Calcium Fluctuations Research Articles

A Digital Realization of Neuroglial Interaction Model and Its Network Structure

Astrocyte cells, the most existing abundant cells in central nervous system, play an essential role in modulating the neuronal activities, information processing, and regulating the synaptic plasticity through calcium (Ca2+) fluctuations of ions hemostasis by using a feedback mechanism. The pathophysiological and hypersynchronous neuronal activity can lead to the epileptic seizures, which is known as one of the neurodegenerative disorders in the field of neuroscience. This paper presents a modified neuron-astrocyte interaction (tripartite synapse) model based on leaky and integrate fire neuron and the astrocyte-synapse models using an area-efficient hardware approach called stochastic computing paradigm. The proposed model is synthesized physically on field-programmable gate array as a proof of concept. The implementation results of the presented model can mimic the bidirectional communication in biological minimal network of pre-postsynaptic and Ca2+-based model for astrocyte with considerably lower hardware cost. The influence of astrocytes on neural network behavioral has been investigated by providing a proper feedback mechanism and considering the role of gap junction coupling and the various coefficients on desynchronizing the impaired synchronization of the coupled neurons.

ORAI1 regulates sustained cytosolic free calcium fluctuations during breast cancer cell apoptosis and apoptotic resistance via a STIM1 independent pathway.

Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice <em>In Vivo</em>

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the α-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the α-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

An integrated strategy for the identification and screening of anti-allergy components from natural products based on calcium fluctuations and cell extraction coupled with HPLC-Q-TOF-MS.

Allergic diseases are a significant public health problem worldwide. Traditional Chinese medicines (TCMs) with reported anti-allergy effects may be important sources for the development of new anti-allergy drugs. Thus, establishing an analytical method that can simultaneously identify and screen anti-allergic compounds in TCMs is important. The increased concentrations of intracellular calcium ions resulting in mast cell degranulation releasing active mediators play a key role in allergic diseases, which can be used as a potential index to identify anti-allergic herbs and compounds. In this study, we provide a new strategy that was applied to screening natural anti-allergic compounds based on fluorescence calcium ion (Ca2+) fluctuation integrated with cell extract and high-performance liquid chromatography-mass spectrometry (HPLC-MS). A low-cost, convenient fluorescence detection Ca2+ signaling method was established and successfully applied to identify three herbs. Then, the method was integrated with biospecific cell fishing and HPLC-MS to screen potential active components that have the effect of stabilizing the cell membrane of rat basophilic leukemia granulocytes (RBL-2H3). Seven components, namely, albiflorin and paeoniflorin from Radix Paeoniae Alba, ononin and formononetin from Radix Astragali, cimifugin, 4'-O-β-D-glucosyl-5-O-methylvisamminol, and prim-O-glucosylcimifugin from Radix Saposhnikoviae were fished. These seven compounds have the effect of inhibiting cell Ca2+ influx. 4'-O-β-D-Glucosyl-5-O-methylvisamminol, prim-O-glucosylcimifugin, paeoniflorin, ononin, and formononetin significantly inhibit the release of β-hexosaminidase, which is equivalent to the positive drug. In conclusion, the integrated strategy of fluorescence detection calcium ion kinetic method binding with biospecific cell fishing was an effective mode to identify and screen natural anti-allergic compounds.

BIOCHEMICAL PARAMETERS CHANGES OF JAW BONE REMODELING IN EXPERIMENTAL ANIMALS UNDER THE INFLUENCE OF THE SUGGESTED PASTE, INTENDED FOR LOCAL TREATMENT OF CHRONIC GRANULATING PERIODONTITIS OF PERMANENT TEETH IN CHILDREN

