Ca Influx Research Articles

Mitochondrial calcium uniporter is essential for hearing and hair cell preservation in congenic FVB/NJ mice

Mitochondrial Ca2+ regulates a wide range of cell processes, including morphogenesis, metabolism, excitotoxicity, and survival. In cochlear hair cells, the activation of mechano-electrical transduction and voltage-gated Ca2+ channels result in a large influx of Ca2+. The intracellular rise in Ca2+ is partly balanced by the mitochondria which rapidly uptakes Ca2+ via a highly selective channel comprised of the main pore-forming subunit, the mitochondrial Ca2+ uniporter (MCU), and associated regulatory proteins. MCU thus contributes to Ca2+ buffering, ensuring cytosolic homeostasis, and is posited to have a critical role in hair cell function and hearing. To test this hypothesis, Ca2+ homeostasis in hair cells and cochlear function were investigated in FVB/NJ mice carrying the knockout allele of Mcu (Mcu+/− or Mcu−/−). The Mcu knockout allele, which originated in C57BL/6 strain cosegregated along with Cdh23ahl allele to the FVB/NJ strain, due to the close proximity of these genes. Neither Mcu+/− nor Mcu−/− genotypes affected cochlear development, morphology, or Ca2+ homeostasis of auditory hair cells in the first two postnatal weeks. However, Mcu−/− mice displayed high-frequency hearing impairment as early as 3 weeks postnatal, which then progressed to profound hearing loss at all frequencies in about 6 months. In Mcu+/− mice, significantly elevated ABR thresholds were observed at 6 months and 9 months of age only at 32 kHz frequency. In three-month-old Mcu−/− mice, up to 18% of the outer hair cells and occasionally some inner hair cells were missing in the mid-cochlear region. In conclusion, mitochondrial Ca2+ uniporter is not required for the development of cochlea in mice, but is essential for hearing and hair cell preservation in congenic FVB/NJ mice.

Inactivation of Staphylococcus aureus using ultrasound in combination with thyme essential oil nanoemulsions and its synergistic mechanism

The present study investigated the antimicrobial effect and mechanism of ultrasound (US) with thyme essential oil nanoemulsions (TEON) on the inactivation of Staphylococcus aureus (S. aureus) cells. A remarkable increase in reducing the population of S. aureus was observed when cells were subjected to combined US (3.33 W/mL, 3 min) and TEON (0.375 mg/mL) treatment, which exhibited synergistic inactivation efficiency and caused a 3.21-log reduction in viability. The most severe damage to cell morphology was visualized in US + TEON group using scanning electron microscopy. Other proposed mechanisms involved that ultrasound reduced the membrane hydrophobicity, which increased the exposure of cell membrane to TEON. Moreover, the greatest degree of membrane permeabilization was evaluated, along with the massive leakage of intracellular contents. Depolarization in membrane potential was also observed and confirmed by the excessive outflow of K+ and influx of Ca2+. On the other hand, a significant decrease in membrane fluidity was monitored and further verified by the modifications in bacterial membrane fatty acid profile. This study also highlighted the impact of US + TEON treatment on bacterial membrane compositions involving proteins, nucleic acids, and phospholipids by infrared spectroscopy. Current study provides a proof-of-concept into the synergistic mechanisms of the integrated technology for enhancing S. aureus inactivation.

Lactation and gestation controls on calcium isotopic compositions in a mammalian model.

Lactation and gestation are among the physiological events that trigger the most intense changes in body calcium (Ca) fluxes. Along with the composition of the animal 2021 diet, these events are suspected to impact the Ca isotopic composition of Ca body reservoirs but their dynamics are poorly understood. In this study, we monitored a group of domestic sows across a full reproduction cycle. We collected tissues and fluids (blood, urine, milk, colostrum, umbilical blood, adult and piglet bones) at different steps of gestation and lactation, and analyzed their Ca isotopic compositions (i.e. δ44/42Ca) by means of multi-collector inductively coupled plasma mass spectrometry. Among other results, we report the first observations of Ca isotopic fractionation between maternal and umbilical blood (Δ44/42Caumbilical blood-sow blood = -0.18±0.11‰, n=3). Our data also highlight that gestation and lactation periods are characterized by small diet-bone Ca isotopic offsets (Δ44/42Cabone-diet = -0.28±0.11‰, n=3), with 44Ca-enriched blood compositions during nursing (Δ44/42Canursing blood-gestation blood = $+ 0.42{\rm{\,\,}}_{ - 0.12}^{ + 0.11}$‰, n=3). Under the light of an up-to-date mammalian box model, we explored different scenarios of gestation and lactation Ca fluxes experienced by a sow-like animal. These simulations suggest that gestation changes on body δ44/42Ca values may result from the intensification of Ca absorption by the animal, whereas the production of 44Ca-depleted milk is the main driver for the 44Ca enrichment in blood during lactation. In addition, our results also support that bone mineralization could be associated with a more restricted Ca isotopic fractionation than previously envisioned. Together, these results refine the framework of Ca isotope applications, notably regarding the monitoring of human bone balance and the study of species and ecosystems from the present and the past.

