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The study of the respiratory microbiota has revealed that the lungs of healthy and diseased individuals harbour distinct microbial communities. Imbalances in these communities can contribute to the pathogenesis of lung disease. How these imbalances occur and establish is largely unknown. This review is focused on the genetically inherited condition of Cystic Fibrosis (CF). Understanding the microbial and host-related factors that govern the establishment of chronic CF lung inflammation and pathogen colonisation is essential. Specifically, dissecting the interplay in the inflammation–pathogen–host axis. Bile acids are important host derived and microbially modified signal molecules that have been detected in CF lungs. These bile acids are associated with inflammation and restructuring of the lung microbiota linked to chronicity. This community remodelling involves a switch in the lung microbiota from a high biodiversity/low pathogen state to a low biodiversity/pathogen-dominated state. Bile acids are particularly associated with the dominance of Proteobacterial pathogens. The ability of bile acids to impact directly on both the lung microbiota and the host response offers a unifying principle underpinning the pathogenesis of CF. The modulating role of bile acids in lung microbiota dysbiosis and inflammation could offer new potential targets for designing innovative therapeutic approaches for respiratory disease.

Clinical expression of Ellis‐van Creveld syndrome (EvC) is variable and mild phenotypes have been described, including patients with mostly cardiac and limb involvement. Whether these cases are part of the EvC phenotypic spectrum or separate conditions is disputed. Herein, we describe a family with vertical transmission of atrioventricular canal defect (AVCD), common atrium, and postaxial polydactyly. Targeted sequencing of EVC, EVC2, WDR35, DYNC2LI1, and DYNC2H1 identified different compound heterozygosity in EVC genotypes in the two affected members, consisting of a nonsense (p.Arg622Ter) and a missense (p.Arg663Pro) variant in the father, and the same nonsense variant and a noncanonical splice‐site in‐frame change (c.1316–7A>G) in the daughter. Complementary DNA sequencing, immunoblot, and immunofluorescence experiments using patient‐derived fibroblasts and Evc–/– mouse embryonic fibroblasts showed that p.Arg622Ter is a loss‐of‐function mutation, whereas p.Arg663Pro and the splice‐site change c.1316–7A>G are hypomorphic variants resulting in proteins that retain, in part, the ability to complex with EVC2. Our molecular and functional data demonstrate that at least in some cases the condition characterized as “common atrium/AVCD with postaxial polydactyly” is a mild form of EvC due to hypomorphic EVC mutations, further supporting the occurrence of genotype‐phenotype correlations in this syndrome., This study was supported by funding from the Italian Ministry of Health (RC‐2019) to Alessandro De Luca, Fondazione Bambino Gesù (Vite Coraggiose) to Marco Tartaglia, and the Spanish Ministry of Science, Innovation and Universities to Victor L. Ruiz‐Perez (SAF2016‐75434‐R (AEI/FEDER, UE) and PID2019‐105620RB‐I00/AEI/10.13039/501100011033).

Background: Cystic fibrosis (CF) is a hereditary disorder in which persistent unresolved inflammation and recurrent airway infections play major roles in the initiation and progression of the disease. Little is known about triggering factors modulating the transition to chronic microbial infection and inflammation particularly in young children. Cystic fibrosis respiratory disease starts early in life, with the detection of inflammatory markers and infection evident even before respiratory symptoms arise. Thus, identifying factors that dysregulate immune responsiveness at the earliest stages of the disease will provide novel targets for early therapeutic intervention. Methods: We evaluated the clinical significance of bile acid detection in the bronchoalveolar lavage fluid of clinically stable preschool-aged children diagnosed with CF. Results: We applied an unbiased classification strategy to categorize these specimens based on bile acid profiles. We provide clear associations linking the presence of bile acids in the lungs with alterations in the expression of inflammatory markers. Using multiple regression analysis, we also demonstrate that clustering based on bile acid profiles is a meaningful predictor of the progression of structural lung disease. Conclusions: Altogether, our work has identified a clinically relevant host-derived factor that may participate in shaping early events in the aetiology of CF respiratory disease.

