"It's about how you take in things with your brain" - young people's perspectives on mental health and help seeking: an interview study.

INTRODUCTION: Poor mental health in young people has become a growing problem globally over the past decades. However, young people have also been shown to underutilize available healthcare resources. The World Health Organisation (WHO) has formulated guidelines for youth-friendly health services (YFHSs) to increase youth participation in healthcare. Still, little is known about how young people using these services perceive mental health, indicating a knowledge gap concerning the subjective evaluation of their mental health. AIM: To investigate how young people visiting youth health clinics (YHC) perceive the concept of mental health and factors they view as central to maintaining mental health. METHODS: In total 21 interviews were carried out, 16 in 2018, and 5 in 2023 to assure no changes in findings after the COVID-19 pandemic. Subjects were recruited during visits to youth health clinics (YHCs) in mid-Sweden and were aged 15-23 years. Recruitment strived to achieve heterogeneity in the sample concerning gender, sexual orientation, gender identity and age. Interviews were transcribed and analysed using qualitative content analysis. FINDINGS: Findings of the analysis revealed two themes, "Mental health is helped and hindered by the surroundings" and "Mental health is difficult to understand and difficult to achieve". The participants described their health as highly dependent on their social surroundings, and that these are important to maintaining health but may also affect health negatively. They described mixed experiences of the health care services and mentioned prerequisites for seeking care for mental health problems such as accessibility and respect for their integrity, including the right to turn down offered treatment. The informants also viewed mental health as an ongoing undertaking that one must work for, and that it is sometimes difficult to know what constitutes mental health. They also expressed a need from healthcare services to enquire about their health, and to show an active interest in how they are doing. CONCLUSIONS: Findings underline the need of young people's individual needs to be met in the healthcare system and their vulnerability to their social surroundings. Health status assessments in young people should consider social and individual factors to fully capture mental health.

Development and usability evaluation of an electronic health report form to assess health in young people: a mixed-methods approach.

BACKGROUND: Electronic Patient-Reported Outcomes (ePROs) have potential to improve health outcomes and healthcare. The development of health-technology applications, such as ePROs, should include the potential users and be theoretically grounded. Swedish Youth Health Clinics (YHCs) offer primarily sexual and psychological healthcare for young people aged 12 to 25 years old. Young people in healthcare settings are considered a vulnerable group. The development of a collection of Patient-Reported Outcomes (PROs) in an Electronic Health Report Form (eHRF) for identifying health and health-related problems in young people, was preceded by a qualitative interview study, exploring young people's views on using an eHRF at YHCs and which questions about health an eHRF should contain. The aim of the current study was to develop and evaluate the usability of an eHRF prototype for identifying health and health-related problems in young people visiting YHCs. METHODS: This study used a participatory design. During the development, an expert panel consisting of eight researchers and one Information Technology worker, participated. A wide literature search was performed to find PROs to construct an eHRF prototype to cover health areas. A mixed methods usability evaluation included 14 participants (young people, healthcare professionals, and an expert panel). RESULTS: The development resulted in an eHRF prototype, containing ten reliable and valid health questionnaires addressing mental-, physical-, and sexual health and social support, a self-efficacy question, and background questions, in total 74 items. The interviews in the usability evaluation resulted in three categories describing the usability of the eHRF: 'Captures the overall health of young people but needs clarification', 'Fun, easy, and optional and will keep young people's interest', and 'Potential contribution to improve the health consultation'. The quantitative results support the usability of the eHRF for YHCs. CONCLUSIONS: The participatory approach contributed to development of the eHRF prototype to cover health areas adapted for the target population. The usability evaluation showed that the eHRF was usable and had the potential for self-reflection and contributions to cooperation between young people and healthcare professionals during the health consultation.

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species.

