Potential to grow carp oedema virus (genogroup I) in monolayers of carp-derived primary cells with further implication in cell analysis.

Carp oedema virus (CEV) has distinct molecularly identified genogroups of viral mutations, denoted as I, IIa, and IIb. Failure to propagate CEV in vitro limits studies towards understanding its interactions with host cells. Here, virus isolates belonging to genogroup I collected during natural outbreaks in the Czech Republic were employed for routine CEV cultivation in monolayers of carp-derived primary cells, common carp brain (CCB) cells, and epithelioma papulosum cyprinid (EPC) cells. Induction of cytopathic effects (CPEs) was observed and recorded in affected cells. Cell survival rate was evaluated under serial dilutions of the CEV inoculum. Virus cell entry was quantified and visualized by qPCR and transmission electron microscopy, respectively. Study findings indicate primary gills epithelia likely present the most suitable matrix for CEV growth in vitro. Cells of the head kidney and spleen facilitate virus entry with microscopically confirmed CPEs and the presence of cytoplasmic pleomorphic virus particles. Cells of the trunk kidney and gonads are unlikely to permit virus cell entry and CPEs development. Although CEV cultivation in cell lines was inconclusive, EPC cells were CEV permissible. Monolayers of carp-derived primary cells show promise for CEV cultivation that could enable elaborate study of mechanisms underlying cellular binding and responses.

Efficacy and safety of the novel GlyT1 inhibitor BI 425809 in Alzheimer's dementia: a randomized controlled trial.

BACKGROUND: This phase II proof-of-concept study assessed the efficacy and safety of BI 425809, a novel selective glycine transporter-1 inhibitor, for the treatment of cognitive impairment associated with probable Alzheimer's disease dementia. METHODS: This 12-week, multicenter, double-blind, placebo-controlled, parallel-group study randomized (1:1:1:1:1) patients with mild-to-moderate probable Alzheimer's disease dementia to BI 425809 2, 5, 10, and 25 mg or placebo once daily. The primary efficacy endpoint was the change from baseline in Alzheimer's Disease Assessment Scale-Cognitive Subscale 11-item total score after 12 weeks of treatment. Safety was also assessed. RESULTS: Six hundred and ten male and female patients were randomized to BI 425809 2 mg (n = 123), 5 mg (n = 122), 10 mg (n = 122), and 25 mg (n = 123) or placebo (n = 120). Approximately 47% (n = 286) were male; the mean (standard deviation) age was 72.9 (7.7) years. Treatment compliance was above 97% for all dose groups. The Mini-Mental State Examination category on the median score was < 22 in 47% (n = 287) of patients and ≥ 22 in 53% (n = 322) of patients. No significant, non-flat dose-response relationship was detected for the primary endpoint (adjusted p-value > 0.76 for all models). BI 425809 was generally well-tolerated. Overall, 47.9% (n = 292) of patients reported at least one adverse event during the trial; the frequency of patients with investigator-defined drug-related adverse events was similar in all treatment groups, ranging from 15.4 to 19.5% across the BI 425809 treatment groups and 15.8% for placebo. CONCLUSIONS: No clinically meaningful changes from baseline were observed following treatment with BI 425809 in patients with mild-to-moderate probable Alzheimer's disease dementia. TRIAL REGISTRATION: ClinicalTrials.gov NCT02788513 (1346-0023). Registered on June 2, 2016. EU Clinical Trials Register 2015-005438-24. Registered on May 6, 2016.

Partial fads2 Gene Knockout Diverts LC-PUFA Biosynthesis via an Alternative Δ8 Pathway with an Impact on the Reproduction of Female Zebrafish (Danio rerio).

