High nitrogen concentration causes G2/M arrest in Hanseniaspora vineae.

Yeasts have been widely used as a model to better understand cell cycle mechanisms and how nutritional and genetic factors can impact cell cycle progression. While nitrogen scarcity is well known to modulate cell cycle progression, the relevance of nitrogen excess for microorganisms has been overlooked. In our previous work, we observed an absence of proper entry into the quiescent state in Hanseniaspora vineae and identified a potential link between this behavior and nitrogen availability. Furthermore, the Hanseniaspora genus has gained attention due to a significant loss of genes associated with DNA repair and cell cycle. Thus, the aim of our study was to investigate the effects of varying nitrogen concentrations on H. vineae's cell cycle progression. Our findings demonstrated that nitrogen excess, regardless of the source, disrupts cell cycle progression and induces G2/M arrest in H. vineae after reaching the stationary phase. Additionally, we observed a viability decline in H. vineae cells in an ammonium-dependent manner, accompanied by increased production of reactive oxygen species, mitochondrial hyperpolarization, intracellular acidification, and DNA fragmentation. Overall, our study highlights the events of the cell cycle arrest in H. vineae induced by nitrogen excess and attempts to elucidate the possible mechanism triggering this absence of proper entry into the quiescent state.

Biology and physiology of Hanseniaspora vineae: metabolic diversity and increase flavour complexity for food fermentation.

Apiculate yeasts belonging to the genus Hanseniaspora are predominant on grapes and other fruits. While some species, such as Hanseniaspora uvarum, are well known for their abundant presence in fruits, they are generally characterized by their detrimental effect on fermentation quality because the excessive production of acetic acid. However, the species Hanseniaspora vineae is adapted to fermentation and currently is considered as an enhancer of positive flavour and sensory complexity in foods. Since 2002, we have been isolating strains from this species and conducting winemaking processes with them. In parallel, we also characterized this species from genes to metabolites. In 2013, we sequenced the genomes of two H. vineae strains, being these the first apiculate yeast genomes determined. In the last 10 years, it has become possible to understand its biology, discovering very peculiar features compared to the conventional Saccharomyces yeasts, such as a natural and unique G2 cell cycle arrest or the elucidation of the mandelate pathway for benzenoids synthesis. All these characteristics contribute to phenotypes with proved interest from the biotechnological point of view for winemaking and the production of other foods.

The association between SARS-CoV-2 infection and preterm delivery: a prospective study with a multivariable analysis.

BACKGROUND: To determine whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the cause of COVID-19 disease) exposure in pregnancy, compared to non-exposure, is associated with infection-related obstetric morbidity. METHODS: We conducted a multicentre prospective study in pregnancy based on a universal antenatal screening program for SARS-CoV-2 infection. Throughout Spain 45 hospitals tested all women at admission on delivery ward using polymerase-chain-reaction (PCR) for COVID-19 since late March 2020. The cohort of positive mothers and the concurrent sample of negative mothers was followed up until 6-weeks post-partum. Multivariable logistic regression analysis, adjusting for known confounding variables, determined the adjusted odds ratio (aOR) with 95% confidence intervals (95% CI) of the association of SARS-CoV-2 infection and obstetric outcomes. MAIN OUTCOME MEASURES: Preterm delivery (primary), premature rupture of membranes and neonatal intensive care unit admissions. RESULTS: Among 1009 screened pregnancies, 246 were SARS-CoV-2 positive. Compared to negative mothers (763 cases), SARS-CoV-2 infection increased the odds of preterm birth (34 vs 51, 13.8% vs 6.7%, aOR 2.12, 95% CI 1.32-3.36, p = 0.002); iatrogenic preterm delivery was more frequent in infected women (4.9% vs 1.3%, p = 0.001), while the occurrence of spontaneous preterm deliveries was statistically similar (6.1% vs 4.7%). An increased risk of premature rupture of membranes at term (39 vs 75, 15.8% vs 9.8%, aOR 1.70, 95% CI 1.11-2.57, p = 0.013) and neonatal intensive care unit admissions (23 vs 18, 9.3% vs 2.4%, aOR 4.62, 95% CI 2.43-8.94, p <  0.001) was also observed in positive mothers. CONCLUSION: This prospective multicentre study demonstrated that pregnant women infected with SARS-CoV-2 have more infection-related obstetric morbidity. This hypothesis merits evaluation of a causal association in further research.

