Mutations in FKBP10 cause recessive osteogenesis imperfecta and Bruck syndrome.

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by bone fragility and alteration in synthesis and posttranslational modification of type I collagen. Autosomal dominant OI is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Bruck syndrome is a recessive disorder featuring congenital contractures in addition to bone fragility; Bruck syndrome type 2 is caused by mutations in PLOD2 encoding collagen lysyl hydroxylase, whereas Bruck syndrome type 1 has been mapped to chromosome 17, with evidence suggesting region 17p12, but the gene has remained elusive so far. Recently, the molecular spectrum of OI has been expanded with the description of the basis of a unique posttranslational modification of type I procollagen, that is, 3-prolyl-hydroxylation. Three proteins, cartilage-associated protein (CRTAP), prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene), and the prolyl cis-trans isomerase cyclophilin-B (PPIB), form a complex that is required for fibrillar collagen 3-prolyl-hydroxylation, and mutations in each gene have been shown to cause recessive forms of OI. Since then, an additional putative collagen chaperone complex, composed of FKBP10 (also known as FKBP65) and SERPINH1 (also known as HSP47), also has been shown to be mutated in recessive OI. Here we describe five families with OI-like bone fragility in association with congenital contractures who all had FKBP10 mutations. Therefore, we conclude that FKBP10 mutations are a cause of recessive osteogenesis imperfecta and Bruck syndrome, possibly Bruck syndrome Type 1 since the location on chromosome 17 has not been definitely localized.

Cost-effectiveness of pharmacotherapies for nicotine dependence in primary care settings: a multinational comparison.

OBJECTIVE: To estimate the incremental cost-effectiveness of the first-line pharmacotherapies (nicotine gum, patch, spray, inhaler, and bupropion) for smoking cessation across six Western countries-Canada, France, Spain, Switzerland, the United States, and the United Kingdom. DESIGN AND STUDY POPULATION: A Markov-chain cohort model to simulate two cohorts of smokers: (1) a reference cohort given brief cessation counselling by a general practitioner (GP); (2) a treatment cohort given counselling plus pharmacotherapy. Effectiveness expressed as odds ratios for quitting associated with pharmacotherapies. Costs based on the additional physician time required and retail prices of the medications. INTERVENTIONS: Addition of each first-line pharmacotherapy to GP cessation counselling. MAIN OUTCOME MEASURES: Cost per life-year saved associated with pharmacotherapies. RESULTS: The cost per life-year saved for counselling only ranged from US190 dollars in Spain to 773 dollars in the UK for men, and from 288 dollars in Spain to 1168 dollars in the UK for women. The incremental cost per life-year saved for gum ranged from 2230 dollars for men in Spain to 7643 dollars for women in the US; for patch from 1758 dollars for men in Spain to 5131 dollars for women in the UK; for spray from 1935 dollars for men in Spain to 7969 dollars for women in the US; for inhaler from 3480 dollars for men in Switzerland to 8700 dollars for women in France; and for bupropion from 792 dollars for men in Canada to 2922 dollars for women in the US. In sensitivity analysis, changes in discount rate, treatment effectiveness, and natural quit rate had the strongest influences on cost-effectiveness. CONCLUSIONS: The cost-effectiveness of the pharmacotherapies varied significantly across the six study countries, however, in each case, the results would be considered favourable as compared to other common preventive pharmacotherapies.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

HLA Alleles Influence the Clinical Signature of Amoxicillin-Clavulanate Hepatotoxicity.

BACKGROUND AND AIM The genotype-phenotype interaction in drug-induced liver injury (DILI) is a subject of growing interest. Previous studies have linked amoxicillin-clavulanate (AC) hepatotoxicity susceptibility to specific HLA alleles. In this study we aimed to examine potential associations between HLA class I and II alleles and AC DILI with regards to phenotypic characteristics, severity and time to onset in Spanish AC hepatotoxicity cases. METHODS High resolution genotyping of HLA loci A, B, C, DRB1 and DQB1 was performed in 75 AC DILI cases and 885 controls. RESULTS The distributions of class I alleles A*3002 (P/Pc = 2.6E-6/5E-5, OR 6.7) and B*1801 (P/Pc = 0.008/0.22, OR 2.9) were more frequently found in hepatocellular injury cases compared to controls. In addition, the presence of the class II allele combination DRB1*1501-DQB1*0602 (P/Pc = 5.1E-4/0.014, OR 3.0) was significantly increased in cholestatic/mixed cases. The A*3002 and/or B*1801 carriers were found to be younger (54 vs 65 years, P = 0.019) and were more frequently hospitalized than the DRB1*1501-DQB1*0602 carriers. No additional alleles outside those associated with liver injury patterns were found to affect potential severity as measured by Hy's Law criteria. The phenotype frequencies of B*1801 (P/Pc = 0.015/0.42, OR 5.2) and DRB1*0301-DQB1*0201 (P/Pc = 0.0026/0.07, OR 15) were increased in AC DILI cases with delayed onset compared to those corresponding to patients without delayed onset, while the opposite applied to DRB1*1302-DQB1*0604 (P/Pc = 0.005/0.13, OR 0.07). CONCLUSIONS HLA class I and II alleles influence the AC DILI signature with regards to phenotypic expression, latency presentation and severity in Spanish patients.

