Cheese whey: a cost-effective alternative for hyaluronic acid production by Streptococcus zooepidemicus

This study focuses on the optimization of cheese whey formulated media for the production of hyaluronic acid HA by Streptococcus zooepidemicus. Culture media containing whey (W; 2.1 g/L) or whey hydrolysate (WH; 2.4 g/L) gave the highest HA productions. Both W and WH produced high yields on protein consumed, suggesting cheese whey is a good nitrogen source for S. zooepidemicus production of HA. Polysaccharide concentrations of 4.0 g/L and 3.2 g/L were produced in W and WH in a further scale-up to 5 L bioreactors, confirming the suitability of the low-cost nitrogen source. Cheese whey culture media provided high molecular weight (> 3000 kDa) HA products. This study revealed replacing the commercial peptone by the low-cost alternative could reduce HA production costs by up to a 70%compared to synthetic media.Isabel Rodriguez was funded by a postdoctoral contract from the Xunta de Galicia, Spain (Plan I2C, 2012). This research was financially supported by projects: MAT 2010-21509-C03-01 (Ministerio de Economia y Competitividad, Spain) and FP7 Project BiValBi. Biotechnologies to Valorise the regional food Biodiversity in Latin America (PIRSES-GA-2013-611493). The authors want to thank Benigno Pereira, the manager of Queizuar S.L. (A Coruna, Spain) for providing the cheese whey utilised to conduct this research. We also wish to thank Ana Duran and Margarita Nogueira (IIM-CSIC) for their excellent technical assistance

3D printed functional cookies fortified with Arthrospira platensis: Evaluation of its antioxidant potential and physical-chemical characterization

In the last few decades, consumers' growing attention to the close relationship between health and nutrition is emerging as a new trend, mostly regarding the incorporation of natural ingredients into food. Among those ingredients, microalgae are considered as innovative and promising compounds, rich in valuable nutrients and bioactive molecules. In the present work, 3D printed cookies were fortified with the microalga Arthrospira platensis aiming at developing a new functional food with antioxidant properties. A. platensis antioxidants were recovered using ultrasound-assisted extraction in hydroalcoholic solutions. Ethanol/water and biomass/solvent ratios were optimised through a Design of Experiments (DOE) approach, using the antioxidant activity (ORAC and ABTS) and total phenolic content (TPC) as response variables. The highest ORAC, ABTS and TPC values were observed in the extract obtained with 0% ethanol and 2.0% biomass; thus, this extract was chosen to be incorporated into a printable cookie dough. Three different incorporation approaches were followed: (1) dried biomass, (2) freeze-dried antioxidant extract and (3) antioxidant extract encapsulated into alginate microbeads to enhance the stability to heat, light, and oxygen during baking and further storage. All dough formulations presented shape fidelity with the 3D model. The cookies had aw values low enough to be microbiologically stable, and the texture remained constant after 30 days of storage. Moreover, the extract encapsulation promoted an improvement in the ORAC value and colour stability when compared to all other formulations, revealing the potential of A. platensis for the development of a functional 3D food-ink.This work was funded by the European Union INTERREG Atlantic Area Programme and the European Regional Development Fund (ERDF) through the project “Enhance Microalgae: High added-value industrial opportunities for microalgae in the Atlantic Area” (Ref. EAPA_338/2016).info:eu-repo/semantics/publishedVersio

Bio-based nanoparticles as a carrier of β-carotene: production, characterisation and in vitro gastrointestinal digestion

