Mycobiome of the external ear canal of healthy cows.

Malassezia yeasts belong to the normal skin microbiota of a wide range of warm-blooded animals. However, their significance in cattle is still poorly understood. In the present study, the mycobiota of the external ear canal of 20 healthy dairy Holstein cows was assessed by cytology, culture, PCR, and next-generation sequencing. The presence of Malassezia was detected in 15 cows by cytology and PCR. The metagenomic analysis revealed that Ascomycota was the predominant phylum but M. pachydermatis the main species. The Malassezia phylotype 131 was detected in low abundance. Nor M. nana nor M. equina were detected in the samples.

The mycobiota of the external ear canal of healthy cows was assessed by cytology, culture, PCR, and NGS. The presence of Malassezia was detected by cytology and PCR. Ascomycota was the main phylum and M. pachydermatis the main species. The Malassezia phylotype 131 was also detected in the samples.

GreeNC 2.0: a comprehensive database of plant long non-coding RNAs.

The Green Non-Coding Database (GreeNC) is one of the reference databases for the study of plant long non-coding RNAs (lncRNAs). Here we present our most recent update where 16 species have been updated, while 78 species have been added, resulting in the annotation of more than 495 000 lncRNAs. Moreover, sequence clustering was applied providing information about sequence conservation and gene families. The current version of the database is available at: http://greenc.sequentiabiotech.com/wiki2/Main_Page.

PRGdb 4.0: an updated database dedicated to genes involved in plant disease resistance process.

The Plant Resistance Genes database (PRGdb; http://prgdb.org/prgdb4/) has been greatly expanded, keeping pace with the increasing amount of available knowledge and data (sequenced proteomes, cloned genes, public analysis data, etc.). The easy-to-use style of the database website has been maintained, while an updated prediction tool, more data and a new section have been added. This new section will contain plant resistance transcriptomic experiments, providing additional easy-to-access experimental information. DRAGO3, the tool for automatic annotation and prediction of plant resistance genes behind PRGdb, has been improved in both accuracy and sensitivity, leading to more reliable predictions. PRGdb offers 199 reference resistance genes and 586.652 putative resistance genes from 182 sequenced proteomes. Compared to the previous release, PRGdb 4.0 has increased the number of reference resistance genes from 153 to 199, the number of putative resistance genes from 177K from 76 proteomes to 586K from 182 sequenced proteomes. A new section has been created that collects plant-pathogen transcriptomic data for five species of agricultural interest. Thereby, with these improvements and data expansions, PRGdb 4.0 aims to serve as a reference to the plant scientific community and breeders worldwide, helping to further study plant resistance mechanisms that contribute to fighting pathogens.

Next-Generation Probiotics for Inflammatory Bowel Disease.

Engineered probiotics represent a cutting-edge therapy in intestinal inflammatory disease (IBD). Genetically modified bacteria have provided a new strategy to release therapeutically operative molecules in the intestine and have grown into promising new therapies for IBD. Current IBD treatments, such as corticosteroids and immunosuppressants, are associated with relevant side effects and a significant proportion of patients are dependent on these therapies, thus exposing them to the risk of relevant long-term side effects. Discovering new and effective therapeutic strategies is a worldwide goal in this research field and engineered probiotics could potentially provide a viable solution. This review aims at describing the proceeding of bacterial engineering and how genetically modified probiotics may represent a promising new biotechnological approach in IBD treatment.

Exploring the Healthy Eye Microbiota Niche in a Multicenter Study.

