The selection of a wound dressing is crucial for successful wound management. Conventional dressings are preferable for the treatment of simple wounds. However, a bioactive wound dressing that supports wound management and accelerates the healing process is required when it comes to treating non-self-healing wounds.2-deoxy-D-ribose (2dDR) is a small deoxy sugar that naturally occurs in human body. Although we have previously demonstrated that 2dDR can be used to induce neovascularisation and accelerates wound healing in vitro and in vivo, the literature on small sugars is conflicting, and the knowledge on how 2dDR achieves its biological activity is very limited.In this study, several small sugars including D-glucose (DG), 2-deoxy-D-glucose (2dDG), 2deoxy-L-ribose (2dLR) were compared to 2dDR by investigating their effects on the metabolic activities of both human dermal microvascular endothelial cells (HDMECs) and human dermal fibroblasts (HDFs). Then, for the first time, a two-dimensional (2D) scratch wound healing model was used to explore the migratory response of HDFs in response to 2dDR treatment. Finally, 2dDR was incorporated into Poly (PHBV) polymer fibres via electrospinning, and the metabolic activity of both types of cells in vitro was investigated in response to sugar release via Alamar Blue assay.The results demonstrated that 2dDR was the only sugar, among others, that enhances the metabolic activity of both HDMECs and HDFs and the migratory response of HDFs in a 2D scratch assay in a dose-dependent manner. In addition to direct administration, 2dDR was also found to increase the metabolic activity of HDMECs and HDFs over 7 days when released from polymer fibres. It is concluded that 2dDR is a potential pro-angiogenic agent that has a positive impact not only on endothelial cells but also fibroblasts, which take a key role in wound healing. It could easily be introduced into polymeric scaffolds to be released quickly to enhance the metabolic activity and the migratory response of cells that are associated with angiogenesis and wound healing. Skin plays an important role in the prevention of the body against microorganism invasion , from deRegular healing steps of a superficial wound includes six main stages: (i) haemostasis, (ii) inflammation, (iii) granular tissue formation, (iv) proliferation, (v) neovascularisation, and (vi) remodelling . HoweverThe physiological tissue/wound healing process is strictly controlled by cell-cell and cell-extracellular matrix (ECM) interactions . As a rePlatelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) are the commonly used growth factors either to induce angiogenesis or to improve wound healing . Growth 2-deoxy-D-ribose (2dDR) is a deoxy pentose sugar that is found in the DNA’s nucleic acid structure in the human body. It is an endogenous metabolite and naturally formed as a result of the degradation of thymidine to thymine by thymidine phosphorylase (TP)  Priestl. The angAlongside 2dDR, three other small sugars (2-deoxy-L-ribose (2dLR), 2-deoxy-D-glucose (2dDG), and D-glucose (DG)) were tested for their potential pro-angiogenic activity. These sugars were selected initially due to being involved in angiogenesis-related studies (either in a positive or negative way) in the literature. However, the rationale behind selecting (i) 2dLR was to investigate whether this effect is structure-dependent or not as 2dLR is the stereoisomer of 2dDR and to explore how conformational change affects the biological activity of a sugar, (ii) DG was to see if the effect is due to the metabolisation of sugar and again if it is independent of the sugar-origin or not, and (iii) 2dDG was to see what would happen if we use a sugar which recapitulates the function of DG but cannot undergo further glycolysis.Wound dressings are made of both synthetic and natural materials. Natural materials are advantageous due to their superior biological activity in supporting the adhesion and functionality of cells. However, all bioactive components should be removed to avoid undesired host response upon implantation . On the To date, various synthetic polymers have been reported as biomaterials for their use in angiogenesis, tissue engineering, and wound healing research. These polymers include but not limited to polycaprolactone (PCL) , polylacAccordingly, in this study, the effect of different doses of 2dDR on the metabolic activity of human dermal microvascular endothelial cells (HDMECs) that has a key role in wound healing  was evalA total of 37% formaldehyde solution, AlamarBlue cell viability assay, amphotericin, penicillin/streptomycin, dimethyl sulphoxide (DMSO), dichloromethane (DCM), methanol, fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), phosphate-buffered saline (PBS), Poly3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), and trypsin EDTA were purchased from Sigma Aldrich. Human Dermal Microvascular Endothelial Cells (HDMECs), Endothelial Cell Growth Medium (EC GM) and EC GM Supplement Pack were purchased from PromoCell. Human Dermal Fibroblasts (HDFs) and Fibroblast Growth Medium (FGM) BulletKit were purchased from Lonza.A total of 10 mM stock solutions of 2dDR, 2dLR, 2dDG and DG were prepared by dissolving the sugars in low serum (2% FCS) EC GM and filtered using a 0.2 μm syringe filter. The stock sugar solutions were then diluted 10× and 100× in sterile EC GM to prepare 1 mM and 100 μM sugar solutions. 80 ng/mL VEGF solution was prepared in sterile EC GM and used as a positive control. Low serum EC GM was used as a control group in the metabolic activity experiments.2. The growth medium was replaced every 3 days until they reached ~80%–90% confluency.HDMECs, between passages 2 and 6, were cultured in EC GM supplemented with a total of 10% FBS and growth medium 2 kit supplements at 37 °C and 5% CO4 cells/cm2) HDMECs were then cultured with EC GM either supplemented with 100 μM, 1 mM and 10 mM concentrations of 2dDR, 2dLR, 2dDG, DG, or VEGF or as non-supplemented (control). AlamarBlue readings were taken on days 1, 4, and 7 to track the metabolic activity changes in response to sugar treatments. Briefly, 0.1 mM AlamarBlue solution was prepared in EC GM. The growth medium of cells was removed, and the cells were washed with PBS once. Then, 1 mL of AlamarBlue solution was added to each well and incubated at 37°C for 4 h. After the incubation, 200 μL of the solution was transferred into a 96-well plate, and the fluorescence readings were taken at 540 nm (excitation) and 635 nm (emission).AlamarBlue Cell Viability Assay was used to measure the metabolic activity of HDMECs over 7 days in response to 100 μM, 1 mM and 10 mM concentrations of 2dDR, 2dLR, 2dDG, and DG as described previously . Once HD2. The growth medium was replaced every 3 days until they reached ~80%–90% confluency.Following the evaluations of different small sugars’ effects on metabolic activity change of HDMECs, further doses of 2dDR  were assessed to see the effect of it on increasing the metabolic activity of HDFs. For this, HDFs, between passages 3 and 8, were cultured in FGM supplemented with a total of 10% FBS and BulletKit supplements at 37°C and 5% CO4 cells/cm2). HDFs were cultured with FGM either supplemented with 1 μM, 100 μM, 1 mM, and 10 mM of 2dDR or non-supplemented (control). AlamarBlue readings were taken on days 1, 4, and 7 to track the metabolic activity changes in response to sugar treatments as described in Section 3.1.AlamarBlue Cell Viability Assay was used to measure the metabolic activity of HDFs over 7 days in response to 1 μM, 100 μM, 1 mM, and 10 mM concentrations of 2dDR. Once HDFs reached confluence, they were collected, prepared into a cell suspension, and added into 48-well plates  was replenished either supplemented with 2dDR (1 μM and 1 mM), EGF (50 ng/mL) or as non-supplemented (control). The well-plates were returned to a cell culture incubator and incubated at 37 °C, 5% COAn inverted microscope  was used to take digital images of the wound area. The images were taken at the beginning of the experiment and after 12, 48, and 60 h. Adobe Photoshop CS6 software  was used to convert the raw image to a black and white image to facilitate image processing. For this, wound areas were drawn manually by the magic wand (quick selection) tool. The areas outside the wound zone were coloured black, while the wound zone was coloured with white. Black and white images were then imported to ImageJ software . The white zones corresponding to the wound area were quantified using the histogram function of ImageJ software, where histograms of each image were analysed, and the white areas in the histograms were tabulated to Microsoft Excel software to calculate the percentage of wound reduction for each group. Normalised percentage wound closure rates were determined as the percentage wound area reduction per the time point that the image was acquired.Electrospinning was used to fabricate 2dDR releasing PHBV fibres as bioactive wound dressings . BrieflyThe surface morphology of 2dDR releasing wound dressings was investigated under a scanning electron microscope (SEM)  . Fibre dA spectrophotometric method was used for the detection of 2dDR, as described previously  . BrieflFollowing the fabrication of 2dDR releasing PHBV dressings, they were cut into 10 mm diameter circles using a biopsy punch and disinfected in 70% ethanol for 45 min. Then, the dressings were washed in PBS for 1 h  and placed into 12-well plates for cell seeding.4 cells/cm2. Scaffolds were kept at 37 °C for 60 min to allow cells to attach. Then, 4 mL of EC GM (for HDMECs) and FGM (for HDFs) were added to each well. PHBV dressings were kept in culture for 7 days by changing the culture medium every 2–3 days.HDMECs and HDFs were cultured as described in the previous sections, and once they reached to confluence, they were trypsinised and seeded onto 2dDR releasing PHBV scaffolds with a seeding density of 4 × 10The metabolic activity of HDMECs and HDFs seeded onto 2dDR releasing PHBV fibres were tracked over 7 days using Alamar Blue Cell