Wine yeast selection in the Iberian Peninsula: Saccharomyces and non-Saccharomyces as drivers of innovation in Spanish and Portuguese wine industries.

Yeast selection for the wine industry in Spain started in 1950 for the understanding of the microbial ecology, and for the selection of optimal strains to improve the performance of alcoholic fermentation and the overall wine quality. This process has been strongly developed over the last 30 years, firstly on Saccharomyces cerevisiae, and, lately, with intense activity on non-Saccharomyces. Several thousand yeast strains have been isolated, identified and tested to select those with better performance and/or specific technological properties. The present review proposes a global survey of this massive ex-situ preservation of eukaryotic microorganisms, a reservoir of biotechnological solutions for the wine sector, overviewing relevant screenings that led to the selection of strains from 12 genera and 22 species of oenological significance. In the first part, the attention goes to the selection programmes related to relevant wine-producing areas (i.e. Douro, Extremadura, Galicia, La Mancha and Uclés, Ribera del Duero, Rioja, Sherry area, and Valencia). In the second part, the focus shifted on specific non-Saccharomyces genera/species selected from different Spanish and Portuguese regions, exploited to enhance particular attributes of the wines. A fil rouge of the dissertation is the design of tailored biotechnological solutions for wines typical of given geographic areas.

Murine Model for the Study of Influenza D Virus.

A novel genus within the Orthomyxoviridae family was identified in the United States and named influenza D virus (IDV). Bovines have been proposed to be the primary host, and three main viral lineages (D/OK-like, D/660-like, and D/Japan-like) have been described. Experimental infections had previously been performed in swine, ferrets, calves, and guinea pigs in order to study IDV pathogenesis. We developed a murine experimental model to facilitate the study of IDV pathogenesis and the immune response. DBA/2 mice were inoculated with 105 50% tissue culture infective dose (TCID50) of D/bovine/France/5920/2014 (D/OK-like). No clinical signs or weight loss were observed. Viral replication was observed mainly in the upper respiratory tract (nasal turbinates) but also in the lower respiratory tract of infected mice, with a peak at 4 days postinfection. Moreover, the virus was also detected in the intestines. All infected mice seroconverted by 14 days postinfection. Transcriptomic analyses demonstrated that IDV induced the activation of proinflammatory genes, such as gamma interferon (IFN-γ) and CCL2. Inoculation of NF-κB-luciferase and Ifnar1-/- mice demonstrated that IDV induced mild inflammation and that a type I interferon response was not necessary in IDV clearance. Adaptation of IDV by serial passages in mice was not sufficient to induce disease or increased pathogenesis. Taken together, present data and comparisons with the calf model show that our mouse model allows for the study of IDV replication and fitness (before selected viruses may be inoculated on calves) and also of the immune response.IMPORTANCE Influenza D virus (IDV), a new genus of Orthomyxoviridae family, presents a large host range and a worldwide circulation. The pathogenicity of this virus has been studied in the calf model. The mouse model is frequently used to enable a first assessment of a pathogen's fitness, replication, and pathogenesis for influenza A and B viruses. We showed that DBA/2 mice are a relevant in vivo model for the study of IDV replication. This model will allow for rapid IDV fitness and replication evaluation and will enable phenotypic comparisons between isolated viruses. It will also allow for a better understanding of the immune response induced after IDV infection.

Influence of freezing temperatures prior to freeze-drying on viability of yeasts and lactic acid bacteria isolated from wine.

AIMS: To determine the effect of three different freezing temperatures on post-freeze-drying survival rates of wine yeasts and lactic acid bacteria (LAB). To know if a similar freeze-drying protocol can be used for both micro-organisms. METHODS AND RESULTS: Cells from liquid culture media were recovered and concentrated in appropriate lyoprotectants. Aliquots of each strain were frozen at -20, -80 and -196°C before vacuum drying. Viable cell counts were done before freezing and after freeze-drying. Survival rates were calculated. Freezing temperatures differently affected yeast and bacteria survival. The highest survival rates were obtained at -20 and -80°C for yeasts, but at -196°C for LAB. Major differences in survival rates were recorded among freeze-dried yeasts, but were less drastic for LAB. Yeasts Pichia membranifaciens, Starmerella bacillaris and Metschnikowia pulcherrima, and LAB Lactobacillus paracasei, Pediococcus parvulus and Lactobacillus mali, were the most tolerant species to freeze-drying, regardless of freezing temperature. CONCLUSIONS: Yeast and LAB survival rates differed for each tested freezing temperature. For yeasts, -20°C ensured the highest post-freeze-drying viability and -196°C for LAB. SIGNIFICANCE AND IMPACT OF THE STUDY: Freezing temperature to freeze-dry cells is a crucial factor for ensuring good wine yeast and LAB survival. These results are important for appropriately preserving micro-organisms and for improving starter production processes.

