Effects of medium pH on the yeast plasma membrane potential.

The effects of different pH incubation values and K+ on yeast plasma membrane potential (PMP) were studied both by the fluorescence changes and the accumulation of thioflavin T (ThT), a method that has been shown most adequate for both procedures. By the changes in fluorescence of ThT, the qualitative observation of PMP at the 3 evaluated pHs indicated that cells at pH 4.0 maintain a PMP lower, but close to the observed at pH 6.0 and 7.0. By measuring the accumulation of ThT and applying the Nernst equation on the different concentrations in and out, the values of PMP could also be estimated at the different pHs, resulting in values in mV, in agreement with our observations by following the fluorescence. Yeast cells at their native niches, or during fermentations must cope with low pHs, so the importance to maintain a robust PMP to survive. The contribution of bicarbonate, derived from the fermentation to the establishment of the PMP is also described. The experiments showed once more the efficacy of the methods used with this dye.

The high osmolarity glycerol (HOG) pathway in fungi†.

While fungi are widely occupying nature, many species are responsible for devastating mycosis in humans. Such niche diversity explains how quick fungal adaptation is necessary to endow the capacity of withstanding fluctuating environments and to cope with host-imposed conditions. Among all the molecular mechanisms evolved by fungi, the most studied one is the activation of the phosphorelay signalling pathways, of which the high osmolarity glycerol (HOG) pathway constitutes one of the key molecular apparatus underpinning fungal adaptation and virulence. In this review, we summarize the seminal knowledge of the HOG pathway with its more recent developments. We specifically described the HOG-mediated stress adaptation, with a particular focus on osmotic and oxidative stress, and point out some lags in our understanding of its involvement in the virulence of pathogenic species including, the medically important fungi Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, compared to the model yeast Saccharomyces cerevisiae. Finally, we also highlighted some possible applications of the HOG pathway modifications to improve the fungal-based production of natural products in the industry.

Chemical Characterization of Plant Extracts and Evaluation of their Nematicidal and Phytotoxic Potential.

Nacobbus aberrans ranks among the "top ten" plant-parasitic nematodes of phytosanitary importance. It causes significant losses in commercial interest crops in America and is a potential risk in the European Union. The nematicidal and phytotoxic activities of seven plant extracts against N. aberrans and Solanum lycopersicum were evaluated in vitro, respectively. The chemical nature of three nematicidal extracts (EC50,48h ≤ 113 µg mL-1) was studied through NMR analysis. Plant extracts showed nematicidal activity on second-stage juveniles (J2): (≥87%) at 1000 µg mL-1 after 72 h, and their EC50 values were 71.4-468.1 and 31.5-299.8 µg mL-1 after 24 and 48 h, respectively. Extracts with the best nematicidal potential (EC50,48h < 113 µg mL-1) were those from Adenophyllum aurantium, Alloispermum integrifolium, and Tournefortia densiflora, which inhibited L. esculentum seed growth by 100% at 20 µg mL-1. Stigmasterol (1), ß-sitosterol (2), and α-terthienyl (3) were identified from A. aurantium, while 1, 2, lutein (4), centaurin (5), patuletin-7-ß-O-glucoside (6), pendulin (7), and penduletin (8) were identified from A. integrifolium. From T. densiflora extract, allantoin (9), 9-O-angeloyl-retronecine (10), and its N-oxide (11) were identified. The present research is the first to report the effect of T. densiflora, A. integrifolium, and A. aurantium against N. aberrans and chemically characterized nematicidal extracts that may provide alternative sources of botanical nematicides.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Retraction Note to: Acridine yellow. A novel use to estimate and measure the plasma membrane potential in Saccharomyces cerevisiae.

The authors have retracted this article [1]. After publication the dye used in this study was analysed by NMR and mass spectroscopy and found not to be acridine yellow, but rather, was identified as thioflavin T.

Acridine yellow. A novel use to estimate and measure the plasma membrane potential in Saccharomyces cerevisiae.