Viability Assay as described in previous sections . The metStatistical analysis was performed using a statistical analysis software . The analysis was conducted either using one-way or two-way analysis of variance (ANOVA). Error bars indicate standard deviations unless otherwise stated.The results of the AlamarBlue assay demonstrated that 2dDR, 2dLR and DG did not cause any change in the metabolic activity of HDMECs compared to controls at any doses tested, while VEGF increased the metabolic activity significantly. In contrast, 2dDG at 1 mM and 10 mM doses was found to decrease the cellular activity even on day 1.2dDR at 100 μM and 1 mM doses and VEGF at 80 ng/mL (as the positive control) has been found effective in terms of increasing the activity of HDMECs on days 4 and 7. In contrast to 100 μM and 1 mM doses, the highest concentration (10 mM) of 2dDR was found to decrease the activity significantly on days 4 and 7. A similar trend was observed for 2dLR at 10 mM dose, whereas lower doses (100 μM and 1 mM) were ineffective at any time points, and no statistically meaningful change was observed compared to controls. For 2dDG, the reductions in the metabolic activity of HDMECs on day 4 and 7 were more dramatic compared to 2dDR and 2dLR, including the mid-dose (1 mM) alongside the highest dose of 10 mM. However, 100 μM dose of 2dDG caused a significant reduction in the activity only on day 7 but not on days 1 and 4 (no significant change was observed compared to controls). Throughout the metabolic activity measurements, DG did not show any change in the metabolic activity of HDMECs at any concentration on any days compared to controls. The metabolic activity changes of HDMECs over 7 days in response to 100 μM, 1 mM, and 10 mM doses of DG , 2dDR F, 2dLR F, and 2dDTake home messages from this sugar comparison study were as follows: (i) 2dDR (at 100 μM and 1 mM) was the only sugar that enhances the metabolic activity of HDMECs significantly at the end of 7 days, (ii) 2dDR , 2dLR  and 2dDG  caused a dramatic decrease in the activity of HDMECs due to probable dose-dependent toxicity. For 2dDG, this may not be surprising be explained as 2dDG has previously been demonstrated as a potential anti-angiogenic agent . HoweverThe metabolic activity measurements of HDFs over 7 days showed that similar to that observed on HDMECs, 2dDR treatment at 100 μM and 1 mM doses also increased the metabolic activity of HDFs by days 4 and 7, while there was no statistically meaningful difference on day 1. Although 1 μM dose of 2dDR caused a slight increase by days 4 and 7, it was not statistically different compared to controls. The light microscope images of HDFs at the end of day 7 and the results of the AlamarBlue metabolic activity assay in response to 2dDR treatment over 7 days are given in One thing to note is that, similar to HDMECs, a significant decrease was observed in the activity of HDFs when treated with 10 mM 2dDR. However, it seems like HDFs were more tolerable to the highest dose of 2dDR as the rate of decrease was lower compared to that of HDMECs. In addition, light microscope images also revealed that there were still some HDFs attached to the well plate in contrast to endothelial cells, which has previously been reported to detach completely from the well plate when treated with 10 mM of 2dDR .2D scratch assay is a convenient and inexpensive protocol for the analysis of cell migration corresponding to wound healing in vitro . When asFollowing the metabolic activity studies of HDFs, the scratch wound healing assay also showed that 1 mM of 2dDR caused an increase in the migratory response of HDFs in 60 h compared to controls . AlthougWhen ormalized wound closure rates were investigated, only EGF and 1 mM 2dDR treatments have been found to increase the closure rates by 12, 48, and 60 h but no other groups. EGF treatment increased the wound closure rate by approximately 1,3-fold at all time points . Similarly, 1 mM 2dDR treatment caused an increase of 1.1-fold, 1.2-fold and 1.1-fold by 12, 48, and 60 h, respectively, in the wound closure rates .The light microscope and the processed images (given in the top right of each image) showing the migration of HDFs corresponding to 2D scratch wound closure over 60 h, and the quantified graphs of percentage wound closure and normalised wound closure rates are given in SEM images of the 2dDR releasing PHBV dressings are given in The releases of 2dDR from PHBV fibres was measured over 10 days, as shown in Alongside the direct addition of 2dDR to the growth media, the 2dDR released from the PHBV dressings were also found to increase the metabolic activity of HDMECs and HDFs over 7 days .By day 1, the metabolic activity of HDFs was higher than that of HDMECs attached to plain PHBV dressing. Although there was no statistically meaningful difference, the activity of HDMEC and HDFs went up by 1.6-fold and 1.1-fold, respectively, when seeded onto 2dDR releasing PHBV dressings. On day 4, by the release of 2dDR from the PHBV dressings, the metabolic activity of HDMECs and HDFs were increased significantly by approximately 1.6-fold and 1.3-fold, respectively. By day 7, cell activity went up by 1.5-fold and 1.8-fold for HDFs and HDMECs seeded onto the 2dDR-releasing PHBV fibres, respectively.Acute wounds can follow the physiological healing steps successfully and heal with an expected rate and within a predicted time. However, chronic, deeper and complex wounds where usually more than one layer of skin is damaged, and a constant infection occurs due to delayed healing requires special attention and a bioactive approach for successful healing.Physiological wound healing is a complicated process where cell-cell, cell-ECM interactions and growth factors play key roles. The main steps of wound healing are haemostasis, inflammation, granular tissue formation, proliferation, neovascularization, and remodelling  Kawasum. EnsurinThe use of growth factors is a well-established and effective way of developing active wound patches to induce angiogenesis and accelerate wound healing. Among many growth factors, VEGF is considered the most effective stimulator of angiogenesis . However. In addition, we investigated its potential to accelerate the healing of chronic wounds using a diabetic rat wound healing model (2dDR is a small deoxy pentose sugar that is naturally found in the human body in the nucleic acid structure. During the dynamic metabolic processes of the DNA, 2dDR is formed by the TP activity as a side product of the enzymatic degradation of thymidine into thymine . Startinng model  and showng model . Althougng model  and growng model . Thus, tAccordingly, in this study, three potential doses of 2dDR were compared with other small sugars, 2dLR, 2dDG, and DG to investigate its specificity to HDMECs, a small-diameter blood vessel endothelial cells sourced from human skin, which is a great representative cell type that can be used in vitro angiogenesis and wound healing studies . Then, t2dLR, 2dDG, and DG sugars were selected not only due to being involved in angiogenesis studies in literature but also in an attempt to simply clarify whether the biological activity of 2dDR is structure dependent or is it due to the metabolism of any sugar by the cells. This allowed us to partly investigate the specificity of 2dDR as a potential pro-angiogenic agent to wound healing related cells. Our current knowledge on small sugars and how they are involved in angiogenesis pathways is quite conflicting. For instance, 2dLR has previously been reported as a promoter  and inhi2dDR is highly soluble in water and organic solvents, which makes it easy to incorporate into polymeric fibres. The addition of 2dDR into the PHBV solution did not alter the microstructure of the electrospun fibres. A smooth and beadless morphology was obtained from all PHBV electrospinning groups. Both 2dDR concentrations slightly increased the average fibre diameter of the scaffolds but did not change the average pore size. Although studying the release kinetics of 2dDR would extend our knowledge on what would be an ideal release behaviour for the acceleration of wound healing and how does the release of 2dDR from PHBV fibres fits this profile, exploration of the release kinetics of 2dDR was beyond the merits of this study. Thus, the main objective of this work was kept as simple as possible to investigate whether 2dDR can be loaded into PHBV fibres and released from them in a certain period of time to show the biological activity. The release behaviour of 2dDR from the PHBV fibres was observed over 10 days from both concentrations. More than 90% of the 2dDR was released just in 7 days. We have previously observed a similar release behaviour from a variety of biomaterials  , which hTo date, no studies have been conducted to investigate the effect of 2dDR on the migratory and proliferative response of fibroblast cells, which take important roles in physiological wound healing process . This stTo conclude, among the small sugars investigated in the scope of this study, 2dDR was the only small sugar that increases the metabolic activity of HDMECs and HDFs when used as a supplement in the growth medium. In addition, 2dDR has also been shown, for the first time, to stimulate the migratory response of HDFs in a 2D wound healing assay when used at 1 mM concentration. It has been found approximately 85% effective in terms of accelerating the wound closure rate compared to EGF, positive control. Alongside the direct addition to the growth medium, 2dDR was also effective in increasing the metabolic activity of both cell types when released from PHBV fibres over 7 days. The release of 2dDR improved the metabolic activity of HDFs and HDMECs growing on polymer fibres by 7 days. This study demonstrates that the introduction of 2dDR into the biomaterials offers a promising approach that can be used to enhance not only the metabolic activity of endothelial cells but also the metabolic activity and the migratory response of fibroblasts, which play a key role in wound healing.


