Correction: The Swine Plasma Metabolome Chronicles "Many Days" Biological Timing and Functions Linked to Growth.

[This corrects the article DOI: 10.1371/journal.pone.0145919.].

Regulatory T cells and IFN-γ-producing Th1 cells play a critical role in the pathogenesis of Sjögren's Syndrome.

Objectives: Sjögren's Disease (SjD) is an autoimmune disorder characterized by progressive dysfunction, inflammation and destruction of salivary and lacrimal glands, and by extraglandular manifestations. Its etiology and pathophysiology remain incompletely understood, though a role for autoreactive B cells has been considered key. Here, we investigated the role of effector and regulatory T cells in the pathogenesis of SjD. Methods: Histological analysis, RNA-sequencing and flow cytometry were conducted on glands, lungs, eyes and lymphoid tissues of mice with regulatory T cell-specific deletion of stromal interaction proteins (STIM) 1 and 2 ( Stim1/2 Foxp3 ), which play key roles in calcium signaling and T cell function. The pathogenicity of T cells from Stim1/2 Foxp3 mice was investigated through adoptively transfer into lymphopenic host mice. Additionally, single-cell transcriptomic analysis was performed on peripheral blood mononuclear cells (PBMCs) of patients with SjD and control subjects. Results: Stim1/2 Foxp3 mice develop a severe SjD-like disorder including salivary gland (SG) and lacrimal gland (LG) inflammation and dysfunction, autoantibodies and extraglandular symptoms. SG inflammation in Stim1/2 Foxp3 mice is characterized by T and B cell infiltration, and transcriptionally by a Th1 immune response that correlates strongly with the dysregulation observed in patients with SjD. Adoptive transfer of effector T cells from Stim1/2 Foxp3 mice demonstrates that the SjD-like disease is driven by interferon (IFN)-γ producing autoreactive CD4 + T cells independently of B cells and autoantiboodies. scRNA-seq analysis identifies increased Th1 responses and attenuated memory Treg function in PBMCs of patients with SjD. Conclusions: We report a more accurate mouse model of SjD while providing evidence for a critical role of Treg cells and IFN-γ producing Th1 cells in the pathogenesis of SjD, which may be effective targets for therapy.

Loss of STIM1 and STIM2 in salivary glands disrupts ANO1 function but does not induce Sjogren's disease.

Sjogren's disease (SjD) is an autoimmune disease characterized by xerostomia (dry mouth), lymphocytic infiltration into salivary glands and the presence of SSA and SSB autoantibodies. Xerostomia is caused by hypofunction of the salivary glands and has been involved in the development of SjD. Saliva production is regulated by parasympathetic input into the glands initiating intracellular Ca 2+ signals that activate the store operated Ca 2+ entry (SOCE) pathway eliciting sustained Ca 2+ influx. SOCE is mediated by the STIM1 and STIM2 proteins and the ORAI1 Ca 2+ channel. However, there are no studies on the effects of lack of STIM1/2 function in salivary acini in animal models and its impact on SjD. Here we report that male and female mice lacking Stim1 and Stim2 ( Stim1/2 K14Cre ) in salivary glands showed reduced intracellular Ca 2+ levels via SOCE in parotid acini and hyposalivate upon pilocarpine stimulation. Bulk RNASeq of the parotid glands of Stim1/2 K14Cre mice showed a decrease in the expression of Stim1/2 but no other Ca 2+ associated genes mediating saliva fluid secretion. SOCE was however functionally required for the activation of the Ca 2+ activated chloride channel ANO1. Despite hyposalivation, ageing Stim1/2 K14Cre mice showed no evidence of lymphocytic infiltration in the glands or elevated levels of SSA or SSB autoantibodies in the serum, which may be linked to the downregulation of the toll-like receptor 8 ( Tlr8 ). By contrast, salivary gland biopsies of SjD patients showed increased STIM1 and TLR8 expression, and induction of SOCE in a salivary gland cell line increased the expression of TLR8 . Our data demonstrate that SOCE is an important activator of ANO1 function and saliva fluid secretion in salivary glands. They also provide a novel link between SOCE and TLR8 signaling which may explain why loss of SOCE does not result in SjD.

Na+/Ca2+ exchange in enamel cells is dominated by the K+-dependent NCKX exchanger.

