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1.
Voltage-gated calcium channel nanodomains: molecular composition and function.
Gandini, MA, Zamponi, GW
The FEBS journal. 2022;(3):614-633
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Abstract
Voltage-gated calcium (CaV ) channels and their regulation by proteins at the synaptic cleft play a critical role in neurotransmission. These interactions fine-tune the synaptic response through the regulation of Ca2+ entry into the presynaptic terminal and trigger the fusion of vesicles filled with neurotransmitters and peptides. Regulation of CaV channel intrinsic properties and their numbers at the active zones shape the timing and strength of synaptic function. Here, we provide an overview of a number of proteins reported to be part of CaV channel nanodomains at the synaptic cleft and the repercussions of these interactions for CaV channel trafficking, tethering at the active zone, and regulation of their biophysical properties. We summarize the current state of knowledge by which CaV channels are regulated at presynaptic sites.
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Serum Iba-1, GLUT5, and TSPO in Patients With Diabetic Retinopathy: New Biomarkers for Early Retinal Neurovascular Alterations? A Pilot Study.
Trotta, MC, Gesualdo, C, Petrillo, F, Cavasso, G, Corte, AD, D'Amico, G, Hermenean, A, Simonelli, F, Rossi, S
Translational vision science & technology. 2022;(3):16
Abstract
PURPOSE This study explored the possibility of highlighting early retinal neurovascular alterations of diabetic retinopathy (DR) by monitoring in DR patients the serum levels of microglial biomarkers ionized calcium-binding adapter molecule 1 (Iba-1), glucose transporter 5 (GLUT5), and translocator protein (TSPO), along with serum changes of the endothelial dysfunction marker arginase-1. METHODS Serum markers were determined by enzyme-linked immunosorbent assay in 50 patients: 12 non-diabetic subjects, 14 diabetic patients without DR, 13 patients with non-proliferative DR (NPDR), and 11 patients with proliferative DR (PDR). The results were correlated with hyperreflective retinal spots (HRS), observed with optical coherence tomography (OCT). RESULTS Although HRS were absent in diabetic patients without DR, NPDR patients showed an average of 4 ± 1 HRS, whereas the highest presence was detected in PDR patients, with 8 ± 1 HRS (P < 0.01 vs. NPDR). HRS were positively correlated (P < 0.01) with serum levels of arginase-1 (r = 0.91), Iba-1 (r = 0.96), GLUT5 (r = 0.94), and TSPO (r = 0.88). Moreover, serum proinflammatory cytokines and chemokines showed a positive correlation (P < 0.01) with HRS number and the serum markers analyzed. CONCLUSIONS Serum markers of microglial activation positively correlate with retinal HRS in NPDR and PDR patients. TRANSLATIONAL RELEVANCE These data corroborate the possibility of highlighting early retinal neurovascular changes due to diabetes by monitoring circulating microglial markers.
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An Expanded Genome-Wide Association Study of Fructosamine Levels Identifies RCN3 as a Replicating Locus and Implicates FCGRT as the Effector Transcript.
Riveros-Mckay, F, Roberts, D, Di Angelantonio, E, Yu, B, Soranzo, N, Danesh, J, Selvin, E, Butterworth, AS, Barroso, I
Diabetes. 2022;(2):359-364
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Abstract
Fructosamine is a measure of short-term glycemic control, which has been suggested as a useful complement to glycated hemoglobin (HbA1c) for the diagnosis and monitoring of diabetes. To date, a single genome-wide association study (GWAS) including 8,951 U.S. White and 2,712 U.S. Black individuals without a diabetes diagnosis has been published. Results in Whites and Blacks yielded different association loci, near RCN3 and CNTN5, respectively. In this study, we performed a GWAS on 20,731 European-ancestry blood donors and meta-analyzed our results with previous data from U.S. White participants from the Atherosclerosis Risk in Communities (ARIC) study (Nmeta = 29,685). We identified a novel association near GCK (rs3757840, βmeta = 0.0062; minor allele frequency [MAF] = 0.49; Pmeta = 3.66 × 10-8) and confirmed the association near RCN3 (rs113886122, βmeta = 0.0134; MAF = 0.17; Pmeta = 5.71 × 10-18). Colocalization analysis with whole-blood expression quantitative trait loci data suggested FCGRT as the effector transcript at the RCN3 locus. We further showed that fructosamine has low heritability (h2 = 7.7%), has no significant genetic correlation with HbA1c and other glycemic traits in individuals without a diabetes diagnosis (P > 0.05), but has evidence of shared genetic etiology with some anthropometric traits (Bonferroni-corrected P < 0.0012). Our results broaden knowledge of the genetic architecture of fructosamine and prioritize FCGRT for downstream functional studies at the established RCN3 locus.