For as-grown QDs, low laser repetition rates favor the generation of the higher Larmor frequency spin component individually, while the lower Larmor frequency spin component can be enhanced by increasing the laser repetition rates. We hope that the explored methods can inspire further investigations of spin dynamics and related photophysical processes in colloidal nanostructures.The coexistence of two spin components with different Larmor frequencies in colloidal CdSe and CdS quantum dots (QDs) leads to the entanglement of spin signals, complicating the analysis of dynamic processes and hampering practical applications. Here, we explored several methods, including varying the types of hole acceptors, air or anaerobic atmosphere and laser repetition rates, in order to facilitate the obtention of one single Larmor frequency in the coherent spin dynamics using time-resolved ellipticity spectroscopy at room temperature. In an air or nitrogen atmosphere, manipulating the photocharging processes by applying different types of hole acceptors, e.g., Li[Et High laser repetition rates can enhance the spin signal of the v1 component, whereas low laser repetition rates are convenient for obtaining the v2 component alone.In this work, we investigated the spin signals of colloidal CdSe and CdS QDs in toluene using time-resolved ellipticity spectroscopy under different photocharging conditions, such as addition of different types of hole acceptors, changing laser repetitions as well as air and nitrogen  and ole3BH] , purchasThe coherent spin dynamics of colloidal QDs were measured using time-resolved ellipticity spectroscopy ,19,20. TIn the pump−probe measurements as shown in B of 500 and 300 mT, respectively. The corresponding fast Fourier transform (FFT) spectra are shown in the right panels of v1 = 7.32 GHz and v2 = 11.11 GHz. Two g factor values of g1 = 1.05 and g2 = 1.59 are correspondingly derived from these frequencies by the equation h, g factor values are in line with the size dependence of g factors reported in the literature  hole acceptors prepared in a N2 atmosphere. The presence of the hole acceptor Li[Et3BH] strongly increases the ellipticity signals both for CdSe and CdS QDs. As depicted in the insets of v1 component is remarkably enhanced, while the v2 component disappears. This indicates that addition of Li[Et3BH] hole acceptors in a N2 atmosphere is conducive to separating the entangled spin signals by increasing the v1 component and suppressing the v2 component. The reason is that the Li[Et3BH] hole acceptor, as a strong chemical reducing agent, can capture the photogenerated holes and leave the electrons in the dot core . With eng state ) is thusng state ,24) in ang-lived . Under tv1 component is to modify colloidal QDs with Li[Et3BH] hole acceptors in an anaerobic atmosphere. The spin signals of the v1 component are considerably amplified, which also improves the signal-to-noise ratio significantly.Consequently, an effective strategy to achieve pure states of the v1 spin component by modifying QDs surface with an appropriate hole acceptor in a N2 atmosphere, we investigated the effect of adding another OT hole acceptor to QDs prepared in an air atmosphere. The comparison of the time-resolved ellipticity signals of CdSe QDs with and without OT prepared in an air atmosphere is shown in v2 component increases to about 8 times that of as-grown QDs. The inset of v2 enlarges, while v1 disappears. A similar phenomenon can be found in the measurements in as-grown CdS QDs and QDs with OT prepared in an air atmosphere, as depicted in v2 component significantly increases and brings the v2 component into being separately. Note that the laser repetition for CdS QDs with OT is 10 kHz. Since with the higher laser repetition, i.e., with 50 kHz laser repetition, the spin signal of the v1 component is apparent as a result of the pile-up effect from previous pulses. The v1 component can appear at high laser repetition rates, and the higher the laser repetition rate, the stronger the intensity of the v1 component, which will be illustrated below. For measurements with different laser repetition rates, a normalization of both pulse energies and laser powers is requisite. The spin signal of CdS QDs with OT measured at 10 kHz laser repetition rates in the main panel has already been multiplied by a factor of 5; equivalently, both the pulse energy and laser power are normalized to be the same between the measurements with 10 and 50 kHz laser repetition rates.Inspired by the success of individually obtainment of the v2 spin component by using OT hole acceptors. OT molecules are linked to the QD surface by replacing native ligands and capture the photogenerated holes in QDs. Therefore, the captured holes are still in nearby the QD surface. Due to the Coulombic attraction, the electron also tends to stay at nearby the QD surface. This charging state contributes to the spin signal of the v2 component  or OT, in a N2 atmosphere is beneficial to obtain the pure spin signal of the v1 component.Addition of an appropriate molar ratio of hole acceptors, Li and OT, with a proper molar ratio in N2 or air atmosphere, the two spin components can be selectively enhanced or suppressed, leaving one pure spin component. Low laser repetition rates are beneficial to the appearance of the v2 component solely. Higher laser repetition rates significantly contribute to the enhancement of the v1 spin component. Consequently, those results imply that addition of hole acceptors either in an air or anaerobic atmosphere and adjusting laser repetition rates provide effective strategies to manipulate the spin signals in colloidal QDs, which is helpful for in-depth investigation of spin dynamics and practical applications.In conclusion, the coherent spin dynamics of CdSe and CdS colloidal QDs were studied using time-resolved ellipticity spectroscopy. Two spin components with different Larmor frequencies typically coexist in QDs with native ligands when measured at high laser repetition rates. In the presence of appropriate hole acceptors, e.g., Li[Et


It is reported that BALB/c mice have inherently higher airway responsiveness, but show no different contractive response of tracheal ring as compared to C57BL/6 mice. However, whether the different airway responsiveness is due to the different extents of small airway contraction, and what’s underlying mechanism remains unknown. Here, we assess agonist-induced small airway contraction and Ca2+ oscillations in ASM cells between BALB/c and C57BL/6 mice by using precision-cut lung slices (PCLS). We found that BALB/c mice showed an intrinsically stronger extent of small airway narrowing and faster Ca2+ oscillations in ASM cells in response to agonists. These differences were associated with a higher magnitude of Ca2+ influx via store-operated Ca2+ entry (SOCE), as a result of increased expression of SOCE components  in the ASM cells of small airway of BALB/c mice. An established mathematical model and experimental results suggested that the increased SOC current could result in increased agonist-induced Ca2+ oscillations. Therefore, the inherently higher SOC underlies the increased Ca2+ oscillation frequency in ASM cells and stronger small airway contraction in BALB/c mice, thus higher airway responsiveness in BALB/c than C57BL/6 mouse strain.BALB/c and C57BL/6 mouse strains are widely used as animal model in studies of respiratory diseases, such as asthma. Asthma is characterized by airway hyperresponsiveness, which is eventually resulted from the excessive airway smooth muscle (ASM) contraction mediated by Ca Therefore, the maintenance of agonist-induced Ca2+ oscillations and ASM contraction requires an influx of extracellular Ca2+. We has demonstrated that this influx occurs mainly via store-operated calcium entry (SOCE)  membrane-spanning Ca2+-sensing protein called stromal interaction molecule 1 (STIM1) , a phenomenon of excessive airway narrowing in response to agonists. Although AHR might result from hypertrophy and hyperplasia of airway smooth muscle (ASM) induced by airway inflammation , the contraction . Notablyy (SOCE) , which i (STIM1) .2+ oscillations regulated by the magnitude of SOCE contributes to these differences.Mouse is commonly proposed as an experimental animal model to investigate the pathogenesis of AHR. Different mouse strains were used to measure airway reactivity, therefore, the inherent differences among mouse strains have been observed. For example, two widely used mouse strains, BALB/c and C57BL/6, have been demonstrated to display substantial differences in the distribution of inflammatory cells, expression of proinflammatory cytokines, ASM mass, and subepithelial collagen and fibronectin deposition after acute or chronic exposure to allergens . It is w2+ oscillations of ASM cells in BALB/c and C57BL/6 mice. We report that BALB/c mice show an inherently stronger extent of small airway narrowing and a higher frequency of Ca2+ oscillation in ASM cells in response to agonists, which might be associated with an increased Ca2+ influx via SOCE.In this study, we used an established precision-cut lung slices (PCLS) to access the agonist-induced contraction of the small airway and CaAge matched female C57BL/6 mice and BALB/c mice (8–12 weeks with bodyweight of 19–22 g) were purchased from Charles River Breeding Laboratories  and GemPharmatech Co. Ltd . The mice were bred in a specific pathogen free environment. The adequate number of mice was estimated by a power analysis. All protocols were in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of University of Massachusetts Medical School and West China Hospital .2+ solution (0-Ca2+ sHBSS) was prepared with no-Ca2+ HBSS supplemented with 1 mM Na2H2-EGTA, 20 mM HEPES and 0.9 mM MgSO4. Oregon Green 488 BAPTA-1 a.m. was purchased from Life Technologies . Ryanodine was purchased from Abcam Inc , other reagents were purchased from either Sigma-Aldrich  or Thermo Fisher Scientific .Hanks’ Balanced Salt Solution (HBSS) was