Is the effect of inguinal field block with 0.5 % bupivacaine on postoperative pain after hernia repair enhanced by addition of ketorolac or S(+) ketamine ?

Résumé Malgré l'apparition de nouvelles techniques chirurgicales dites « sans tension », l'antalgie post-opératoire après cure de hernie inguinale reste un défi pour les anesthésiologistes. Récemment on a suggéré que l'addition de ketamine ou d'un anti-inflammatoire non-stéroïdien (AINS) à un anesthésique local pourrait améliorer et prolonger l'analgésie postopératoire. Le but de cette étude, à laquelle ont participé 36 patients ASA I-II, était d'évaluer si la coadministration de S(+) ketamine ou de ketorolac renforcerait les effets analgésiques de la bupivacaïne après cure ambulatoire de hernie inguinale sous anesthésie générale. L'analgésie a consisté en une infiltration de la plaie associé à un bloc inguinal avec soit 30 ml de bupivacaïne 0,5 % (n=12), soit 27 ml de bupivacaïne 0,5 % + 3 ml de S(+) ketamine (75 mg) (n=12), soit 28 ml de bupivacaïne 0,5 % + 2 ml de ketorolac (60 mg) (n=12). La prise orale d'antalgique en phase postopératoire était standardisée. L'intensité des douleurs a été évaluée au moyen d'une échelle visuelle analogique (EVA), d'un score verbal d'estimation et par algométrie de pression respectivement 2, 4, 6, 24 et 48 heures après l'intervention. Les trois groupes de patients ont présenté le score de douleur évalué par EVA le plus élevé à 24 heures, score significativement différent de ceux mesurés à 6 et 48 heures (P <0.05). A part une sensation de douleurs significativement moindre (score verbal d'estimation) dans le groupe ketorolac à 24 et 48 heures et seulement à 48 heures dans le groupe ketamine, il n'y avait aucune autre différence entre les groupes pour la durée de l'étude (48 heures) en ce qui concerne les scores de douleur, les seuils de douleur à la pression ou la prise postopératoire d'antalgiques « de secours ». En conclusion, l'addition de S(+) ketamine ou de ketorolac, n'améliore que marginalement l'effet analgésique de la bupivacaïne. Ceci peut-être mis en relation avec la technique de cure de hernie « sans tension » induisant un bas niveau de douleur postopératoire. Abstract Objective: The aim of the study was to assess whether coadministration of S(±) ketamine or ketorolac would enhance or prolong local analgesic effect of bupivacaine after inguinal hernia repair. Design: Prospective double-blind randomized study evaluating pain intensity after surgery under general anesthesia. Setting: Outpatient facilities of the University Hospital of Lausanne. Patient: Thirty-six ASA I-II outpatients scheduled for elective day-case inguinal herniorraphy. Intervention: Analgesia strategy consisted of a wound infiltration and an inguinal field block either with 30 mL bupivacairie (0.5%) or with the same volume of a mixture of 27 mL bupivacaine (0.5%) + 3 mL S(+) ketamine (75 mg) or a 28 mL bupivacaine (0.5%) + 2 ML ketorolac (60 mg). Postoperative analgesic regimen was standardized. Outcome Measures: Pain intensity was assessed with a Visual Analog Seale, a verbal rating score, and by pressure algometry 2, 4, 6, 24, and 48 hours after surgery. Results: The 3 groups of patients experienced the highest Visual Analog Scale pain score at 24 hours, which was different from those at 6 and 48 hours (P < 0.05). Apart from a significantly lower pain sensation (verbal rating score) in the ketorolac group at 24 and 48 hours and only at 48 hours with ketamine, there were no other differences in pain scores, pain pressure thresholds, or rescue analgesic consumption between groups throughout the 48-hour study period. Conclusion: The addition of S (+)-ketamine or ketorolac only minimally improves the analgesic effect of bupivacaine. This may be related to the tension-free hernia repair technique associated with low postoperative pain.

