Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages

open access articlePurpose: The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on. Materials and methods: Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage–HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen. Results: Our data highlighted that the biomimetic HA nanocrystal–bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions. Conclusion: Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin

African genomes illuminate the early history and transition to selfing in Arabidopsis thaliana

Over the past 20 y, many studies have examined the history of the plant ecological and molecular model, Arabidopsis thaliana, in Europe and North America. Although these studies informed us about the recent history of the species, the early history has remained elusive. In a large-scale genomic analysis of African A. thaliana, we sequenced the genomes of 78 modern and herbarium samples from Africa and analyzed these together with over 1,000 previously sequenced Eurasian samples. In striking contrast to expectations, we find that all African individuals sampled are native to this continent, including those from sub-Saharan Africa. Moreover, we show that Africa harbors the greatest variation and represents the deepest history in the A. thaliana lineage. Our results also reveal evidence that selfing, a major defining characteristic of the species, evolved in a single geographic region, best represented today within Africa. Demographic inference supports a model in which the ancestral A. thaliana population began to split by 120-90 kya, during the last interglacial and Abbassia pluvial, and Eurasian populations subsequently separated from one another at around 40 kya. This bears striking similarities to the patterns observed for diverse species, including humans, implying a key role for climatic events during interglacial and pluvial periods in shaping the histories and current distributions of a wide range of species.Peer Reviewe

The Union is Strength: The Synergic Action of Long Fatty Acids and a Bacteriophage against Xanthomonas campestris Biofilm

Xanthomonas campestris pv. campestris is known as the causative agent of black rot disease, which attacks mainly crucifers, severely lowering their global productivity. One of the main virulence factors of this pathogen is its capability to penetrate and form biofilm structures in the xylem vessels. The discovery of novel approaches to crop disease management is urgent and a possible treatment could be aimed at the eradication of biofilm, although anti-biofilm approaches in agricultural microbiology are still rare. Considering the multifactorial nature of biofilm, an effective approach against Xanthomonas campestris implies the use of a multi-targeted or combinatorial strategy. In this paper, an anti-biofilm strategy based on the use of fatty acids and the bacteriophage (Xccφ1)-hydroxyapatite complex was optimized against Xanthomonas campestris mature biofilm. The synergic action of these elements was demonstrated and the efficient removal of Xanthomonas campestris mature biofilm was also proven in a flow cell system, making the proposed approach an effective solution to enhance plant survival in Xanthomonas campestris infections. Moreover, the molecular mechanisms responsible for the efficacy of the proposed treatment were explored

A two-step adaptive walk rewires nutrient transport in a challenging edaphic environment

Most well-characterized cases of adaptation involve single genetic loci. Theory suggests that multilocus adaptive walks should be common, but these are challenging to identify in natural populations. Here, we combine trait mapping with population genetic modeling to show that a two-step process rewired nutrient homeostasis in a population of Arabidopsis as it colonized the base of an active stratovolcano characterized by extremely low soil manganese (Mn). First, a variant that disrupted the primary iron (Fe) uptake transporter gene (IRT1) swept quickly to fixation in a hard selective sweep, increasing Mn but limiting Fe in the leaves. Second, multiple independent tandem duplications occurred at NRAMP1 and together rose to near fixation in the island population, compensating the loss of IRT1 by improving Fe homeostasis. This study provides a clear case of a multilocus adaptive walk and reveals how genetic variants reshaped a phenotype and spread over space and time

Italian Guidelines in diagnosis and treatment of alopecia areata

Alopecia areata (AA) is an organ-specific autoimmune disorder that targets anagen phase hair follicles. The course is unpredictable and current available treatments have variable efficacy. Nowadays, there is relatively little evidence on treatment of AA from well-designed clinical trials. Moreover, none of the treatments or devices commonly used to treat AA are specifically approved by the Food and Drug Administration. The Italian Study Group for Cutaneous Annexial Disease of the Italian Society of dermatology proposes these Italian guidelines for diagnosis and treatment of Alopecia Areata deeming useful for the daily management of the disease. This article summarizes evidence-based treatment associated with expert-based recommendations

Ancient pigs reveal a near-complete genomic turnover following their introduction to Europe

