Maturation and antioxidant activity of 'Giombo' and 'Rama Forte' persimmons produced in the Brazilian semiarid.

The objective of this work was characterizing persimmons of the 'Giombo' and 'Rama Forte' cultivars harvested at different ripening stages in the Brazilian semiarid. Fruits were harvested at three ripening stages - green, semi-ripe and ripe - then evaluated for the following characteristics: fruit weight and diameter, skin and pulp color, fruit firmness, pulp pH, soluble solids content, titratable acidity, SSC/TA ratio, total soluble sugars, reducing sugars, astringency index, and the contents of tannin, vitamin C, carotenoid, ß-carotene, and total extractable polyphenols. Also, total antioxidant activity by the DPPH and ABTS methods and pectin methylesterase, and polygalacturonase enzyme activities were evaluated. Two experiments were carried out in a completely randomized design, one for each cultivar, with treatments consisting of different stages of maturation, with five replications of three fruits each. Data were submitted to analysis of variance and the differences between the means were compared using the Tukey test at 5% probability. Fruit firmness and soluble solids content did not vary between maturation stages for any of the cultivars. However, the skin color index increased with advancing maturation for both 'Giombo' and 'Rama Forte'. The astringency index, the content of total extractable polyphenols, soluble tannins and the antioxidant capacity were lower in fruits harvested at the ripe stage, for both cultivars. It can be concluded that persimmons of the 'Giombo' and 'Rama Forte' cultivars present better physicochemical quality characteristics when harvested when ripe, with a totally yellow skin.

ITS2 as a molecular marker for the identification of Diatraea saccharalis and D. flavipennella and possible infection with Cotesia spp.

In Brazil, the species Diatraea flavipennella and D. saccharalis play an important role in the sugar and alcohol agribusiness by causing many damages in sugarcane fields. The egg, larva, pupa, and adult stages are very morphologically similar between these species, and the identification can be confused. The internal transcribed spacer 2 (ITS 2) from ribosomal DNA has important features as evolutionary divergence. It is a good marker for species identification, participates in the rDNA processing, and has been applied in phylogenetic and population studies. This study aimed to make available a molecular marker to assist on the identification method of pests' species of Diatraea and to identify possible traces of Cotesia in the resistant host. The DNA was extracted from the egg, larva, and adult samples. PCR amplicons were purified and sequenced. The sequences were analyzed in MEGA 5.01. The ITS 2 length was 410 bp in D. flavipennella and 448 bp in D. saccharalis. The GC content was similar between the species. Three microsatellite loci were present in D. saccharalis and absent in D. flavipennella, contributing to differences in ITS 2 length in the species. An additional 367-bp band was attributed to Cotesia spp. The differences among ITS 2 from D. flavipennella, D. saccharalis, and Cotesia sp were sufficient to identify them on electrophoresis gel and sequencing. The presence of Cotesia sp traits in adult D. flavipennella showed possible host refractoriness, but further studies are necessary.

High intracellular trehalase activity prevents the storage of trehalose in the yeast Dekkera bruxellensis.

UNLABELLED: Dekkera bruxellensis hit the spotlight in the past decade mostly due to its rather high ability to adapt to several different fermentation processes. This yeast relies on different genetic and physiological aspects to achieve and preserve its high industrial fitness and some of these traits are shared with Saccharomyces cerevisiae. We have previously described that D. bruxellensis is unable to make use of accumulating trehalose as a strategy for cell adaptation and survival in the industrial scenario, as opposed to S. cerevisiae. Since trehalose is often involved in mechanisms related to cell protection, we aimed to investigate both cause and effect of the absence of this metabolite in the cell adaptive capacity in the industrial environment. Our results indicate that the major cause for the nonaccumulation of trehalose is the high constitutive activity of neutral trehalase. Therefore, the rate of trehalose degradation could be higher than its rate of synthesis, preventing accumulation. Altogether, our data elucidate the mechanisms involved in the lack of trehalose accumulation in D. bruxellensis as well as evaluates the implications of this feature. SIGNIFICANCE AND IMPACT OF THE STUDY: Dekkera bruxellensis can successfully take advantage of its peculiar physiological and genetic traits in order to adapt and survive in fermentation processes. So far, tolerance to stress has been credited to trehalose synthesis. The data presented in this work provided information on the underlying mechanism that prevents trehalose accumulation and corroborated the recent information that trehalose itself is not implicated in yeast stress tolerance. Second, it showed that D. bruxellensis responds differently to Saccharomyces cerevisiae to excess of sugar, which may explain its preference for respiration (oxidative metabolism) over fermentation (reductive metabolism) even at limited oxygen supply. These findings help to understand the drop on ethanol production in processes overtaken by this yeast.