Aequorin as a Useful Calcium-Sensing Reporter in Candida albicans.

In Candida albicans, calcium ions (Ca2+) regulate the activity of several signaling pathways, especially the calcineurin signaling pathway. Ca2+ homeostasis is also important for cell polarization, hyphal extension, and plays a role in contact sensing. It is therefore important to obtain accurate tools with which Ca2+ homeostasis can be addressed in this fungal pathogen. Aequorin from Aequorea victoria has been used in eukaryotic cells for detecting intracellular Ca2+. A codon-adapted aequorin Ca2+-sensing expression system was therefore designed for probing cytosolic Ca2+ flux in C. albicans. The availability of a novel water-soluble formulation of coelenterazine, which is required as a co-factor, made it possible to measure bioluminescence as a readout of intracellular Ca2+ levels in C. albicans. Alkaline stress resulted in an immediate influx of Ca2+ from the extracellular medium. This increase was exacerbated in a mutant lacking the vacuolar Ca2+ transporter VCX1, thus confirming its role in Ca2+ homeostasis. Using mutants in components of a principal Ca2+ channel (MID1, CCH1), the alkaline-dependent Ca2+ spike was greatly reduced, thus highlighting the crucial role of this channel complex in Ca2+ uptake and homeostasis. Exposure to the antiarrhythmic drug amiodarone, known to perturb Ca2+ trafficking, resulted in increased cytoplasmic Ca2+ within seconds that was abrogated by the chelation of Ca2+ in the external medium. Ca2+ import was also dependent on the Cch1/Mid1 Ca2+ channel in amiodarone-exposed cells. In conclusion, the aequorin Ca2+ sensing reporter developed here is an adequate tool with which Ca2+ homeostasis can be investigated in C. albicans.

Annexins A1 and A2 Accumulate and Are Immobilized at Cross-Linked Membrane-Membrane Interfaces.

Rapid membrane repair is required to ensure cell survival after rupture of the plasma membrane. The annexin family of proteins is involved in plasma membrane repair (PMR) and is activated by the influx of Ca2+ from the extracellular medium at the site of injury. Annexins A1 and A2 (ANXA1 and ANXA2, respectively) are structurally similar and bind to negatively charged phosphatidylserine (PS) to induce membrane cross-linking and to promote fusion, which are both essential processes that occur during membrane repair. The degree of annexin accumulation and the annexin mobility at cross-linked membranes are important aspects of ANXA1 and ANXA2 function in repair. Here, we quantify ANXA1- and ANXA2-induced membrane cross-linking between giant unilamellar vesicles (GUVs). Time-lapse measurements show that ANXA1 and ANXA2 can induce membrane cross-linking on a time scale compatible with PMR. Cross-linked membrane-membrane interfaces between the GUVs persist in time without fusion, and quantification of confocal microscopy images demonstrates that ANXA1, ANXA2, and, to a lesser extent, PS lipids accumulate at the double membrane interface. Fluorescence recovery after photobleaching shows that the annexins are fully immobilized at the double membrane interface, whereas PS lipids display a 75% decrease in mobility. In addition, the complete immobilization of annexins between two membranes indicates a high degree of network formation between annexins, suggesting that membrane cross-linking is mainly driven by protein-protein interactions.

Ca2+ Microdomains, Calcineurin and the Regulation of Gene Transcription.