Archaeological evidence indicates that pig domestication had begun by ~10,500 y before the present (BP) in the Near East, and mitochondrial DNA (mtDNA) suggests that pigs arrived in Europe alongside farmers ~8,500 y BP. A few thousand years after the introduction of Near Eastern pigs into Europe, however, their characteristic mtDNA signature disappeared and was replaced by haplotypes associated with European wild boars. This turnover could be accounted for by substantial gene flow from local Euro-pean wild boars, although it is also possible that European wild boars were domesticated independently without any genetic con-tribution from the Near East. To test these hypotheses, we obtained mtDNA sequences from 2,099 modern and ancient pig samples and 63 nuclear ancient genomes from Near Eastern and European pigs. Our analyses revealed that European domestic pigs dating from 7,100 to 6,000 y BP possessed both Near Eastern and European nuclear ancestry, while later pigs possessed no more than 4% Near Eastern ancestry, indicating that gene flow from European wild boars resulted in a near-complete disappearance of Near East ancestry. In addition, we demonstrate that a variant at a locus encoding black coat color likely originated in the Near East and persisted in European pigs. Altogether, our results indicate that while pigs were not independently domesticated in Europe, the vast majority of human-mediated selection over the past 5,000 y focused on the genomic fraction derived from the European wild boars, and not on the fraction that was selected by early Neolithic farmers over the first 2,500 y of the domestication process

LACTOFERRIN ADSORBED ONTO BIOMIMETIC HYDROXYAPATITE: A MULTIFUNCTIONAL ANTIMICROBIAL MOLECULE

Lactoferrin (LF), is an iron-binding protein, belonging to transferrin family. It is found in the mucosal secretions (tears, saliva, milk, and colostrums) of the majority of mammalian species, including humans. LF is a multitask molecule: it partecipates to iron absorption and distribution, but also displays anti-inflammatory, antioxidant, anticarcinogenic, and antimicrobial properties. Hydroxyapatite (HA) plays an important role in the formation of the bony skeleton and bone remodeling. Alteration of HA metabolism can cause serious clinical consequences, such as arterial calcification, chronic kidney disease or osteoporosis. HA is also used in numerous bioengineering and biomedical applications - due to its bioactivity and osteoconductivity – and also as vehicle for drug targeting, bone scaffolds and implant coating materials, filler in polymeric matrices. In this project, the biological properties (such as antimicrobial, immunomodulatory and antioxidant activities) of lactoferrin adsorbed onto biomimetic hydroxyapatatite nanocrystals (LF-HA) were evaluated. After the biological characterization, the complex was embedded in an active packaging system (on a cling film) - by electrodeposition - to estimate the antioxidant property of this active packaging system. Packaging protects food from external pathogen contamination and environmental factors (oxygen, moisture and heat), providing longer product shelf life and avoiding the quality decay. Shelf life prolongation is possible by controlling enzymatic and microbial activities through the control of temperature, addition of salt, or by removing oxygen. In recent years, foods preserved with natural additives have become very popular due to the great consumer awareness and concern regarding synthetic chemical additives. The tests carried out showed the antimicrobial and antioxidant efficacy of LF-HA versus the lactoferrin alone, when tested at the same concentrations, and the effectiveness of this molecule embedded in a food packaging system. The results obtained permit to conclude that the chemical manipulation of the lactoferrin significantly improved the antimicrobial and antioxidant activities of the native molecule. The main characteristics of the food packaging system obtained by electrodeposition of LF-HA on cling film are: uniform coating with LF-HA obtained throughout a new method of electrodeposition; long lasting stability, when stored for six months or longer at room temperature before being used to protect the food; stable to deep freezing, for six months or more

Data from: Adaptation of perennial flowering phenology across the European range of Arabis alpina