Distribution of Dekkera bruxellensis in a sugarcane-based fuel ethanol fermentation plant.

UNLABELLED: We investigated the presence of the yeast Dekkera bruxellensis in samples collected at three points surrounding the industrial alcoholic fermentation plants of two distilleries where there are often cases of contamination caused by this yeast: this involved sugar cane wash water, feeding sugar cane juice and vinasse from the treatment pond. Total yeast was isolated in WLN medium with bromocresol green and cycloheximide and further selected on the basis of its ability to grow in synthetic medium containing nitrate. Following this, colonies were selected from the distribution on nitrate plates and identified by amplification with species-specific primers and DNA sequencing of the 26S-D1/D2 locus. The results showed that D. bruxellensis is introduced through the feeding substrate, which suggests that its cells originated with the harvested cane. Subsequently, its population circulates as a result of the reuse of water for washing the cane, in a continuous re-inoculation of the plant with yeasts. Furthermore, the yeast population is formed in the vinasse by the addition of wash water into the treatment ponds and then reintroduced to the culture fields by fertigation, so that the process can be renewed in the following season. It is now possible to adopt sanitation procedures that can prevent the entry of the contamination to the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of the yeast Dekkera bruxellensis is sometimes attributed to a decline in the industrial productivity of ethanol since it has a more limited fermentation capacity than Saccharomyces cerevisiae. Although its adaptability to the industrial environment has been noted, so far, there has been no evidence to determine the source of this contamination. In this study, we provide evidence to show that D. bruxellensis comes from the fields together with the harvested cane and is then accumulated and recirculated. It might be possible to prevent the accumulation of this yeast by carrying out sanitation controls during the harvesting season.

First Report of Mango Anthracnose Caused by Colletotrichum karstii in Brazil.

From April to June 2010, mango fruits (Mangifera indica L.) (cv. Tommy Atkins) showing post-harvest anthracnose symptoms were collected during a survey conducted in São Francisco Valley, northeastern Brazil. Fruits affected by anthracnose showed sunken, prominent, dark brown to black decay spots. Small pieces (4 to 5 mm) of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter-1 streptomycin sulfate. Plates were incubated at 25°C in the dark for 5 to 7 days and colonies that were morphologically similar to species of Colletotrichum were transferred to PDA (1). Identification was made using morphological characteristics and phylogenetic analysis. Two isolates (CMM 4101 and CMM 4102) presented colonies that had white aerial mycelia and orange conidial mass, varying between colorless and pale orange in reverse. Conidia were hyaline, cylindrical, and aseptate 14.52 (10.40 to 20.20) µm long and 4.90 (3.80 to 6.50) µm wide, length/width ratio = 3.0. Mycelial growth rate was 5.20 mm per day at 25°C. Morphological and cultural characterizations were consistent with the description of Colletotrichum karstii (3). PCR amplification by universal primers (ITS1/ITS4) and DNA sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted to confirm the identifications. Analysis of representative sequences (GenBank Accession Nos. HM585409 and HM585406) suggested that the isolated pathogen was C. karstii. Using published ITS data for C. karstii (3), a phylogenetic analysis was made via Bayesian inference, which shows that the isolated fungi belong to the C. karstii clade. Sequences of the isolates obtained in this study were deposited in GenBank (KC295235 and KC295236), and cultures were deposited in the Culture Collection of Phytopathogenic Fungi of the Universidade Federal Rural de Pernambuco (CMM, Recife, Brazil). Pathogenicity tests were conducted with the C. karstii strains on mango fruits cv. Tommy Atkins. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were applied in shallow wounds (0.4 cm in diameter) at the medium region of the each fruit. PDA discs without fungal growing were used as controls. Inoculated fruits were placed in plastic containers lined with paper towels wetted in distilled water. The containers were partially sealed with plastic bags to maintain high humidity and incubated at 25°C in the dark. The plastic bags and paper towels were removed after 24 h, and fruits were kept at the same temperature. The experiment was arranged in a completely randomized design with four replicates per treatment (isolate) and four fruits per replicate. Typical anthracnose symptoms were observed after 10 days in mango fruits. C. karstii was successfully reisolated from symptomatic mango fruits to fulfill Koch's postulates. C. karstii was previously described from Orchidaceae in southwest China and the United States (2,3). To our knowledge, this is the first report of C. karstii causing mango anthracnose in Brazil and worldwide. References: (1) U. Damm et al. Stud. Mycol. 73:1, 2012. (2) I. Jadrane et al. Plant Dis. 96:1227, 2012. (3) Yang et al. Cryptogamie Mycol. 32:229, 2011.

