Doppler Ultrasound and Resistive Indices in the Diagnosis of Ureteropelvic Junction Obstruction in the Pediatric Population.

OBJECTIVES: Mercapto acetyl tri-glycine renogram (MAG3) scan has been the gold standard assessment of pediatric ureteropelvic junction obstruction (UPJO) but requires intravenous access and radiation exposure. While Doppler ultrasound measurements of resistive indices (RI) of the arcuate arteries have been proposed as an alternative assessment of obstruction, they have not been widely adopted in the pediatric population. We hypothesized that RI of the main renal artery (RA) is more strongly correlated with MAG3 findings than arcuate RI. METHODS: Pediatric patients with unilateral Society for Fetal Urology grade 3-4 hydronephrosis undergoing concomitant RUS and MAG3 were recruited. Doppler ultrasound peak systolic velocity (PSV); RI of bilateral RA at the origin, middle, and hilum; and RI of the superior, middle, and lower pole arcuate arteries were obtained. MAG3 differential renal function (DRF) and T½ were recorded. Differences in RI measurements (DRI) between the affected and normal kidney were calculated and compared with DRF and T½. RESULTS: 31 patients (median 4.6-month-old) were enrolled. Only RA RI at the origin differed between affected and normal kidneys (p < .001). DRI RA at the origin showed weak evidence for an association with MAG3 DRF < 40% (p .07). DRI was not associated with T½ > 20 minutes. CONCLUSION: There was weak evidence for an association between RA DRI at the origin and DRF but not with T½. These findings suggest that RA DRI may provide additional data in the evaluation of patients with UPJO to tailor the use of MAG3 and associated risk of radiation exposure to those patients most at risk for concomitant renal function impairment.

Predictive evolution of metabolic phenotypes using model-designed environments.

Adaptive evolution under controlled laboratory conditions has been highly effective in selecting organisms with beneficial phenotypes such as stress tolerance. The evolution route is particularly attractive when the organisms are either difficult to engineer or the genetic basis of the phenotype is complex. However, many desired traits, like metabolite secretion, have been inaccessible to adaptive selection due to their trade-off with cell growth. Here, we utilize genome-scale metabolic models to design nutrient environments for selecting lineages with enhanced metabolite secretion. To overcome the growth-secretion trade-off, we identify environments wherein growth becomes correlated with a secondary trait termed tacking trait. The latter is selected to be coupled with the desired trait in the application environment where the trait manifestation is required. Thus, adaptive evolution in the model-designed selection environment and subsequent return to the application environment is predicted to enhance the desired trait. We experimentally validate this strategy by evolving Saccharomyces cerevisiae for increased secretion of aroma compounds, and confirm the predicted flux-rerouting using genomic, transcriptomic, and proteomic analyses. Overall, model-designed selection environments open new opportunities for predictive evolution.

Importance of micronutrients and organic nitrogen in fermentations with Torulaspora delbrueckii and Saccharomyces cerevisiae.

The current use of non-Saccharomyces yeasts in mixed fermentations increases the relevance of the interactions between yeast species. In this work, the interactions between Saccharomyces cerevisiae and Torulaspora delbrueckii were analyzed. For this purpose, fermentations with and without contact between strains of those yeast species were performed in synthetic must. Fermentation kinetics, yeast growth and dynamics were measured over time. Additionally, the effects of nitrogen and other nutrient supplementations on the mixed fermentations were determined. Our results showed that S. cerevisiae did not always dominate the sequential fermentations, and experiments without yeast contact (in which T. delbrueckii cells were removed from the medium before inoculating S. cerevisiae at 48 h) resulted in stuck fermentations except when the inoculum size was increased (from 2 × 106 to 108 cells/mL) or there was a supplementation of thiamine, zinc and amino acids at the same concentration as initially found in the synthetic must. Our findings highlight the importance of inoculum size and ensuring the availability of enough micronutrients for all yeast species, especially in sequential fermentations.

Unexpected challenges faced by caregivers of children with neurogenic bladder: A qualitative study.