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Role of Store-Operated Ca2+ Entry in the Pulmonary Vascular Remodeling Occurring in Pulmonary Arterial Hypertension.
Masson, B, Montani, D, Humbert, M, Capuano, V, Antigny, F
Biomolecules. 2021;(12)
Abstract
Pulmonary arterial hypertension (PAH) is a severe and multifactorial disease. PAH pathogenesis mostly involves pulmonary arterial endothelial and pulmonary arterial smooth muscle cell (PASMC) dysfunction, leading to alterations in pulmonary arterial tone and distal pulmonary vessel obstruction and remodeling. Unfortunately, current PAH therapies are not curative, and therapeutic approaches mostly target endothelial dysfunction, while PASMC dysfunction is under investigation. In PAH, modifications in intracellular Ca2+ homoeostasis could partly explain PASMC dysfunction. One of the most crucial actors regulating Ca2+ homeostasis is store-operated Ca2+ channels, which mediate store-operated Ca2+ entry (SOCE). This review focuses on the main actors of SOCE in human and experimental PASMC, their contribution to PAH pathogenesis, and their therapeutic potential in PAH.
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Circulating EGFL7 distinguishes between IUGR and PE: an observational case-control study.
Massimiani, M, Salvi, S, Tiralongo, GM, Moresi, S, Stuhlmann, H, Valensise, H, Lanzone, A, Campagnolo, L
Scientific reports. 2021;(1):17919
Abstract
Isolated intrauterine growth restriction (IUGR) and preeclampsia (PE) share common placental pathogenesis. Differently from IUGR, PE is a systemic disorder which may also affect liver and brain. Early diagnosis of these conditions may optimize maternal and fetal management. Aim of this study was to assess whether Epidermal Growth Factor-Like domain 7 (EGFL7) dosage in maternal blood discriminates between isolated IUGR and PE. A total of 116 women were enrolled in this case-control study: 12 non-pregnant women, 34 healthy pregnant women, 34 women presenting with isolated IUGR and 36 presenting with PE. Levels of circulating EGFL7 and other known pro- and anti-angiogenic factors were measured by ELISA at different gestational ages (GA). Between 22-25 weeks of gestation, EGFL7 levels in early-onset PE (e-PE) plasma samples were significantly higher than those measured in controls or isolated IUGR samples (69.86 ± 6.17 vs. 19.8 ± 2.5 or 18.8 ± 2.8 µg/ml, respectively). Between 26-34 weeks, EGFL7 levels remained significantly higher in e-PE compared to IUGR. At term, circulating and placental EGFL7 levels were comparable between IUGR and late-onset PE (l-PE). In contrast, circulating levels of PlGF were decreased in both IUGR- and PE- complicated pregnancies, while levels of both sFLT-1 and sENDOGLIN were increased in both conditions. In conclusion, EGFL7 significantly discriminates between isolated IUGR and PE.
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Inflammasome Genetic Variants, Macrophage Function, and Clinical Outcomes in Cystic Fibrosis.