The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus, and A. steynii) have been whole-genome PacBio sequenced to provide genetic references in three Aspergillus sections. A. taichungensis and A. candidus also were sequenced for SM elucidation. Thirteen Aspergillus genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, A. novofumigatus was compared with the pathogenic species A. fumigatus This suggests that A. novofumigatus can produce most of the same allergens, virulence, and pathogenicity factors as A. fumigatus, suggesting that A. novofumigatus could be as pathogenic as A. fumigatus Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris, and A. steynii, respectively, and novofumigatonin, ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus Our study delivers six fungal genomes, showing the large diversity found in the Aspergillus genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of A. novofumigatus pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.

Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells.

Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high-level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.

Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis.

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions to further elucidate the role of the GSH synthesis pathway. Two strategies were used: the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Over time of the bioprocess, a correlation between intracellular GSH and product titer has been observed. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO-treated cells and nontreated cells has highlighted a slowdown of the tricarboxylic acid cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the GSH-related proteome has been observed with an upregulation of the regulatory subunit of glutamate-cysteine ligase and a downregulation of a specific GSH transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global downregulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it could be the missing piece of the puzzle to finally elucidate the link between GSH synthesis and productivity.

Influence of Infant Cleft Dimensions on Velopharyngeal Function in 5-Year-Old Danish Children Born With Unilateral Cleft Lip and Palate.

AIM: To examine the association of cleft severity at infancy and velopharyngeal competence in preschool children with unilateral cleft lip and palate operated with early or delayed hard palate repair. DESIGN: Subgroup analysis within a multicenter randomized controlled trial of primary surgery (Scandcleft). SETTING: Tertiary health care. One surgical center. PATIENTS AND METHODS: One hundred twenty-five infants received cheilo-rhinoplasty and soft palate repair at age 3 to 4 months and were randomized to hard palate closure at age 12 or 36 months. Cleft size and cleft morphology were measured 3 dimensionally on digital models, obtained by laser surface scanning of preoperative plaster models (mean age: 1.8 months). MAIN OUTCOME MEASUREMENTS: Velopharyngeal competence (VPC) and hypernasality assessed from a naming test (VPC-Sum) and connected speech (VPC-Rate). In both scales, higher scores indicated a more severe velopharyngeal insufficiency. RESULTS: No difference between surgical groups was shown. A low positive correlation was found between posterior cleft width and VPC-Rate (Spearman = .23; P = .025). The role of the covariate "cleft size at tuberosity level" was confirmed in an ordinal logistic regression model (odds ratio [OR] = 1.17; 95% confidence interval [CI]:1.01-1.35). A low negative correlation was shown between anteroposterior palatal length and VPC-Sum (Spearman = -.27; P = .004) and confirmed by the pooled scores VPC-Pooled (OR = 0.82; 95% CI: 0.69-0.98) and VPC-Dichotomic (OR = 0.82; 95% CI: 0.68-0.99). CONCLUSIONS: Posterior cleft dimensions can be a modest indicator for the prognosis of velopharyngeal function at age 5 years, when the soft palate is closed first, independently on the timing of hard palate repair. Antero-posterior palatal length seems to protect from velopharyngeal insufficiency and hypernasality. However, the association found was significant but low.

Genus level analysis of PKS-NRPS and NRPS-PKS hybrids reveals their origin in Aspergilli.

