Challenges and prospects for interpreting long‐term phytoplankton diversity changes in Lake Zurich (Switzerland)

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Anthelmintic efficacy on UK Thoroughbred stud farms

Anthelmintic drugs have been applied indiscriminately to control horse nematodes for over 40 years. We undertook a comprehensive study to investigate efficacy of the four available broad-spectrum anthelmintic drugs on 16 Thoroughbred stud farms using the faecal egg count reduction test. Efficacy against strongyles was determined by calculating the percentage of reduction in faecal egg count between the group mean at Day 0 and Days 14–17 post-treatment and the 95% lower confidence intervals estimated by non-parametric bootstrapping. Individual strongyle faecal egg count reduction tests (n = 429) were performed in which 179, 131, 89 and 30 horses were administered ivermectin, moxidectin, pyrantel and fenbendazole, respectively. Moxidectin was efficacious in all tests (faecal egg count reduction range: 99.8–100%; 95% lower confidence intervals range: 96.8–100%) and reduced efficacy of ivermectin (faecal egg count reduction range: 85.7–100%; 95% lower confidence intervals range: 65–100%) was observed in one group of yearlings. Reduced pyrantel efficacy was observed in five groups of yearlings (faecal egg count reduction range: 0–73%; 95% lower confidence intervals range: 0–59.5%), but pyrantel was found to be efficacious when administered to mares (faecal egg count reduction range: 98–99.4%; 95% lower confidence intervals range: 91.8–99.3%). Low efficacy of fenbendazole was always observed (faecal egg count reduction range: 0.4–41%; 95% lower confidence intervals not calculable). Two further methods for estimating efficacy were applied and outputs obtained using all methodologies were in agreement. Efficacy against Parascaris equorum was assessed on four farms: fenbendazole had acceptable efficacy (faecal egg count reduction range: 97.5–99.9%; 95% lower confidence intervals range: 96.3–99.1%), but reduced efficacy of ivermectin was observed (faecal egg count reduction range: 25.5–91.2%; 95% lower confidence intervals range: 6.7–82.4%). Strongyle faecal egg count were analysed at approximately 2 week intervals for up to 12 weeks after anthelmintic drug administration to determine the egg reappearance period for moxidectin, ivermectin and pyrantel. The egg reappearance period for all three anthelmintic drugs was shorter than previously observed. Overall, our results indicate that ivermectin and moxidectin administration provided acceptable efficacy at 14 days; however, egg reappearance period results suggest that these products are working less effectively than measured previously. As shortened egg reappearance period is believed to be an early indicator of resistance, this highlights the issue of impending multi-drug resistance in strongyles on stud farms.     

Bacterial Nonhomologous End Joining Requires Teamwork

All living organisms must repair DNA double-stranded breaks (DSBs) in order to survive. Many bacteria rely on nonhomologous end joining (NHEJ) when only a single copy of the genome is available and maintain NHEJ pathways with a minimum of two proteins. In this issue, Bhattarai and colleagues identify additional factors that can work together to aid in survival of stationary-phase cells with chromosomal breaks.     

Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV

Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.     

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~ 27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency > 90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.     

Structure and Biochemical Properties of the Alkene Producing Cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 Bacterium

The production of hydrocarbons in nature has been documented for only a limited set of organisms, with many of the molecular components underpinning these processes only recently identified. There is an obvious scope for application of these catalysts and engineered variants thereof in the future production of biofuels. Here we present biochemical characterization and crystal structures of a cytochrome P450 fatty acid peroxygenase: the terminal alkene forming OleT_JE (CYP152L1) from Jeotgalicoccus sp. 8456. OleT_JE is stabilized at high ionic strength, but aggregation and precipitation of OleT_JE in low salt buffer can be turned to advantage for purification, because resolubilized OleT_JE is fully active and extensively dissociated from lipids. OleT_JE binds avidly to a range of long chain fatty acids, and structures of both ligand-free and arachidic acid-bound OleT_JE reveal that the P450 active site is preformed for fatty acid binding. OleT_JE heme iron has an unusually positive redox potential (−103 mV versus normal hydrogen electrode), which is not significantly affected by substrate binding, despite extensive conversion of the heme iron to a high spin ferric state. Terminal alkenes are produced from a range of saturated fatty acids (C12–C20), and stopped-flow spectroscopy indicates a rapid reaction between peroxide and fatty acid-bound OleT_JE (167 s⁻¹ at 200 μm H₂O₂). Surprisingly, the active site is highly similar in structure to the related P450_BSβ, which catalyzes hydroxylation of fatty acids as opposed to decarboxylation. Our data provide new insights into structural and mechanistic properties of a robust P450 with potential industrial applications.     

