here we describe a comprehensive protocol for the preparation and utilization of acute pancreas tissue slices in mice, the use of high-resolution confocal microscopy for observation of glucose-stimulated calcium dynamics in beta cells, and the computational analysis for objective evaluation of calcium signals.