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Circadian rhythmicity of gene expression is a conserved feature of cell physiology. This involves fine-tuning between transcriptional and post-transcriptional mechanisms and strongly depends on the metabolic state of the cell. Together these processes guarantee an adaptive plasticity of tissue-specific genetic programs. However, it is unclear how the epigenome and RNA Pol II rhythmicity are integrated. Here we show that the PcG protein EZH1 has a gateway bridging function in postmitotic skeletal muscle cells. On the one hand, the circadian clock master regulator BMAL1 directly controls oscillatory behavior and periodic assembly of core components of the PRC2-EZH1 complex. On the other hand, EZH1 is essential for circadian gene expression at alternate Zeitgeber times, through stabilization of RNA Polymerase II preinitiation complexes, thereby controlling nascent transcription. Collectively, our data show that PRC2-EZH1 regulates circadian transcription both negatively and positively by modulating chromatin states and basal transcription complex stability., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

The purpose of this study is to characterize the ocular surface microbiota of patients with an anophthalmic cavity. An eNAT with 1 mL of Liquid Amies Medium was used to collect samples. Microbial DNA from anophthalmic socket and healthy fellow control eye samples was isolated and sequenced. Raw reads were analyzed with GAIA (v 2.02). The richness and Shannon alpha diversity metrics, as well as Bray-Curtis beta diversity and Wilcoxon signed-rank test values, were computed with R packages such as phyloseq, mia, or DESeq2 to allow for microbiome analysis. Principal coordinate analysis (PCoA) was performed using the function plotReducedDim from the R package scater. The different taxonomic profiles were described under the concept of eye community state type (ECST). The microbiomes of both eyes from 25 patients with an anophthalmic cavity were analyzed in this study. While the microbial communities of paired eyes from the same patients showed notable dissimilarity, no consistent patterns emerged when comparing healthy eyes to anophthalmic sockets. Alpha diversity values did not significantly differ between healthy eyes and anophthalmic socket samples, though there was considerable variability within each group. Notably, anophthalmic socket samples generally exhibited lower abundances of genera such as Staphylococcus , Enterococcus , Paenibacillus , and Sediminibacterium compared to their healthy counterparts. Microbial variability between healthy eyes and anophthalmic sockets may be due to anatomical differences. Further research is needed to determine whether patients without anophthalmic sockets exhibit similar microbiome patterns in both eyes.

Objective: The present study is aimed at introducing and evaluating MaterniCode, a state-of-the-art bioinformatic pipeline for noninvasive prenatal testing (NIPT) that leverages the Ion Torrent semiconductor sequencing platform. The initiative strives to revolutionize prenatal diagnostics by offering a rapid and cost-effective method without sacrificing accuracy. Methods: Two distinct bioinformatic strategies were employed for fetal sex determination, one of which achieved 100% accuracy. We analyzed 1225 maternal blood samples for fetal aneuploidies, benchmarking against the industry standard Illumina VeriSeq™ NIPT Solution v2. The capability of MaterniCode to detect and characterize complex chromosomal anomalies was also assessed. Results: MaterniCode achieved near-perfect accuracy in fetal sex determination through chromosome Y (chrY )-specific gene analysis, whereas the alternative method, employing the ratio of high-quality mapped reads on chrY relative to all reads, delivered 100% accuracy. For fetal aneuploidy detection, both the integrated WisecondorX and NIPTeR algorithms demonstrated a 100% sensitivity and specificity rate, consistent with Illumina VeriSeq™ NIPT Solution v2. The pipeline also successfully identified and precisely mapped significant chromosomal abnormalities, exemplified by a 2.4 Mb deletion on chromosome 13 and a 3 Mb duplication on chromosome 2. Conclusion: MaterniCode has proven to be an innovative and highly efficient tool in the domain of NIPT, demonstrating excellent sensitivity and specificity. Its robust capability to effectively detect a wide range of complex chromosomal aberrations, including rare and subtle variations, positions it as a promising and valuable addition to prenatal diagnostic technologies. This enhancement to diagnostic precision significantly aids clinicians in making informed decisions during pregnancy management., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Federico Gabrielli et al.)