Cloning and characterization of a new laccase from Lactobacillus plantarum J16 CECT 8944 catalyzing biogenic amines degradation.

In our search for degrading activities of biogenic amines (BAs) in lactic acid bacteria, a protein annotated as laccase enzyme was identified in Lactobacillus plantarum J16 (CECT 8944). In this study, the gene of this new laccase was cloned and heterologously overexpressed in Escherichia coli. The recombinant laccase protein was purified and characterized biochemically. The purified laccase showed characteristic spectroscopic properties of blue multicopper oxidases. The enzyme has a molecular weight of ∼ 62.5 kDa and activity toward typical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (2,6-DMP). The pH optima on ABTS and 2,6-DMP were 3.5 and 7.0, respectively. Kinetic constants Km and Vmax were of 0.21 mM and 0.54 U/mg for ABTS and 1.67 mM and 0.095 U/mg for 2,6-DMP, respectively. The highest oxidizing activity toward 2,6-DMP was obtained at 60 °C. However, after a preincubation step at 85 °C for 10 min, no residual activity was detected. It has been demonstrated that recombinant L. plantarum laccase oxidizes biogenic amines, mainly tyramine, and thus presents new biotechnological potential for the enzyme in eliminating toxic compounds present in fermented food and beverages.

Assessing the potential of the European Atlantic sturgeon Acipenser sturio to control bivalve invasions in Europe.

This pilot study explored the potential of juvenile European Atlantic sturgeon Acipenser sturio to feed on two invasive bivalve species, the Asian clam Corbicula fluminea and the Eurasian zebra mussel Dreissena polymorpha. Preliminary results indicate that native A. sturio were feeding on D. polymorpha at a very limited rate and their potential to prevent the establishment of invasive bivalve species, in new and previously invaded areas, is considered limited.

Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine.

The main objectives of this study were the search for enzymatic activities responsible for biogenic amine (BA) degradation in lactic acid bacteria (LAB) strains isolated from wine, their identification, and the evaluation of their applicability for reducing BAs in wine. Fifty-three percent of the 76 LAB cell extracts showed activity against a mixture of histamine, tyramine, and putrescine when analyzed in-gel. The quantification of the degrading ability for each individual amine was tested in a synthetic medium and wine. Most of the bacteria analyzed were able to degrade the three amines in both conditions. The highest percentages of degradation in wine were those of putrescine: up to 41% diminution in 1 week. Enzymes responsible for amine degradation were isolated and purified from Lactobacillus plantarum J16 and Pediococcus acidilactici CECT 5930 strains and were identified as multicopper oxidases. This is the first report of an efficient BA reduction in wine by LAB. Furthermore, the identity of the enzymes involved has been revealed.

Glycemic control and prescription profiles in internal medicine inpatients: The role of frailty.

AIM: This work aims to characterize the clinical profile of individuals with frailty syndrome, diabetes mellitus (DM), and hyperglycemia during hospitalization in regard to glycemic control and treatment regimen. METHODS: This cross-sectional multicentric study included patients with DM or hyperglycemia at admission. Demographic data, blood glucose values, treatment administered during hospitalization, and treatment indicated at discharge were analyzed. The sample was divided into three groups according to score on a frailty questionnaire. Generalized additive models were used to describe the relationship between either glycemic variability (GV) or minimum capillary blood glucose and hypoglycemia. Models were adjusted for age, comorbidity, and sarcopenia. RESULTS: A total of 1,137 patients were analyzed. Patients with frailty syndrome had more comorbidity and sarcopenia, worse renal function, and lower albumin and lymphocyte levels. A GV between 21% and 60% was related to a higher probability of hypoglycemia, especially in patients with frailty. Regarding minimum capillary blood glucose, patients with frailty had the highest probability of hypoglycemia. This probability remained significant even in the group with frailty in which, with a reference value of 200 mg/dl, the adjusted odds ratio of a minimum capillary blood glucose of 151 mg/dL was 1.08 (95% confidence interval (1.12-1.05)). Baseline treatments showed a significant predominance of insulin use in the frailest groups. CONCLUSIONS: Patients with frailty had more sarcopenia and undernourishment. These patients were managed in a similar manner during hospitalization to patients without frailty, despite their higher risk of hypoglycemia according to GV or minimum capillary blood glucose levels.