prepared by supplementing 20 mM HEPES buffer and adjusted to pH 7.4 (sHBSS). Hanks’ 0-Ca2 and 37°C for up to 2 days, within the periods the contractility of small airway remains unchanged. The experiments were performed at 37°C, and repeated at room temperature, in a custom-made, temperature-controlled microscope enclosure as described previously  of MCh/5-HT sequentially with washing-out of agonists by sHBSS between each dose. The extent of airway narrowing in response to the agonists was determined by the change in lumen area, which was recorded by a CCD camera at 1 image per 2 s on an inverted microscope (Nikon Diaphot or Olympus IX71). The area of the lumen was calculated by using ImageJ, and then normalized by the formula: Area (%) = 100% * Lumen area at a certain time/Initial lumen area.To compare the extent of airway narrowing between BALB/c and C57BL/6 mice, the extent of airway contraction was defined and calculated by the formula: airway contraction (%) = 100% */Initial lumen area. Then a dose-response curve was plotted by using the extent of airway contraction in response to increasing doses of agonists at sequential timepoints  of the contraction assay. For each assay, 6 BALB/c mice and 6 C57BL/6 mice were used, 1–2 PCLS were obtained from each mouse, 2 randomly selected airways in each PCLS were assessed, and specific sample sizes were reported in the figure legends.2+ signaling of a single ASM cell in the PCLS was recorded by a custom-built, video-rate scanning 2-photon laser microscope at a rate of 15 images per second. The change in fluorescence intensity in an interest region (8 × 10 pixels) of an ASM cell was analyzed by Video Savant 4  with custom-written code. The fluorescence intensity at a particular time (Ft) was normalized to the initial time (F0) as Ft/F0. For each assay, five to six BALB/c mice and five to six C57BL/6 mice were used, 1–2 PCLS were obtained from each mouse, two to three randomly selected ASM cells in each PCLS were assessed, and specific sample sizes were reported in the figure legends.The detailed protocol has been previously described . The PCL2+ concentration in SR ([Ca2+]SR), activating SOCE to induce Ca2+ influx. The SOCE-activated PCLS remains viable for at least 5 h, during this time the [Ca2+]SR is kept in a low level due to the persistent opening of RyR, and consequently the activation of SOCE is sustained and irreversible. As a result, the [Ca2+]i is determined by the extracellular Ca2+ concentration ([Ca2+]e) via SOC influx.The validation of SOCE-activated PCLS has been reported . Briefly+/K+ ATPase , α-smooth muscle actin , STIM1 , and Orai1  were applied to the lung sections respectively for 2 h at room temperature. Then horseradish peroxidase (HRP)-labeled rabbit secondary antibodies were applied to the lung sections for 20 min, followed by incubation with TSA opal fluorophore  for 10 min at room temperature. The antibody-TSA complex was removed through a heat-mediated antigen retrieval process using Tris/EDTA buffer (pH 9.0) during each staining cycle. Then the lung sections were counterstained with DAPI . The immunofluorescence images were captured by Nikon A1R MP confocal microscope. We conducted a negative control to rule out the possibility of spontaneous fluorescence and non-specific signals by staining lung sections with vehicle instead of primary antibodies. The α-SMA  was used to identify ASM cells. The fluorescence intensity of NKA  was used as an internal control. We then quantified the fluorescence density of Orai1 and STIM1  by normalizing the fluorescence intensity of Orai1/STIM1 to that of NKA in the same region of interest of ASM cells using ImageJ. Three BALB/c mice and three C57BL/6 mice were used for each staining, and two to three lung sections were obtained from each mouse. Three randomly selected ASM cells in each lung section were analyzed in a blinded manner, the specific sample sizes were reported in the figure legends.Mice were anesthetized with intraperitoneal injection of 2.5% sodium pentobarbital, and subsequently euthanized through exsanguination from the right ventricle to collect lung tissue. The left lung was inflated and preserved with 10% formaldehyde solution, followed by paraffin embedding and slicing. We employed tyramide signal amplification (TSA) principle for immunofluorescence staining, by using a multiplex fluorescence kit and following the guidelines provided by the manufacturer . Briefly, the lung sections underwent dewaxing and rehydration, followed by antigen retrieval. Protein blocking was performed using peroxidase blocking solution for 30 min before each of the three staining cycles. Primary antibodies against Nat-test or Mann-Whitney test. For the comparison of dose-response curves, nonlinear regression analysis was performed first, and then the best-fits values, including the bottom, top and concentration for 50% of the maximal effect (EC50) of the curves, were compared between groups. Data are presented as the mean ± SD, the sample sizes and numbers of mice used in experiments were reported in the figure legends; a value of p < 0.05 was considered significant.Data were statistically analyzed and plotted by GraphPad Prism 7.00 . For the comparison of two groups, the distribution of data was first tested by the Shapiro-Wilk normality test or KS normality test, and then the data were compared using the Unpaired 2+ oscillations in the ASM of PCLS (2+ concentration (c = [Ca2+]i) and the SR Ca2+ concentration (cs = [Ca2+]SR) are modelled by:Jin accounts for the Ca2+ influx, primarily through SOCE, while JPMCA and JSERCA represent the Ca2+ fluxes through the PMCA and SR Ca2+ ATPases (SERCA) pumps, respectively. Jrel, on the other hand, models the efflux of Ca2+ from the SR through inositol trisphosphate (IP3) receptors (IP3R) and RyR. The comprehensive description of the mathematical model is provided in the supplement.The mathematical model has been reported previously and well validated in stimulating the dynamic structure of Ca of PCLS . In brieTo assess the extent of “small airway” narrowing, we distinguished airway generation as reported previously : the firThe extent of small airway narrowing in PCLS derived from BALB/c and C57BL/6 mice was visualized  and anal2+ oscillations  reduced the frequency of Ca2+ oscillation. This prediction was validated in experiment that the blockage of SOCE by 1 μM and 10 μM GSK7975A, a specific inhibitor targets SCOE , Ca2+ efflux from SR through RyR (PRyR), Ca2+ uptake to the SR by SERCA (PSERCA), and intercellular Ca2+ efflux through plasma membrane Ca2+ ATPase (PCMA) and Na+/Ca2+ exchanger (NCX)  to the SOCE-activated ASM cells firstly to decrease the [Ca2+]i and [Ca2+]SR. As shown in 2+ sHBSS induced decreased [Ca2+]i within 5 min. As previously described, the [Ca2+]i is primarily determined by the SOC influx in the SOCE-activated ASM cells, we then added back 1.3 mM Ca2+ to assess the transient Ca2+ influx via SOCE  has been well demonstrated . STIM1 sSM cells . PreviouSM cells . In addiSM cells . FurtherSM cells . In the 2+ oscillations of ASM cells. Previously, we reported that both the Ca2+ oscillation frequency and the Ca2+ sensitivity of ASM cells contribute to innate ASM contractility (2+ sensitivity of ASM cells in BALB/c mice differs from that in C57BL/6 mice is still unknown and worth further investigation. In addition, it is reported that different SERCA expression (3R receptor density (2+ oscillation frequency, whether BALB/c and C57BL/6 mice have different SERCA or IP3R expression might need further investigation. Furthermore, it has been reported that the contractile protein content in ASM cells (It is worth noting that agonist-induced airway contraction is not only determined by the Caactility , whetherpression , IP3R re density  and imba density  could afSM cells , the tonSM cells , and theSM cells  also exe2+ oscillation frequency, which might result from higher SOCE influx due to increased expression of Orai1 and STIM1 in ASM cells. These results suggest that an increased SOC current in ASM itself might lead to a higher Ca2+ oscillation frequency and stronger airway contraction, highlighting the importance of SOCE in understanding the mechanisms of AHR.Taken together, our findings demonstrate that, as compared to C57BL/6 mice, BALB/c mice have inherently higher small airway contraction along with increased agonist-induced Ca


TCa is routinely adjusted for albumin using several formulae  to more closely reflect Ca2+. Here, we derive a novel formula to estimate Ca2+ and compare its performance to established formulae.Free ionized calcium (Ca2+Cohort for prediction of Ca: 2806 serum samples (TCa) taken contemporaneously with blood gas samples (Ca2+) at Imperial College Healthcare NHS Trust were used to derive formulae to estimate Ca2+ using multivariable linear regression. Cohort for prediction of PTH: Performance of novel and existing formulae to predict PTH in 5510 patients was determined by Spearman correlation.2+ prediction CohortCa: Adjusted calcium (r2 = 0.269) was less strongly associated with Ca2+, than TCa (r2 = 0.314). Prediction of Ca2+ from a newly derived formula incorporating TCa, potassium, albumin, and hematocrit had an improved r2 of 0.327, whereas inclusion of all available parameters increased the r2 further to 0.364. Of the established formulae, James performed best in predicting Ca2+ (r2 = 0.27). PTH prediction cohort: Berry resulted in higher whereas Orell in lower adjusted calcium levels. Prediction of PTH was strongest in the setting of hypercalcemia, with James having the highest Spearman correlation coefficient (+0.496) similar to including all parameters (+0.499).2+. Further prospective studies are needed to optimise adjustment of TCa and to establish bounds for validity.Adjustment of calcium for albumin using established formulae does not always outperform unadjusted TCa in the reflection of Ca Calcium is a ubiquitous mineral that has a critical role across several physiological processes , 