Performance of Saccharomyces cerevisiae strains against the application of adaptive laboratory evolution strategies for butanol tolerance.

Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds.

Antimicrobial potential of oregano essential oil vehiculated in Pickering cellulose nanofibers-stabilized emulsions.

Essential oils show several biological properties, such as antimicrobial activity, but have limitations regarding their availability and stability. To maximize their antimicrobial effect and protection against environmental conditions, Pickering-type emulsions were used to vehiculate oregano essential oil (OEO) using cellulose nanofibers (CNF) as emulsion stabilizer. Enzymatic hydrolysis was used to produce CNF from a food industry waste (cassava peel), obtaining an environmentally sustainable emulsion stabilizer. It was evaluated how the different properties of the nanofibers affected the stability of the emulsions. Furthermore, the composition of the dispersed phase was varied (different ratios of OEO and sunflower oil-SO) in view of the target application in biodegradable active coatings. Even at very low concentration (0.01 % w/w), CNF was able to form kinetically stable emulsions with small droplet sizes using oil mixtures (OEO + SO). The stabilization mechanism was not purely Pickering, as there was a reduction in interfacial tension. Excellent antimicrobial activity was observed against bacteria and the fungus Alternaria alternata, demonstrating the ability to apply these emulsions in active systems such as coatings and films. An improvement in the stability of emulsions was observed when using a mixture of oils, which is extremely advantageous considering costs and stability to heat treatments, since the desired antimicrobial activity is maintained for the final application.

Genomic-wide analysis of Salmonella enterica strains isolated from peanuts in Brazil.

Peanut-based products have been associated with Salmonella foodborne outbreaks and/or recalls worldwide. The ability of Salmonella to persist for a long time in a low moisture environment can contribute to this kind of contamination. The objective of this study was to analyse the genome of five S. enterica enterica strains isolated from the peanut supply chain in Brazil, as well as to identify genetic determinants for survival under desiccation and validate these findings by phenotypic test of desiccation stress. The strains were in silico serotyped using the platform SeqSero2 as Miami (M2851), Javiana (M2973), Oranienburg (M2976), Muenster (M624), and Glostrup/Chomedey (M7864); with phylogenomic analysis support. Based on Multilocus Sequence Typing (MLST) the strains were assigned to STs 140, 1674, 321, 174, and 2519. In addition, eight pathogenicity islands were found in all the genomes using the SPIFinder 2.0 (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, SPI-13, SPI-14). The absence of a SPI-4 may indicate a loss of this island in the surveyed genomes. For the pangenomic analysis, 49 S. enterica genomes were input into the Roary pipeline. The majority of the stress related genes were considered as soft-core genes and were located on the chromosome. A desiccation stress phenotypic test was performed in trypticase soy broth (TSB) with four different water activity (aw) values. M2976 and M7864, both isolated from the peanut samples with the lowest aw, showed the highest OD570nm in TSB aw 0.964 and were statistically different (p < 0.05) from the strain isolated from the peanut sample with the highest aw (0.997). In conclusion, genome analyses have revealed signatures of desiccation adaptation in Salmonella strains, but phenotypic analyses suggested the environment influences the adaptive ability of Salmonella to overcome desiccation stress.

The Complexity of Decisions in Genetics: Annotation of Three Novel Variants in the PKD1 and PKD2 Genes.