Meeting report: Emerging respiratory viral infections and nonhuman primate case reports.

A workshop on Emerging Respiratory Viral Infections and Spontaneous Diseases in nonhuman primates was sponsored by the concurrent Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology, held December 1-5, 2012, in Seattle, Washington. The session had platform presentations from Drs Karen Terio, Thijs Kuiken, Guy Boivin, and Robert Palermo that focused on naturally occurring influenza, human respiratory syncytial virus, and metapneumovirus in wild and zoo-housed great apes; the molecular biology and pathology of these viral respiratory diseases in nonhuman primate (NHP) models; and the therapeutic and vaccine approaches to prevention and control of these emerging respiratory viral infections. These formal presentations were followed by presentations of 14 unique case studies of rare or newly observed spontaneous lesions in NHPs (see online files for access to digital whole-slide images corresponding to each case report at http://scanscope.com/ACVP%20Slide%20Seminars/2012/Primate%20Pathology/view.apml). The session was attended by meeting participants that included students, pathology trainees, and experienced pathologists from academia and industry with an interest in respiratory and spontaneous diseases of NHPs.

Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus.

While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k(cat). While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (∼23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

Amino acids and volatile compounds in wines from Cabernet Sauvignon and Tempranillo varieties subjected to malolactic fermentation in barrels.

The aim of the present paper is to compare the behaviour of industrial lactic bacteria and indigenous bacteria of the cellar when malolactic fermentation was carried out in barrels. The effects of these bacteria on the concentration of metabolised amino acids during malolactic fermentation and on the composition of volatile compounds both before and after malolactic fermentation are studied. The experiment was performed with wines of the Tempranillo and Cabernet Sauvignon varieties. An analysis has been made of the easily extractable volatile compounds of the wood and the compounds from the grapes, and the action of the yeasts during the alcoholic fermentation. Acetoin and diacetyl decreased during the malolactic fermentation in barrels and the concentrations of furfural and its derivatives were up to 100 times higher in wines not subjected to malolactic fermentation. Most of the volatile phenols increased during the malolactic fermentation in wines of the Tempranillo variety, while only guaiacol (p < 0.05) and t-isoeugenol increased in the Cabernet Sauvignon wines. The decrease in amino acids during the malolactic fermentation depends much more on the variety than on the bacterial strain which carries out the malolactic fermentation.

Characterization of an Atlantic salmon Salmo salar stream at the southern limit of its eastern Atlantic distribution.

Ten reaches of an Atlantic stream located in north-west Spain were sampled intensively during one summer to characterize the conditions where Atlantic salmon Salmo salar have been re-introduced along the stream. Fish species richness and diversity showed a downstream increase, which was mainly attributed to the higher number of cyprinid species found in the lower reaches. Moreover, except for brown trout Salmo trutta that appeared to be the most ubiquitous species, the densities of the other species was higher in the lower than in the upper stream reaches. Redundancy analysis showed that the pattern of fish assemblages observed along the studied stream was mainly related to the expected gradient observed in the levels of dissolved oxygen, discharge and mean current speed. There was a significant differentiation between midstream and downstream reaches, both in terms of the composition of their fish assemblages and the freshwater habitat. This study emphasizes the importance of describing the variations in fish assemblages and habitat characteristics along a river to explore its relation to potential changes in the survival of fish populations. In particular, the development of habitat-fish relationships may be a useful tool for water managers to assess the effects of development and restoration projects on the habitat of S. salar.

Regulation of hdc expression and HDC activity by enological factors in lactic acid bacteria.

