Sphingolipid-Induced Bone Regulation and Its Emerging Role in Dysfunction Due to Disease and Infection.

The human skeleton is a metabolically active system that is constantly regenerating via the tightly regulated and highly coordinated processes of bone resorption and formation. Emerging evidence reveals fascinating new insights into the role of sphingolipids, including sphingomyelin, sphingosine, ceramide, and sphingosine-1-phosphate, in bone homeostasis. Sphingolipids are a major class of highly bioactive lipids able to activate distinct protein targets including, lipases, phosphatases, and kinases, thereby conferring distinct cellular functions beyond energy metabolism. Lipids are known to contribute to the progression of chronic inflammation, and notably, an increase in bone marrow adiposity parallel to elevated bone loss is observed in most pathological bone conditions, including aging, rheumatoid arthritis, osteoarthritis, and osteomyelitis. Of the numerous classes of lipids that form, sphingolipids are considered among the most deleterious. This review highlights the important primary role of sphingolipids in bone homeostasis and how dysregulation of these bioactive metabolites appears central to many chronic bone-related diseases. Further, their contribution to the invasion, virulence, and colonization of both viral and bacterial host cell infections is also discussed. Many unmet clinical needs remain, and data to date suggest the future use of sphingolipid-targeted therapy to regulate bone dysfunction due to a variety of diseases or infection are highly promising. However, deciphering the biochemical and molecular mechanisms of this diverse and extremely complex sphingolipidome, both in terms of bone health and disease, is considered the next frontier in the field.

In vitro and in vivo calibration of low affinity genetic Ca2+ indicators.

Calcium is a universal intracellular messenger and proper Ca2+concentrations ([Ca2+]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca2+ homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca2+ levels result in malfunction and disease. Selective intraorganellar Ca2+measurements are best achieved by using targeted genetically encoded Ca2+ indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca2+] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca2+]ER/SR based on imaging of fluorescence after heating the tissue at 50-52 °C, since this value coincides with that obtained in the absence of Ca2+ (Rmin). Knowledge of the dynamic range (Rmax/Rmin) and the Ca2+-affinity (KD) of the indicator permits calculation of [Ca2+] by applying a simple algorithm. We have validated this procedure in vitro using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as in vivo in Drosophila. Moreover, this methodology is applicable to other low Ca2+ affinity green and red GECIs.

Relationships between medical student wellness, self-efficacy, and academic performance during the "post-COVID" period.

This study was a part of a longitudinal study investigating the relationships between medical student wellness, self-efficacy, and performance. Eighty-two eligible students were asked to complete online surveys during their second (M2) and third (M3) years. Performance outcomes included scores on various summative assessments during the M.D. program. Wellness survey results indicated that the sample of 38 M2 and 28 M3 students were overall well and self-efficacious, and they broadly maintained similar wellness characteristics across their medical education despite COVID-19 disruptions. Twenty-three students completed both surveys, and a paired analysis for this subgroup showed modest increases in stress and burnout in the M3 year. Notable correlations were observed between self-efficacy for academic work and a whole range of wellness variables for M2 students. M2 academic performance was modestly correlated to self-efficacy (rs = 0.38, P = 0.02, n = 38) and student burnout (rs = -0.34, P = 0.04, n = 38). In contrast, for the M3 students there was little correlation between wellness, clinical self-efficacy, and clinical performance, with the only significant relationships observed to be between overall clinical self-efficacy and the strength of social networks (rs = 0.41, P = 0.03, n = 28) and between scores for postencounter notes during Objective Structure Clinical Examinations (OSCEs) and self-efficacy in evidence-based medicine (rs = 0.44, P = 0.02, n = 28). In conclusion, 1) students remained generally well throughout the post-COVID period, and 2) self-efficacy for academic work is a good predictor of student wellness and performance during the preclerkship period but not during clinical training.NEW & NOTEWORTHY This study followed a group of medical students through the "post-COVID" period to assess their wellness as they transitioned from basic sciences to clinical training. We found that their wellness and belief in their ability to succeed (self-efficacy) remained strong, showing their resiliency. We observed correlations between self-efficacy and their level of wellness and academic performance during basic science classes but not during clinical training.