Translocation of ions and other molecules across the plasma membrane of yeast requires the electric potential generated by a H+-ATPase. We measured under different conditions fluorescence changes and accumulation of acridine yellow, looking for qualitative and quantitative estimations of the PMP in Saccharomyces cerevisiae in various conditions. Fluorescence changes indicated an accumulation of the dye requiring a substrate, and accumulation and quenching by mitochondria that could be released by an uncoupler. K+ produced a decrease of the fluorescence that was much lower upon the addition of Na+. These changes were confirmed by images of the cells under the microscope. The dye accumulation under different conditions showed changes consistent with the physiological situation of the cells. Since it accumulates due to the PMP, but a large part of it binds to the internal components, we permeabilized the cells with chitosan to subtract this factor and correct the accumulation data. Both raw and corrected values of PMP are different to those obtained before by other authors and our group, showing acridine yellow as a promising indicator to follow changes of the PMP by the fluorescence changes, but also by its accumulation. Under conditions described, the dye is a low cost monitor to define and follow qualitative and quantitative changes of PMP in yeast. Acridine yellow can also be used to follow changes of the mitochondrial membrane potential.

Mechanisms involved in the inhibition of glycolysis by cyanide and antimycin A in Candida albicans and its reversal by hydrogen peroxide. A common feature in Candida species.

In Candida albicans, cyanide and antimycin A inhibited K(+) transport, not only with ethanol-O2 as the substrate, but also with glucose. The reason for this was that they inhibited not only respiration, but also fermentation, decreasing ATP production. Measurements of oxygen levels in cell suspensions allowed identification of the electron pathways involved. NADH fluorescence levels increased in the presence of the inhibitors, indirectly indicating lower levels of NAD(+) and so pointing to glyceraldehyde-3-phosphate dehydrogenase as the limiting step responsible for the inhibition of glycolysis, which was confirmed by the levels of glycolytic intermediaries. The cyanide effect could be reversed by hydrogen peroxide, mainly due to an activity by which H2O2 can be reduced by electrons flowing from NADH through a pathway that can be inhibited by antimycin A, and appears to be a cytochrome c peroxidase. Therefore, the inhibition of glycolysis by the respiratory inhibitors seems to be due to the decreased availability of NAD(+), resulting in a decreased activity of glyceraldehyde-3-phosphate dehydrogenase. Compartmentalization of pyridine nucleotides in favor of the mitochondria can contribute to explaining the low fermentation capacity of C. albicans. Similar results were obtained with three C. albicans strains, Candida dubliniensis and, to a lower degree, Candida parapsilosis.

Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae stopped by maintaining the pH of the medium in a pH-stat at pH 8.0 or 9.0. Studying its main physiological capacities and comparing cells after incubation at pH 6.0 vs. 8.0 or 9.0, we found that (a) fermentation was moderately decreased by high pH and respiration was similar and sensitive to the addition of an uncoupler, (b) ATP and glucose-6-phosphate levels upon glucose addition increased to similar levels and (c) proton pumping and K(+) transport were also not affected; all this indicating that energy mechanisms were preserved. Growth inhibition at high pH was also not due to a significant lower amino acid transport by the cells or incorporation into proteins. The cell cycle stopped at pH 9.0, probably due to an arrest as a result of adjustments needed by the cells to contend with the changes under these conditions, and microarray experiments showed some relevant changes to this response.

Laboratory Population Parameters and Field Impact of the Larval Endoparasitoid Pseudapanteles dignus (Hymenoptera: Braconidae) on its Host Tuta absoluta (Lepidoptera: Gelechiidae) in Tomato Crops in Argentina.

