Metabolic engineering of Pseudomonas taiwanensis VLB120 for rhamnolipid biosynthesis from biomass-derived aromatics.

Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of Pseudomonas taiwanensis VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was actP, coding for a cation/acetate symporter, and the other gene was paaA coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmolrhl/Cmol4-coumarate) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmolrhl/Cmolferulate), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.

Rational Engineering of Phenylalanine Accumulation in Pseudomonas taiwanensis to Enable High-Yield Production of Trans-Cinnamate.

Microbial biocatalysis represents a promising alternative for the production of a variety of aromatic chemicals, where microorganisms are engineered to convert a renewable feedstock under mild production conditions into a valuable chemical building block. This study describes the rational engineering of the solvent-tolerant bacterium Pseudomonas taiwanensis VLB120 toward accumulation of L-phenylalanine and its conversion into the chemical building block t-cinnamate. We recently reported rational engineering of Pseudomonas toward L-tyrosine accumulation by the insertion of genetic modifications that allow both enhanced flux and prevent aromatics degradation. Building on this knowledge, three genes encoding for enzymes involved in the degradation of L-phenylalanine were deleted to allow accumulation of 2.6 mM of L-phenylalanine from 20 mM glucose. The amino acid was subsequently converted into the aromatic model compound t-cinnamate by the expression of a phenylalanine ammonia-lyase (PAL) from Arabidopsis thaliana. The engineered strains produced t-cinnamate with yields of 23 and 39% Cmol Cmol-1 from glucose and glycerol, respectively. Yields were improved up to 48% Cmol Cmol-1 from glycerol when two enzymes involved in the shikimate pathway were additionally overexpressed, however with negative impact on strain performance and reproducibility. Production titers were increased in fed-batch fermentations, in which 33.5 mM t-cinnamate were produced solely from glycerol, in a mineral medium without additional complex supplements. The aspect of product toxicity was targeted by the utilization of a streamlined, genome-reduced strain, which improves upon the already high tolerance of P. taiwanensis VLB120 toward t-cinnamate.

High-Yield Production of 4-Hydroxybenzoate From Glucose or Glycerol by an Engineered Pseudomonas taiwanensis VLB120.

Aromatic compounds such as 4-hydroxybenzoic acid are broadly applied in industry for a myriad of applications used in everyday life. However, their industrial production currently relies heavily on fossil resources and involves environmentally unfriendly production conditions, thus creating the need for more sustainable biotechnological alternatives. In this study, synthetic biology was applied to metabolically engineer Pseudomonas taiwanensis VLB120 to produce 4-hydroxybenzoate from glucose, xylose, or glycerol as sole carbon sources. Genes encoding a 4-hydroxybenzoate production pathway were integrated into the host genome and the flux toward the central precursor tyrosine was enhanced by overexpressing genes encoding key enzymes of the shikimate pathway. The flux toward tryptophan biosynthesis was decreased by introducing a P290S point mutation in the trpE gene, and degradation pathways for 4-hydroxybenzoate, 4-hydroxyphenylpyruvate and 3-dehydroshikimate were knocked out. The resulting production strains were tailored for the utilization of glucose and glycerol through the rational modification of central carbon metabolism. In batch cultivations with a completely mineral medium, the best strain produced 1.37 mM 4-hydroxybenzoate from xylose with a C-mol yield of 8% and 3.3 mM from glucose with a C-mol yield of 19.0%. Using glycerol as a sole carbon source, the C-mol yield increased to 29.6%. To our knowledge, this is the highest yield achieved by any species in a fully mineral medium. In all, the efficient conversion of bio-based substrates into 4-hydroxybenzoate by these deeply engineered P. taiwanensis strains brings the renewable production of aromatics one step closer.

[Cluster-randomized, controlled evaluation of stress management training for high school students]. / Clusterrandomisierte kontrollierte Untersuchung eines Stressbewältigungs-Trainings in der gymnasialen Oberstufe.

