Improving enzyme functional annotation by integrating in vitro and in silico approaches: The example of histidinol phosphate phosphatases.

Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function-determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof-of-principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species.

Micropollutants as internal probe compounds to assess UV fluence and hydroxyl radical exposure in UV/H2O2 treatment.

Organic micropollutants (MPs) are increasingly detected in water resources, which can be a concern for human health and the aquatic environment. Ultraviolet (UV) radiation based advanced oxidation processes (AOP) such as low-pressure mercury vapor arc lamp UV/H2O2 can be applied to abate these MPs. During UV/H2O2 treatment, MPs are abated primarily by photolysis and reactions with hydroxyl radicals (•OH), which are produced in situ from H2O2 photolysis. Here, a model is presented that calculates the applied UV fluence (Hcalc) and the •OH exposure (CT•OH,calc) from the abatement of two selected MPs, which act as internal probe compounds. Quantification of the UV fluence and hydroxyl radical exposure was generally accurate when a UV susceptible and a UV resistant probe compound were selected, and both were abated at least by 50 %, e.g., iopamidol and 5-methyl-1H-benzotriazole. Based on these key parameters a model was developed to predict the abatement of other MPs. The prediction of abatement was verified in various waters (sand filtrates of rivers Rhine and Wiese, and a tertiary wastewater effluent) and at different scales (laboratory experiments, pilot plant). The accuracy to predict the abatement of other MPs was typically within ±20 % of the respective measured abatement. The model was further assessed for its ability to estimate unknown rate constants for direct photolysis (kUV,MP) and reactions with •OH (k•OH,MP). In most cases, the estimated rate constants agreed well with published values, considering the uncertainty of kinetic data determined in laboratory experiments. A sensitivity analysis revealed that in typical water treatment applications, the precision of kinetic parameters (kUV,MP for UV susceptible and k•OH,MP for UV resistant probe compounds) have the strongest impact on the model's accuracy.

[Behavioural and physiological indicators of pain and distress in calves following tail docking]. / Verhaltensassoziierte und physiologische Indikatoren für Schmerz und Unwohlsein bei Kälbern nach Schwanzamputation.

OBJECTIVES: According to the German law on animal welfare (§ 6, section 3, No. 3), the prophylactic amputation of beef calves' tail end using elastic rubber rings requires authorities' permission. Removal of the tail end is performed in order to avoid tail tip necrosis. Other invasive management procedures such as dehorning and castration are associated with pain reactions and stress in calves. Also, more proximal docking of the tail between 2 vertebrae has a proven impact on the animal's wellbeing. However, little is known concerning the occurrence of pain and stress during docking the soft tail tip in beef calves. For this reason, this pilot study aimed to evaluate pain reactions of calves during tail docking using rubber rings. MATERIAL AND METHODS: Sixteen male Fleckvieh and Fleckvieh-crossbreed calves 8-10 weeks of age were involved using a double-blind study. On day 0, a rubber ring was placed 3 cm proximally to the tip of the tail in the calves in group A (amputation), whereas calves in the group K (control) underwent a similar control handling. Following the procedure, blood plasma concentrations of cortisol and substance P were determined by ELISA. Parameters concerning behavior and activity were collected using video-assisted observation and recording of pedometer data. RESULTS: Treatment had no impact on substance P levels. The development of serum cortisol levels showed a decrease in group K compared to baseline values whereas cortisol levels remained constant in group A. On day 0, calves in group A exhibited higher step counts, tail shaking frequency as well as grooming of tail and hind quarters than calves in group K. CONCLUSION: Increased step count may be interpreted as restlessness. Frequent tail shaking as well as repetitive tail and rear quarter grooming may be associated with unpleasant sensation of the rubber rings' pressure. Alterations in physiological behavior, as well as a constant level of cortisol on day 0 and day + 1 may be indicative for pain perception provoked by the rubber ring application. CLINICAL RELEVANCE: In the first place, improvement of animal welfare is warranted for a reduction in incidence of tail tip necrosis, especially taking husbandry conditions into account. However, if tail docking is deemed necessary, based on the presented results the administration of a non-steroidal anti-inflammatory drug is recommended in order to reduce peri-procedural pain.