Air Pollution and Lung Cancer: Contributions of Extracellular Vesicles as Pathogenic Mechanisms and Clinical Utility.

PURPOSE OF REVIEW: This review addresses the pressing issue of air pollution's threat to human health, focusing on its connection to non-small cell lung cancer (NSCLC) development. The aim is to explore the role of extracellular vesicles (EVs) as potential pathogenic mechanisms in lung cancer, including NSCLC, induced by air pollutants. RECENT FINDINGS: Recent research highlights EVs as vital mediators of intercellular communication and key contributors to cancer progression. Notably, this review emphasizes the cargo of EVs released by both cancerous and non-cancerous lung cells, shedding light on their potential role in promoting various aspects of tumor development. The review underscores the importance of comprehending the intricate interplay between air pollution, biological damage mechanisms, and EV-mediated communication during NSCLC development. Major takeaways emphasize the significance of this understanding in addressing air pollution-related lung cancer. Future research avenues are also highlighted, aiming to enhance the applicability of EVs for diagnosis and targeted therapies, ultimately mitigating the inevitable impact of air pollution on NSCLC development and treatment.

Fungal Endophthalmitis Masquerading as Sympathetic Ophthalmia.

PURPOSE: To describe the ocular pathology of a patient with fungal endophthalmitis with features mimicking sympathetic ophthalmia. METHODS: Review of medical records and histopathology of a single patient. RESULTS: A 72-year-old male who sustained penetrating injury to the left eye with an agave plant presented to our clinic 16 months after the initial injury. Prior to presentation, the patient had developed endophthalmitis and had undergone anterior chamber washout, vitrectomy, and intravitreal steroids, antibiotics, antifungals, and anti-vascular endothelial growth factor (VEGF) therapy. At presentation, the patient had a blind, painful eye and subsequently underwent enucleation. Histopathology demonstrated granulomatous inflammation with multinucleated giant cells in the iris and Dalen Fuchs nodules with CD68 positive epithelioid histiocytes associated with the retinal pigment epithelium (RPE) sparing the choriocapillaris. These findings were initially attributed to sympathetic ophthalmia. The fellow eye did not have any signs of inflammation, and additional fungal PAS stains were positive for filamentous fungal elements, leading to a diagnosis of fungal endophthalmitis. CONCLUSIONS: Fungal endophthalmitis may develop histopathologic features that are similar to those seen in sympathetic ophthalmia. Recognition of the overlap between the histopathologic features of these diseases may reduce the possibility of misdiagnosis and unnecessary treatment of the fellow eye.

Associations of fitness and physical activity with specific abdominal fat depots in children with overweight/obesity.

OBJECTIVES: To examine the relationship between physical fitness and physical activity (PA) with specific abdominal fat depots and their potential implications for cardiometabolic risk and insulin resistance (IR) in children with overweight/obesity. MATERIALS AND METHODS: A total of 116 children with overweight/obesity (10.7 ± 1.1 year, 54% girls) participated in the study. Abdominal visceral (VAT), subcutaneous (ASAT), and intermuscular abdominal adipose tissue (IMAAT) were assessed by magnetic resonance imaging. The cardiometabolic risk (MetS) score and the insulin resistance homeostasis model assessment (HOMA-IR) were calculated. Health-related physical fitness components (treadmill test, and 20 m shuttle run, handgrip, standing broad jump and 4 × 10 m tests) were evaluated, and PA was measured (accelerometry). Children were categorized as fit or unfit for each specific fitness test, and as active or inactive. RESULTS: Higher VAT, ASAT, and IMAAT were associated with higher MetS score and HOMA-IR (all p < 0.02). A better performance in all fitness tests and total and vigorous PA were strongly associated with lower VAT (all p < 0.04), ASAT (all p < 0.005), and IMAAT (all p < 0.005). Fit or active children had lower VAT, ASAT, and IMAAT (all p < 0.03) than their unfit or inactive counterparts. CONCLUSION: These results reinforce the importance of having adequate fitness and PA levels to reduce abdominal fat accumulation in children. Given that VAT, ASAT, and IMAAT are associated with higher risk of developing cardiovascular diseases and type 2 diabetes, the improvement of physical fitness by the promotion of PA should be goals of lifestyle interventions for improving health in children with overweight/obesity.

Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca2+ homeostasis with adipose tissue lipolysis.

Appropriate cristae remodeling is a determinant of mitochondrial function and bioenergetics and thus represents a crucial process for cellular metabolic adaptations. Here, we show that mitochondrial cristae architecture and expression of the master cristae-remodeling protein OPA1 in proopiomelanocortin (POMC) neurons, which are key metabolic sensors implicated in energy balance control, is affected by fluctuations in nutrient availability. Genetic inactivation of OPA1 in POMC neurons causes dramatic alterations in cristae topology, mitochondrial Ca2+ handling, reduction in alpha-melanocyte stimulating hormone (α-MSH) in target areas, hyperphagia, and attenuated white adipose tissue (WAT) lipolysis resulting in obesity. Pharmacological blockade of mitochondrial Ca2+ influx restores α-MSH and the lipolytic program, while improving the metabolic defects of mutant mice. Chemogenetic manipulation of POMC neurons confirms a role in lipolysis control. Our results unveil a novel axis that connects OPA1 in POMC neurons with mitochondrial cristae, Ca2+ homeostasis, and WAT lipolysis in the regulation of energy balance.

Regulation of Cas9 by viral proteins Tat and Rev for HIV-1 inactivation.

While combined antiretroviral therapy (cART) has had a great impact on the treatment of HIV-1 infection, the persistence of long-lived cells with an intact provirus precludes virus eradication and sterilizing cure. CRISPR/Cas9 genome editing has become an efficient tool to eradicate HIV-1 genome or prevent replication. Furthermore, regulation of Cas9 gene expression by HIV can induce mutations that could inactivate the proviral genome, making a gene therapy safe by preventing the induction of non-specific mutations, which could compromise the integrity of healthy cells. In this study, isolated HIV-1 LTR, INS and RRE sequences were used to regulate Cas9 expression in HEK293 cells, and guide RNAs (gRNAs) were designed to target mutations in HIV-1 conserved regions such as tat and rev regulatory genes. We demonstrate that Cas9 expression in our system is controlled by the HIV-1 Tat and Rev proteins, leading to self-regulation of gene edition, and showing a strong antiviral effect by inactivating HIV-1 replication. Sequencing analysis confirmed that viral genome was partially excised by multiplex editing (90% efficiency), and viral capsid protein (CA-p24) was undetectable. In conclusion, the self-regulated CRISPR/Cas9 system may be a reliable and accurate strategy for eliminating HIV-1 infection whose effect will be restricted to infected cells.

Direct monitoring of ER Ca2+ dynamics reveals that Ca2+ entry induces ER-Ca2+ release in astrocytes.

Excitability in astroglia is controlled by Ca2+ fluxes from intracellular organelles, mostly from the endoplasmic reticulum (ER). Astrocytic ER possesses inositol 1,4,5-trisphosphate receptors (InsP3R) that can be activated upon stimulation through a vast number of metabotropic G-protein-coupled receptors. By contrast, the role of Ca2+-gated Ca2+ release channels is less explored in astroglia. Here we address this process by monitoring Ca2+ dynamics directly in the cytosol and the ER of astroglial cells. Cultured astrocytes exhibited spontaneous and high-K-evoked cytosolic Ca2+ transients, both of them reversibly abolished by external Ca2+ removal, addition of plasma membrane channel blockers or ER Ca2+ depletion with SERCA inhibitors. Resting astrocyte [Ca2+]ER averaged 400 µM and maximal stimulation with ATP provoked a complete and reversible ER discharge. Direct monitoring of Ca2+ in the lumen of ER showed that high-K induced a Ca2+ release from the ER, and its amplitude was proportional to the [K]. Furthermore, by combining the low affinity GAP3 indicator targeted to the ER with the high affinity cytosolic Rhod-2, we simultaneously imaged ER- and cytosolic-Ca2+ signals, in astrocytes in culture and in situ. Plasma membrane Ca2+ entry triggered a fast ER Ca2+ release coordinated with an increase in cytosolic Ca2+. Thus, we identify a Ca2+-induced Ca2+-release (CICR) mechanism that is likely to participate in spontaneous astroglial oscillations, providing a graded amplification of the cytosolic Ca2+ signal.