Calcium (Ca2+) extrusion is an essential function of the enamel-forming ameloblasts, providing Ca2+ for extracellular mineralization. The plasma membrane Ca2+ ATPases (PMCAs) remove cytosolic Ca2+ (cCa2+) and were recently shown to be efficient when ameloblasts experienced low cCa2+ elevation. Sodium-calcium (Na+/Ca2+) exchange has higher capacity to extrude cCa2+, but there is limited evidence on the function of the two main families of Na+/Ca2+ exchangers in enamel formation. The purpose of this study was to analyze the function of the NCX (coded by SLC8) and the K+-dependent NCKX (coded by SLC24) exchangers in rat ameloblasts and to compare their efficacy in the two main stages of enamel formation: the enamel forming secretory stage and the mineralizing or maturation stage. mRNA expression profiling confirmed the expression of Slc8 and Slc24 genes in enamel cells, Slc24a4 being the most highly upregulated transcript during the maturation stage, when Ca2+ transport increases. Na+/Ca2+ exchange was analyzed in the Ca2+ influx mode in Fura-2 AM-loaded ameloblasts. We show that maturation-stage ameloblasts have a higher Na+/Ca2+ exchange capacity than secretory-stage cells. We also show that Na+/Ca2+ exchange in both stages is dominated by NCKX over NCX. The importance of NCKX function in ameloblasts may partly explain why mutations in the SLC24A4 gene, but not in SLC8 genes, result in enamel disease. Our results demonstrate that Na+/Ca2+ exchangers are fully operational in ameloblasts and that their contribution to Ca2+ homeostasis increases in the maturation stage, when Ca2+ transport need is higher.

The Ca2+ channel ORAI1 is a regulator of oral cancer growth and nociceptive pain.

Oral cancer causes pain associated with cancer progression. We report here that the function of the Ca2+ channel ORAI1 is an important regulator of oral cancer pain. ORAI1 was highly expressed in tumor samples from patients with oral cancer, and ORAI1 activation caused sustained Ca2+ influx in human oral cancer cells. RNA-seq analysis showed that ORAI1 regulated many genes encoding oral cancer markers such as metalloproteases (MMPs) and pain modulators. Compared with control cells, oral cancer cells lacking ORAI1 formed smaller tumors that elicited decreased allodynia when inoculated into mouse paws. Exposure of trigeminal ganglia neurons to MMP1 evoked an increase in action potentials. These data demonstrate an important role of ORAI1 in oral cancer progression and pain, potentially by controlling MMP1 abundance.

High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

PMCA Ca2+ clearance in dental enamel cells depends on the magnitude of cytosolic Ca2.

Enamel formation (amelogenesis) is a two-step process whereby crystals partially grow during the secretory stage followed by a significant growth expansion during the maturation stage concurrent with an increase in vectorial Ca2+ transport. This requires tight regulation of cytosolic Ca2+ (c Ca2+ ) concentration in the enamel forming ameloblasts by controlling Ca2+ influx (entry) and Ca2+ extrusion (clearance). Gene and protein expression studies suggest that the plasma membrane Ca2+ -ATPases (PMCA1-4) are likely involved in c Ca2+ extrusion in ameloblasts, yet no functional analysis of these pumps has been reported nor whether their activity changes across amelogenesis. PMCAs have high Ca2+ affinity and low Ca2+ clearance which may be a limiting factor in their contribution to enamel formation as maturation stage ameloblasts handle high Ca2+ loads. We analyzed PMCA function in rat secretory and maturation ameloblasts by blocking or potentiating these pumps. Low/moderate elevations in c Ca2+ measured using the Ca2+ probe Fura-2-AM show that secretory ameloblasts clear Ca2+ faster than maturation stage cells through PMCAs. This process was completely inhibited by an external alkaline (pH 9.0) solution or was significantly delayed by the PMCA blockers vanadate and caloxin 1b1. Eliciting higher c Ca2+ transients via the activation of the ORAI1 Ca2+ channel showed that the PMCAs of maturation ameloblasts were more efficient. Inhibiting PMCAs decreased the rate of Ca2+ influx via ORAI1 but potentiation with forskolin had no effect. Our findings suggest that PMCAs are functional Ca2+ pumps during amelogenesis regulating c Ca2+ upon low and/or moderate Ca2+ stimulus in secretory stage, thus participating in amelogenesis.

Overexpression of RCAN1, a Gene on Human Chromosome 21, Alters Cell Redox and Mitochondrial Function in Enamel Cells.