Ca2+ ions function as second messengers regulating many intracellular events, including neurotransmitter release, exocytosis, muscle contraction, metabolism and gene transcription. Cells of a multicellular organism express a variety of cell-surface receptors and channels that trigger an increase of the intracellular Ca2+ concentration upon stimulation. The elevated Ca2+ concentration is not uniformly distributed within the cytoplasm but is organized in subcellular microdomains with high and low concentrations of Ca2+ at different locations in the cell. Ca2+ ions are stored and released by intracellular organelles that change the concentration and distribution of Ca2+ ions. A major function of the rise in intracellular Ca2+ is the change of the genetic expression pattern of the cell via the activation of Ca2+-responsive transcription factors. It has been proposed that Ca2+-responsive transcription factors are differently affected by a rise in cytoplasmic versus nuclear Ca2+. Moreover, it has been suggested that the mode of entry determines whether an influx of Ca2+ leads to the stimulation of gene transcription. A rise in cytoplasmic Ca2+ induces an intracellular signaling cascade, involving the activation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin and various protein kinases (protein kinase C, extracellular signal-regulated protein kinase, Ca2+/calmodulin-dependent protein kinases). In this review article, we discuss the concept of gene regulation via elevated Ca2+ concentration in the cytoplasm and the nucleus, the role of Ca2+ entry and the role of enzymes as signal transducers. We give particular emphasis to the regulation of gene transcription by calcineurin, linking protein dephosphorylation with Ca2+ signaling and gene expression.

Real-time temperature measurement of living cells exposed to microwaves using a temperature-dependent fluorescent dye

Real-time measurement of temperature in living cells exposed to microwaves is challenging because of the low spatial resolution of conventional temperature probes. Here, we established a noninvasive method by using a temperature-dependent fluorescent dye, Rhodamine B (Rho-B) to stain living cells that were exposed to a customized microwave exposure device. The relationship between Rho-B fluorescence and the temperature of living cells, and between Fluo4/AM and the inward inflow of Ca2+ were established. The results showed that the fluorescence intensity changed by 7.9%/°C as the temperature rose from 25 °C to 37 °C. The spatial resolution and temporal resolution of this method were 225 × 225 µm 2 and 1.5 s, respectively. In conclusion, the real-time temperature measurement technique is simple to use and requires no specialized equipment beyond a LSCM. This method can provide real-time information of the temperature changes in cells simultaneously with the investigation of calcium concentration.

CBD activation of TRPV1 induces oxidative signaling and subsequent ER stress in breast cancer cell lines.

Endoplasmic reticulum (ER) stress is an imbalance between the protein-folding load and capacity of ER. It can be induced by various physiological conditions, activating the unfolded protein response (UPR) to re-establish homeostasis, promoting cell survival. Under severe or chronic stress, apoptosis is induced. Normal cells generally do not experience continuous ER stress induction. The stressful conditions experienced in the tumor microenvironment facilitate chronic ER stress and UPR activation, which plays a pivotal role in tumour survival. Exacerbation of pre-existing ER stress can trigger cancer cell death, with a minimal effect on normal cells. Current literature suggests that cannabinoid treatment may induce cancer cell death via ER stress; however, little is known about the mechanisms of induction. This study proposed that cannabidiol (CBD) mechanism occurred through the influx of Ca2+ via the TRPV1 receptor, and increasing reactive oxygen species (ROS) production affects protein folding and induces ER stress. ER stress was induced, and detection and quantification were completed using Thioflavin T staining and GRP78 by western blot analysis. The effect of cannabinoid treatment on ROS production and Ca2+ influx was measured. CBD was the most potent ER stress inducer, significantly increasing Ca2+ and ROS accumulation. Concomitant treatment with CBD and an antioxidant significantly increased cell viability and decreased ER stress induction in the MCF7 cell line. Concomitant treatment with a TRPV1 antagonist increased viability in this cell line. In conclusion, the data suggested that CBD induced ER stress via Ca2+ influx through the TRPV1 receptor, thereby elevating intracellular ROS levels and disrupting protein folding.