Effects of Crypthecodiniumcohnii, Chlorela spp. e Isochrysisgalbana addition to milk replacer on goat kids and lamb growth.

The effects of Crypthecodinium cohnii (Cryp.), Chlorela spp. (Chlo.) and Isochrysis galbana (Iso.) addition to milk replacer on goat kids and lambs growth were evaluated. About 80 Majorera goat kids (males and females) and 80 Canarian sheep lambs were randomly assigned into four different groups (by specie) according to diet. Control groups were fed with a commercial milk replacer at 16% (w/w); Cryp. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of C. cohnii; Chlo. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of Chlorela spp.; Iso. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of I. galbana. After colostrum period, animals were individually bottle-fed twice daily (8 am and 8 pm) ad libitum with the corresponding diet until day 60 of life. Animals were weighted every week at 8 am and liquid diet intake was recorded weekly. No effects of microseaweed addition were observed, neither growth nor milk replacer intake.

International analysis of the countries where Brazilian franchise chains operate

This paper aims to demonstrate which external environment factors are involved in the international commitment of Brazilian franchise chains. Our objectives herein are to understand which external country characteristics lead to international franchising operations and to ascertain the influence of such characteristics in the commitment of franchise chains in each country they operate in. The database has 54 Brazilian franchise chains with international operations in 26 countries, which implies in 157 franchises operating abroad. Regarding external environment factors, the independent variables form a group divided into market opportunity and business efficiency – trust and ease to doing business. The result of the three distinct clusters show how the market opportunity and the business efficiency (trust and ease in doing business) work as drivers to the international operation of Brazilian franchises. The paper shows that the franchise chains operating in the USA (cluster 3) have an inferior international commitment in comparison with the franchises which operate in developed countries and in small Latin American countries (cluster 2). It is also possible to notice a large number of franchises that operate in underdeveloped countries from Latin America and Africa (cluster 1) with worse business efficiency due to the advantage of learning how to operate in a country that could have some similarities with Brazil

IVA in addition to BMD can change the osteoporosis management in 25% of clinical routine patients

Introduction Vertebral fracture is one of the major osteoporoticfractures which are unfortunately very often undetected. In addition,it is well known that prevalent vertebral fracture increases dramaticallythe risk of future additional fracture. Instant Vertebral Assessment(IVA) has been introduced in DXA device a couple of years ago toease the detection of such fracture when routine DXA are performed.To correctly use such tool, ISCD provided clinical recommendationon when and how to use it. The aim of our study was to evaluate theISCD guidelines in clinical routine patients and see how often itmay change of patient management.Methods During two months (March and April 2010), a medicalquestionnaire was systematically given to our clinical routine patientto check the validity of ISCD IVA recommendations in our population.In addition, all women had BMD measurement at AP spine,femur and 1/3 radius using a Discovery A System (Hologic, Waltham,USA). When appropriate, IVA measurement had been performedon the same DXA system and had been centrally evaluated by twotrained doctors for fracture status according to the semi-quantitativemethod of Genant. The reading had been performed when possiblebetween L5 and T4.Results Out of 210 women seen in the consultation, 109 (52 %)of them (mean age 68.2 ± 11.5 years) fulfilled the necessary criteriato have an IVA measurement. Out of these 109 women, 43 (incidence39.4 %) had osteoporosis at one of the three skeletal sitesand 31 (incidence 28.4 %) had at least one vertebral fracture. 14.7 %of women had both osteoporosis and at least one vertebral fractureclassifying them as "severe osteoporosis" while 46.8 % did not haveosteoporosis and no vertebral fracture. 24.8 % of the women hadosteoporosis but no vertebral fracture while 13.8 % of women didhave osteoporosis but vertebral fracture (clinical osteoporosis).Conclusions In 52 % of our patients, IVA was needed accordingto ISCD criteria. In half of them the IVA test influenced of patientmanagement either may changing the type of treatment of simplyby classifying patient as "clinical osteoporosis". IVA appears to bean important tool in clinical routine but unfortunately is not yetvery often use in most of the centers.