PURPOSE: This study aims to explore and characterize healthy eye microbiota. METHODS: Healthy subjects older than 18 years were selected for this descriptive cross-sectional study. Samples were collected with an eSwab with 1 mL of Liquid Amies Medium (Copan Brescia, Italy). Following DNA extraction, libraries preparation, and amplification, PCR products were purified and end-repaired for barcode ligation. Libraries were pooled to a final concentration of 26 pM. Template preparation was performed with Ion Chef according to Ion 510, Ion 520, and Ion 530 Kit-Chef protocol. Sequencing of the amplicon libraries was carried out on a 520 or 530 chip using the Ion Torrent S5 system (Thermo Fisher; Waltham, MA, USA). Raw reads were analyzed with GAIA (v 2.02). RESULTS: Healthy eye microbiota is a low-diversity microbiome. The vast majority of the 137 analyzed samples were highly enriched with Staphylococcus, whereas only in a few of them, other genera such as Bacillus, Pseudomonas, and Corynebacterium predominate. We found an average of 88 genera with an average Shannon index of 0.65. CONCLUSION: We identified nine different ECSTs. A better understanding of healthy eye microbiota has the potential to improve disease diagnosis and personalized regimens to promote health.

isoCNV: in silico optimization of copy number variant detection from targeted or exome sequencing data.

BACKGROUND: Accurate copy number variant (CNV) detection is especially challenging for both targeted sequencing (TS) and whole-exome sequencing (WES) data. To maximize the performance, the parameters of the CNV calling algorithms should be optimized for each specific dataset. This requires obtaining validated CNV information using either multiplex ligation-dependent probe amplification (MLPA) or array comparative genomic hybridization (aCGH). They are gold standard but time-consuming and costly approaches. RESULTS: We present isoCNV which optimizes the parameters of DECoN algorithm using only NGS data. The parameter optimization process is performed using an in silico CNV validated dataset obtained from the overlapping calls of three algorithms: CNVkit, panelcn.MOPS and DECoN. We evaluated the performance of our tool and showed that increases the sensitivity in both TS and WES real datasets. CONCLUSIONS: isoCNV provides an easy-to-use pipeline to optimize DECoN that allows the detection of analysis-ready CNV from a set of DNA alignments obtained under the same conditions. It increases the sensitivity of DECoN without the need for orthogonal methods. isoCNV is available at https://gitlab.com/sequentiateampublic/isocnv .

External ear canal mycobiome of some rabbit breeds.

The genus Malassezia is part of the normal skin mycobiota of a wide range of warm-blooded animals. In this genus, M. cuniculi is the only species described from rabbits. However, Malassezia species are rarely studied in lagomorphs. In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples. Although no growth was observed in the cultured plates, cytological examination revealed the presence of round cells similar to those of Malassezia yeasts. For metagenomics analysis, the D1/D2 domain of the large subunit of the ribosomal DNA (LSU rDNA) was PCR amplified and the resulting reads were mapped against a custom-made cured database of 26S fungal sequences. NGS analysis revealed that Basidiomycota was the most abundant phylum in all the samples followed by Ascomycota. Malassezia was the most common genus presenting the highest abundance in the external ear canal. Malassezia phylotype 131 and M. cuniculi were the main sequences detected in the external auditory canal of rabbits. The study included both lop-eared and erect-eared rabbits and no differences were observed in the results when comparing both groups. This is the first attempt to study the external ear canal mycobiome of rabbits of different breeds using NGS. LAY SUMMARY: In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples.

Phytotherapics in COVID19: Why palmitoylethanolamide?

At present, googling the search terms "COVID-19" and "Functional foods" yields nearly 500,000,000 hits, witnessing the growing interest of the scientific community and the general public in the role of nutrition and nutraceuticals during the COVID-19 pandemic. Many compounds have been proposed as phytotherapics in the prevention and/or treatment of COVID-19. The extensive interest of the general public and the enormous social media coverage on this topic urges the scientific community to address the question of whether which nutraceuticals can actually be employed in preventing and treating this newly described coronavirus-related disease. Recently, the Canadian biotech pharma company "FSD Pharma" received the green light from the Food and Drug Administration to design a proof-of-concept study evaluating the effects of ultramicronized palmitoylethanolamide (PEA) in COVID-19 patients. The story of PEA as a nutraceutical to prevent and treat infectious diseases dates back to the 1970s where the molecule was branded under the name Impulsin and was used for its immunomodulatory properties in influenza virus infection. The present paper aims at analyzing the potential of PEA as a nutraceutical and the previous evidence suggesting its anti-inflammatory and immunomodulatory properties in infectious and respiratory diseases and how these could translate to COVID-19 care.