Prevalence of agenesis of the palmaris longus muscle in Brazil and its clinics correlation

Introduction: The palmaris longus muscle it is considered one of the most variable muscles in the human body, and its agenesis is the most frequent anatomic variation. The aim of this study was to determine the prevalence of unilateral and bilateral agenesis of palmaris longus muscle for the subjects in the city of Jataí, Goiás. Material and methods: 740 subjects, 279 men and 461 women, in the age of 10-70 years old, were observed through clinical inspection in both forearms to absence of the tendon of palmaris. The data were described in percentage values and the absence of the muscle was analyzed statistically (Chi square test; P < 0.05). Results: The bilateral presence was identified in 73.5%, the absence on the left side in 7.8%, on the right in 6.5% and bilateral absence was observed in 12.2% of subjects. Among women and men the percentage values was 9.3%, 7.3%, 13.1% and 5.4%, 5%, 10.7%, respectively to agenesis in left side, in right side and bilateral agenesis. The unilateral/bilateral agenesis was more frequent in women (29.7%) than in men (21.1%; P < 0.05). Bilateral absence was 12.4% in right handed subjects and 9.1% in left handed subjects (P > 0.05). Left and right handed subjects showed absence of muscle similar in both forearms (P > 0.05). Conclusion: most subjects studied showed bilateral presence of palmaris longus muscle, the agenesis was significantly more common in women and with similar percentage in right and left handed subjects.

Participation of CWI, HOG and Calcineurin pathways in the tolerance of Saccharomyces cerevisiae to low pH by inorganic acid.

AIMS: The present work aimed at identifying the metabolic response to acid stress and the mechanisms that lead to cell tolerance and adaptation. METHODS AND RESULTS: Two strategies were used: screening deletion mutants for cell growth at neutral and acid pH compared to wild type and measurement by qPCR of the expression of yeast genes involved in different pathways. CONCLUSIONS: The results complement our previous findings and showed that the Cell Wall Integrity pathway is the main mechanism for cell tolerance to acid pH, and this damage triggers the protein kinase C (PKC) pathway mainly via the Wsc1p membrane sensor. In addition, cell wall injury might mimic the effects of high osmotic shock and activates the High Osmolarity Glycerol pathway, which amplifies the signal in the upper part of PKC pathway and leads to the activation of Ca(2+) channels by SLT2 overexpression and this Ca(2+) influx further activates calcineurin. Together, these mechanisms induce the expression of genes involved in cell cycle regulation and cell wall regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: These interactions are responsible for long-term adaptation of yeast cells to the acidic environment, and the results could drive future work on the genetic modification of yeast strains for high tolerance to the stresses of the bioethanol fermentation process.

Cold fronts and reservoir limnology: an integrated approach towards the ecological dynamics of freshwater ecosystems.

In this paper the authors discuss the effects of cold fronts on the dynamics of freshwater ecosystems of southeast South America. Cold fronts originating from the Antarctic show a monthly frequency that promotes turbulence and vertical mixing in reservoirs with a consequence to homogenize nutrient distribution, dissolved oxygen and temperature. Weak thermoclines and the athelomixis process immediately before, during and after the passage of cold fronts interfere with phytoplankton succession in reservoirs. Cyanobacteria blooms in eutrophic reservoirs are frequently connected with periods of stratification and stability of the water column. Cold fronts in the Amazon and Pantanal lakes may produce fish killings during the process of "friagem" associated mixing events. Further studies will try to implement a model to predict the impact of cold fronts and prepare management procedures in order to cope with cyanobacteria blooms during warm and stable water column periods. Changes in water quality of reservoirs are expected during circulation periods caused by cold fronts.

Physiological and molecular analysis of the stress response of Saccharomyces cerevisiae imposed by strong inorganic acid with implication to industrial fermentations.

AIMS: This work aimed to identify the molecular mechanism that allows yeast cells to survive at low pH environments such as those of bioethanol fermentation. METHODS AND RESULTS: The industrial strain JP1 cells grown at pH 2 was evaluated by microarray analysis showing that most of the genes induced at low pH were part of the general stress response (GSR). Further, an acid-tolerant yeast mutant was isolated by adaptive selection that was prone to grow at low pH in inorganic but weak organic acid. It showed higher viability under acid-temperature synergistic treatment. However, it was deficient in some physiological aspects that are associated with defects in protein kinase A (PKA) pathway. Microarray analysis showed the induction of genes involved in inhibition of RNA and protein synthesis. CONCLUSIONS: The results point out that low pH activates GSR, mainly heat shock response, that is important for long-term cell survival and suggest that a fine regulatory PKA-dependent mechanism that might affect cell cycle in order to acquire tolerance to acid environment. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings might guide the construction of a high-fermentative stress-tolerant industrial yeast strain that can be used in complex industrial fermentation processes.

Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process.

Monitoring for wild yeast contaminants is an essential component of the management of the industrial fuel ethanol manufacturing process. Here we describe the isolation and molecular identification of 24 yeast species present in bioethanol distilleries in northeast Brazil that use sugar cane juice or cane molasses as feeding substrate. Most of the yeast species could be identified readily from their unique amplification-specific polymerase chain reaction (PCR) fingerprint. Yeast of the species Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis, as well as a species of Candida that belongs to the C. intermedia clade, were found to be involved in acute contamination episodes; the remaining 20 species were classified as adventitious. Additional physiologic data confirmed that the presence of these major contaminants cause decreased bioethanol yield. We conclude that PCR fingerprinting can be used in an industrial setting to monitor yeast population dynamics to early identify the presence of the most important contaminant yeasts.

Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation.

AIMS: To identify and characterize the main contaminant yeast species detected in fuel-ethanol production plants in Northeast region of Brazil by using molecular methods. METHODS AND RESULTS: Total DNA from yeast colonies isolated from the fermentation must of industrial alcohol plants was submitted to PCR fingerprinting, D1/D2 28S rDNA sequencing and species-specific PCR analysis. The most frequent non-Saccharomyces cerevisiae isolates were identified as belonging to the species Dekkera bruxellensis, and several genetic strains could be discriminated among the isolates. The yeast population dynamics was followed on a daily basis during a whole crop harvesting period in a particular industry, showing the potential of D. bruxellensis to grow faster than S. cerevisiae in industrial conditions, causing recurrent and severe contamination episodes. CONCLUSIONS: The results showed that D. bruxellensis is one of the most important contaminant yeasts in distilleries producing fuel-ethanol from crude sugar cane juice, specially in continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Severe contamination of the industrial fermentation process by Dekkera yeasts has a negative impact on ethanol yield and productivity. Therefore, early detection of D. bruxellensis in industrial musts may avoid operational problems in alcohol-producing plants.

Chromosome instability in industrial strains of Saccharomyces cerevisiae batch cultivated under laboratory conditions.

Industrial ethanol fermentation is a complex microbiological process to which yeast cells must adapt for survival. One of the mechanisms for adaptation is thought to involve chromosome rearrangements. We found that changes in chromosome banding patterns measured by pulsed-field gel electrophoresis can also be produced in laboratory media under simulated industrial conditions. Based on analysis of their generational variation, we found that these chromosome changes were specific to the genetic backgrounds of the initial strains. We conclude that chromosome rearrangements could be one of the factors involved in yeast cell adaptation to the industrial environment.

Contaminant yeast detection in industrial ethanol fermentation must by rDNA-PCR.

AIMS: The present work focuses on the possibility to use conserved primers that amplify yeast ITS1-5.8S-ITS2 ribosomal DNA locus (rDNA) to detect the presence of non-Saccharomyces cerevisiae yeast in fermentation must of bioethanol fermentation process. METHODS AND RESULTS: Total DNA was extracted from pure or mixed yeast cultures containing different cell concentrations and different contaminant/fermenting yeast concentrations and submitted to PCR. Upon improvement of detection limits and DNA extraction protocol, must samples of distillery were checked for the presence of contaminant yeast. Contaminant rDNA bands were detected only in industrial samples during contamination episodes, but not in noncontaminated must. CONCLUSIONS: The method described here could detect the presence of contaminant yeast from industrial must in eight hours after sampling. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved procedure may help to avoid severe contamination episodes at fermentation industries by decreasing the detection time from 5 days to 8 h and possible quantification of contaminant yeasts that can impose economical loss to the process.

Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein.

The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

Effect of culture conditions on lipase production by Fusarium solani in batch fermentation.

Lipase (Glycerol ester hydrolase EC 3.1.1.3.) from a Brazilian strain of Fusarium solani FSI has been investigated. The effect of different carbon sources and trace elements added to basal medium was observed with the aim of improving enzyme production. Lipase specific activity was highest (0.45 U mg(-1)) for sesame oil. When this medium was supplemented with trace elements using olive oil, corn oil and sesame oil the lipase specific activity increased to 0.86, 1.89 and 1.64 U mg(-1), respectively, after 96 h cultivation without any considerable biomass increase. The Km of this lipase using pNPP (p-nitrophenylpalmitate) as substrate, was 1.8 mM with a Vmax of 1.7 micromol min(-1) mg protein(-1). Lipase activity increased in the presence of increasing concentrations of hexane and toluene. In contrast, incubation of this enzyme with water-soluble solvents decreased its activity after 10% concentration (v/v) of the solvent. The lipase activity was stable below 35 degrees C but above this temperature activity losses were observed.

