A Simple Polymicrobial Biofilm Keratinocyte Colonization Model for Exploring Interactions Between Commensals, Pathogens and Antimicrobials

© 2020 Jordana-Lluch, Garcia, Kingdon, Singh, Alexander, Williams and Hardie. Skin offers protection against external insults, with the skin microbiota playing a crucial defensive role against pathogens that gain access when the skin barrier is breached. Linkages between skin microbes, biofilms and disease have not been well established although single-species biofilm formation by skin microbiota in vitro has been extensively studied. Consequently, the purpose of this work was to optimize and validate a simple polymicrobial biofilm keratinocyte model for investigating commensal, pathogen and keratinocyte interactions and for evaluating therapeutic agents or health promoting interventions. The model incorporates the commensals (Staphylococcus epidermidis and Micrococcus luteus) and pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) which form robust polymicrobial biofilms on immortalized keratinocytes (HaCat cells). We observed that the commensals reduce the damage caused to the keratinocyte monolayer by either pathogen. When the commensals were combined with P. aeruginosa and S. aureus, much thinner biofilms were observed than those formed by the pathogens alone. When P. aeruginosa was inoculated with S. epidermidis in the presence or absence of M. luteus, the commensals formed a layer between the keratinocytes and pathogen. Although S. aureus completely inhibited the growth of M. luteus in dual-species biofilms, inclusion of S. epidermidis in triple or quadruple species biofilms, enabled M. luteus to retain viability. Using this polymicrobial biofilm keratinocyte model, we demonstrate that a quorum sensing (QS) deficient S. aureus agr mutant, in contrast to the parent, failed to damage the keratinocyte monolayer unless supplied with the exogenous cognate autoinducing peptide. In addition, we show that treatment of the polymicrobial keratinocyte model with nanoparticles containing an inhibitor of the PQS QS system reduced biofilm thickness and P. aeruginosa localization in mono- and polymicrobial biofilms

Robust and scalable barcoding for massively parallel long‑read sequencing

Nucleic-acid barcoding is an enabling technique for many applications, but its use remains limited in emerging long-read sequencing technologies with intrinsically low raw accuracy. Here, we apply so-called NS-watermark barcodes, whose error correction capability was previously validated in silico, in a proof of concept where we synthesize 3840 NS-watermark barcodes and use them to asymmetrically tag and simultaneously sequence amplicons from two evolutionarily distant species (namely Bordetella pertussis and Drosophila mojavensis) on the ONT MinION platform. To our knowledge, this is the largest number of distinct, non-random tags ever sequenced in parallel and the frst report of microarray-based synthesis as a source for large oligonucleotide pools for barcoding. We recovered the identity of more than 86% of the barcodes, with a crosstalk rate of 0.17% (i.e., one misassignment every 584 reads). This falls in the range of the index hopping rate of established, highaccuracy Illumina sequencing, despite the increased number of tags and the relatively low accuracy of both microarray-based synthesis and long-read sequencing. The robustness of NS-watermark barcodes, together with their scalable design and compatibility with low-cost massive synthesis, makes them promising for present and future sequencing applications requiring massive labeling, such as long-read single-cell RNA-Seq.Fil: Ezpeleta, Joaquín. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina.Fil: Labari, Ignacio Garcia. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina.Fil: Bulacio, Pilar. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina.Fil: Tapia, Elizabeth. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina.Fil: Ezpeleta, Joaquín. Universidad Nacional de Rosario. Facultad de Ciencias Exactas, Ingeniería y Agrimensura; Argentina.Fil: Bulacio, Pilar. Universidad Nacional de Rosario. Facultad de Ciencias Exactas, Ingeniería y Agrimensura; Argentina.Fil: Tapia, Elizabeth. Universidad Nacional de Rosario. Facultad de Ciencias Exactas, Ingeniería y Agrimensura; Argentina.Fil: Villanova, Gabriela Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Lavista Llanos, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Villanova, Gabriela Vanina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática. Centro Científico Tecnológico y Educativo Acuario del Río Paraná; Argentina.Fil: Posner, Victoria María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática. Centro Científico Tecnológico y Educativo Acuario del Río Paraná; Argentina.Fil: Arranz, Silvia Eda. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática. Centro Científico Tecnológico y Educativo Acuario del Río Paraná; Argentina.Fil: Krsticevic, Flavia. The Hebrew University of Jerusalem. Robert H Smith Faculty of Agriculture, Food and Environment; Israel

The quorum sensing system of Yersinia enterocolitica 8081 regulates swimming motility, host cell attachment, and virulence plasmid maintenance