Engineered Lactobacillus paracasei Producing Palmitoylethanolamide (PEA) Prevents Colitis in Mice.

Palmitoylethanolamide (PEA) is an N-acylethanolamide produced on-demand by the enzyme N-acylphosphatidylethanolamine-preferring phospholipase D (NAPE-PLD). Being a key member of the larger family of bioactive autacoid local injury antagonist amides (ALIAmides), PEA significantly improves the clinical and histopathological stigmata in models of ulcerative colitis (UC). Despite its safety profile, high PEA doses are required in vivo to exert its therapeutic activity; therefore, PEA has been tested only in animals or human biopsy samples, to date. To overcome these limitations, we developed an NAPE-PLD-expressing Lactobacillus paracasei F19 (pNAPE-LP), able to produce PEA under the boost of ultra-low palmitate supply, and investigated its therapeutic potential in a murine model of UC. The coadministration of pNAPE-LP and palmitate led to a time-dependent release of PEA, resulting in a significant amelioration of the clinical and histological damage score, with a significantly reduced neutrophil infiltration, lower expression and release of pro-inflammatory cytokines and oxidative stress markers, and a markedly improved epithelial barrier integrity. We concluded that pNAPE-LP with ultra-low palmitate supply stands as a new method to increase the in situ intestinal delivery of PEA and as a new therapeutic able of controlling intestinal inflammation in inflammatory bowel disease.

PRGdb 3.0: a comprehensive platform for prediction and analysis of plant disease resistance genes.

The Plant Resistance Genes database (PRGdb; http://prgdb.org) has been redesigned with a new user interface, new sections, new tools and new data for genetic improvement, allowing easy access not only to the plant science research community but also to breeders who want to improve plant disease resistance. The home page offers an overview of easy-to-read search boxes that streamline data queries and directly show plant species for which data from candidate or cloned genes have been collected. Bulk data files and curated resistance gene annotations are made available for each plant species hosted. The new Gene Model view offers detailed information on each cloned resistance gene structure to highlight shared attributes with other genes. PRGdb 3.0 offers 153 reference resistance genes and 177 072 annotated candidate Pathogen Receptor Genes (PRGs). Compared to the previous release, the number of putative genes has been increased from 106 to 177 K from 76 sequenced Viridiplantae and algae genomes. The DRAGO 2 tool, which automatically annotates and predicts (PRGs) from DNA and amino acid with high accuracy and sensitivity, has been added. BLAST search has been implemented to offer users the opportunity to annotate and compare their own sequences. The improved section on plant diseases displays useful information linked to genes and genomes to connect complementary data and better address specific needs. Through, a revised and enlarged collection of data, the development of new tools and a renewed portal, PRGdb 3.0 engages the plant science community in developing a consensus plan to improve knowledge and strategies to fight diseases that afflict main crops and other plants.

The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives.

Here, we report the draft genome sequence of Solanum commersonii, which consists of ∼830 megabases with an N50 of 44,303 bp anchored to 12 chromosomes, using the potato (Solanum tuberosum) genome sequence as a reference. Compared with potato, S. commersonii shows a striking reduction in heterozygosity (1.5% versus 53 to 59%), and differences in genome sizes were mainly due to variations in intergenic sequence length. Gene annotation by ab initio prediction supported by RNA-seq data produced a catalog of 1703 predicted microRNAs, 18,882 long noncoding RNAs of which 20% are shown to target cold-responsive genes, and 39,290 protein-coding genes with a significant repertoire of nonredundant nucleotide binding site-encoding genes and 126 cold-related genes that are lacking in S. tuberosum. Phylogenetic analyses indicate that domesticated potato and S. commersonii lineages diverged ∼2.3 million years ago. Three duplication periods corresponding to genome enrichment for particular gene families related to response to salt stress, water transport, growth, and defense response were discovered. The draft genome sequence of S. commersonii substantially increases our understanding of the domesticated germplasm, facilitating translation of acquired knowledge into advances in crop stability in light of global climate and environmental changes.

