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Objective: To evaluate the effects of a multidisciplinary obesity treatment programme on fecal microbiota composition and immunoglobulin-coating bacteria in overweight and obese adolescents and their relationship to weight loss. Design: Longitudinal intervention study based on both a calorie-restricted diet (calorie reduction=10-40%) and increased physical activity (calorie expenditure=15-23 kcal/kg body weight per week) for 10 weeks. Participants: Thirty-nine overweight and obese adolescents (BMI mean 33.1 range 23.7-50.4; age mean 14.8 range, 13.0-16.0). Measurements: BMI, BMI z-scores and plasma biochemical parameters were measured before and after the intervention. Fecal microbiota was analyzed by fluorescent in situ hybridization. Immunoglobulin-coating bacteria were detected using fluorescent-labelled F(ab')2 antihuman IgA, IgG and IgM. Results: Reductions in Clostridium histolyticum and E. rectale-C. coccoides proportions significantly correlated with weight and BMI z-score reductions in the whole adolescent population. Proportions of C. histolyticum, C. lituseburense and E. rectale-C. coccoides dropped significantly whereas those of the Bacteroides-Prevotella group increased after the intervention in those adolescents who lost more than 4 kg. Total fecal energy was almost significantly reduced in the same group of adolescents but not in the group that lost less than 2.5 kg. IgA-coating bacterial proportions also decreased significantly in participants who lost more than 6 kg after the intervention, paralleled to reductions in C. histolyticum and E. rectale-C. coccoides populations. E. rectale-C. coccoides proportions also correlated with weight loss and BMI z-score reduction in participants whose weight loss exceeded 4 kg. Conclusions: Specific gut bacteria and an associated IgA response were related to body weight changes in adolescents under lifestyle intervention. These results suggest interactions between diet, gut microbiota and host metabolism and immunity in obesity.

Store-operated Ca²⁺ entry (SOCE) is a major pathway for Ca²⁺ entry that regulates several cellular functions. SOCE remodeling mediated by changes in the expression and/or function of the Orai channels results in the reorganization of intracellular Ca 2+ homeostasis leading to a variety of pathologies, including cancer. Notably, a significant alteration of Orai function has been reported in breast cancer cells, where the dysregulation of the Notch1 signaling pathway plays a role in the development and progression of cancer hallmarks. Here, we have investigated the possible role of Notch1 in the regulation of the expression of Orai1 and Orai3 in different breast cancer cell lines. Expression of the active form of Notch1, as well as cell stimulation with the Notch1 agonist Jagged-1 (Jag-1), demonstrates a differential role of Notch1 in the regulation of Orai expression in non-tumoral breast epithelial cells and triple negative or luminal breast cancer cells. The role of Notch1 was confirmed using DAPT, a γ-secretase inhibitor that prevents activation of the Notch pathway. Modulation of Orai1 and Orai3 expression by Notch1 was paralleled by changes in SOCE. The effect in Orai expression mediated by activation of Notch1 signaling pathway was mimicked by the expression of HEY1 or the non-phosphorylatable HEY1-S68A mutant; by contrast, expression of the phosphomimetic HEY1-S68D mutant was without effect on Orai expression. Understanding the Notch1-HEY1-Orai axis might provide insights into the development of subtype-specific therapeutic strategies targeting breast cancer.

(1) Background: Regucalcin or senescence marker protein 30 (SMP30) is a Ca2+ binding protein discovered in 1978 with multiple functions reported in the literature. However, the impact of exercise training on SMP30 in humans has not been analyzed. Aging is associated with many detrimental physiological changes that affect body composition, functional capacity, and balance. The present study aims to investigate the effects of whole body vibration (WBV) in postmenopausal women. (2) Methods: A total of 13 women (aged 54.3 ± 3.4 years) participated in the study. SMP30, body composition (fat mass, lean mass, and bone mass) and physical fitness (balance, time up and go (TUG) and 6-min walk test (6MWT)) were measured before and after the 12 weeks of WBV training. (3) Results: The WBV training program elicited a significant increase in SPM30 measured in plasma (27.7%, p = 0.004) and also in 6MWT (12.5%, p < 0.001). The WBV training also significantly reduced SPM30 measured in platelets (38.7%, p = 0.014), TUG (23.1%, p < 0.001) and total body fat mass (4.4%, p = 0.02). (4) Conclusions: There were no significant differences in balance, lean mass or bone mass. The present study suggests that 12 weeks of WBV has the potential to improve SPM30, fat mass, TUG and 6MWT in postmenopausal women.