Although Yersinia enterocolitica genomes are highly heterogeneous, they contain a conserved N-acylhomoserine lactone-dependent (AHL) quorum sensing (QS) system consisting of the luxR and luxI orthologs yenR and yenI respectively. Certain hypervirulent strains also contain a putative orphan luxR gene, ycoR, that is not linked to an AHL synthase. To explore the contribution of yenR/yenI/ycoR to QS-dependent phenotypes in Yersinia enterocolitica strain 8081, single and multiple mutants were constructed. AHL profiling identified N-(3-oxohexanoyl) homoserine lactone, N-hexanoylhomoserine lactone, and N-(3-oxoseptanoyl) homoserine lactone as the most abundant. The AHL profiles of the yenR, ycoR and yenR/ycoR mutants were similar to the parent suggesting that the two LuxR homologues do not regulate AHL production while the yenI mutants were AHL-negative. A role for QS in swimming motility and cell attachment was demonstrated. Down-regulation of the virulence plasmid partition gene, spyA, in yenI and yenI/yenR/ycoR mutants is consistent with the greater loss of the Y. enterocolitica pYVe virulence plasmid in the yenI mutant during serial passage at 37 C but not at 22 C. A role for QS-regulated spyA in virulence plasmid maintenance is suggested

The Opa1-Dependent Mitochondrial Cristae Remodeling Pathway Controls Atrophic, Apoptotic and Ischemic Tissue Damage

SummaryMitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo

Age-Associated Loss of OPA1 in Muscle Impacts Muscle Mass, Metabolic Homeostasis, Systemic Inflammation, and Epithelial Senescence

Mitochondrial dysfunction occurs during aging, but its impact on tissue senescence is unknown. Here, we find that sedentary but not active humans display an age-related decline in the mitochondrial protein, optic atrophy 1 (OPA1), that is associated with muscle loss. In adult mice, acute, muscle-specific deletion of Opa1 induces a precocious senescence phenotype and premature death. Conditional and inducible Opa1 deletion alters mitochondrial morphology and function but not DNA content. Mechanistically, the ablation of Opa1 leads to ER stress, which signals via the unfolded protein response (UPR) and FoxOs, inducing a catabolic program of muscle loss and systemic aging. Pharmacological inhibition of ER stress or muscle-specific deletion of FGF21 compensates for the loss of Opa1, restoring a normal metabolic state and preventing muscle atrophy and premature death. Thus, mitochondrial dysfunction in the muscle can trigger a cascade of signaling initiated at the ER that systemically affects general metabolism and aging

Multifaceted role of TREX2 in the skin defense against UV-induced skin carcinogenesis

TREX2 is a 3'-DNA exonuclease specifically expressed in keratinocytes. Here, we investigated the relevance and mechanisms of TREX2 in ultraviolet (UV)-induced skin carcinogenesis. TREX2 expression was up-regulated by chronic UV exposure whereas it was de-regulated or lost in human squamous cell carcinomas (SCCs). Moreover, we identified SNPs in the TREX2 gene that were more frequent in patients with head and neck SCCs than in healthy individuals. In mice, TREX2 deficiency led to enhanced susceptibility to UVB-induced skin carcinogenesis which was preceded by aberrant DNA damage removal and degradation as well as reduced inflammation. Specifically, TREX2 loss diminished the up-regulation of IL12 and IFNγ, key cytokines related to DNA repair and antitumor immunity. In UV-treated keratinocytes, TREX2 promoted DNA repair and passage to late apoptotic stages. Notably, TREX2 was recruited to low-density nuclear chromatin and micronuclei, where it interacted with phosphorylated H2AX histone, which is a critical player in both DNA repair and cell death. Altogether, our data provide new insights in the molecular mechanisms of TREX2 activity and establish cell autonomous and non-cell autonomous functions of TREX2 in the UVB-induced skin response

A DNA barcode-assisted annotated checklist of the spider (Arachnida, Araneae) communities associated to white oak woodlands in Spanish National Parks

Background: A large scale semi-quantitative biodiversity assessment was conducted in white oak woodlands in areas included in the Spanish Network of National Parks, as part of a project aimed at revealing biogeographic patterns and identify biodiversity drivers. The semiquantitative COBRA sampling protocol was conducted in sixteen 1-ha plots across six national parks using a nested design. All adult specimens were identified to species level based on morphology. Uncertain delimitations and identifications due to either limited information of diagnostic characters or conflicting taxonomy were further investigated using DNA barcode information. New information: We identified 376 species belonging to 190 genera in 39 families, from the 8,521 adults found amongst the 20,539 collected specimens. Faunistic results include the discovery of 7 new species to the Iberian Peninsula, 3 new species to Spain and 11 putative new species to science. As largely expected by environmental features, the southern parks showed a higher proportion of Iberian and Mediterranean species than the northern parks, where the Palearctic elements were largely dominant. The analysis of approximately 3,200 DNA barcodes generated in the present study, corroborated and provided finer resolution to the morphologically based delimitation and identification of specimens in some taxonomically challenging families. Specifically, molecular data confirmed putative new species with diagnosable morphology, identified overlooked lineages that may constitute new species, confirmed assignment of specimens of unknown sexes to species and identified cases of misidentifications and phenotypic polymorphisms