As nephrology practice is evolving toward precision medicine, and genetic tests are becoming widely available, basic genetic literacy is increasingly required for clinical nephrologists. Yet, decisions based on results of genetic tests are seldom straightforward. We report a 37-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) who was referred for medically assisted reproduction with monogenic preimplantation genetic testing (PGT-M). The PKD1 and PKD2 genes were screened for pathogenic variants. Sequencing analysis revealed the presence of three novel missense single nucleotide variants, two in the PKD1 gene - c.349T>G, p.(Leu117Val) and c.1736C>T, p.(Pro579Leu); and the third in the PKD2 gene - c.1124A>G, p.(Asn375Ser). Bioinformatic predictions of the functional effects of those three missense variants were inconsistent across different software tools. The family segregation analysis, which was mandatory to identify the relevant variant(s) for PGT-M, strongly supported that the disease-causing variant was PKD1 c.349T>G p.(Leu117Val), while the other two were nonpathogenic or, at most, phenotypic modulators. Proving the pathogenicity of novel variants is often complex but is critical to guide genetic counseling and screening, particularly when discussing reproductive alternatives for primary prevention in the progeny of at-risk couples. The family reported herein illustrates those challenges in the setting of ADPKD, and the invaluable importance of a detailed family history and segregation analysis for proper clinical annotation of novel variants. Basic genetic knowledge and proper clinical annotation of novel allelic variants in genes associated with hereditary kidney disorders are increasingly necessary for the contemporary practice of clinical nephrology.

Essential oils: An eco-friendly alternative for controlling toxigenic fungi in cereal grains.

Fungi are widely disseminated in the environment and are major food contaminants, colonizing plant tissues throughout the production chain, from preharvest to postharvest, causing diseases. As a result, grain development and seed germination are affected, reducing grain quality and nutritional value. Some fungal species can also produce mycotoxins, toxic secondary metabolites for vertebrate animals. Natural compounds, such as essential oils, have been used to control fungal diseases in cereal grains due to their antimicrobial activity that may inhibit fungal growth. These compounds have been associated with reduced mycotoxin contamination, primarily related to reducing toxin production by toxigenic fungi. However, little is known about the mechanisms of action of these compounds against mycotoxigenic fungi. In this review, we address important information on the mechanisms of action of essential oils and their antifungal and antimycotoxigenic properties, recent technological strategies for food industry applications, and the potential toxicity of essential oils.

Rituximab therapy for childhood onset idiopathic nephrotic syndrome: experience of a Portuguese tertiary center / Terapia com Rituximabe para síndrome nefrótica idiopática de início na infância: experiência de um centro terciário português

ABSTRACT Introduction: Rituximab (RTX) is a therapeutic option in pediatric difficult-to-treat idiopathic nephrotic syndrome (NS). We aimed to assess the efficacy and safety of RTX use in these patients. Method: A retrospective study of all patients with idiopathic NS treated with RTX was conducted in a pediatric nephrology division of a tertiary hospital. Demographic, anthropometric, clinical and analytical data were collected prior to treatment and at 6, 12, and 24 months. Results: Sixteen patients were included (11 males), with a median (25th-75th percentile, P25-P75) age at diagnosis of 2 (2.0-2.8) years. Fifteen were steroid-sensitive and 1 was steroid-resistant and sensitive to cyclosporine. The median age at administration of RTX was 10 (6.3-14.0) years. Throughout a median follow-up time of 2.5 (1.0-3.0) years, 6 (37.5%) patients achieved partial remission and 7 (43.8%) had no relapses and were not taking any immunosuppressants at the 24-month follow-up visit. Regarding complications, 1 patient presented persistent hypogammaglobulinemia. Compared with the 12-month period before RTX, there was a decrease in the median number of relapses at 6 and 12 months [3 (3.0-4.0) vs 0 (0-0.8) and 0.50 (0-1.0), respectively; p = 0.001] and in the daily steroids dose (mg/kg/day) at 6, 12, and 24 months [0.29 (0.15-0.67)vs [0.10 (0.07-0.13); p = 0.001], [0.12 (0.05-0.22); p = 0.005] and [0.07(0.04-0.18); p = 0.021]], respectively. There was also a reduction in the median BMI z score at 24 months [2.11 (0.45-3.70) vs. 2.93 (2.01-3.98); p = 0.049]. Conclusion: Our results confirm the efficacy and safety of RTX use in pediatric idiopathic NS and highlight its' potential cardiometabolic benefits.

