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Introduction: A microdeletion including the SNORD116gene (SNORD116MD) has been shown to drive the Prader-Willi syndrome (PWS) features. PWS is a neurodevelopmental disorder clinically characterized by endocrine impairment, intellectual disability and psychiatric symptoms such as a lack of emotional regulation, impulsivity, and intense temper tantrums with outbursts. In addition, this syndrome is associated with a nutritional trajectory characterized by addiction-like behavior around food in adulthood. PWS is related to the genetic loss of expression of a minimal region that plays a potential role in epigenetic regulation. Nevertheless, the role of the SNORD116MD in DNA methylation, as well as the impact of the oxytocin (OXT) on it, have never been investigated in human neurons. Methods: We studied the methylation marks in induced pluripotent stem-derived dopaminergic neurons carrying a SNORD116MD in comparison with those from an age-matched adult healthy control. We also performed identical neuron differentiation in the presence of OXT. We performed a genome-wide DNA methylation analysis from the iPSC-derived dopaminergic neurons by reduced-representation bisulfite sequencing. In addition, we performed RNA sequencing analysis in these iPSC-derived dopaminergic neurons differentiated with or without OXT. Results: The analysis revealed that 153,826 cytosines were differentially methylated between SNORD116MD neurons and control neurons. Among the differentially methylated genes, we determined a list of genes also differentially expressed. Enrichment analysis of this list encompassed the dopaminergic system with COMTand SLC6A3. COMTdisplayed hypermethylation and under-expression in SNORD116MD, and SLC6A3displayed hypomethylation and over-expression in SNORD116MD. RT-qPCR confirmed significant over-expression of SLC6A3in SNORD116 MDneurons. Moreover, the expression of this gene was significantly decreased in the case of OXT adjunction during the differentiation. Conclusion: SNORD116MD dopaminergic neurons displayed differential methylation and expression in the COMTand SLC6A3genes, which are related to dopaminergic clearance.

Late‐life aging is often associated with appetite reduction and weight loss. Physical activity (PA) may prevent these processes, but the molecular mechanisms involved remain elusive. The present study investigated the putative mediating aspect of growth differentiation factor 15 (GDF‐15), a stress signalling protein involved in aging, exercise and appetite control, on the association between PA and late‐life‐associated weight loss. One thousand eighty‐three healthy adults (63.8% women) aged 70 years and over who participated in the Multidomain Alzheimer Preventive Trial were included. Bodyweight (kg) and PA levels (square root of metabolic equivalent of task‐min/week) were assessed repeatedly from baseline to the 3‐year visit, whereas plasma GDF‐15 (pg/mL) was measured at the 1‐year visit. Multiple linear regressions were performed to test the association between first‐year mean PA level, 1‐year visit GDF‐15 concentration and subsequent bodyweight changes. Mediation analyses were used to investigate whether GDF‐15 mediated the association between first‐year mean PA levels and consecutive bodyweight changes. Multiple regression analyses demonstrated that higher first‐year mean PA levels significantly predicted lower GDF‐15 and bodyweight at 1 year (B = −2.22; SE = 0.79; P= 0.005). In addition, higher 1‐year visit GDF‐15 levels were associated with faster subsequent bodyweight loss (Time × GDF‐15 interaction B = −0.0004; SE = 0.0001; P= 0.003). Mediation analyses confirmed that GDF‐15 mediated the association between first‐year mean PA levels and subsequent bodyweight changes (mediated effect ab = 0.0018; bootstrap SE = 0.001; P< 0.05) and revealed that mean PA had no direct effect on subsequent bodyweight changes (c′ = 0.006; SE = 0.008; P> 0.05). This study suggests that GDF‐15 may be one of the molecules mediating the link between PA and late‐life weight loss, but mechanistic studies are necessary to further support the present findings.

