Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica

The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can then be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl-CoA was obtained by introducing a point mutation into the α-subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2p^I1220F). The intracellular accumulation of myristic acid increased 8.4-fold. Next, fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6 mg L^-1 Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene's overexpression led to a further 1.5-fold titer increase, reaching 93.9 ± 11.7 mg L^-1 in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1 ± 13.4 mg L^-1 of Z11-14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation.     

Interaction of dietary resveratrol with animal-associated bacteria

The recent decline in the effectiveness of some azole fungicides in controlling the wheat pathogen Mycosphaerella graminicola has been associated with mutations in the CYP51 gene encoding the azole target, the eburicol 14α-demethylase (CYP51), an essential enzyme of the ergosterol biosynthesis pathway. In this study, analysis of the sterol content of M. graminicola isolates carrying different variants of the CYP51 gene has revealed quantitative differences in sterol intermediates, particularly the CYP51 substrate eburicol. Together with CYP51 gene expression studies, these data suggest that mutations in the CYP51 gene impact on the activity of the CYP51 protein.     

Escherichia coli STb toxin binding to sulfatide and its inhibition by carragenan

Escherichia coli heat-STb is an important cause of diarrhea in piglets. STb was shown to interact specifically with sulfatide (3'-sulfogalactosyl-ceramide) present on the surface of epithelial cells of piglet jejunum. Basic data are lacking on STb binding to sulfatide in solution and more precisely on the possible inhibition of this interaction. Using surface plasmon resonance technology, we compare binding of STb to sulfatide and other glycoshingolipids previously shown, with a multiplate-binding assay, to also interact to various degrees with the enterotoxin. In addition, inhibition of STb-sulfatide binding was studied using free galactose, galactose-sulfate residues and a polymer of sulfated galactans known as carragenan. We determined a dissociation constant of 2.4±0.61 nM for the STb-sulfatide interaction. These data indicated that STb was binding to sulfatide with greater affinity than previously determined using radiolabeled toxin. Much lower affinities were observed for lactoceramide and glucoceramide. The binding of STb to sulfatide was clearly inhibited by λ-carragenan but not by galactose, 4-SO₄-galactose or 6-SO₄-galactose. Inhibition of STb binding to its receptor was achieved using λ-carragenan at picomolar concentrations. Then, using IPEC-J2 cells in culture and flow cytometry, we showed that λ-carragenan was able to inhibit the permeabilization process associated with STb.     

Effect of water activity and temperature on Tenuazonic acid production byAlternaria alternata on sunflower seeds

Alternaria alternata is isolated in high frequency in sunflower seeds both in the field and storage. This species produces several toxic metabolites among them alternariol, alternariol monomethyl ether and tenuazonic acid. The accumulation of mycotoxins is regulated by physical, chemical and biological factors and for their production in many commodities nothing is known with regards to these conditions. In sunflower seeds the optimal conditions of temperature and water activity for tenuazonic acid production are unknown. The aim of this work was to investigate the effect of temperature and water activity on tenuazonic acid production byAlternaria alternata ITEM 539 in sunflower seeds. The temperature conditions evaluated were: 20, 25, and 30 °C and the water activities were 0.97, 0.90, 0.87, 0.80. The tenuazonic acid determinations were carried out during the incubation period at intervals of 7 days. Under conditions of constant temperature 25 °C) and variable water activities, 0.90 was the optimal value for tenuazonic acid production. At this water activity of the optimal temperature for tenuazonic acid production was 25 °C.     

Novel interactions of CLN5 support molecular networking between Neuronal Ceroid Lipofuscinosis proteins

Background  

Neuronal ceroid lipofuscinoses (NCLs) comprise at least eight genetically characterized neurodegenerative disorders of childhood. Despite of genetic heterogeneity, the high similarity of clinical symptoms and pathology of different NCL disorders suggest cooperation between different NCL proteins and common mechanisms of pathogenesis. Here, we have studied molecular interactions between NCL proteins, concentrating specifically on the interactions of CLN5, the protein underlying the Finnish variant late infantile form of NCL (vLINCL_Fin).     

NMDA-induced stimulation of glycolysis in developing hippocampal cell cultures

Developmental changes in energy metabolism of primary hippocampal cell cultures from newborn rats were investigated during the first 3 weeks. These changes were measured by intensity of and number of cells exhibiting NAD(P)H fluorescence in response to NMDA-induced activation of neuronal activity. We observed gradual changes of stimulation-evoked NAD(P)H signaling over the first 3 weeks, such that at day 7 and 16, this stimulation is minimal, while at 5 and 12 days, it is maximal. These results describe a biphasic pattern that was similar to earlier findings from experiments investigating developmental changes in population spike amplitudes or glutamate release in young rats. Inhibition of mitochondrial respiration by KCN revealed that the NMDA-evoked stimulation of energy metabolism is mainly due to increased glycolytic activity.     

Commensal and pathogenic biofilms differently modulate peri‐implant oral mucosa in an organotypic model

The impact of oral commensal and pathogenic bacteria on peri‐implant mucosa is not well understood, despite the high prevalence of peri‐implant infections. Hence, we investigated responses of the peri‐implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri‐implant mucosa‐biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri‐implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin‐6 (IL‐6), interleukin‐8 (CXCL8), and monocyte chemoattractant protein‐1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor‐alpha (TNF‐α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri‐implant mucosa‐biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri‐implant disease.     

In vivo sun protection factor and UVA protection factor determination using (hybrid) diffuse reflectance spectroscopy and a multi-lambda-LED light source

The sun protection factor (SPF) values are currently determined using an invasive procedure, in which the volunteers are irradiated with ultraviolet (UV) light. Non-invasive approaches based on hybrid diffuse reflectance spectroscopy (HDRS) have shown a good correlation with conventional SPF testing. Here, we present a novel compact and adjustable DRS test system. The in vivo measurements were performed using a multi-lambda-LED light source and an 84-channel imaging spectrograph with a fiber optic probe for detection. A transmission spectrum was calculated based on the reflectance measured with sunscreen and the reflectance measured without sunscreen. The preexposure in vitro spectrum was fitted to the in vivo spectrum. Each of the 11 test products was investigated on 10 volunteers. The SPF and UVA-PF values obtained by this new approach were compared with in vivo SPF results determined by certified test institutes. A correlation coefficient R² = 0.86 for SPF, and R² = 0.92 for UVA-PF were calculated. Having examined various approaches to apply the HDRS principle, the method we present was found to produce valid and reproducible results, suggesting that the multi-lambda-LED device is suitable for in-vivo SPF testing based on the HDRS principle as well as for in-vivo UVA-PF measurements.