2. Intr2+)  separates into diffusible ~55%) and non-diffusible fractions (~45%) 5% and no, dependi2+)  and the 2+) . McLean omponent , 10, witomponent . Crucialecretion –14. Thus2+ concentrations requires direct ion selective electrodes (ISE) that are not universally available in the biochemistry laboratory (2+) remains unaltered  and TCa  to evaluate the effect of parameters including gender, age, creatinine, total protein, albumin, globulin, phosphate, sodium, potassium, creatinine, hemoglobin, and hematocrit, that could affect prediction of Ca2+ from TCa. We also identified boundaries for these parameters beyond which the relationship between TCa and Ca2+ was lost e.g., extremes of albumin.This study aims to assess whether routinely measured analytes and baseline factors can be used to better predict Ca2+ and having derived a novel formula to predict Ca2+, we then evaluated the performance of this formula  to assess their ability to predict PTH in samples from a second cohort of patients with known vitamin D and PTH levels.Thereafter, having evaluated the performance of existing formulae to reflect CaThis work was conducted as part of a service evaluation of calcium estimation at Imperial College Healthcare NHS Trust (ICHT), reference number: ASM_044.2+ Prediction Cohort) being used to derive a novel formula to predict Ca2+ from TCa , and the second (PTH Prediction Cohort) being used to evaluate the performance of the newly derived formula (as well as established formulae) in the prediction of PTH. Data for both cohorts were extracted from laboratory data for the period 1 June 2016 to 29 June 2019.Data were obtained from two cohorts, with the first cohort (Ca2+ Prediction Cohort), we collated data from 2806 patients who had contemporaneous collection of serum samples (providing TCa) and blood gas samples (providing Ca2+) in order to estimate the relationship between Ca2+ and TCa. As blood gas analysis was usually conducted in the acute setting, we also repeated the analysis in a restricted subset of this cohort of 537 patients with complete data, who had normal pH (7.35-7.45), creatinine (<120 μmol/L), albumin (>15 g/L), total protein (>40 g/L) and calcium (2.2-2.6 mmol/L) levels to exclude those with markedly perturbed results due to significant ill health. TCa, adjusted calcium (as reported by the ICHT pathology lab based on the formula: TCa + 0.013*(40 – Alb) which was derived by J. Meek from internal data using Abbott bromocresol-purple (BCP) albumin assay & calcium method), creatinine, albumin, globulin, phosphate, total protein, sodium, potassium, hemoglobin, and hematocrit were measured in the clinical chemistry lab. Although gender, and age, were available, data on individual patient diagnoses/medications were not available. The relationship between Ca2+ and these other parameters was determined from this cohort using multivariable linear regression.For the first cohort  comprised of 5510 patients with the same laboratory measures available as in the derivation cohort, but with the addition of PTH and vitamin D levels. This second cohort was more commonly outpatient based as vitamin D and PTH are not usually measured acutely, whereas Ca2+ was measured on the GEM 2000 blood gas analyser . The intra- and inter-assay coefficients of variation for all assays were <5% within the reportable range.TCa (Arsenazo III method), creatinine (Jaffe method), albumin (Bromo-cresol purple method), phosphate (phosphomolybdate UV method), total protein (Biuret method), serum sodium (Indirect ISE method), and serum potassium (Indirect ISE method) were measured on the Architect 2000 platform . Haematocrit and haemoglobin were measured using Sysmex XE2100 analysers at Imperial College Healthcare NHS Trust hospitals. Capost hoc Tukey’s multiple comparison test, or Kruskal-Wallis test with post hoc Dunn’s test, as appropriate. Multivariable linear regression was used for prediction of Ca2+. Spearman correlation was used to assess correlations between calcium and PTH stratified by vitamin D category and calcium below and above 2.5 mmol/L.Analysis was performed using Prism v9.3 (GraphPad), and Stata v13.0 (StataCorp). Evidence for non-parametric distribution was assessed using the D’Agostino-Pearson test. Data are presented as mean ± standard deviation ( ± SD) for parametrically distributed data, or median  for non-parametrically distributed data. Statistical comparison across multiple groups was performed using one-way ANOVA with Baseline characteristics of both the first and second cohorts are summarized in TCa and 2+Ca was altered for available biochemical variables. The relationship was altered in those with pH values <7.24, albumin concentrations ≤15 g/L, total protein <40 g/L, and creatinine >120 μmol/L .We firstly assessed the boundaries for which the relationship between 2+ correlated similarly with TCa (r=0.66) compared to adjusted calcium by the local adjustment formula (J.Meek) (r=0.65)  than with TCa (r=0.36).A correlation matrix was used to present univariable Spearman Correlation Coefficients for available parameters in both the unrestricted  and each other variable in turn . This r2 of our new formula F2 was only marginally increased by the addition of any other single variable to a maximum r2 of 0.326. In the restricted cohort  or established formulae for the adjustment of TCa for albumin. Baseline characteristics for this cohort are presented in In order to interpret PTH levels appropriately, it is necessary to accurately categorize patients according to their calcium status i.e. as either being normocalcemic or hypercalcemic. This is crucial for correct clinical interpretation and subsequent management. To assess the impact of different adjustments of calcium for albumin on the categorization of patients as normocalcemic or hypercalcemic, we selected patients with borderline TCa levels (i.e. 2.59-2.61 mmol/L) and assessed the proportion of patients that would be classified as hypercalcemic (>2.6 mmol/L) using each of the albumin-adjustment calcium formulae. The proportion of these borderline cases (n=58) that would be categorized as hypercalcemic was: 22.4% for TCa, 29.3% if using ICHT adjusted Calcium formula, 29.3% by James formula, 1.7% by Orell formula, 31.0% by Payne and 81.0% by Berry. If we select patients with TCa levels between 2.55-2.65 mmol/L (n=109), then the proportion of patients that would be classified as hypercalcemic (>2.6 mmol/L) was: 31.5% for TCa, 43.5% if using ICHT adjusted Calcium formula, 45.4% by James formula, 13.9% by Orell formula, 51.9% by Payne, and 84.3% by Berry.In order to better understand PTH ranges at different calcium levels, PTH values were plotted according to categories of TCa level. PTH levels had a U-shaped relationship with TCa levels, with PTH levels reaching a nadir at a TCa of between 2.40-2.49 mmol/L  Table 3.2+ is considered the gold standard representation of biological calcium activity. However, measurement of Ca2+ is not practical, as most commercial vendors do not currently offer Ca2+ assay on large-scale automated instruments  hyperparathyroidism had both normal TCa and Ca2+ levels, further demonstrating the benefits of Ca2+ assessment to clarify diagnosis  similar to previous studies , and in the prediction of PTH in the second cohort in both hypocalcemic (r -0.11) and hypercalcemic patients (r +0.496), with similar performance to the newly derived formula (F1) incorporating all available biochemical parameters (F1) (r +0.499). Additionally, our study highlights that it may be necessary to derive bespoke reference ranges and formulae to adjust calcium in local centres to account for variations in assays used and population demographics, as well as to update these from time-to-time to take account of changes in performance of the assays (Of the existing formulae tested, the James formula appeared to perform best in both the prediction of Cae assays , 37.2+ being predominantly assessed in an acute setting, and PTH and vitamin D in the non-acute outpatient setting, it was not possible to directly assess both Ca2+ and PTH in the same cohort. Thus, we used the first cohort to derive a formula to estimate Ca2+, and then used a second cohort to correlate the estimated Ca2+ against PTH and vitamin D. Future prospective studies with concomitant measurement of Ca2+ and all other potentially relevant parameters e.g. FGF23 and patient histories in a controlled setting could enable more definitive exposition of the relationships between TCa and Ca2+ to enable more precise formulae to be derived. Additionally, urine calcium measures were not routinely available, to enable the exclusion of hypercalciuria as a cause of unexplained hyperparathyroidism.Strengths of the study include that this was a biochemistry laboratory centred audit enabling extraction of a large number of biochemical values analysed using the same analysers, however conversely a limitation is that granular clinical data on individual patient diagnoses/medications were not available. It is important to note that albumin measurements can differ depending on the assay used. For example, Roche BCP yields lower albumin readings than Abbott and differs from BCG and immunoturbidimetric methods. Consequently, it is important to note that older formulas such as Payne from 1973  used a B2+ in an unrestricted dataset and that the relationship between PTH and calcium is strongest in the setting of hypercalcaemia. Of the existing formulae, the James formula performed best at predicting PTH levels in the setting of both high and low calcium levels.We find that adjustment of TCa for albumin does not outperform unadjusted values in the prediction of CaThe original contributions presented in the study are included in the article/MP wrote the original draft. MP and AC contributed equally as first authors to this manuscript. AS, ML, PE, SC, PM analysed the data. TT, JC, WD, AA edit, review and finalise the manuscript. All authors contributed to the article and approved the submitted version.


