Cystine-mediated oxidative defence in Lactobacillus reuteri BR11

Lactobacillus reuteri BR11 possesses an abundant cystine uptake (Cyu) ABC-transporter that was previously found to be involved in a novel mechanism of oxidative defence mediated by cystine. The current study aimed to elucidate this mechanism with a focus on the role of the co-transcribed cystathionine ã-lyase (Cgl). Growth studies of wild-type L. reuteri BR11 and mutants inactivated in cgl and the cystine-binding protein encoding gene cyuC showed that in contrast to the Cyu transporter, whose inactivation led to growth arrest in aerated cultures, Cgl is not crucial for oxidative defence. However, the role of Cgl in oxidative defence became apparent in the presence of severe oxidative damage and cysteine deprivation. Cysteine was found to be protective against oxidative stress, and the action of Cgl in both cysteine biosynthesis and degradation poses a seemingly futile pathway that deprives the intracellular cysteine pool. To further characterise the relationship between Cgl activity and cysteine and their roles in oxidative defence, enzymatic assays were performed on purified Cgl, and intracellular concentrations of cysteine, cystathionine and methionine were determined. Cgl was highly active towards cystine and cystathionine and less active towards cysteine in vitro, suggesting the main function of Cgl to be cysteine biosynthesis. Cysteine was found at high concentrations in the cell, but the levels were not significantly affected by inactivation of cgl or growth under aerobic conditions. It was concluded that both anabolic and catabolic activities of Cgl towards cysteine contribute to oxidative defence, the former by maintaining an intracellular reservoir of thiol analogous to glutathione, and the latter by producing H2S which is readily secreted, thus creating a reducing extracellular environment. The significance of the Cyu transporter to the physiology of L. reuteri BR11 prompted a phylogenetic study to determine its presence in bacteria. Orthologs of the Cyu transporter that are closest matches to the Cyu transporter are only limited to several species of Lactobacillus and Leuconostoc. Outside the Lactobacillales order, the closest matching orthologs belong to Proteobacteria, and there are more orthologs in Proteobacteria than non-Lactobacillales Firmicutes, suggesting that the Cyu transporter locus was present in the ancestor of the Proteobacteria and Firmicutes, and over evolutionary time has been lost or diverged in many Firmicutes. The clustering of the Cyu transporter locus with a gene encoding a Cgl family protein is even rarer. It was only found in L. reuteri, Lactobacillus vaginalis, Weissella paramesenteroides, the Lactobacillus casei group, and several Campylobacter sp. An accompanying phylogenetic study of L. reuteri BR11 using multi-locus sequence analysis showed that L. reuteri BR11 had diverged from more than 100 strains of L. reuteri isolated from various hosts and geographical locations. However, comparison with other Lactobacillus species supported the current classification of BR11 as L. reuteri. The most closely related species to L. reuteri is L. vaginalis or Lactobacillus antri, depending on the housekeeping gene used for analysis. The close evolutionary relationship of L. vaginalis to L. reuteri and the high degree of sequence identity between the cgl-cyuABC loci in both species suggest that the Cyu system is highly likely to perform similar functions in L. vaginalis. In search of other genes that function in oxidative defence, a number of mutants which were inactivated in genes that confer increased resistance to oxidative stress in other bacteria were constructed. The genes targeted were ahpC (peroxidase component of the alkyl hydroperoxide reductase system), tpx (thiol peroxidase), osmC (osmotically induced protein C), mntH (Mn2+/Fe2+ transporter), gshA (ã-glutamylcysteine synthetase) and msrA (methionine sulfoxide reductase). The ahpC and mntH mutants had slightly lower minimum inhibitory concentrations of organic peroxides, suggesting these genes might be involved in resistance to organic peroxides in L. reuteri. However, none of the mutants exhibited growth defects in aerated cultures, in stark contrast to the cyuC mutant. This may be due to compensatory functions of other genes, a hypothesis which cannot be tested until a robust protocol for constructing markerless multiple gene deletion mutants in L. reuteri is developed. These results highlight the importance of the Cyu transporter in oxidative defence and provide a foundation for extending the research of this system in other bacteria

Culture-independent bacterial community profiling of carbon dioxide treated raw milk