BACKGROUND: Management of the neurogenic bladder is variable, complex, and often requires a demanding bladder care regimen which may present caregiver burdens that are unique among chronic disease. While research into patient quality of life is increasing, parallel study of the caregiver experience is scant. Existing research primarily comprises survey data using validated instruments originally developed for non-urologic conditions, such as dementia. These surveys may detect high caregiver burden and decreased quality of life amongst caregivers but are limited in their ability to understand the underlying causes. OBJECTIVE: To characterize the experience of those caring for children with neurogenic bladders, with a focus on unexpected burdens and challenges. METHODS: In light of limited existing research, a qualitative research methodology was selected to explore the caregiver experience. Semi-structured phone interviews were conducted with primary caregivers of children with neurogenic bladder, all of whom were patients in the pediatric urology department of a single tertiary pediatric referral center. Purposive sampling was used to ensure diverse representation. Interviews were recorded, transcribed, and professionally translated if needed. Transcripts were analyzed using a team-based inductive grounded-theory approach, facilitated by ATLAS. ti software. Member-checking focus groups were held to validate the results. RESULTS: Twenty-five caregivers were interviewed (20 in English, 5 in Spanish), at which point thematic saturation was reached. Three primary themes emerged surrounding the topic of unexpected challenges: 1. High caregiver burden, 2. Challenges with catheterization and supplies, 3. Urinary tract infections. Member-checking focus groups validated the thematic analysis and provided additional insights into mitigating factors for these challenges. A child's independence with his or her health care regimen was cited as particularly important for decreasing caregiver burden. DISCUSSION: Caregivers of children with neurogenic bladder report their role is more difficult than they anticipated it would be. Catheterization represents a particularly burdensome task, and recurrent infections are an unexpected and persistent medical challenge. Understanding unexpected challenges that caregivers face will help pediatric urologists target modifiable factors to decrease caregiver burden, address current gaps in counseling and expectation-setting, and set the stage for more complete shared decision-making. CONCLUSIONS: This study represents an initial qualitative characterization of the experience caring for a child with neurogenic bladder. This is a key first step in understanding how caregivers make decisions for their children and their families. This initial study is foundational to a larger project to create a decision aid for caregivers of children with neurogenic bladder.

Feasibility of establishing a multi-center research database using the electronic health record: The PURSUIT network.

BACKGROUND: Although multi-center research is needed in pediatric urology, collaboration is impeded by differences in physician documentation and research resources. Electronic health record (EHR) tools offer a promising avenue to overcome these barriers. OBJECTIVE: To assess the accuracy, completeness, and utilization of structured data elements across multiple practices. STUDY DESIGN: A standardized template was developed and implemented at five academic pediatric urology practices to document clinic visits for patients with congenital hydronephrosis and/or vesicoureteral reflux. Data from standardized elements in the template and from pre-existing EHR fields were extracted into a secure database. A 20% random sample of infants with data from structured elements from 1/1/2020 and 4/30/2021 were identified and compared to manual chart review at sites with >100 charts; all other sites reviewed at least 20 charts. Manual chart review was standardized across sites and included: clinic and operative notes, orders linked to the clinic encounter, radiology results, and active medications. Accuracy of data extraction was evaluated by computing the kappa statistic and percentage agreement. For sites that had adopted the templates prior to 6/1/2019 (early adopters), a list of eligible patients with an initial clinic visit from 1/1/2020-7/27/2020 was generated using standardized reporting techniques and confirmed by manual chart review. Physician utilization of the template was then calculated by comparing patients with data obtained from the note template to the generated list of eligible patients. RESULTS: 230 patient records met study criteria. Agreement between manual chart review and data extracted from the EHR was high (>85%). Race, ethnicity and insurance data were misclassified in about 10-15% of cases; this was due to site-specific differences in how these fields were coded. Renal ultrasound was misclassified 12% of the time; this was primarily due to outside images documented in radiology results but not included in the clinical note. All other data elements had >90% agreement (Figure). Template utilization for early adopters was >75% (75.5-87.5%). DISCUSSION: This is the first study in urology to demonstrate that use of structured data elements can support multi-center research. Limitations include: inclusion of only academic sites with the Epic EHR and lack of data on utilization and sustainability at sites without a prior history of structured template use. CONCLUSIONS: Multi-center research collaboration using EHR-based data collection tools is feasible with generally high accuracy compared to manual chart review. Additionally, sites with a long history of template adoption have high levels of provider utilization.