Addictive disorders have been much investigated and many studies have underlined the role of environmental factors such as social interaction in the vulnerability to and maintenance of addictive behaviors. Research on addiction pathophysiology now suggests that certain behavioral disorders are addictive, one example being food addiction. Yet, despite the growing body of knowledge on addiction, it is still unknown why only some of the individuals exposed to a drug become addicted to it. This observation has prompted the consideration of genetic heritage, neurodevelopmental trajectories, and gene-environment interactions in addiction vulnerability. Prader–Willi syndrome (PWS) is a rare neurodevelopmental disorder in which children become addicted to food and show early social impairment. PWS is caused by the deficiency of imprinted genes located on the 15q11–q13 chromosome. Among them, the SNORD116gene was identified as the minimal gene responsible for the PWS phenotype. Several studies have also indicated the role of the Snord116gene in animal and cellular models to explain PWS pathophysiology and phenotype (including social impairment and food addiction). We thus present here the evidence suggesting the potential involvement of the SNORD116gene in addictive disorders.

Background: Achondroplasia is the most common short-limbed skeletal dysplasia resulting from gain-of-function pathogenic variants in fibroblast growth factor receptor 3 (FGFR3) gene, a negative regulator of endochondral bone formation. Most treatment options are symptomatic, targeting medical complications. Infigratinib is an orally bioavailable, FGFR1–3 selective tyrosine kinase inhibitor being investigated as a direct therapeutic strategy to counteract FGFR3 overactivity in achondroplasia.Objectives: The main objective of PROPEL is to collect baseline data of children with achondroplasia being considered for future enrollment in interventional studies sponsored by QED Therapeutics. The objectives of PROPEL 2 are to obtain preliminary evidence of safety and efficacy of oral infigratinib in children with achondroplasia, to identify the infigratinib dose to be explored in future studies, and to characterize the pharmacokinetic (PK) profile of infigratinib and major metabolites.Design: PROPEL (NCT04035811) is a prospective, noninterventional clinical study designed to characterize the natural history and collect baseline data of children with achondroplasia over 6−24 months. PROPEL 2 (NCT04265651), a prospective, phase II, open-label study of infigratinib in children with achondroplasia, consists of a dose-escalation, dose-finding, and dose-expansion phase to confirm the selected dose, and a PK substudy.Methods and analysis: Children aged 3−11 years with achondroplasia who completed ⩾6 months in PROPEL are eligible for PROPEL 2. Primary endpoints include treatment-emergent adverse events and change from baseline in annualized height velocity. Four cohorts at ascending dose levels are planned for dose escalation. The selected dose will be confirmed in the dose-expansion phase.Ethics: PROPEL and PROPEL 2 are being conducted in accordance with the International Conference on Harmonization Good Clinical Practice guidelines, principles of the Declaration of Helsinki, and relevant human clinical research and data privacy regulations. Protocols have been approved by local health authorities, ethics committees, and institutions as applicable. Parents/legally authorized representatives are required to provide signed informed consent; signed informed assent by the child is also required, where applicable.Discussion: PROPEL and PROPEL 2 will provide preliminary evidence of the safety and efficacy of infigratinib as precision treatment of children with achondroplasia and will inform the design of future studies of FGFR-targeted agents in achondroplasia.Registration: ClinicalTrials.gov: NCT04035811; NCT04265651.

During pregnancy, mineral concentrations, of calcium and phosphorus in particular, are maintained at a high level in fetal blood so that the developing skeleton may accrete adequate mineral content. The placenta actively transports minerals for this purpose. Maternal intestinal absorption increases in order to meet the fetal demand for calcium, which is only partly dependent on calcitriol. Mineral regulation is essentially dependent on parathyroid hormone (PTH) and PTH-related protein (PTHrP). The calcium-sensing receptor (CaSR) regulates PTH and PTHrP production. If calcium intake is insufficient, the maternal skeleton will undergo resorption due to PTHrP. After birth, a switch from fetal to neonatal homeostasis occurs through increase in PTH and calcitriol, and developmental adaptation of the kidneys and intestines with bone turnover contributing additional mineral to the circulation. Calcium absorption becomes progressively active and dependent on calcitriol. The postnatal skeleton can transiently present with osteoposis but adequate mineral diet usually allows full restoration. Cases of primary osteoporosis must be identified. Loss of trabecular mineral content occurs during lactation in order to provide calcium to the newborn. This programmed bone loss is dependent on a “brain-breast-bone” circuit. The physiological bone resorption during reproduction does not normally cause fractures or persistent osteoporosis. Women who experience fracture are likely to have other causes of bone loss.