AIMS: The aim of this work was to study the influence of enological factors on the histidine decarboxylase gene (hdc) expression and on histidine decarboxylase enzyme (HDC) activity in Lactobacillus hilgardii, Pediococcus parvulus and Oenococcus oeni. METHODS AND RESULTS: Cell extracts and whole cells were used. Glucose, fructose, malic acid and citric acid diminished the hdc expression. Ethanol did not increase hdc expression or activity in cells, but increased HDC activity. Temperature and pH had effect on the activity of HDC but not on hdc expression. Tartaric acid and l-lactic acid, and sulphur dioxide (SO(2)) had no effect on enzyme synthesis and activity. Bacterial species differ in the relative enzymatic activity but all the factors affected similarly to L. hilgardii, P. parvulus and O. oeni. CONCLUSIONS: The hdc gene expression was lowered by glucose, fructose, malic acid, and citric acid, whereas ethanol enhanced the HDC enzyme activity. The conditions that normally occur during malolactic fermentation and later on, could favour histamine production. SO(2) could prevent bacterial growth, but does not diminish the HDC enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on hdc expression and HDC activity can contribute to the prevention of histamine formation during wine production and storage.

Factors affecting the production of putrescine from agmatine by Lactobacillus hilgardii XB isolated from wine.

AIMS: To elucidate and characterize the metabolic putrescine synthesis pathway from agmatine by Lactobacillus hilgardii X(1)B. METHODS AND RESULTS: The putrescine formation from agmatine by resting cells (the normal physiological state in wine) of lactic acid bacteria isolated from wine has been determined for the first time. Agmatine deiminase and N-carbamoylputrescine hydrolase enzymes, determined by HPLC and LC-Ion Trap Mass Spectrometry, carried out the putrescine synthesis from agmatine. The influence of pH, temperature, organic acids, amino acids, sugars and ethanol on the putrescine formation in wine was determined. CONCLUSIONS: Resting cells of Lact. hilgardii X(1)B produce putrescine in wine. The putrescine production was carried out from agmatine through the agmatine deiminase system. SIGNIFICANCE AND IMPACT OF THE STUDY: These results have significance from two points of view, wine quality and toxicological and microbiological aspects, taking account that putrescine, which origin is still controversial, is quantitatively the main biogenic amine found in wine.

Comparative survey of putrescine production from agmatine deamination in different bacteria.

This article aims to study putrescine production in Lactobacillus hilgardii strain X(1)B, an agmatine degrader isolated from wine, and to compare it with three other different species, previously reported as putrescine producers from agmatine: Pseudomonas aeruginosa PAO1, Enterococcus faecalis ATCC11700 and Bacillus cereus CECT 148(T). The effect of different biogenic amines, organic acids, cofactors, amino acids and sugars on putrescine production was evaluated. In some cases, a similar effect was found in all the strains studied but the magnitude differed. Arginine, glucose and fructose showed an inhibitory effect, whereas the presence of agmatine induced the production of putrescine in all microorganisms. In other cases, the effect differed between P. aeruginosa PAO1 and the other microorganisms. Histamine and tyramine poorly influenced the utilization of agmatine, although a small increase in putrescine production was observed in P. aeruginosa PAO1. Succinate, spermidine and spermine also led to an increase in putrescine production in P. aeruginosa PAO1, whereas the succinate had no effect in the other microorganisms. Spermine and spermidine always produced a diminution in agmatine deamination. In this work, we have also demonstrated that pyridoxal 5-phosphate, Mg(2+) and Mn(2+) had no effect on putrescine production from agmatine. Results presented in this paper indicate differences in regulation mechanisms of agmatine deiminase pathway among P. aeruginosa PAO1 and L. hilgardii X(1)B, E. faecalis ATCC11700 and B. cereus CECT 148(T). These results are significant from two points of view, first food quality, and second the toxicological and microbiological aspects. It should be taken into account that putrescine, whose origin is still controversial, is quantitatively the main biogenic amine found in food.

Thermoplastic starch-nanohybrid films with polyhedral oligomeric silsesquioxane.

Thermoplastic starch forms packaging films that have low gas permeability, but they are more permeable to water vapour and they are attacked by water. Our approach was to create surface and internal localised hydrophobicity using added reactive nano-materials to form nano-silica hybrids with emphasis on enhancing surface water resistance. Functionalization was via epoxy-POS, that were further linked to hydrophobic erucamide or an amphiphilic poly(oxyethylene-co-oxypropylene) mono-amine. High amylose thermoplastic starch was combined with mono-functionalised hepta-isobutyl polyhedral oligomeric silsesquioxane (POS). POS modified thermoplastic starch increased water resistance of TPS film. Wettability kinetics was a function of two distinct mechanisms each with independent linear behaviour. Surface water resistance increased and is proposed to be due to preferential location of the POS derivatives at the surface with associated increase of hydrophobicity due a surface change.

Fe3O4@Au@mSiO2 as an enhancing nanoplatform for Rose Bengal photodynamic activity.