Pyeloplasty with ureteral stent placement in children: Do prophylactic antibiotics serve a purpose?

OBJECTIVES: Ureteral stents are commonly used during pyeloplasty to ensure drainage and anastomotic healing. Antibiotic prophylaxis is often used due to concerns for urinary tract infection (UTI). Although many surgeons prescribe prophylactic antibiotics following pyeloplasty, practices vary widely due to lack of clear evidence-based guidelines. We hypothesize that the rate of stent UTI does not significantly vary between children who receive antibiotics and those who do not. METHODS: We reviewed the medical records of 741 patients undergoing pyeloplasty between January 2010 and July 2018 across seven institutions. Exclusion criteria were: age older than 22 years, no stent placed, externalized stents used, and incomplete records. Surgical approach, age, antibiotic use, stent duration, Foley duration, and urine culture results were recorded. Patients were categorized into two groups, those younger than four years of age and those four years and older as proxy for likely diaper use. Univariate logistic regression was conducted to identify variables associated with UTI. Multivariable backward stepwise logistic regression was used to identify the best model with Akaike information criterion as model selection criteria. The selected model was used to calculate odds ratios and 95% confidence intervals summarizing the association between prophylactic antibiotics and stent UTI while controlling for age, gender, and intra-operative urine cultures. RESULTS: 672 patients were included; 338 received antibiotic prophylaxis and 334 did not. These groups differed in mean age (3.91 vs. 6.91 years, P < .001), mean stent duration (38.5 vs. 35.32 days, P < .001), and surgical approach (53.25% vs. 32.04% open vs. laparoscopic, P < .001). The incidence of stent UTI was low overall (7.59%) and similar in both groups: 31/338 (9.17%) in the prophylaxis group and 20/334 (5.99%) in the non-prophylaxis group (P = .119). Although female gender, likely diaper use, and positive intra-operative urine culture were each associated with significantly higher odds of stent UTI, prophylactic antibiotic use was not associated with significant reduction in stent UTI in any of these groups. Surgical approach, stent duration, and Foley duration were not associated with stent UTI. CONCLUSION: Incidence of stent UTI is low overall following pyeloplasty. Prophylactic antibiotics are not associated with lower rates of stent UTI following pyeloplasty even after controlling for risk factors of female gender, likely diaper use, and positive intra-operative urine culture. Routine administration of prophylactic antibiotics after pyeloplasty does not appear to be beneficial, and may be best reserved for those with multiple risk factors for UTI.

Protective Effects of Melatonin on Saccharomyces cerevisiae under Ethanol Stress.

During alcoholic fermentation, Saccharomyces cerevisiae is subjected to several stresses, among which ethanol is of capital importance. Melatonin, a bioactive molecule synthesized by yeast during alcoholic fermentation, has an antioxidant role and is proposed to contribute to counteracting fermentation-associated stresses. The aim of this study was to unravel the protective effect of melatonin on yeast cells subjected to ethanol stress. For that purpose, the effect of ethanol concentrations (6 to 12%) on a wine strain and a lab strain of S. cerevisiae was evaluated, monitoring the viability, growth capacity, mortality, and several indicators of oxidative stress over time, such as reactive oxygen species (ROS) accumulation, lipid peroxidation, and the activity of catalase and superoxide dismutase enzymes. In general, ethanol exposure reduced the cell growth of S. cerevisiae and increased mortality, ROS accumulation, lipid peroxidation and antioxidant enzyme activity. Melatonin supplementation softened the effect of ethanol, enhancing cell growth and decreasing oxidative damage by lowering ROS accumulation, lipid peroxidation, and antioxidant enzyme activities. However, the effects of melatonin were dependent on strain, melatonin concentration, and growth phase. The results of this study indicate that melatonin has a protective role against mild ethanol stress, mainly by reducing the oxidative stress triggered by this alcohol.

