Epigenetic modification of the FMR1 gene in fragile X syndrome is associated with differential response to the mGluR5 antagonist AFQ056.

Fragile X syndrome (FXS) is an X-linked condition associated with intellectual disability and behavioral problems. It is caused by expansion of a CGG repeat in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. This mutation is associated with hypermethylation at the FMR1 promoter and resultant transcriptional silencing. FMR1 silencing has many consequences, including up-regulation of metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. mGluR5 receptor antagonists have shown promise in preclinical FXS models and in one small open-label study of FXS. We examined whether a receptor subtype-selective inhibitor of mGluR5, AFQ056, improves the behavioral symptoms of FXS in a randomized, double-blind, two-treatment, two-period, crossover study of 30 male FXS patients aged 18 to 35 years. We detected no significant effects of treatment on the primary outcome measure, the Aberrant Behavior Checklist-Community Edition (ABC-C) score, at day 19 or 20 of treatment. In an exploratory analysis, however, seven patients with full FMR1 promoter methylation and no detectable FMR1 messenger RNA improved, as measured with the ABC-C, significantly more after AFQ056 treatment than with placebo (P < 0.001). We detected no response in 18 patients with partial promoter methylation. Twenty-four patients experienced an adverse event, which was mostly mild to moderately severe fatigue or headache. If confirmed in larger and longer-term studies, these results suggest that blockade of the mGluR5 receptor in patients with full methylation at the FMR1 promoter may show improvement in the behavioral attributes of FXS.

Development and Demonstration of a Freezing Drizzle Algorithm for Roadway Environmental Sensing Systems, Final Report, October 2012

The primary goal of this project is to demonstrate the accuracy and utility of a freezing drizzle algorithm that can be implemented on roadway environmental sensing systems (ESSs). The types of problems related to the occurrence of freezing precipitation range from simple traffic delays to major accidents that involve fatalities. Freezing drizzle can also lead to economic impacts in communities with lost work hours, vehicular damage, and downed power lines. There are means for transportation agencies to perform preventive and reactive treatments to roadways, but freezing drizzle can be difficult to forecast accurately or even detect as weather radar and surface observation networks poorly observe these conditions. The detection of freezing precipitation is problematic and requires special instrumentation and analysis. The Federal Aviation Administration (FAA) development of aircraft anti-icing and deicing technologies has led to the development of a freezing drizzle algorithm that utilizes air temperature data and a specialized sensor capable of detecting ice accretion. However, at present, roadway ESSs are not capable of reporting freezing drizzle. This study investigates the use of the methods developed for the FAA and the National Weather Service (NWS) within a roadway environment to detect the occurrence of freezing drizzle using a combination of icing detection equipment and available ESS sensors. The work performed in this study incorporated the algorithm developed initially and further modified for work with the FAA for aircraft icing. The freezing drizzle algorithm developed for the FAA was applied using data from standard roadway ESSs. The work performed in this study lays the foundation for addressing the central question of interest to winter maintenance professionals as to whether it is possible to use roadside freezing precipitation detection (e.g., icing detection) sensors to determine the occurrence of pavement icing during freezing precipitation events and the rates at which this occurs.

Thyroid hormones stimulate expression and modification of cytoskeletal protein during rat sciatic nerve regeneration.

Peripheral neurons can regenerate after axotomy; in this process, the role of cytoskeletal proteins is important because they contribute to formation and reorganization, growth, transport, stability and plasticity of axons. In the present study, we examined the effects of thyroid hormones (T3) on the expression of major cytoskeletal proteins during sciatic nerve regeneration. At various times after sciatic nerve transection and T3 local administration, segments of operated nerves from T3-treated rats and control rats were examined by Western blotting for the presence of neurofilament, tubulin and vimentin. Our results revealed that, during the first week after surgery, T3 treatment did not significantly alter the level of NF subunits and tubulin in the different segments of operated nerves compared to control nerves. Two or 4 weeks after operation, the concentration of NF-H and NF-M isoforms was clearly increased by T3 treatment. Moreover, under T3-treatment, NF proteins appeared more rapidly in the distal segment of operated nerves. Likewise, the levels of betaIII, and of acetylated and tyrosinated tubulin isotypes, were also up-regulated by T3-treatment during regeneration. However, only the tyrosinated tubulin form appeared earlier in the distal nerve segments. At this stage of regeneration, T3 had no effect on the level of vimentin expression. In conclusion, thyroid hormone improves and accelerates peripheral nerve regeneration and exerts a positive effect on cytoskeletal protein expression and transport involved in axonal regeneration. These results help us to understand partially the mechanism by which thyroid hormones enhance peripheral nerve regeneration. The stimulating effect of T3 on peripheral nerve regeneration may have considerable therapeutic potential.