Dietary fibre positively influences gut microbiome composition, enhancing the metabolism of dietary flavonoids to produce bioactive metabolites. These synergistic activities facilitate the beneficial effects of dietary flavonoids on cardiometabolic health parameters. The aims of this study were to investigate whether isoquercetin (a major dietary flavonoid) and inulin (soluble fibre), either alone or in combination could improve features of the metabolic syndrome. Following a 1 week acclimatization, male C57BL6 mice (6–8 weeks) were randomly assigned to; (i) normal chow diet (n = 10), (ii) high fat (HF) diet (n = 10), (iii) HF diet + 0.05% isoquercetin (n = 10), (iv) HF diet + 5% inulin, or (v) HF diet + 0.05% isoquercetin + 5% inulin (n = 10). Body weight and food intake were measured weekly. At 12 weeks, glucose and insulin tolerance tests were performed, and blood, faecal samples, liver, skeletal muscle and adipose tissue were collected. At 12 weeks, mice on the HF diet had significantly elevated body weights as well as impaired glucose tolerance and insulin sensitivity compared to the normal chow mice. Supplementation with either isoquercetin or inulin had no effect, however mice receiving the combination had attenuated weight gain, improved glucose tolerance and insulin sensitivity, reduced hepatic lipid accumulation, adipocyte hypertrophy, circulating leptin and adipose FGF21 levels, compared to mice receiving the HF diet. Additionally, mice on the combination diet had improvements in the composition and functionality of their gut microbiome as well as production of short chain fatty acids. In conclusion, long-term supplementation with the dietary flavonoid isoquercetin and the soluble fibre inulin can attenuate development of the metabolic syndrome in mice fed a high fat diet. This protective effect appears to be mediated, in part, through beneficial changes to the microbiome.

// John P. Phelan 1 , F. Jerry Reen 1 , Jose A. Caparros-Martin 3 , Rosemary O’Connor 2 and Fergal O’Gara 1, 3 1 BIOMERIT Research Centre, School of Microbiology, University College Cork - National University of Ireland, Cork, T12 YN60, Ireland 2 School of Biochemistry and Cell Biology, University College Cork, National University of Ireland, Cork, T12 YN60, Ireland 3 Human Microbiome Programme, School of Biomedical Science, Curtin Health Innovation Research Institute, Curtin University, Perth, WA 6102, Australia Correspondence to: Fergal O’Gara, email: f.ogara@ucc.ie Keywords: bile acids; microbiome; gut-axis; cancer; dysbiosis Received: June 21, 2017 Accepted: October 27, 2017 Published: December 01, 2017 ABSTRACT Dietary factors, probiotic agents, aging and antibiotics/medicines impact on gut microbiome composition leading to disturbances in localised microbial populations. The impact can be profound and underlies a plethora of human disorders, including the focus of this review; cancer. Compromised microbiome populations can alter bile acid signalling and produce distinct pathophysiological bile acid profiles. These in turn have been associated with cancer development and progression. Exposure to high levels of bile acids, combined with localised molecular/genome instability leads to the acquisition of bile mediated neoplastic alterations, generating apoptotic resistant proliferation phenotypes. However, in recent years, several studies have emerged advocating the therapeutic benefits of bile acid signalling in suppressing molecular and phenotypic hallmarks of cancer progression. These studies suggest that in some instances, bile acids may reduce cancer phenotypic effects, thereby limiting metastatic potential. In this review, we contextualise the current state of the art to propose that the bile acid/gut microbiome axis can influence cancer progression to the extent that classical in vitro cancer hallmarks of malignancy (cell invasion, cell migration, clonogenicity, and cell adhesion) are significantly reduced. We readily acknowledge the existence of a bile acid/gut microbiome axis in cancer initiation, however, in light of recent advances, we focus exclusively on the role of bile acids as potentially beneficial molecules in suppressing cancer progression. Finally, we theorise that suppressing aggressive malignant phenotypes through bile acid/gut microbiome axis modulation could uncover new and innovative disease management strategies for managing cancers in vulnerable cohorts.