Genetic and transcriptomic evidences suggest ARO10 genes are involved in benzenoid biosynthesis by yeast.

Benzenoids are compounds associated with floral and fruity flavours in flowers, fruits and leaves and present a role in hormonal signalling in plants. These molecules are produced by the phenyl ammonia lyase pathway. However, some yeasts can also synthesize them from aromatic amino acids using an alternative pathway that remains unknown. Hanseniaspora vineae can produce benzenoids at levels up to two orders of magnitude higher than Saccharomyces species, so it is a model microorganism for studying benzenoid biosynthesis pathways in yeast. According to their genomes, several enzymes have been proposed to be involved in a mandelate pathway similar to that described for some prokaryotic cells. Among them, the ARO10 gene product could present benzoylformate decarboxylase activity. This enzyme catalyses the decarboxylation of benzoylformate into benzaldehyde at the end of the mandelate pathway in benzyl alcohol formation. Two homologous genes of ARO10 were found in the two sequenced H. vineae strains. In this study, nine other H. vineae strains were analysed to detect the presence and per cent homology of ARO10 sequences by PCR using specific primers designed for this species. Also, the copy number of the genes was estimated by quantitative PCR. To verify the relation of ARO10 with the production of benzyl alcohol during fermentation, a deletion mutant in the ARO10 gene of Saccharomyces cerevisiae was used. The two HvARO10 paralogues were analysed and compared with other α-ketoacid decarboxylases at the sequence and structural level.

The Mandelate Pathway, an Alternative to the Phenylalanine Ammonia Lyase Pathway for the Synthesis of Benzenoids in Ascomycete Yeasts.

Benzenoid-derived metabolites act as precursors for a wide variety of products involved in essential metabolic roles in eukaryotic cells. They are synthesized in plants and some fungi through the phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) pathways. Ascomycete yeasts and animals both lack the capacity for PAL/TAL pathways, and metabolic reactions leading to benzenoid synthesis in these organisms have remained incompletely known for decades. Here, we show genomic, transcriptomic, and metabolomic evidence that yeasts use a mandelate pathway to synthesize benzenoids, with some similarities to pathways used by bacteria. We conducted feeding experiments using a synthetic fermentation medium that contained either 13C-phenylalanine or 13C-tyrosine, and, using methylbenzoylphosphonate (MBP) to inhibit benzoylformate decarboxylase, we were able to accumulate intracellular intermediates in the yeast Hanseniaspora vineae To further confirm this pathway, we tested in separate fermentation experiments three mutants with deletions in the key genes putatively proposed to form benzenoids (Saccharomyces cerevisiaearo10Δ, dld1Δ, and dld2Δ strains). Our results elucidate the mechanism of benzenoid synthesis in yeast through phenylpyruvate linked with the mandelate pathway to produce benzyl alcohol and 4-hydroxybenzaldehyde from the aromatic amino acids phenylalanine and tyrosine, as well as sugars. These results provide an explanation for the origin of the benzoquinone ring, 4-hydroxybenzoate, and suggest that Aro10p has benzoylformate and 4-hydroxybenzoylformate decarboxylase functions in yeast.IMPORTANCE We present here evidence of the existence of the mandelate pathway in yeast for the synthesis of benzenoids. The link between phenylpyruvate- and 4-hydroxyphenlypyruvate-derived compounds with the corresponding synthesis of benzaldehydes through benzoylformate decarboxylation is demonstrated. Hanseniaspora vineae was used in these studies because of its capacity to produce benzenoid derivatives at a level 2 orders of magnitude higher than that produced by Saccharomyces Contrary to what was hypothesized, neither ß-oxidation derivatives nor 4-coumaric acid is an intermediate in the synthesis of yeast benzenoids. Our results might offer an answer to the long-standing question of the origin of 4-hydroxybenzoate for the synthesis of Q10 in humans.