GREENC: a Wiki-based database of plant lncRNAs.

Long non-coding RNAs (lncRNAs) are functional non-translated molecules greater than 200 nt. Their roles are diverse and they are usually involved in transcriptional regulation. LncRNAs still remain largely uninvestigated in plants with few exceptions. Experimentally validated plant lncRNAs have been shown to regulate important agronomic traits such as phosphate starvation response, flowering time and interaction with symbiotic organisms, making them of great interest in plant biology and in breeding. There is still a lack of lncRNAs in most sequenced plant species, and in those where they have been annotated, different methods have been used, so making the lncRNAs less useful in comparisons within and between species. We developed a pipeline to annotate lncRNAs and applied it to 37 plant species and six algae, resulting in the annotation of more than 120 000 lncRNAs. To facilitate the study of lncRNAs for the plant research community, the information gathered is organised in the Green Non-Coding Database (GreeNC, http://greenc.sciencedesigners.com/).

Maize RNA PolIV affects the expression of genes with nearby TE insertions and has a genome-wide repressive impact on transcription.

BACKGROUND: RNA-directed DNA methylation (RdDM) is a plant-specific epigenetic process that relies on the RNA polymerase IV (Pol IV) for the production of 24 nucleotide small interfering RNAs (siRNA) that guide the cytosine methylation and silencing of genes and transposons. Zea mays RPD1/RMR6 gene encodes the largest subunit of Pol IV and is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs) and transcriptional regulation of specific alleles. RESULTS: In this study we applied a total RNA-Seq approach to compare the B73 and rpd1/rmr6 leaf transcriptomes. Although previous studies indicated that loss of siRNAs production in RdDM mutants provokes a strong loss of CHH DNA methylation but not massive gene or TEs transcriptional activation in both Arabidopsis and maize, our total RNA-Seq analysis of rpd1/rmr6 transcriptome reveals that loss of Pol IV activity causes a global increase in the transcribed fraction of the maize genome. Our results point to the genes with nearby TE insertions as being the most strongly affected by Pol IV-mediated gene silencing. TEs modulation of nearby gene expression is linked to alternative methylation profiles on gene flanking regions, and these profiles are strictly dependent on specific characteristics of the TE member inserted. Although Pol IV is essential for the biogenesis of siRNAs, the genes with associated siRNA loci are less affected by the pol IV mutation. CONCLUSIONS: This deep and integrated analysis of gene expression, TEs distribution, smallRNA targeting and DNA methylation levels, reveals that loss of Pol IV activity globally affects genome regulation, pointing at TEs as modulator of nearby gene expression and indicating the existence of multiple level epigenetic silencing mechanisms. Our results also suggest a predominant role of the Pol IV-mediated RdDM pathway in genome dominance regulation, and subgenome stability and evolution in maize.

Effects of Mecp2 loss of function in embryonic cortical neurons: a bioinformatics strategy to sort out non-neuronal cells variability from transcriptome profiling.

BACKGROUND: Mecp2 null mice model Rett syndrome (RTT) a human neurological disorder affecting females after apparent normal pre- and peri-natal developmental periods. Neuroanatomical studies in cerebral cortex of RTT mouse models revealed delayed maturation of neuronal morphology and autonomous as well as non-cell autonomous reduction in dendritic complexity of postnatal cortical neurons. However, both morphometric parameters and high-resolution expression profile of cortical neurons at embryonic developmental stage have not yet been studied. Here we address these topics by using embryonic neuronal primary cultures from Mecp2 loss of function mouse model. RESULTS: We show that embryonic primary cortical neurons of Mecp2 null mice display reduced neurite complexity possibly reflecting transcriptional changes. We used RNA-sequencing coupled with a bioinformatics comparative approach to identify and remove the contribution of variable and hard to quantify non-neuronal brain cells present in our in vitro cell cultures. CONCLUSIONS: Our results support the need to investigate both Mecp2 morphological as well as molecular effect in neurons since prenatal developmental stage, long time before onset of Rett symptoms.