BACKGROUND: Filamentous fungi produce a vast amount of bioactive secondary metabolites (SMs) synthesized by e.g. hybrid polyketide synthase-nonribosomal peptide synthetase enzymes (PKS-NRPS; NRPS-PKS). While their domain structure suggests a common ancestor with other SM proteins, their evolutionary origin and dynamics in fungi are still unclear. Recent rational engineering approaches highlighted the possibility to reassemble hybrids into chimeras - suggesting molecular recombination as diversifying mechanism. RESULTS: Phylogenetic analysis of hybrids in 37 species - spanning 9 sections of Aspergillus and Penicillium chrysogenum - let us describe their dynamics throughout the genus Aspergillus. The tree topology indicates that three groups of PKS-NRPS as well as one group of NRPS-PKS hybrids developed independently from each other. Comparison to other SM genes lead to the conclusion that hybrids in Aspergilli have several PKS ancestors; in contrast, hybrids are monophyletic when compared to available NRPS genes - with the exception of a small group of NRPSs. Our analysis also revealed that certain NRPS-likes are derived from NRPSs, suggesting that the NRPS/NRPS-like relationship is dynamic and proteins can diverge from one function to another. An extended phylogenetic analysis including bacterial and fungal taxa revealed multiple ancestors of hybrids. Homologous hybrids are present in all sections which suggests frequent horizontal gene transfer between genera and a finite number of hybrids in fungi. CONCLUSION: Phylogenetic distances between hybrids provide us with evidence for their evolution: Large inter-group distances indicate multiple independent events leading to the generation of hybrids, while short intra-group distances of hybrids from different taxonomic sections indicate frequent horizontal gene transfer. Our results are further supported by adding bacterial and fungal genera. Presence of related hybrid genes in all Ascomycetes suggests a frequent horizontal gene transfer between genera and a finite diversity of hybrids - also explaining their scarcity. The provided insights into relations of hybrids and other SM genes will serve in rational design of new hybrid enzymes.

Strategies to establish the link between biosynthetic gene clusters and secondary metabolites.

Filamentous fungi produce a vast number of bioactive secondary metabolites (SMs), some of which have found applications in the pharmaceutical industry including as antibiotics and immunosuppressants. As more and more species are whole genome sequenced the number of predicted clusters of genes for SM biosynthesis is ever increasing - holding a promise of novel useful bioactive SMs. To be able to fully utilize the potential of novel SMs, it is necessary to link the SM and the genes responsible for producing it. This can be challenging, but many strategies and tools have been developed for this purpose. Here we provide an overview of the methods used to establish the link between SM and biosynthetic gene cluster (BGC) and vice versa, along with the challenges and advantages of each of the methods. Part I of the review, associating BCG with SM, is divided into gene manipulations native strain and heterologous expression strategies, depending on the fungal species. Part II, associating SM with BGC, is divided into three main approaches: (1) homology search (2) retro-biosynthesis and (3) comparative genomics.

Application of a curated genome-scale metabolic model of CHO DG44 to an industrial fed-batch process.

CHO cells have become the favorite expression system for large scale production of complex biopharmaceuticals. However, industrial strategies for upstream process development are based on empirical results, due to a lack of fundamental understanding of intracellular activities. Genome scale models of CHO cells have been reconstructed to provide an economical way of analyzing and interpreting large-omics datasets, since they add cellular context to the data. Here the most recently available CHO-DG44 genome-scale specific model was manually curated and tailored to the metabolic profile of cell lines used for industrial protein production, by modifying 601 reactions. Generic changes were applied to simplify the model and cope with missing constraints related to regulatory effects as well as thermodynamic and osmotic forces. Cell line specific changes were related to the metabolism of high-yielding production cell lines. The model was semi-constrained with 24 metabolites measured on a daily basis in n = 4 independent industrial 2L fed batch cell culture processes for a therapeutic antibody production. This study is the first adaptation of a genome scale model for CHO cells to an industrial process, that successfully predicted cell phenotype. The tailored model predicted accurately both the exometabolomics data (r2 ≥ 0.8 for 96% of the considered metabolites) and growth rate (r2 = 0.91) of the industrial cell line. Flux distributions at different days of the process were analyzed for validation and suggestion of strategies for medium optimization. This study shows how to adapt a genome scale model to an industrial process and sheds light on the metabolic specificities of a high production process. The curated genome scale model is a great tool to gain insights into intracellular fluxes and to identify possible bottlenecks impacting cell performances during production process. The general use of genome scale models for modeling industrial recombinant cell lines is a long-term investment that will highly benefit process development and speed up time to market.

Reprogramming AA catabolism in CHO cells with CRISPR/Cas9 genome editing improves cell growth and reduces byproduct secretion.