Genomic and Transcriptomic Basis of Hanseniaspora vineae's Impact on Flavor Diversity and Wine Quality.

Hanseniaspora is the main genus of the apiculate yeast group that represents approximately 70% of the grape-associated microflora. Hanseniaspora vineae is emerging as a promising species for quality wine production compared to other non-Saccharomyces species. Wines produced by H. vineae with Saccharomyces cerevisiae consistently exhibit more intense fruity flavors and complexity than wines produced by S. cerevisiae alone. In this work, genome sequencing, assembling, and phylogenetic analysis of two strains of H. vineae showed that it is a member of the Saccharomyces complex and it diverged before the whole-genome duplication (WGD) event from this clade. Specific flavor gene duplications and absences were identified in the H. vineae genome compared to 14 fully sequenced industrial S. cerevisiae genomes. The increased formation of 2-phenylethyl acetate and phenylpropanoids such as 2-phenylethyl and benzyl alcohols might be explained by gene duplications of H. vineae aromatic amino acid aminotransferases (ARO8 and ARO9) and phenylpyruvate decarboxylases (ARO10). Transcriptome and aroma profiles under fermentation conditions confirmed these genes were highly expressed at the beginning of stationary phase coupled to the production of their related compounds. The extremely high level of acetate esters produced by H. vineae compared to that by S. cerevisiae is consistent with the identification of six novel proteins with alcohol acetyltransferase (AATase) domains. The absence of the branched-chain amino acid transaminases (BAT2) and acyl coenzyme A (acyl-CoA)/ethanol O-acyltransferases (EEB1) genes correlates with H. vineae's reduced production of branched-chain higher alcohols, fatty acids, and ethyl esters, respectively. Our study provides sustenance for understanding and potentially utilizing genes that determine fermentation aromas.IMPORTANCE The huge diversity of non-Saccharomyces yeasts in grapes is dominated by the apiculate genus Hanseniaspora Two native strains of Hanseniaspora vineae applied to winemaking because of their high oenological potential in aroma and fermentation performance were selected to obtain high-quality genomes. Here, we present a phylogenetic analysis and the complete transcriptome and aroma metabolome of H. vineae during three fermentation steps. This species produced significantly richer flavor compound diversity than Saccharomyces, including benzenoids, phenylpropanoids, and acetate-derived compounds. The identification of six proteins, different from S. cerevisiae ATF, with diverse acetyltransferase domains in H. vineae offers a relevant source of native genetic variants for this enzymatic activity. The discovery of benzenoid synthesis capacity in H. vineae provides a new eukaryotic model to dilucidate an alternative pathway to that catalyzed by plants' phenylalanine lyases.

GqqA, a novel protein in Komagataeibacter europaeus involved in bacterial quorum quenching and cellulose formation.