<p>Perennial <em>Arabis alpina</em> has a wide geographic distribution and is adapted to local environments. However, the traits that underlie adaptation are unknown. Flowering phenology is an adaptive trait in other species, but its geographic variation has not been systematically studied in herbaceous perennials.</p> <p>Accessions of <em>A. alpina</em> were collected across the European range. Their flowering behavior was tested in controlled conditions, in experimental common-garden plantations at native sites and <em>in-situ</em> in natural populations. Also, genetic diversity within and among populations was examined.</p> <p>French Alpine and Scandinavian accessions varied in timing and duration of flowering. By contrast, in controlled conditions and <em>in-situ</em>, all Spanish accessions were obligate vernalization-requiring with a short duration of flowering. Nevertheless, Spanish populations were as genetically diverse as French Alpine populations and more so than Scandinavian populations. Furthermore, <em>perpetual flowering 1</em>, a mutant that shows no vernalization requirement and a long duration of flowering, showed higher mortality and poorer performance than local accessions at Spanish experimental sites.</p> <p>We propose that in this perennial species, the vernalization requirement and short duration of flowering are under selection in Spain as a strategy to survive exposure to longer, warmer growing seasons.</p><p>Funding provided by: Deutsche Forschungsgemeinschaft<br>Crossref Funder Registry ID: https://ror.org/018mejw64<br>Award Number: SPP1529</p><p>Funding provided by: Deutsche Forschungsgemeinschaft<br>Crossref Funder Registry ID: https://ror.org/018mejw64<br>Award Number: CRC TRR341</p><p>Funding provided by: Swedish Research Council<br>Crossref Funder Registry ID: http://dx.doi.org/10.13039/501100004359<br>Award Number: </p><h4>Genotyping and whole-genome sequencing</h4> <p class="Normal1">Genetic variation and differentiation of <em>A. alpina</em> populations were analyzed with genotype and whole-genome sequence data. First, DNA was extracted from fresh leaves of 31 individuals from 9 populations representing Spanish, French Alpine and Scandinavian populations (using Qiagen DNeasy Plant Maxi Kit, Qiagen) and whole genomes were sequenced on an Illumina HiSeq2000 at the Max Planck Genome Centre Cologne and at FASTERIS sequencing service in Switzerland (<a href="http://www.fasteris.com">www.fasteris.com</a>). From these sequences, 35788 unambiguous biallelic SNPs were identified (details in Supporting Information). A subset of 253 widely spread, intergenic SNPs was used for genotyping on a Custom GoldenGate SNP-panel. The SNP set was chosen to be informative across the entire sampled geographic range and/or within regions and populations. Then, 892 accessions were genotyped using DNA extracted from leaf material collected <em>in-situ </em>(Supporting Information). These accessions include: i) a range-wide collection; ii) an evenly subsampled set of 50 accessions from each of three populations from each of the three main regions; iii) a total of 187 Spanish accessions from 10 populations to analyze in detail population structure in Spain. </p> <p class="Normal1">In addition, genetic variation was analyzed genome-wide on an evenly subsampled set of the whole genome sequences, comprising eight individuals from two populations in both Spain and the French Alps, and eleven individuals from four populations in Scandinavia, for a total of 27 individuals. Average coverage for these samples varied between 7.2X and 23.1X (mean across samples = 15.9X).</p> <h4>Phenotyping experiments</h4> <p class="Normal1">Flowering and fitness traits were scored by phenotyping plants in controlled greenhouse conditions and at experimental sites established within or next to native populations of <em>A. alpina</em>. Some traits were also scored <em>in situ</em>. Details are given in Supporting Information. </p> <h4>Greenhouse experiments</h4> <p class="Normal1">Flowering behavior was scored under greenhouse conditions following two different experimental treatments. i) Experiment without vernalization: Plants were grown under long days (LD: 16h/8h day/night cycle, 22°/16°C) and scored for flowering traits twice a week, for 26 to 51 weeks after germination across four replicates of the experiment (Table S1) ii) Experiment with vernalization: Plants were grown in long days for 6 weeks, then vernalized for about 14 weeks (short days, SD: 8h/16h day/night cycle, 4°C), and then transferred back into LD and scored for 19 weeks. In each experiment, most natural populations were represented by at least 20 maternal families and at least 8 siblings per family, except experiment 4 in which smaller family sizes were used (Table S1). As controls, the reference accession <em>Pajares </em>from Spain and the early-flowering <em>pep1-1</em> mutant were used in all experiments. All plants were randomized in all experiments.