Background Statins are a class of therapeutics used to regulate serum cholesterol and reduce the risk of heart disease. Although statins are highly effective in removing cholesterol from the blood, their consumption has been linked to potential adverse effects in some individuals. The most common events associated with statin intolerance are myopathy and increased risk of developing type 2 diabetes mellitus. However, the pathological mechanism through which statins cause these adverse effects is not well understood. Results Using a murine model, we describe for the first time profound changes in the microbial composition of the gut following statin treatment. This remodelling affected the diversity and metabolic profile of the gut microbiota and was associated with reduced production of butyrate. Statins altered both the size and composition of the bile acid pool in the intestine, tentatively explaining the observed gut dysbiosis. As also observed in patients, statin-treated mice trended towards increased fasting blood glucose levels and weight gain compared to controls. Statin treatment affected the hepatic expression of genes involved in lipid and glucose metabolism. Using gene knockout mice, we demonstrated that the observed effects were mediated through pregnane X receptor (PXR). Conclusion This study demonstrates that statin therapy drives a profound remodelling of the gut microbiota, hepatic gene deregulation and metabolic alterations in mice through a PXR-dependent mechanism. Since the demonstrated importance of the intestinal microbial community in host health, this work provides new perspectives to help prevent the statin-associated unintended metabolic effects. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0312-4) contains supplementary material, which is available to authorized users.

Cardiovascular disease (CVD) is the leading cause of death worldwide. The human body is populated by a diverse community of microbes, dominated by bacteria, but also including viruses and fungi. The largest and most complex of these communities is located in the gastrointestinal system and, with its associated genome, is known as the gut microbiome. Gut microbiome perturbations and related dysbiosis have been implicated in the progression and pathogenesis of CVD, including atherosclerosis, hypertension, and heart failure. Although there have been advances in the characterization and analysis of the gut microbiota and associated bacterial metabolites, the exact mechanisms through which they exert their action are not well understood. This review will focus on the role of the gut microbiome and associated functional components in the development and progression of atherosclerosis. Potential treatments to alter the gut microbiome to prevent or treat atherosclerosis and CVD are also discussed.

et al., Autosomal recessive osteogenesis imperfecta (AR-OI) is an inherited condition which in recent years has been shown with increasing genetic and clinical heterogeneity. In this article, we performed clinical assessment and sought mutations in patients from 10 unrelated families with AR-OI, one of whom was presented with the additional features of Bruck syndrome (BS). Pathogenic changes were identified in five different genes: three families had mutations in FKBP10, three in SERPINF1, two in LEPRE1, one in CRTAP, and one in PPIB. With the exception of a FKBP10 mutation in the BS case, all changes are novel. Of note, insertion of an AluYb8 repetitive element was detected in exon 6 of SERPINF1. Since the studied patients had variable manifestations and some distinctive features, genotype/phenotype correlations are suggested., Funded by: Spanish Ministry of Science and Innovation. Grant Number: SAF2010-17901and Centro de Investigación Biomédica en Red de Enfermedades Raras (Programa de Investigación de Enfermedades Pediátricas and ACCI 2012).

[Background]: Osteogenesis imperfecta (OI) is a heterogeneous bone disorder characterized by recurrent fractures. Although most cases of OI have heterozygous mutations in COL1A1 or COL1A2 and show autosomal dominant inheritance, during the last years there has been an explosion in the number of genes responsible for both recessive and dominant forms of this condition. Herein, we have analyzed a cohort of patients with OI, all offspring of unaffected parents, to determine the spectrum of variants accounting for these cases. Twenty patients had nonrelated parents and were sporadic, and 21 were born to consanguineous relationships., [Methods]: Mutation analysis was performed using a next‐generation sequencing gene panel, homozygosity mapping, and whole exome sequencing (WES)., [Results]: Patients offspring of nonconsanguineous parents were mostly identified with COL1A1 or COL1A2 heterozygous changes, although there were also a few cases with IFITM5 and WNT1 heterozygous mutations. Only one sporadic patient was a compound heterozygote for two recessive mutations. Patients offspring of consanguineous parents showed homozygous changes in a variety of genes including CRTAP,FKBP10,LEPRE1,PLOD2,PPIB,SERPINF1,TMEM38B, and WNT1. In addition, two patients born to consanguineous parents were found to have de novo COL1A1 heterozygous mutations demonstrating that causative variants in the collagen I structural genes cannot be overlooked in affected children from consanguineous couples. Further to this, WES analysis in probands lacking mutations in OI genes revealed deleterious variants in SCN9A,NTRK1, and SLC2A2, which are associated with congenital indifference to pain (CIP) and Fanconi–Bickel syndrome (FBS)., [Conclusion]: This work provides useful information for clinical and genetic diagnosis of OI patients with no positive family history of this disease. Our data also indicate that CIP and FBS are conditions to be considered in the differential diagnosis of OI and suggest a positive role of SCN9A and NTRK1 in bone development., Spanish Ministry of Economy and Competitiveness (Grant/Award Number: ‘SAF2013-43365-R’), CIBERER (Grant/Award Number: ‘ER14-PR04-ACCI13-760’).