BACKGROUND: We report on the functional screening and identification of an active quorum quenching (QQ) gene in the Komagataeibacter europaeus strain CECT 8546, which is a member of the acetic acid bacteria (AAB). RESULTS: Using a previously published screening protocol (Schipper et al., in Appl Environ Microbiol 75:224-233, 2009. doi: 10.1128/AEM.01389-08 ) for QQ genes, we identified a single gene, designated gqqA, that interfered strongly with bacterial quorum sensing (QS) in various reporter strains. It encodes for a 281-amino acid protein with a molecular mass of 30 kDa. Although the GqqA protein is similar to predicted prephenate dehydratases, it does not complement Escherichia coli mutants of the pheA gene, thus indicating a potentially different function. Recombinant GqqA protein attenuated QS-dependent pyocyanin production and swarming motility in the Pseudomonas aeruginosa strain PAO1. Moreover, GqqA quenched the QS response of the Agrobacterium tumefaciens NTL4 and the Chromobacterium violaceum CV026 reporter strains. Interestingly, the addition of recombinant GqqA protein to growing cultures of the Komagataeibacter europaeus strain CECT 8546 altered the cellulose production phenotype of CECT 8546 and other AAB strains. In the presence of GqqA protein, cells were planktonic, and no visible cellulose biofilms formed. The addition of low levels of N-acylhomoserine lactones maintained the biofilm formation phenotype. CONCLUSIONS: Our data provide evidence for an interconnection between QS and AAB cellulose biofilm formation as well as QQ activity of the GqqA protein.

Cellulose production and cellulose synthase gene detection in acetic acid bacteria.

The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.

Single site laparoscopy for fertility preservation: a cohort study.

STUDY OBJECTIVE: To compare operative and postoperative results of ovarian cortex retrieval by conventional laparoscopy (1cm umbilical site and 3 accessory 5-mm-reusable working ports) (HASS) versus single site laparoscopy (SSL). DESIGN: Prospective cohort study. SETTING: Fertility Preservation Programme at La Fe University Hospital-University of Valencia, Valencia, Spain, 2011 to 2012. Fertility Preservation Programme at La Fe University Hospital of Valencia, Valencia, Spain. PATIENTS: Twenty-one patients with cancer (breast cancer: n = 17; Hodgkin's lymphoma: n = 3; and non-Hodgkin's lymphoma: n = 1). INTERVENTION: Ovarian cortex retrieval either by conventional laparoscopy using an umbilical Hasson port and 3 accessory ports (HASS group: n = 11) or by SSL (SSL group: n = 10). MEASUREMENTS AND MAIN RESULTS: Operative length, blood loss, postoperative pain (visual analog scale for pain at 6, 24, and 48 hours), need of additional analgesia, quality of life (European Quality of Life-5 Dimensions), cosmesis of the scar, and patient's self-perception were assessed at 24 and 48 hours and 3 months after surgery. Baseline characteristics were similar between groups. Estimated blood loss, operative length, and postoperative pain did not differ between groups. The start of chemotherapy was not delayed in either group, and cosmesis and image self-perception were also similar. CONCLUSION: The SSL approach can be considered a safe option compared with the classic multisite approach.

Acetic acid bacteria from biofilm of strawberry vinegar visualized by microscopy and detected by complementing culture-dependent and culture-independent techniques.

Acetic acid bacteria (AAB) usually develop biofilm on the air-liquid interface of the vinegar elaborated by traditional method. This is the first study in which the AAB microbiota present in a biofilm of vinegar obtained by traditional method was detected by pyrosequencing. Direct genomic DNA extraction from biofilm was set up to obtain suitable quality of DNA to apply in culture-independent molecular techniques. The set of primers and TaqMan--MGB probe designed in this study to enumerate the total AAB population by Real Time--PCR detected between 8 × 10(5) and 1.2 × 10(6) cells/g in the biofilm. Pyrosequencing approach reached up to 10 AAB genera identification. The combination of culture-dependent and culture-independent molecular techniques provided a broader view of AAB microbiota from the strawberry biofilm, which was dominated by Ameyamaea, Gluconacetobacter, and Komagataeibacter genera. Culture-dependent techniques allowed isolating only one genotype, which was assigned into the Ameyamaea genus and which required more analysis for a correct species identification. Furthermore, biofilm visualization by laser confocal microscope and scanning electronic microscope showed different dispositions and cell morphologies in the strawberry vinegar biofilm compared with a grape vinegar biofilm.

Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation.

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography-mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.

Acetobacter malorum and Acetobacter cerevisiae identification and quantification by Real-Time PCR with TaqMan-MGB probes.