Cluster-randomized, controlled evaluation of stress management training for high school students Abstract. OBJECTIVE: One fifth of German adolescents show elevated levels of mental health problems. The development of mental health problems is often closely related to experiences of stress. Hence, fostering competences in coping with stress offers an approach for counteracting the development of psychological problems. The aim of the present study is to test a stress management training of 3 x 90 minutes in a German high school, 11th grade. METHOD: A cluster-randomized, controlled design was used. The primary outcome was stress. Secondary outcome criteria were knowledge about stress, coping behaviour, emotional and behavioural problems as well as health-related quality of life. Students from twenty-one courses from four schools participated in the study. RESULTS: The sample comprised 286 students (58.25 % girls; mean age 16.58 ± 0.65). Changes in stress levels from pre- to post-evaluation did not differ significantly between intervention and control group. With regard to secondary criteria, apart from a significant increase in knowledge no further effects were observed. An exploratory moderation analysis hints at a positive association between baseline levels of stress and effectiveness of the training. CONCLUSIONS: The results question whether the universal application of a brief cognitive behavioural intervention is appropriate to prevent stress in this population. Future research should, inter alia, investigate the potential of indicated prevention regarding stress.

Metabolic engineering of Pseudomonas taiwanensis VLB120 with minimal genomic modifications for high-yield phenol production.

Aromatic chemicals are important building blocks for the production of a multitude of everyday commodities. Currently, aromatics production relies almost exclusively on petrochemical processes. To achieve sustainability, alternative synthesis methods need to be developed. Here, we strived for an efficient production of phenol, a model aromatic compound of industrial relevance, from renewable carbon sources using the solvent-tolerant biocatalyst Pseudomonas taiwanensis VLB120. First, multiple catabolic routes for the degradation of aromatics and related compounds were inactivated, thereby obtaining the chassis strain P. taiwanensis VLB120Δ5 incapable of growing on 4-hydroxybenzoate (ΔpobA), tyrosine (Δhpd), and quinate (ΔquiC, ΔquiC1, ΔquiC2). In this context, a novel gene contributing to the quinate catabolism was identified (quiC2). Second, we employed a combination of reverse- and forward engineering to increase metabolic flux towards the product, using leads obtained from the analysis of aromatics producing Pseudomonas putida strains previously generated by mutagenesis. Phenol production was enabled by the heterologous expression of a codon-optimized and chromosomally integrated tyrosine phenol-lyase encoding gene from Pantoea agglomerans AJ2985 (PaTPL2). The genomic modification of endogenous genes encoding TrpEP290S, AroF-1P148L, and PheAT310I, and the deletion of pykA improved phenol production 17-fold, while also minimizing the burden caused by plasmids and auxotrophies. The additional overexpression of known bottleneck enzymes (AroGfbr, TyrAfbr) derived from Escherichia coli further enhanced phenol titers. The best producing strain P. taiwanensis VLB120Δ5-TPL36 reached yields of 15.8% and 18.5% (Cmol/Cmol) phenol from glucose and glycerol, respectively, in a mineral medium without addition of complex nutrients. This is the highest yield ever reported for microbially produced phenol.

[School absenteeism: Preliminary developments and maintaining persisting challenges]. / Schulabsentismus: Entwicklungen und fortbestehende Herausforderungen.

A first step when considering school absenteeism is to understand the meaning and definition of the term. School absenteeism encompasses several terms such as school refusal, truancy and school phobia, all of which have been used inconsistently and confusingly in the past. Furthermore, the question of how many days of absence can be seen as problematic remains unclear. Due to these definitional problems, available data is inconsistent. Therefore, the prevalence rates of school absenteeism can only be estimated (about 5 % of all students). School absenteeism affects not only individual students, but also family, school and society structures. In order to establish appropriate support and intervention programs, a multimodal as well as an individual approach should be considered to address this interdependency. The primary goal, however, should be the students' resumption of a regular school attendance, which requires a strong cooperation between parents, schools, youth welfare services and psychotherapeutic offers. If therapeutic interventions are required, it is highly recommended to start with outpatient treatment. If school attendance still remains irregular an inpatient treatment should follow.

Rigorously modeling self-stabilizing fault-tolerant circuits: An ultra-robust clocking scheme for systems-on-chip.