Piezo1 channel activation in response to mechanobiological acoustic radiation force in osteoblastic cells

Mechanobiological stimuli, such as low-intensity pulsed ultrasound (LIPUS), have been shown to promote bone regeneration and fresh fracture repair, but the fundamental biophysical mechanisms involved remain elusive. Here, we propose that a mechanosensitive ion channel of Piezo1 plays a pivotal role in the noninvasive ultrasound-induced mechanical transduction pathway to trigger downstream cellular signal processes. This study aims to investigate the expression and role of Piezo1 in MC3T3-E1 cells after LIPUS treatment. Immunofluorescence analysis shows that Piezo1 was present on MC3T3-E1 cells and could be ablated by shRNA transfection. MC3T3-E1 cell migration and proliferation were significantly increased by LIPUS stimulation, and knockdown of Piezo1 restricted the increase in cell migration and proliferation. After labeling with Fluo-8, MC3T3-E1 cells exhibited fluorescence intensity traces with several high peaks compared with the baseline during LIPUS stimulation. No obvious change in the fluorescence intensity tendency was observed after LIPUS stimulation in shRNA-Piezo1 cells, which was similar to the results in the GsMTx4-treated group. The phosphorylation ratio of ERK1/2 in MC3T3-E1 cells was significantly increased (P < 0.01) after LIPUS stimulation. In addition, Phalloidin-iFluor-labeled F-actin filaments immediately accumulated in the perinuclear region after LIPUS stimulation, continued for 5 min, and then returned to their initial levels at 30 min. These results suggest that Piezo1 can transduce LIPUS-induced mechanical signals into intracellular calcium. The influx of Ca2+ serves as a second messenger to activate ERK1/2 phosphorylation and perinuclear F-actin filament polymerization, which regulate the proliferation of MC3T3-E1 cells.

The Negative Allosteric Modulator EU1794-4 Reduces Single-Channel Conductance and Ca2+ Permeability of GluN1/GluN2A N-Methyl-d-Aspartate Receptors.

NMDA receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic currents. These receptors are involved in several important brain functions, including learning and memory, and have also been implicated in neuropathological conditions and acute central nervous system injury, which has driven therapeutic interest in their modulation. The EU1794 series of positive and negative allosteric modulators of NMDA receptors has structural determinants of action near the preM1 helix that is involved in channel gating. Here, we describe the effects of the negative allosteric modulator EU1794-4 on GluN1/GluN2A channels studied in excised outside-out patches. Coapplication of EU1794-4 with a maximally effective concentration of glutamate and glycine increases the fraction of time the channel is open by nearly 1.5-fold, yet reduces single-channel conductance by increasing access of the channel to several subconductance levels, which has the net overall effect of reducing the macroscopic current. The lack of voltage-dependence of negative modulation suggests this is unrelated to a channel block mechanism. As seen with other NMDA receptor modulators that reduce channel conductance, EU1794-4 also reduces the Ca2+ permeability relative to monovalent cations of GluN1/GluN2A receptors. We conclude that EU1794-4 is a prototype for a new class of NMDA receptor negative allosteric modulators that reduce both the overall current that flows after receptor activation and the flux of Ca2+ ion relative to monovalent cations. SIGNIFICANCE STATEMENT: NMDA receptors are implicated in many neurological conditions but are challenging to target given their ubiquitous expression. Several newly identified properties of the negative allosteric modulator EU1794-4, including reducing Ca2+ flux through NMDA receptors and attenuating channel conductance, explain why this modulator reduces but does not eliminate NMDA receptor function. A modulator with these properties could have therapeutic advantages for indications in which attenuation of NMDA receptor function is beneficial, such as neurodegenerative disease and acute injury.

Abstract P785: The B Cell Response to Extracellular Glutamate in the Ischemic Brain