[EN] This work presents the isolation and the biochemical characterization of the Arabidopsis thaliana gene AtSgpp. This gene shows homology with the Arabidopsis low molecular weight phosphatases AtGpp1 and AtGpp2 and the yeast counterpart GPP1 and GPP2, which have a high specificity for dl-glycerol-3-phosphate. In addition, it exhibits homology with DOG1 and DOG2 that dephosphorylate 2-deoxy-d-glucose-6-phosphate. Using a comparative genomic approach, we identified the AtSgpp gene as a conceptual translated haloacid dehalogenase-like hydrolase HAD protein. AtSgpp (locus tag At2g38740), encodes a protein with a predicted Mw of 26.7 kDa and a pI of 4.6. Its sequence motifs and expected structure revealed that AtSgpp belongs to the HAD hydrolases subfamily I, with the C1-type cap domain. In the presence of Mg2+ ions, the enzyme has a phosphatase activity over a wide range of phosphosugars substrates (pH optima at 7.0 and K (m) in the range of 3.6-7.7 mM). AtSgpp promiscuity is preferentially detectable on d-ribose-5-phosphate, 2-deoxy-d-ribose-5-phosphate, 2-deoxy-d-glucose-6-phosphate, d-mannose-6-phosphate, d-fructose-1-phosphate, d-glucose-6-phosphate, dl-glycerol-3-phosphate, and d-fructose-6-phosphate, as substrates. AtSgpp is ubiquitously expressed throughout development in most plant organs, mainly in sepal and guard cell. Interestingly, expression is affected by abiotic and biotic stresses, being the greatest under Pi starvation and cyclopentenone oxylipins induction. Based on both, substrate lax specificity and gene expression, the physiological function of AtSgpp in housekeeping detoxification, modulation of sugar-phosphate balance and Pi homeostasis, is provisionally assigned., We acknowledge Professors Montserrat Pages (CSIC Barcelona, Spain), Thomas Kupke (University of Heidelberg, Germany) and Manuel Hernandez (University Polytechnic of Valencia, Spain) for their warm support. We also thank the advice and provision of plasmid pSBETa by Dr. Florence Vignols and Yves Meyer (University of Perpignan, France); the computer software helps by Ramon Nogales-Rangel and Alexis Gonzalez-Policarpo; Eugenio Grau-Ferrando for kind advice and help for sequencing. This work was funded by the 10 month research contract MEC-FEDER to J.A.C.-M., 10 month research contract JAE-DOC to I.M.-S. and by the research project BIO2006-10138 from the MEC-FEDER of Spain to F.A.C.-M. In memoriam of Dr. Mari Cruz Cutanda-Perez.

We have isolated two Arabidopsis thaliana genes, AtGpp1 and AtGpp2, showing homology with the yeast low molecular weight phosphatases GPP1 and GPP2, which have a high specificity for DL-glycerol- 3-phosphate, and moreover homology with DOG1 and DOG2 that dephosphorylate 2-deoxyglucose-6-phos- phate. Using a comparative genomic approach, the corresponding genes were identified as conceptual translated haloacid dehalogenase-like hydrolase pro- teins. AtGpp1 (gi 18416631) and AtGpp2 (gi 18423981), encode proteins that share 95% identity, with a pre- dicted Mw of 33 and 27 kDa and a pI of 7.8 and 5.6, respectively. Both isoforms have a high specificity for DL-glycerol-3-phosphate, pH optima at 7.0, and Km in the range of 3.5-5.2 mM. AtGpp1 and AtGpp2 are ex- pressed throughout development in all plant organs, most strongly in siliqua, and expression is not affected by osmotic, ionic or oxidative stress. A putative chlo- roplast transit peptide cTP-containing sequence is ap- pended to the AtGpp1 N-terminus while AtGpp2, devoid of this tail, is predicted to be in the extraplasti- dial cytosol; this compartmenting was further confirmed by subcellular fractionation. An immunohystochemical localization study, using anti-AtGpp2 antibodies, indi- cates that the AtGpp proteins are mainly restricted to the meristem of immature flower and vascular elements of the root, shoot, leave, siliqua and developing embryo. Considerable immunoreaction was observed in the cytoplasm as well as in plastid compartments of distinct cells types from different heterotrophic Ara- bidopsis tissues, and particularly localised within phloem companion cells. Transgenic Arabidopsis plants, with gain of AtGpp2 function, show altered phosphatase activity rates and improved tolerance to salt, osmotic and oxidative stress.