The aim of the research was to study in an experiment on rats the biochemical parameters of the jaw bone remodeling under the influence of the developed paste, intended for the local treatment of chronic granulating periodontitis of permanent teeth in children.
 The experimental research was performed on 36 young rats (males) of the Wistar line (130-150g body weight), which were kept in the vivarium. The animals were divided into 2 groups: an experimental and a control one - 18 rats each. They were operated in sterile conditions under ketamine anesthesia. After applying the alcohol to the skin, a scalpel was used to make an incision along the lower edge of the mandible in the area of the canines and a longitudinal incision of the buccal muscle, then access to the bone was bluntly created. Using a sharp dental probe, a bone defect was modeled, in which the animals of the experimental group were injected with a paste based on comfrey tincture and calcium hydroxide. The control group was operated without the application of paste, the bone defect healed under a blood clot. The surgical wound was sutured in layers with polyamide thread and treated with 1% alcohol solution of diamond green.
 Blood sampling in experimental animals of both groups (6 rats each) was performed on 7th, 14th and 28th day after surgery. Animals were removed from the experiment by decapitation under ketamine anesthesia. Experimental animals were operated on and removed from the experiment during one day, under the same conditions.
 Determination of the amount of calcium and inorganic phosphorus in the blood serum of rats was carried out using standard kits of research by "SIMKO Ltd” company, Lviv. The activity of acidic and alkaline phosphatases in the serum was determined using kits of research by BIO-Ld-TEST company, Lachem (Czech Republic) and "Simbj Ltd", Lviv, respectively. In order to determine these parameters in the serum of normal rats, we took blood from 6 healthy young animals that did not participate in the experiment and were on the diet of the vivarium.
 The research found out that in the healing of artificial bone defects in the lower jaws of rats of the experimental group there were significant fluctuations in calcium and phosphorus levels and changes in the activity of acid phosphatase and alkaline phosphatase in the serum. The amount of Ca and P in the blood of rats decreased sufficiently. Alkaline phosphatase activity increased in both groups on the 7th and 14th day of the experiment, but the difference between the indicators in the groups was 38.35% (p <0.05) and 13.16% (p <0, 01) respectively. This was probably due to the rapid remission of inflammation at the site of surgery in the experimental group, as the paste we developed, which was used to fill a bone defect in this group of animals, has anti-inflammatory properties. Subsequent changes in alkaline phosphatase activity occurred similarly in both groups. This testified to the intensification of bone remodeling processes, as well as bone regeneration and mineralization under the influence of the paste we have developed.
 Our biochemical researches of the blood of experimental animals confirmed the previously obtained results of morphological and densitometric studies and showed that the recovery of bone wounds of the lower jaws of rats occurs almost equally in the experimental and control groups, and paste based on comfrey tincture and calcium hydroxide promotes bone regeneration at the site of the defect and stimulates osteosynthesis.

Glial cells have more important role in tDCS-induced brain activities rather than neurons

Transcranial Direct Current Stimulation (tDCS) is a non-invasive and sub-threshold method for altering the excitability of neurons to achieve target conditions. Many clinical applications of tDCS are in progress including treatment of neurological disease. Despite the therapeutic and practical potentials of tDCS, the precise mechanism of its impact is still unclear to a large extent. The initial perspective about the effect of tDCS on synaptic plasticity was that electric stimulation of the brain degrades the threshold of neural firing, thus facilitating electrical transmission and improving conductivity between neurons and ultimately leading to changes in the processing of information. However, new results of some experimental studies have changed the initial perspective. Recently, calcium imaging has shown sudden calcium fluctuations, followed by a change in cortical metaplasticity under tDCS. These pieces of evidence pinpoint the important role of astrocytes in describing the effect of tDCS.Astrocytes are glial cells that are traditionally viewed with only a supportive and nutritional role, but recent research expresses that they are active and contribute to processing and regulating synaptic information. We hypothesize that considering astrocytes as an important player in tDCS effect instead of the direct effect on voltage membrane of neurons is closer to the reality of tDCS effect mechanism. This hypothesis is examined here based on the experimental evidence published so far, which indicates astroglia involvement in tDCS-induced activities and results of our proposed computational model.