The short variant of the polymorphism within the promoter region of the serotonin transporter gene is a risk factor for late onset Alzheimer's disease.

We analyzed a deletion/insertion polymorphism within the promoter region of the serotonin transporter gene (5-HTTPLR) in 81 patients with late onset Alzheimer's (AD) disease (mean age 70.02 +/- 8.13 years). Control groups included 81 normal subjects with comparable age (mean age 75.6 +/- 10.2) and 82 younger normal subjects (mean age 37.4 +/- 9.1). Statistical analysis showed a significant difference in the genotype and gene frequencies between the AD group and normal controls (chi 2 = 9.021; 2 d.f. and chi 2 = 5.59, 1 d.f., respectively, P < 0.05) due to the higher frequency of the L allele and the lower frequency of the s allele in controls than among AD patients. However, no differences were found in the genotype frequencies in older as compared to younger normal control groups (chi 2 = 0.337, 2 d.f. and P > 0.05). The present study confirms, in a different population, that the short variant of the 5-HTTPLR polymorphism may be a risk factor for late onset AD.

Searching for a functional analogy between yeast Pso4 and bacterial RecA proteins in induced mitotic recombination.

The pso4-1 mutant of S. cerevisiae is phenotypically similar to the recA mutant of E. coli; it is sensitive to DNA cross-linking agents and defective in both recombination and mutagenesis. In this paper we have measured the effect of the recA gene expression on the frequency of mitotic crossing-over and mitotic gene conversion in response to DNA damage induced by photoactivated 8-methoxypsoralen (8-MOP + UVA), ultraviolet radiation (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The diploid pso4-1 mutant and the repair wild type strain were transformed with the multicopy plasmid carrying the recA gene placed under the control of the ADH1 promoter. The results showed that RecA is not able to restore block in induced mitotic recombination in pso4-1 cells after DNA damaging agents used. Thus RecA protein is not able to substitute Pso4 protein in homologous mitotic recombination indicating that they have probably different functions in this process.

Further characterization of the yeast pso4-1 mutant: interaction with rad51 and rad52 mutants after photoinduced psoralen lesions.

The pso4-1 mutant was characterized as deficient in some types of recombination, including gene conversion, crossing over, and intrachromosomal recombination. The mode of interaction between pso4-1 and rad51 and between pso4-1 and rad52 mutants indicated that the PSO4 gene belongs to the RAD52 epistasis group for strand-break repair. Moreover, the presence of the pso4-1 mutation decreased 8-MOP-photoinduced mutagenesis of the rad51 and rad52 mutants. Complementation tests using heterozygous diploid strains showed that the pso4 protein might interact with the rad52 protein during repair of 8-mop photolesions. The pso4-1 mutant, even though defective in inter- and intra-chromosomal recombination, conserves the ability for plasmid integration of circular and linear plasmid DNA. On the other hand, similar to the rad51 mutant, pso4-1 was able to incise but did not restore high-molecular-weight DNA during the repair of cross links induced by 8-MOP plus UVA. These results, together with those of previous reports, indicate that the PSO4 gene belongs to the RAD52 DNA repair group and its product participates in the DNA rejoining step of the repair of cross-link lesions, which are crucial for induced mutagenesis and recombinogenesis.

Biological consequences of E.coli RecA protein expression in the repair defective pso4-1 and rad51::URA3 mutants of S. cerevisiae after treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

RecA protein of E.coli is a multifunctional protein participating in genetic recombination, recombinational repair and mutagenesis. We used E.coli recA gene as a probe for complementation of repair defects after treatment of N-methyl-N'-nitro-N-nitrosoguanidine in the pso4-1 and rad51::URA3 mutants of S. cerevisiae. We tried to find the role of the RecA protein in S. cerevisiae mutants defective in different repair pathways. The RecA protein had no effect on survival of haploid and diploid pso4-1 mutants, but it had a significant effect on MNNG induced mutagenesis, which was increased to the wild type level. No effect of the RecA protein on survival was observed in rad51::URA3 mutant after MNNG treatment. However, in this case the RecA protein decreased the induced mutagenesis. We can suppose that the RecA protein, with its multifunctional enzymatic activity, can bind to special intermediates and initiate function of different repair pathways depending on repair defects of the mutants studied.