BACKGROUND: To perform a detailed morphological analysis of the inorganic portion of two different clinical presentations of calcium-based deposits retrieved from subjects with SSc and identify a chemical dissolution of these deposits suitable for clinical use.
METHODS: Chemical analysis using Fourier Transform IR spectroscopy (\'FTIR\'), Raman microscopy, Powder X-Ray Diffraction (\'PXRD\'), and Transmission Electron Microscopy (\'TEM\') was undertaken of two distinct types of calcinosis deposits: paste and stone. Calcinosis sample titration with ethylenediaminetetraacetic acid (\'EDTA\') assessed the concentration at which the EDTA dissolved the calcinosis deposits in vitro.
RESULTS: FTIR spectra of the samples displayed peaks characteristic of hydroxyapatite, where signals attributable to the phosphate and carbonate ions were all identified. Polymorph characterization using Raman spectra were identical to a hydroxyapatite reference while the PXRD and electron diffraction patterns conclusively identified the mineral present as hydroxyapatite. TEM analysis showed differences of morphology between the samples. Rounded particles from stone samples were up to a few micron in size, while needle-like crystals from paste samples reached up to 0.5 µm in length. Calcium phosphate deposits were effectively dissolved with 3% aqueous solutions of EDTA, in vitro. Complete dissolution of both types of deposit was achieved in approximately 30 min using a molar ratio of EDTA/HAp of ≈ 300.
CONCLUSIONS: Stone and paste calcium-based deposits both comprise hydroxyapatite, but the constituent crystals vary in size and morphology. Hydroxyapatite is the only crystalline polymorph present in the SSc-related calcinosis deposits. Hydroxyapatite can be dissolved in vitro using a dosage of EDTA considered safe for clinical application. Further research is required to establish the optimal medium to develop the medical product, determine the protocol for clinical application, and to assess the effectiveness of EDTA for local treatment of dystrophic calcinosis.

This study investigated the characteristics of Lactobacillus helveticus-derived whey-calcium chelate (LHWCC) and its effect on the calcium absorption and bone health of rats. Fourier-transform infrared spectroscopy showed that carboxyl oxygen atoms, amino nitrogen atoms, and phosphate ions were the major binding sites with calcium in LHWCC, which has a sustained release effect in simulated in vitro digestion. LHWCC had beneficial effects on serum biochemical parameters, bone biomechanics, and the morphological indexes of the bones of calcium-deficient rats when fed at a dose of 40 mg Ca/kg BW for 7 weeks. In contrast to the inorganic calcium supplement, LHWCC significantly upregulated the gene expression of transient receptor potential cation V5 (TRPV5), TRPV6, PepT1, calcium-binding protein-D9k (Calbindin-D9k), and a calcium pump (plasma membrane Ca-ATPase, PMCA1b), leading to promotion of the calcium absorption rate, whereas Ca3(PO4)2 only upregulated the TRPV6 channel in vivo. These findings illustrate the potential of LHWCC as an organic calcium supplement.

Oncology treatments cause infertility, and ovarian tissue cryopreservation and transplantation (OTCT) is the only option for fertility preservation in prepubertal girls with cancer. However, OTCT is associated with massive follicle loss. Here, we aimed to determine the effect of supplementation of slow freezing and vitrification media with BAPTA-AM and melatonin alone and in combination on ovarian tissue viability, reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), and follicular morphology and viability. Our results indicated that BAPTA-AM and melatonin can significantly improve ovarian tissue viability and the TAC/ROS ratio and reduce ROS generation in frozen-thawed ovarian tissues in slow freezing and vitrification procedures. BAPTA-AM was also found to be less effective on TAC compared to melatonin in vitrified ovarian tissue. While supplementation of slow freezing and vitrification media with BAPTA-AM and/or melatonin could increase the percentage of morphologically intact follicles in cryopreserved ovarian tissues, the differences were not significant. In conclusion, supplementation of cryopreservation media with BAPTA-AM or melatonin improved the outcome of ovarian tissue cryopreservation in both vitrification and slow freezing methods. Our data provide some insight into the importance of modulating redox balance and intracellular Ca2+ levels during ovarian tissue cryopreservation to optimize the current cryopreservation methods.

The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly cryo-tolerant. Nevertheless, with regard to poultry semen, even brief in vitro storage periods can lead to a notable decline in fertility, despite the in vivo capacity to maintain fertility for several weeks when within the hen\'s sperm storage tubules. For fertility in sperm, intracellular calcium ions ([Ca2+]i) play a key role in signaling towards modifying energy metabolism. While reducing [Ca2+]i has been found to enhance the preservation of sperm fertility in some mammals, the connection between semen fertility and calcium availability in avian sperm has received limited attention. In this study, we demonstrate that the use of extracellular and intracellular calcium chelators in liquid semen extenders, specifically EGTA and EGTA-AM, has distinct effects on prolonging the fertility of chicken sperm. These results were validated through in vivo fertility tests. Mechanistically, the effects observed were linked to coordination of mitochondrial metabolism and ATP catabolism. Despite both calcium chelators inducing hypoxia, they differentially regulated mitochondrial respiration and ATP accumulation. This regulation was closely linked to a bimodal control of dynein ATPase activity; a direct initial activation with reduction in [Ca2+]i, and subsequent suppression by cytoplasmic acidification caused by lactic acid. These findings not only contribute to advancing poultry liquid semen preservation techniques, but also elucidates biologically relevant mechanisms that may underlie storage within the female reproductive tract in birds.