Due to technical simplicity and strong inhibition against the growth of psychrotrophic bacteria in milk, CO treatment has emerged as an attractive processing aid to increase the storage time of raw milk before downstream processing. However, it is yet to be adopted by the industry. In order to further explore the suitability of CO treatment for raw milk processing, the bacterial populations of carbonated raw milk collected locally from five different sources in Australia were analysed with next-generation sequencing. Growth inhibition by CO was confirmed, with spoilage delayed by at least 7\ua0days compared with non-carbonated controls. All non-carbonated controls were spoiled by Gammaproteobacteria, namely Pseudomonas fluorescens group bacteria, Serratia and Erwinia. Two out of the five carbonated samples shared the same spoilage bacteria as their corresponding controls. The rest of the three carbonated samples were spoiled by the lactic acid bacterium (LAB) Leuconostoc. This is consistent with higher tolerance of LAB towards CO and selection of LAB in meat products stored in CO-enriched modified atmosphere packaging. No harmful bacteria were found to be selected by CO. LAB are generally regarded as safe (GRAS), thus the selection for Leuconostoc by CO in some of the samples poses no safety concern. In addition, we have confirmed previous findings that 454 pyrosequencing and Illumina sequencing of 16S rRNA gene amplicons from the same sample yield highly similar results. This supports comparison of results obtained with the two different sequencing platforms, which may be necessary considering the imminent discontinuation of 454 pyrosequencing

A genetic diversity study of antifungal Lactobacillus plantarum isolates

Lactobacillus plantarum is a lactic acid bacterium commonly found on fruits and vegetables and also used in a variety of food fermentations. Strains from this species are also regularly reported as having antifungal or probiotic activity. Genotyping methods can be used to differentiate strains of the same species thus determining if strains are related or not. However for L.\ua0plantarum, the currently used methods have limitations including DNA band profile interpretation difficulty and cost. In this study, a new genotyping method based on multi-locus variable number tandem repeat analysis (MLVA) was developed and compared to a previously reported randomly amplified polymorphic DNA-PCR (RAPD-PCR) method for L.\ua0plantarum. With a selection of 13 antifungal strains of L.\ua0plantarum isolated from heterogeneous sources (cheese, silage, sauerkraut, vegetables and a probiotic product), RAPD-PCR revealed 9 different profiles resulting in a Hunter-Gaston discrimination index (D-value) of 0.94. The new MLVA method which compares the lengths of 4 repetitive regions within LPXTG motif-containing surface protein genes differentiated the 13\ua0L.\ua0plantarum strains into 10 different subtypes leading to a D-value of 0.95. Interestingly 11 additional L.\ua0plantarum isolates obtained in a previous study during a screen for antifungal activity against the common cheese spoilage mould Penicillium commune all possessed the same RAPD-PCR and MLVA profile as each other and the commercial probiotic strain L.\ua0plantarum 299v. This study demonstrates that the new MLVA method can be used to simply and inexpensively differentiate L.\ua0plantarum strains and provide information regarding strain relatedness and thus potential insight into strain properties

Inhibition of bacterial growth in sweet cheese whey by carbon dioxide as determined by culture-independent community profiling

Whey is a valuable co-product from cheese making that serves as a raw material for a wide range of products. Its rich nutritional content lends itself to rapid spoilage, thus it typically needs to be pasteurised and refrigerated promptly. Despite the extensive literature on milk spoilage bacteria, little is known about the spoilage bacteria of whey. The utility of carbon dioxide (CO) to extend the shelf-life of raw milk and cottage cheese has been well established, but its application in whey preservation has not yet been explored. This study aims to characterise the microbial populations of fresh and spoiled sweet whey by culture-independent community profiling using 454 pyrosequencing of 16S rRNA gene amplicons and to determine whether carbonation is effective in inhibiting bacterial growth in sweet whey. The microbiota of raw Cheddar and Mozzarella whey was dominated by cheese starter bacteria. After pasteurisation, two out of the three samples studied became dominated by diverse environmental bacteria from various phyla, with Proteobacteria being the most dominant. Diverse microbial profiles were maintained until spoilage occurred, when the entire population was dominated by just one or two genera. Whey spoilage bacteria were found to be similar to those of milk. Pasteurised Cheddar and Mozzarella whey was spoiled by Bacillus sp. or Pseudomonas sp., and raw Mozzarella whey was spoiled by Pseudomonas sp., Serratia sp., and other members of the Enterobacteriaceae family. CO was effective in inhibiting bacterial growth of pasteurised Cheddar and Mozzarella whey stored at 15°C and raw Mozzarella whey stored at 4°C. The spoilage bacteria of the carbonated samples were similar to those of the non-carbonated controls

Comparison of next generation technologies and bioinformatics pipelines for capsular typing of Streptococcus pneumoniae

Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. In silico serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (n = 17; 14.3%), 14 (n = 10; 8.4%), and 15B/C (n = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.This study has been funded in part by Fondo Europeo de Desarrollo Regional (FEDER) and the Ministry of Science and Innovation, Instituto de Salud Carlos III (ISCIII) through the project "PI19/00104" (Principal Investigator: C.M.A.), the predoctoral Contract for Training in Research into Health “FI17/00248” (Recipient: D.H.), and the grant “PID2020–119298RB-I00“ (Recipient: J.Y.). CMA also received a research grant from Pfizer laborato ries and Fundación Godia paid to the Sant Joan de Déu foundation. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S