We present the first implementation of a distributed clock generation scheme for Systems-on-Chip that recovers from an unbounded number of arbitrary transient faults despite a large number of arbitrary permanent faults. We devise self-stabilizing hardware building blocks and a hybrid synchronous/asynchronous state machine enabling metastability-free transitions of the algorithm's states. We provide a comprehensive modeling approach that permits to prove, given correctness of the constructed low-level building blocks, the high-level properties of the synchronization algorithm (which have been established in a more abstract model). We believe this approach to be of interest in its own right, since this is the first technique permitting to mathematically verify, at manageable complexity, high-level properties of a fault-prone system in terms of its very basic components. We evaluate a prototype implementation, which has been designed in VHDL, using the Petrify tool in conjunction with some extensions, and synthesized for an Altera Cyclone FPGA.

[School absenteeism in Germany: prevalence of excused and unexcused absenteeism and its correlation with emotional and behavioural problems]. / Schulabsentismus in Deutschland--Die Prävalenz von entschuldigten und unentschuldigten Fehlzeiten und ihre Korrelation mit emotionalen und Verhaltensauffälligkeiten.

Data about the prevalence of school absenteeism and its correlation with emotional and behavioural problems in Germany is scarce, in particular regarding excused absenteeism. This study aims to close the gap by examining a sample of 2,679 pupils attending the different types of secondary school (Hauptschule, Realschule, Gymnasium), who participated in a clinical trial for the prevention of truancy (WE-STAY-Project). Pupils' mean age was 14 years (M = 13.94, SD = 0.85, Range = 11-19) and gender distribution was balanced (49.35% males, 50.65% females). Using a self-report questionnaire, pupils where asked on how many days they had missed school on average per month during the last school year (excused and unexcused). Emotional and behavioural problems were measured by using the "Strengths and Difficulties Questionnaire" (SDQ). 4.1% of the pupils reported to have missed school without a valid excuse on more than four days per month (unexcused absenteeism). 6.1% had missed school having an excuse on more than ten days per month (excused absenteeism). Both, unexcused and excused absenteeism, showed an increase of emotional and behavioural problems dependent on the intensity of absenteeism. In conclusion, these findings show the relevance of school absenteeism in Germany. In the future, more attention should be given to pupils with also excused absenteeism.

Specific music therapy techniques in the treatment of primary headache disorders in adolescents: a randomized attention-placebo-controlled trial.

UNLABELLED: Migraine and tension-type headache have a high prevalence in children and adolescents. In addition to common pharmacologic and nonpharmacologic interventions, music therapy has been shown to be efficient in the prophylaxis of pediatric migraine. This study aimed to assess the efficacy of specific music therapy techniques in the treatment of adolescents with primary headache (tension-type headache and migraine). A prospective, randomized, attention-placebo-controlled parallel group trial was conducted. Following an 8-week baseline, patients were randomized to either music therapy (n = 40) or a rhythm pedagogic program (n = 38) designed as an "attention placebo" over 6 sessions within 8 weeks. Reduction of both headache frequency and intensity after treatment (8-week postline) as well as 6 months after treatment were taken as the efficacy variables. Treatments were delivered in equal dose and frequency by the same group of therapists. Data analysis of subjects completing the protocol showed that neither treatment was superior to the other at any point of measurement (posttreatment and follow-up). Intention-to-treat analysis revealed no impact of drop-out on these results. Both groups showed a moderate mean reduction of headache frequency posttreatment of about 20%, but only small numbers of responders (50% frequency reduction). Follow-up data showed no significant deteriorations or improvements. PERSPECTIVE: This article presents a randomized placebo-controlled trial on music therapy in the treatment of adolescents with frequent primary headache. Music therapy is not superior to an attention placebo within this study. These results draw attention to the need of providing adequate controls within therapeutic trials in the treatment of pain.

Distributed algorithms for sensor networks.

Distributed algorithms are an established tool for designing protocols for sensor networks. In this paper, we discuss the relation between distributed computing theory and sensor network applications. We also present a few basic and illustrative distributed algorithms.