Chinese hamster ovary (CHO) cells are the preferred host for producing biopharmaceuticals. Amino acids are biologically important precursors for CHO metabolism; they serve as building blocks for proteogenesis, including synthesis of biomass and recombinant proteins, and are utilized for growth and cellular maintenance. In this work, we studied the physiological impact of disrupting a range of amino acid catabolic pathways in CHO cells. We aimed to reduce secretion of growth inhibiting metabolic by-products derived from amino acid catabolism including lactate and ammonium. To achieve this, we engineered nine genes in seven different amino acid catabolic pathways using the CRISPR-Cas9 genome editing system. For identification of target genes, we used a metabolic network reconstruction of amino acid catabolism to follow transcriptional changes in response to antibody production, which revealed candidate genes for disruption. We found that disruption of single amino acid catabolic genes reduced specific lactate and ammonium secretion while specific growth rate and integral of viable cell density were increased in many cases. Of particular interest were Hpd and Gad2 disruptions, which show unchanged AA uptake rates, while having growth rates increased up to 19%, and integral of viable cell density as much as 50% higher, and up to 26% decrease in specific ammonium production and to a lesser extent (up to 22%) decrease in lactate production. This study demonstrates the broad potential of engineering amino acid catabolism in CHO cells to achieve improved phenotypes for bioprocessing.

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.

Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms.

The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host- or biopharmaceutical-specific product quality obstacles. In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools. We focus on genes for knockout/knockdown and overexpression for meaningful approaches to improve biopharmaceutical PTMs and discuss their applicability as well as future trends in the field.

Scandcleft Project, Trial 1: Comparison of Speech Outcome in Relation to Timing of Hard Palate Closure in 5-Year-Olds With UCLP.

OBJECTIVE: To compare in-depth speech results in Scandcleft Trial 1 as well as reference data from peers without cleft palate (CP). DESIGN: A prospective randomized clinical trial. SETTING: A Danish and a Swedish CP center. PARTICIPANTS: 143 of 148 randomized 5-year-olds with unilateral cleft lip and palate. All received lip and velum closure at 4 months, and hard palate closure at 12 months (arm A) or 36 months (arm B). MAIN OUTCOME MEASURES: A composite measure based on velopharyngeal competence (VPC) or velopharyngeal incompetence (VPI), an overall assessment of VPC from connected speech (VPC-Rate), Percentage of Consonants Correct (PCC-score), and consonant errors. Speech therapy visits, average hearing thresholds, and secondary pharyngeal surgeries documented burden of treatment. RESULTS: Across the trial, 61.5% demonstrated VPC and 38.5% VPI. Twenty-two percent of participants achieved age appropriate PCC-scores. There were no statistically significant differences between arms or centers for these measures. In the Danish center, arm B: achieved lower PCC-scores (P = .01); obtained PCC-scores without s-errors below 79% (P = .002); produced ≥3 active oral cleft speech characteristics (P = .004) than arm A. In both centers, arm B attended more speech visits. CONCLUSIONS: At age 5, differences between centers and treatment arms were not statistically significant for VPC/VPI, but consonant proficiency differed between treatment arms in the Danish center. Poor speech outcomes were seen for both treatment arms. Variations between centers were observed. As the Swedish center had few participants, intercenter comparisons should be interpreted with caution.

Influence of timing of delayed hard palate closure on articulation skills in 3-year-old Danish children with unilateral cleft lip and palate.