Background: Neuronal networks require significant neurotrophic support for functional plasticity after stroke. We showed that B cells exhibit a cell-specific migration pattern in the post-stroke brain. Post-stroke B cell depletion impedes neurogenesis, increases anxiety, and exacerbates memory deficits in mice; deficits generally mediated by brain regions occurring outside the initial infarct. We hypothesize that the post-stroke microenvironment can enhance neurotrophic capacities of B cells to promote plasticity. Methods: Splenic B cells were isolated from 3-5 mo-old male C57Bl/6J mice. B cell N-methyl-D-aspartate receptor (NMDAR) subunits were identified by confocal microscopy. The acute (8 min) Ca 2+ response to 1uM glutamate (glu) +/- NMDAR antagonists (10uM DAPV (competitive NMDAR inhibitor), 30uM ifenprodil (ifen., GluN2B subunit inhibitor), and 10uM TCN201 (GluN2A subunit inhibitor)) was assessed via flow cytometry in B cells (+/- 5ug/mL LPS). B cell viability and neurotrophin (NT)-related genes were assessed by flow cytometry and qPCR, respectively, in B cells (+/- LPS) treated with glu +/- NMDAR antagonists for 24h. Data were analyzed in Graphpad Prism. Results: B cells express functional GluN2A- and GluN2B-containing NMDARs that influx Ca 2+ in response to extracellular glu (*p&lt;0.05). While LPS did not impact NMDAR-dependent Ca 2+ influx in most B cell subsets, Ca 2+ influx was significantly reduced by NMDAR antagonists in LPS-stimulated B cells (Effector B cells (DAPV *p&lt;0.05, ifen **p&lt;0.01), Bregs (DAPV *p&lt;0.05, Ifen *p&lt;0.05), B220 + antibody-secreting cells (ifen *p&lt;0.05, TCN201 *p&lt;0.05)). Furthermore, a 24h glu treatment increased NT (BDNF: 2.28-fold, IL-10: 27.16-fold), NT receptor (TrkB: 1.33-fold) and NMDAR (GluN2A: 2.01-fold, GluN2B: 1.27-fold) expression in LPS-stimulated B cells (vs. untreated controls). Conclusions: Our studies show that B cells respond to glu via NMDARs. Our data suggests that exposure to physiologic levels of glu enhance NMDAR-dependent signaling and upregulate NTs and NT receptors. These results are the first to indicate a glu-induced neurotrophic role for B cells in the ischemic brain. Future studies will determine whether B cell-derived NTs can protect neurons after stroke.

Rising Copper Exposure Effects on Nutrient Uptake in Two Species with Distinct Copper Tolerance

Excessive copper concentrations affect nutrient uptake in sensible species. However, the effects of copper on nutrient uptake in tolerant species have not been studied equally well. Thus, the main objective of this study was to determine the effect of rising Cu exposure on nutrient uptake rates and root/shoot nutrient contents in two species with distinct copper tolerance: Acacia caven (Mol.) Mol. and Helianthus annuus L. To this end, a hydroponic experiment was conducted. Copper treatments were applied at concentrations of 0, 2.0, 3.9, 7.9, and 15.7 μM. Relative root elongation was chosen as the endpoint for Cu toxicity assessment. The results of our study demonstrate that A. caven and H. annuus differ greatly in their tolerance to Cu. Copper concentration in the solution associated with the calculated EC25 was found to be six times higher in A. caven (15 μM) than in H. annuus (2.3 μM). When effective concentrations were instead based on the measured root Cu concentrations, the difference observed was ten times greater (1044 and 98 mg/kg for A. caven and H. annuus, respectively). Both species showed equal Cu uptake kinetics, with root absorbing power (α) of 7.5 ± 0.7 × 10–6 and 7.8 ± 0.5 × 10–6 cm s–1 for A. caven and H. annuus, respectively. Rising Cu concentrations in the exposure solution progressively diminished the influx of Ca into the roots of H. annuus, whereas no significant effect was noted for A. caven.

English

The effect of extracellular calcium on isolated uterine muscle contractions stimulated by acetone leaf extract of Anogeissus leiocarpa was investigated in a rat. About 12 m segment of the uterine muscle strip was mounted initially in a thermostatically regulated organ bath (37&deg;C) containing normal Physiological Salt Solution (PSS; De Jalon) and later inphysiological salt solution devoid of CaCl2. The extract contracted the uterine muscle preparation in a concentration-related manner in normal PSS, with 0.53 mg/ml as the lowest active concentration. This contractile response was abolished by the calcium channel blocker, verapamil HCl (2 &micro;g/ml). The extract however, did not evoke any contractile activity on the isolated tissue in a calcium-free media (PSS), even when caffeine (2 mmol) known to release calcium via the calcium induced-calcium release (CICR) mechanism was added to the perfusate. The results demonstrated the requirement for extracellular calcium for the extract mediated contractions and the inability of the extract to access calcium from intracellular storage sites such as the sarcoplasmic reticulum and the mitochondria. Therefore, the influx of Ca2+ into the cell cytosol is a pre-requisite for the extract-mediated uterine muscle contraction. Key word: Anogeissus leiocarpa, thermostatically organ bath, verapamil, caffeine, uterus.