To describe the presenting features, bone characteristics and molecular genetics in a large monocentric cohort of children and young adults with idiopathic primary osteoporosis.

HCV core induces loss of polarity and down-regulates SHIP2 and Dlg1 expression. SHIP2 and PtdIns(3,4)P2 are localized at the basolateral membrane of polarized cells. SHIP2 siRNA and its catalytically inactive mutant disrupt epithelial polarity, and SHIP2 rescues core-induced loss of polarity through RhoA activation.

Abstract

Loss-of-function variants in the calcium-sensing receptor (CASR) gene are known to be involved in a clinical spectrum ranging from asymptomatic familial hypocalciuric hypercalcemia (FHH) to neonatal severe hyperparathyroidism (NSHPT). Homozygous or compound heterozygous variants are usually responsible for severe neonatal forms, whereas heterozygous variants cause benign forms. One recurrent pathogenic variant, p.Arg185Gln, has been reported in both forms, in a heterozygous state. This variant can be a de novooccurrence or can be inherited from a father with FHH.

OBJECTIVE: To investigate the associations of molecular markers of joint tissue turnover with clinical and radiological variables in patients with hip osteoarthritis (OA). METHODS: Patients of the ECHODIAH trial cohort (60% female; mean age 63 yrs, disease duration 5 yrs) fulfilling the American College of Rheumatology criteria for hip OA were studied. Pain was assessed using a 100 mm visual analog scale, and the presence of night pain and morning stiffness was observed as the index of joint inflammation. Joint space width (JSW) and subchondral bone sclerosis were assessed on hip radiographs. Ten markers were measured, 8 in serum: N-propeptides of collagen type I (PINP) and type III (PIIINP), cartilage oligomeric matrix protein (COMP), YKL-40, hyaluronan (HA), matrix metalloproteases (MMP1 and MMP3), and ultrasensitive C-reactive protein (CRP); and 2 in urine: C-terminal crosslinking telopeptides of collagen type I (CTX-I) and type II (CTX-II). Analyses of 376 patients with measurements of all the markers included principal component analyses to identify independent clusters of markers; followed by stepwise multivariate regressions to determine associations between markers, clinical variables, and radiographic signs of joint damage. RESULTS: Markers could be segregated into independent clusters: CTX-II, PINP, and CTX-I for cartilage degradation and bone turnover; COMP, PIIINP, and HA as potential markers of synovitis; and CRP and YKL-40, which are likely to indicate systemic inflammation; plus MMP1 and MMP3. After adjustment for age, sex, and body mass index, pain was significantly associated with CTX-II (p = 0.0095) and CRP (p = 0.046) and joint inflammation with COMP (p = 0.013). Radiographic signs of joint damage were associated with CTX-II (p = 0.001 for JSW; p = 0.007 for bone sclerosis). CONCLUSION: This cross-sectional study of OA molecular markers in a large cohort may provide biological evidence of different pathophysiological processes involved in hip OA. Among the markers measured, CTX-II showed the most consistent association with the symptoms and joint damage of OA.

Exosomes are small vesicles secreted from multivesicular bodies, which are able to stimulate the immune system leading to tumour cell eradication. We have analysed lipids of exosomes secreted either upon stimulation from rat mast cells (RBL-2H3 cells), or constitutively from human dendritic cells. As compared with parent cells, exosomes displayed an enrichment in sphingomyelin, but not in cholesterol. Phosphatidylcholine content was decreased, but an enrichment was noted in disaturated molecular species as in phosphatidylethanolamines. Lyso(bis)phosphatidic acid was not enriched in exosomes as compared with cells. Fluorescence anisotropy demonstrated an increase in exosome-membrane rigidity from pH 5 to 7, suggesting their membrane reorganization between the acidic multivesicular body compartment and the neutral outer cell medium. NMR analysis established a bilayer organization of exosome membrane, and ESR studies using 16-doxyl stearic acid demonstrated a higher flip-flop of lipids between the two leaflets as compared with plasma membrane. In addition, the exosome membrane exhibited no asymmetrical distribution of phosphatidylethanolamines. Therefore exosome membrane displays a similar content of the major phospholipids and cholesterol, and is organized as a lipid bilayer with a random distribution of phosphatidylethanolamines. In addition, we observed tight lipid packing at neutral pH and a rapid flip-flop between the two leaflets of exosome membranes. These parameters could be used as a hallmark of exosomes.