Transposon Insertions, Structural Variations, and SNPs Contribute to the Evolution of the Melon Genome.

The availability of extensive databases of crop genome sequences should allow analysis of crop variability at an unprecedented scale, which should have an important impact in plant breeding. However, up to now the analysis of genetic variability at the whole-genome scale has been mainly restricted to single nucleotide polymorphisms (SNPs). This is a strong limitation as structural variation (SV) and transposon insertion polymorphisms are frequent in plant species and have had an important mutational role in crop domestication and breeding. Here, we present the first comprehensive analysis of melon genetic diversity, which includes a detailed analysis of SNPs, SV, and transposon insertion polymorphisms. The variability found among seven melon varieties representing the species diversity and including wild accessions and highly breed lines, is relatively high due in part to the marked divergence of some lineages. The diversity is distributed nonuniformly across the genome, being lower at the extremes of the chromosomes and higher in the pericentromeric regions, which is compatible with the effect of purifying selection and recombination forces over functional regions. Additionally, this variability is greatly reduced among elite varieties, probably due to selection during breeding. We have found some chromosomal regions showing a high differentiation of the elite varieties versus the rest, which could be considered as strongly selected candidate regions. Our data also suggest that transposons and SV may be at the origin of an important fraction of the variability in melon, which highlights the importance of analyzing all types of genetic variability to understand crop genome evolution.

Flowering and trichome development share hormonal and transcription factor regulation.

Gibberellins (GAs) and cytokinins (CKs) are plant hormones that act either synergistically or antagonistically during the regulation of different developmental processes. In Arabidopsis thaliana, GAs and CKs overlap in the positive regulation of processes such as the transition from the vegetative to the reproductive phase and the development of epidermal adaxial trichomes. Despite the fact that both developmental processes originate in the rosette leaves, they occur separately in time and space. Here we review how, as genetic and molecular mechanisms are being unraveled, both processes might be closely related. Additionally, this shared genetic network is not only dependent on GA and CK hormone signaling but is also strictly controlled by specific clades of transcription factor families. Some key flowering genes also control other rosette leaf developmental processes such as adaxial trichome formation. Conversely, most of the trichome activator genes, which belong to the MYB, bHLH and C2H2 families, were found to positively control the floral transition. Furthermore, three MADS floral organ identity genes, which are able to convert leaves into floral structures, are also able to induce trichome proliferation in the flower. These data lead us to propose that the spatio-temporal regulation and integration of diverse signals control different developmental processes, such as floral induction and trichome formation, which are intimately connected through similar genetic pathways.

Spermatozoa from infertile patients exhibit differences of DNA methylation associated with spermatogenesis-related processes: an array-based analysis.

The influence of aberrant sperm DNA methylation on the reproductive capacity of couples has been postulated as a cause of infertility. This study compared the DNA methylation of spermatozoa of 19 fertile donors and 42 infertile patients using the Illumina 450K array. Clustering analysis of methylation data arranged fertile and infertile patients into two groups. Bivariate clustering analysis identified a differential distribution of samples according to the characteristics of seminogram and age, suggesting a possible link between these parameters and specific methylation profiles. The study identified 696 differentially methylated cytosine-guanine dinucleotides (CpG) associated with 501 genes between fertile donors and infertile patients. Ontological enrichment analysis revealed 13 processes related to spermatogenesis. Data filtering identified a set of 17 differentially methylated genes, some of which had functions relating to spermatogenesis. A significant association was identified between RPS6KA2 hypermethylation and advanced age (P = 0.016); APCS hypermethylation and oligozoospermia (P = 0.041); JAM3/NCAPD3 hypermethylation and numerical chromosome sperm anomalies (P = 0.048); and ANK2 hypermethylation and lower pregnancy rate (P = 0.040). This description of a set of differentially methylated genes provides a framework for further investigation into the influence of such variation in male fertility in larger patient cohorts.

Next-generation sequencing, FISH mapping and synteny-based modeling reveal mechanisms of decreasing dysploidy in Cucumis.