et al., Most patients with Ellis-van Creveld syndrome (EvC) are identified with pathogenic changes in EVC or EVC2, however further genetic heterogeneity has been suggested. In this report we describe pathogenic splicing variants in WDR35, encoding retrograde intraflagellar transport protein 121 (IFT121), in three families with a clinical diagnosis of EvC but having a distinctive phenotype. To understand why WDR35 variants result in EvC, we analysed EVC, EVC2 and Smoothened (SMO) in IFT-A deficient cells. We found that the three proteins failed to localize to Wdr35−/− cilia, but not to the cilium of the IFT retrograde motor mutant Dync2h1−/−, indicating that IFT121 is specifically required for their entry into the ciliary compartment. Furthermore expression of Wdr35 disease cDNAs in Wdr35−/− fibroblasts revealed that the newly identified variants lead to Hedgehog signalling defects resembling those of Evc−/− and Evc2−/− mutants. Together our data indicate that splicing variants in WDR35, and possibly in other IFT-A components, underlie a number of EvC cases by disrupting targeting of both the EvC complex and SMO to cilia., This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2013-43365-R) and by funding from the Italian Ministry of Health (RF-2010-2310935, RC-2014 to A.D.L.).

Osteogenesis imperfecta or >brittle bone disease> is a collagen type I-related condition associated with osteoporosis and increased risk of bone fractures. Using a combination of homozygosity mapping and candidate gene approach we identified a homozygous single basepair deletion (c.1052delA) in SP7/Osterix (OSX) in a consanguineous patient with recessive osteogenesis imperfecta. The clinical findings of this patient include recurrent fractures, mild bone deformities, delayed tooth eruption, normal hearing, and white sclera. OSX encodes a transcription factor containing three Cys2-His2 zinc-finger DNA-binding domains at its C-terminus, which in mice has been shown to be essential for bone formation. The frameshift caused by the c.1052delA deletion removes the last 81 amino acids of the protein including the third zinc-finger motif. This finding adds a new locus to the spectrum of genes associated with osteogenesis imperfecta and reveals that SP7/OSX also plays a key role in human bone development.

[EN] The Arabidopsis thaliana gene AtSgpp (locus tag At2g38740), encodes a protein whose sequence motifs and expected structure reveal that it belongs to the HAD hydrolases subfamily I, with the C1-type cap domain (Caparrs-Martin et al. in Planta 237:943-954, 2013). In the presence of Mg2+ ions, the enzyme has a phosphatase activity over a wide range of phosphosugar substrates. AtSgpp promiscuity is preferentially detectable on d-ribose-5-phosphate, 2-deoxy-d-ribose-5-phosphate, 2-deoxy-d-glucose-6-phosphate, d-mannose-6-phosphate, d-fructose-1-phosphate, d-glucose-6-phosphate, dl-glycerol-3-phosphate, and d-fructose-6-phosphate. Site-directed mutagenesis analysis of the putative signature sequence motif-5 (IAGKH), which defines its specific chemistry, brings to light the active-site residues Ala-69 and His-72. Mutation A69M, changes the pH dependence of AtSgpp catalysis, and mutant protein AtSgpp-H72K was inactive in phosphomonoester dephosphorylation. It was also observed that substitutions I68M and K71R slightly affect the substrate specificity, while the replacement of the entire motif for that of homologous dl-glycerol-3-phosphatase AtGpp (MMGRK) does not switch AtSgpp activity to the specific targeting for dl-glycerol-3-phosphate., We acknowledge Professors Montserrat Pages (CSIC Barcelona, Spain), Thomas Kupke (University of Heidelberg, Germany) and Manuel Hernandez (University Polytechnic of Valencia, Spain) for their warm support. We also thank the advice and provision of plasmid pSBETa by Dr Florence Vignols and Yves Meyer (University of Perpignan, France); the computer software helps by Ramon Nogales-Rangel and Alexis Gonzalez-Policarpo; the Eugenio Grau-Ferrando kind advice and help for sequencing; the Dr Angela Batcheller support in the English edition of the manuscript. This work was funded by the 10-month research contract MEC-FEDER to J. A. C.-M., 10-month research contract JAE-DOC to I. M.-S., and by the research project BIO2006-10138 from the MEC-FEDER of Spain to F. A. C.-M.