Polar body emission is a special form of cytokinesis in oocyte meiosis that ensures the correct number of chromosomes in reproduction-competent eggs. The molecular mechanism of the last step, polar body abscission, is poorly understood. While it has been proposed that Ca2+ signaling plays important roles in embryonic cytokinesis, to date transient increases in intracellular free Ca2+ have been difficult to document in oocyte meiosis except for the global Ca2+ wave induced by sperm at fertilization. Here, we find that microinjection of the calcium chelator dibromo-BAPTA inhibits polar body abscission in Xenopus laevis oocytes. Using a novel, microtubule-targeted ratio-metric calcium sensor, we detected a calcium transient that is focused at the contractile ring-associated plasma membrane and which occurred after anaphase and constriction of the contractile ring but prior to abscission. This calcium transient was confirmed by mobile calcium probes. Further, the Ca2+-sensitive protein kinase Cβ C2 domain transiently translocated to the contractile ring-associated membrane simultaneously with the calcium transient. Collectively, these results demonstrate that a calcium transient, apparently originating at the contractile ring-associated plasma membrane, promotes polar body abscission.

Neuroactive amino acids derivatised at their carboxylate groups with a photolabile nitroindolinyl group are highly effective reagents for the sub-µs release of neuroactive amino acids in physiological solutions. However, the same does not apply in the case of calcium ion chelators. In this study, nitroindolinyl-caged BAPTA is found to be completely photostable, whereas nitroindolinyl-caged EDTA photolyses only when saturated with calcium ions.

To identify whether root canal irrigants with calcium chelation ability play a role in the removal of calcium hydroxide (CH) from the root canals when compared to non-chelators.
The protocol is registered in the Open Science Framework registry (doi 10.17605/OSF.IO/CHG2Q). PubMed, Scopus, Embase, Cochrane Library, ProQuest, Google Scholar, Science direct and open grey databases were searched until March 2021. Laboratory studies comparing the effectiveness of calcium chelators in the removal of CH with non-chelators delivered using needle irrigation, irrigation agitation or instrumentation techniques were included. The quality of included studies was appraised using a modified Joanna Briggs Institute critical appraisal checklist for a randomised clinical trial. Two independent reviewers were involved in study selection, data extraction, appraising the quality of studies. Any disagreements were resolved by a third reviewer.
The current review included 17 studies, with 16 being of \"moderate\" quality and one of \"low\" quality. Due to methodological differences within the included studies, quantitative analysis was not performed. Laboratory studies were only included in the current review because no clinical study exists on this topic. Evidence from the review indicates that calcium chelators are superior to non-chelators in the removal of CH when used with needle irrigation, passive ultrasonic irrigation and instrumentation techniques.
Calcium chelators are superior in the removal of CH from the root canal system over non-chelators.

OBJECTIVE: To compare 3 methods for creating ethylenediaminetetraacetic acid (EDTA) solution using readily available Vacutainer tubes for the treatment of band keratopathy.
METHODS: All 3 protocols used commercially available Vacutainer blood collection tubes coated with K2EDTA. An osmometer was used to measure and compare the concentration of EDTA created using 3 different protocols. The time required for preparation of the solution was measured and compared to evaluate its efficiency for everyday clinical use. In addition, volume of EDTA solution obtained was measured for method 1. The most promising protocol for clinical use was then used for treatment of a series of patients.
RESULTS: Average osmolarity was 532, 285, and 422 for methods 1, 2, and 3, respectively (ANOVA P < 0.01, all Tukey honestly significant difference P < 0.01). For the respective mixtures, average concentration was 65, 35, and 52 mg/mL, and average time to create solution was 189, 38, and 83 seconds (ANOVA P < 0.01, all Tukey honestly significant difference P < 0.01). The most promising, method 3, was found to be safe and effective in removing calcium from the corneal stroma in a series of 5 patients with 6 eyes treated. It also yielded 25% more solution for clinical use than method 1.
CONCLUSIONS: Method 3 using a single 10-mL Vacutainer tube with 18 mg of K2-EDTA had the best balance of effective concentration of EDTA, time to preparation, and simplicity of methodology, when compared with previously published methods 1 and 2. It also yielded a greater final volume of solution.

The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca2+ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca2+ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca2+ entry are scarce. Even more, to date the presence of a store-operated Ca2+ channel (SOC) has not been reported. Despite the apparent absence of Orai and STIM-like proteins in these parasites, in the present work we demonstrate the presence of a store-operated Ca2+-entry (SOCE) in T. equiperdum, using physiological techniques. This Ca2+-entry is induced by thapsigargin (TG) and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), and inhibited by 2-aminoethoxydiphenyl borate (2APB). Additionally, the use of bioinformatics techniques allowed us to identify putative transient receptor potential (TRP) channels, present in members of the Trypanozoon family, which would be possible candidates responsible for the SOCE described in the present work in T. equiperdum.

Ca2+ handling by mitochondria is implicated in energy production, shaping of cytosolic Ca2+ rises, and determination of cell fate. It is therefore of crucial interest for researchers to directly measure mitochondrial Ca2+ concentration [Ca2+] in living cells. Synthetic fluorescent Ca2+ indicators, providing a straightforward loading technique, represents a tempting strategy. Recently, we developed a new highly selective mitochondria-targeted Ca2+ indicator named mt-fura-2 , obtained by coupling two triphenylphosphonium cation-containing groups to the molecular backbone of the cytosolic ratiometric Ca2+ indicator fura-2 .The protocols we describe here cover all the significant steps that are necessary to characterize the probe and apply it to biologically relevant contexts. The procedures reported are referred to mt-fura-2 but could in principle be applied to characterize other mitochondria-targeted Ca2+ probes . More in general, with the due modifications, this chapter can be considered as a handbook for the characterization and/or application of mitochondria-targeted chemical Ca2+ probes .