Transient receptor potential channels convey signaling information from a number of stimuli to a wide variety of cellular functions, mainly by inducing changes in cytosolic Ca2+ concentration. Different members of the TRPC, TRPM and TRPV subfamilies have been reported to play a role in tumorigenesis. Here we show that the estrogen receptor positive and triple negative breast cancer cell lines, MCF7 and MDA-MB-231, respectively, exhibit enhanced expression of the TRPC6 channel as compared to the non-tumoral MCF10A cell line. In vitro TRPC6 knockdown using shRNA impaired MCF7 and MDA-MB-231 cell proliferation, migration and invasion detected by BrdU incorporation, wound healing and Boyden chamber assays, respectively. Using RNAi-mediated TRPC6 silencing as well as overexpression of the pore-dead dominant-negative TRPC6 mutant we have found that TRPC6 plays a relevant role in the activation of store-operated Ca2+ entry in the breast cancer cell lines but not in non-tumoral breast cells. Finally, we have found that TRPC6 interacts with Orai1 and Orai3 in MCF7 and MDA-MB-231 cells and is required for the translocation of Orai1 and Orai3 to the plasma membrane in MDA-MB-231 and MCF7 cells, respectively, upon Ca2+ store depletion. These findings introduce a novel mechanism for the modulation of Ca2+ influx and the development of different cancer hallmarks in breast cancer cells.

Breast cancer is a heterogeneous disease from the histological and molecular expression point of view, and this heterogeneity determines cancer aggressiveness. Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is significantly remodeled in cancer cells and plays an important role in the development and support of different cancer hallmarks. The store-operated CRAC (Ca2+ release-activated Ca2+) channels are predominantly comprised of Orai1 but the participation of Orai2 and Orai3 subunits has been reported to modulate the magnitude of Ca2+ responses. Here we provide evidence for a heterogeneous expression of Orai2 among different breast cancer cell lines. In the HER2 and triple negative breast cancer cell lines SKBR3 and BT20, respectively, where the expression of Orai2 was greater, Orai2 modulates the magnitude of SOCE and sustain Ca2+ oscillations in response to carbachol. Interestingly, in these cells Orai2 modulates the activation of NFAT1 and NFAT4 in response to high and low agonist concentrations. Finally, we have found that, in cells with high Orai2 expression, Orai2 knockdown leads to cell cycle arrest at the G0-G1 phase and decreases apoptosis resistance upon cisplatin treatment. Altogether, these findings indicate that, in breast cancer cells with a high Orai2 expression, Orai2 plays a relevant functional role in agonist-evoked Ca2+ signals, cell proliferation and apoptosis resistance.

(1) Background: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been reported. (2, 3) Methods and results: We used genetic and biochemical approaches to identify the molecular mechanism downstream of σ2R/TMEM97. The novel σ2R/TMEM97 fluorescent ligand, NO1, reduced the proliferation and survival of the triple negative breast cancer cell lines (TNBC: MDA-MB-231 and MDA-MB-468 cell lines), due to NO1-induced apoptosis. Greater bioaccumulation and faster uptake of NO1 in MDA-MB-231 cells compared to MCF10A or MCF7 cell lines were also shown. Accordingly, elevated σ2R/TMEM97 expression was confirmed by Western blotting. In contrast to NO1, other σ2R/TMEM97 ligands, such as SM21 and PB28, enhanced MDA-MB-231 cell proliferation and migration. Store-operated calcium entry (SOCE) is crucial for different cancer hallmarks. Here, we show that NO1, but not other σ2R/TMEM97 ligands, reduced SOCE in MDA-MB-231 cells. Similarly, TMEM97 silencing in MDA-MB-231 cells also impaired SOCE. NO1 administration downregulated STIM1-Orai1 interaction, probably by impairing the positive regulatory effect of σ2R/TMEM97 on STIM1, as we were unable to detect interaction with Orai1. (4) Conclusion: σ2R/TMEM97 is a key protein for the survival of triple negative breast cancer cells by promoting SOCE; therefore, NO1 may become a good pharmacological tool to avoid their proliferation.

The STIM family of proteins plays a crucial role in a plethora of cellular functions through the regulation of store-operated Ca2+ entry (SOCE) and, thus, intracellular calcium homeostasis. The two members of the mammalian STIM family, STIM1 and STIM2, are transmembrane proteins that act as Ca2+ sensors in the endoplasmic reticulum (ER) and, upon Ca2+ store discharge, interact with and activate the Orai/CRACs in the plasma membrane. Dysregulation of Ca2+ signaling leads to the pathogenesis of a variety of human diseases, including neurodegenerative disorders, cardiovascular diseases, cancer, and immune disorders. Therefore, understanding the mechanisms underlying Ca2+ signaling pathways is crucial for developing therapeutic strategies targeting these diseases. This review focuses on several rare conditions associated with STIM1 mutations that lead to either gain- or loss-of-function, characterized by myopathy, hematological and immunological disorders, among others, and due to abnormal activation of CRACs. In addition, we summarize the current evidence concerning STIM2 allele duplication and deletion associated with language, intellectual, and developmental delay, recurrent pulmonary infections, microcephaly, facial dimorphism, limb anomalies, hypogonadism, and congenital heart defects.