The identification and quantification of Acetobacter malorum and Acetobacter cerevisiae in wine and vinegar were performed using the Real-Time PCR (RT-PCR) with two TaqMan-MGB probes designed to amplify the internal transcribed spacer (ITS) region between the 16S-23S rRNA genes. The primers and probes were highly specific, with a detection limit of 10² cells/ml for both species, and the efficiency of the technique was >80%. The RT-PCR technique with these two new TaqMan-MGB probes, together with the five (Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans, Gluconacetobacter hansenii and Gluconacetobacter europaeus) that are already available (Torija et al., 2010), were validated on known concentrations of Acetic Acid Bacteria (AAB) grown in glucose medium (GY) and in inoculated matrices of wine and vinegar. Furthermore, this technique was applied to evaluate the AAB population in real wine samples collected in the Canary Islands. PCR enrichment performed prior to RT-PCR increased the accuracy of quantification and produced results similar to those detected with SYBR-Green. In real wine samples, the total AAB enumeration ranged from 9 × 10² to 106 cells/ml, and the seven AAB species tested were detected in more than one sample. However, AAB recovery on plates was poor; the isolates obtained on plates were A. malorum, G. oxydans, A. cerevisiae and A. pasteurianus species. RT-PCR with TaqMan-MGB probes is an accurate, specific and fast method for the identification and quantification of AAB species commonly found in wine and vinegar.

A peculiar cell cycle arrest at g2/m stage during the stationary phase of growth in the wine yeast Hanseniaspora vineae.

Yeasts of the genus Hanseniaspora gained notoriety in the last years due to their contribution to wine quality, and their loss of several genes, mainly related to DNA repair and cell cycle processes. Based on genomic data from many members of this genus, they have been classified in two well defined clades: the "faster-evolving linage" (FEL) and the "slower-evolving lineage" (SEL). In this context, we had detected that H. vineae exhibited a rapid loss of cell viability in some conditions during the stationary phase compared to H. uvarum and S. cerevisiae. The present work aimed to evaluate the viability and cell cycle progression of representatives of Hanseniaspora species along their growth in an aerobic and discontinuous system. Cell growth, viability and DNA content were determined by turbidity, Trypan Blue staining, and flow cytometry, respectively. Results showed that H. uvarum and H. opuntiae (representing FEL group), and H. osmophila (SEL group) exhibited a typical G1/G0 (1C DNA) arrest during the stationary phase, as S. cerevisiae. Conversely, the three strains studied here of H. vineae (SEL group) arrested at G2/M stages of cell cycle (2C DNA), and lost viability rapidly when enter the stationary phase. These results showed that H. vineae have a unique cell cycle behavior that will contribute as a new eukaryotic model for future studies of genetic determinants of yeast cell cycle control and progression.

Obstetric Outcomes of SARS-CoV-2 Infection in Asymptomatic Pregnant Women.

Around two percent of asymptomatic women in labor test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Spain. Families and care providers face childbirth with uncertainty. We determined if SARS-CoV-2 infection at delivery among asymptomatic mothers had different obstetric outcomes compared to negative patients. This was a multicenter prospective study based on universal antenatal screening for SARS-CoV-2 infection. A total of 42 hospitals tested women admitted for delivery using polymerase chain reaction, from March to May 2020. We included positive mothers and a sample of negative mothers asymptomatic throughout the antenatal period, with 6-week postpartum follow-up. Association between SARS-CoV-2 and obstetric outcomes was evaluated by multivariate logistic regression analyses. In total, 174 asymptomatic SARS-CoV-2 positive pregnancies were compared with 430 asymptomatic negative pregnancies. No differences were observed between both groups in key maternal and neonatal outcomes at delivery and follow-up, with the exception of prelabor rupture of membranes at term (adjusted odds ratio 1.88, 95% confidence interval 1.13-3.11; p = 0.015). Asymptomatic SARS-CoV-2 positive mothers have higher odds of prelabor rupture of membranes at term, without an increase in perinatal complications, compared to negative mothers. Pregnant women testing positive for SARS-CoV-2 at admission for delivery should be reassured by their healthcare workers in the absence of symptoms.

