An important step forward for the future development of an easy and fast procedure for identifying the most dangerous wine spoilage yeast, Dekkera bruxellensis, in wine environment

Dekkera bruxellensis is the main reason for spoilage in the wine industry. It renders the products unacceptable leading to large economic losses. Fluorescence In Situ Hybridisation (FISH) technique has the potential for allowing its specific detection. Nevertheless, some experimental difficulties can be encountered when FISH technique is applied in the wine environment (e.g. matrix and cells autofluorescence, fluorophore inadequate selection and probes low specificity to the target organisms). An easy and fast in-suspension RNA-FISH procedure was applied for the first time for identifying D. bruxellensis in wine. A previously designed RNA-FISH probe to detect D. bruxellensis (26S D. brux.5.1) was used and the matrix and cells fluorescence interferences, the influence of three fluorophores in FISH performance and the probe specificity were evaluated. The results revealed that to apply RNA-FISH technique in the wine environment a red-emitting fluorophore should be used. Good probe performance and specificity was achieved with 25% of formamide. The resulting RNA-FISH protocol was applied in wine samples artificially inoculated with D. bruxellensis. This spoilage microorganism was detected in wine at cell densities lower than those associated with phenolic off-flavours. Thus, the RNA-FISH procedure described in this work represents an advancement to facilitate early detection of the most dangerous wine spoilage yeast and, consequently, to reduce the economic losses caused by this yeast to the wine industry.This work was co-financed by Foundation for Science and Technology (FCT) and the European Union through the European Regional Development Fund ALENTEJO 2020 through the projects PTDC/BBB-IMG/0046/2014 and ALT20-03-0145-FEDER-000015, respectively. Marina González-Pérez acknowledges FCT for the economic support through the post-doctoral grant SFRH/BPD/100754/2014

Use of superheated liquids for the extraction of non-volatile compounds from wood: HPLC studies

A study of the extraction of oak wood compounds using superheated water-ethanol mixtures ranging from 10 to 60% ethanol is reported. Identification and characterization of the extracted compounds have been made by high performance liquid chromatography. The extraction has been performed using the static mode by single or repetitive cycles. The variables affecting the extraction process have been studied and their optimum values established (extraction time: 50 min; pressure: 40 atm; extraction temperature: 180º C). The study allows to compare the non-volatile polyphenol fractions obtained in this way with those present in commercial samples with fully agreement between them. In addition, the method allows manipulation of the extract composition by changing the working pressure, temperature and water-ethanol ratio

Distinct roles of Hoxa2 and Krox20 in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening

<p>Abstract</p> <p>Background</p> <p>Little is known about the involvement of molecular determinants of segmental patterning of rhombomeres (r) in the development of rhythmic neural networks in the mouse hindbrain. Here, we compare the phenotypes of mice carrying targeted inactivations of <it>Hoxa2</it>, the only <it>Hox </it>gene expressed up to r2, and of <it>Krox20</it>, expressed in r3 and r5. We investigated the impact of such mutations on the neural circuits controlling jaw opening and breathing in newborn mice, compatible with Hoxa2-dependent trigeminal defects and direct regulation of <it>Hoxa2 </it>by Krox20 in r3.</p> <p>Results</p> <p>We found that <it>Hoxa2 </it>mutants displayed an impaired oro-buccal reflex, similarly to <it>Krox20 </it>mutants. In contrast, while <it>Krox20 </it>is required for the development of the rhythm-promoting parafacial respiratory group (pFRG) modulating respiratory frequency,<it> Hoxa2 </it>inactivation did not affect neonatal breathing frequency. Instead, we found that <it>Hoxa2</it>^-/-but not <it>Krox20</it>^-/-mutation leads to the elimination of a transient control of the inspiratory amplitude normally occurring during the first hours following birth. Tracing of r2-specific progenies of <it>Hoxa2 </it>expressing cells indicated that the control of inspiratory activity resides in rostral pontine areas and required an intact r2-derived territory.</p> <p>Conclusion</p> <p>Thus, inspiratory shaping and respiratory frequency are under the control of distinct <it>Hox</it>-dependent segmental cues in the mammalian brain. Moreover, these data point to the importance of rhombomere-specific genetic control in the development of modular neural networks in the mammalian hindbrain.</p

Transcriptomics reveal an integrative role for maternal thyroid hormones during zebrafish embryogenesis

Thyroid hormones (THs) are essential for embryonic brain development but the genetic mechanisms involved in the action of maternal THs (MTHs) are still largely unknown. As the basis for understanding the underlying genetic mechanisms of MTHs regulation we used an established zebrafish monocarboxylic acid transporter 8 (MCT8) knock-down model and characterised the transcriptome in 25hpf zebrafish embryos. Subsequent mapping of differentially expressed genes using Reactome pathway analysis together with in situ expression analysis and immunohistochemistry revealed the genetic networks and cells under MTHs regulation during zebrafish embryogenesis. We found 4,343 differentially expressed genes and the Reactome pathway analysis revealed that TH is involved in 1681 of these pathways. MTHs regulated the expression of core developmental pathways, such as NOTCH and WNT in a cell specific context. The cellular distribution of neural MTH-target genes demonstrated their cell specific action on neural stem cells and differentiated neuron classes. Taken together our data show that MTHs have a role in zebrafish neurogenesis and suggest they may be involved in cross talk between key pathways in neural development. Given that the observed MCT8 zebrafish knockdown phenotype resembles the symptoms in human patients with Allan-Herndon-Dudley syndrome our data open a window into understanding the genetics of this human congenital condition.Portuguese Fundacao para Ciencia e Tecnologia (FCT) [PTDC/EXPL/MARBIO/0430/2013]; CCMAR FCT Plurianual financing [UID/Multi/04326/2013]; FCT [SFRH/BD/111226/2015, SFRH/BD/108842/2015, SFRH/BPD/89889/2012]; FCT-IF Starting Grant [IF/01274/2014]info:eu-repo/semantics/publishedVersio