Interação medicamentosa em pacientes com câncer: revisão integrativa da literatura / Drug interaction in cancer patients: an integrative literature review

Introdução: O câncer é uma patologia considerada como doença crônica não transmissível, que na sociedade atual, se encontra entre as 7 principais causas de morte no mundo, segundo estimativas da Organização Mundial da Saúde (OMS). Pacientes com neoplasias comumente realizam tratamentos paralelos além dos realizados para combater o câncer, como para tratar: outras doenças crônicas pré - existentes, reações adversas a medicamentos causadas pelo tratamento primário além da auto medicação o que causa as interações medicamentosas as quais desencadeiam efeitos prejudiciais à saúde do paciente, como intoxicação medicamentosa, efeito nulo do medicamento, não tratamento da doença e ênfase nos efeitos adversos daqueles medicamentos Objetivo: objetivou-se analisar as incidência de interação medicamentosa em pacientes com câncer, submetidos ao tratamento quimioterápico. Metodologia: Foi realizada uma revisão bibliográfica por meio da pesquisa de artigos científicos. As bases de dados utilizadas foram BVS, Scielo, Lilacs e PubMed, e foram utilizados como descritores para a pesquisa: “interação medicamentosa e câncer”, “interação medicamentosa, oncologia e quimioterapia” “potenciais interações medicamentosas, neoplasias e agentes anticâncer” “drug interaction and cancer”, “drug interaction oncology and chemotherapy” e “potential drug interactions neoplasms anticancer agents”. Resultados: Foram incluídos os artigos originais completos de acordo com o tema proposto. Foram selecionados quatorze artigos após aplicação dos critérios de inclusão e exclusão. O número médio de medicamentos ministrados por paciente foi entre 7 a 11,7 e as bases de dados variaram entre: Micromedex, Epocrats, Drug Interactions Facts, MedScape e Lexi Interact. O medicamento que foi comumente envolvido nas interações com agentes anticâncer foi a dexametasona, um agente indutor do Citocromo P450 (CYP 450). Considerações finais: Esta revisão demonstrou uma alta prevalência de interações medicamentosa devido à complexidade da farmacoterapia, sendo essencial que se adote métodos para minimização desses dados, enfatizando a necessidade de um foco global intensificado na prevenção de interação em doenças como o câncer

Definite and indeterminate nonalcoholic steatohepatitis share similar clinical features and prognosis: A longitudinal study of 1893 biopsy-proven nonalcoholic fatty liver disease subjects

[Background and Aim] Histological score systems may not fully capture the essential nonalcoholic steatohepatitis (NASH) features, which is one of the leading causes of screening failure in clinical trials. We assessed the NASH distribution and its components across the fibrosis stages and their impact on the prognosis and their relationship with the concept of metabolic-associated fatty liver disease (MAFLD).[Methods] Spanish multicenter study including 1893 biopsy-proven nonalcoholic fatty liver disease (NAFLD) patients from HEPAmet registry. NASH was diagnosed by NAS score ≥4 (including steatosis, ballooning and lobular inflammation) and fibrosis by Kleiner score. The presence of MAFLD was determined. Progression to cirrhosis, first episode of decompensated cirrhosis and death were collected during the follow-up (4.7 ± 3.8 years).[Results] Fibrosis was F0 34.3% (649/1893), F1 27% (511/1893), F2 16.5% (312/1893), F3 15% (284/1893) and F4 7.2% (137/1893). NASH diagnosis 51.9% (982/1893), and its individual components (severe steatosis, ballooning and lobular inflammation), increased from F0 (33.6%) to F2 (68.6%), and decreased significantly in F4 patients (51.8%) (P = .0001). More than 70% of non-NASH patients showed some inflammatory activity (ballooning or lobular inflammation), showing a similar MAFLD rate than NASH (96.2% [945/982] vs. 95.2% [535/562]) and significantly higher than nonalcoholic fatty liver (NAFL) subjects (89.1% [311/349]) (P < .0001). Progression to cirrhosis was similar between NASH (9.5% [51/539]) and indeterminate NASH (7.9% [25/316]), and higher than steatosis (5% [14/263]) (logRank 8.417; P = .015). Death and decompensated cirrhosis were similar between these.[Conclusions] The prevalence of steatohepatitis decreased in advanced liver disease. However, most of these patients showed some inflammatory activity histologically and had metabolic disturbances. These findings should be considered in clinical trials whose main aim is to prevent cirrhosis progression and complications, liver transplant and death.This project has been partially funded by the ‘Consejería de Salud de la Junta de Andalucía’ (PI-0075-2014) and the ‘Spanish Ministry of Economy, Innovation and Competition, Instituto de Salud Carlos III’ (PI19/01404, PI16/01842, PI17/00535 and GLD19/00100).Peer reviewe