Oxidation of histone H3 at lysine 4 (H3K4ox) is catalyzed by lysyl oxidase homolog 2 (LOXL2). This histone modification is enriched in heterochromatin in triple-negative breast cancer (TNBC) cells and has been linked to the maintenance of compacted chromatin. However, the molecular mechanism underlying this maintenance is still unknown. Here, we show that LOXL2 interacts with RuvB-Like 1 (RUVBL1), RuvB-Like 2 (RUVBL2), Actin-like protein 6A (ACTL6A), and DNA methyltransferase 1associated protein 1 (DMAP1), a complex involved in the incorporation of the histone variant H2A.Z. Our experiments indicate that this interaction and the active form of RUVBL2 are required to maintain LOXL2-dependent chromatin compaction. Genome-wide experiments showed that H2A.Z, RUVBL2, and H3K4ox colocalize in heterochromatin regions. In the absence of LOXL2 or RUVBL2, global levels of the heterochromatin histone mark H3K9me3 were strongly reduced, and the ATAC-seq signal in the H3K9me3 regions was increased. Finally, we observed that the interplay between these series of events is required to maintain H3K4ox-enriched heterochromatin regions, which in turn is key for maintaining the oncogenic properties of the TNBC cell line tested (MDA-MB-231)., (© 2024 Federation of European Biochemical Societies.)

Background: The increase in temperatures due to the current climate change dramatically affects crop cultivation, resulting in yield losses and altered fruit quality. Tomato is one of the most extensively grown and consumed horticultural products, and although it can withstand a wide range of climatic conditions, heat stress can affect plant growth and development specially on the reproductive stage, severely influencing the final yield. In the present work, the heat stress response mechanisms of one thermotolerant genotype (E42) were investigated by exploring its regulatory gene network. This was achieved through a promoter analysis based on the identification of the heat stress elements (HSEs) mapping in the promoters, combined with a gene co-expression network analysis aimed at identifying interactions among heat-related genes., Results: Results highlighted 82 genes presenting HSEs in the promoter and belonging to one of the 52 gene networks obtained by the GCN analysis; 61 of these also interact with heat shock factors (Hsfs). Finally, a list of 13 candidate genes including two Hsfs, nine heat shock proteins (Hsps) and two GDSL esterase/lipase (GELPs) were retrieved by focusing on those E42 genes exhibiting HSEs in the promoters, interacting with Hsfs and showing variants, compared to Heinz reference genome, with HIGH and/or MODERATE impact on the translated protein. Among these, the Gene Ontology annotation analysis evidenced that only LeHsp100 (Solyc02g088610) belongs to a network specifically involved in the response to heat stress., Conclusions: As a whole, the combination of bioinformatic analyses carried out on genomic and trascriptomic data available for tomato, together with polymorphisms detected in HS-related genes of the thermotolerant E42 allowed to determine a subset of candidate genes involved in the HS response in tomato. This study provides a novel approach in the investigation of abiotic stress response mechanisms and further studies will be conducted to validate the role of the highlighted genes., (© 2024. The Author(s).)

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 Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

For almost two decades, clinicians have overlooked the diagnostic potential of CD34 neg hematopoietic stem cells because of their limited homing capacity relative to CD34 pos HSCs when injected intravenously. This has contributed to the lack of appeal of using umbilical cord blood in HSC transplantation because its stem cell count is lower than bone marrow. The present study reveals that the homing and engraftment of CD34 neg HSCs can be improved by adding the Sialyl Lewis X molecule via α1,3-fucosylation. This unlocks the potential for using this more primitive stem cell to treat blood disorders because our findings show CD34 neg HSCs have the capacity to regenerate cells in the bone marrow of mice for several months. Furthermore, our RNA sequencing analysis revealed that CD34 neg HSCs have unique adhesion pathways, downregulated in CD34 pos HSCs, that facilitate interaction with the bone marrow niche. Our findings suggest that CD34 neg cells will best thrive when the HSC resides in its microenvironment., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Alternative splicing (AS) is an important mechanism contributing to stress-induced regulation of gene expression and proteome diversity. Massive sequencing technologies allow the identification of transcripts generated via stress-responsive AS, potentially important for adaptation to stress conditions. Several bioinformatics tools have been developed to identify differentially expressed alternative splicing events/transcripts from RNA-sequencing results. This chapter describes a detailed protocol for differential alternative splicing analysis using the rMATS tool. In addition, we provide guidelines for validation of the detected splice variants by qRT-PCR based on the obtained output files., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