Serum Calcium Levels in Adult Patients With Chronic Hypoparathyroidism Treated With rhPTH(1–84) Compared With a Historical Control Cohort

Adult patients (pts) with chronic hypoparathyroidism (HypoPT) have wide fluctuations in albumin-corrected serum calcium (Ca) measurements.1 This study assessed Ca levels over a 5-yr period in adult pts with chronic HypoPT treated with or without recombinant human parathyroid hormone (1–84), rhPTH(1–84). The rhPTH(1–84)-treated pt cohort was from NCT01297309 (RACE) and NCT01199614 (HEXT) clinical trials. A historical control pt cohort with chronic HypoPT who did not receive rhPTH(1–84) or rhPTH(1–34) came from the US Explorys electronic medical record database (Jan 2007−Aug 2019); selection criteria were similar to those used for the rhPTH(1–84)-treated cohort. The index date was the day after initiation of treatment for the rhPTH(1–84) cohort and the day after the first calcitriol prescription for the control cohort. Pts were required to have ≥1 pair of serum albumin and Ca values occurring on the same date during the 6 months before index and 5 yrs (±6 months) after index. For pts from RACE, baseline and study visit data after rhPTH(1–84) initiation were collected from the antecedent trials. Specified ranges for albumin-corrected serum Ca values were: <7.5 mg/dL (<1.875 mmol/L); ≥7.5−<8.0 mg/dL (≥1.875−<2.0 mmol/L); ≥8.0−<9.0 mg/dL (≥2.0−<2.25 mmol/L); ≥9.0−<10.2 mg/dL (≥2.25−<2.55 mmol/L); and ≥10.2 mg/dL (≥2.55 mmol/L). Changes in Ca levels were assessed using multivariable regression models. There were 71 pts in the rhPTH(1–84) cohort and 119 pts in the control. Before the index date, rhPTH(1–84)-treated pts, compared with the control, were younger (mean±SD, 47.8±10.8 vs 54.9±15.5 years; P<0.001) and a lower proportion had acute manifestations of HypoPT (22.5% vs 64.7%; P<0.001). Over a 5-yr period, in adjusted analyses rhPTH(1–84)-treated pts, compared with the control, had a similar mean proportion of <7.5 mg/dL Ca measurements per pt (13.1% vs 13.1%; P=0.41), a higher proportion of ≥7.5−<8.0 mg/dL Ca measurements per pt (18.8% vs 10.6%; P<0.001), a similar proportion of ≥8.0−<9.0 mg/dL Ca measurements per pt (50.7% vs 48.5%; P=0.68), a lower proportion of ≥9.0−<10.2 mg/dL Ca measurements per pt (15.6% vs 24.1%; P<0.001), and a lower proportion of ≥10.2 mg/dL Ca measurements per pt (1.9% vs 3.7%; P=0.27). The rhPTH(1–84) cohort, compared with the control, had a higher proportion of pts with target range Ca measurements ≥7.5−<9.0 mg/dL (≥1.875−<2.25 mmol/L) for at least 50% of their values (88.7% vs 62.2%; P<0.001). Data interpretation is limited by the differing pt management (ie, trial protocols for the rhPTH[1–84] cohort and clinical practice for the control cohort). Over a 5-yr period, per pt serum Ca levels fluctuated in pts with chronic HypoPT, but levels were more stable in pts treated with rhPTH(1–84) and a lower proportion had hypercalcemia, compared with controls. 1. Ayodele O, et al. ASBMR 2020, 11−15 Sep 2020.

Novel Heterozygous Calcium Sensing Receptor (CASR) Genetic Variant in Child with Unique Phenotype: Hypocalcemia, Mandibular Hypoplasia, Renal Cysts and Type E Brachydactyly

