Priming of natural scene categorization during continuous flash suppression.

Continuous Flash Suppression (CFS) reduces conscious awareness of stimuli. Whether stimuli suppressed by CFS are processed at categorical or semantic levels is still debated. Here, we approached this question using a large set of indoor and outdoor scene photographs in a priming paradigm. Perceptually suppressed primes were followed by visible targets. Participants rapidly reported whether the targets showed an indoor or an outdoor scene. Responses were faster (and fast responses more accurate) when primes and targets came from a congruent superordinate category (e.g., both were outdoor scenes). During CFS, priming effects were relatively small (up to 10 ms) and modulated by prime visibility and stimulus onset asynchrony (SOA) of prime and target. Without CFS, the stimuli elicited consistent and more robust priming effects (about 24 ms). Our results imply that scene category is processed during CFS, although some residual prime visibility is likely necessary for significant priming effects to occur.

Transcriptomic response of Saccharomyces cerevisiae to octanoic acid production.

The medium-chain fatty acid octanoic acid is an important platform compound widely used in industry. The microbial production from sugars in Saccharomyces cerevisiae is a promising alternative to current non-sustainable production methods, however, titers need to be further increased. To achieve this, it is essential to have in-depth knowledge about the cell physiology during octanoic acid production. To this end, we collected the first RNA-Seq data of an octanoic acid producer strain at three time points during fermentation. The strain produced higher levels of octanoic acid and increased levels of fatty acids of other chain lengths (C6-C18) but showed decreased growth compared to the reference. Furthermore, we show that the here analyzed transcriptomic response to internally produced octanoic acid is notably distinct from a wild type's response to externally supplied octanoic acid as reported in previous publications. By comparing the transcriptomic response of different sampling times, we identified several genes that we subsequently overexpressed and knocked out, respectively. Hereby we identified RPL40B, to date unknown to play a role in fatty acid biosynthesis or medium-chain fatty acid tolerance. Overexpression of RPL40B led to an increase in octanoic acid titers by 40%.

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations.

The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.

Treatment of hypertension and obstructive sleep apnea counteracts cognitive decline in common neurocognitive disorders in diagnosis-related patterns.

The aim of this study was to investigate the effect of arterial hypertension (AH) and of obstructive sleep apnea (OSA) on cognitive course in the neurocognitive disorder (NCD) cohort RIFADE which enrolled patients with NCD due to Alzheimer's disease (AD), vascular NCD (vNCD), and mixed NCD (AD + vNCD = mNCD). Multiple risk factors (RF), including AH and OSA, that contribute to the development of various kinds of dementia have been identified in previous studies. Studies that observed AH lacked investigation of long-term effects and did not isolate it from other RF. Studies involving OSA as a risk factor did not include participants with all stages of NCD. 126 subjects were screened for AH and OSA. Repeated cognitive measurements were performed with the DemTect as primary outcome and the clock drawing test as secondary outcome measure. 90 patients had AH (71.4%) and 40 patients had OSA (31.7%). RF-status had a significant effect on cognitive outcome in models with RF as single factors (AH p = 0.027, OSA p < 0.001), a 2-factor analysis with AH × OSA (AH as main factor p = 0.027) as well as a model including the 3 factors AH × OSA × diagnosis (p = 0.038). Similarly, a 3-factor model was significant for the clock-drawing test, whereas single factor-models remained insignificant. AH and OSA appear to be risk factors in common NCD and cognitive decline can be mitigated by treatment of these RF.

High-Throughput Screening of an Octanoic Acid Producer Strain Library Enables Detection of New Targets for Increasing Titers in Saccharomyces cerevisiae.

Octanoic acid is an industrially relevant compound with applications in antimicrobials or as a precursor for biofuels. Microbial biosynthesis through yeast is a promising alternative to current unsustainable production methods. To increase octanoic acid titers in Saccharomyces cerevisiae, we use a previously developed biosensor that is based on the octanoic acid responsive pPDR12 promotor coupled to GFP. We establish a biosensor strain amenable for high-throughput screening of an octanoic acid producer strain library. Through development, optimization, and execution of a high-throughput screening approach, we were able to detect two new genetic targets, KCS1 and FSH2, which increased octanoic acid titers through combined overexpression by about 55% compared to the parental strain. Neither target has yet been reported to be involved in fatty acid biosynthesis. The presented methodology can be employed to screen any genetic library and thereby more genes involved in improving octanoic acid production can be detected in the future.