Modification of Ca-Montmorillonite by Low-Temperature Heat Treatment, HR-106, 1964

Objectives of this investigation were to measure the effects of moderate heat treatments (below the dehydroxylation temperature) on physical and chemical properties of a calcium-montmorillonite clay. Previous workers have noted the reduction in cation exchange capacity and swelling property after heating in the range 200 to 400°C, and have suggested several possible explanations, such as hysteresis effect, increased inter-layer attractions due to removal of inter-layer water, or changes in the disposition of inter-layer or layer surface ions. The liquid limits of Ca-montmorillonite were steadily decreased with increased temperature of treatment, levelling at about 450°C. The plastic limit decreased slightly up to 350°C, above which samples could no longer be rolled into threads. The gradual change is in contrast with sudden major changes noted for weight loss (maximum rates of change at l00°C and 500°C), glycol retention surface area (520°C), and d001 diffraction peak intensity (17.7 A spacing) and breadth after glycolation (530°C). Other properties showing more gradual reductions with heat treatment were amount of exchangeable calcium (without water soaking), cation exchange capacity by NH4AC method, and d001 intensity (21 A spacing) after storing at 100% r.h. one month and re-wetting with water. Previous water soaking allowed much greater release of fixed Ca++ up to 450°C. Similar results were obtained with cation exchange capacities when samples were treated with N CaCl2 solution. The 21.0 A peak intensity curve showed close similarity to the liquid limit and plastic index curves in the low temperature range, and an explanation is suggested.

Altered expression and posttranslational modification of neurofilaments in methylmalonic aciduria

Neurofilaments (NF), the main components of axonal cytoskeleton, are known to be involved in several neurodegenerative diseases. It has been reported that methylmalonate and propionate affect phosphorylation of NFs. In an in vitro model for methylmalonic aciduria our group has recently shown that 2- methylcitrate (2-MCA) is the most toxic metabolite for developing brain cells. Here, we studied the effects of repetitive administration of 1mM 2- MCA every 12 hours over 3 days on the development of NFs in 3D organotypic rat brain cell cultures. By immunohistochemistry with antibodies specific for the different NF subunits (light NFL, medium NFM, heavy NFH) as well as for phosphorylated (p) and glycosylated (g) forms of NFs, we observed a decrease of axonal labeling and a disorganized axonal pattern. Interestingly, signal retention of p-NFM and g-NFM was observed in neuronal soma. Western blotting showed the decrease of NFL and NFH subunits. Taken together, our data show that 2-MCA alters expression of the different NF subunits as well as their post-translational modifications. This likely results in disturbed NF assembly, abnormal accumulation of NF in neuronal cell bodies and impairment of axonal development.We conclude thatNF are involved in 2-MCA-induced neurodegeneration in methylmalonic aciduria.

Surface modification of biomaterials for conjugation with human fetal osteoblasts.

Surface functionalization of hydroxyapatite (HA) and beta-tricalcium phosphate (TCP) bioceramics with chemical ligands containing a pyrrogallol moiety was developed to improve the adhesion of bone cell precursors to the biomaterials. Fast and biocompatible copper-free click reaction with azido-modified human fetal osteoblasts resulted in improved cell binding to both HA and TCP bioceramics, opening the way for using this methodology in the preparation of cell-engineered bone implants.

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.

Transjugular liver biopsy: prospective evaluation of the angle formed between the hepatic veins and the vena cava main axis and modification of a semi-automated biopsy device in cases of an unfavorable angle.

In cases of transjugular liver biopsies, the venous angle formed between the chosen hepatic vein and the vena cava main axis in a frontal plane can be large, leading to technical difficulties. In a prospective study including 139 consecutive patients who underwent transjugular liver biopsy using the Quick-Core biopsy set, the mean venous angle was equal to 49.6 degrees. For 21.1% of the patients, two attempts at hepatic venous catheterization failed because the venous angle was too large, with a mean of 69.7 degrees. In all of these patients, manual reshaping of the distal curvature of the stiffening metallic cannula, by forming a new mean angle equal to 48 degrees , allowed successful completion of the procedure in less than 10 min.

