Diversity of Saccharomyces cerevisiae yeasts associated to spontaneously fermenting grapes from an Italian "heroic vine-growing area".

The main aim of this work was to analyse the diversity of wild Saccharomyces cerevisiae isolated from spontaneous fermentations of grapes collected from heroic vine-growing area. A first screening based on several technological traits was used to select 39 strains among 132 isolates. By using three molecular typing techniques (evaluation of cell wall gene polymorphisms, mtDNA restriction analysis, inter-delta amplification analysis) a significant genetic variability was found. The analysis of principal aromatic compounds produced during inoculated fermentation of two grape musts demonstrated the strain impact on wine flavour and a significant influence of grape must on strain metabolic behavior. One selected strain was used in fermentation at cellar level and the analysis of inter-delta region on yeast colonies isolated during the process revealed the high-implantation ability of this strain. The obtained results demonstrate the usefulness of different molecular and technological markers for the evaluation of natural biodiversity among S. cerevisiae strains. This study represents an essential step towards the exploitation and the preservation of biodiversity of strains isolated from heroic vine-growing area. Selected S. cerevisiae strains could represent starter cultures available for winemakers addressed to production of quality premium wines maintaining differential properties of their own area.

Co-inoculation of different Saccharomyces cerevisiae strains and influence on volatile composition of wines.

Wine is the result of the performance of different yeast strains throughout the fermentation in both spontaneous and inoculated processes. 22 Saccharomyces cerevisiae strains were characterized by microsatellite fingerprinting, selecting 6 of them to formulate S. cerevisiae mixed cultures. The aim of this study was to ascertain a potential benefit to use mixed cultures to improve wine quality. For this purpose yeasts behavior was studied during co-inoculated fermentations. Aromatic composition of the wines obtained was analyzed, and despite the fact that only one strain dominated at the end of the process, co-cultures released different concentrations of major volatile compounds than single strains, especially higher alcohols and acetaldehydes. Nevertheless, no significant differences were found in the type and quantity of the amino acids assimilated. This study demonstrates that the final wine composition may be modulated and enhanced by using suitable combinations of yeast strains.

An algorithm based on OmniView technology to reconstruct sagittal and coronal planes of the fetal brain from volume datasets acquired by three-dimensional ultrasound.

OBJECTIVE: To describe a novel algorithm, based on the new display technology 'OmniView', developed to visualize diagnostic sagittal and coronal planes of the fetal brain from volumes obtained by three-dimensional (3D) ultrasonography. METHODS: We developed an algorithm to image standard neurosonographic planes by drawing dissecting lines through the axial transventricular view of 3D volume datasets acquired transabdominally. The algorithm was tested on 106 normal fetuses at 18-24 weeks of gestation and the visualization rates of brain diagnostic planes were evaluated by two independent reviewers. The algorithm was also applied to nine cases with proven brain defects. RESULTS: The two reviewers, using the algorithm on normal fetuses, found satisfactory images with visualization rates ranging between 71.7% and 96.2% for sagittal planes and between 76.4% and 90.6% for coronal planes. The agreement rate between the two reviewers, as expressed by Cohen's kappa coefficient, was > 0.93 for sagittal planes and > 0.89 for coronal planes. All nine abnormal volumes were identified by a single observer from among a series including normal brains, and eight of these nine cases were diagnosed correctly. CONCLUSIONS: This novel algorithm can be used to visualize standard sagittal and coronal planes in the fetal brain. This approach may simplify the examination of the fetal brain and reduce dependency of success on operator skill.

First-trimester umbilical vein blood flow in pregnancies with low serum pregnancy-associated plasma protein-A levels: an early predictor of fetal growth restriction.

OBJECTIVE: To investigate umbilical vein blood flow (UVBF) during the first trimester in pregnancies with low serum pregnancy-associated plasma protein-A (PAPP-A) levels and to relate umbilical vein (UV) diameter, time-averaged maximum velocity (TAMXV) and UVBF values to the subsequent development of fetal intrauterine growth restriction (IUGR). METHODS: UVBF assessment was performed at 11 + 0 to 13 + 6 weeks' gestation in 102 singleton pregnancies with PAPP-A concentrations of < 0.3 multiples of the median. UV diameter, UV-TAMXV and UVBF were calculated and analyzed in relation to pregnancy outcome. RESULTS: Pregnancy outcomes were: 51 pregnancies with birth weight ≥ 10(th) centile (Group A), 30 pregnancies with birth weight < 10(th) centile with normal Doppler in the umbilical artery throughout gestation (Group B) and 21 pregnancies with birth weight < 10(th) centile and abnormal umbilical artery Doppler later in gestation (Group C). No differences were found in PAPP-A levels between groups. Group C fetuses exhibited significantly lower values of UV-TAMXV (z-score - 1.99 SDs, t = 8.527, P ≤ 0.0001) and UVBF (z-score - 0.97 SDs, t = 7.420, P ≤ 0.0001) in comparison with normal reference ranges, while no differences were found in Groups A or B. CONCLUSIONS: Decreased UV-TAMXV and UVBF at 11 + 0 to 13 + 6 weeks' gestation identify fetuses at risk of developing IUGR among pregnancies with low levels of PAPP-A.