The tomato moth, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is a key pest of tomato, Lycopersicon esculentum L., crops in Central and South America. At present it is dispersing rapidly in Africa and Eurasian continents as an invasive pest, threatening worldwide tomato production. Pseudapanteles dignus (Muesebeck) (Hymenoptera: Braconidae) is an American endoparasitoid reported as the main natural enemy of T. absoluta in commercial tomato. To gain knowledge of the potential role of P. dignus in the biological control of this pest, we determined its population parameters in laboratory and the parasitoid's impact on T. absoluta in the field. In laboratory, lifetime fecundity was 193 eggs per female, and longevity was 24 and 26 d for female and male, respectively. The finite rate of increase (λ) was 1.15 per female per day and the intrinsic rate of natural increase (r(m)) was 0.14. The net reproductive rate (R(0)) was found to be 51.2, and generation time (T) 28.8 d. The time for doubling the population (DT) was 5 d. Furthermore, field parasitism of T. absoluta varied between 33 and 64% in the different years studied. Population parameters estimated in this study can be considered baseline information for a mass-rearing protocol of this parasitoid. Moreover, growth rates of P. dignus, particularly r(m), and its impact on field populations of T. absoluta indicated that this parasitoid is a valuable candidate for biological control of this pest.

Conservation biological control in strawberry: effect of different pollen on development, survival, and reproduction of Neoseiulus californicus (Acari: Phytoseiidae).

Wild vegetation surrounding crops may provide temporary habitat and potential food sources for phytoseiids in different seasons. Monthly vegetation samples of wild plants adjacent to strawberry plants and wild plants in a vegetation strip close to the crop were taken. The frequency of Neoseiulus californicus, Tetranychus urticae and other mites and insects was recorded. In addition, in a laboratory assay, the survival, developmental time and fecundity of females fed on pollen of strawberry and pollen of wild plants where N. californicus was recorded during their flowering, were estimated. Pollen from Urtica urens, Lamium amplexicaule, Convolvulus arvensis, Sonchus oleraceous, Galega officinalis, and Fragaria x ananassa (strawberry) allowed development of N. californicus to adult, but not reproduction. Survival was 70-80 % when fed on pollen from S. oleraceus, G. officinalis and C. arvensis, 80-90 % when fed on pollen from U. urens and F. x ananassa, and more than 90 % when fed on T. urticae and on pollen from L. amplexicaule. In autumn and winter, U. urens, L. amplexicaule and S. oleraceous could promote the persistence of N. californicus when prey density in strawberry is low, offering T. urticae, thrips and pollen. In summer, pollen of C. arvensis and G. officinalis would contribute to the persistence of N. californicus when the strawberry crop is ending and offers scarce food resources. Although the pollen of these plants would not enable the predator population to increase, the presence of these plants in the vicinity of strawberry could contribute to the persistence of N. californicus population and help to limit T. urticae growth when this pest begins to colonize the crop.

Short and long-term effects of three neurotoxic insecticides on biological and behavioural attributes of the orb-web spider Alpaida veniliae (Araneae, Araneidae): implications for IPM programs.

Soybean pest control in Argentina is done just by chemical control using broad-spectrum pesticides. Alpaida veniliae (Araneae, Araneidae) is one of the most abundant spider species of the orb web weaver guild in soybean, and it is considered a very important polyphagous predator, attacking different insects' families. The objective of this study was to determine if neurotoxic insecticides commonly used in soybean crops and a new active ingredient registered in Argentina (spinosad) adversely affected survival, prey consumption, mating behaviour, web building and reproductive capacity of A. veniliae females, under standard laboratory conditions. Spinosad was the most harmful insecticide due to high acute toxicity, even at lower concentrations than those registered for its field use and for its sublethal effects also. Cypermethrin caused several sublethal effects although its acute toxicity on spider was lower than other insecticides. It reduced prey consumption, affected web building, caused abnormalities in eggs sacs and decreased drastically the fecundity and fertility at sublethal concentrations. Endosulfan did not reduce prey consumption but it affected web building, caused abnormalities in eggs sacs and egg masses, and decreased the fecundity and fertility. Spinosad was also the compound with the most drastic effect on web building, it did not reduce prey consumption and fecundity, but fertility was reduced and abnormalities in egg sacs and egg masses were observed. The use of these insecticides in IPM programs according to their potential toxicity on spider communities is discussed.

Purification and characterization of structural and functional properties of two lectins from a marine sponge Spheciospongia vesparia.