Sarcoplasmic reticulum Ca2+ decreases with age and correlates with the decline in muscle function in Drosophila.

Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca2+ peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum (SR) Ca2+ concentration ([Ca2+]SR) is at the origin of this loss of Ca2+ homeostasis. We engineered Drosophila melanogaster to express the Ca2+ indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca2+]SRin vivo [Ca2+]SR fell with age from ∼600 µM to 50 µM in close correlation with muscle function, which declined monotonically when [Ca2+]SR was <400 µM. [Ca2+]SR results from the pump-leak steady state at the SR membrane. However, changes in expression of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump and of the ryanodine receptor leak were too modest to explain the large changes seen in [Ca2+]SR Instead, these changes are compatible with increased leakiness through the ryanodine receptor as the main determinant of the [Ca2+]SR decline in aging muscle. In contrast, there were no changes in endoplasmic reticulum [Ca2+] with age in brain neurons.This article has an associated First Person interview with the first author of the paper.

Imaging of Endoplasmic Reticulum Ca2+ in the Intact Pituitary Gland of Transgenic Mice Expressing a Low Affinity Ca2+ Indicator.

The adenohypophysis contains five secretory cell types (somatotrophs, lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs), each secreting a different hormone, and controlled by different hypothalamic releasing hormones (HRHs). Exocytic secretion is regulated by cytosolic Ca2+ signals ([Ca2+]C), which can be generated either by Ca2+ entry through the plasma membrane and/or by Ca2+ release from the endoplasmic reticulum (ER). In addition, Ca2+ entry signals can eventually be amplified by ER release via calcium-induced calcium release (CICR). We have investigated the contribution of ER Ca2+ release to the action of physiological agonists in pituitary gland. Changes of [Ca2+] in the ER ([Ca2+]ER) were measured with the genetically encoded low-affinity Ca2+ sensor GAP3 targeted to the ER. We used a transgenic mouse strain that expressed erGAP3 driven by a ubiquitous promoter. Virtually all the pituitary cells were positive for the sensor. In order to mimick the physiological environment, intact pituitary glands or acute slices from the transgenic mouse were used to image [Ca2+]ER. [Ca2+]C was measured simultaneously with Rhod-2. Luteinizing hormone-releasing hormone (LHRH) or thyrotropin releasing hormone (TRH), two agonists known to elicit intracellular Ca2+ mobilization, provoked robust decreases of [Ca2+]ER and concomitant rises of [Ca2+]C. A smaller fraction of cells responded to thyrotropin releasing hormone (TRH). By contrast, depolarization with high K+ triggered a rise of [Ca2+]C without a decrease of [Ca2+]ER, indicating that the calcium-induced calcium-release (CICR) via ryanodine receptor amplification mechanism is not present in these cells. Our results show the potential of transgenic ER Ca2+ indicators as novel tools to explore intraorganellar Ca2+ dynamics in pituitary gland in situ.

Bowel obstruction due to the migration of the deflated intragastric balloon, a rare and potentially lethal complication.

Obesity is a worldwide epidemic that carries significant morbidity and mortality. There are many weight loss strategies available, yet to this date, none are risk-free. Intragastric balloons have been used for decades as a temporary measure for weight reduction and can be a useful approach for specific patients. Serious complications related to the device remain rare; however, prompt intervention is usually needed when they arise. We present the case of a 38-year-old female patient, she was using an intragastric balloon to treat her obesity. Regretfully, the balloon deflated causing intestinal migration and obstruction. After a successful surgery, the patient completely recovered.

Cecum and appendix perforation due to inadvertent ingestion of two toothpicks.