Glycolytic Proteins Interact With Intracellular Melatonin in Saccharomyces cerevisiae.

Melatonin is a bioactive compound that is present in fermented beverages and synthesized by yeast during alcoholic fermentation. Many studies have shown that melatonin interacts with some mammalian proteins, such as sirtuins or orphan receptor family proteins. The aim of this study was to determine the intracellular synthesis profile of melatonin in Saccharomyces cerevisiae and to identify the proteins that may interact with this molecule in yeast cells. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry, and proteins bound to melatonin were immunopurified by melatonin-IgG-Dynabeads. Melatonin was produced intracellularly in the lag phase of yeast growth and was exported to the extracellular media during the stationary phase. During this period, melatonin was bound to six proteins with molecular weights from 55 to 35 kDa. Sequence analysis showed that most proteins shared high levels of homology with glycolytic enzymes. An RNA-binding protein was also identified, the elongation alpha factor, which is related to mitochondria. This study reports for the first time the interaction of melatonin and proteins inside yeast cells. These results highlight the possible role of melatonin as a signal molecule and provide a new perspective for understanding its role in yeast.

Effects of melatonin and tryptophol addition on fermentations carried out by Saccharomyces cerevisiae and non-Saccharomyces yeast species under different nitrogen conditions.

During wine fermentation, yeasts produce metabolites that are known growth regulators. The relationship between certain higher alcohols derived from aromatic amino acid metabolism and yeast signalling has previously been reported. In the present work, tryptophol (TrpOH) or melatonin (MEL), which are putative growth regulators, were added to alcoholic fermentations. Fermentations were performed with three different inocula, combining Saccharomyces cerevisiae and four non-Saccharomyces yeast species, under two nitrogen conditions. The combinations tested were: (i) only S. cerevisiae; (ii) the mixture of four non-Saccharomyces species; and (iii) the combination of all five species together. The results revealed that the TrpOH and MEL addition caused changes in fermentation kinetics, viability and species distribution during fermentation, but it was dependent on the nitrogen present in the media and the composition of the inocula. Low nitrogen condition seemed to favour the presence of non-Saccharomyces species until mid-fermentation, although at the end of fermentation the imposition of Saccharomyces was higher in this condition. The presence of high concentrations of TrpOH resulted in limited growth and a delay in fermentation, noticeably significant in fermentations performed with S. cerevisiae inocula. These effects were reversed by the presence of non-Saccharomyces yeast in the medium. Low TrpOH concentration allowed faster fermentation with mixed non-Saccharomyces and Saccharomyces inocula. Moreover, in the absence of S. cerevisiae, a low concentration of TrpOH increased the presence of Torulaspora delbrueckii during fermentation with high nitrogen availability but not under low nitrogen conditions, when the population of S. bacillaris was higher than that in the control. The effects of MEL were particularly evident at the beginning and end of the process, primarily favouring the growth of non-Saccharomyces strains, especially the first hours after inoculation.

Results of the Andalusian Cystic Fibrosis Neonatal Screening Program, 5 Years After Implementation. / Resultados del programa de screening neonatal de fibrosis quística en Andalucía tras 5 años de su implantación.