We have evaluated the effect of melatonin on apoptosis evoked by increases in [Ca²⁺] c in human leukocytes. Our results show that treatment of neutrophils with the calcium mobilizing agonist FMLP or the specific inhibitor of calcium reuptake thapsigargin induced a transient increase in [Ca²⁺] c . Our results also show that FMLP and thapsigargin increased caspase-9 and -3 activities and the active forms of both caspases. The effect of FMLP and thapsigargin on caspase activation was time-dependent. Similar results were obtained when lymphocytes were stimulated with thapsigargin. This stimulatory effect was accompanied by induction of mPTP, activation of the proapoptotic protein Bax and release of cytochrome c. However, when leukocytes were pretreated with melatonin, all of the apoptotic features indicated above were significantly reversed. Our results suggest that melatonin reduces caspase-9 and -3 activities induced by increases in [Ca²⁺] c in human leukocytes, which are produced through the inhibition of both mPTP and Bax activation.

Background: Regucalcin, or senescence marker protein-30 (SMP30), is a Ca2+-binding protein with multiple functions reported in the literature. Physical exercise has been shown to improve aging markers; nevertheless, SMP30 in humans has not been extensively researched. Older adults experience a decline in functional capacity and body composition. The purpose of this study was to examine the effects of a multicomponent training (MCT) program on SMP30 and its regulation of walking ability and body composition in functionally limited, frail, and pre-frail older adults. Methods: A total of 34 older adults (aged 80.3 ± 6.1 years) were divided into an intervention group (IG = 20) and control group (CG = 14). The IG performed a supervised MCT (strength, endurance, balance, coordination, and flexibility) program for 6 months, 3 days per week, whereas the CG continued their normal lives without any specific physical training. SMP30 was analyzed in plasma after 3 and 6 months of MCT, while some physical fitness variables (Timed Up and Go (TUG) and 6-min walk test (6MWT)) and body composition (fat mass and lean mass) were measured at baseline, as well as after 3 months and 6 months of MCT. Results: No significant changes were observed in SPM30 between the IG (877.5 a.u. to 940.5 a.u., respectively) and CG (790.4 a.u. to 763.8 a.u., respectively). Moreover, no SMP30 differences were found between groups after 3 and 6 months of MCT. The IG improved significantly in the 6MWT after 3 months (472.2 ± 84.2 m) compared to baseline (411.2 ± 75.2 m). The IG also significantly enhanced their TUG performance after 3 months (7.6 ± 1.6 s) and 6 months (7.3 ± 1.8 s) of training compared to baseline (9.3 ± 3.2 s) (all, p < 0.001). There were no significant differences in body composition between the IG and CG through the 6 months of MCT. Conclusions: The present study suggests that MCT did not change SMP30 levels from 3 to 6 months, where there were changes in neither walking ability nor body composition; however, MCT was effective in improving 6MWT and TUG performance from baseline to 3 months.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a central role in the intracellular signaling of P2X7 nucleotide receptors in neurons and glial cells. Fine spatio-temporal tuning of mitogen-activated protein (MAP) kinases is essential to regulate their biological activity. MAP kinase phosphatases (MKPs) are dual specificity protein phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues in MAP kinases. This study focuses on how DUSP, DUSP6/MKP3, a phosphatase specific for ERK1/2 is regulated by the P2X7 nucleotide receptor in cerebellar granule neurons and astrocytes. Stimulation with the specific P2X7 agonist, BzATP, or epidermal growth factor (EGF) (positive control for ERK activation) regulates the levels of DUSP6 in a time dependent manner. Both agonists promote a decline in DUSP6 protein, reaching minimal levels after 30 min yet recovering to basal levels after 1 h. The initial loss of protein occurs through proteasomal degradation, as confirmed in experiments with the proteasome inhibitor, MG-132. Studies carried out with Actinomycin D demonstrated that the enhanced transcription of the gene is responsible for recovering the DUSP6 protein levels. Interestingly, ERK1/2 proteins are involved in the biphasic regulation of the protein phosphatase, being required for both the degradation and the recovery phase. We show that direct Ser phosphorylation of DUSP6 by ERK1/2 proteins could be part of the mechanism regulating their cytosolic levels, at least in glial cells. Thus, the ERK1/2 activated by P2X7 receptors exerts positive feedback on these kinase's own activity, promoting the degradation of one of their major inactivators in the cytosolic compartment, DUSP6, both in granule neurons and astrocytes. This feedback loop seems to function as a common universal mechanism to regulate ERK signaling in neural and non-neural cells.