Resumo Introdução: Rituximabe (RTX) é uma opção terapêutica na síndrome nefrótica (SN) idiopática pediátrica de difícil tratamento. Visamos avaliar eficácia e segurança do uso de RTX nestes pacientes. Método: Realizou-se estudo retrospectivo de todos os pacientes com SN idiopática tratados com RTX, em uma unidade de nefrologia pediátrica de um hospital terciário. Dados demográficos, antropométricos, clínicos e analíticos foram coletados antes do tratamento e aos 6, 12 e 24 meses. Resultados: Incluímos 16 pacientes (11 do sexo masculino), com idade mediana (percentil 25-75, P25-P75) de 2 (2,0-2,8) anos ao diagnóstico. Quinze eram sensíveis a esteroides, e 1 resistente a esteroides e sensível à ciclosporina.A idade mediana na administração do RTX foi 10 (6,3-14,0) anos. Durante um tempo mediano de acompanhamento de 2,5(1,0-3,0) anos, 6 (37,5%) pacientes alcançaram remissão parcial e 7 (43,8%) não tiveram recidivas e não estavam tomando imunossupressor no acompanhamento aos 24 meses. Quanto às complicações,1 paciente apresentou hipogamaglobulinemia persistente. Comparado ao período de12 meses anterior ao RTX, houve diminuição no número mediano de recidivas em 6 e 12 meses [3 (3,0-4,0) vs 0 (0-0,8) e 0,50 (0-1,0), respectivamente; p = 0,001] e na dose diária de esteroides (mg/kg/dia) aos 6, 12 e 24 meses [0,29 (0,15-0,67) >vs [0,10 (0,07-0,13); p = 0,001], [0,12 (0,05-0,22); p = 0,005] e [0,07 (0,04-0,18); p = 0,021], respectivamente. Houve também redução na mediana do escore z do IMC aos 24 meses [2,11 (0,45-3,70) vs 2,93 (2,01-3,98);p = 0,049]. Conclusões: Nossos resultados confirmam a eficácia e segurança do uso de RTX em SN idiopática pediátrica, destacando seus potenciais benefícios cardiometabólicos.

Quantitative PCR (qPCR) for estimating the presence of Fusarium and its mycotoxins in barley grains.

Members within the Fusarium sambucinum species complex (FSAMSC) are able to produce mycotoxins, such as deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN) and enniatins (ENNs) in food products. Consequently, alternative methods for assessing the levels of these mycotoxins are relevant for quick decision-making. In this context, qPCR based on key mycotoxin biosynthetic genes could aid in determining the toxigenic fungal biomass, and could therefore infer mycotoxin content. The aim of this study was to verify the use of qPCR as a technique for estimating DON, NIV, ENNs and ZEN, as well as Fusarium graminearum sensu lato (s.l.) and F. poae in barley grains. For this purpose, 53 barley samples were selected for mycobiota, mycotoxin and qPCR analyses. ENNs were the most frequent mycotoxins, followed by DON, ZEN and NIV. 83% of the samples were contaminated by F. graminearum s.l. and 51% by F. poae. Pearson correlation analysis showed significant correlations for TRI12/15-ADON with DON, ESYN1 with ENNs, TRI12/15-ADON and ZEB1 with F. graminearum s.l., as well as ESYN1 and TRI12/NIV with F. poae. Based on the results, qPCR could be useful for the assessment of Fusarium presence, and therefore, provide an estimation of its mycotoxins' levels from the same sample.

Virulence factors, antimicrobial resistance and phylogeny of bovine mastitis-associated Streptococcus dysgalactiae.

We carried out a thorough genetic evaluation of Streptococcus dysgalactiae isolated from clinical bovine mastitis cases and performed a phylogenetic analysis to represent the evolutionary relationship between S. dysgalactiae sequences. A total of 35 S. dysgalactiae strains were isolated from cases of clinical mastitis identified at a large commercial dairy farm located near Ithaca, New York. Whole-genome sequencing identified twenty-six antibiotic resistance genes, four of which were acquired genes, in addition to fifty virulence genes. Multi-locus sequence typing detected three new sequence types (STs). We conclude that a high proportion of this microorganism carries multiple virulence determinants and resistance genes, and that this indicates its potential to cause mastitis. Eight different STs were identified, of which ST453 (n = 17) was the most prevalent and ST714, ST715, ST716 were novel STs.