Mutations in BMP1 cause osteogenesis imperfect (OI) type XIII (MIM 614856), a probably not so uncommon form of autosomal recessive OI. (Editor's Note: See Chapters 1 and 2 for discussion of OI types related to specific mutations.) We have studied an Egyptian consanguineous family with two children initially diagnosed as OI type III. They had blue sclera, severe kyphoscoliosis and large umbilical hernia. Genetic analysis of this family performed by homozygosity mapping showed lack of linkage to any of the previously known AR-OI loci but identified a homozygous 10.27. Mb DNA segment on chromosome 8p in both affected children comprising the type I collagen C-propeptide protease gene BMP1. Direct sequencing of BMP1 in the proband revealed a Phe249Leu homozygous missense change within the BMP1/mTLD astacin protease domain. Phe249 is a residue which is invariable in all invertebrate and vertebrate members of the astacin family of metalloproteases. A few months later, using a combination of whole-exome sequencing and filtering for homozygous stretches of identified variants, another mutation was reported by other authors in two affected sibs of a consanguineous family from Turkey. They showed increased bone mineral density and multiple recurrent fractures. The mutation in BMP1, a 34. G>C transversion in exon 1 resulting in a Gly12Arg (G12R) substitution within the signal peptide, impaired protein's localization to the endoplasmic reticulum, the correct post-translational glycosylation and its secretion. BMP1 expands the number of growing genes involved in AR-OI and demonstrates the high genetic complexity of the collagen I pathway disorders.

et al., Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2ο43) caused mislocalization of Evc/Evc2ο43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2-/- chondrocytes. Moreover, Evc silencing in Sufu-/- cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation. © The Author 2012. Published by Oxford University Press. All rights reserved., This work was funded by the Spanish Ministry of Science and Innovation (SAF-17901), the European Union (LSHM-CT-2007-03741) and the Ramón Areces Foundation.

PLOD2 and FKBP10 are genes mutated in Bruck syndrome (BS), a condition resembling osteogenesis imperfecta (OI), but that is also typically associated with congenital joint contractures. Herein, we sought mutations in six consanguineous BS families and detected changes in either PLOD2 or FKBP10 in all cases. Two probands were found with a homozygous frameshift mutation in the alternative exon 13a of PLOD2, indicating that specific inactivation of the longer protein isoform encoded by this gene is sufficient to cause BS. In addition, by homozygosity mapping, followed by a candidate gene approach, we identified a homozygous donor splice site mutation in PLOD2 in a patient with autosomal-recessive OI (AR-OI). Screening of additional samples also revealed compound heterozygous mutations in PLOD2 in two brothers, one affected with mild AR-OI and the other with mild BS. Thus, PLOD2 in addition to causing BS is also associated with AR-OI phenotypes of variable severity. © 2012 Wiley Periodicals, Inc., Contract grant sponsor: The Spanish Ministry of Science and Innovation SAF2010-17901; the Centro de Investigación Biomédica en Red de Enfermedades Raras Programa de Investigación de Enfermedades Pedíatricas.

Ellis-van Creveld syndrome protein homolog (Evc) was previously shown to mediate expression of Indian hedgehog (Ihh) downstream targets in chondrocytes. Consequently disruption of the Ihh/Pthrp axis was demonstrated in Evc(-/-) mice, but the full extent of Evc involvement in endochondral development was not totally characterized. Herein we have examined further the Evc(-/-) growth plate in a homogeneous genetic background and show that Evc promotes chondrocyte proliferation, chondrocyte hypertrophy and the differentiation of osteoblasts in the perichondrium, hence implicating Evc in both Pthrp-dependent and Pthrp-independent Ihh functions. We also demonstrate that Evc, which localizes to osteoblast primary cilia, mediates Hedgehog (Hh) signaling in the osteoblast lineage. In spite of this, bone collar development is mildly affected in Evc(-/-) mutants. The onset of perichondrial osteoblastogenesis is delayed at the initial stages of endochondral ossification in Evc(-/-) mice, and in later stages, the leading edge of expression of osteoblast markers and Wnt/ß-catenin signaling components is located closer to the primary spongiosa in the Evc(-/-) perichondrium owing to impaired osteoblast differentiation. Additionally we have used Ptch1-LacZ reporter mice to learn about the different types of Hh-responsive cells that are present in the perichondrium of normal and Evc(-/-) mice. Evc mediates Hh target gene expression in inner perichondrial cells, but it is dispensable in the external layers of the perichondrium. Finally, we report cranial base defects in Evc(-/-) mice and reveal that Evc is essential for intrasphenoidal synchondrosis development., This work was funded by the Spanish Ministry of Science and Innovation (SAF-62291 and SAF-17901), the European Union (LSHM-CT-2007-03741) and the Ramón Areces Foundation.