The regulator of calcineurin (RCAN1) has been implicated in the pathogenesis of Down syndrome (DS). Individuals with DS show dental abnormalities for unknown reasons, and RCAN1 levels have been found to be elevated in several tissues of DS patients. A previous microarray analysis comparing cells of the two main formative stages of dental enamel, secretory and maturation, showed a significant increase in RCAN1 expression in the latter. Because the function of RCAN1 during enamel formation is unknown, there is no mechanistic evidence linking RCAN1 with the dental anomalies in individuals with DS. We investigated the role of RCAN1 in enamel by overexpressing RCAN1 in the ameloblast cell line LS8 (LS8+RCAN1). We first confirmed that RCAN1 is highly expressed in maturation stage ameloblasts by qRT-PCR and used immunofluorescence to show its localization in enamel-forming ameloblasts. We then analyzed cell redox and mitochondrial bioenergetics in LS8+RCAN1 cells because RCAN1 is known to impact these processes. We show that LS8+RCAN1 cells have increased reactive oxygen species (ROS) and decreased mitochondrial bioenergetics without changes in the expression of the complexes of the electron transport chain, or in NADH levels. However, LS8+RCAN1 cells showed elevated mitochondrial Ca2+ uptake and decreased expression of several enamel genes essential for enamel formation. These results provide insight into the role of RCAN1 in enamel and suggest that increased RCAN1 levels in the ameloblasts of individuals with DS may impact enamel formation by altering both the redox environment and mitochondrial function, as well as decreasing the expression of enamel-specific genes.

Ca2+-activated chloride channel ANO1: A new regulator of osteoclast function.

A paper by Sun et al. identified the Ca2+-activated Cl- channel anoctamin 1 or ANO1 (TMEM16A) as an important regulator of osteoclast function by interacting with RANKL activating signaling pathways involved in bone resorption. Although Cl- transporters (e.g. ClC7, CLIC5) have been known to be involved in the active process of bone resorption, ANO1 appears to control osteoclast differentiation and function to levels beyond those of other Cl-transporters. Regulating ANO1 function might be a useful target for therapeutics in osteoporosis.

On the Connections between TRPM Channels and SOCE.

Plasma membrane protein channels provide a passageway for ions to access the intracellular milieu. Rapid entry of calcium ions into cells is controlled mostly by ion channels, while Ca2+-ATPases and Ca2+ exchangers ensure that cytosolic Ca2+ levels ([Ca2+]cyt) are maintained at low (~100 nM) concentrations. Some channels, such as the Ca2+-release-activated Ca2+ (CRAC) channels and voltage-dependent Ca2+ channels (CACNAs), are highly Ca2+-selective, while others, including the Transient Receptor Potential Melastatin (TRPM) family, have broader selectivity and are mostly permeable to monovalent and divalent cations. Activation of CRAC channels involves the coupling between ORAI1-3 channels with the endoplasmic reticulum (ER) located Ca2+ store sensor, Stromal Interaction Molecules 1-2 (STIM1/2), a pathway also termed store-operated Ca2+ entry (SOCE). The TRPM family is formed by 8 members (TRPM1-8) permeable to Mg2+, Ca2+, Zn2+ and Na+ cations, and is activated by multiple stimuli. Recent studies indicated that SOCE and TRPM structure-function are interlinked in some instances, although the molecular details of this interaction are only emerging. Here we review the role of TRPM and SOCE in Ca2+ handling and highlight the available evidence for this interaction.

Mitochondria modulate ameloblast Ca2+ signaling.

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca2+ (m Ca2+ ) uptake. However, m Ca2+ extrusion via Na+ -Li+ -Ca2+ exchanger was more prominent in maturation. To address if m Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (c Ca2+ ) buffering, we stimulated Ca2+ influx via the store-operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented c Ca2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher m Ca2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and m Ca2+ uptake are complementary processes in biological mineralization.

Calcium Transport in Specialized Dental Epithelia and Its Modulation by Fluoride.

Most cells use calcium (Ca2+) as a second messenger to convey signals that affect a multitude of biological processes. The ability of Ca2+ to bind to proteins to alter their charge and conformation is essential to achieve its signaling role. Cytosolic Ca2+ (cCa2+) concentration is maintained low at ~100 nM so that the impact of elevations in cCa2+ is readily sensed and transduced by cells. However, such elevations in cCa2+ must be transient to prevent detrimental effects. Cells have developed a variety of systems to rapidly clear the excess of cCa2+ including Ca2+ pumps, exchangers and sequestering Ca2+ within intracellular organelles. This Ca2+ signaling toolkit is evolutionarily adapted so that each cell, tissue, and organ can fulfill its biological function optimally. One of the most specialized cells in mammals are the enamel forming cells, the ameloblasts, which also handle large quantities of Ca2+. The end goal of ameloblasts is to synthesize, secrete and mineralize a unique proteinaceous matrix without the benefit of remodeling or repair mechanisms. Ca2+ uptake into ameloblasts is mainly regulated by the store operated Ca2+ entry (SOCE) before it is transported across the polarized ameloblasts to reach the insulated enamel space. Here we review the ameloblasts Ca2+ signaling toolkit and address how the common electronegative non-metal fluoride can alter its function, potentially addressing the biology of dental fluorosis.

Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP3 receptor function.

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca2+) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Cav blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca2+]cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca2+ pools, and Ca2+ influx by ORAI channels have been associated with the increase in [Ca2+]cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil's effect on [Ca2+]cyt. To address these questions, we monitored changes in [Ca2+]cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 µM to 100 µM). We show that mibefradil elicits an increase in [Ca2+]cyt at concentrations above 10 µM (IC50 around 50 µM) and a fast Ca2+ increase capacity at 100 µM. We found that inhibiting IP3 receptors, depleting the ER-Ca2+ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca2+]cyt. Moreover, the transient application of 100 µM mibefradil triggered Ca2+ influx by store-operated Ca2+ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP3R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

Mitochondrial Function in Enamel Development.

Enamel is the most calcified tissue in vertebrates. Enamel formation and mineralization is a two-step process that is mediated by ameloblast cells during their secretory and maturation stages. In these two stages, ameloblasts are characterized by different morphology and function, which is fundamental for proper mineral growth in the extracellular space. Ultrastructural studies have shown that the mitochondria in these cells localize to different subcellular regions in both stages. However, limited knowledge is available on the role/s of mitochondria in enamel formation. To address this issue, we analyzed mitochondrial biogenesis and respiration, as well as the redox status of rat primary enamel cells isolated from the secretory and maturation stages. We show that maturation stage cells have an increased expression of PGC1α, a marker of mitochondrial biogenesis, and of components of the electron transport chain. Oxygen consumption rate (OCR), a proxy for mitochondrial function, showed a significant increase in oxidative phosphorylation during the maturation stage, promoting ATP production. The GSH/GSSG ratio was lower in the maturation stage, indicative of increased oxidation. Because higher oxidative phosphorylation can lead to higher ROS production, we tested if ROS affected the expression of AmelX and Enam genes that are essential for enamel formation. The ameloblast cell line LS8 treated with H2O2 to promote ROS elicited significant expression changes in AmelX and Enam. Our data highlight important metabolic and physiological differences across the two enamel stages, with higher ATP levels in the maturation stage indicative of a higher energy demand. Besides these metabolic shifts, it is likely that the enhanced ETC function results in ROS-mediated transcriptional changes.

Short and long period growth markers of enamel formation distinguish European Pleistocene hominins.

Characterizing dental development in fossil hominins is important for distinguishing between them and for establishing where and when the slow overall growth and development of modern humans appeared. Dental development of australopiths and early Homo was faster than modern humans. The Atapuerca fossils (Spain) fill a barely known gap in human evolution, spanning ~1.2 to ~0.4 million years (Ma), during which H. sapiens and Neandertal dental growth characteristics may have developed. We report here perikymata counts, perikymata distributions and periodicities of all teeth belonging to the TE9 level of Sima del Elefante, level TD6.2 of Gran Dolina (H. antecessor) and Sima de los Huesos. We found some components of dental growth in the Atapuerca fossils resembled more recent H. sapiens. Mosaic evolution of perikymata counts and distribution generate three distinct clusters: H. antecessor, Sima de los Huesos and H. sapiens.

TRPM7 activation potentiates SOCE in enamel cells but requires ORAI.

