Danio rerio embryos on Prozac – Effects on the detoxification mechanism and embryo development

In the past decade the presence of psychopharmaceuticals, including fluoxetine (FLU), in the aquaticenvironment has been associated with the increasing trend in human consumption of these substances.Aquatic organisms are usually exposed to chronic low doses and, therefore, risk assessments shouldevaluate the effects of these compounds in non-target organisms. Teleost fish possess an array of activedefence mechanisms to cope with the deleterious effects of xenobiotics. These include ABC transporters,phase I and II of cellular detoxification and oxidative stress enzymes. Hence, the present study aimed atcharacterising the effect of FLU on embryo development of the model teleost zebrafish (Danio rerio) con-comitantly with changes in the detoxification mechanisms during early developmental phases. Embryoswere exposed to different concentrations of FLU (0.0015, 0.05, 0.1, 0.5 and 0.8-M) for 80 hours post fer-tilization. Development was screened and the impact in the transcription of key genes, i.e., abcb4, abcc1,abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat, ahr, pxr, ppar˛, pparˇ, ppar-, rxraa, rxrab, rxrbb, rxrga, rxrgb,raraa, rarab, rarga evaluated. In addition, accumulation assays were performed to measure the activity ofABC proteins and antioxidant enzymes (CAT and Cu/ZnSOD) after exposure to FLU. Embryo developmentwas disrupted at the lowest FLU concentration tested (0.0015 -M), which is in the range of concen-trations found in WWTP effluents. Embryos exposed to higher concentrations of FLU decreased Cu/ZnSOD, and increased CAT (0.0015 and 0.5 -M) enzymatic activity. Exposure to higher concentrations ofFLU decreased the expression of most genes belonging to the detoxification system and upregulated catat 0.0015 -M of FLU. Most of the tested concentrations downregulated ppar˛, pparˇ, ppar-, and raraa,rxraa, rxrab, rxrbb rxrgb and ahr gene expression while pxr was significantly up regulated at all testedconcentrations. In conclusion, this study shows that FLU can impact zebrafish embryo development, atconcentrations found in effluents of WWTPs, concomitantly with changes in antioxidant enzymes, andthe transcription of key genes involved in detoxification and development. These finding raises additionalconcerns supporting the need to monitor the presence of this compound in aquatic reservoirs

Streptomyces natalensis programmed cell death and morphological differentiation are dependenton oxidative stress

Streptomyces are aerobic Gram-positive bacteria characterized by a complex life cycle that includes hyphae differentiation and spore formation. Morphological differentiation is triggered by stressful conditions and takes place in a pro-oxidant environment, which sets the basis for an involvement of the oxidative stress response in this cellular process. Characterization of the phenotypic traits of Streptomycesnatalensis ΔkatA1 (mono-functional catalase) and ΔcatR (Fur-like repressor of katA1 expression) strains in solid medium revealed that both mutants had an impaired morphological development process. The sub-lethal oxidative stress caused by the absence of KatA1 resulted in the formation of a highly proliferative and undifferentiated vegetative mycelium, whereas de-repression of CatR regulon, from which KatA1 is the only known representative, resulted in the formation of scarce aerial mycelium. Both mutant strains had the transcription of genes associated with aerial mycelium formation and biosynthesis of the hyphae hydrophobic layer down-regulated. The first round of the programmed cell death (PCD) was inhibited in both strains which caused the prevalence of the transient primary mycelium (MI) over secondary mycelium (MII). Our data shows that the first round of PCD and morphological differentiation in S. natalensis is dependent on oxidative stress in the right amount at the right time.This work was funded by: "NORTE-07-0124-FEDER-000003 - Cell homeostasis tissue organization and organism biology" project co-funded by FEDER funds through the Operational North Region Programme (ON.2 - O Novo Norte) under National Strategic Reference Framework (QREN) and by National funds through FCT - Fundacao para a Ciencia e Tecnologia/MEC - Ministerio da Educacao e Ciencia and when applicable co-funded by FEDER funds within the partnership agreement PT2020 related with the research unit number 4293. TB was supported by a post-doctoral fellowship under the PEst-C/SAU/LA0002/2013 (FCOMP-01-0124-FEDER-037277) project; PO, MVM and SDSP were supported by the FCT fellowships SFRH/BPD/74894/2010, SFRH/BPD/95683/2013 and SFRH/BD/66367/2009, respectively. We thank Rui Fernandes, Hugo Osorio and Catarina Santos for excellent technical assistance in the preparation of samples for confocal microscopy, protein identification and in silico analysis of the S. natalensis genome, respectively

Effect of hyperbaric stress on yeast morphology: Study by automated image analysis

The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO2.CAPES and CNPq (Brazil). Fundação para a Ciência e Tecnologia (Portugal)

Yarrowia lipolytica growth under increased air pressure: influence on enzymes production

Improvement of microbial cell cultures oxygenation can be achieved by the increase of total air pressure, which increases oxygen solubility in the medium. In this work, a pressurized bioreactor was used for Yarrowia lipolytica batch cultivation under increased air pressure from 1 to 6 bar. Cell growth was strongly enhanced by the pressure rise. Fivefold and 3.4-fold increases in the biomass production and in specific growth rate, respectively, were observed under 6 bar. The increase of oxygen availability caused the induction of the antioxidant enzyme superoxide dismutase, which indicates that the defensive mechanisms of the cells against oxidative stress were effective and cells could cope with increased pressure. The pregrowth of Y. lipolytica under increased pressure conditions did not affect the lipase production ability of the cells. Moreover, the extracellular lipase activity increased 96% using a 5-bar air pressure instead of air at 1- bar pressure during the enzyme production phase. Thus, air pressure increase in bioreactors is an effective mean of cell mass and enzyme productivity enhancement in bioprocess based in Y. lipolytica cultures

