Effect of the mGluR5-NAM Basimglurant on Behavior in Adolescents and Adults with Fragile X Syndrome in a Randomized, Double-Blind, Placebo-Controlled Trial: FragXis Phase 2 Results.

Preclinical data suggest that inhibition of the metabotropic glutamate receptor 5 (mGluR5) receptor might hold therapeutic benefits in Fragile X syndrome (FXS). Treatment of Fmr1 knockout mice with mGluR5-negative allosteric modulators (NAMs) has been reported to correct a broad range of phenotypes related to FXS. The early short-term clinical trials with mGluR5 NAMs, including basimglurant, assessing the effects in individuals with FXS, were supportive of further exploration in larger, well-controlled trials. We evaluated basimglurant, a potent and selective mGluR5 NAM, in a 12-week, double-blind, parallel-group study of 183 adults and adolescents (aged 14-50, mean 23.4 years) with FXS. Individuals with an FMR1 full mutation were randomized to placebo or one of two doses of basimglurant. The primary efficacy endpoint was the change from baseline in behavioral symptoms using the Anxiety Depression and Mood Scale (ADAMS) total score. All treatment arms showed marked behavioral improvements from baseline to week 12 with less improvement in the basimglurant 1.5 mg arm than placebo; however, basimglurant 0.5 mg was inferior to placebo in the ADAMs total score. Treatment with basimglurant was overall well-tolerated. A higher incidence of adverse events classified as psychiatric disorders were reported in patients treated with basimglurant, including three patients with hallucinations or psychosis. In this phase 2 clinical trial, basimglurant did not demonstrate improvement over placebo. Evaluation of the overall risk-benefit in younger patient populations is an important consideration for the design of potential further investigations of efficacy with this class of medications.

An Analysis of Two Genome-wide Association Meta-analyses Identifies a New Locus for Broad Depression Phenotype.

BACKGROUND: The genetics of depression has been explored in genome-wide association studies that focused on either major depressive disorder or depressive symptoms with mostly negative findings. A broad depression phenotype including both phenotypes has not been tested previously using a genome-wide association approach. We aimed to identify genetic polymorphisms significantly associated with a broad phenotype from depressive symptoms to major depressive disorder. METHODS: We analyzed two prior studies of 70,017 participants of European ancestry from general and clinical populations in the discovery stage. We performed a replication meta-analysis of 28,328 participants. Single nucleotide polymorphism (SNP)-based heritability and genetic correlations were calculated using linkage disequilibrium score regression. Discovery and replication analyses were performed using a p-value-based meta-analysis. Lifetime major depressive disorder and depressive symptom scores were used as the outcome measures. RESULTS: The SNP-based heritability of major depressive disorder was 0.21 (SE = 0.02), the SNP-based heritability of depressive symptoms was 0.04 (SE = 0.01), and their genetic correlation was 1.001 (SE = 0.2). We found one genome-wide significant locus related to the broad depression phenotype (rs9825823, chromosome 3: 61,082,153, p = 8.2 × 10-9) located in an intron of the FHIT gene. We replicated this SNP in independent samples (p = .02) and the overall meta-analysis of the discovery and replication cohorts (1.0 × 10-9). CONCLUSIONS: This large study identified a new locus for depression. Our results support a continuum between depressive symptoms and major depressive disorder. A phenotypically more inclusive approach may help to achieve the large sample sizes needed to detect susceptibility loci for depression.

Acute tumour response to a bispecific Ang-2-VEGF-A antibody: insights from multiparametric MRI and gene expression profiling.

BACKGROUND: To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A). METHODS: Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg(-1) dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 10(6) mm(2) s(-1)), vascular perfusion/permeability (K(trans), min(-1)) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed. RESULTS: Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour K(trans) (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis. CONCLUSIONS: Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.

Biomarker for Spinal Muscular Atrophy: Expression of SMN in Peripheral Blood of SMA Patients and Healthy Controls.

Spinal muscular atrophy is caused by a functional deletion of SMN1 on Chromosome 5, which leads to a progressive loss of motor function in affected patients. SMA patients have at least one copy of a similar gene, SMN2, which produces functional SMN protein, although in reduced quantities. The severity of SMA is variable, partially due to differences in SMN2 copy numbers. Here, we report the results of a biomarker study characterizing SMA patients of varying disease severity. SMN copy number, mRNA and Protein levels in whole blood of patients were measured and compared against a cohort of healthy controls. The results show differential regulation of expression of SMN2 in peripheral blood between patients and healthy subjects.