Diversity in composition of scarlet (S. aethiopicum) and gboma (S. macrocarpon) eggplants and of interspecific hybrids between S. aethiopicum and common eggplant (S. melongena)

[EN] Scarlet (Solanum aethiopicum) and gboma (S. macrocarpon) eggplants are cultivated vegetable crops native to Africa, for which no comprehensive reports exist on composition and diversity. We have evaluated diversity in composition of three varieties of scarlet eggplant and four varieties of gboma eggplant, as well as of four interspecific hybrids between scarlet and common eggplant (S. melongena) and their respective parents. With the exception of moisture (between 85.8 and 88.3 g/100 g) and pH (between 5.32 and 5.89), there was a wide diversity among varieties within each of the species for the composition traits evaluated, revealing ample possibilities for selection of varieties with improved fruit composition. Scarlet eggplant varieties evaluated presented, on average, lower content than gboma eggplant varieties for carbohydrates (3.60 vs 6.48 g/100 g), starch (3.18 vs 6.15 g/100 g), vitamin C (11.6 vs 18.9 mg/100 g), and total phenolics (24.4 vs 144 mg/100 g) and higher values for soluble sugars content and for the ascorbic/dehydroascorbic acid ratio. Interspecific hybrids between scarlet and gboma eggplants presented moisture content (79.0 g/100 g) and pH (5.15) values below those of any of the parents. For the rest of traits, values were intermediate between both parents, although much more similar to the scarlet eggplant parent. (C) 2015 Elsevier Inc. All rights reserved.This work was partially financed by the Ministerio de Ciencia y Tecnologı´a and FEDER (AGL2012-34213) and project OTRI-UCMFundacio´n Sabor y Salud (323-2012).San José, R.; Plazas Ávila, MDLO.; Sánchez-Mata, MC.; Cámara Hurtado, MM.; Prohens Tomás, J. (2016). Diversity in composition of scarlet (S. aethiopicum) and gboma (S. macrocarpon) eggplants and of interspecific hybrids between S. aethiopicum and common eggplant (S. melongena). Journal of Food Composition and Analysis. 45:130-140. https://doi.org/10.1016/j.jfca.2015.10.009S1301404

A novel procedure to measure the antioxidant capacity of Yerba maté extracts

Yerba maté extracts have in vitro antioxidant capacity attributed to the presence of polyphenolic compounds, mainly chlorogenic acids and dicaffeoylquinic acid derivatives. DPPH is one of the most used assays to measure the antioxidant capacity of pure compounds and plant extracts. It is difficult to compare the results between studies because this assay is applied in too many different conditions by the different research groups. Thus, in order to assess the antioxidant capacity of yerba maté extracts, the following procedure is proposed: 100 µL of an aqueous dilution of the extracts is mixed in duplicate with 3.0 mL of a DPPH 'work solution in absolute methanol (100 µM.L-1), with an incubation time of 120 minutes in darkness at 37 ± 1 °C, and then absorbance is read at 517 nm against absolute methanol. The results should be expressed as ascorbic acid equivalents or Trolox equivalents in mass percentage (g% dm, dry matter) in order to facilitate comparisons. The AOC of the ethanolic extracts ranged between 12.8 and 23.1 g TE % dm and from 9.1 to 16.4 g AAE % dm. The AOC determined by the DPPH assay proposed in the present study can be related to the total polyphenolic content determined by the Folin-Ciocalteu assay

The extinct Sicilian wolf shows a complex history of isolation and admixture with ancient dogs

The Sicilian wolf remained isolated in Sicily from the end of the Pleistocene until its extermination in the 1930s–1960s. Given its long-term isolation on the island and distinctive morphology, the genetic origin of the Sicilian wolf remains debated. We sequenced four nuclear genomes and five mitogenomes from the seven existing museum specimens to investigate the Sicilian wolf ancestry, relationships with extant and extinct wolves and dogs, and diversity. Our results show that the Sicilian wolf is most closely related to the Italian wolf but carries ancestry from a lineage related to European Eneolithic and Bronze Age dogs. The average nucleotide diversity of the Sicilian wolf was half of the Italian wolf, with 37–50% of its genome contained in runs of homozygosity. Overall, we show that, by the time it went extinct, the Sicilian wolf had high inbreeding and low-genetic diversity, consistent with a population in an insular environmen