Graustein, AD, Berrington, WR, Buckingham, KJ, Nguyen, FK, Joudeh, LL, Rosenfeld, M, Bamshad, MJ, Gibson, RL, Hawn, TR, Emond, MJ
American journal of respiratory cell and molecular biology. 2021;(2):157-166
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Abstract
Cystic fibrosis (CF) is characterized by chronic airway infection, inflammation, and tissue damage that lead to progressive respiratory failure. NLRP3 and NLRC4 are cytoplasmic pattern recognition receptors that activate the inflammasome, initiating a caspase-1-mediated response. We hypothesized that gain-of-function inflammasome responses are associated with worse outcomes in children with CF. We genotyped nonsynonymous variants in NLRP3 and the NLRC4 pathway from individuals in the EPIC (Early Pseudomonas Infection Control) Observational Study cohort and tested for association with CF outcomes. We generated knockouts of NLRP3 and NLRC4 in human macrophage-like cells and rescued knockouts with wild-type or variant forms of NLRP3 and NLRC4. We identified a SNP in NLRP3, p.(Q705K), that was associated with a higher rate of P. aeruginosa colonization (N = 609; P = 0.01; hazard ratio, 2.3 [Cox model]) and worsened lung function over time as measured by forced expiratory volume in 1 second (N = 445; P = 0.001 [generalized estimating equation]). We identified a SNP in NLRC4, p.(A929S), that was associated with a lower rate of P. aeruginosa colonization as part of a composite of rare variants (N = 405; P = 0.045; hazard ratio, 0.68 [Cox model]) and that was individually associated with protection from lung function decline (P < 0.001 [generalized estimating equation]). Rescue of the NLRP3 knockout with the p.(Q705K) variant produced significantly more IL-1β in response to NLRP3 stimulation than rescue with the wild type (P = 0.020 [Student's t test]). We identified a subset of children with CF at higher risk of early lung disease progression. Knowledge of these genetic modifiers could guide therapies targeting inflammasome pathways.
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Variants in Genes of Calpain System as Modifiers of Spinocerebellar Ataxia Type 3 Phenotype.
Martins, AC, Rieck, M, Leotti, VB, Saraiva-Pereira, ML, Jardim, LB
Journal of molecular neuroscience : MN. 2021;(9):1906-1913
Abstract
Calpain-mediated proteolysis has been proposed to modulate the pathogenesis of spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), a disorder due to a CAG repeat expansion (CAGexp) at ATXN3. We aimed to investigate if single-nucleotide polymorphisms (SNPs) at calpain gene CAPN2 and at calpastatin gene CAST modulate the age at onset (AO) and disease progression in SCA3/MJD. A total of 287 SCA3/MJD symptomatic subjects (151 families) were included. AO was analyzed and controlled by the CAG repeat length of expanded allele and family. Candidate polymorphisms were chosen based on the literature and on a priori criteria. The CAG repeat length and SNPs were genotyped according to standard methods. AO of carriers of AA and AG + GGrs1559085 genotypes in CAST and with the median value of 75 repeats at the expanded allele were 34.23 (33.07-35.38) and 36.42 years (34.50-38.34), respectively (p = 0.049, mixed model treating the expanded CAG repeat size as fixed effect and family as random effect). Carriers of haplotype Crs27852/Grs1559085 had mean AO of 37.23 (12.76) and 33.42 years (12.20) (p = 0.047, Student's t test). Our data suggest an association between allele Grs1559085 and haplotype Crs27852/Grs1559085 at CAST and variations in the AO of SCA3/MJD subjects, independent from the effects of the CAGexp and family. The present results support the potential role of calpain cleavage pathway over modulation of SCA3/MJD phenotype.
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Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression.
Weickert, MT, Hecker, JS, Buck, MC, Schreck, C, Rivière, J, Schiemann, M, Schallmoser, K, Bassermann, F, Strunk, D, Oostendorp, RAJ, et al
Scientific reports. 2021;(1):5944
Abstract
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.
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A low cartilage formation and repair endotype predicts radiographic progression of symptomatic knee osteoarthritis.