Background: There are over 230 disease-causing variants in the calcium-sensing receptor gene (CaSR). Gain-of-function missense mutations in CaSR cause Autosomal Dominant Hypocalcemia (ADH) characterized by hypocalcemia (hCa), hypoparathyroidism (hPTH), and hypercalciuria. Patients with ADH are sensitive to fluctuations in serum calcium (Ca); and supplementation with Ca and vitamin D can cause inappropriate renal calcium retention leading to hypercalcemic events and long-term renal complications. Clinical Case: A 15-year-old adopted (at age 18 months) Korean female was initially diagnosed with hPTH and chronic hCa after presenting with hCa seizures. Laboratory values showed hCa (7.7 mg/dL), hyperphosphatemia (7.6 mg/dL) and hPTH (< 3 pg/mL.) Initially, she was treated with Ca supplementation (20 mg/kg/day elemental Ca), and calcitriol (0.01 mcg/kg/day). She presented at age 4 with hematuria and was found to have obstructive nephrolithiasis requiring operative intervention. Renal ultrasound (US) showed bilateral medullary nephrocalcinosis. She continued treatment with Ca and calcitriol. At age 6, a thiazide diuretic and potassium citrate supplement were added due to hypercalciuria. She had recurrent nephrolithiasis and persistent nephrocalcinosis. Follow-up renal US also showed bilateral renal cysts. Biweekly laboratory evaluation demonstrated an exuberant response to calcium supplementation. Serum Ca levels oscillated between 7.0 -10 mg/dL, but she showed minimal symptoms of hCa. At age 14, she was also recognized to have submandibular hypoplasia and brachydactyly of the 4th and 5th metacarpals and metatarsals bilaterally and genetic testing for CaSR gene mutation was requested. Sshe developed acute kidney injury and hypercalcemia, possibly precipitated by viral illness. However, 3 weeks before, calcitriol dose was increased to 1.25 mcg twice a day (0.07 mcg/kg/day). At admission, serum Ca was 12.7 mg/dL, iPTH 5.2 mg/dL, phosphorus 4.5 mg/dL, BUN 36 mg/dL, creatinine 1.85 mg/dL. Symptoms included headache, muscle spasm and throat spasm. She received intravenous fluids and recovered, but had an extended hospital stay. Targeted genetic analysis of the CaSR gene was completed, and identified a heterozygous variant (c.2506G>T, p.V836L) which is predicted to be likely pathogenic and cause ADH. After CaSR gene mutation identification, the calcitriol and also elemental Ca dosing were decreased to achieve a low Ca level (~7 mg/dL) with normal urine Ca/creatinine ratio. Patient remains asymptomatic. Conclusion: This is the first case of a novel mutation in the CaSR (c.2506G>T, p.V836L) associated with ADH, brachydactyly, renal cysts, and mandibular hypoplasia. Timely genetic testing for ADH in patients with newly diagnosed hPTH can lead to changes in therapy and improved prognosis.

Calcium-responsive contrast agents for functional magnetic resonance imaging.

Calcium ions represent one of the key second messengers accompanying neural activity and synaptic signaling. Accordingly, dynamic imaging of calcium fluctuations in living organisms represents a cornerstone technology for discovering neural mechanisms that underlie memory, determine behavior, and modulate emotional states as well as how these mechanisms are perturbed by neurological disease and brain injury. While optical technologies are well established for high resolution imaging of calcium dynamics, physical limits on light penetration hinder their application for whole-brain imaging in intact vertebrates. Unlike optics, magnetic resonance imaging (MRI) enables noninvasive large-scale imaging across vertebrates of all sizes. This has motivated the development of several sensors that leverage innovative physicochemical mechanisms to sensitize MRI contrast to intracellular and extracellular changes in calcium. Here, we review the current state-of-the-art in MRI-based calcium sensors, focusing on fundamental aspects of sensor performance, in vivo applications, and challenges related to sensitivity. We also highlight how innovations at the intersection of reporter gene technology and gene delivery open potential opportunities for mapping calcium activity in genetically targeted cells, complementing the benefits of small molecule probes and nanoparticle sensors.

Calcium-Sensing Receptor (CaSR), Its Impact on Inflammation and the Consequences on Cardiovascular Health.

The calcium Sensing Receptor (CaSR) is a cell surface receptor belonging to the family of G-protein coupled receptors. CaSR is mainly expressed by parathyroid glands, kidneys, bone, skin, adipose tissue, the gut, the nervous system, and the cardiovascular system. The receptor, as its name implies is involved in sensing calcium fluctuations in the extracellular matrix of cells, thereby having a major impact on the mineral homeostasis in humans. Besides calcium ions, the receptor is also activated by other di- and tri-valent cations, polypeptides, polyamines, antibiotics, calcilytics and calcimimetics, which upon binding induce intracellular signaling pathways. Recent studies have demonstrated that CaSR influences a wide variety of cells and processes that are involved in inflammation, the cardiovascular system, such as vascular calcification, atherosclerosis, myocardial infarction, hypertension, and obesity. Therefore, in this review, the current understanding of the role that CaSR plays in inflammation and its consequences on the cardiovascular system will be highlighted.

Distracting stimuli evoke ventral tegmental area responses in rats during ongoing saccharin consumption.