Biological diversity of Saccharomyces yeasts of spontaneously fermenting wines in four wine regions: comparative genotypic and phenotypic analysis.

Combination of molecular genetic analysis (karyotyping, PCR-RFLP of MET2, the ITS1-ITS2 region and the NTS region) and physiological examination (melibiose and mannitol utilization, sugar-, ethanol- and copper tolerance, killer activity, fermentation vigor and production of metabolites) of yeasts isolated from spontaneously fermenting wines in four wine regions revealed very high diversity in the Saccharomyces cerevisiae populations. Practically each S. cerevisiae isolate showed a unique pattern of properties. Although the strains originating from the same wine were quite similar in certain traits, they showed diversity in other properties. These results indicate that alcoholic fermentation in grape wines is performed by highly diverse yeast consortia rather than by one or two dominating strains. The less frequent Saccharomyces uvarum strains were less diverse, showed lower karyotype variability, were Mel(+), Man(+), more sensitive to 60% sugar, and ethanol or copper in the medium. They produced less acetic acid and fermented better at 14 degrees C than most of the S. cerevisiae isolates, but certain S. cerevisiae strains showed comparably high fermentation rates at this temperature, indicating that it is not a general rule that S. uvarum ferments better than S. cerevisiae at low temperatures. The segregation of certain traits (melibiose utilization, mannitol utilization and copper resistance) in both species indicates that the genomes can easily change during vegetative propagation. The higher diversity among the S. cerevisiae isolates suggests that the S. cerevisiae genome may be more flexible than the S. uvarum genome and may allow more efficient adaptation to the continuously changing environment in the fermenting wine.

"Pecorino di Filiano" cheese as a selective habitat for the yeast species, Debaryomyces hansenii.

The composition of yeast microflora in artisanal "Pecorino di Filiano" cheese, a typical product of the Basilicata region of Southern Italy, was studied during ripening. The isolates were identified by restriction analysis of the 18S rDNA amplified region with the combined use of Hinf I and Cfo I enzymes. The majority of the isolates were identified as Debaryomyces hansenii, whereas two yeasts were identified as Kluyveromyces lactis and one as Dekkera anomala. To evaluate natural biodiversity, D. hansenii "Pecorino di Filiano" isolates were submitted to genetic and technological characterization. RAPD-PCR analysis with P80 (5CGCGTGCCCA3) primer revealed significant polymorphism among D. hansenii isolates. About 30% of the isolates showed single molecular profiles, whereas the other D. hansenii yeasts were separated into three main patterns, differing for both the ripening time and the isolation source. Furthermore, the yeasts showed significant variability in their, "proteolytic activity". This work demonstrated the high predominance of D. hansenii among the yeast population of "Pecorino di Filiano" cheese, probably in consequence of the traditional salting process, which was selected for this salt tolerant species. This preliminary study allowed us to isolate autochthonous D. hansenii yeasts potentially useful as starters for the production of this artisanal cheese.

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts.

AIMS: The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. METHODS AND RESULTS: A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. CONCLUSIONS: A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.

Molecular and technological approaches to evaluate strain biodiversity in Hanseniaspora uvarum of wine origin.

AIMS: The characterization by molecular and physiological methods of wild apiculate strains, isolated from 'Aglianico del Vulture' grape must. METHODS AND RESULTS: The restriction analysis of 18S rDNA allowed the identification of strains at the species level, which were predominantly Hanseniaspora uvarum. The RAPD analysis and the evaluation of technological traits, such as the metabolic and enzymatic activities, were useful to evaluate the polymorphism of this species. CONCLUSIONS: The RAPD analysis clustered the wild H. uvarum strains in four main genetic groups and a very high phenotypic variability confirmed this genetic polymorphism. The technological variables, which determined the strain biodiversity differed significantly, demonstrating that these technological traits are strain dependent. A certain correlation was found between the strain behaviour and its isolation zone, indicating the influence of the environment on the genetic patrimony of the population. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological biodiversity recorded among H. uvarum wild strains represents the basis for organizing a collection of apiculate strains exhibiting oenological characteristics at different levels, such as high/low production of secondary compounds, and, therefore, potentially useful for a selection programme.