VPS13 is a lipid transfer protein family conserved among Eukaryotes and playing roles in fundamental processes involving vesicular transport and membrane expansion including autophagy and organelle biogenesis. VPS13 folds into a long hydrophobic tunnel, allowing lipid transport, decorated by distinct domains involved in protein localization and regulation. Whereas VPS13 organization and function have been extensively studied in yeast and mammals, information in organisms originating from primary endosymbiosis is scarce. In the higher plant Arabidopsis thaliana , four paralogs, AtVPS13S, X, M1, and M2, were identified, AtVPS13S playing a role in the regulation of root growth, cell patterning, and reproduction. In this work, we performed phylogenetic, as well as domain and structural modeling of VPS13 proteins in Archaeplastida in order to understand their general organization and evolutionary history. We confirmed the presence of human VPS13B orthologues in some phyla and described two new VPS13 families presenting a particular domain arrangement: VPS13R in Rhodophytes and VPS13Y in Chlorophytes and Streptophytes. By focusing on Viridiplantae, we were able to draw the evolutionary history of these proteins made by multiple gene gains and duplications as well as domain rearrangements. We showed that some Chlorophytes have only three (AtVPS13M, S, Y) whereas some Charophytes have up to six VPS13 paralogs (AtVPS13M1, M2, S, Y, X, B). We also highlighted specific structural features of VPS13M and X paralogs. This study reveals the complex evolution of VPS13 family and opens important perspectives for their functional characterization in photosynthetic organisms., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBD) that affect the gastrointestinal tract. Changes in the microbiome and its interaction with the immune system are thought to play a key role in their development. The aim of this study was to determine whether metagenomic analysis is a feasible non-invasive diagnostic tool for IBD in paediatric patients. A pilot study of oral and faecal microbiota was proposed with 36 paediatric patients divided in three cohorts [12 with CD, 12 with UC and 12 healthy controls (HC)] with 6 months of follow-up. Finally, 30 participants were included: 13 with CD, 11 with UC and 8 HC (6 dropped out during follow-up). Despite the small size of the study population, a differential pattern of microbial biodiversity was observed between IBD patients and the control group. Twenty-one bacterial species were selected in function of their discriminant accuracy, forming three sets of potential markers of IBD. Although IBD diagnosis requires comprehensive medical evaluation, the findings of this study show that faecal metagenomics or a reduced set of bacterial markers could be useful as a non-invasive tool for an easier and earlier diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Monleón-Getino, Pujol-Muncunill, Méndez Viera, Álvarez Carnero, Sanseverino, Paytuví-Gallart and Martín de Carpí.)

The chloroplast (cp) genome diversity has been used in phylogeny studies, breeding, and variety protection, and its expression has been shown to play a role in stress response. Smooth- and curly-leafed endives ( Cichorium endivia var. latifolium and var. crispum ) are of nutritional and economic importance and are the target of ever-changing breeding programmes. A reference cp genome sequence was assembled and annotated (cultivar 'Confiance'), which was 152,809 base pairs long, organized into the angiosperm-typical quadripartite structure, harboring two inverted repeats separated by the large- and short- single copy regions. The annotation included 136 genes, 90 protein-coding genes, 38 transfer, and 8 ribosomal RNAs and the sequence generated a distinct phyletic group within Asteraceae with the well-separated C. endivia and intybus species. SSR variants within the reference genome were mostly of tri-nucleotide type, and the cytosine to uracil (C/U) RNA editing recurred. The cp genome was nearly fully transcribed, hence sequence polymorphism was investigated by RNA-Seq of seven cultivars, and the SNP number was higher in smooth- than curly-leafed ones. All cultivars maintained C/U changes in identical positions, suggesting that RNA editing patterns were conserved; most cultivars shared SNPs of moderate impact on protein changes in the ndhD , ndhA , and psbF genes, suggesting that their variability may have a potential role in adaptive response. The cp transcriptome expression was investigated in leaves of plants affected by pre-harvest rainfall and rainfall excess plus waterlogging events characterized by production loss, compared to those of a cycle not affected by extreme rainfall. Overall, the analyses evidenced stress- and cultivar-specific responses, and further revealed that genes of the Cytochrome b6/f, and PSI-PSII systems were commonly affected and likely to be among major targets of extreme rain-related stress.

