Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments

[EN] Deep investigation of the microbiome of food-production and foodprocessing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.SIThis work was funded by the European Commission under the European Union’s Horizon 2020 research and innovation program under grant agreement no. 818368 (MASTER). C.B. is grateful to Junta de Castilla y León and the European Social Fund for awarding her a pre-doctoral grant (BOCYL-D-07072020-6). A.P. is grateful to Ministerio de Ciencia e Innovación for awarding her a pre-doctoral grant (PRE2021-098910). N.M.Q. is currently funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 101034371. We thank AV Star Systems for their role in creating the Supplementary Video, and M. Coakley and S. Mortensen for their help in its preparation

Orbital Meningoencephalocele Due to Extraventricular Neurocytoma: Case Report

Extraventricular Neurocytoma (EVN) is a rare primary tumor of Central Nervous System (CNS). To date, no cases have been reported in International Literature, about EVN associated to meningoencephalocele as manifestation of subacute increased intracranial pressure.We report the first case of EVN that manifested with diplopia and ocular motor disorders due to intraorbital meningoencephalocele through a bone gap in the orbital roof.Diagnosis of EVN is challenging and its differential diagnosis with more aggressive lesions is mandatory. Complete surgical removal does not need any adiuvant therapies and permits to control tumor growth and symptoms related to intracranial pressure.</p

Discordance between Genotypic and Phenotypic Drug Resistance Profiles in Human Immunodeficiency Virus Type 1 Strains Isolated from Peripheral Blood Mononuclear Cells

The aim of the study was to analyze the relationship between genotypic and phenotypic drug resistance profiles of human immunodeficiency virus type 1 (HIV-1) strains isolated from patients during double-analogue nucleoside therapy. A drug-resistant HIV strain was isolated from 20 out of 25 patients, with 16 (64%) subjects carrying a virus with multiple drug resistance mutations. The most frequent resistance mutations were M184V (18 isolates) and M41L (7 isolates). Discordance between the genotypic and phenotypic profile for at least one drug was detected in 16 out of 25 strains. Particularly, eight isolates had a discordant genotypic-phenotypic resistance pattern for two drugs and one isolate had such a pattern for three drugs. A genotypic resistance pattern with a phenotypic sensitivity profile was detected in six isolates (four resistant to zidovudine and two resistant to lamivudine). On the other hand for several strains a genotypic pattern of sensitivity pattern to abacavir (10 strains), didanosine (7 strains), stavudine (3 strains), zidovudine (2 strains), and lamivudine (1 strain) with a phenotypic resistance profile was detected. After a follow-up period of 8 months, an impairment of virological and immunological parameters was detected only in subjects with an HIV-1 isolate with a phenotypic resistance profile in despite of the genotypic results. Predicting resistance phenotype from genotypic data has important limitations. Despite the low number of patients and the short follow-up period, this study suggests that during failing therapy with analogue nucleosides, a phenotypic analysis could be performed in spite of an HIV genotypic sensitivity pattern

Cardioprotective Effects of Nanoemulsions Loaded with Anti-Inflammatory Nutraceuticals against Doxorubicin-Induced Cardiotoxicity

Doxorubicin is a highly active antineoplastic agent, but its clinical use is limited because of its cardiotoxicity. Although nutraceuticals endowed with anti-inflammatory properties exert cardioprotective activity, their bioavailability and stability are inconsistent. In an attempt to address this issue, we evaluated whether bioavailable nanoemulsions loaded with nutraceuticals (curcumin and fresh and dry tomato extracts rich in lycopene) protect cardiomyoblasts (H9C2 cells) from doxorubicin-induced toxicity. Nanoemulsions were produced with a high-pressure homogenizer. H9C2 cells were incubated with nanoemulsions loaded with different nutraceuticals alone or in combination with doxorubicin. Cell viability was evaluated with a modified MTT method. The levels of the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxy-2-butanone (4-HNA), and of the cardiotoxic-related interleukins IL-6, IL-8, IL-1&beta; and IL-10, tumor necrosis factor-alpha (TNF-&alpha;), and nitric oxide were analyzed in cardiomyoblasts. The hydrodynamic size of nanoemulsions was around 100 nm. Cell viability enhancement was 35&ndash;40% higher in cardiomyoblasts treated with nanoemulsion + doxorubicin than in cardiomyoblasts treated with doxorubicin alone. Nanoemulsions also protected against oxidative stress as witnessed by a reduction of MDA and 4-HNA. Notably, nanoemulsions inhibited the release of IL-6, IL-8, IL-1&beta;, TNF-&alpha; and nitric oxide by around 35&ndash;40% and increased IL-10 production by 25&ndash;27% versus cells not treated with emulsions. Of the nutraceuticals evaluated, lycopene-rich nanoemulsions had the best cardioprotective profile. In conclusion, nanoemulsions loaded with the nutraceuticals described herein protect against cardiotoxicity, by reducing inflammation and lipid oxidative stress. These results set the stage for studies in preclinical models