Disruptions in attention, salience and increased distractibility are implicated in multiple psychiatric conditions. The ventral tegmental area (VTA) is a potential site for converging information about external stimuli and internal states to be integrated and guide adaptive behaviours. Given the dual role of dopamine signals in both driving ongoing behaviours (e.g., feeding) and monitoring salient environmental stimuli, understanding the interaction between these functions is crucial. Here, we investigate VTA neuronal activity during distraction from ongoing feeding. We developed a task to assess distraction exploiting self‐paced licking in rats. Rats trained to lick for saccharin were given a distraction test, in which three consecutive licks within 1 s triggered a random distractor (e.g. light and tone stimulus). On each trial they were quantified as distracted or not based on the length of their pauses in licking behaviour. We expressed GCaMP6s in VTA neurons and used fibre photometry to record calcium fluctuations during this task as a proxy for neuronal activity. Distractor stimuli caused rats to interrupt their consumption of saccharin, a behavioural effect which quickly habituated with repeat testing. VTA neural activity showed consistent increases to distractor presentations and, furthermore, these responses were greater on distracted trials compared to non‐distracted trials. Interestingly, neural responses show a slower habituation than behaviour with consistent VTA responses seen to distractors even after they are no longer distracting. These data highlight the complex role of the VTA in maintaining ongoing appetitive and consummatory behaviours while also monitoring the environment for salient stimuli.

Establishment of MHHi001-A-5, a GCaMP6f and RedStar dual reporter human iPSC line for in vitro and in vivo characterization and in situ tracing of iPSC derivatives.

Transgenic hiPSC lines carrying reporter genes represent valuable tools for functional characterization of iPSC derivatives, disease modelling and clinical evaluation of cell therapies. Here, the hiPSC line 'Phoenix' (Haase et al., 2017) was genetically engineered using TALEN-based integration of the calcium sensor GCaMP6f and RedStarnuc reporter into the AAVS1 site. Characterization of undifferentiated cells and functional investigation of hiPSC-derived cardiomyocytes-containing BCTs showed a strong intracellular calcium transient-dependent GCaMP6f and eminent RedStarnuc signal. Therefore, our dual reporter line provides an excellent tool to facilitate monitoring of engraftment, calcium fluctuations and coupling of iPSC derivatives such as cardiomyocytes in vitro and in vivo in animal models.

Chronic BDNF simultaneously inhibits and unmasks superficial dorsal horn neuronal activity

Brain-derived neurotrophic factor (BDNF) is critically involved in the pathophysiology of chronic pain. However, the mechanisms of BDNF action on specific neuronal populations in the spinal superficial dorsal horn (SDH) requires further study. We used chronic BDNF treatment (200 ng/ml, 5–6 days) of defined-medium, serum-free spinal organotypic cultures to study intracellular calcium ([Ca2+]i) fluctuations. A detailed quantitative analysis of these fluctuations using the Frequency-independent biological signal identification (FIBSI) program revealed that BDNF simultaneously depressed activity in some SDH neurons while it unmasked a particular subpopulation of ‘silent’ neurons causing them to become spontaneously active. Blockade of gap junctions disinhibited a subpopulation of SDH neurons and reduced BDNF-induced synchrony in BDNF-treated cultures. BDNF reduced neuronal excitability assessed by measuring spontaneous excitatory postsynaptic currents. This was similar to the depressive effect of BDNF on the [Ca2+]i fluctuations. This study reveals novel regulatory mechanisms of SDH neuronal excitability in response to BDNF.

Management of hypocalcemia after modified bilateral radical neck dissection followed with total thyroidectomy: a case report

Hypocalcemia is a common complication after total thyroidectomy due to acute hypoparathyroidsm post-surgery. The incident varies from 7%-69% depends on the surgery center. The management is divided based on the onset and symptoms. Carpal-pedal spasm, seizure, bronchospasms are the emergency symptoms of hypocalcemia that require administration of intravenous calcium. However the management of asymptomatic hypocalcemia with oral calcium, vitamin D and hydrochlorothiazide can be difficult due to wide fluctuations of serum calcium and the risks of complication. Hereby we report a 14-year-old girl with hypocalcemia due to hypoparathyroidsm post modified bilateral radical neck dissection and total thyroidectomy. She had papillary thyroid carcinoma and lymph node metastases. After surgery, she developed classic sign of hypocalcemia, numbness in extremities which progressed to cramps. Physical examination revealed chvostek’s sign. Laboratory examination showed critical value of hypocalcemia with normal level of albumin, hypoparathyroidsm and hypovitaminosis D. The initial treatment was started by giving intravenous calcium gluconate 10% and calcitriol. Monitoring level of calcium and phosphate were performed regularly. Calcium level was maintained by giving high dose of elemental calcium, vitamin D3 and hydrochlorothiazide followed.