</p> <h4>Experimental sites   </h4> <p class="Normal1">Flowering behavior and fitness of plants grown at experimental sites were scored in Spain, the French Alps and Scandinavia, close to or within native populations, and in Germany outside the natural range. Plants were pre-grown in small plugs for about 4 weeks in a greenhouse to ensure high establishment success. The seedlings were then transplanted into natural soil at the experimental sites in spring. Apart from watering on the first few days after planting, the plants grew under natural environmental conditions and in native soil, and they were scored at the end of the growing season the year of plantation and the following year. Each population was represented by about 20 maternal families and 10 siblings per family (Table S1). As controls, we used the reference accession <em>Pajares</em> from Spain and the early-flowering <em>pep1-1</em> mutant. The individuals were planted in a randomized block design.</p> <h4>Observations <em>in-situ</em> </h4> <p class="Normal1">Plants in natural populations were labelled, photographed and scored for various traits 2-5 times per year over several years. To estimate the duration of flowering the photographic documentation was evaluated for a period of five years (Supporting Information).</p> <h4>Phenotypic traits</h4> <p class="Normal1">For flowering behavior under controlled greenhouse conditions, the following traits were scored: i) the onset of flowering without vernalization, which was the time between germination and appearance of the first open flower, ii) the onset of flowering after vernalization, which was the time from the end of the vernalization treatment to the appearance of the first open flower, iii) the duration of flowering after vernalization, which was the time between appearance of the first and the last open flower and iv) the proportion of flowering plants, which was the proportion of plants that produced visible open flowers. Plants were assigned to three categories for the onset of flowering without vernalization: "Early-onset" plants flowered within four months of germination (this reflects the average snow-free period in the Scandinavian and French Alpine habitats). "Late-onset" plants required more than four months from germination to flowering. "Not flowering" plants produced no open flowers without vernalization during the experiment (up to 51 weeks).</p> <p class="Normal1">At experimental sites, we scored survival, as the percentage of surviving plants, and the proportion of flowering plants, inferred by the proportion of plants bearing siliques. Scoring was done at the end of the growing season the year of plantation and the following year. At the Spanish site we additionally scored the number of rosettes for vegetative performance, and the average number of siliques as an indication of fecundity and fitness (Supporting Information).</p> <p class="Normal1">In natural populations (<em>in-situ</em>) individual plants were labeled, visited 2-5 times per year, photographed and scored for whether or not they were flowering. The proportion of plants showing an extended (perpetual) flowering duration was estimated at the end of the growing season, typically late September to early October.</p> <h4>Weather data</h4> <p class="Normal1">Air temperature was measured with logging intervals ≤ 45 minutes by sensors installed about 20 cm above the soil surface in a subset of the natural populations and at all experimental sites. Records were standardized to hourly means from which daily means were calculated. Periods of snow coverage were estimated based on oscillations ≤ 1°C in daily temperature, indicating that the sensor was covered by snow. The growing season was defined as the number of days between the midpoints of the first and last snow-free, 5-day period with mean temperature > 5°C, following the definition of (Sletvold & Agren, 2015).</p> <p class="Normal1">Temperature data were analyzed using custom made scripts and the R-package 'seas' (Toews<em> et al.</em>, 2007). Precipitation data was obtained from public weather stations near the field sites. </p&gt

Phage therapy: a crosslink between human and plants bacterial infection.

Bacteria are under constant attack by bacteriophages (phages), the most abundant life forms in the biosphere. This work describes how the phage therapy is an efficient way to contrast the bacterial infection regardless the host. Here we describe the activity of phage in both plant and chicken meat. All the experiment carried out are with phage complex whit the hydroxyapatite, since many years, hydroxyapatite is being explored as vehicle for drug targeting, transfection, bone scaffolds and implant coating materials and, for these reasons we evaluated the effect of this mineral for the bacteriophage therapy. At first, we validated the antimicrobial activity of the phage and its possible direct administration into the plant xylem. We next performed both in vivo and in vitro experiments to assess the activity of the bacteriophage in association whit hydroxyapatite; seconds, we demonstrate that the hydroxyapatite/bacteriophage complex was able to reduce the bacterial load of Salmonella Rissen in previously infected minced meat, respect to bacteriophage or hydroxyapatite alone