Effect of Gliding Arc Plasma Jet on the Mycobiota and Deoxynivalenol Levels in Naturally Contaminated Barley Grains.

Fusarium graminearum and Fusarium meridionale are primary contaminants of barley, capable of producing several mycotoxins, mainly type B trichothecenes and zearalenone. Cold plasma decontamination has been gaining prominence, seeking to control the fungal and mycotoxin contamination of food and feed and to improve product quality. To reach this objective, the present study was divided into two parts. In the first part, F. meridionale and F. graminearum strains were exposed to gliding arc plasma jet (GAPJ). Cell viability tests showed the inactivation of F. meridionale after 15-min treatment, whereas F. graminearum showed to be resistant. In the second part, barley grains were treated by GAPJ for 10, 20, and 30 min, demonstrating a reduction of about 2 log CFU/g of the barley's mycobiota, composed of yeasts, strains belonging to the F. graminearum species complex, Alternaria, and Aspergillus. A decrease in DON levels (up to 89%) was observed after exposure for 20 min. However, an increase in the toxin Deoxynivalenol-3-glucoside (D3G) was observed in barley grains, indicating a conversion of DON to D3G.

Virulence and antimicrobial resistance genes profiles of spa type t605 methicillin-susceptible Staphylococcus aureus isolated from subclinical bovine mastitis.

AIMS: Staphylococcus aureus is one of the most common pathogens associated with mastitis in dairy herds worldwide. This study evaluated the profile of virulence and antimicrobial resistance genes of spa type t605 methicillin-susceptible Staphylococcus aureus isolated from subclinical bovine mastitis in São Paulo, Brazil. METHODS AND RESULTS: A total of 57 S. aureus strains were screened by conventional PCR (Polymerase Chain Reaction) for 49 virulence genes. The most prevalent virulence genes detected were icaD (94.7%), fib (93%), fnbA (82.5%), clfA (80.7%), bap (78.9%), clfB (73.7%), icaA (66.7%), see (64.9%), and sed (61.4%). The blaZ (94.7%), aac6'aph2' (15.8%), and ant4 (12.3%) genes were the most common antimicrobial resistance genes; however, mecA and mecC genes were not found. All methicillin-susceptible S. aureus (MSSA) strains were characterized through spa and agr typing. The spa type t605 was found in all isolates. By agr typing, the most prevalent were type II (56.1%). Antimicrobial resistance was determined by the disk diffusion method, and 93% showed resistance to at least one antibiotic. Penicillin resistance was the most prevalent (87.7%), followed by tetracycline (12.3%), oxacillin (10.5%), and gentamicin (10.5%) resistance. CONCLUSION: Our study confirmed the spa type t605 as endemic, carrying a wide variety of virulence factors and high-level penicillin resistance. The profile seems to be associated with the colonization of MSSA and its persistence in subclinical mastitis.

Deoxynivalenol and zearalenone in Brazilian barley destined for brewing.

Barley is an important cereal worldwide. However, fungal contamination during pre and postharvest is a recurrent problem for barley production, causing a direct impact on the quality of the grains and their by-products due to spoilage and mycotoxin accumulation. The Fusarium graminearum species complex is the main contaminant during preharvest and some species can produce deoxynivalenol and zearalenone, important mycotoxins that represent a risk to human and animal health. This study evaluated the fungal diversity and the levels of DON and ZEN in barley grains produced in Brazil. The results showed high frequency (60%) of Fusariumcontamination in barley grains. Additionally, mycotoxin levels ranged from 46 to 2074 µg/kg for DON and from 74 to 556 µg/kg for ZEN. Co-occurrence of DON and ZEN was observed in 40% of the samples and 30% of barley samples had DON and ZEN levels higher than the maximum levels established by Brazilian and European legislations. .

Dry surface biofilms in the food processing industry: An overview on surface characteristics, adhesion and biofilm formation, detection of biofilms, and dry sanitization methods.