The purification, structural and functional characterization of two different lectins (named Svl-1 and Svl-2) has been reported from the marine sponge Spheciospongia vesparia. Purification procedure includes ammonium sulfate precipitation, combined with chromatography including Octyl-Sepharose-(NH4)SO4 hydrophobic column and DEAE-Toyopearl anion-exchange column using a high performance liquid chromatography. The similarities in function, specificity for saccharides, molecular weight, amino acid content and the N-terminal sequence of two lectins suggest that these proteins are isolectins. Amino acid composition and fluorescence analyses reveal that they contain an intrachain disulfide bridge, which might contribute to their high thermal stability. Furthermore, the purified lectins exhibit antibacterial activity against the gram-negative bacteria Pseudomonas aeruginosa and E. coli, indicating that they may be involved in a recognition strategy and may play a role in the defense response function of the sponge. This is the first report on the isolation of lectins from the S. vesparia. The purified lectins represent a potential possible candidate for future application in the recognition or treatment of cancer cells.

[Relationship between the oxidative profile and the diet diversity index in older adults in an urban-marginal area of Costa Rica]. / Relación entre el perfil oxidativo y el índice de diversidad de la dieta en adultos mayores de una zona urbano-marginal de Costa Rica.

BACKGROUND AND OBJECTIVE: The older we get, the greater the production of reactive oxygen species and therefore the greater the oxidative stress, which is related to the deterioration of the health of older adults. This study analyzed the relationship between the oxidative profile and the dietary diversity index in an urban-marginal population of older adults in Costa Rica. METHODS: Eighty-eight older adults were studied and various markers of oxidative stress, serum glucose levels, lipid profile, and some micronutrients were determined. In addition, the body mass index (BMI) was calculated and the dietary diversity index (DDI) was determined. RESULTS: Lipid peroxidation and DNA oxidation, a mean plasma antioxidant capacity percentage of 39.54±10.67%, which decreased with age, were evidenced. 67% of the participants had alterations in glycemia, 73% had one or more alterations in blood lipid levels, 55% had insufficient vitamin D levels, and 68.6% were overweight. The average IDD was 4.91 points, indicating that the diet was not very diverse. No relationship was found between IDD and nutritional status, between nutritional status and oxidative stress, nor between biochemical variables and oxidative stress. CONCLUSION: The adults studied presented high oxidative stress, a high percentage of overweight, and a low IDD. A higher IDD was associated with a lower blood concentration of MDA and a higher % PAC.

The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains.

The use of the cationic, dye thioflavin T (ThT), to estimate the electric plasma membrane potential difference (PMP) via the fluorescence changes and to obtain its actual values from the accumulation of the dye, considering important correction factors by its binding to the internal components of the cell, was described previously for baker's yeast. However, it was considered important to explore whether the method developed could be applied to other yeast strains. Alternative ways to estimate the PMP by using flow cytometry and a multi-well plate reader are also presented here. The methods were tested with other strains of Saccharomyces cerevisiae (W303-1A and FY833), as well as with non-conventional yeasts: Debaryomyces hansenii, Candida albicans, Meyerozyma guilliermondii, and Rhodotorula mucilaginosa. Results of the estimation of the PMP via the fluorescence changes under different conditions were adequate with all strains. Consistent results were also obtained with several mutants of the main monovalent transporters, validating ThT as a monitor for PMP estimation.

Neo-functionalization in Saccharomyces cerevisiae: a novel Nrg1-Rtg3 chimeric transcriptional modulator is essential to maintain mitochondrial DNA integrity.