A prospective survey study of lower urinary tract dysfunction in childhood cancer survivors after vincristine and/or doxorubicin chemotherapy.

BACKGROUND: Two chemotherapeutic agents used widely in pediatric oncology are vincristine (VCR) and doxorubicin (DOX), which may cause neuropathy and myopathy, respectively. The study hypothesis is that neurotoxic effects of VCR and/or myotoxic effects of DOX affect bladder physiology and manifest clinically as lower urinary tract dysfunction (LUTD). PROCEDURE: Based on a priori power analysis, 161 children divided evenly by gender were recruited. Children aged 5-10 years completed the dysfunctional voiding scoring system (DVSS) survey. The study cohort comprised cancer survivors treated with VCR and/or DOX. Healthy controls were recruited from well-child clinic visits. Exclusion criteria included pelvic-based malignancy, pelvic irradiation, pre-existing LUTD, neurologic abnormalities, and treatment with cyclophosphamide/ifosfamide. DVSS scores and presence of LUTD, defined as DVSS scores above gender-specific thresholds (males ≥9, females ≥6), were compared across cohorts. RESULTS: Median DVSS scores were higher in the study cohort (6 vs. 4, p = .003). Moreover, children in the study cohort were more likely to exceed threshold scores for LUTD (38.8% vs. 21%, p = .014; OR 1.8). Subanalysis by gender revealed female cancer survivors are more likely to report LUTD than controls (57.5% vs. 30%, p = .013, OR 1.9). This did not hold true for males (20% vs. 12.2%, p = .339). CONCLUSIONS: Childhood cancer survivors who received VCR and/or DOX reported higher rates of LUTD than controls. Female cancer survivors appear more likely to suffer from LUTD than males. Further study with a positive control cohort of cancer survivors who received non-VCR, non-DOX chemotherapy is underway to elucidate the contribution of a cancer diagnosis to LUTD.

Treatment of infants with ureteropelvic junction obstruction: findings from the PURSUIT network.

PURPOSE: Studies based on administrative databases show that infant pyeloplasty is associated with minority race/ethnicity but lack clinical data that may influence treatment. Our objective was to identify clinical and demographic factors associated with pyeloplasty in infants from three large tertiary centers. METHODS: We reviewed infants with unilateral Society for Fetal Urology (SFU) grade 3-4 hydronephrosis seen at three tertiary centers from 2/1/2018 to 9/30/2019. Patients were excluded if > 6 months old or treated surgically prior to the initial visit. Outcomes were: pyeloplasty < age 1 year and SFU grade on most recent ultrasound (US) within the first year. Covariables included: age at the initial visit, race/ethnicity, treating site, insurance type, febrile UTI, and initial imaging findings. Univariable and multivariable analyses were performed using log-rank tests and Cox proportional hazards models, respectively. RESULTS: 197 patients met study criteria; 19.3% underwent pyeloplasty. Pyeloplasty was associated with: treating site (p = 0.03), SFU 4 on initial US (p = 0.001), MAG-3 (p < 0.001), and T½ > 20 min (p < 0.001) in patients undergoing a MAG-3 (n = 107). MAG-3 (p < 0.001) and location (p = 0.08) were associated with earlier time to pyeloplasty on multivariable Cox analysis. In infants with follow-up US (n = 115), initial SFU grade, MAG-3 evaluation or findings, and pyeloplasty were not associated with improvement of hydronephrosis. CONCLUSIONS: We found that infant pyeloplasty rates vary between sites. Prolonged T½ was associated with surgery despite prior studies suggesting this is a poor predictor of worsening dilation or function. These findings suggest the need to standardize evaluation and indications for intervention in infants with suspected UPJ obstruction.

Effect of a Multistarter Yeast Inoculum on Ethanol Reduction and Population Dynamics in Wine Fermentation.