A Yeast-Based Biosensor for Screening of Short- and Medium-Chain Fatty Acid Production.

Short- and medium-chain fatty acids (SMCFA) are important platform chemicals currently produced from nonsustainable resources. The engineering of microbial cells to produce SMCFA, however, lacks high-throughput methods to screen for best performing cells. Here, we present the development of a whole-cell biosensor for easy and rapid detection of SMCFA. The biosensor is based on a multicopy yeast plasmid containing the SMCFA-responsive PDR12 promoter coupled to GFP as the reporter gene. The sensor detected hexanoic, heptanoic and octanoic acid over a linear range up to 2, 1.5, and 0.75 mM, respectively, but did not show a linear response to decanoic and dodecanoic acid. We validated the functionality of the biosensor with culture supernatants of a previously engineered Saccharomyces cerevisiae octanoic acid producer strain and derivatives thereof. The biosensor signal correlated strongly with the octanoic acid concentrations as determined by gas chromatography. Thus, this biosensor enables the high-throughput screening of SMCFA producers and has the potential to drastically speed up the engineering of diverse SMCFA producing cell factories.

Elucidation of the Two H3K36me3 Histone Methyltransferases Set2 and Ash1 in Fusarium fujikuroi Unravels Their Different Chromosomal Targets and a Major Impact of Ash1 on Genome Stability.

In this work, we present a comprehensive analysis of the H3K36 histone methyltransferases Set2 and Ash1 in the filamentous ascomycete Fusarium fujikuroi In Saccharomyces cerevisiae, one single methyltransferase, Set2, confers all H3K36 methylation, while there are two members of the Set2 family in filamentous fungi, and even more H3K36 methyltransferases in higher eukaryotes. Whereas the yeast Set2 homolog has been analyzed in fungi previously, the second member of the Set2 family, designated Ash1, has not been described for any filamentous fungus. Western blot and ChIP-Seq analyses confirmed that F. fujikuroi Set2 and Ash1 are H3K36-specific histone methyltransferases that deposit H3K36me3 at specific loci: Set2 is most likely responsible for H3K36 methylation of euchromatic regions of the genome, while Ash1 methylates H3K36 at the subtelomeric regions (facultative heterochromatin) of all chromosomes, including the accessory chromosome XII. Our data indicate that H3K36me3 cannot be considered a hallmark of euchromatin in F. fujikuroi, and likely also other filamentous fungi, making them different to what is known about nuclear characteristics in yeast and higher eukaryotes. We suggest that the H3K36 methylation mark exerts specific functions when deposited at euchromatic or subtelomeric regions by Set2 or Ash1, respectively. We found an enhanced level of H3K27me3, an increased instability of subtelomeric regions and losses of the accessory chromosome XII over time in Δash1 mutants, indicating an involvement of Ash1 in DNA repair processes. Further phenotypic analyses revealed a role of H3K36 methylation in vegetative growth, sporulation, secondary metabolite biosynthesis, and virulence in F. fujikuroi.

Establishment of the Inducible Tet-On System for the Activation of the Silent Trichosetin Gene Cluster in Fusarium fujikuroi.

The PKS-NRPS-derived tetramic acid equisetin and its N-desmethyl derivative trichosetin exhibit remarkable biological activities against a variety of organisms, including plants and bacteria, e.g., Staphylococcus aureus. The equisetin biosynthetic gene cluster was first described in Fusarium heterosporum, a species distantly related to the notorious rice pathogen Fusarium fujikuroi. Here we present the activation and characterization of a homologous, but silent, gene cluster in F. fujikuroi. Bioinformatic analysis revealed that this cluster does not contain the equisetin N-methyltransferase gene eqxD and consequently, trichosetin was isolated as final product. The adaption of the inducible, tetracycline-dependent Tet-on promoter system from Aspergillus niger achieved a controlled overproduction of this toxic metabolite and a functional characterization of each cluster gene in F. fujikuroi. Overexpression of one of the two cluster-specific transcription factor (TF) genes, TF22, led to an activation of the three biosynthetic cluster genes, including the PKS-NRPS key gene. In contrast, overexpression of TF23, encoding a second Zn(II)2Cys6 TF, did not activate adjacent cluster genes. Instead, TF23 was induced by the final product trichosetin and was required for expression of the transporter-encoding gene MFS-T. TF23 and MFS-T likely act in consort and contribute to detoxification of trichosetin and therefore, self-protection of the producing fungus.