Bowel perforation due to inadvertent ingestion of foreign objects is, fortunately, a rare event. However, it can lead to deadly complications when it occurs. Thin, sharp and pointed objects like toothpicks are more likely to pierce the bowel wall. Diagnosing toothpick ingestion and perforation is difficult since most patients do not recall swallowing the toothpick, symptoms and physical examinations are nonspecific, the symptoms can resemble many abdominal pathologies, and since a toothpick has a radiolucent nature that makes it difficult to detect through X-ray imaging. Due to this, most of the cases are identified during the transoperative period. We present the case of a 27-year-old male who presented with symptoms clinically indistinguishable from acute appendicitis. During surgery, two toothpicks were discovered that compromised the cecum and the appendix. After successful removal of the foreign objects, the patient underwent a full recovery.

Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca2+ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10-7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3-1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR.

Mesenteric Adipose-derived Stromal Cells From Crohn's Disease Patients Induce Protective Effects in Colonic Epithelial Cells and Mice With Colitis.

Mesenteric adipose tissue hyperplasia is a hallmark of Crohn's disease (CD). Recently, we showed that mesenteric adipose-derived stromal cells (ADSCs) from CD, ulcerative colitis, and control patients synthesize and release adipokines in a disease-dependent manner. Here we examined the expression profiles of CD and control patient-derived mesenteric ADSCs and studied the effects of their extracellular mediators on colonocyte signaling in vitro and experimental colitis in vivo. ADSCs were isolated from mesenteric fat of control and CD patients. Microarray profiling and network analysis were performed in ADSCs and human colonocytes treated with conditioned media from cultured ADSCs. Mice with acute colitis received daily injections of conditioned media from patient-derived ADSCs, vehicle, or apolactoferrin. Proliferative responses were evaluated in conditioned media-treated colonocytes and mouse colonic epithelium. Total protein was isolated from cultured colonocytes after treatment with apolactoferrin for Western blot analysis of phosphorylated intracellular signaling kinases. Microarray profiling revealed differential mRNA expression in CD patient-derived ADSCs compared with controls, including lactoferrin. Administration of CD patient-derived medium or apolactoferrin increased colonocyte proliferation compared with controls. Conditioned media from CD patient-derived ADSCs or apolactoferrin attenuated colitis severity in mice and enhanced colonocyte proliferation in vivo. ADSCs from control and CD patients show disease-dependent inflammatory responses and alter colonic epithelial cell signaling in vitro and in vivo. Furthermore, we demonstrate lactoferrin production by adipose tissue, specifically mesenteric ADSCs. We suggest that mesenteric ADSC-derived lactoferrin may mediate protective effects and participate in the pathophysiology of CD by promoting colonocyte proliferation and the resolution of inflammation.

Using aequorin probes to measure Ca2+ in intracellular organelles.

Aequorins are excellent tools for measuring intra-organellar Ca2+ and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal. We also focus on bioluminescent Ca2+ imaging in nucleus or mitochondria of living cells. Finally, recent advances with a new chimeric GFP-aequorin protein (GAP), which can be used either as luminescent or fluorescent Ca2+ probe, are presented. GAP is robustly expressed in transgenic flies and mice, where it has proven to be a suitable Ca2+ indicator for monitoring physiological Ca2+ signaling ex vivo and in vivo.

Measuring Ca2+ inside intracellular organelles with luminescent and fluorescent aequorin-based sensors.

GFP-Aequorin Protein (GAP) can be used to measure [Ca2+] inside intracellular organelles, both by luminescence and by fluorescence. The low-affinity variant GAP3 is adequate for ratiometric imaging in the endoplasmic reticulum and Golgi apparatus, and it can be combined with conventional synthetic indicators for simultaneous measurements of cytosolic Ca2+. GAP is bioorthogonal as it does not have mammalian homologues, and it is robust and functionally expressed in transgenic flies and mice, where it can be used for Ca2+ measurements ex vivo and in vivo to explore animal models of health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca(2+) Dynamics in High-Ca(2+) Organelles.