The zebrafish (Danio rerio) genome contains a single gene fads2 encoding a desaturase (FADS2) with both Δ6 and Δ5 activities, the key player in the endogenous biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs), which serve essential functions as membrane components, sources of energy and signaling molecules. LC-PUFAs include the precursors of eicosanoids and are thus predicted to be indispensable molecules for reproductive health in virtually all vertebrates. In mice, an amniotic vertebrate, fads2 deletion mutants, both males and females, have been confirmed to be sterile. In anamniotic vertebrates, such as fish, there is still no information available on the reproductive (in)ability of fads2 mutants, although zebrafish have become an increasingly important model of lipid metabolism, including some aspects of the generation of germ cells and early embryonic development. In the present study, we apply the CRISPR/Cas9 genome editing system to induce mutations in the zebrafish genome and create crispants displaying a degree of fads2 gene editing within the range of 50-80%. Focusing on adult G0 crispant females, we investigated the LC-PUFA profiles of eggs. Our data suggest an impaired pathway of the LC-PUFA biosynthesis of the ω6 and ω3 series in the first-rate limiting steps of the conversion of linoleic acid (LA) into γ-linolenic acid (GLA), and α-linolenic acid (ALA) into stearidonic acid (SDA), respectively, finally resulting in bad-quality eggs. Our data suggest the existence of an alternative Δ8 pathway, which bypasses the first endogenous LC-PUFA biosynthetic step in zebrafish in vivo, and suggest that the zebrafish bifunctional FADS2 enzyme is actually a trifunctional Δ6/Δ5/Δ8 desaturase.

Assessment of Fatty Acid Desaturase (Fads2) Structure-Function Properties in Fish in the Context of Environmental Adaptations and as a Target for Genetic Engineering.

Fatty acid desaturase 2 (Fads2) is the key enzyme of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis. Endogenous production of these biomolecules in vertebrates, if present, is insufficient to meet demand. Hence, LC-PUFA are considered as conditionally essential. At present, however, LC-PUFA are globally limited nutrients due to anthropogenic factors. Research attention has therefore been paid to finding ways to maximize endogenous LC-PUFA production, especially in production species, whereby deeper knowledge on molecular mechanisms of enzymatic steps involved is being generated. This review first briefly informs about the milestones in the history of LC-PUFA essentiality exploration before it focuses on the main aim-to highlight the fascinating Fads2 potential to play roles fundamental to adaptation to novel environmental conditions. Investigations are summarized to elucidate on the evolutionary history of fish Fads2, providing an explanation for the remarkable plasticity of this enzyme in fish. Furthermore, structural implications of Fads2 substrate specificity are discussed and some relevant studies performed on organisms other than fish are mentioned in cases when such studies have to date not been conducted on fish models. The importance of Fads2 in the context of growing aquaculture demand and dwindling LC-PUFA supply is depicted and a few remedies in the form of genetic engineering to improve endogenous production of these biomolecules are outlined.

Investigating the effects of nintedanib on biomarkers of extracellular matrix turnover in patients with IPF: design of the randomised placebo-controlled INMARK®trial.

INTRODUCTION: A feature of the pathogenesis of idiopathic pulmonary fibrosis (IPF) is the excess accumulation of extracellular matrix (ECM) in the lungs. Cleavage of the ECM by metalloproteinases (MMPs) generates free-circulating protein fragments known as neoepitopes. The PROFILE study suggested that changes in ECM turnover proteins may be of value as markers of disease progression in patients with IPF. Nintedanib is an approved treatment for IPF that slows disease progression by reducing decline in forced vital capacity (FVC). METHODS AND ANALYSIS: The INMARK® trial is evaluating the effect of nintedanib on the rates of change of biomarkers of ECM turnover in patients with IPF, the value of changes in these biomarkers as predictors of disease progression and whether nintedanib affects the associations between changes in these biomarkers and disease progression. Following a screening period, 347 patients with IPF and FVC ≥80% predicted were randomised 1:2 to receive nintedanib 150 mg two times a day or placebo for 12 weeks, followed by an open-label period in which all patients will receive nintedanib for 40 weeks. The primary endpoint is the rate of change in C reactive protein degraded by MMP-1/8 from baseline to week 12. ETHICS AND DISSEMINATION: This trial is being conducted in compliance with the protocol, the ethical principles detailed in the Declaration of Helsinki and in accordance with the International Conference on Harmonisation Harmonised Tripartite Guideline for Good Clinical Practice. The results of the trial will be presented at national and international meetings and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT02788474.

Nicoboxil/nonivamide cream effectively and safely reduces acute nonspecific low back pain - a randomized, placebo-controlled trial.