β-carotene loaded bio-based nanoparticles (NPs) were produced by the solvent-displacement method using two polymers: zein and ethylcellulose. The production of NPs was optimised through an experimental design and characterised in terms of average size and polydispersity index. The processing conditions that allowed to obtain NPs (<100 nm) were used for β-carotene encapsulation. Then β-carotene loaded NPs were characterised in terms of zeta potential and encapsulation efficiency. Transmission electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction analysis were performed for further morphological and chemical characterisation. In the end, a static in vitro digestion following the INFOGEST protocol was performed and the bioaccessibility of β-carotene encapsulated in both NPs was determined. Results show that the best conditions for a size-controlled production with a narrow size distribution are lower polymer concentrations and higher antisolvent concentrations. The encapsulation of β-carotene in ethylcellulose NPs resulted in nanoparticles with a mean average size of 60 ± 9 nm and encapsulation efficiency of 74 ± 2%. β-carotene loaded zein-based NPs resulted in a mean size of 83 ± 8 nm and encapsulation efficiency of 93 ± 4%. Results obtained from the in vitro digestion showed that β-carotene bioaccessibility when encapsulated in zein NPs is 37 ± 1%, which is higher than the value of 8.3 ± 0.1% obtained for the ethylcellulose NPs.This research was funded by “MobFood—Mobilizing scientific and technological knowledge in response to the challenges of the agri-food market” (POCI-01-0247-FEDER-024524; LISBOA-01-0247-FEDER024524), by “MobFood” Consortium, and financed by European Regional Development Fund (ERDF), through the Incentive System to Research and Technological development, within the Portugal2020 Competitiveness and Internationalization Operational Program. The research also received funding from the European Union’s H2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement N 778388 (H2020MSCA-RISE-2017 project Food for Diabetes and Cognition (FODIAC), and grant agreement N 713640 (MSCA2015-COFUND-FP), and MICRODIGEST project (grant agreement 037716) co-funded by FCT and ERDF through COMPETE2020.info:eu-repo/semantics/publishedVersio

Regulation of histone H2A.Z expression is mediated by sirtuin 1 in prostate cancer

Histone variants seem to play a major role in gene expression regulation. In prostate cancer, H2A.Z and its acetylated form are implicated in oncogenes’ upregulation. SIRT1, which may act either as tumor suppressor or oncogene, reduces H2A.Z levels in cardiomyocytes, via proteasome-mediated degradation, and this mechanism might be impaired in prostate cancer cells due to sirtuin 1 downregulation. Thus, we aimed to characterize the mechanisms underlying H2A.Z and SIRT1 deregulation in prostate carcinogenesis and how they interact. We found that H2AFZ and SIRT1 were up- and downregulated, respectively, at transcript level in primary prostate cancer and high-grade prostatic intraepithelial neoplasia compared to normal prostatic tissues. Induced SIRT1 overexpression in prostate cancer cell lines resulted in almost complete absence of H2A.Z. Inhibition of mTOR had a modest effect on H2A.Z levels, but proteasome inhibition prevented the marked reduction of H2A.Z due to sirtuin 1 overexpression. Prostate cancer cells exposed to epigenetic modifying drugs trichostatin A, alone or combined with 5-aza-2’-deoxycytidine, increased H2AFZ transcript, although with a concomitant decrease in protein levels. Conversely, SIRT1 transcript and protein levels increased after exposure. ChIP revealed an increase of activation marks within the TSS region for both genes. Remarkably, inhibition of sirtuin 1 with nicotinamide, increased H2A.Z levels, whereas activation of sirtuin 1 by resveratrol led to an abrupt decrease in H2A.Z. Finally, protein-ligation assay showed that exposure to epigenetic modifying drugs fostered the interaction between sirtuin 1 and H2A.Z. We concluded that sirtuin 1 and H2A.Z deregulation in prostate cancer are reciprocally related. Epigenetic mechanisms, mostly histone post-translational modifications, are likely involved and impair sirtuin 1-mediated downregulation of H2A.Z via proteasome-mediated degradation. Epigenetic modifying drugs in conjunction with enzymatic modulators are able to restore the normal functions of sirtuin 1 and might constitute relevant tools for targeted therapy of prostate cancer patient

Superparamagnetic iron oxide nanoparticles decorated mesoporous silica nanosystem for combined antibiofilm therapy

A crucial challenge to face in the treatment of biofilm-associated infection is the ability of bacteria to develop resistance to traditional antimicrobial therapies based on the administration of antibiotics alone. This study aims to apply magnetic hyperthermia together with controlled antibiotic delivery from a unique magnetic-responsive nanocarrier for a combination therapy against biofilm. The design of the nanosystem is based on antibiotic-loaded mesoporous silica nanoparticles (MSNs) externally functionalized with a thermo-responsive polymer capping layer, and decorated in the outermost surface with superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs are able to generate heat upon application of an alternating magnetic field (AMF), reaching the temperature needed to induce a change in the polymer conformation from linear to globular, therefore triggering pore uncapping and the antibiotic cargo release. The microbiological assays indicated that exposure of E. coli biofilms to 200 µg/mL of the nanosystem and the application of an AMF (202 kHz, 30 mT) decreased the number of viable bacteria by 4 log10 units compared with the control. The results of the present study show that combined hyperthermia and antibiotic treatment is a promising approach for the effective management of biofilm-associated infections.Depto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

Evaluation of antimicrobial effectiveness of pimaricin-loaded thermosensitive nanohydrogels in grape juice