Groundwater discharge impacts marine isotope budgets of Li, Mg, Ca, Sr, and Ba

Groundwater-derived solute fluxes to the ocean have long been assumed static and subordinate to riverine fluxes, if not neglected entirely, in marine isotope budgets. Here we present concentration and isotope data for Li, Mg, Ca, Sr, and Ba in coastal groundwaters to constrain the importance of groundwater discharge in mediating the magnitude and isotopic composition of terrestrially derived solute fluxes to the ocean. Data were extrapolated globally using three independent volumetric estimates of groundwater discharge to coastal waters, from which we estimate that groundwater-derived solute fluxes represent, at a minimum, 5% of riverine fluxes for Li, Mg, Ca, Sr, and Ba. The isotopic compositions of the groundwater-derived Mg, Ca, and Sr fluxes are distinct from global riverine averages, while Li and Ba fluxes are isotopically indistinguishable from rivers. These differences reflect a strong dependence on coastal lithology that should be considered a priority for parameterization in Earth-system models.

STUDY OF CLINICAL CASES OF MYOCHE’S MYOPATHY AND DIFFERENTIAL DIAGNOSIS WITH OTHER TYPES OF ADVERSE MYOPATHIES

Markesbery-Griggs myopathy, Miyoshi type (MM) is a rare type of myopathy, a form muscular dystrophy with the main involvement of the lower girdle and distal parts of the legs. Due to complexity of genetic testing, the diagnosis is mainly made on the neurological examination of the patient, which adds value to this case report. The childhood or adolescence onset of the disease is characterized initially by the calf muscles` wasting, accompanied by the severe elevation of the serum creatine kinase, as well as a slowly progressive ascending course. The disease refers to dysferlinopathies with various mutations in the DYSF gene. The dysferlin protein is localized in the plasma membrane and in the T-tubule system of skeletal muscles. Physiologically, skeletal muscles are constantly exposed to micromembrane lesions. Depending on the severity, these damages are restored using various complexes. One of the main reparative complexes is the dysferlin-dependent mechanism. Mutations can lead to a defect in the membrane repair, causing the influx of Ca 2+ into the cell, which leads to a cell`s destruction. There are three genetically identifiable types of Miyoshi myopathy: MMD1, MMD2, MMD3. The main clinical signs of the disease are the muscle weakness and atrophy, with predominant involvement of the distal parts of the lower limbs, especially in the gastrocnemius and plantar muscles. The MM causes tip toe walking disturbances and difficulties in climbing the stairs. Progression of the disease and further atrophy leads to the wasting of the lower girdle muscles, mainly gluteal ones. Peculiarity of these myopathies is the absence of cardiomyopathy, due to the immunity of cardiomyocytes to a deficiency of the protein dysferelin. Diagnosis is made on the basis of muscle biopsy and molecular genetic testing. The gold standard is immunoblotting or immunohistochemistry. One of treatment methods is the use of improperly folded dysferlin (treatment with a proteasome inhibitor MG-132) in fibroblasts with restoration of membrane sealing. The aim of this case report is to present an example of a possible clinical diagnosis of MM in a young man, in the absence of opportunities for molecular genetic testing.

Post-Translational Modification of Cysteines: A Key Determinant of Endoplasmic Reticulum-Mitochondria Contacts (MERCs).

Cells must adjust their redox state to an ever-changing environment that could otherwise result in compromised homeostasis. An obvious way to adapt to changing redox conditions depends on cysteine post-translational modifications (PTMs) to adapt conformation, localization, interactions and catalytic activation of proteins. Such PTMs should occur preferentially in the proximity of oxidative stress sources. A particular concentration of these sources is found near membranes where the endoplasmic reticulum (ER) and the mitochondria interact on domains called MERCs (Mitochondria-Endoplasmic Reticulum Contacts). Here, fine inter-organelle communication controls metabolic homeostasis. MERCs achieve this goal through fluxes of Ca2+ ions and inter-organellar lipid exchange. Reactive oxygen species (ROS) that cause PTMs of mitochondria-associated membrane (MAM) proteins determine these intertwined MERC functions. Chronic changes of the pattern of these PTMs not only control physiological processes such as the circadian clock but could also lead to or worsen many human disorders such as cancer and neurodegenerative diseases.