Bacterial biofilm formation in low moisture food processing (LMF) plants is related to matters of food safety, production efficiency, economic loss, and reduced consumer trust. Dry surfaces may appear dry to the naked eye, however, it is common to find a coverage of thin liquid films and microdroplets, known as microscopic surface wetness (MSW). The MSW may favor dry surface biofilm (DSB) formation. DSB formation is similar in other industries, it occurs through the processes of adhesion, production of extracellular polymeric substances, development of microcolonies and maturation, it is mediated by a quorum sensing (QS) system and is followed by dispersal, leading to disaggregation. Species that survive on dry surfaces develop tolerance to different stresses. DSB are recalcitrant and contribute to higher resistance to sanitation, becoming potential sources of contamination, related to the spoilage of processed products and foodborne disease outbreaks. In LMF industries, sanitization is performed using physical methods without the presence of water. Although alternative dry sanitizing methods can be efficiently used, additional studies are still required to develop and assess the effect of emerging technologies, and to propose possible combinations with traditional methods to enhance their effects on the sanitization process. Overall, more information about the different technologies can help to find the most appropriate method/s, contributing to the development of new sanitization protocols. Thus, this review aimed to identify the main characteristics and challenges of biofilm management in low moisture food industries, and summarizes the mechanisms of action of different dry sanitizing methods (alcohol, hot air, UV-C light, pulsed light, gaseous ozone, and cold plasma) and their effects on microbial metabolism.

Rituximab therapy for childhood onset idiopathic nephrotic syndrome: experience of a Portuguese tertiary center.

INTRODUCTION: Rituximab (RTX) is a therapeutic option in pediatric difficult-to-treat idiopathic nephrotic syndrome (NS). We aimed to assess the efficacy and safety of RTX use in these patients. METHOD: A retrospective study of all patients with idiopathic NS treated with RTX was conducted in a pediatric nephrology division of a tertiary hospital. Demographic, anthropometric, clinical and analytical data were collected prior to treatment and at 6, 12, and 24 months. RESULTS: Sixteen patients were included (11 males), with a median (25th-75th percentile, P25-P75) age at diagnosis of 2 (2.0-2.8) years. Fifteen were steroid-sensitive and 1 was steroid-resistant and sensitive to cyclosporine. The median age at administration of RTX was 10 (6.3-14.0) years. Throughout a median follow-up time of 2.5 (1.0-3.0) years, 6 (37.5%) patients achieved partial remission and 7 (43.8%) had no relapses and were not taking any immunosuppressants at the 24-month follow-up visit. Regarding complications, 1 patient presented persistent hypogammaglobulinemia. Compared with the 12-month period before RTX, there was a decrease in the median number of relapses at 6 and 12 months [3 (3.0-4.0) vs 0 (0-0.8) and 0.50 (0-1.0), respectively; p = 0.001] and in the daily steroids dose (mg/kg/day) at 6, 12, and 24 months [0.29 (0.15-0.67)vs [0.10 (0.07-0.13); p = 0.001], [0.12 (0.05-0.22); p = 0.005] and [0.07(0.04-0.18); p = 0.021]], respectively. There was also a reduction in the median BMI z score at 24 months [2.11 (0.45-3.70) vs. 2.93 (2.01-3.98); p = 0.049]. CONCLUSION: Our results confirm the efficacy and safety of RTX use in pediatric idiopathic NS and highlight its' potential cardiometabolic benefits.

Posterior Reversible Encephalopathy Syndrome in a Pediatric Intensive Care Unit: A Case Series.

Posterior reversible encephalopathy syndrome (PRES) is a reversible clinical-radiographic abnormality. It is characterized by headache, altered consciousness, seizures, and visual disruption, in addition to characteristic white matter edema lesions in the parieto-occipital areas of the brain. Early detection and treatment are crucial to prevent irreversible damage. This paper presents the cases of three patients with PRES with concurrent diagnoses of glomerulonephritis, Guillain-Barré syndrome, and sickle cell disease. All patients experienced systemic hypertension, seizures, and altered consciousness. All patients were admitted to intensive care for decreased level of awareness or status epilepticus requiring invasive mechanical ventilation. Anticonvulsants and antihypertensive therapy were essential. No chronic complications were recorded.