Ab initio benchmark study for the oxidative addition of CH4 to Pd: importance of basis-set flexibility and polarization

To obtain a state-of-the-art benchmark potential energy surface (PES) for the archetypal oxidative addition of the methane C-H bond to the palladium atom, we have explored this PES using a hierarchical series of ab initio methods (Hartree-Fock, second-order Møller-Plesset perturbation theory, fourth-order Møller-Plesset perturbation theory with single, double and quadruple excitations, coupled cluster theory with single and double excitations (CCSD), and with triple excitations treated perturbatively [CCSD(T)]) and hybrid density functional theory using the B3LYP functional, in combination with a hierarchical series of ten Gaussian-type basis sets, up to g polarization. Relativistic effects are taken into account either through a relativistic effective core potential for palladium or through a full four-component all-electron approach. Counterpoise corrected relative energies of stationary points are converged to within 0.1-0.2 kcal/mol as a function of the basis-set size. Our best estimate of kinetic and thermodynamic parameters is -8.1 (-8.3) kcal/mol for the formation of the reactant complex, 5.8 (3.1) kcal/mol for the activation energy relative to the separate reactants, and 0.8 (-1.2) kcal/mol for the reaction energy (zero-point vibrational energy-corrected values in parentheses). This agrees well with available experimental data. Our work highlights the importance of sufficient higher angular momentum polarization functions, f and g, for correctly describing metal-d-electron correlation and, thus, for obtaining reliable relative energies. We show that standard basis sets, such as LANL2DZ+ 1f for palladium, are not sufficiently polarized for this purpose and lead to erroneous CCSD(T) results. B3LYP is associated with smaller basis set superposition errors and shows faster convergence with basis-set size but yields relative energies (in particular, a reaction barrier) that are ca. 3.5 kcal/mol higher than the corresponding CCSD(T) values

Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves.

Physical damage and disease are known to lead to changes in the oxylipin signature of plants. We searched for oxylipins produced in response to both wounding and pathogenesis in Arabidopsis leaves. Linoleic acid 9- and 13-ketodienes (KODEs) were found to accumulate in wounded leaves as well as in leaves infected with the pathogen Pseudomonas syringae pv. tomato (Pst). Quantification of the compounds showed that they accumulated to higher levels during the hypersensitive response to Pst avrRpm1 than during infection with a Pst strain lacking an avirulence gene. KODEs are Michael addition acceptors, containing a chemically reactive alpha,beta-unsaturated carbonyl group. When infiltrated into leaves, KODEs were found to induce expression of the GST1 gene, but vital staining indicated that these compounds also damaged plant cells. Several molecules typical of lipid oxidation, including malonaldehyde, also contain the alpha,beta-unsaturated carbonyl reactivity feature, and, when delivered in a volatile form, powerfully induced the expression of GST1. The results draw attention to the potential physiological importance of naturally occurring Michael addition acceptors in plants. In particular, these compounds could act directly, or indirectly via cell damage, as powerful gene activators and might also contribute to host cell death.

KnotProt: a database of proteins with knots and slipknots.

The protein topology database KnotProt, http://knotprot.cent.uw.edu.pl/, collects information about protein structures with open polypeptide chains forming knots or slipknots. The knotting complexity of the cataloged proteins is presented in the form of a matrix diagram that shows users the knot type of the entire polypeptide chain and of each of its subchains. The pattern visible in the matrix gives the knotting fingerprint of a given protein and permits users to determine, for example, the minimal length of the knotted regions (knot's core size) or the depth of a knot, i.e. how many amino acids can be removed from either end of the cataloged protein structure before converting it from a knot to a different type of knot. In addition, the database presents extensive information about the biological functions, families and fold types of proteins with non-trivial knotting. As an additional feature, the KnotProt database enables users to submit protein or polymer chains and generate their knotting fingerprints.

Fusion of the human gene for the polyubiquitination co-effector UEV-1 with Kua, a newly identified gene

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon–intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua–UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua–UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination.[Kua cDNAs isolated by RT-PCR and described in this paper have been deposited in the GenBank data library under accession nos. AF1155120 (H. sapiens) and AF152361 (D. melanogaster). Genomic clones containing UEV genes: S. cerevisiae, YGL087c (accession no. Z72609); S. pombe, c338 (accession no. AL023781); P. falciparum, MAL3P2 (accession no. AL034558); A. thaliana, F26F24 (accession no. AC005292); C. elegans, F39B2 (accession no. Z92834); D. melanogaster, AC014908; and H. sapiens, 1185N5 (accession no. AL034423). Accession numbers for Kua cDNAs in GenBank dbEST: M. musculus, AA7853; T. cruzi, AI612534. Other Kua-containing sequences: A. thaliana genomic clones F10M23 (accession no. AL035440), F19K23 (accession no. AC000375), and T20K9 (accession no. AC004786).