Modification of the monomer composition of polyhydroxyalkanoate synthesized in Saccharomyces cerevisiae expressing variants of the beta-oxidation-associated multifunctional enzyme.

Expression by Saccharomyces cerevisiae of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the Brassica napus isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA. The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the B. napus ICL to target foreign proteins to the peroxisome of S. cerevisiae was demonstrated with green fluorescent protein fusions. PHA synthesis was found to be dependent on the presence of both the enzymes generating the beta-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding PEX5 gene, demonstrating the requirement for a functional peroxisome and a beta-oxidation cycle for PHA synthesis. Using a variant of the S. cerevisiae beta-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons.

Novel insulated gamma and lentis retroviral vectors towards safer genetic modification of stem cells

In otherwise successful gene therapy trials insertional mutagenesis has resulted in leukemia. The identification of new short synthetic genetic insulator elements (GIE) which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts with e.g. b-globin HS4, have met with poor efficacy and genetic instability. We have investigated potential improvement with two new candidate synthetic GIEs in SIN-gamma and lentiviral vectors. With each constructs two internal promoters have been tested: either the strong Fr- MuLV-U3 or the housekeeping hPGK.We could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro and lentivectors. In target cells a dramatic shift of expression is observed with an homogenous profile the level of which strictly depends on the promoter strength. These data remain stable in both HeLa cells over three months and cord blood HSCs for two months, irrespective of the multiplicity of infection (MOI). In comparison, control native and SIN vectors expression levels show heterogeneous, depend on the MOI and prove unstable. We have undertaken genotoxicity assessment in comparing integration patterns ingenuity in human target cells sampled over three months using high-throughput pyro-sequencing. Data will be presented. Further genotoxicity assessment will include in vivo studies. We have established insulated vectors which harbour both boundary and enhancer-blocking effect and show stable in prolonged in vitro culture conditions. Work performed with support of EC-DG research FP6-NoE, CLINIGENE: LSHB-CT-2006-018933

NAD+-dependent post-translational modification of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein reported to be a target of several covalent modifications in many organisms. In a previous study we showed that enterohemorragic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains secrete GAPDH and that this protein binds to human plasminogen and fibrinogen. Here we report that GAPDH of these pathogens is ADP-ribosylated either in the cytoplasm or in the extracellular medium. GAPDH catalyzes its own modification which involves Cys149 at the active site. ADP-ribosylation of extracellular GAPDH may play important role in the interaction with the host as it has been proposed in other pathogens.

Managing the Sick Child in the Era of Declining Malaria Transmission: Development of ALMANACH, an Electronic Algorithm for Appropriate Use of Antimicrobials.

OBJECTIVE: To review the available knowledge on epidemiology and diagnoses of acute infections in children aged 2 to 59 months in primary care setting and develop an electronic algorithm for the Integrated Management of Childhood Illness to reach optimal clinical outcome and rational use of medicines. METHODS: A structured literature review in Medline, Embase and the Cochrane Database of Systematic Review (CDRS) looked for available estimations of diseases prevalence in outpatients aged 2-59 months, and for available evidence on i) accuracy of clinical predictors, and ii) performance of point-of-care tests for targeted diseases. A new algorithm for the management of childhood illness (ALMANACH) was designed based on evidence retrieved and results of a study on etiologies of fever in Tanzanian children outpatients. FINDINGS: The major changes in ALMANACH compared to IMCI (2008 version) are the following: i) assessment of 10 danger signs, ii) classification of non-severe children into febrile and non-febrile illness, the latter receiving no antibiotics, iii) classification of pneumonia based on a respiratory rate threshold of 50 assessed twice for febrile children 12-59 months; iv) malaria rapid diagnostic test performed for all febrile children. In the absence of identified source of fever at the end of the assessment, v) urine dipstick performed for febrile children <2 years to consider urinary tract infection, vi) classification of 'possible typhoid' for febrile children >2 years with abdominal tenderness; and lastly vii) classification of 'likely viral infection' in case of negative results. CONCLUSION: This smartphone-run algorithm based on new evidence and two point-of-care tests should improve the quality of care of <5 year children and lead to more rational use of antimicrobials.