Effect of supercritical CO2 fractionation of Tahiti lemon (Citrus latifolia Tanaka) essential oil on its antifungal activity against predominant molds from pan bread.

This study aimed to verify the in vitro antifungal activity of Tahiti lemon essential oil (LEO) and its fractions, obtained by supercritical CO2 fractionation, against Penicillium sumatrense and Aspergillus niger isolated from pan bread. For this, LEO was solubilized (20 MPa and 40 °C) and fractionated (10 MPa and 40 °C) in supercritical CO2, resulting in soluble (SF) and precipitated (PF) fractions. LEO, SF and PF volatile compounds were identified by gas chromatography coupled to mass spectrometry (GC-MS) and semiquantified by gas chromatography with a flame ionization detector (GC-FID). To evaluate the in vitro antifungal activity of the essential oils (LEO, SF and PF), the minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) were determined using the 96-well plate methodology. For this, pan breads ware prepared with no preservatives and stored for seven days at 25 °C, and their pH, water activity and moisture contents were determined. Then, two predominant species (Penicillium sumatrense and Aspergillus niger) were isolated from pan breads, characterized according to their morphological and molecular characteristics, and were used in the antifungal activity studies. LEO and its fractions presented monoterpenes, sesquiterpenes and their oxygenated derivatives in their composition. Specifically, limonene was the major component identified in the essential oils. SF showed greater antifungal potential than PF and LEO, showing that supercritical CO2 fractionation could improve the antifungal efficiency of LEO. The results suggest that LEO and its fractions may contribute to the inhibition of Aspergillus niger and Penicillium sumatrense growth in pan breads.

Extraction optimization, biological activities, and application in O/W emulsion of deep eutectic solvents-based phenolic extracts from olive pomace.

This study evaluated the phenolic compound extraction from olive pomace with deep eutectic solvents (DES) prepared with choline chloride ([Ch]Cl) and four (poly-)carboxylic acids. Temperature, water addition in the solvent, and solid-liquid ratio were evaluate in total phenolic content and antioxidant activity of extracts obtained with DES and ethanol, as control. Moreover, the antimicrobial activities of solvents and extracts were evaluated. Oil-in-water emulsion with DES extract was prepared, characterized and its oxidative stability analyzed. The extract with the highest total phenolic content was obtained with [Ch]Cl:malonic acid. Under optimal conditions, DES extracted 9 % more total phenolic content than ethanol. DES extract showed superior antibacterial activity to the ethanolic extract, and its presence in oil-in-water emulsion increased the induction time in 10-fold when compared to the one prepared with water. These results reinforce that DES are a potential solvent for phenolic compound extraction from olive pomace with antibacterial and technological benefits.

Rapid assessment of enniatins in barley grains using near infrared spectroscopy and chemometric tools.

Barley is an important crop worldwide, and it can be affected by various fungi, among them Fusarium is one of the most relevant due to the economic losses caused by mycotoxin contamination. Enniantins (ENNs) are one of the emergent group of mycotoxins that have been found in grains around the world. Nowadays, the main analytical tools available to evaluate these contaminants are based on chromatographic techniques that are efficient but time-consuming and expensive. In this context, the present study aimed to assess the performance of near infrared (NIR) spectroscopy to detect and/or classify the enniatin (ENN) content on barley grains. Sixty samples of barley grains from three different regions of Brazil were investigated and the ENN content determined by UPLC-MS/MS. The levels found were then used to develop multivariate models based on infrared spectral data. The results indicated high incidence off ENN presence in the samples (>70 %) and the PLS-DA model determined by NIR data showed adequate values of sensitivity and sensibility (100 % and 94.2 %, respectively) distinguishing between contaminated and non-contaminated barley samples, demonstrating NIR as a promising tool to monitoring this emergent mycotoxin.