Dynamics of mixed bursting in coupled pre-Bötzinger complex

The pre-Bötzinger complex, which is located at a ventrolateral medulla of human and mammal, is considered to be the center for the generation of respiratory rhythms. In a normal state, the respiratory rhythm is uniform and orderly. Otherwise, the respiratory rhythm will change to a pathological state. Therefore, the monitoring of respiratory rhythm is of great significance in monitoring the health. In this paper, according to a two-coupled model of pre-Bötzinger complex with calcium ion current, we investigate the generation and transition mechanism of anti-phase bursting synchronization by using phase-plane analysis, bifurcation and fast-slow decomposition. It is found that the pre-Bötzinger complex model can exhibit mixed bursting when calcium ion concentration is at steady state, which indicates that the oscillation of calcium is not a necessary condition for the generation of mixed bursting. This is quite different from the results obtained in previous studies, indicating that the mixed bursting is caused by the periodic fluctuations of calcium. The methods used in this paper can provide a new idea for investigating the dynamics of mixed bursting, and it can also be applied to the study of other neuronal systems on a multiple time scale.

Self-Organized Criticality Governs Dynamic Equilibrium in Bacterial Calcium

How dynamic bacterial calcium is maintained, with kinetics faster than known mechanisms of cellular adaptation, is unknown. Herein, we discover bacterial calcium fluctuations are temporal fractals resulting from calcium dynamics self-organizing at a phase transition, a property known as self-organized criticality (SOC). SOC processes are poised at a transition between chaotic and ordered dynamics and are observed in many anthropogenic and natural systems, including neural circuits. SOC emerges due to calcium channel coupling which is mediated via membrane voltage. Environmental or genetic perturbations elicit different calcium dynamics, though always maintaining SOC, suggesting a continuum of critical attractors. We find moving along this continuum alters the collective information capacity of single-cell communities. In summary, we discover dynamic calcium equilibrium is maintained not by biological sensing, but rather by the physics of phase transitions, and our work connects ionic homeostasis in bacteria to the architecture of nervous systems in metazoans.

Hypoparathyroidism as the single major component for decades of autoimmune polyglandular syndrome type 1.

SummaryAutoimmune polyglandular syndrome type 1 (APS-1) is a very rare autoimmune entity, accounting for about 400 cases reported worldwide. It is characterized by the presence of at least two of three cardinal components: chronic mucocutaneous candidiasis (CMC), hypoparathyroidism and Addison’s disease. It typically manifests in childhood with CMC and years later with hypoparathyroidism. A 50-year-old man was referred to the Endocrinology outpatient clinic due to irregular follow-up of primary hypoparathyroidism diagnosed at age 7. Previous analysis reported frequent fluctuations of calcium and phosphate levels and persistent hypercalciuria. He presented several comorbidities, including bilateral cataracts, other ocular disorders, transient alopecia and chronic gastritis. Due to weight loss, fatigue, gastrointestinal complaints and the findings at objective examination, Addison’s disease and CMC were investigated and confirmed. Antifungal therapy and hormonal replacement were started with evident clinical improvement. Regarding hypoparathyroidism, calcium-phosphate product decreased and other extraskeletal calcifications were diagnosed, such as nephrolithiasis and in basal ganglia. Further evaluation by genetic analysis revealed homozygosity for a frameshift mutation considered to be a pathogenic variant. It was reported only in two Asian siblings in compound heterozygosity. This case highlights the broad phenotypic spectrum of APS-1 and the significative intra-familial phenotype variability. A complete clinical history taking and high index of suspicion allowed the diagnosis of this rare entity. This case clarifies the need for regular long-term follow-up. In the specific case of hypoparathyroidism and Addison’s disease in combination, the management of APS-1 can be complex.Learning points:Autoimmune polyglandular syndrome type 1 (APS-1) is a deeply heterogeneous genetic entity with a broad spectrum of clinical manifestations and a significant intra-family phenotypic variability.Early diagnosis of APS-1 is challenging but clinically relevant, as endocrine and non-endocrine manifestations may occur during its natural history.APS-1 should be considered in cases of acquired hypoparathyroidism, and even more so with manifestations with early onset, family history and consanguinity.APS-1 diagnosis needs a high index of suspicion. Key information such as all the comorbidities and family aspects would never be valued in the absence of a complete clinical history taking.Especially in hypoparathyroidism and Addison’s disease in combination, the management of APS-1 can be complex and is not a matter of simply approaching individually each condition.Regular long-term monitoring of APS-1 is essential. Intercalary contact by phone calls benefits the control of the disease and the management of complications.