In Saccharomyces cerevisiae, the transcriptional repressor Nrg1 (Negative Regulator of Glucose-repressed genes) and the ß-Zip transcription factor Rtg3 (ReTroGrade regulation) mediate glucose repression and signalling from the mitochondria to the nucleus, respectively. Here, we show a novel function of these two proteins, in which alanine promotes the formation of a chimeric Nrg1/Rtg3 regulator that represses the ALT2 gene (encoding an alanine transaminase paralog of unknown function). An NRG1/NRG2 paralogous pair, resulting from a post-wide genome small-scale duplication event, is present in the Saccharomyces genus. Neo-functionalization of only one paralog resulted in the ability of Nrg1 to interact with Rtg3. Both nrg1Δ and rtg3Δ single mutant strains were unable to use ethanol and showed a typical petite (small) phenotype on glucose. Neither of the wild-type genes complemented the petite phenotype, suggesting irreversible mitochondrial DNA damage in these mutants. Neither nrg1Δ nor rtg3Δ mutant strains expressed genes encoded by any of the five polycistronic units transcribed from mitochondrial DNA in S. cerevisiae. This, and the direct measurement of the mitochondrial DNA gene complement, confirmed that irreversible damage of the mitochondrial DNA occurred in both mutant strains, which is consistent with the essential role of the chimeric Nrg1/Rtg3 regulator in mitochondrial DNA maintenance.

Preliminary Analysis of Unsaturated Fatty Acid Profiles of Coffea arabica L., in Samples with a Denomination of Origin and Speciality of Oaxaca, Mexico.

In February 2020, Coffea arabica L. grown on the coast and in the Southern Sierra Madre of the state of Oaxaca, Mexico obtained the denomination of origin. Which does not have data on color and chemical composition, the first associated with the degree of roasting and the second with lipids (17-18%), as the group of compounds responsible, in part, for flavor, consistency, and may contribute to health benefits. In the present work, color was determined on the CIE L*a*b* scale and the unsaturated fatty acids by Nuclear Magnetic Resonance (NMR) of 1H and 13C in samples of medium roasted specialty coffee from the "Pluma" coffee-growing region, Oaxaca, Mexico. The average value of L* luminosity in ground coffee was 42.1 ± 0.1 reported for a light roast. Unsaturated fatty acids were quantified from the lipid fraction of the gr1 ound grain by NMR 1H and 13C, obtaining on average the highest abundance of linoleic (41.7 ± 0.5 by 1 H and 41.24 ± 0.5 by 13C), followed by oleic (9.2 ± 0.2 by 1H and 7.4 ± 0.2 by 13C) and linolenic (1.5 ± 0.1 by H and 1.1 ± 0.2 by 13C). This study indicates that 1H and 13C NMR spectroscopy is a useful tool for the quantification of linolenic, linoleic, and oleic fatty acids by the method of key signal shifts of these acids found in lipid samples in roasted coffee grains.

Ketoconazole and miconazole alter potassium homeostasis in Saccharomyces cerevisiae.

The effects of ketoconazole and miconazole uptake on K(+) transport and the internal pH of Saccharomyces cerevisiae were studied. The uptake of both drugs was very fast, linear with concentration and not dependent on glucose, indicating entrance by diffusion and concentrating inside. Low (5.0µM) to intermediate concentrations (40µM) of both drugs produced a glucose-dependent K(+) efflux; higher ones also produced a small influx of protons, probably through a K(+)/H(+) exchanger, resulting in a decrease of the internal pH of the cells and the efflux of material absorbing at 260nm and phosphate. The cell membrane was not permeabilized. The K(+) efflux with miconazole was dependent directly on the medium pH. This efflux results in an increased membrane potential, responsible for an increased Ca(2+) uptake and other effects. These effects were not observed with two triazolic antifungals. A decrease of the Zeta (ζ) potential was observed at low concentrations of miconazole. Although the main effect of these antifungals is the inhibition of ergosterol synthesis, K(+) efflux is an important additional effect to be considered in their therapeutic use. Under certain conditions, the use of single mutants of several transporters involved in the movements of K(+) allowed to identify the participation of several antiporters in the efflux of the cation.

Characterization of glycolytic metabolism and ion transport of Candida albicans.