The notebook contents are organized to optimize data analysis workflow and efficiency. The method is demonstrated through application to several different Ca2+ signaling experiment types.Calcium signaling data analysis has become increasing complex as the size of acquired datasets increases. In this paper we present a Ca This transformation process is invariably computer based and has become increasingly complex, working with ever larger quantities of data. Not only does the analysis process have to accommodate large amounts of data, but there is also the challenge of moving from a qualitative to a quantitative interpretation.2+ signaling experiments. Often contemporary imaging methods are designed to acquire information with high temporal and spatial fidelity from numerous individual cells and/or from multiple intracellular regions of interest. Interrogating these increasingly large data sets to provide quantitative information relating to latency, magnitude of response, frequency of events and spatial homogeneity is required to yield mechanistic insight into cellular processes controlling Ca2+ homeostasis and is increasingly labor intensive. In our experience, processing methods for Ca2+ signaling datasets share some common requirements but can also be highly specific to the needs of a particular lab and no current off-the-shelf software tool meets all these needs.  identification and post-processing includes notebooks for signal peak identification, frequency analysis and movie making. Detected ROIs are used to isolate and extract subsets of image data that are associated with individual cells under study. Peak analysis, in various forms, is used to quantify CaOur notebooks have been designed to automate repetitive steps and offer users the flexibility of diagnostic interactive data exploration. Notebook script features include generating plots, saving raw summary data in comma-separated-values (csv) files, and creating time-stamped directories for organizing results.2.We have developed a collection of eight notebooks, namely:ROI_DetectionRatiometric_ROI_DetectionPeak_DetectionCSV_Peak_DetectionCSV_Peak_Detection_SOCECSV_Peak_Detection_InhibitorFrequency_AnalysisMovie_Making2+ signaling experiments. Each experiment in our laboratory uses two or more of these notebooks for data analysis.These notebooks provide a flexible collection of analysis tools which can be applied to a broad range of CaThe notebooks employ a number of numerical methods, many of which originate from within computational computer graphics. For example, Canny edge detection  is used 3.2+ signaling, experiments that quantify store-operated calcium entry (SOCE), and experiments to define the efficacy of inhibitors of Ca2+ signaling pathways. Note that, with time varying fluorescence microscopy imaging of biological samples, it is essential to be able isolate specific cellular regions-of-interest in the image stack sequence for further analysis and this is a common requirement in each of our experiment types. Automated region of interest (ROI) detection is a key feature of our notebooks. This process was formerly highly labor-intensive and time consuming.The experiments in our laboratory fall broadly onto four types: single wavelength indicator experiments, experiments that quantify agonist induced Cahttps://github.com/jrugis/cell_tools/blob/master/Supplemental_Notes.pdfThis section gives an overview of how we employed the notebooks to analyze data from each of our different experiment types. A more detailed step-by-step outline of notebook usage is given in the supplemental notes that can be found at the link: 3.1.2+ indicators are now commonly used [2+ affinities useful for probing both bulk cytosolic and local [Ca2+]. Next, we describe a series of notebooks designed to analyze and present data generated using a single excitation wavelength indicator.A wide variety of so-called “single wavelength” or “single excitation” Canly used . These pnly used  are avai3.1.1.The analysis of gCamp experiments requires the use of the ROI_Detection, Peak_Detection and optionally the Frequency_Analysis and Movie_Making notebooks. Although the notebooks are described using the Mistgcamp-3 dataset, user-determined parameters offer flexibility in the types of single wavelength indicator data that can be processed.2+ indicator gCamp6F, specifically in exocrine cells  [2+], often from numerous cells in a field of view and captured over time with fast frame rates. Here we describe notebooks to first assign regions of interest (ROIs) to cells and subsequently to quantify various parameters associated with increases in [Ca2+].A host of hormones, neurotransmitters and growth factors signaling through phospholipase C lead to an increase in cytosolic [Ca2+] , 13. A c3.2.1.2+ release from the endoplasmic reticulum (ER) into the cytosol of the cell [2+] is commonly measured by changes in the ratiometric indicator, Fura-2 [2+] signal differ between stimulation with CCh or trypsin. The analysis of both the CCh and trypsin experiments is facilitated using the Ratiometric_ROI_Detection and CSV_Peak_Detection notebooks.Carbachol (CCh) and trypsin bind to muscarinic and protease-activated receptors, respectively, couple through Gαq to cause an increase in inositol 1,4,5-trisphosphate concentration and thus induce Cathe cell . The  in the cytosol, which is related to the rate of SOCE. Therefore, it is critical to accurately identify the maximum point of the concentration curves. Thus, unlike in CSV_Peak_Detection, CSV_Peak_Detection_SOCE does not rely on peak detection to find the peak of the concentration curve, as the rise can sometimes be too gradual for a peak detection method to work reliably. Instead, the maximum point within the stimulation zone is used. The latency point is still identified by the point at which the traces go above a user adjustable threshold level.When analyzing this type of experiment, it is important to measure the slope of the increase in [Ca2+ concentrations up to the point in which extracellular Ca2+ is depleted, at which point cytosolic Ca2+ concentrations gradually return to baseline levels. The subsequent exposure to CPA in Ca2+ imaging buffer, as seen from 900–1074 s in 2+ again causes the cytosolic Ca2+ concentrations to rise. With no limitation of extracellular Ca2+ concentration, this concentration will not return to baseline before the end of the experiment. The slope and latency of both increases are important measurements in these experiments.As seen in Note that, specifically, the rise time, slope, and maximum results from the CSV_Peak_Detection_SOCE notebook allow quantitative analysis of the extent to which the ATPases of the cell are inhibited. Using multiple stimulation zones (indicated with white background color in 3.4.2+ signaling machinery. Here, as an example, we analyzed the effect of the ORAI inhibitor, GSK7975a [2+ following exposure to CPA, as described in the previous section. The notebook is however designed to yield important parameters from a wide variety of experimental situations investigating the potency, efficacy, and kinetics of inhibitors of Ca2+ signaling.A frequent experimental question is the effect of an inhibitor of the CaGSK7975a , on the 3.4.1.2+ concentration is an important measurement in experiments to assess the effects of inhibitors. In addition, the difference between the initial cytosolic Ca2+ concentrations and lower cytosolic Ca2+ concentrations following exposure is an important parameter.The latency and slope of the decrease in cytosolic Ca2+ in the cytosol. Therefore, the minimum point within the stimulation zone is identified and used in the slope calculation. The latency point is determined as the point at which the trace goes below the calculated threshold level as opposed to above it with the other notebooks. The result is the ability to measure the downward slope of the trace.Analysis begins with ROI identification, the details of which can be found in the supplemental notes. Analysis continues using the CSV_Peak_Detection_Inhibitor notebook. This notebook allows for analysis of antagonist experiments, which calls for negative latency thresholds and negative slope measurements. When analyzing inhibitor experiments, it is important to measure the slope of the decrease in concentration of Ca2+ concentration. Another