Abstract 17071: Cell Communication Inference in Macrophages

Introduction: Tissue macrophages play diverse roles in the cardiovascular system during health and disease. They have diverse functions within tissues, but our understanding of their dynamics is limited because most macrophage characterization assays are destructive and have low temporal resolution. We asked whether these cells are dynamic and interconnected. Methods: Here, we describe experimental and analytical methods for measuring cell dynamics and inferring communication between cells in vitro and in vivo. We created a mouse (Csf1r-Cre x GCaMP5) expressing the Cre-inducible genetically encoded calcium indicator GCaMP5 under the regulation of the innate immune promoter, Csf1r, to non-destructively quantify high-frequency cell dynamics and differentiated them in culture using m-CSF. We developed custom image analysis routines and parameterization strategies for classifying calcium responses. Results: Our studies revealed that calcium reporter BMDMs display minimal fluctuations at baseline but exhibit a dynamic response to immunogenic DNA sensing. DNA-induced isolated cell injury and death, which precipitated cell communication that spread with a velocity of [9μm/s], consistent with an extracellular diffusion mechanism. We developed quantitative image analysis methods that corrected for random calcium fluctuations and identified statistically significant areas of correlated calcium changes suggestive of communication. An analytical pipeline enabled quantification of calcium spike dynamics and correlations of dynamic calcium profiles of single cell sharing a local microenvironment. This resulted in an “improbable synchrony” metric that allowed localization of communication in time and space. We adapted the pipeline for in vivo studies and tested them in a dorsal window chamber model using intravital microscopy. At 2Hz sampling frequency, we identified 27 potential communication events as they responded to complex microenvironmental cues in vivo. Conclusion: The experimental and analytical methods for inferring cell communication provide a new quantitative toolkit for investigating known as-yet undiscovered cell communication pathways.

Quantifying the efficacy of checkpoint inhibitors on CD8+ cytotoxic T cells for immunotherapeutic applications via single-cell interaction

The inhibition of the PD1/PDL1 pathway has led to remarkable clinical success for cancer treatment in some patients. Many, however, exhibit little to no response to this treatment. To increase the efficacy of PD1 inhibition, additional checkpoint inhibitors are being explored as combination therapy options. TSR-042 and TSR-033 are novel antibodies for the inhibition of the PD1 and LAG3 pathways, respectively, and are intended for combination therapy. Here, we explore the effect on cellular interactions of TSR-042 and TSR-033 alone and in combination at the single-cell level. Utilizing our droplet microfluidic platform, we use time-lapse microscopy to observe the effects of these antibodies on calcium flux in CD8+ T cells upon antigen presentation, as well as their effect on the cytotoxic potential of CD8+ T cells on human breast cancer cells. This platform allowed us to investigate the interactions between these treatments and their impacts on T-cell activity in greater detail than previously applied in vitro tests. The novel parameters we were able to observe included effects on the exact time to target cell killing, contact times, and potential for serial-killing by CD8+ T cells. We found that inhibition of LAG3 with TSR-033 resulted in a significant increase in calcium fluctuations of CD8+ T cells in contact with dendritic cells. We also found that the combination of TSR-042 and TSR-033 appears to synergistically increase tumor cell killing and the single-cell level. This study provides a novel single-cell-based assessment of the impact these checkpoint inhibitors have on cellular interactions with CD8+ T cells.