Genetic segregation of natural Saccharomyces cerevisiae strains derived from spontaneous fermentation of Aglianico wine.

AIMS: Investigation of the meiotic segregation of karyotypes and physiological traits in indigenous Saccharomyces strains isolated from Aglianico (South Italy) red wine. METHODS AND RESULTS: Segregation was studied in F1 and F2 descendants. Tetrads were isolated from sporulating cultures by micromanipulation. The spore clones were subjected to karyotype analysis by pulse-field gel electrophoresis (Bio-Rad model CHEF-DR II) and to various physiological tests. Certain chromosomes of the isolates showed 2:2 segregation patterns in F1 but proved to be stable in F2. The ability of cells to utilize maltose also segregated in a 2 : 2 manner in F1 and did not segregate in F2. Resistance to CuSO4, SO2 tolerance, the fermentative power and the production of certain metabolites segregated in both F1 and F2 generations and showed patterns indicating the involvement of polygenic regulation. CONCLUSIONS: The analysis revealed a high degree of genetic instability and demonstrated that meiosis can improve chromosomal and genetic stability. SIGNIFICANCE AND IMPACT OF THE STUDY: Winemaking is critically dependent on the physiological properties and genetic stability of the fermenting Saccharomyces yeasts. Selection of clones from F2 or later generations can be a method of reduction of genetic instability.

Function of yeast species and strains in wine flavour.

The diversity and the composition of the yeast micropopulation significantly contribute to the sensory characteristics of wine. The growth of each wine yeast species is characterized by a specific metabolic activity, which determines concentrations of flavour compounds in the final wine. However, it must be underlined that, within each species, significant strain variability has been recorded. The wide use of starter cultures, mainly applied to reduce the risk of spoilage and unpredictable changes of wine flavour, can ensure a balanced wine flavour, but it may also cause a loss of characteristic aroma and flavour determinants. Thus, the beneficial contribution from the yeast increases when starter cultures for winemaking are selected on the basis of scientifically verified characteristics and are able to complement and optimise grape quality and individual characteristics. Here we report the characterization of a large number of strains of different wine yeast species, isolated from spontaneous wine fermentations and included in the culture collection of the Basilicata University.

Molecular typing techniques as a tool to differentiate non-Saccharomyces wine species.

A total of 32 yeast strains belonging to four non-Saccharomyces species associated with winemaking was characterized by different molecular techniques. The PCR amplification of 18S rRNA-coding DNA and nontranscribed spacer, followed by restriction analysis with the endonucleases HaeIII and MspI, and PCR fingerprinting with microsatellite primers (GAC)(5) and (GTG)(5) were used. The methods used provided species-specific profiles and proved to be fast and reliable for monitoring the evolution of the four non-Saccharomyces yeast populations throughout wine fermentation.

An increased expression of the mGlu5 receptor protein following LTP induction at the perforant path-dentate gyrus synapse in freely moving rats.

The involvement of metabotropic glutamate (mGlu) receptors in the induction of long-term potentiation (LTP) in vivo has been consistently documented. We have investigated whether LTP induction in the dentate gyrus of rats leads to changes in expression of mGlu2/3 or -5 receptor subtypes in the hippocampus. LTP was induced at the medial perforant path-dentate gyrus synapses, and mGlu receptor expression was examined by Western blot or in situ hybridization. An up-regulation of mGlu5 receptors was observed in the hippocampus both 24 and 48 h following LTP induction. This effect was restricted to the dentate gyrus and CA1 region, whereas no changes in mGlu5 receptor protein (but an increase in mRNA levels) were observed in the CA3 region. The increased expression of mGlu5 receptors was directly related to the induction of LTP, because it was not observed when tetanic stimulation was carried out in animals treated with the NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5). Western blot analysis also showed a reduced expression of mGlu2/3 receptors in the whole hippocampus 24 h after LTP induction, indicating that the increased expression of mGlu5 receptors was specific. These data suggest that an up-regulation of mGlu5 receptors is a component of the plastic changes that follow the induction of LTP at the perforant path-dentate gyrus synapse.

Typing of Saccharomyces cerevisiae and Kloeckera apiculata strains from Aglianico wine.