stimulation zone could have been selected, say from 300–400 s, and the automatic baseline from this stimulation zone could have been compared to the starting baseline to measure the difference in initial cytosolic Ca2+ concentrations and lower cytosolic Ca2+ concentrations caused by GSK exposure.2+ concentrations. By using multiple stimulation zones and automatic baselines, it is also possible to quantify the change in baseline Ca2+ levels in the cytosol as a result of ORAI inhibition.Note that, specifically, the slope and fall time measurements output by the CSV_Peak_Detection_Inhibitor notebook allow for a quantitative analysis of the effects of ORAI inhibition on cytosolic Ca4.We found that the notebook paradigm is a good conceptual fit in a laboratory environment which contributes to ease of use.Why didn’t we use the well-known software tool ImageJ ? In fact2+ signaling. However, in our case, because of a priori knowledge of cell size and shape, we were able to employ a very much less complex computational approach.Other researchers, such as, for example, Leigh, et al. , have de2+ signaling data is often qualitative and at least somewhat subjective. However, given mathematically well-defined criteria and consistent parameters, repeatable analysis is possible. The resultant consistent quantitative analysis largely eliminates the subjective component.Initial interpretation of CaIn practice, we found that peak identification, as opposed to frequency analysis, is preferred by users for characterizing response plots. This is probably due the fact that peak locations are more immediately intuitively meaningful than are relatively abstract frequency plots. We found that in general, our notebook collection, as a toolset and template, easily accommodates differing experiment requirements.Because our Jupyter notebooks contain script source code in readable plain text, nothing is hidden, the analysis process is transparent. The notebook collection is available for use by other research groups as publicly hosted downloadable open-source code in a repository on GitHub . InstrucIt does take some time to acquire a working familiarity with Python and Jupyter notebooks. However, both were designed with ease-of-use in mind and there are readily available online learning resources. We hope that other experimentalists will find these notebooks useful either as they are, or as a starting template for further bespoke customization. The learning curve for navigating and modifying Jupyter notebooks is modest but not trivial. In our experience the benefit far outweighs the “cost”.


We have shown by quantitative PCR analysis, that 5-HT1BR and 5-HT2BR mRNAs levels are almost equal in HUVEC. Immunofluorescent staining demonstrated, that 5-HT1BR and 5-HT2BR are expressed both in plasma membrane and inside the cells. Intracellular 5-HT1BR are localized mainly in the nuclear region, whereas 5-HT2BR receptors are almost evenly distributed in HUVEC. 5-HT, 5-HT1BR agonist CGS12066B, or 5-HT2BR agonist BW723C86 added to HUVEC caused a slight increase in [Ca2+]i, which was much lower than that of histamine, ATP, or SFLLRN, an agonist of protease-activated receptors (PAR1). However, activation of 5-HT1BR with CGS12066B followed by activation of 5-HT2BR with BW723C86 manifested a synergism of response, since several-fold higher rise in [Ca2+]i occurred. CGS12066B caused more than a 5-fold increase in [Ca2+]i rise in HUVEC in response to 5-HT. This 5-HT induced [Ca2+]i rise was abolished by 5-HT2BR antagonist RS127445, indicating that extracellular 5-HT acts through 5-HT2BR. Synergistic [Ca2+]i rise in response to activation of 5-HT1BR and 5-HT2BR persisted in a calcium-free medium. It was suppressed by the phospholipase C inhibitor U73122 and was not inhibited by the ryanodine and NAADP receptors antagonists dantrolene and NED-19. [Ca2+]i measurements in single cells demonstrated that activation of 5-HT2BR alone by BW723C86 caused single asynchronous [Ca2+]i oscillations in 19.8 ± 4.2% (n = 3) of HUVEC that occur with a long delay . On the contrary, histamine causes a simultaneous and almost immediate increase in [Ca2+]i in all the cells. Pre-activation of 5-HT1BR by CGS12066B led to a 3–4 fold increase in the number of HUVEC responding to BW723C86, to synchronization of their responses with a delay shortening, and to the bursts of [Ca2+]i oscillations in addition to single oscillations. In conclusion, to get a full rise of [Ca2+]i in HUVEC in response to 5-HT, simultaneous activation of 5-HT1BR and 5-HT2BR is required. 5-HT causes an increase in [Ca2+]i via 5-HT2BR while 5-HT1BR could be activated by the membrane-permeable agonist CGS12066B. We hypothesized that CGS12066B acts via intracellular 5-HT1BR inaccessible to extracellular 5-HT. Intracellular 5-HT1BR might be activated by 5-HT which could be accumulated in EC under certain pathological conditions.The aim of this work was to explore the involvement of 5-HT Endothelial cells (EC) are the first target of 5-HT in blood vessels and are in the frontline protecting vascular wall from elevated concentrations of 5-HT in plasma. However, little is known about the effect of 5-HT on [Ca2+]i in EC and the role of the individual receptors in this process. Vascular EC express 5-HT2BR coupled with Gq/11 protein and phospholipase C, 5-HT1BR coupled with Gi protein and adenylyl cyclase (AC) inhibition, and 5-HT4 receptors coupled with Gs protein and AC activation [2+]i elevation in HUVEC. The authors suggested that 5-HT-induced secretion of von Willebrand factor in HUVEC is mediated by a decrease in cAMP level due to activation of 5-HT1BR. On the other hand, it has been shown that in EC from human coronary arteries, activation of 5-HT1BR and 5-HT2BR causes an increase in [Ca2+]i [2BR agonist BW723C86 causes an increase in [Ca2+]i in human pulmonary artery EC by activating ryanodine-sensitive reticulum channels, while 5-HT1BR is not involved in the regulation of calcium metabolism [1BR and 5-HT2BR in the presence of hydrogen peroxide markedly elevate [Ca2+]i in HUVEC [2O2 attenuates histamine-induced [Ca2+]i rise in these cells i . Ullmer  [Ca2+]i  demonstrin HUVEC . On the se cells .2+]i via 5-HT1BR and 5-HT2BR in HUVEC and to compare the effects of their agonists with the effects of histamine and agonists of other receptors that cause an increase in [Ca2+]i in EC. CGS12066B and BW723C86 were used to stimulate 5-HT1BR and 5-HT2BR, respectively. Since both 5-HT1BR and 5-HT2BR might be involved in the action of 5-HT on calcium metabolism in HUVEC, we investigated what would be the changes in [Ca2+]i when they are simultaneously activated by their selective agonists. Surprisingly, it has been found that their action on [Ca2+]i in HUVEC exhibits a strong synergy. Registration of the responses of individual cells has demonstrated that simultaneous stimulation of 5-HT1BR and 5-HT2BR results in an increase in the number of the cells in which [Ca2+]i rise occurs and initiates the bursts of [Ca2+]i oscillations in addition to single oscillations. We have also demonstrated that 5-HT induces very weak [Ca2+]i rise in HUVEC via 5-HT2BR, and 5-HT1BR agonist CGS12066B greatly enhances the 5-HT effect.The aim of this work was to further explore the regulation of [Ca1BR and 5-HT2BR in HUVEC [1BR and 5-HT2BR in these cells  were recorded. As shown in 2+]i rise. The 5-HT2BR antagonist RS127445 i induced by BW723C86 in the presence of added CGS12066B. Dependence on extracellular calcium manifests itself in the second slow phase of the response to BW723C86, which indicates an inward current of Ca2+, apparently by the mechanism of store-operated calcium entry. It has been shown that EC of the human mesenteric artery and immortalized EA.hy926 endothelial-derived cells express RyR3, whereas RyR1 and RyR2 were not detected in these cells [2+]i in HUVEC in response to BW723C86 alone and together with CGS12066B  activated by NAADP. One of the functions of these channels is to trigger global calcium release by recruiting CICR channels at lysosomal–endoplasmic reticulum (ER) junctions [2+]i under the influence of CGS12066B and BW723C86, although attenuated histamine-induced [Ca2+]i elevation  (2+]i were performed in three wells with the average number of HUVEC in the field of view 116 ± 3 (mean ± SEM). Within 5 min after the addition of