Body size and physical activity in relation to incidence of chronic obstructive pulmonary disease

Background:

Limited evidence suggests that adiposity and lack of physical activity may increase the risk of chronic obstructive pulmonary disease (COPD). We investigated the relation of body size and physical activity with incidence of COPD.

Methods:

We obtained data on anthropometric measurements and physical activity from 113 279 participants in the National Institutes of Health–AARP Diet and Health Study who reported no diagnosis of COPD at baseline (1995–1996). We estimated associations between these measurements and subsequent diagnosis of COPD between 1996 and 2006, with extensive adjustment for smoking and other potentially confounding variables.

Results:

Participants reported 3648 new COPD diagnoses during follow-up. The incidence of COPD was higher in both severely obese (body mass index [BMI]D≥ 35) and underweight (BMID< 18.5) participants, but after adjustment for waist circumference, only underweight remained positively associated with COPD (relative risk [RR]D1.56, 95% confidence interval [CI]D1.15–2.11). Larger waist circumference (highest v. normal categories, adjusted RRD1.72, 95% CID1.37–2.16) and higher waist–hip ratio (highest v. normal categories, adjusted RRD1.46, 95% CID1.23–1.73) were also positively associated with COPD. In contrast, hip circumference (highest v. normal categories, adjusted RR 0.78, 95% CI 0.62–0.98) and physical activity (≥ 5 v. 0 times/wk, adjusted RRD0.71, 95% CID0.63–0.79) were inversely associated with COPD.

Interpretation:

Obesity, in particular abdominal adiposity, was associated with an increased risk of COPD, and increased hip circumference and physical activity were associated with a decreased risk of COPD. These findings suggest that following guidelines for a healthy body weight, body shape and physical activity decrease the risk of COPD.Chronic obstructive pulmonary disease (COPD) is a progressive, irreversible condition that severely affects quality of life¹ and ability to work.² Direct and indirect annual costs of COPD, including inpatient and outpatient care, medication and loss of productivity, sum to $50 billion in the United States³ and R39 billion (about US$50 billion) in Europe.⁴Chronic obstructive pulmonary disease may be prevented by avoidance of tobacco smoke, occupational dust and other environmental air pollution.⁵ Body mass index (BMI) and physical activity are established correlates of disease progression among patients with COPD,^6,7 but data relating body size or physical activity to incident COPD are sparse. The few studies available are based on small samples and show inverse relations of both BMI^8,9 and physical activity^10,11 to incidence of COPD. Data are lacking regarding waist or hip circumference in relation to COPD incidence. We therefore examined BMI, waist circumference, hip circumference, waist–hip ratio and physical activity in relation to incidence of COPD in a large cohort of women and men in the US.     

Application of QbD Principles for the Evaluation of Empty Hard Capsules as an Input Parameter in Formulation Development and Manufacturing

Understanding the product and process variable on the final product performance is an essential part of the quality-by-design (QbD) principles in pharmaceutical development. The hard capsule is an established pharmaceutical dosage form used worldwide in development and manufacturing. The empty hard capsules are supplied as an excipient that is filled by pharmaceutical manufacturers with a variety of different formulations and products. To understand the potential variations of the empty hard capsules as an input parameter and its potential impact on the finished product quality, a study was performed investigating the critical quality parameters within and in between different batches of empty hard gelatin capsules. The variability of the hard capsules showed high consistency within the specification of the critical quality parameters. This also accounts for the disintegration times, when automatic endpoint detection was used. Based on these data, hard capsules can be considered as a suitable excipient for product development using QbD principles.