Luo, Y, Samuels, J, Krasnokutsky, S, Byrjalsen, I, Kraus, VB, He, Y, Karsdal, MA, Abramson, SB, Attur, M, Bay-Jensen, AC
Journal of orthopaedics and traumatology : official journal of the Italian Society of Orthopaedics and Traumatology. 2021;(1):10
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Abstract
BACKGROUND Osteoarthritis (OA) is a disease with multiple endotypes. A hallmark of OA is loss of cartilage; however, it is evident that the rate of cartilage loss differs among patients, which may partly be attributed to differential capacity for cartilage repair. We hypothesize that a low cartilage repair endotype exists and that such endotypes are more likely to progress radiographically. The aim of this study is to examine the associations of level of cartilage formation with OA severity and radiographic OA progression. We used the blood-based marker PRO-C2, reflecting type II collagen formation, to assess levels of cartilage formation. MATERIALS AND METHODS The type II collagen propeptide PRO-C2 was measured in the serum/plasma of knee OA subjects from New York University (NYU, n = 106) and a subcohort of the phase III oral salmon calcitonin (sCT) trial SMC021-2301 (SMC, n = 147). Risk of radiographic medial joint space narrowing (JSN) over 24 months was compared between quartiles (very low, low, moderate, and high) of PRO-C2. Associations were adjusted for age, gender, BMI, race, baseline pain levels, and baseline joint space width. RESULTS In both the NYU and SMC cohorts, subjects with low PRO-C2 levels had greater JSN compared with subjects with high PRO-C2. Mean difference in JSN between subjects with very low and high levels of PRO-C2 was 0.65 mm (p = 0.002), corresponding to a 3.4 (1.4-8.6)-fold higher risk of progression. There was no significant effect of sCT treatment, compared with placebo, on JSN over 2 years before stratification based on baseline PRO-C2. However, there were proportionately fewer progressors in the sCT arm of the very low/low PRO-C2 group compared with the moderate/high group (Chi squared = 6.5, p = 0.011). CONCLUSION Serum/plasma level of type II collagen formation, PRO-C2, may be an objective indicator of a low cartilage repair endotype, displaying radiographic progression and superior response to a proanabolic drug. LEVEL OF EVIDENCE Level III post hoc exploratory analysis of one longitudinal cohort and a sub-study from one phase III clinical trial.
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Development of antibodies against the notch ligand Delta-Like-1 by phage display with activity against breast cancer cells.
Sales-Dias, J, Ferreira, A, Lamy, M, Domenici, G, Monteiro, SMS, Pires, A, Lemos, AR, Kucheryava, K, Nobre, LS, Sousa, PMF, et al
New biotechnology. 2021;:17-26
Abstract
Notch signalling is a well-established oncogenic pathway, and its ligand Delta-like 1 (DLL1) is overexpressed in estrogen receptor-positive (ER+) breast cancers and associated with poor patient prognosis. Hence, DLL1 has become an interesting therapeutic target for breast cancer. Here, the development of specific functional blocking anti-DLL1 antibodies with potential activity against ER+ breast cancer cells is reported. Human DLL1 proteins, containing the essential regions for binding to the Notch receptor and Notch signalling activation, were produced and used to select specific scFv antibody fragments by phage display. Fifteen unique scFvs were identified and reformatted into full IgGs. Characterization of these antibodies by ELISA, surface plasmon resonance and flow cytometry enabled selection of three specific anti-DLL1 IgGs, sharing identical VH regions, with nM affinities. Cellular assays on ER+ breast cancer MCF-7 cells showed that one of the IgGs (IgG-69) was able to partially impair DLL1-mediated activation of the Notch pathway, as determined by Notch reporter and RT-qPCR assays, and to attenuate cell growth. Treatment of MCF-7 cells with IgG-69 reduced mammosphere formation, suggesting that it decreases the breast cancer stem cell subpopulation. These results support the use of this strategy to develop and identify potential anti-DLL1 antibodies candidates against breast cancer.