Engineering of factors determining alpha-amylase and cyclodextrin glycosyltranferase specificity in the cyclodextrin glycosyltransferase form Thermoanaerobacterium thermosulfurigenes EM1

The starch-degrading enzymes alpha-amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of alpha-amylases (A, B and C). Amino acid residue 196 (Thermoanaerobacterium thermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active-site cleft. All alpha-amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved. Characterization of the F196G mutant CGTase of T. thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted [mutant F196G(Delta'DE)]. This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa). The mutant protein was more similar to an alpha-amylase protein in terms of the identity of residue 196, and in the domain structure containing, however some additional C-terminal structure. The mutant showed a strongly reduced temperature optimum. Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced. Mutant F196G(Delta'DE) displayed a strongly reduced raw-starch-binding capacity. similar to the situation in most alpha-amylases that lack a raw-starch-binding E domain. Compared to wild-type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(Delta'DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase. Under industrial production process conditions, wild-type CGTase converted starch into 35% cyclodextrins and 11% linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(Delta'DE) converted starch into 21% cyclodextrins and 18% into linear oligosaccharides. These biochemical characteristics indicate a clear shift from CGTase to alpha-amylase specificity

Production and excretion of secondary metabolites by plant cell cultures of Tagetes

In this thesis, the results are presented of several approaches to improve the production and excretion of thiophenes by cell cultures or hairy roots of Tagetes spp.In chapter one, most of the techniques to improve the production and/or excretion of secondary metabolites with plant cell cultures are discussed in a literature survey. From this chapter, it becomes clear that to date no general rules exist that apply for all plant species. The methods described in this chapter are the introduction of transformed cells in the form of hairy roots, the use of elicitors, the immobilization of plant cells, the use of two-phase cultures and the use of selected fermenters; all have as aim to improve the growth, production and excretion conditions for the plant cell cultures. All these methods have also been tested in the work described in this thesis, with the aim to obtain a higher production of thiophenes for less costs (meaning faster growth and better excretion).Chapter two is the description of a side-step towards the extension of an immobilization technique for large scale operation that was introduced a few years before, the resonance nozzle technique. Until then, it had only been tested with alginate, but in this chapter, K -carrageenan, agar and gellan gum were tested as well. The gel formation with these materials is improved when the drops are caught in an organic phase, so the reactions of four cell types (bacteria, yeast, plant cells and insect cells) to several organic solvents and the immobilization were examined. The various cell types appeared to react differently to the applied conditions, but in general hydrophobic solvents proved to be more suitable than hydrophilic solvents.The latter phenomenon was elaborated in more detail in the third chapter. Cells of Tagetes minuta , in free or immobilized state, were grown in the presence of various organic solvents and their respiration activity was followed. The introduction of organic solvents can have advantageous effects, like the selective removal of the product from the cells or the aqueous phase, thereby combining production and downstream processing, while the removal of the product can relieve product inhibition effects. There appeared to be a relation between the hydrophobicity of the solvent and the residual activity of the cells - the more hydrophobic the solvent was, the more active the cells were in its presence. The immobilization of the cells provided a slight protection against harmful solvents. Although these Tagetes minuta cells were easy to work with (a fast growing, fine cell suspension), we changed to hairy root cultures of Tagetes patula , transformed with Agrobacterium rhizogenes LBA 9402, because those cells did produce thiophenes, in contrast to the former. These hairy root cultures are more difficult to work with, because they grow as one clump of interconnected roots, so circulation is difficult and samples cannot be taken. Therefore, in chapter four methods are compared that determine the hairy root biomass present in a fermenter in an indirect way. From the three methods extensively tested, i.e. volume/weight balance, oxygen consumption and conductivity measurement, the latter proved to be the simplest and easiest to handle when the roots were in good condition.In chapter five, the sensitivity of the hairy roots to organic solvents is described. The results were comparable to those obtained with the Tagetes minuta cell suspensions. An interesting phenomenon was observed with respect to the excretion of thiophenes. In one-phase cultures with only aqueous medium, the excretion is always practically zero. In the presence of some of the solvents tested, e.g. hexadecane, part of the thiophenes produced were excreted into the solvent. When experiments were performed with these two-phase cultures in bioreactors with volumes up to 6 litres, the excretion could rise to about 70% of the total thiophenes produced. Obviously, this is an important result, because it can facilitate the downstream processing of the thiophenes to a great extent. Of the two types of bioreactors tested, the stirred tank reactor and the bubble column, the latter gave the best results for this system.In chapter six, similar experiments are described in aqueous two-phase systems. Again, the hairy roots were able to grow and to produce in the aqueous two-phase systems tested, i.e. in shake flasks and bioreactors. In this case, the excretion was stimulated up to 10% of the total thiophene production. In chapter seven, the results are presented of elicitation studies with the Tagetes patula hairy root cultures. The biotic elicitors applied were bacteria, yeast and fungi, while abiotic elicitors were used in the form of extreme pH, Ca 2+addition and the addition of Ethephon, a precursor of ethene. Nearly all elicitor treatments resulted in a higher thiophene production. The highest increase was obtained when the pH was adjusted to 12 (280% of the control production), but the cells were no longer viable after this treatment. The elicitation with extracts of Aspergillus niger led to a production that raised up to 185% of the control, without loss of viability.In chapter eight, the combined effects of elicitor treatment and of the use of a solid second phase, XAD-7, is described. Here, the excretion was affected by the addition of the resin, while growth and total production were not influenced, either in the presence or the absence of elicitor and/or XAD.Finally, in the general discussion, the obtained results are shortly evaluated