Clinically relevant effectiveness of focused extracorporeal shock wave therapy in the treatment of chronic plantar fasciitis: a randomized, controlled multicenter study.

BACKGROUND: The effectiveness of extracorporeal shock wave therapy in the treatment of plantar fasciitis is controversial. The objective of the present study was to test whether focused extracorporeal shock wave therapy is effective in relieving chronic heel pain diagnosed as plantar fasciitis. METHODS: Two hundred and fifty subjects were enrolled in a prospective, multicenter, double-blind, randomized, and placebo-controlled U.S. Food and Drug Administration trial. Subjects were randomized to focused extracorporeal shock wave therapy (0.25 mJ/mm(2)) or placebo intervention, with three sessions of 2000 impulses in weekly intervals. Primary outcomes were both the percentage change of heel pain on the visual analog scale composite score (pain during first steps in the morning, pain with daily activities, and pain with a force meter) and the Roles and Maudsley score at twelve weeks after the last intervention compared with the scores at baseline. RESULTS: Two hundred and forty-six patients (98.4%) were available for intention-to-treat analysis at the twelve-week follow-up. With regard to the first primary end point, the visual analog scale composite score, there was a significant difference (p = 0.0027, one-sided) in the reduction of heel pain in the extracorporeal shock wave therapy group (69.2%) compared with the placebo therapy group (34.5%). Extracorporeal shock wave therapy was also significantly superior to the placebo therapy for the Roles and Maudsley score (p = 0.0006, one-sided). Temporary pain and swelling during and after treatment were the only device-related adverse events observed. CONCLUSIONS: The results of the present study provide proof of the clinically relevant effect size of focused extracorporeal shock wave therapy without local anesthesia in the treatment of recalcitrant plantar fasciitis, with success rates between 50% and 65%. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R(2) = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

Radial extracorporeal shock wave therapy (rESWT) induces new bone formation in vivo: results of an animal study in rabbits.

The aim of this study was to investigate if radial extracorporeal shock wave therapy (rESWT) induces new bone formation and to study the time course of ESWT-induced osteogenesis. A total of 4000 impulses of radial shock waves (0.16 mJ/mm²) were applied to one hind leg of 13 New Zealand white rabbits with the contralateral side used for control. Treatment was repeated after 7 days. Fluorochrome sequence labeling of new bone formation was performed by subcutaneous injection of tetracycline, calcein green, alizarin red and calcein blue. Animals were sacrificed 2 weeks (n = 4), 4 weeks (n = 4) and 6 weeks (n = 5) after the first rESWT and bone sections were analyzed by fluorescence microscopy. Deposits of fluorochromes were classified and analyzed for significance with the Fisher exact test. rESWT significantly increased new bone formation at all time points over the 6-week study period. Intensity of ossification reached a peak after 4 weeks and declined at the end of the study. New bone formation was significantly higher and persisted longer at the ventral cortex, which was located in the direction to the shock wave device, compared with the dorsal cortex, emphasizing the dose-dependent process of ESWT-induced osteogenesis. No traumata, such as hemorrhage, periosteal detachment or microfractures, were observed by histologic and radiologic assessment. This is the first study demonstrating low-energy radial shock waves to induce new bone formation in vivo. Based on our results, repetition of ESWT in 6-week intervals can be recommended. Application to bone regions at increased fracture risk (e.g., in osteoporosis) are possible clinical indications.

The dynamic locking screw (DLS) can increase interfragmentary motion on the near cortex of locked plating constructs by reducing the axial stiffness.

BACKGROUND: The plate-screw interface of an angular stable plate osteosynthesis is very rigid. So far, all attempts to decrease the stiffness of locked plating construct, e.g. the bridged plate technique, decrease primarily the bending stiffness. Thus, the interfragmentary motion increases only on the far cortical side by bending the plate. To solve this problem, the dynamic locking screw (DLS) was developed. MATERIALS AND METHODS: Comparison tests were performed with locking screws (LS) and DLS. Axial stiffness, bending stiffness and interfragmentary motion were compared. For measurements, we used a simplified transverse fracture model, consisting of POM C and an 11-hole LCP3.5 with a fracture gap of 3 mm. Three-dimensional fracture motion was detected using an optical measurement device (PONTOS 5 M/GOM) consisting of two CCD cameras (2,448 x 2,048 pixel) observing passive markers. RESULTS: The DLS reduced the axial stiffness by approximately 16% while increasing the interfragmentary motion at the near cortical side significantly from 282 microm (LS) to 423 microm (DLS) applying an axial load of 150 N. CONCLUSION: The use of DLS reduces the stiffness of the plate-screw interface and thus increases the interfragmentary motion at the near cortical side without altering the advantages of angular stability and the strength.