BACKGROUND/OBJECTIVE: Low back pain affects many patients and has a high socioeconomic impact. Topical capsaicinoids have been used for decades to treat musculoskeletal pain. This study investigated the effects of the fixed dose combination (FDC) of nonivamide (a capsaicinoid) and nicoboxil (a nicotinic acid ester) cream in the treatment of acute nonspecific low back pain. MATERIALS AND METHODS: This phase III randomized, double-blind, placebo-controlled, multinational, multi-center trial investigated efficacy, safety, and tolerability of topical nicoboxil 1.08%/nonivamide 0.17% (Finalgon® cream) in treatment of acute nonspecific low back pain with the endpoints: pain intensity (PI) difference between pre-dose baseline and 8 hours after first application and the end of treatment, mobility score, and efficacy score. RESULTS: Patients (n=138), 21-65 years of age, were treated for up to 4 days with FDC or placebo cream. Mean baseline PI was 6.8 on a 0-10 point numerical rating scale. After 8 hours, pain was more reduced with the FDC than with placebo (adjusted means: 2.824 vs. 0.975 points; p<0.0001). On the last treatment day, mean pain reduction by the FDC was stronger than with placebo (adjusted means: 5.132 vs. 2.174 points; p<0.0001). Mobility on Day 1 was in favor of the FDC when compared to placebo (odds ratio [95% confidence interval {CI}]: 7.200 [3.609, 14.363], p<0.0001). At the end of treatment, patients treated with the FDC rated efficacy significantly higher than placebo (odds ratio [95% CI]: 11.370 [5.342, 24.199], p<0.0001). Both treatments were tolerated well. No serious adverse events were reported. CONCLUSION: Nicoboxil/nonivamide cream is an effective and safe treatment for acute nonspecific low back pain, adding a promising treatment option.

The Effect of Tiotropium in Symptomatic Asthma Despite Low- to Medium-Dose Inhaled Corticosteroids: A Randomized Controlled Trial.

BACKGROUND: Tiotropium, a once-daily long-acting anticholinergic bronchodilator, has demonstrated efficacy in patients with asthma who were symptomatic despite treatment with medium- to high-dose inhaled corticosteroids (ICS). OBJECTIVE: The objective of this study was to evaluate the efficacy and safety of once-daily tiotropium Respimat (5 µg or 2.5 µg), compared with placebo Respimat, as add-on therapy to low- to medium-dose ICS for adults with symptomatic asthma. METHODS: A phase III, double-blind, placebo-controlled trial was conducted (NCT01316380). Adults with symptomatic asthma receiving low- to medium-dose ICS (200-400 µg budesonide or equivalent dose) and a pre-bronchodilator forced expiratory volume in 1 second (FEV1) ≥60% and ≤90% of predicted normal were randomized to 12 weeks of treatment with once-daily tiotropium Respimat 5 µg or 2.5 µg, or placebo Respimat, as add-on therapy to ICS. The primary endpoint was peak FEV1(0-3h) response. RESULTS: In total, 464 patients were randomized (61% female; mean age 43 years; mean baseline FEV1 78% of predicted normal). After 12 weeks, both tiotropium Respimat doses were superior to placebo (adjusted mean difference from placebo: 5 µg, 128 mL; 2.5 µg, 159 mL; both P < .001). Both doses of tiotropium Respimat were also superior to placebo with regard to the secondary endpoints of adjusted mean trough FEV1 and FEV1 area under the curve(0-3h) responses, and the other endpoints of morning and evening peak expiratory flow. Adverse events were comparable across the treatment groups. CONCLUSIONS: Once-daily tiotropium Respimat add-on therapy to low- to medium-dose ICS in adults with symptomatic asthma is an efficacious bronchodilator, and its safety and tolerability are comparable with those of placebo Respimat.

Dose-finding evaluation of once-daily treatment with olodaterol, a novel long-acting ß2-agonist, in patients with asthma: results of a parallel-group study and a crossover study.