A previous study demonstrated that cross-desensitization experiments performed with the lysophosphatidic acid (LPA) analogues (R)- and (S)-N-palmitoyl-norleucinol 1-phosphate (PNPAs) inhibited LPA-induced platelet aggregation without any stereospecificity. Here we report opposite biological effects of the two enantiomers on mitogenesis of IMR-90 fibroblasts in relation to their respective metabolism. (R)PNPA was proliferative, while (S)PNPA induced apoptosis by specifically inhibiting phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by cholinephosphotransferase. This effect was not direct but required dephosphorylation of PNPAs by ecto-lipid phosphate phosphatase before cellular uptake of the generated N-palmitoyl-norleucinols (PNOHs). Inhibition of cholinephosphotransferase by the derivative (S)PNOH was confirmed by an in vitro assay. (S)PNPA proapoptotic effects led us to clarify the mechanism linking cholinephosphotransferase inhibition to apoptosis. Three proapoptotic responses were observed: the activation of caspase-3, the production of ceramides from newly synthesized pools (as demonstrated by the inhibitor Fumonisin B1) and finally the activation of stress-activated protein kinase, p38 and c-Jun N-terminal kinases 1/2, as a result of ceramide increase. Thus our data demonstrate that synthetic analogues of LPA might display stereospecific effects leading to apoptosis independently of classical LPA-activated pathways.

The lipid mediator sphingosine 1-phosphate (S1P) may alter the proliferation of mesangial cells during pathophysiological processes. Here, S1P stimulated proliferation of rat mesangial cells and phosphorylation of MAPKs at subconfluent cell density. Both effects were inhibited by pertussis toxin treatment. Mesangial cells expressed several S1P receptors of theendothelial differentiation gene family: EDG-1, -3, -5, and -8. Conversely, S1P induced apoptosis at low cell density (2 × 104cells/cm2), which was demonstrated by flow cytometry and Hoechst staining. Apoptosis was observed also in quiescent or growing cells and was not reverted by lysophosphatidic acid or platelet-derived growth factor. S1P enhanced phosphorylation of SAPKs. Incubation with [33P]S1P, [3H]S1P, and [3H]sphingosine demonstrated increased S1P hydrolysis, resulting in enhanced intracellular sphingosine levels and decreased S1P levels. A rise in total ceramide levels was also observed; however, ceramide did not originate from [3H]sphingosine, and S1P-induced apoptosis was not inhibited by fumonisin B, precluding involvement of de novoceramide synthesis in apoptosis. Therefore, we suggest that sphingosine accumulation and decreased S1P are primarily responsible for S1P-induced apoptosis. In conclusion, incubation of low-density mesangial cells with S1P results in apoptosis, presumably due to increased S1P hydrolysis.

The ubiquitous form of the sodium–hydrogen exchanger, NHE1, is devoted to the regulation of intracellular pH and cell volume. In addition, NHE1 activity is stimulated by growth factors and increased NHE rates are found in both circulating and immortalized cells during diabetes or diabetic nephropathy. In this context, we searched for polymorphisms of the 5′-flanking regulatory region of NHE1 gene in subjects with type-I diabetes. We identified a C/T transition 696 bases upstream the translation initiation start site which disrupts a repeated palindromic GC sequence. The TT genotype was significantly more frequent in type-1 diabetics and may have functional importance. Genetic linkage between NHE1 and diabetes has been previously described in NOD mice strains with consequences on NHE rates. Hence, the polymorphism described hereby may act as a predisposition factor to type-I diabetes or to diabetic complications, and may be useful to investigate the genetic involvement of NHE1 in human pathophysiology.

Hyperactive SHP2 promotes inflammation-driven insulin resistance by skewing macrophage balance in metabolic tissues and may be a target in T2D.