Proper functioning of organelles such as the ER or the Golgi apparatus requires luminal accumulation of Ca(2+) at high concentrations. Here we describe a ratiometric low-affinity Ca(2+) sensor of the GFP-aequorin protein (GAP) family optimized for measurements in high-Ca(2+) concentration environments. Transgenic animals expressing the ER-targeted sensor allowed monitoring of Ca(2+) signals inside the organelle. The use of the sensor was demonstrated under three experimental paradigms: (1) ER Ca(2+) oscillations in cultured astrocytes, (2) ex vivo functional mapping of cholinergic receptors triggering ER Ca(2+) release in acute hippocampal slices from transgenic mice, and (3) in vivo sarcoplasmic reticulum Ca(2+) dynamics in the muscle of transgenic flies. Our results provide proof of the suitability of the new biosensors to monitor Ca(2+) dynamics inside intracellular organelles under physiological conditions and open an avenue to explore complex Ca(2+) signaling in animal models of health and disease.

Corticotropin-Releasing Hormone Receptor 2 Signaling Promotes Mucosal Repair Responses after Colitis.

The corticotropin-releasing hormone family mediates functional responses in many organs, including the intestine. Activation of corticotropin-releasing hormone receptor 2 (CRHR2) in the colonic mucosa promotes inflammation during acute colitis but inhibits inflammation during chronic colitis. We hypothesized that specific modulation of CRHR2 signaling in the colonic mucosa can promote restoration of the epithelium through stimulation of cell proliferative, migratory, and wound healing responses. Mucosal repair was assessed after dextran sodium sulfate (DSS)-induced colitis in mice receiving intracolonic injections of a CRHR2 antagonist or vehicle and in Crhr2(-/-) mice. Histologic damage, cytokine expression, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and Ki-67 immunoreactivity were evaluated. Cell viability, proliferation, and migration were compared between parental and CRHR2-overexpressing colonic epithelial cells. Protein lysates were processed for phosphoprotein assays and a wound healing assay performed in vitro. Administration of a CRHR2 antagonist after DSS-induced colitis increased disease activity, delayed healing, and decreased epithelial cell proliferation in vivo. Colons from these mice also showed increased apoptosis and proinflammatory cytokine expression. Compared with controls, Crhr2(-/-) mice showed increased mortality in the DSS healing protocol. CRHR2-overexpressing cells had increased proliferation and migration compared with parental cells. Wound healing and signal transducer and activator of transcription 3 activity were elevated in CRHR2-overexpressing cells after urocortin 2 and IL-6 treatment, suggesting advanced healing progression. Our results suggest that selective CRHR2 activation may provide a targeted approach to enhance mucosal repair pathways after colitis.

A new low-Ca²âº affinity GAP indicator to monitor high Ca²âº in organelles by luminescence.

We have recently described a new class of genetically encoded Ca(2+) indicators composed of two jellyfish proteins, a variant of green fluorescent protein (GFP) and the calcium binding protein apoaequorin, named GAP (Rodriguez-García et al., 2014). GAP is a unique dual-mode Ca(2+) indicator, able to function either as a fluorescent or a luminescent probe, depending on whether the photoprotein aequorin is in its apo-state or reconstituted with its cofactor coelenterazine. We describe here a novel application of GAP as a low affinity bioluminescent indicator, suitable for measurements of [Ca(2+)] in ER or in Golgi apparatus. We used the low affinity variant, GAP1, which carries mutations in two EF-hands of aequorin, reconstituted with coelenterazine n. In comparison to previous bioluminescent aequorin fusions, the decay rate of GAP1 was decreased 8 fold and the affinity for Ca(2+) was lowered one order of magnitude. This improvement allows long-term measurements in high Ca(2+) environments avoiding fast aequorin consumption. GAP1 was targeted to the ER of various cell types, where it monitored resting Ca(2+) concentrations in the range from 400 to 600 µM. ER could be emptied of calcium by stimulation with ATP, carbachol or histamine in intact cells, and by challenge with inositol tris-phosphate in permeabilized cells. GAP1 was also targeted to the Golgi apparatus where it was able to precisely monitor long-term calcium dynamics. GAP1 provides a novel and robust indicator applicable to bioluminescent high-throughput quantitative assays.