BW723C86, secondary single oscillations of [Ca2+]i occurred in 2.3 ± 0.5% (n = 3) of cells. After preincubation with CGS12066B, the cells responded to BW723C86 not only with single oscillations but also with bursts of oscillations. Responses to BW723C86 occurred in 69.7 ± 12.4% (n = 3) cells. Secondary single oscillations or bursts of oscillations in response to BW723C86 added after CGS12066B occurred in 39.7 ± 13.2% (n = 3) of HUVEC. Calcium responses of HUVEC to BW723C86 after preincubation with CGS12066B resembled those to histamine (n = 3) of HUVEC.Theoretically observed potentiation of the response to BW723C86 in a population of HUVEC can occur for several reasons: as a result of an increase in the number of responding cells; synchronization of i oscillations arising in response to VW723C86, to BW723C86 in the presence of CGS12066B, and to histamine. The values of maximal [Ca2+]i rise at the peaks of oscillations (max ΔF/Fo), and the maximum rate of [Ca2+]i increase (ΔF/Fo per s) during oscillations were calculated , 0.414 ± 0.0678 , and 0.5369 ± 0.0054 . The maximum rates of [Ca2+]i rise (ΔF/Fo per second) were 0.118 ± 0.0061 s−1 for BW723C86 , 0.171 ± 0.004 s−1  for BW723C86 with CGS12066B, and for histamine 0.4165 ± 0.0078 s−1 . The presented data indicate that the increase in [Ca2+]i under the combined action of CGS12066B and BW723C86 occurs as a result of an increase in the number of reacting cells, synchronization of their responses, the appearance of bursts of oscillations and repeated oscillations, and an increase in the rate of [Ca2+]i rise.We determined the parameters of i but increases the calcium signal from 5-HT2BR in HUVEC several-fold. The potentiating effect of CGS12066B begins to manifest itself at concentrations of up to 10 µM, and at 50 µM, the effect of CGS12066B reaches its maximum. According to previously published data [35S]GTPγS to rat striatal membranes with an EC50 of 1.2 µM and has a maximum effect at concentrations of 20 µM. These values are close to concentrations of CGS12066B, potentiating the calcium response to BW723C86 in HUVEC. Of note, the IC50 value for CGS12066B at the 5HT1BR recognition site is 51 nM, as determined using the binding of [3H]5HT in the presence of 1 μM spiperone i rise in response to BW723C86 or 5-HT can be excluded since it is suppressed by 5-HT2BR antagonist RS127445. We hypothesize that CGS12066B enhances the response to BW723C86 and 5-HT by activating intracellular 5-HT1BR. CGS12066B is a lipophilic compound capable of diffusing through the plasma membrane and crossing the blood–brain barrier i and how the response to BW723C86 is potentiated. It has been shown previously that in EC from the human pulmonary artery, this compound via 5-HT2BR stimulates calcium release from intracellular stores through a pathway, which involves activation of ryanodine receptors and is independent of PI-hydrolysis [2+]i in response to BW723C86, while the RyR inhibitor dantrolene does not affect it. This suggests that 5-HT2BR activates the release of calcium ions through InsP3-activated channels (InsP3R). InsP3R is a calcium ion-permeable channel formed by four protein subunits that, similar to RyR, are additionally activated by calcium ions [2+-binding sites that modulate the activity of the channel. According to [2+]i above the basal level increases the probability of channel open state, reduces the time of the closed state, and ensures their simultaneous opening in the cluster. Thus, there is an increase both in the intensity and duration of the current of calcium ions through the cluster formed by InsP3Rs. We suggest that the potentiation of the calcium signal from 5-HT2BR under the action of the 5-HT1BR agonist CGS12066B can be mediated by Ca2+-induced activation of InsP3R. This hypothesis is consistent with our data on an increase in the [Ca2+]i rise rate and peak height of BW723C86-induced calcium oscillations in single cells under the influence of CGS12066B. At the first stage of the calcium response to BW723C86 added after CGS1206B, [Ca2+]i elevates due to its mobilization from intracellular depots since the maximal rise and the rate of [Ca2+]i elevation does not depend on the presence of calcium ions in the extracellular medium. After emptying the intracellular depots, the entry of calcium ions from the outside by the SOCE mechanism, the refilling of the reticulum, and repeated oscillations appear. We assume that CGS12066B locally elevates [Ca2+]i and, in this way, potentiates InsP3-dependent Ca2+ release from the reticulum induced by BW723C86. The presence of a lag phase before the onset of Ca2+ oscillations in HUVEC in response to activation of 5-HT2BR can apparently be explained by a slow increase in the local [Ca2+]i to the threshold level required to open InsP3-sensitive channels. CGS12066B causes a slight increase in [Ca2+]i in HUVEC. The intracellular source from which calcium ions are released in response to CGS12066B has not been established. We have shown that NED-19, a blocker of NAADP-stimulated two-pore channels, does not reduce the effect of CGS12066B, suggesting that acid endolysosomal vesicles are not involved in this process. The question of how the synergism is realized with the simultaneous activation of 5-HT1BR and 5-HT2BR needs further investigation.The next question concerns the mechanism by which 5-HTium ions . In the rding to , the act2+]i is a trigger of many physiological and pathological processes. It has been shown that, normally, 5-HT through 5-HT2BR regulates differentiation and proliferation during development as well as cardiac structure and function in adults i in the EC, leading to their damage. The risk of pulmonary hypertension in humans is increased under the action of dexfenfluramine, which is an active metabolite and an agonist of 5-HT2BR. Bloodworth et al. i caused by the activation of 5-HT1BR, and 5-HT2BR is significantly increased by the addition of H2O2 [2+]i induced by histamine [1BR and 5-HT2BR upon activation of the rise in [Ca2+]i. In the vascular bed, a potent vasoconstrictor and an activator of smooth muscle cell proliferation endothelin-1 (ET-1) is produced by EC and released preferentially to the basal side of endothelium [2+]i leads to endothelial hypersecretion of ET-1, and this may be an additional factor in the pathogenesis of hypertension. The increase in the calcium response of EC to 5-HT via 5-HT2BR upon activation of 5-HT1BR should apparently be taken into account as a potential cause of 5-HT undesirable effects.An increase in i was performed in physiological salt solution (PSS) containing NaCl (145 mM), KCl (5 mM), MgCl2 (1 mM), CaCl2 (1 mM), HEPES (5 mM), and D-glucose (10 mM) at pH 7.4. Fluorescence was registered at 485 nm (excitation) and 530 nm (emission) at 25 °C using a Synergy 4 Microplate Reader . The changes in [Ca2+]i in HUVEC are presented as the ratio (F − Fo)/Fo or ΔF/Fo, where Fo is the basal fluorescence, and F is the fluorescence at time points during recording. Each curve in the graph is a superposition of five to ten curves recorded independently.HUVEC grown in 96-well plates were loaded with 1 µM CalciumGreen/AM dissolved with 0.02% Pluronic F-127 in M199 during 1 h at 37 °C in a CO2 incubator . Measurement of [Ca2+]i in single cells was carried out using a Leica DMI 6000 fluorescent microscope  using an HCX PL FLUOTAR L 20.0 × 0.40 DRY objective , diode illuminator with wavelengths of 480/40 nm and fluorescent filter L5 ET . HUVEC were grown in 24-well plates and loaded with 1 µM CalciumGreen/AM, as described above. Fluorescence was recorded from the area of 334 × 447 microns in the center of the well. Fluorescence measurements were taken at 1 s intervals. The resulting images were converted to tiff files and analyzed using the CellProfiler program i during the oscillation was determined from the increase in ΔF/Fo during one second during the development of the oscillation using Microsoft Excel 2010.Measurement of i in HUVEC via 5-HT2BR when 5-HT1BR are activated by the membrane-permeable agonist CGS12066B. We hypothesize that the potentiation of [Ca2+]i rise by CGS12066B is mediated by intracellular 5-HT1BR, which are inaccessible to 5-HT added from the outside. We assume that intracellular 5-HT1BR are activated by 5-HT when it is accumulated in EC under certain pathological conditions.HUVEC express 5-HT