The Hydrogeochemical Stratigraphy of Brines and Its Implications on Water Management in the Central Jordan-Dead Sea Rift Valley, Israel

The exploratory borehole Megiddo-Jezre’el 1 (MJ1) was drilled in Israel, in the Bet She’an Valley which branches out from the Central Jordan Rift. It reached the depth of 5060 m and bottomed within the Upper Triassic Mohilla Fm. Following the increase of groundwater exploitation, the Cl- concentrations increased and ionic ratios changed indicating inflow of Ca2+-Cl- brines, the origins of which were hitherto unknown. Data from the new MJ1 borehole revealed that rock porosities decrease with depth. Lowermost values of about 3% were interpreted from logs in Lower Jurassic and Triassic strata. The highest shut-in pressures were measured in the Upper Jurassic sequence raising the water much higher than the ground surface. Along the drilled section, there is a continuous downward increase in Cl- concentrations in the range of 12-186 g Cl-/l and a very clear stratification of brines. Data from the MJ1 borehole and from other exploration wells indicate that in the subsurface of the area, there are two definite source brines: Triassic brine and the Late Tertiary (so-called) Rift brine. Brines encountered in Jurassic and Cretaceous beds represent ancient mixtures of the two source brines involving various water-rock chemical transformations. Evidence of very high pressures in deep boreholes Devora 2A, Rosh Pinna 1, and MJ1 revealed the existence of a mechanism in which the deep brines are “piston-driven” upwards and possibly also laterally. The ongoing salinization of groundwater in the area is due to the inflow of the Late Tertiary Ca2+-Cl- Rift brines and not that of the Jurassic or Triassic brines. The hydrogeological and hydrochemical data from borehole MJ1 is of major importance for the management of groundwater resources in the Central Jordan Rift Valley and in the adjacent geologically connected areas.

Effects of mound building Lasius flavus on organic carbon and nutrient fluxes in soils of temperate grassland ecosystems

Ants that build long-lasting mounds and move large amounts of mineral soil may influence the composition and fluxes of soil solutions, and thus soil nutrient cycling. However, studies quantifying such effects are scarce. In central Europe, the yellow meadow ant (Lasius flavus) is a common species that changes the character of extensively managed grasslands by its mound building activity.In total, sixteen mound-control pairs were selected within four plots on two grassland sites in Thuringia, Central Germany and monitored from 2013 to 2016. On each plot, bulk precipitation and soil solutions were collected via two open funnel samplers and eight suction plates to determine input and loss fluxes of dissolved organic carbon (DOC), nitrogen (DN), and base cations under L. flavus mounds and control locations. Effects of L. flavus mounds on soil microclimatic conditions were also investigated.In comparison to the controls, L. flavus mounds were characterised by slightly lower soil temperatures and always lower soil moisture. The main driver of lower fluxes of DOC and DN from mounds was related to the reduced seepage water flux under mounds compared to the adjacent grassland. Lasius flavus activity increased potassium (K) concentrations and fluxes in soil solutions from mounds in comparison to the controls, independent from plot-specific soil and seepage water flux variability. Increased concentrations of calcium (Ca) and magnesium (Mg) were measured in the soil solutions of the mounds but were generally not associated with higher Ca or Mg fluxes. In conclusion, L. flavus present unique micro-sites within grassland ecosystems by creating mounds that may function as ecosystem control points for the cycling of certain elements, such as K.