Measurement of the Tau Lepton Polarisation at LEP2

A first measurement of the average polarisation P_tau of tau leptons produced in e+e- annihilation at energies significantly above the Z resonance is presented. The polarisation is determined from the kinematic spectra of tau hadronic decays. The measured value P_tau = -0.164 +/- 0.125 is consistent with the Standard Model prediction for the mean LEP energy of 197 GeV.A first measurement of the average polarisation Pτ of tau leptons produced in e + e − annihilation at energies significantly above the Z resonance is presented. The polarisation is determined from the kinematic spectra of tau hadronic decays. The measured value Pτ=−0.164±0.125 is consistent with the Standard Model prediction for the mean LEP energy of 197 GeV.A first measurement of the average polarisation P_tau of tau leptons produced in e+e- annihilation at energies significantly above the Z resonance is presented. The polarisation is determined from the kinematic spectra of tau hadronic decays. The measured value P_tau = -0.164 +/- 0.125 is consistent with the Standard Model prediction for the mean LEP energy of 197 GeV

National trends in total cholesterol obscure heterogeneous changes in HDL and non-HDL cholesterol and total-to-HDL cholesterol ratio : a pooled analysis of 458 population-based studies in Asian and Western countries

Background: Although high-density lipoprotein (HDL) and non-HDL cholesterol have opposite associations with coronary heart disease, multi-country reports of lipid trends only use total cholesterol (TC). Our aim was to compare trends in total, HDL and nonHDL cholesterol and the total-to-HDL cholesterol ratio in Asian and Western countries. Methods: We pooled 458 population-based studies with 82.1 million participants in 23 Asian and Western countries. We estimated changes in mean total, HDL and non-HDL cholesterol and mean total-to-HDL cholesterol ratio by country, sex and age group. Results: Since similar to 1980, mean TC increased in Asian countries. In Japan and South Korea, the TC rise was due to rising HDL cholesterol, which increased by up to 0.17 mmol/L per decade in Japanese women; in China, it was due to rising non-HDL cholesterol. TC declined in Western countries, except in Polish men. The decline was largest in Finland and Norway, at similar to 0.4 mmol/L per decade. The decline in TC in most Western countries was the net effect of an increase in HDL cholesterol and a decline in non-HDL cholesterol, with the HDL cholesterol increase largest in New Zealand and Switzerland. Mean total-to-HDL cholesterol ratio declined in Japan, South Korea and most Western countries, by as much as similar to 0.7 per decade in Swiss men (equivalent to similar to 26% decline in coronary heart disease risk per decade). The ratio increased in China. Conclusions: HDL cholesterol has risen and the total-to-HDL cholesterol ratio has declined in many Western countries, Japan and South Korea, with only a weak correlation with changes in TC or non-HDL cholesterol.Peer reviewe

Repositioning of the global epicentre of non-optimal cholesterol

High blood cholesterol is typically considered a feature of wealthy western countries(1,2). However, dietary and behavioural determinants of blood cholesterol are changing rapidly throughout the world(3) and countries are using lipid-lowering medications at varying rates. These changes can have distinct effects on the levels of high-density lipoprotein (HDL) cholesterol and non-HDL cholesterol, which have different effects on human health(4,5). However, the trends of HDL and non-HDL cholesterol levels over time have not been previously reported in a global analysis. Here we pooled 1,127 population-based studies that measured blood lipids in 102.6 million individuals aged 18 years and older to estimate trends from 1980 to 2018 in mean total, non-HDL and HDL cholesterol levels for 200 countries. Globally, there was little change in total or non-HDL cholesterol from 1980 to 2018. This was a net effect of increases in low- and middle-income countries, especially in east and southeast Asia, and decreases in high-income western countries, especially those in northwestern Europe, and in central and eastern Europe. As a result, countries with the highest level of non-HDL cholesterol-which is a marker of cardiovascular riskchanged from those in western Europe such as Belgium, Finland, Greenland, Iceland, Norway, Sweden, Switzerland and Malta in 1980 to those in Asia and the Pacific, such as Tokelau, Malaysia, The Philippines and Thailand. In 2017, high non-HDL cholesterol was responsible for an estimated 3.9 million (95% credible interval 3.7 million-4.2 million) worldwide deaths, half of which occurred in east, southeast and south Asia. The global repositioning of lipid-related risk, with non-optimal cholesterol shifting from a distinct feature of high-income countries in northwestern Europe, north America and Australasia to one that affects countries in east and southeast Asia and Oceania should motivate the use of population-based policies and personal interventions to improve nutrition and enhance access to treatment throughout the world.Peer reviewe