Microbiological strategies are currently being considered as methods for reducing the ethanol content of wine. Fermentations started with a multistarter of three non-Saccharomyces yeasts (Metschnikowia pulcherrima (Mp), Torulaspora delbrueckii (Td) and Zygosaccharomyces bailii (Zb)) at different inoculum concentrations. S. cerevisiae (Sc) was inoculated into fermentations at 0 h (coinoculation), 48 h or 72 h (sequential fermentations). The microbial populations were analyzed by a culture-dependent approach (Wallerstein Laboratory Nutrient (WLN) culture medium) and a culture-independent method (PMA-qPCR). The results showed that among these three non-Saccharomyces yeasts, Td became the dominant non-Saccharomyces yeast in all fermentations, and Mp was the minority yeast. Sc was able to grow in all fermentations where it was involved, being the dominant yeast at the end of fermentation. We obtained a significant ethanol reduction of 0.48 to 0.77% (v/v) in sequential fermentations, with increased concentrations of lactic and acetic acids. The highest reduction was achieved when the inoculum concentration of non-Saccharomyces yeast was 10 times higher (107 cells/mL) than that of S. cerevisiae. However, this reduction was lower than that obtained when these strains were used as single non-Saccharomyces species in the starter, indicating that interactions between them affected their performance. Therefore, more combinations of yeast species should be tested to achieve greater ethanol reductions.

Pectinatus spp. - Unpleasant and recurrent brewing spoilage bacteria.

Traditionally, beer has been recognised as a beverage with high microbiological stability because of the hostile growth environment posed by beer and increasing attention being paid to brewery hygiene. However, the microbiological risk has increased in recent years because of technological advances toward reducing oxygen in beers, besides the increase in novel beer styles production, such as non-pasteurised, flash pasteurised, cold sterilised, mid-strength, and alcoholic-free beer, that are more prone to spoilage bacteria. Moreover, using innovative beer ingredients like fruits and vegetables is an added cause of microbial spoilage. To maintain quality and good brand image, beer spoilage microorganisms are a critical concern for breweries worldwide. Pectinatus and Megasphaera are Gram-negative bacteria mostly found in improper brewing environments, leading to consumer complaints and financial losses. Because of the lack of compiled scientific knowledge on Pectinatus spoilage ability, this review provides a comprehensive overview of the occurrence, survival mechanisms, and the factors affecting beer spoilage Pectinatus species in the brewing process.

Transcriptomic Insights into the Effect of Melatonin in Saccharomyces cerevisiae in the Presence and Absence of Oxidative Stress.

Melatonin is a ubiquitous indolamine that plays important roles in various aspects of biological processes in mammals. In Saccharomyces cerevisiae, melatonin has been reported to exhibit antioxidant properties and to modulate the expression of some genes involved in endogenous defense systems. The aim of this study was to elucidate the role of supplemented melatonin at the transcriptional level in S. cerevisiae in the presence and absence of oxidative stress. This was achieved by exposing yeast cells pretreated with different melatonin concentrations to hydrogen peroxide and assessing the entry of melatonin into the cell and the yeast response at the transcriptional level (by microarray and qPCR analyses) and the physiological level (by analyzing changes in the lipid composition and mitochondrial activity). We found that exogenous melatonin crossed cellular membranes at nanomolar concentrations and modulated the expression of many genes, mainly downregulating the expression of mitochondrial genes in the absence of oxidative stress, triggering a hypoxia-like response, and upregulating them under stress, mainly the cytochrome complex and electron transport chain. Other categories that were enriched by the effect of melatonin were related to transport, antioxidant activity, signaling, and carbohydrate and lipid metabolism. The overall results suggest that melatonin is able to reprogram the cellular machinery to achieve tolerance to oxidative stress.

Viability-PCR Allows Monitoring Yeast Population Dynamics in Mixed Fermentations Including Viable but Non-Culturable Yeasts.