[EN] Dual-specificity phosphatases (DSPs) constitute a large protein tyrosine phosphatase (PTP) family, with examples in distant evolutive phyla. PFA-DSPs (Plant and Fungi Atypical DSPs) are a group of atypical DSPs present in plants, fungi, kinetoplastids, and slime molds, the members of which share structural similarity with atypical- and lipid phosphatase DSPs from mammals. The analysis of the PFA-DSPs from the plant Arabidopsis thaliana (AtPFA-DSPs) showed differential tissue mRNA expression, substrate specificity, and catalytic activity for these proteins, suggesting different functional roles among plant PFA-DSPs. Bioinformatic analysis revealed the existence of novel PFA-DSP-related proteins in fungi (Oca1, Oca2, Oca4 and Oca6 in Saccharomyces cerevisiae) and protozoa, which were segregated from plant PFADSPs. The closest yeast homolog for these proteins was the PFA-DSP from S. cerevisiae ScPFA-DSP1/Siw14/Oca3. Oca1, Oca2, Siw14/Oca3, Oca4, and Oca6 were involved in the yeast response to caffeine and rapamycin stresses. Siw14/Oca3 was an active phosphatase in vitro, whereas no phosphatase activity could be detected for Oca1. Remarkably, overexpression of Siw14/Oca3 suppressed the caffeine sensitivity of oca1, oca2, oca4, and oca6 deleted strains, indicating a genetic linkage and suggesting a functional relationship for these proteins. Functional studies on mutations targeting putative catalytic residues from the A. thaliana AtPFA-DSP1/At1g05000 protein indicated the absence of canonical amino acids acting as the general acid/base in the phosphor-ester hydrolysis, which suggests a specific mechanism of reaction for PFA-DSPs and related enzymes. Our studies demonstrate the existence of novel phosphatase protein families in fungi and protozoa, with active and inactive enzymes linked in common signaling pathways. This illustrates the catalytic and functional complexity of the expanding family of atypical dual-specificity phosphatases in non-metazoans, including parasite organisms responsible for infectious human diseases., This work was supported in part by grants SAF2006-08319 from Ministerio de Educacion y Ciencia, SAF2009-10226 from Ministerio de Ciencia e Innovacion (Spain and Fondo Europeo de Desarrollo Regional, FEDER; Plan de estimulo a la economia y el empleo, Plan E), AP-117/08, ACOMP2009/363 and ACOMP2010/222 from Generalitat Valenciana (Spain) (to R. P.), G0701233 from Medical Research Council (U.K.) (to L. T.), BIO2007-67299 from Ministerio de Ciencia e Innovacion (Spain) (to M. M.), and by European Union Research Training Network MRTN-CT-2006-035830. C. Roma-Mateo and A. Sacristan-Reviriego have been recipients of predoctoral fellowships from Ministerio de Educacion y Ciencia (Spain). We thank Peter Sudbery and Jose Luis Revuelta for providing cDNAs, the Arabidopsis Biological Resource Center (USA; donors: SSP Consortium, Sakis Theologis, Joe Ecker) for providing plasmids, and Isabel Rogla and Charis Saville for expert technical assistance.