The effects of COVID-19 on child mental health:Biannual assessments up to April 2022 in a clinical and two general population samples

BACKGROUND: The COVID-19 pandemic has had an acute impact on child mental and social health, but long-term effects are still unclear. We examined how child mental health has developed since the start of the COVID-19 pandemic up to 2 years into the pandemic (April 2022).METHODS: We included children (age 8-18) from two general population samples (N = 222-1333 per measurement and N = 2401-13,362 for pre-covid data) and one clinical sample receiving psychiatric care (N = 334-748). Behavioral questionnaire data were assessed five times from April 2020 till April 2022 and pre-pandemic data were available for both general population samples. We collected parent-reported data on internalizing and externalizing problems with the Brief Problem Monitor and self-reported data on Anxiety, Depressive symptoms, Sleep-related impairments, Anger, Global health, and Peer relations with the Patient-Reported Outcomes Measurement Information System (PROMIS®).RESULTS: In all samples, parents reported overall increased internalizing problems, but no increases in externalizing problems, in their children. Children from the general population self-reported increased mental health problems from before to during the pandemic on all six PROMIS domains, with generally worst scores in April 2021, and scores improving toward April 2022 but not to pre-pandemic norms. Children from the clinical sample reported increased mental health problems throughout the pandemic, with generally worst scores in April 2021 or April 2022 and no improvement. We found evidence of minor age effects and no sex effects.CONCLUSIONS: Child mental health in the general population has deteriorated during the first phase of the COVID-19 pandemic, has improved since April 2021, but has not yet returned to pre-pandemic levels. Children in psychiatric care show worsening of mental health problems during the pandemic, which has not improved since. Changes in child mental health should be monitored comprehensively to inform health care and policy.</p

ENIGMA and global neuroscience: A decade of large-scale studies of the brain in health and disease across more than 40 countries

This review summarizes the last decade of work by the ENIGMA (Enhancing NeuroImaging Genetics through Meta Analysis) Consortium, a global alliance of over 1400 scientists across 43 countries, studying the human brain in health and disease. Building on large-scale genetic studies that discovered the first robustly replicated genetic loci associated with brain metrics, ENIGMA has diversified into over 50 working groups (WGs), pooling worldwide data and expertise to answer fundamental questions in neuroscience, psychiatry, neurology, and genetics. Most ENIGMA WGs focus on specific psychiatric and neurological conditions, other WGs study normal variation due to sex and gender differences, or development and aging; still other WGs develop methodological pipelines and tools to facilitate harmonized analyses of "big data" (i.e., genetic and epigenetic data, multimodal MRI, and electroencephalography data). These international efforts have yielded the largest neuroimaging studies to date in schizophrenia, bipolar disorder, major depressive disorder, post-traumatic stress disorder, substance use disorders, obsessive-compulsive disorder, attention-deficit/hyperactivity disorder, autism spectrum disorders, epilepsy, and 22q11.2 deletion syndrome. More recent ENIGMA WGs have formed to study anxiety disorders, suicidal thoughts and behavior, sleep and insomnia, eating disorders, irritability, brain injury, antisocial personality and conduct disorder, and dissociative identity disorder. Here, we summarize the first decade of ENIGMA's activities and ongoing projects, and describe the successes and challenges encountered along the way. We highlight the advantages of collaborative large-scale coordinated data analyses for testing reproducibility and robustness of findings, offering the opportunity to identify brain systems involved in clinical syndromes across diverse samples and associated genetic, environmental, demographic, cognitive, and psychosocial factors