The use of controlled mixed inocula of Saccharomyces cerevisiae and non-Saccharomyces yeasts is a common practice in winemaking, with Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima being the most commonly used non-Saccharomyces species. Although S. cerevisiae is usually the dominant yeast at the end of mixed fermentations, some non-Saccharomyces species are also able to reach the late stages; such species may not grow in culture media, which is a status known as viable but non-culturable (VBNC). Thus, an accurate methodology to properly monitor viable yeast population dynamics during alcoholic fermentation is required to understand microbial interactions and the contribution of each species to the final product. Quantitative PCR (qPCR) has been found to be a good and sensitive method for determining the identity of the cell population, but it cannot distinguish the DNA from living and dead cells, which can overestimate the final population results. To address this shortcoming, viability dyes can be used to avoid the amplification and, therefore, the quantification of DNA from non-viable cells. In this study, we validated the use of PMAxx dye (an optimized version of propidium monoazide (PMA) dye) coupled with qPCR (PMAxx-qPCR), as a tool to monitor the viable population dynamics of the most common yeast species used in wine mixed fermentations (S. cerevisiae, T. delbrueckii, L. thermotolerans and M. pulcherrima), comparing the results with non-dyed qPCR and colony counting on differential medium. Our results showed that the PMAxx-qPCR assay used in this study is a reliable, specific and fast method for quantifying these four yeast species during the alcoholic fermentation process, being able to distinguish between living and dead yeast populations. Moreover, the entry into VBNC status was observed for the first time in L. thermotolerans and S. cerevisiae during alcoholic fermentation. Further studies are needed to unravel which compounds trigger this VBNC state during alcoholic fermentation in these species, which would help to better understand yeast interactions.

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

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

Effect of Several Nutrients and Environmental Conditions on Intracellular Melatonin Synthesis in Saccharomyces cerevisiae.

Melatonin is a bioactive compound that is present in fermented beverages and has been described to be synthesized by yeast during alcoholic fermentation. The aim of this study was to assess the capacity of intracellular and extracellular melatonin production by different Saccharomyces strains from diverse food origin and to study the effects of different fermentation parameters, such as sugar and nitrogen concentration, temperature or initial population, on melatonin production using a synthetic grape must medium. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry. Intracellular melatonin synthesis profile did not present differences between yeast strains. However, extracellular melatonin production depended on the yeast origin. Thus, we suggest that melatonin production and secretion during the different yeast growth phases follows a species-specific pattern. Other parameters that affected the fermentation process such as sugar content and low temperature had an impact on intracellular melatonin production profile, as well as the melatonin content within the cell. This study reports the effect of several conditions on the melatonin synthesis profile, highlighting its possible role as a signal molecule.

Occurrence of melatonin and indolic compounds derived from l-tryptophan yeast metabolism in fermented wort and commercial beers.

Melatonin and serotonin are bioactive compounds present in foods and beverages and related to neuroprotection and anti-angiogenesis, among other activities. They have been described in wines and the role of yeast in their formation is clear. Thus, this study evaluates the content of these bioactives and other related indolic compounds in beer. For this purpose, commercial beers were analyzed by a validated UHPLC-HRMS method and sample treatment optimized due to the low concentrations expected. Moreover, a wort was fermented with different commercial beer yeast (Abbaye, Diamond, SafAle, SafLager) in order to monitor the formation of these bioactives during the elaboration process. Results show that indolic compounds such as N-acetylserotonin and 3-indoleacetic acid are produced during the alcoholic fermentation of wort. Moreover, the occurrence of four indolic compounds (5-hydroxytryptophan, N-acetylserotonin, 3-indoleacetic acid, l-tryptophan ethyl ester) in commercial beers is reported for the first time.

A Rapid Method for Selecting Non-Saccharomyces Strains with a Low Ethanol Yield.