CoA is required for many synthetic and degradative reactions in intermediary metabolism and is the principal acyl carrier in prokaryotic and eukaryotic cells. CoA is synthesized in five steps from pantothenate, and recently, the CoA biosynthetic genes of Arabidopsis have all been identified and characterized. Here, we demonstrate the biochemical and physiological characterization of a pyrophosphatase from Arabidopsis thaliana, called AtCoAse (locus tag At5g45940), cleaving CoA to 4'-phosphopantetheine and 3',5'-adenosine-diphosphate in the presence of Mg 2+ /Mn 2+ ions. The CoA cleaving enzyme is a member of the Nudix hydrolases, pyrophosphatases that hydrolyze nucleoside diphosphates, already described as CoAse and now further characterized in detail by us. Mutagenesis of residues of the so-called Nudix and NuCoA motifs drastically reduced the hydrolase activity. AtCoAse is not absolute specific for CoA, and in the presence of Mn 2+ ions, a minor hydrolyzing activity was observed with NADH as substrate. The AtCoAse expression is ubiquitous, strongly in flower and unaffected by abiotic stress. The immunohistochemical localization indicates that the AtCoAse protein is observed in the cytoplasm of distinct cells types from different heterotrophic Arabidopsis tissues, mainly restricted to the vascular elements of the root and shoot and in flower and developing embryo. Transgenic Arabidopsis plants, with increased AtCoAse expression, show altered growth rates and development, expanding their live cycle far away from the wild-type.

We have isolated two Arabidopsis thaliana genes, AtGppl and AtGpp2, showing homology with the yeast low molecular weight phosphatases GPP1 and GPP2, which have a high specificity for DL-glycerol-3-phosphate, and moreover homology with DOG1 and DOG2 that dephosphorylate 2-deoxyglucose-6-phosphate. Using a comparative genomic approach, the corresponding genes were identified as conceptual translated haloacid dehalogenase-like hydrolase proteins. AtGppl (gi 18416631) and AtGpp2 (gi 18423981), encode proteins that share 95% identity, with a predicted Mw of 33 and 27 kDa and a pI of 7.8 and 5.6, respectively. Both isoforms have a high specificity for DL-glycerol-3-phosphate, pH optima at 7.0, and Km in the range of 3.5-5.2 mM. AtGppl and AtGpp2 are expressed throughout development in all plant organs, most strongly in siliqua, and expression is not affected by osmotic, ionic or oxidative stress. A putative chloroplast transit peptide cTP-containing sequence is appended to the AtGppl N-terminus while AtGpp2, devoid of this tail, is predicted to be in the extraplastidial cytosol; this compartmenting was further confirmed by subcellular fractionation. An immunohystochemical localization study, using anti-AtGpp2 antibodies, indicates that the AtGpp proteins are mainly restricted to the meristem of immature flower and vascular elements of the root, shoot, leave, siliqua and developing embryo. Considerable immunoreaction was observed in the cytoplasm as well as in plastid compartments of distinct cells types from different heterotrophic Arabidopsis tissues, and particularly localised within phloem companion cells. Transgenic Arabidopsis plants, with gain of AtGpp2 function, show altered phosphatase activity rates and improved tolerance to salt, osmotic and oxidative stress.

et al., We report that hypofunctional alleles of WNT1 cause autosomal-recessive osteogenesis imperfecta, a congenital disorder characterized by reduced bone mass and recurrent fractures. In consanguineous families, we identified five homozygous mutations in WNT1: one frameshift mutation, two missense mutations, one splice-site mutation, and one nonsense mutation. In addition, in a family affected by dominantly inherited early-onset osteoporosis, a heterozygous WNT1 missense mutation was identified in affected individuals. Initial functional analysis revealed that altered WNT1 proteins fail to activate canonical LRP5-mediated WNT-regulated β-catenin signaling. Furthermore, osteoblasts cultured in vitro showed enhanced Wnt1 expression with advancing differentiation, indicating a role of WNT1 in osteoblast function and bone development. Our finding that homozygous and heterozygous variants in WNT1 predispose to low-bone-mass phenotypes might advance the development of more effective therapeutic strategies for congenital forms of bone fragility, as well as for common forms of age-related osteoporosis., This work was supported by German Federal Ministry of Education and Research grants 01GM1211A (E-RARE network CRANIRARE-2), 01GM1109C (national rare disease network Forschungsverbund Ausgewählter Craniofacialer Entwicklungsstörungen) to B.W., and 01EC1006A (Molecular Pathogenesis of Osteoporosis) to U.K. and S.M., by TÜBITAK grant 112S398 (E-RARE network CRANIRARE-2) to H.K., and by National Institute of Child Health and Human Development (National Institutes of Health) Intramural Research Program funding to S.L. and J.C.M. M.S. holds a Doc female forte scholarship from Austria and is a member of the Berlin-Brandenburg School for Regenerative Therapies.