AIMS: Kloeckera apiculata and Saccharomyces cerevisiae yeast species are dominant, respectively, at the early and at the following stages of wine fermentation. In the present study, PCR fingerprinting and NTS region amplification and restriction were applied as techniques for monitoring yeast population performing Aglianico of Vulture grape must fermentation. METHODS AND RESULTS: Thirty S. cerevisiae and 30 K. apiculata strains were typed by PCR fingerprinting with (GAC)5 and (GTG)5 primers and by complete NTS region amplification followed by restriction with HaeIII and MspI enzymes. S. cerevisiae strains generated two patterns with (GAC)5 primer, while (GTG)5 primer yielded a higher genetic polymorphism. Conversely, in K. apiculata Aglianico wine strains (GAC)5 and (GTG)5 primers generated the same profile for all strains. Restriction analysis of the amplified NTS region gave the same profile for all strains within the same species, except for one strain of S. cerevisiae. CONCLUSIONS: The PCR fingerprinting technique was useful in discriminating at strain level S. cerevisiae, particularly with the primer (GTG)5. RFLP patterns generated from the NTS region of the two species can be more easily compared than the patterns resulting from PCR fingerprinting, thus RFLP is more suitable for the rapid monitoring of the species involved in different stages of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular techniques used allow discrimination of S. cerevisiae at strain level and monitoring of the ratio of S. cerevisiae/K. apiculata during the fermentation process. Thus, their application can assure technological adjustments in a suitable time.

Clinical behavior of 100 cutaneous malignant melanomas.

The last 100 patients with malignant melanoma were studied following actual methodology, with these results: mean age at diagnosis was 55.3 years; mean age at the beginning of disease was 51.8 years. Lesions were more frequent in patients with pale skin; there was no predominance of any color of eyes. Forty-nine percent of the patients were referred with a previous nevus which was confirmed only in 7%. One tumor developed on a giant pigmentary congenital nevus. Lesions showed no preponderance in one sex. Four percent of patients had multiple lesions. Familial background was found in 5%. The localization was most often to the head and neck, trunk, and legs. The most frequent types were SSMM and NMM; the first one on trunk and the second one on legs. The SSMM was observed in younger people than NMM. Level IV was seen in the greatest number of cases, with an average thickness of 3.07 mm. In SSMM, level III predominated, while in NMM, level IV predominated. The average thickness of SSMM was 1.82 mm; that of NMM was 6.25 mm. Our data are representative of malignant melanoma in Argentina. Therapy and evolution of lesions will be analyzed with an appropriate long-term follow-up.

Lepra lepromatosa en la infancia: (a proposito de las tres ultimas observaciones). / Lepromatous leprosy in infancy: Apropos of the 3 last observations

Se comunican tres casos de lepra lepromatosa en la segunda infancia analizandose en base a la bibliografia existente los siguientes items: mecanismo de trasmision, edad, sexo, clinica, bacteriologia, histopatologia e inmunologia. Todos se presentaron a la consulta con caracteristicas lesionales distintas, uno de ellos con eritema nudoso reaccional, no observandose exacerbaciones pre-puberales. Se destaca la poca frecuencia de esta forma clinica en este periodo de la vida

Lepra lepromatosa en la infancia: (a proposito de las tres ultimas observaciones). / Lepromatous leprosy in infancy: Apropos of the 3 last observations

Se comunican tres casos de lepra lepromatosa en la segunda infancia analizandose en base a la bibliografia existente los siguientes items: mecanismo de trasmision, edad, sexo, clinica, bacteriologia, histopatologia e inmunologia. Todos se presentaron a la consulta con caracteristicas lesionales distintas, uno de ellos con eritema nudoso reaccional, no observandose exacerbaciones pre-puberales. Se destaca la poca frecuencia de esta forma clinica en este periodo de la vida

Fagedenismo geometrico. / Geometric phagedenique

A proposito de 5 casos internados en nuestro servicio en los ultimos 6 anos, se realiza una revision y actualizacion bibliografica de la etiopatogenia, clinica, asociaciones, evolucion, cuadro humoral, diagnosticos diferenciales, histopatologia y tratamiento de la entidad

Fagedenismo geometrico. / Geometric phagedenique

A proposito de 5 casos internados en nuestro servicio en los ultimos 6 anos, se realiza una revision y actualizacion bibliografica de la etiopatogenia, clinica, asociaciones, evolucion, cuadro humoral, diagnosticos diferenciales, histopatologia y tratamiento de la entidad