The alcohol content in wine has increased due to external factors in recent decades. In recent reports, some non-Saccharomyces yeast species have been confirmed to reduce ethanol during the alcoholic fermentation process. Thus, an efficient screening of non-Saccharomyces yeasts with low ethanol yield is required due to the broad diversity of these yeasts. In this study, we proposed a rapid method for selecting strains with a low ethanol yield from forty-five non-Saccharomyces yeasts belonging to eighteen species. Single fermentations were carried out for this rapid selection. Then, sequential fermentations in synthetic and natural must were conducted with the selected strains to confirm their capacity to reduce ethanol compared with that of Saccharomyces cerevisiae. The results showed that ten non-Saccharomyces strains were able to reduce the ethanol content, namely, Hanseniaspora uvarum (2), Issatchenkia terricola (1), Metschnikowia pulcherrima (2), Lachancea thermotolerans (1), Saccharomycodes ludwigii (1), Torulaspora delbrueckii (2), and Zygosaccharomyces bailii (1). Compared with S. cerevisiae, the ethanol reduction of the selected strains ranged from 0.29 to 1.39% (v/v). Sequential inoculations of M. pulcherrima (Mp51 and Mp FA) and S. cerevisiae reduced the highest concentration of ethanol by 1.17 to 1.39% (v/v) in synthetic or natural must. Second, sequential fermentations with Z. bailii (Zb43) and T. delbrueckii (Td Pt) performed in natural must yielded ethanol reductions of 1.02 and 0.84% (v/v), respectively.

Nitrogen Preferences during Alcoholic Fermentation of Different Non-Saccharomyces Yeasts of Oenological Interest.

Non-Saccharomyces yeasts have long been considered spoilage microorganisms. Currently, oenological interest in those species is increasing, mostly due to their positive contribution to wine quality. In this work, the fermentative capacity and nitrogen consumption of several non-Saccharomyces wine yeast (Torulaspora delbrueckii, Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, and Metschnikowia pulcherrima) were analyzed. For this purpose, synthetic must with three different nitrogen compositions was used: a mixture of amino acids and ammonium, only organic or inorganic nitrogen. The fermentation kinetics, nitrogen consumption, and yeast growth were measured over time. Our results showed that the good fermentative strains, T. delbrueckii and L. thermotolerans, had high similarities with Saccharomyces cerevisiae in terms of growth, fermentation profile, and nitrogen assimilation preferences, although L. thermotolerans presented an impaired behavior when only amino acids or ammonia were used, being strain-specific. M. pulcherrima was the non-Saccharomyces strain least affected by the nitrogen composition of the medium. The other two poor fermentative strains, H. uvarum and S. bacillaris, behaved similarly regarding amino acid uptake, which occurred earlier than that of the good fermentative species in the absence of ammonia. The results obtained in single non-Saccharomyces fermentations highlighted the importance of controlling nitrogen requirements of the wine yeasts, mainly in sequential fermentations, in order to manage a proper nitrogen supplementation, when needed.

Melatonin and glycolytic protein interactions are related to yeast fermentative capacity.

Melatonin is an indole amine that interacts with some proteins in mammals, such as calreticulin, calmodulin or sirtuins. In yeast, melatonin is synthetized and interacts with glycolytic proteins during alcoholic fermentation in Saccharomyces cerevisiae. Due to its importance as an antioxidant molecule in both Saccharomyces and non-Saccharomyces yeasts, the aim of this study was to determine the intracellular and extracellular synthesis profiles of melatonin in four non-Saccharomyces strains (Torulaspora delbrueckii, Hanseniaspora uvarum, Starmeralla bacillaris and Metschnikowia pulcherrima) and to confirm whether glycolytic enzymes can also interact with this molecule in non-conventional yeast cells. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry, and proteins bound to melatonin were immunopurified by melatonin-IgG-Dynabeads. Melatonin was produced in a similar pattern in all non-Saccharomyces yeast, with M. pulcherrima and S. bacillaris being the highest producers. However, melatonin only bound to proteins in two non-conventional yeasts, S. bacillaris and T. delbrueckii, which specifically had higher fermentative capacities. Sequence analysis showed that most proteins shared high levels of homology with glycolytic enzymes, but an RNA-binding protein, the elongation alpha factor, which is related to mitochondria, was also identified. This study reports for the first time the interaction of melatonin with proteins inside non-Saccharomyces yeast cells. These results reinforce the possible role of melatonin as a signal molecule, likely related to fermentation metabolism and provide a new perspective for understanding its role in yeast.

Glycolytic Proteins Interact With Intracellular Melatonin in Saccharomyces cerevisiae.

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