Endonexin (protein II, 32.5 kDa) has been purified to homogeneity from bovine liver in the following steps: selective extraction by EGTA from membranes precipitated with Triton X‐100/calcium; chromatography on DEAE‐TSK 545 at pH 7.0, endonexin being eluted at 0.1 M NaCl; affinity chromatography on polyacrylamide‐immobilized phosphatidylserine; gel filtration on TSK 3000. The amino acid composition was essentially similar to that previously reported. Using [3H]oleic acid‐labelled Escherichia colimembranes as substrate, endonexin inhibited phospholipase A2from pig pancreas. Maximal inhibition was 55 and 70%, whereas 50% inhibition occurred at 480 and 120 nM endonexin and lipocortin II, respectively. These data could be related to common features shared by both lipocortins/calpactins and endonexin, i.e. the presence of a consensus sequence and the ability to bind to anionic phospholipids in a calcium‐dependent manner.

Endonexin (protein II, 32.5 kDa) has been purified to homogeneity from bovine liver in the following steps: selective extraction by EGTA from membranes precipitated with Triton X-100/calcium; chromatography on DEAE-TSK 545 at pH 7.0, endonexin being eluted at 0.1 M NaCl; affinity chromatography on polyacrylamide-immobilized phosphatidylserine; gel filtration on TSK 3000. The amino acid composition was essentially similar to that previously reported. Using [ 3H]oleic acid-labelled Escherichia colimembranes as substrate, endonexin inhibited phospholipase A 2from pig pancreas. Maximal inhibition was 55 and 70%, whereas 50% inhibition occurred at 480 and 120 nM endonexin and lipocortin II, respectively. These data could be related to common features shared by both lipocortins/calpactins and endonexin, i.e. the presence of a consensus sequence and the ability to bind to anionic phospholipids in a calcium-dependent manner.

From very recent studies, including molecular cloning of cDNA coding for membrane receptors, lysophosphatidic acid (LPA) reached the status of a novel phospholipid mediator with various biological activities. Another strong argument supporting this view was the discovery that LPA is secreted from activated platelets, resulting in its appearance in serum upon blood coagulation. The metabolic pathways as well as the enzymes responsible for LPA production are poorly characterized. However, a survey of literature data indicates some interesting issues which might be used as the basis for further molecular characterization of phospholipases A able to degrade phosphatidic acid.

In order to assess the potential role of the plasma membrane sodium-proton (Na-/H+) exchanger in the pathogenesis of diabetic nephropathy, we investigated 32 insulin dependent (type 1) diabetic patients and 21 control subjects. We tested the Na+/H+ exchange as the rate of amiloride sensitive and sodium dependent volume gain of platelets suspended in sodium propionate. Patients with diabetic nephropathy had significantly increased rates of Na+/H+ exchange (0.31 ± 0.06 s-1 × 10-2) when compared to those without nephropathy (0.24 ± 0.07, p < 0.05) or to a control group (0.23 ± 05, p < 0.05). Nine patients who were classified as hypertensive had a highly significant increase in the Na+/H+ exchange rates when compared to 23 non-hypertensive diabetic patients: 0.33 ± 0.04 versus 0.24 ± 0.06 (p < 0.001). There was no significant correlation between the Na+/H+ exchange rates and age, diabetes duration, glycated hemoglobin or fructosamine levels on the day of the test. In summary, the data presented here demonstrate an increase in the Na+/H+ exchange rate in insulin-dependent diabetic patients with nephropathy and hypertension

We have examined the Acylated Ghrelin (AG)/Gi pathway in different human osteoblastic cell lines. We have found that: 1) AG induces differentiation/mineralization only in mature osteoblasts; 2) the expression of GHS-R1a increases up to the mature cell stage, 3) the action is mediated via the GHS-R/Gi/cAMP pathway only in mature osteoblasts, and 4) osteoblastic cells from adolescent idiopathic scoliosis (AIS) are resistant to the AG/Gi/cAMP pathway. Altogether, these results suggested that AG uses the GHS-R1a/Gi/cAMP pathway to induce differentiation in mature osteoblasts only. This pathway is impaired in AIS osteoblasts. Understanding AG-specific pathways involved in normal and pathological osteoblasts may be useful for developing new treatments for pathologies such as AIS or osteoporosis.

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