BACKGROUND: Olodaterol is a novel, inhaled long-acting ß2-agonist (LABA) with >24-hour duration of action investigated in asthma and chronic obstructive pulmonary disease. METHODS: Two multicentre studies examined the efficacy and safety of 4 weeks' once-daily (QD) olodaterol (2, 5, 10 and 20 µg, with background inhaled corticosteroids) in patients with asthma. One randomised, double-blind, parallel-group study (1222.6; 296 patients) administered treatment in the morning. Pulmonary function tests (PFTs) were performed pre-dose (trough) and ≤3 hours post-dose (weeks 1 and 2), and ≤6 hours post-dose after 4 weeks; primary end point was trough forced expiratory volume in 1 second (FEV1) response (change from baseline mean FEV1) after 4 weeks. A second randomised, double-blind, placebo- and active-controlled (formoterol 12 µg twice-daily) incomplete-block crossover study (1222.27; 198 patients) administered QD treatments in the evening. PFTs were performed over a 24-hour dosing interval after 4 weeks; primary end point was FEV1 area under the curve from 0-24 hours (AUC0-24) response (change from study baseline [mean FEV1] after 4 weeks). RESULTS: Study 1222.6 showed a statistically significant increase in trough FEV1 response with olodaterol 20 µg (0.147 L; 95 % confidence interval [CI]: 0.059, 0.234; p = 0.001) versus placebo, with more limited efficacy and no evidence of dose response compared to placebo across the other olodaterol doses (2, 5 and 10 µg). Study 1222.27 demonstrated increases in FEV1 AUC0-24 responses at 4 weeks with all active treatments (p < 0.0001); adjusted mean (95 % CI) differences from placebo were 0.140 (0.097, 0.182), 0.182 (0.140, 0.224), 0.205 (0.163, 0.248) and 0.229 (0.186, 0.272) L for olodaterol 2, 5, 10 and 20 µg, respectively, and 0.169 (0.126, 0.211) for formoterol, providing evidence of increased efficacy with higher olodaterol dose. Olodaterol was generally well tolerated, with a few events associated with known sympathomimetic effects, mainly with 20 µg. CONCLUSIONS: The LABA olodaterol has >24-hour duration of action. In patients with asthma, evidence of bronchodilator efficacy was demonstrated with statistically and clinically significant improvements in the primary end point of trough FEV1 response measured in clinics over placebo for the highest administered dose of 20 µg in Study 1222.6, and statistically and clinically significant improvements versus placebo in FEV1 AUC0-24 responses at 4 weeks for all doses tested in Study 1222.27, which also exhibited a dose response. Bronchodilator efficacy was seen over placebo for all olodaterol doses for morning and evening peak expiratory flow in both studies. All doses were well tolerated. TRIAL REGISTRATIONS: NCT00467740 (1222.6) and NCT01013753 (1222.27).

Distribution and origin of oxygen-dependent and oxygen-independent forms of Mg-protoporphyrin monomethylester cyclase among phototrophic proteobacteria.

Magnesium-protoporphyrin IX monomethylester cyclase is one of the key enzymes of the bacteriochlorophyll biosynthesis pathway. There exist two fundamentally different forms of this enzyme. The oxygen-dependent form, encoded by the gene acsF, catalyzes the formation of the bacteriochlorophyll fifth ring using oxygen, whereas the oxygen-independent form encoded by the gene bchE utilizes an oxygen atom extracted from water. The presence of acsF and bchE genes was surveyed in various phototrophic Proteobacteria using the available genomic data and newly designed degenerated primers. It was found that while the majority of purple nonsulfur bacteria contained both forms of the cyclase, the purple sulfur bacteria contained only the oxygen-independent form. All tested species of aerobic anoxygenic phototrophs contained acsF genes, but some of them also retained the bchE gene. In contrast to bchE phylogeny, the acsF phylogeny was in good agreement with 16S inferred phylogeny. Moreover, the survey of the genome data documented that the acsF gene occupies a conserved position inside the photosynthesis gene cluster, whereas the bchE location in the genome varied largely between the species. This suggests that the oxygen-dependent cyclase was recruited by purple phototrophic bacteria very early during their evolution. The primary sequence and immunochemical similarity with its cyanobacterial counterparts suggests that acsF may have been acquired by Proteobacteria via horizontal gene transfer from cyanobacteria. The acquisition of the gene allowed purple nonsulfur phototrophic bacteria to proliferate in the mildly oxygenated conditions of the Proterozoic era.