BACKGROUND: Differing results regarding articulation skills in young children with cleft palate (CP) have been reported and often interpreted as a consequence of different surgical protocols. AIMS: To assess the influence of different timing of hard palate closure in a two-stage procedure on articulation skills in 3-year-olds born with unilateral cleft lip and palate (UCLP). Secondary aims were to compare results with peers without CP, and to investigate if there are gender differences in articulation skills. Furthermore, burden of treatment was to be estimated in terms of secondary surgery, hearing and speech therapy. METHODS & PROCEDURES: A randomized controlled trial (RCT). Early hard palate closure (EHPC) at 12 months versus late hard palate closure (LHPC) at 36 months in a two-stage procedure was tested in a cohort of 126 Danish-speaking children born with non-syndromic UCLP. All participants had the lip and soft palate closed around 4 months of age. Audio and video recordings of a naming test were available from 113 children (32 girls and 81 boys) and were transcribed phonetically. Recordings were obtained prior to hard palate closure in the LHPC group. The main outcome measures were percentage consonants correct adjusted (PCC-A) and consonant errors from blinded assessments. Results from 36 Danish-speaking children without CP obtained previously by Willadsen in 2012 were used for comparison. OUTCOMES & RESULTS: Children with EHPC produced significantly more target consonants correctly (83%) than children with LHPC (48%; p < .001). In addition, children with LHPC produced significantly more active cleft speech characteristics than children with EHPC (p < .001). Boys achieved significantly lower PCC-A scores than girls (p = .04) and produced significantly more consonant errors than girls (p = .02). No significant differences were found between groups regarding burden of treatment. The control group performed significantly better than the EHPC and LHPC groups on all compared variables.

Diagnosis-specific self-image predicts longitudinal suicidal ideation in adult eating disorders.

OBJECTIVE: Eating disorders (ED) are prevalent, serious illnesses with elevated mortality, mainly attributable to suicide. Predictors of suicidality include binge/purge symptomatology, impulsivity, and psychiatric comorbidity, as well as personality factors. Recent research has also shown self-image (the Structural Analysis of Social Behavior, SASB, model) to predict manifest suicide attempts in ED, and the study explored suicide risk prediction to increase knowledge of warning signs and intervention targets. METHOD: Participants were adult ED patients registered in the Stepwise clinical database (N = 1537) with anorexia nervosa (AN), bulimia nervosa (BN), binge-eating disorder (BED), or other specified feeding and eating disorder (OSFED). The SASB self-image questionnaire was used in stepwise regressions to predict 12-month suicidal ideation, both self- and clinician-rated, in models both excluding and including baseline clinical variables. RESULTS: Validation analyses showed fair correspondence between outcome variables as well as with suicide attempts. Different variables predicted suicidality in different diagnoses, over and above baseline clinical variables in all but one regression model. Low Self-protection was important in AN and BN, high Self-control in AN, and high Letting go of the self in BN. For BED, self-blame explained variance, and in OSFED, lack of self-love. DISCUSSION: Findings are in line with research showing differential self-image-based prediction of important outcomes in ED, with noteworthy consistencies across diagnoses and suicidality variables. Strengths included the large sample, and limitations pertained to measures, attrition and Type II error risk. Replication is needed, but findings are consistent with some previous work and offers clinical and research implications.

Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors.

Therapeutic monoclonal antibodies (mAbs) are mainly produced by heterologous expression in Chinese hamster ovary (CHO) cells. The glycosylation profile of the mAbs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production. In this study, the effect on IgG N-glycosylation from feeding CHO cells with eight glycosylation precursors during cultivation was investigated. The study was conducted in fed-batch mode in bioreactors with biological replicates to obtain highly controlled and comparable conditions. We assessed charge heterogeneity and glycosylation patterns of IgG. None of the eight feed additives caused statistically significant changes to cell growth or IgG productivity, compared to controls. However, the addition of 20 mM galactose did result in a reproducible increase of galactosylated IgG from 14% to 25%. On the other hand, addition of 20 mM N-acetyl-D-glucosamine (GlcNAc) reduced relative abundance of galactosylated IgG by 4%. Additionally, supplementation with 10 mM mannose slightly reduced GlcNAc occupancy of IgG. Overall, comparing the effects of IgG glycosylation, by supplementing the cell culture medium with glycosylation precursors during cultivation, revealed an application of these glycosylation precursors for modulating N-glycosylation of IgG.

Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.

Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering.

Accurate prediction of secondary metabolite gene clusters in filamentous fungi.