In the large Cucurbitaceae genus Cucumis, cucumber (C. sativus) is the only species with 2n = 2x = 14 chromosomes. The majority of the remaining species, including melon (C. melo) and the sister species of cucumber, C. hystrix, have 2n = 2x = 24 chromosomes, implying a reduction from n = 12 to n = 7. To understand the underlying mechanisms, we investigated chromosome synteny among cucumber, C. hystrix and melon using integrated and complementary approaches. We identified 14 inversions and a C. hystrix lineage-specific reciprocal inversion between C. hystrix and melon. The results reveal the location and orientation of 53 C. hystrix syntenic blocks on the seven cucumber chromosomes, and allow us to infer at least 59 chromosome rearrangement events that led to the seven cucumber chromosomes, including five fusions, four translocations, and 50 inversions. The 12 inferred chromosomes (AK1-AK12) of an ancestor similar to melon and C. hystrix had strikingly different evolutionary fates, with cucumber chromosome C1 apparently resulting from insertion of chromosome AK12 into the centromeric region of translocated AK2/AK8, cucumber chromosome C3 originating from a Robertsonian-like translocation between AK4 and AK6, and cucumber chromosome C5 originating from fusion of AK9 and AK10. Chromosomes C2, C4 and C6 were the result of complex reshuffling of syntenic blocks from three (AK3, AK5 and AK11), three (AK5, AK7 and AK8) and five (AK2, AK3, AK5, AK8 and AK11) ancestral chromosomes, respectively, through 33 fusion, translocation and inversion events. Previous results (Huang, S., Li, R., Zhang, Z. et al., , Nat. Genet. 41, 1275-1281; Li, D., Cuevas, H.E., Yang, L., Li, Y., Garcia-Mas, J., Zalapa, J., Staub, J.E., Luan, F., Reddy, U., He, X., Gong, Z., Weng, Y. 2011a, BMC Genomics, 12, 396) showing that cucumber C7 stayed largely intact during the entire evolution of Cucumis are supported. Results from this study allow a fine-scale understanding of the mechanisms of dysploid chromosome reduction that has not been achieved previously.

Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly.

BACKGROUND: The genome of the melon (Cucumis melo L.) double-haploid line DHL92 was recently sequenced, with 87.5 and 80.8% of the scaffold assembly anchored and oriented to the 12 linkage groups, respectively. However, insufficient marker coverage and a lack of recombination left several large, gene rich scaffolds unanchored, and some anchored scaffolds unoriented. To improve the anchoring and orientation of the melon genome assembly, we used resequencing data between the parental lines of DHL92 to develop a new set of SNP markers from unanchored scaffolds. RESULTS: A high-resolution genetic map composed of 580 SNPs was used to anchor 354.8 Mb of sequence, contained in 141 scaffolds (average size 2.5 Mb) and corresponding to 98.2% of the scaffold assembly, to the 12 melon chromosomes. Over 325.4 Mb (90%) of the assembly was oriented. The genetic map revealed regions of segregation distortion favoring SC alleles as well as recombination suppression regions coinciding with putative centromere, 45S, and 5S rDNA sites. New chromosome-scale pseudomolecules were created by incorporating to the previous v3.5 version an additional 38.3 Mb of anchored sequence representing 1,837 predicted genes contained in 55 scaffolds. Using fluorescent in situ hybridization (FISH) with BACs that produced chromosome-specific signals, melon chromosomes that correspond to the twelve linkage groups were identified, and a standardized karyotype of melon inbred line T111 was developed. CONCLUSIONS: By utilizing resequencing data and targeted SNP selection combined with a large F2 mapping population, we significantly improved the quantity of anchored and oriented melon scaffold genome assembly. Using genome information combined with FISH mapping provided the first cytogenetic map of an inodorus melon type. With these results it was possible to make inferences on melon chromosome structure by relating zones of recombination suppression to centromeres and 45S and 5S heterochromatic regions. This study represents the first steps towards the integration of the high-resolution genetic and cytogenetic maps with the genomic sequence in melon that will provide more information on genome organization and allow for the improvement of the melon genome draft sequence.