Disruption of Yarrowia lipolytica TPS1 Gene Encoding Trehalose-6-P Synthase Does Not Affect Growth in Glucose but Impairs Growth at High Temperature

We have cloned the Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomyces cerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3′half of its coding region due to the overlap of its sequence with the promoter of the essential gene YlTFC1. The Yltps1 mutant grew in glucose although the Y. lipolytica hexokinase is extremely sensitive to inhibition by trehalose-6-P. The presence of a glucokinase, insensitive to trehalose-6-P, that constitutes about 80% of the glucose phosphorylating capacity during growth in glucose may account for the growth phenotype. Trehalose content was below 1 nmol/mg dry weight in Y. lipolytica, but it increased in strains expressing YlTPS1 under the control of the YlTEF1promoter or with a disruption of YALI0D15598 encoding a putative trehalase. mRNA levels of YlTPS1 were low and did not respond to thermal stresses, but that of YlTPS2 (YALI0D14476) and YlTPS3 (YALI0E31086) increased 4 and 6 times, repectively, by heat treatment. Disruption of YlTPS1 drastically slowed growth at 35°C. Homozygous Yltps1 diploids showed a decreased sporulation frequency that was ascribed to the low level of YALI0D20966 mRNA an homolog of the S. cerevisiae MCK1 which encodes a protein kinase that activates early meiotic gene expression

Applications of yeast flocculation in biotechnological processes

A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects – the basics of yeast flocculation, the development of “new” flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer’s yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous β-galactosidase production using a recombinant flocculent Saccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculation bioreactors and discussing potential new uses of these systems.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI - BD11306/97

Simvastatin effects on detoxification mechanisms in Danio rerio embryos

The transcription and protein activity of defence mechanisms such as ABC transporters, phase I and II of cellular detoxification and antioxidant enzymes can be altered in the presence of emerging contaminants such as pharmaceuticals impacting the overall detoxification mechanism. The present work aimed to characterise the effects of simvastatin on the detoxification mechanisms of embryonic stages of Danio rerio. In a first approach, constitutive transcription of key genes involved in detoxification was determined. Embryos were collected at different developmental stages, and transcription patterns of genes coding for ABC transporters, phase I and II and oxidative stress were analysed. With exception of abcc2, all genes seem to be from maternal transfer (0–2 hpf). Embryos were then exposed to different concentrations of simvastatin (5 and 50 μg/L), verapamil and MK571 (10 μM; ABC protein inhibitors) and a combination of simvastatin and ABC inhibitors. mRNA expression levels of abcb4, abcc1, abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat was evaluated. Accumulation assays tomeasure ABC proteins activity and activity of EROD, GST, CAT and Cu/ ZnSOD, were also undertaken. Simvastatin acted as a weak inhibitor of ABC proteins and increased EROD and GST activity, whereas Cu/ZnSOD and CAT activity were decreased. Simvastatin up-regulated abcb4 and cyp3a65 transcription (both concentrations), as well as abcc1 and abcc2 at 50 μg/L, and down-regulated gst, sod, cat at 5 μg/L. In conclusion, our data revealed the interaction of simvastatin with detoxification mechanisms highlighting the importance of monitoring the presence of this emerging contaminant in aquatic environments

The relation between N2 fixation and H2 metabolism in the marine filamentous nonheterocystous cyanobacterium Lyngbya aestuarii CCY 9616

The marine filamentous nonheterocystous nitrogen-fixing cyanobacterium Lyngbya aestuarii (F. K. Mert.) Liebman ex Gomont CCY 9616 was grown under diazotrophic and nondiazotrophic conditions and under an alternating 16:8 light:dark (L:D) regime. Nitrogenase activity appeared just before the onset of the dark period, reaching its maximum 1-2 h in the dark, subsequently decreasing to zero at the beginning of the following light period. Nitrogenase activity was only detected at low levels of O2 (5%) and when the culture was grown in the absence of combined nitrogen. Quantitative reverse transcriptase-PCR (RT-PCR) analysis of one of the structural genes encoding nitrogenase, nifK, showed that the highest levels of transcription preceded the maximum activity of nitrogenase by 2-4 h. nifK transcription was not completely abolished during the remaining time of the 24 h cycle. Even in the presence of nitrate, when nitrogenase activity was undetectable, nifK was still transcribed. The H2-uptake activity seemed to follow the nitrogenase, but the transcription of hupL (gene encoding the large subunit of uptake hydrogenase) preceded the nifK transcription. However, H2-uptake and hupL transcription occurred throughout the 24 h cycle as well as under nondiazotrophic conditions, albeit at much lower levels. The hoxH transcript levels (a structural gene coding for the bidirectional hydrogenase) were similar under diazotrophic or nondiazotrophic conditions but slightly higher during the dark period. All three enzymes investigated are involved in H2 metabolism. It is concluded that the uptake hydrogenase is mainly responsible for H2 uptake. Nevertheless, uptake hydrogenase and nitrogenase do not seem to be coregulated