The main energetic pathways, fermentation and respiration, and the general ion transport properties of Candida albicans were studied. Compared to Saccharomyces cerevisiae, we found that in C. albicans: (a) the cell mass yield when grown in YPD was significantly larger; (b) it required longer times to be starved of endogenous substrates; (c) ethanol production was lower but significant; (d) respiration was also lower; (e) it showed a small activity of an alternative oxidase; (f) fermentation and oxidative phosphorylation seemed to compete for both ADP and NADH; and (g) NADH levels were lower. Regarding ion transport and compared to S. cerevisiae: (a) the general mechanism was similar, with a plasma membrane H(+) -ATPase that generates both a plasma membrane ΔpH and a ΔΨ, the latter being responsible for driving K(+) inside; (b) its acidification capacity is slightly smaller and less sensitive to activation by high pH; and (c) the presence of K(+) results in a large activation of both respiration and fermentation, most probably due to the energy required in the process. ADP produced by H(+) -ATPase stimulation by high pH or the addition of K(+) at low pH results in the increase of both respiration and fermentation.

[Sexual behavior of Alpaida veniliae (Araneae: Araueidae)]. / Comportamiento sexual de Alpaida veniliae (Araneae: Araneidae).

Studies in transgenic soybean crops in Buenos Aires province, Argentina, revealed that Alpaida veniliae is one of the most abundant species in the guild of orb web spiders. This species is an effective natural enemy of insect pests affecting this crop. In the present study we carried out a descriptive and quantitative analysis of sexual behavior (courtship, mating and post-mating) of A. veniliae. The spiders were collected in transgenic soybean crops located in Chivilcoy (35 degrees 01' S - 60 degrees 06' W), Buenos Aires, Argentina, and reared under laboratory conditions. Based on observations of 20 couples (with virgin females), behavioral units of male and female in terms of postures and movements, including details on duration and frequency, were described at all stages of sexual activity (courtship, mating and post-mating). Courtship exhibited the greatest number and duration of behavioral units in both sexes. Male and female had a sequence of 16 and nine units, respectively, being the frequency of repetitions of the units significantly higher in the male. Mating was brief and males used a single palp to fill only one of the female spermathecae, after which the female became unreceptive. Mating had two behavioral units in the male and only one in the female. During post-mating males had three and females two behavioral units. The average duration of the whole sexual behavior was 541.90 +/- 123.1 seconds for the male and 338.20 +/- 74.1 seconds for the female. Alpaida veniliae females rarely accept a second mating with the same or another male (remating), indicating a strict monogamy. In 46% of observed mating, the female cannibalized the male after it. Females became unattractive after mating, since stop producing sex pheromones, causing a reduction of the male vibratory courtship. The high cost of courtship, including the risk of cannibalism, would reinforce the selectivity of males towards receptive virgin females.

[Pelvic lymph node retrieval in cervical cancer]. / Ganglios en histerectomía por cáncer cervical. Propuesta para su recuperación.

BACKGROUND: retrieve the largest number of lymph nodes provides information to know the patient prognosis with invasive cervical cancer undergoing radical hysterectomy. The aim was to evaluate the skill of the attending surgeon to identify and recover lymph nodes. METHODS: pelvic lymphadenectomy specimens obtained from patients with cervical cancer stages IA2-IIA undergoing radical hysterectomy were studied. The surgeon or the pathologist assessed the specimens to determine the number of recovered lymph nodes. The lymph node classification was: right, left, common and total. RESULTS: the surgeon evaluated nine pelvic dissection specimens and pathologist other nine. Right lymph nodes: surgeon 14.8 ± 8, pathology 7.3 ± 3 (p < 0.01). Left lymph nodes: surgeon 14.3 ± 6, pathology 7 ± 2 (p < 0.01). Common lymph nodes: surgeon 6.6 ± 5, pathology 5.8 ± 5 (p ns). Total lymph nodes: surgeon 35.7 ± 17, pathology 20.2 ± 7 (p < 0.01). Specimens with fewer than 20 lymph nodes recovered: surgeon 1, pathologist 6 (p = 0.02). Metastatic lymph nodes: surgery 4, pathologist 2 (p = ns). CONCLUSIONS: the surgeon may retrieve a larger number of lymph nodes of pelvic lymphadenectomy specimens.