Calcium (Ca2+) release-activated Ca2+ (CRAC) channels mediated by STIM1/2 and ORAI (ORAI1-3) proteins form the dominant store-operated Ca2+ entry (SOCE) pathway in a wide variety of cells. Among these, the enamel-forming cells known as ameloblasts rely on CRAC channel function to enable Ca2+ influx, which is important for enamel mineralization. This key role of the CRAC channel is supported by human mutations and animal models lacking STIM1 and ORAI1, which results in enamel defects and hypomineralization. A number of recent reports have highlighted the role of the chanzyme TRPM7 (transient receptor potential melastanin 7), a transmembrane protein containing an ion channel permeable to divalent cations (Mg2+, Ca2+), as a modulator of SOCE. This raises the question as to whether TRPM7 should be considered an alternative route for Ca2+ influx, or if TRPM7 modifies CRAC channel activity in enamel cells. To address these questions, we monitored Ca2+ influx mediated by SOCE using the pharmacological TRPM7 activator naltriben and the inhibitor NS8593 in rat primary enamel cells and in the murine ameloblast cell line LS8 cells stimulated with thapsigargin. We also measured Ca2+ dynamics in ORAI1/2-deficient (shOrai1/2) LS8 cells and in cells with siRNA knock-down of Trpm7. We found that primary enamel cells stimulated with the TRPM7 activator potentiated Ca2+ influx via SOCE compared to control cells. However, blockade of TRPM7 with NS8593 did not decrease the SOCE peak. Furthermore, activation of TRPM7 in shOrai1/2 LS8 cells lacking SOCE failed to elicit Ca2+ influx, and Trpm7 knock-down had no effect on SOCE. Taken together, our data suggest that TRPM7 is a positive modulator of SOCE potentiating Ca2+ influx in enamel cells, but its function is fully dependent on the prior activation of the ORAI channels.

Fluoride exposure alters Ca2+ signaling and mitochondrial function in enamel cells.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca2+ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca2+ stores and store-operated Ca2+ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca2+ homeostasis or increase the expression of ER stress-associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca2+ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump during Ca2+ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

STIM1 R304W in mice causes subgingival hair growth and an increased fraction of trabecular bone.

Calcium signaling plays a central role in bone development and homeostasis. Store operated calcium entry (SOCE) is an important calcium influx pathway mediated by calcium release activated calcium (CRAC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum calcium sensing protein important for SOCE. We generated a mouse model expressing the STIM1 R304W mutation, causing Stormorken syndrome in humans. Stim1R304W/R304W mice showed perinatal lethality, and the only three animals that survived into adulthood presented with reduced growth, low body weight, and thoracic kyphosis. Radiographs revealed a reduced number of ribs in the Stim1R304W/R304W mice. Microcomputed tomography data revealed decreased cortical bone thickness and increased trabecular bone volume fraction in Stim1R304W/R304W mice, which had thinner and more compact bone compared to wild type mice. The Stim1R304W/+ mice showed an intermediate phenotype. Histological analyses showed that the Stim1R304W/R304W mice had abnormal bone architecture, with markedly increased number of trabeculae and reduced bone marrow cavity. Homozygous mice showed STIM1 positive osteocytes and osteoblasts. These findings highlight the critical role of the gain-of-function (GoF) STIM1 R304W protein in skeletal development and homeostasis in mice. Furthermore, the novel feature of bilateral subgingival hair growth on the lower incisors in the Stim1R304W/R304W mice and 25 % of the heterozygous mice indicate that the GoF STIM1 R304W protein also induces an abnormal epithelial cell fate.

Differential regulation of Ca2+ influx by ORAI channels mediates enamel mineralization.

Store-operated Ca2+ entry (SOCE) channels are highly selective Ca2+ channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca2+ fluxes that are critical for many cellular functions. Mutations in the human ORAI1 gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects. In our investigation of the role of ORAI proteins in enamel, we identified enamel defects in a patient with an ORAI1 null mutation. Targeted deletion of the Orai1 gene in mice showed enamel defects and reduced SOCE in isolated enamel cells. However, Orai2-/- mice showed normal enamel despite having increased SOCE in the enamel cells. Knockdown experiments in the enamel cell line LS8 suggested that ORAI2 and ORAI3 modulated ORAI1 function, with ORAI1 and ORAI2 being the main contributors to SOCE. ORAI1-deficient LS8 cells showed altered mitochondrial respiration with increased oxygen consumption rate and ATP, which was associated with altered redox status and enhanced ER Ca2+ uptake, likely due to S-glutathionylation of SERCA pumps. Our findings demonstrate an important role of ORAI1 in Ca2+ influx in enamel cells and establish a link between SOCE, mitochondrial function, and redox homeostasis.

The evolutionary history of the human face.

The face is the most distinctive feature used to identify others. Modern humans have a short, retracted face beneath a large globular braincase that is distinctively different from that of our closest living relatives. The face is a skeletal complex formed by 14 individual bones that houses parts of the digestive, respiratory, visual and olfactory systems. A key to understanding the origin and evolution of the human face is analysis of the faces of extinct taxa in the hominin clade over the last 6 million years. Yet, as new fossils are recovered and the number of hominin species grows, the question of how and when the modern human face originated remains unclear. By examining key features of the facial skeleton, here we evaluate the evolutionary history of the modern human face in the context of its development, morphology and function, and suggest that its appearance is the result of a combination of biomechanical, physiological and social influences.