Biosynthetic pathways of secondary metabolites from fungi are currently subject to an intense effort to elucidate the genetic basis for these compounds due to their large potential within pharmaceutics and synthetic biochemistry. The preferred method is methodical gene deletions to identify supporting enzymes for key synthases one cluster at a time. In this study, we design and apply a DNA expression array for Aspergillus nidulans in combination with legacy data to form a comprehensive gene expression compendium. We apply a guilt-by-association-based analysis to predict the extent of the biosynthetic clusters for the 58 synthases active in our set of experimental conditions. A comparison with legacy data shows the method to be accurate in 13 of 16 known clusters and nearly accurate for the remaining 3 clusters. Furthermore, we apply a data clustering approach, which identifies cross-chemistry between physically separate gene clusters (superclusters), and validate this both with legacy data and experimentally by prediction and verification of a supercluster consisting of the synthase AN1242 and the prenyltransferase AN11080, as well as identification of the product compound nidulanin A. We have used A. nidulans for our method development and validation due to the wealth of available biochemical data, but the method can be applied to any fungus with a sequenced and assembled genome, thus supporting further secondary metabolite pathway elucidation in the fungal kingdom.

Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy.

BACKGROUND: The DHFR negative CHO DXB11 cell line (also known as DUX-B11 and DUKX) was historically the first CHO cell line to be used for large scale production of heterologous proteins and is still used for production of a number of complex proteins. RESULTS: Here we present the genomic sequence of the CHO DXB11 genome sequenced to a depth of 33x. Overall a significant genomic drift was seen favoring GC → AT point mutations in line with the chemical mutagenesis strategy used for generation of the cell line. The sequencing depth for each gene in the genome revealed distinct peaks at sequencing depths of 0x, 16x, 33x and 49x coverage corresponding to a copy number in the genome of 0, 1, 2 and 3 copies. This indicate that 17% of the genes are haploid revealing a large number of genes which can be knocked out with relative ease. This tendency of haploidy was furthermore shown to be present in eight additional analyzed CHO genomes (15-20% haploidy) but not in the genome of the Chinese hamster. The dhfr gene is confirmed to be haploid in CHO DXB11; transcriptionally active and the remaining allele contains a G410C point mutation causing a Thr137Arg missense mutation. We find ~2.5 million single nucleotide polymorphisms (SNP's), 44 gene deletions in the CHO DXB11 genome and 9357 SNP's, which interfere with the coding regions of 3458 genes. Copy number variations for nine CHO genomes were mapped to the chromosomes of the Chinese hamster showing unique signatures for each chromosome. The data indicate that chromosome one and four appear to be more stable over the course of the CHO evolution compared to the other chromosomes thus might presenting the most attractive landing platforms for knock-ins of heterologous genes. CONCLUSIONS: Our studies reveal an unexpected degree of haploidy in CHO DXB11 and CHO cells in general and highlight the chromosomal changes that have occurred among the CHO cell lines sequenced to date.

Combining Stable Isotope Labeling and Molecular Networking for Biosynthetic Pathway Characterization.

Filamentous fungi are a rich source of bioactive compounds, ranging from statins over immunosuppressants to antibiotics. The coupling of genes to metabolites is of large commercial interest for production of the bioactives of the future. To this end, we have investigated the use of stable isotope labeled amino acids (SILAAs). SILAAs were added to the cultivation media of the filamentous fungus Aspergillus nidulans for the study of the cyclic tetrapeptide nidulanin A. Analysis by UHPLC-TOFMS confirmed that the SILAAs were incorporated into produced nidulanin A, and the change in observed m/z could be used to determine whether a compound (known or unknown) incorporated any of the added amino acids. Samples were then analyzed using MS/MS and the data used to perform molecular networking. The molecular network revealed several known and unknown compounds that were also labeled. Assisted by the isotope labeling, it was possible to determine the sequence of several of the compounds, one of which was the known metabolite fungisporin, not previously described in A. nidulans. Several novel analogues of nidulanin A and fungisporin were detected and tentatively identified, and it was determined that these metabolites were all produced by the same nonribosomal peptide synthase. The combination of stable isotope labeling and molecular network generation was shown to very effective for the automated detection of structurally related nonribosomal peptides, while the labeling was effective for determination of the peptide sequence, which could be used to provide information on biosynthesis of bioactive compounds.