A novel nanoplatform composed of three types of materials with different functionalities, specifically core-shell Fe3O4@Au nanoparticles encapsulated near the outer surface of mesoporous silica (mSiO2) nanoparticles, has been successfully synthesised and used to enhance the efficiency of a photosensitiser, namely Rose Bengal, in singlet oxygen generation. Fe3O4 is responsible for the unusual location of the Fe3O4@Au nanoparticle, while the plasmonic shell acts as an optical antenna. In addition, the mesoporous silica matrix firmly encapsulates Rose Bengal by chemical bonding inside the pores, thus guaranteeing its photostability, and in turn making the nanosystem biocompatible. Moreover, the silica surface of the nanoplatform ensures further functionalisation on demand.

Small structural changes of the imidazopyridine diacylglycerol acyltransferase 2 (DGAT2) inhibitors produce an improved safety profile.

Small molecule DGAT2 inhibitors have shown promise for the treatment of metabolic diseases in preclinical models. Herein, we report the first toxicological evaluation of imidazopyridine-based DGAT2 inhibitors and show that the arteriopathy associated with imidazopyridine 1 can be mitigated with small structural modifications, and is thus not mechanism related.

Atherogenic lipid profile of Brazilian near-term newborns.

Cardiovascular disease is the primary cause of death in Brazil. Recent studies have shown that low birth weight and preterm birth are linked to a higher prevalence of cardiovascular disease. The aim of the present study was to compare the levels of lipids and apolipoproteins and atherogenic indexes between term and near-term newborn infants. A sample of umbilical cord blood was obtained from 135 newborns (66 males) divided into two groups: 25 near-term neonates (35-36.6 weeks of gestational age) and 110 term neonates (37-42 weeks of gestational age). The total cholesterol concentrations were higher in the near-term neonates than in the term group (94.04 +/- 8.02 vs 70.42 +/- 1.63 mg/dl, P < 0.01), due to an increase in the LDL-cholesterol fraction in the near-term group (57.76 +/- 6.39 vs 34.38 +/- 1.29 mg/dl, P < 0.001). The atherogenic indexes (total cholesterol/HDL-cholesterol, LDL-cholesterol/HDL-cholesterol and apolipoprotein B/apolipoprotein A-I) were higher in the near-term group (P < 0.001, P < 0.001, and P < 0.05, respectively). The gestational age of the newborns was inversely correlated with total cholesterol and LDL-cholesterol, and also with the total cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol indexes. These findings demonstrate that the lipid profile is worse in the group of near-term neonates compared with the term group. Future studies are needed to determine if this atherogenic profile in near-term neonates can affect body metabolism, increasing the risk for cardiovascular diseases in adult life.

[Two different intra-corneal inlay surgical technique in rabbit eyes]. / Comparación de dos técnicas de implantación de lentes intracorneales en ojos de conejo.

OBJECTIVE: To compare two different surgical techniques of intracorneal lens implantation. To determine the incidence of corneal and lens complications in two groups of rabbits subjected to such surgery. METHOD: The first group (7 rabbits) initially had a keratectomy performed with the microkeratome following which the corneal flap was sutured to the stromal layer. In the second group (7 rabbits), a pocket-shaped stromal dissection was performed manually. Both groups of rabbits had a secondary lens implantation in the corneal inlay. The follow up period was of 2 months duration. RESULTS: The incidence of corneal edema in the first month was higher in group 1 (71.4%) than in group 2 (14.3%) (p = 0.0155); during the second month, the incidence was 42.9% in group 1 and 28.6% in group 2 (p= 0.2927). Flap dislocation and retraction of the flap was seen in 4 of the 7 rabbits in group 1, whereas no complication of this type was observed in the manual pocket group (group 2). The incidence of lens subluxation was 28.6% in group 1 and 0% in group 2. Finally, there was a 71.4% (5/7) loss of the intracorneal lens in the rabbits treated with the microkeratome (group 1) while only 1 of 7 rabbits (14.3%) lost the lens in group 2 (P = 0.0155). DISCUSSION: Eyes in which the corneal pocket shape was made by manual stromal dissection had a higher stability of the lens and less post-operative corneal complications.

Transposons Tn916 and Tn925 can transfer from Enterococcus faecalis to Leuconostoc oenos.

The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oenos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Lactococcus lactis LM2301. Further analysis of L. lactis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no efficient conjugal or transformation systems previously described for this microorganism.