Pimaricin-loaded poly(N-isopropylacrylamide) nanohydrogels with and without acrylic acid, were evaluated as food-spoilage inhibitors in a model system and a real food product: grape juice. Pimaricin was proposed as a non-allergenic alternative to sulphites for protecting juices against recontamination. However, pimaricin may degrade under conditions and treatments (heating, acidification, lighting) commonly applied in producing fresh juices. Nanohydrogel encapsulation may be a feasible procedure to avoid pimaricin degradation, improving its antimicrobial activity. Pimaricin-free nanohydrogels did not affect the growth of the indicator yeast either in the food model system or in grape juice. Conversely, pimaricin-loaded nanohydrogels effectively inhibited the growth of the indicator yeast. In some cases, the inhibition was extended even further than using free pimaricin. For instance, in the food model system, pimaricin-loaded nanohydrogels with acrylic acid (NPPNIPA-20AA(5)) prevented the yeast growth for more than 81 h while free pimaricin was only effective for 12 h. Despite pimaricin-loaded nanohydrogels without acrylic acid (NPPNIPA(5)) were able to reduce maximum yeast growth, as in all treatments with pimaricin, the extent of the inhibitory effect was not significantly (p>0.05) different to that achieved with free pimaricin. In grape juice, both free pimaricin and NPPNIPA-20AA(5) treatment completely inhibited the growth of the indicator yeast until the end of the bioassay. However, the latter provided similar inhibition levels using a smaller amount of pimaricin due to PNIPA-20AA(5) protection and its controlled release from the nanohydrogel. Therefore, nanohydrogel encapsulation may help to optimise antifungal treatments and decrease the incidence of food allergies.Funded by grant (MAT 2006-11662-CO3-CO2-C01/MAT 2010-21509-C03-01/EUI 2008-00115) from the “Ministerio de Educación y Ciencia” (Spain). Grant (SFRH/BPD/87910/2012) from the Fundação para a Ciência e Tecnologia (FCT, Portugal). Marie Curie COFUND Postdoctoral Research Fellow

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.This work was supported by grants from Spanish Health Ministry (FIS) network RIRAAF (RD 07/0064).Ye

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Pediocin SA-1: A selective bacteriocin for controlling Listeria monocytogenes in maize silages

In this study, we assessed the potential as silage additive of a bacteriocin produced by Pediococcus acidilactici Northern Regional Research Laboratory (NRRL) B-5627 (pediocin SA-1). Maize was inoculated either with a bacterial starter alone (I) or in combination with the bacteriocin (IP), and untreated silage served as control. We monitored the products of fermentation (ethanol, and lactic and acetic acids), the microbial population, and the presence of the indicator strain Listeria monocytogenes Colección Española de Cultivos Tipo (CECT) 4032 (1 × 105 cfu/g) after 1, 2, 5, 8, 16, and 30 d of ensiling. Our results indicated antilisterial activity of the bacteriocin, anticipating the disappearance of L. monocytogenes in IP compared with I and control silages. The PCR-denaturing gradient gel electrophoresis analysis revealed the addition of the bacteriocin did not affect the bacterial communities of the spontaneous fermentation, and the inoculant-containing bacteria (Lactobacillus plantarum, Lactobacillus buchneri, and Enterococcus faecium) were found in addition to the bacterial communities of untreated maize silages in I and IP silages. Both treatments increased the concentration of antimicrobial compounds (acetic acid, ethanol, and 1,2-propanodiol) and led to lower residual sugar contents compared with the control, which would provide enhanced aerobic stability. The fact that the identified species L. plantarum, L. buchneri, and E. faecium produce some of these inhibitory compounds, together with their persistence throughout the 30 d of fermentation, suggest these bacteria could actively participate in the ensiling process. According to these results, pediocin SA-1 could be used as an additive to control the presence of L. monocytogenes in maize silages selectively, while improving their fermentative quality and eventually their aerobic stability.Isabel Rodríguez and Clara Fuciños were funded by a postdoctoral contract from the Xunta de Galicia, Spain (Plan I2C in 2012 and 2014, respectively). This research was financially supported by FP7 Project BiValBi (Biotechnologies to Valorise the regional food Biodiversity in Latin America; PIRSES-GA-2013-611493). We thank the Centro de Investigaciones Agrarias de Mabegondo for providing the maize used in this research. We also thank Pioneer Hi-Bred International Inc. for the inoculants and information supplied