The effect of antibacterial acting extracorporeal shockwaves on bacterial cell integrity.

BACKGROUND: Antibacterial effects of extracorporeal shockwaves (ESWs) have been demonstrated in vitro against bacteria under static and dynamic growth conditions. This study assessed the effects of ESWs on the cell wall integrity of bacteria. MATERIAL/METHODS: Standardized suspensions of Staphylococcus aureus were exposed to various shockwave impulses (2000-12,000) of different energy flux densities (EFD, 0.38-0.96 mJ/mm(2)). Bacterial suspensions of equal concentration that had been permeabilized (to >99%) with isopropanol were used as positive controls. The bacteria of all groups were stained with Sytox Green nucleic acid stain. The fluorescence of the shockwave-treated, permeabilized, and untreated suspensions was measured and compared for bacterial survival, quantified by colony-forming units after plating. RESULTS: Although ESWs showed a significant energy-dependent antibacterial effect that reduced CFUs in the treated suspensions by between 56% and 99%, only maximum energies (4000 impulses at 0.96 mJ/mm(2) and 12,000 impulses at 0.59 mJ/mm(2)) were followed by a significant increase in fluorescence compared with the untreated control (p<0.05). However, the fluorescence of these treated groups was still far less than that of the alcohol-permeabilized positive control groups (p<0.05). Lower energies and impulse rates did not show increased intracellular uptake of the fluorescent dye (p>0.05). CONCLUSIONS: This is the first study to assess bacterial cell wall permeability after ESW treatment. It was found that the permeabilization of bacterial cells after ESW treatment was far less than expected due to the corresponding antibacterial effect. Other mechanisms, such as intracellular effects, might be involved in bacterial killing after ESWs and still must be elucidated.

8-year follow-up after cementless hip arthroplasty with a second generation spongy metal total hip replacement.

We studied a consecutive series of 81 cementless total hip arthroplasties in 80 patients using the second generation ESKA cementless spongy metal hip replacement. The study end-point was implant revision and both function as well as satisfaction with treatment outcome were assessed.Mean age at the time of surgery was 50.9 years [range 23-73]. No patient was lost to follow-up and 75 patients (76 hips) could be included in the final analysis at a mean follow-up of 7.9 years [range 7.0-10.0]. Survival rate without loosening as the end-point was 100% for the femoral component and 99% for the acetabular component (one cup revision). Two cups and one stem had to be revised for recurrent dislocation, resulting in a total implant survival at follow-up of 99% for the femoral component and 96% for the acetabular component. Very good functional results were obtained with a mean Merle d' Aubigné score of 15.5 +/- 2.9 at 7.9 years after surgery. Satisfaction with treatment outcome was reported in 88%. 95% of patients would recommend the performed procedure to a friend. Peri-operative complications without revision occurred in eleven patients (14%).We report excellent survival rates of the cementless spongy metal hip arthroplasty at a mean follow-up of eight years, particularly considering the young age of many of the patients.

Energy-dependent stimulatory and inhibitory effects of extracorporeal shock waves on bacteria and on gentamicin activity.

BACKGROUND: Local infection is considered a contraindication for extracorporeal shock wave (ESW) application, although the antibacterial effects of ESW have been clearly demonstrated in vitro. This study aimed to assess the effects of ESW on bacteria under growth-promoting conditions and to evaluate interactions with the activity of gentamicin. MATERIAL/METHODS: Standardized suspensions of Staphylococcus aureus were exposed to 4000 shock wave impulses of various energy flux densities (EFD) both at 37 degrees C in growth medium and at 20 degrees C in saline. Bacterial viability of treatment groups and controls were quantified. Furthermore, the MICs of gentamicin against ESW-treated and untreated suspensions of S. aureus were compared. Finally, suspensions of S. aureus containing graded concentrations of gentamicin were exposed to ESW and bacterial growth was assessed. RESULTS: Antibacterial effects of ESW (0.59-0.96 mJ/mm2) were confirmed with bacteria suspended in normal saline (20 degrees C, p<0.05). However, bacteria suspended in growth medium at 37 degrees C demonstrated significantly increased proliferation (p=0.009) after treatment with shock waves of lower EFD (0.59 mJ/mm2). At higher EFD a significant reduction of bacteria was observed (p=0.009). The MIC of gentamicin against S. aureus was not altered by ESW application. Furthermore, the combination of gentamicin and ESW did not alter gentamicin activity (p>0.05). Nevertheless, a growth-promoting effect of ESW at 0.59 mJ/mm(2) was demonstrated despite simultaneous administration of gentamicin. CONCLUSIONS: This is the first study reporting energy-dependent stimulation of bacterial growth by ESW. Also important is that ESW did not alter the activity of gentamicin.