Abstract PO-026: The mitochondrial calcium uniporter contributes to PDAC development and invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) has an exceptionally poor prognosis, with about 50,000 new cases and 45,000 deaths each year. While novel chemotherapeutics targeted to driver pathways and immune responses have increased the overall survival rate of cancer patients in general, these improvements are rarely seen in PDAC. PDAC often metastasizes early, and metastases ultimately lead to death in many PDAC patients. A stronger understanding of disease mechanisms and new therapeutic modalities are desperately needed to improve outcomes for many patients. Previously, we showed that cancer cell lines of multiple types are “addicted” to constitutive Ca++ flow into mitochondria through the mitochondrial calcium uniporter (MCU) at endoplasmic reticulum-mitochondria contact sites. We hypothesized that mitochondrial Ca++ influx through MCU contributes to cancer cell development, proliferation, and metastasis in PDAC by promoting metabolic activity within mitochondria. We now show that mRNA expression of MCU is elevated in a subset of pancreatic cancer patients, and high MCU protein expression correlates with poor survival outcomes. In addition, MCU gene expression is associated with the expression of a number of genes which associate with metastasis and/or poor survival outcomes, including ADAM proteins, KRAS, and RAC1. To further examine the role of MCU in PDAC, we used the Pdx1cre; KrasLSL-G12D/+; p53fl/+, Rosa26LSL-YFP/LSL-YFP; Mcufl/fl (KPCY) murine model of PDAC. We generated KPCY-McuKO (knockout) animals and cell lines from their YFP-positive tissues for further analysis. Cell lines developed from KPCY-McuKO pancreatic tissues fail to take up Ca++ into mitochondria in a manner that is rescued by stable re-expression of MCU. This Ca++ uptake is associated with an increase in pyruvate dehydrogenase activation. In these cells, MCU expression is also associated with an increase in cell motility, self-renewal capacity, and cell proliferation. Re-expression of MCU in these cells is associated with a morphological change to a more fibroblastic morphology indicative of epithelial to mesenchymal transition (EMT), including decreased surface expression of e-cadherin. These findings suggest that MCU may contribute to growth and metastasis. Indeed, in an immunocompetent, syngeneic orthotopic model of murine PDAC using one of these cell lines, tumor growth and metastasis were greatly ablated. Such results suggest that MCU-mediated influx of mitochondrial Ca++ contributes to PDAC development and metastasis and may present a therapeutic target for cancer treatment. Citation Format: Jillian S. Weissenrieder, Jason R. Pitarresi, Emily Fernandez-Garcia, Ben Z. Stanger, Anil K. Rustgi, J. Kevin Foskett. The mitochondrial calcium uniporter contributes to PDAC development and invasion [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-026.

Calcium influx through the mitochondrial calcium uniporter holocomplex, MCUcx

Ca2+ flux into the mitochondrial matrix through the MCU holocomplex (MCUcx) has recently been measured quantitatively and with milliseconds resolution for the first time under physiological conditions in both heart and skeletal muscle. Additionally, the dynamic levels of Ca2+ in the mitochondrial matrix ([Ca2+]m) of cardiomyocytes were measured as it was controlled by the balance between influx of Ca2+ into the mitochondrial matrix through MCUcx and efflux through the mitochondrial Na+ / Ca2+ exchanger (NCLX). Under these conditions [Ca2+]m was shown to regulate ATP production by the mitochondria at only a few critical sites. Additional functions attributed to [Ca2+]m continue to be reported in the literature. Here we review the new findings attributed to MCUcx function and provide a framework for understanding and investigating mitochondrial Ca2+ influx features, many of which remain controversial. The properties and functions of the MCUcx subunits that constitute the holocomplex are challenging to tease apart. Such distinct subunits include EMRE, MCUR1, MICUx (i.e. MICU1, MICU2, MICU3), and the pore-forming subunits (MCUpore). Currently, the specific set of functions of each subunit remains non-quantitative and controversial. The more contentious issues are discussed in the context of the newly measured native MCUcx Ca2+ flux from heart and skeletal muscle. These MCUcx Ca2+ flux measurements have been shown to be a highly-regulated, tissue-specific with femto-Siemens Ca2+ conductances and with distinct extramitochondrial Ca2+ ([Ca2+]i) dependencies. These data from cardiac and skeletal muscle mitochondria have been examined quantitatively for their threshold [Ca2+]i levels and for hypothesized gatekeeping function and are discussed in the context of model cell (e.g. HeLa, MEF, HEK293, COS7 cells) measurements. Our new findings on MCUcx dependent matrix [Ca2+]m signaling provide a quantitative basis for on-going and new investigations of the roles of MCUcx in cardiac function ranging from metabolic fuel selection, capillary blood-flow control and the pathological activation of the mitochondrial permeability transition pore (mPTP). Additionally, this review presents the use of advanced new methods that can be readily adapted by any investigator to enable them to carry out quantitative Ca2+ measurements in mitochondria while controlling the inner mitochondrial membrane potential, ΔΨm.