INTRODUCTION: Cystic fibrosis neonatal screening (CFNS), based on double determination of immunoreactive trypsinogen ([IRT] [IRT1/IRT2]), has been available in Andalusia since May 2011. If screening is positive, a sweat test is performed, and if that is positive or inconclusive, genetic testing is requested. OBJECTIVE: To analyze CFNS, based on results from the first 4.5 years of the program. MATERIALS AND METHODS: Prospective descriptive study of neonates undergoing CFNS. IRT levels, sweat chloride, and mutations were recorded. Statistical analysis was performed using SPSS 12.0. RESULTS: Between May 2011 and December 2016, 474,953 neonates underwent CFNS. Of these, 1,087 (0.23%) had elevated IRT2. Since CFNS was introduced, 73 cases of cystic fibrosis were diagnosed; 60 were diagnosed by positive CFNS, and 13 were diagnosed by other means. In one case, the patient developed a typical clinical picture of cystic fibrosis, but had not undergone CFNS at the decision of the parents; the remaining 12 had a negative CFNS (false negatives). Of these, one patient was diagnosed before symptoms developed, as his twin brother had a positive CFNS result; another had chloride at the upper limit of normal, and was subsequently diagnosed with genetic testing before symptoms appeared; and 10 patients developed clinical signs and symptoms. Excluding patients with meconium ileus, sensitivity and specificity of the CFNS program were 85.71% and 99.78%, respectively. The incidence of the disease in Andalusia is 1/6,506 live births. CONCLUSION: These results are a basis for reflection on possible areas for improvement of the CFNS algorithm, and thought may be given to the introduction of genetic studies to increase sensitivity and reduce false positives.

Effect of chitosan and SO2 on viability of Acetobacter strains in wine.

Wine spoilage is an important concern for winemakers to preserve the quality of their final product and avoid contamination throughout the production process. The use of sulphur dioxide (SO2) is highly recommended to prevent wine spoilage due to its antimicrobial activity. However, SO2 has a limited effect on the viability of acetic acid bacteria (AAB). Currently, the use of SO2 alternatives is favoured in order to reduce the use of chemicals and improve stabilization in winemaking. Chitosan is a biopolymer that is approved by the European authorities and the International Organization of Vine and Wine to be used as a fining agent and antimicrobial in wines. However, its effectiveness in AAB prevention has not been studied. Two strains of Acetobacter, adapted to high ethanol environments, were analysed in this study. Both chitosan and SO2 effects were compared in artificially contaminated wines. Both molecules reduced the metabolic activity of both AAB strains. Although AAB populations were detected by culture independent techniques, their numbers were reduced with time, and their viability decreased following the application of both products, especially with chitosan.

Whole-Exome Sequencing of Congenital Glaucoma Patients Reveals Hypermorphic Variants in GPATCH3, a New Gene Involved in Ocular and Craniofacial Development.

Congenital glaucoma (CG) is a heterogeneous, inherited and severe optical neuropathy that originates from maldevelopment of the anterior segment of the eye. To identify new disease genes, we performed whole-exome sequencing of 26 unrelated CG patients. In one patient we identified two rare, recessive and hypermorphic coding variants in GPATCH3, a gene of unidentified function, and 5% of a second group of 170 unrelated CG patients carried rare variants in this gene. The recombinant GPATCH3 protein activated in vitro the proximal promoter of CXCR4, a gene involved in embryo neural crest cell migration. The GPATCH3 protein was detected in human tissues relevant to glaucoma (e.g., ciliary body). This gene was expressed in the dermis, skeletal muscles, periocular mesenchymal-like cells and corneal endothelium of early zebrafish embryos. Morpholino-mediated knockdown and transient overexpression of gpatch3 led to varying degrees of goniodysgenesis and ocular and craniofacial abnormalities, recapitulating some of the features of zebrafish embryos deficient in the glaucoma-related genes pitx2 and foxc1. In conclusion, our data suggest the existence of high genetic heterogeneity in CG and provide evidence for the role of GPATCH3 in this disease. We also show that GPATCH3 is a new gene involved in ocular and craniofacial development.

Draft Genome Sequence of Komagataeibacter europaeus CECT 8546, a Cellulose-Producing Strain of Vinegar Elaborated by the Traditional Method.

The present article reports the draft genome sequence of the strain Komagataeibacter europaeus CECT 8546, an acetic acid bacterium characterized by its ability to overproduce cellulose. This species is highly resistant to acetic acid and commonly found during vinegar elaboration.