Safety and effectiveness of extracorporeal shockwave therapy: results of a rabbit model of chronic osteomyelitis.

Extracorporeal shockwave therapy (ESWT) is applied successfully in various orthopedic disorders. Since shockwaves have demonstrated significant bactericidal effectiveness in vitro, safety and effectiveness of ESWT in vivo were evaluated in a rabbit model of osteomyelitis. Chronic osteomyelitis was induced by injecting sodium morrhuate and Staphylococcus aureus into the proximal tibia of 12 New Zealand white rabbits. Four and five wk after the initial operation, soft focused ESWT was applied twice to the infected limbs. Clinical parameters and laboratory values were followed and blood samples were taken for culture before and 30 min after ESWT. Following sacrifice after 8 wk, lungs, spleen and kidneys were studied histologically for signs of sepsis and secondary infection. Tibial osteomyelitis was assessed clinically, and by radiologic, microbiologic and histologic procedures. Signs of bacterial spreading were not detectable after ESWT, neither in blood cultures nor in histologic analyses of representative organs. Temperature, body weight, C-reactive protein and white blood cell levels also remained unchanged after ESWT. Of particular interest, histologic scores of osteomyelitis were significantly decreased in the ESWT-group compared to the untreated control (p = 0.019). However, S. aureus was still detectable in tissue samples of all animals. This is the first study investigating the effects of ESWT applied to infected target areas. ESWT of infected bone did neither induce bacterial spreading nor worsening of infection, and the results suggest the reported treatment protocol of ESWT to be beneficial in the treatment of chronic bone infections.

Enhanced gene trapping in mouse embryonic stem cells.

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.

Evolvability and hierarchy in rewired bacterial gene networks.

Sequencing DNA from several organisms has revealed that duplication and drift of existing genes have primarily moulded the contents of a given genome. Though the effect of knocking out or overexpressing a particular gene has been studied in many organisms, no study has systematically explored the effect of adding new links in a biological network. To explore network evolvability, we constructed 598 recombinations of promoters (including regulatory regions) with different transcription or sigma-factor genes in Escherichia coli, added over a wild-type genetic background. Here we show that approximately 95% of new networks are tolerated by the bacteria, that very few alter growth, and that expression level correlates with factor position in the wild-type network hierarchy. Most importantly, we find that certain networks consistently survive over the wild type under various selection pressures. Therefore new links in the network are rarely a barrier for evolution and can even confer a fitness advantage.

Monitoring conformational changes during the catalytic cycle of OpuAA, the ATPase subunit of the ABC transporter OpuA from Bacillus subtilis.

The ABC transporter (ATP-binding-cassette transporter) OpuA is one of five membrane transport systems in Bacillus subtilis that mediate osmoprotection by importing compatible solutes. Just like all bacterial and archaeal ABC transporters that catalyse the import of substrates, OpuA (where Opu is osmoprotectant uptake) is composed of an ATPase subunit (OpuAA), a transmembrane subunit (OpuAB) and an extracellular substrate-binding protein (OpuAC). In contrast with many well-known ABC-ATPases, OpuAA is composed not only of a catalytic and a helical domain but also of an accessory domain located at its C-terminus. The paradigm of such an architecture is MalK, the ABC-ATPase of the maltose importer of Escherichia coli, for which detailed structural and functional information is available. In the present study, we have applied solution FRET (Förster resonance energy transfer) techniques using two single cysteine mutants to obtain initial structural information on the architecture of the OpuAA dimer in solution. Analysing our results in detail and comparing them with the existing MalK structures revealed that the catalytic and helical domains adopted an arrangement similar to those of MalK, whereas profound differences in the three-dimensional orientation of the accessory domain, which contains two CBS (cystathionine beta-synthetase) domains, were observed. These results shed new light on the role of this accessory domain present in a certain subset of ABC-ATPase in the fine-tuning of three-dimensional structure and biological function.

Engineering ATPase activity in the isolated ABC cassette of human TAP1.

The human transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced.

Insulated piggyBac vectors for insect transgenesis.

BACKGROUND: Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken beta-globin HS4 insulator function in both Drosophila and mammalian cells. RESULTS: To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken beta-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects. CONCLUSION: The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species.