Yeasts and wine off-flavours: a technological perspective

Review article. Part of the special issue "Wine microbiology and safety: from the vineyard to the bottle (Microsafety Wine)", 19-20 Nov. 2009, ItalyIn wine production, yeasts have both beneficial and detrimental activities. Saccharomyces cerevisiae is the yeast mainly responsible for turning grape juice into wine but this species and several others may also show undesirable effects in wines. Among such effects, technologists are particularly concerned with the production of offflavours that may occur during all stages of winemaking. Typical spoiling activities include the production of ethyl acetate by apiculate yeasts before fermentation, hydrogen sulphide by S. cerevisiae during fermentation phases, acetaldehyde by film-forming yeasts during bulk storage, and volatile phenols by Dekkera bruxellensis during storage or after bottling. The occurrence of these hazards depends on the technological operations designed to obtain a given type of wine and most can be avoided by current preventive or curative measures. On the contrary, good manufacturing practices must be strengthened to deal with the problem of volatile phenol production in red wines. Appropriate monitoring of D. bruxellensis populations and quantification of 4-ethylphenol is advised during storage, particularly when oak barrels are used, and absence of viable cells must be guaranteed in bottled wines. This work, which is based on our experience at winery level, aims to provide information on appropriate technological strategies to deal with the problem of off-flavours produced by yeasts

Genome-Wide Search Reveals the Existence of a Limited Number of Thyroid Hormone Receptor Alpha Target Genes in Cerebellar Neurons

Thyroid hormone (T3) has a major influence on cerebellum post-natal development. The major phenotypic landmark of exposure to low levels of T3 during development (hypothyroidism) in the cerebellum is the retarded inward migration of the most numerous cell type, granular neurons. In order to identify the direct genetic regulation exerted by T3 on cerebellar neurons and their precursors, we used microarray RNA hybridization to perform a time course analysis of T3 induced gene expression in primary cultures of cerebellar neuronal cell. These experiments suggest that we identified a small set of genes which are directly regulated, both in vivo and in vitro, during cerebellum post-natal development. These modest changes suggest that T3 does not acts directly on granular neurons and mainly indirectly influences the cellular interactions taking place during development

Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts

Volatile phenols are aromatic compounds and one of the key molecules responsible for olfactory defects in wine. The yeast genus Brettanomyces is the only major microorganism that has the ability to covert hydroxycinnamic acids into important levels of these compounds, especially 4-ethylphenol and 4-ethylguaiacol, in red wine. When 4-ethylphenols reach concentrations greater than the sensory threshold, all wine’s organoleptic characteristics might be influenced or damaged. The aim of this literature review is to provide a better understanding of the physicochemical, biochemical, and metabolic factors that are related to the levels of p-coumaric acid and volatile phenols in wine. Then, this work summarizes the different methods used for controlling the presence of Brettanomyces in wine and the production of ethylphenols

De-Novo Assembly and Analysis of the Heterozygous Triploid Genome of the Wine Spoilage Yeast Dekkera bruxellensis AWRI1499

Despite its industrial importance, the yeast species Dekkera (Brettanomyces) bruxellensis has remained poorly understood at the genetic level. In this study we describe whole genome sequencing and analysis for a prevalent wine spoilage strain, AWRI1499. The 12.7 Mb assembly, consisting of 324 contigs in 99 scaffolds (super-contigs) at 26-fold coverage, exhibits a relatively high density of single nucleotide polymorphisms (SNPs). Haplotype sampling for 1.2% of open reading frames suggested that the D. bruxellensis AWRI1499 genome is comprised of a moderately heterozygous diploid genome, in combination with a divergent haploid genome. Gene content analysis revealed enrichment in membrane proteins, particularly transporters, along with oxidoreductase enzymes. Availability of this assembly and annotation provides a resource for further investigation of genomic organization in this species, and functional characterization of genes that may confer important phenotypic traits

Local hyperthyroidism promotes pancreatic acinar cell proliferation during acute pancreatitis

Proliferation of pancreatic acinar cells is a critical process in the pathophysiology of pancreatic diseases, because limited or defective proliferation is associated with organ dysfunction and patient morbidity. In this context, elucidating the signalling pathways that trigger and sustain acinar proliferation is pivotal to develop therapeutic interventions promoting the regenerative process of the organ.In this study we used genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones to elucidate their role in acinar proliferation following caerulein‐mediated acute pancreatitis in mice. In addition, molecular mechanisms mediating the effects of thyroid hormones were identified by genetic and pharmacological inactivation of selected signalling pathways.In this study we demonstrated that levels of the thyroid hormone 3,3’,5‐triodo‐L‐thyronine (T3) transiently increased in the pancreas during acute pancreatitis. Moreover, by using genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones, we showed that T3 was required to promote proliferation of pancreatic acinar cells, without affecting the extent of tissue damage or inflammatory infiltration.Finally, upon genetic and pharmacological inactivation of selected signalling pathways, we demonstrated that T3 exerted its mitogenic effect on acinar cells via a tightly controlled action on different molecular effectors, including histone deacetylase, AKT, and TGFβ signalling.In conclusion, our data suggest that local availability of T3 in the pancreas is required to promote acinar cell proliferation and provide the rationale to exploit thyroid hormone signalling to enhance pancreatic regeneration