'Corbarino' (COR) and 'Lucariello' (LUC) belong to the family of Mediterranean long shelf-life tomato landraces, producing high quality fruits under low water input cultivation regime in their traditional cultivation area. Understanding the morpho-physiological and molecular details of the peculiar drought stress tolerance of these two genotypes may be key to their valorization as breeding material. RNA sequencing of leaf samples of COR and LUC subjected to drought stress by water withholding in a semi-controlled greenhouse identified 3089 and 2135 differentially expressed genes respectively. These included COR- and LUC-specific annotated genes, as well as genes containing single nucleotide polymorphisms as compared to reference genome. Enriched Gene Ontology categories showed that categories such as response to water, oxidoreductase activity, nucleotide salvation and lipid biosynthesis-related processes were enriched among up-regulated DEGs. By contrast, growth and photosynthesis related genes were down-regulated after drought stress, consistent with leaf gas exchange and biomass accumulation measurements. Genes encoding cell wall degrading enzymes of the pectinase family were also down-regulated in drought stress conditions and upregulated in rewatering, indicating that cell wall composition/hardness is important for drought stress responses. Globally our results contribute to understanding the transcriptomic and physiological responses of representative tomato genotypes from Southern Italy, highlighting a promising set of genes to be investigated to improve tomato tolerance to drought., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular carcinoma (HCC) development remains unclear. In this study, we apply a high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identify 3339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with HCC. We detect 2107 clonally expanded integrations (1817 in tumour and 290 in non-tumour tissues), and a significant enrichment of clonal HBV integrations in mitochondrial DNA (mtDNA) preferentially occurring in the oxidative phosphorylation genes (OXPHOS) and D-loop region. We also find that HBV RNA sequences are imported into the mitochondria of hepatoma cells with the involvement of polynucleotide phosphorylase (PNPASE), and that HBV RNA might have a role in the process of HBV integration into mtDNA. Our results suggest a potential mechanism by which HBV integration may contribute to HCC development., (© 2023. The Author(s).)

The yeast Malassezia pachydermatis is a common inhabitant of the skin and mucosae of dogs. However, under certain circumstances this yeast can overgrow and act as an opportunistic pathogen causing otitis and dermatitis in dogs. Canine pododermatitis is a common disorder in dogs in which M. pachydermatis acts as an opportunistic pathogen. In the present study, the presence of Malassezia yeasts was assessed and quantified in samples collected from the interdigital space of dogs with pododermatitis before and after treatment, and from healthy dogs. The samples were subjected to two different cytological examinations, culture on Sabouraud glucose agar and modified Dixon's agar and a quantitative PCR targeting the internal transcribed spacer (ITS) genomic region. A selection of samples was analyzed by next generation sequencing (NGS) using the D1D2 domain of the large subunit of the ribosomal DNA as target. The pododermatitis samples before treatment showed higher cell counts, colony-forming units and ITS copies than the rest of samples. The NGS analysis revealed that Ascomycota was the main phylum in the healthy and post-treatment samples. However, Basidiomycota and M. pachydermatis was more abundant in the pododermatitis samples before treatment. These results support M. pachydermatis as an opportunistic agent in canine pododermatitis by a variety of methods, and demonstrate the correlation between cytologic and molecular methods for quantification., (© 2022. The Author(s).)

The purpose of this study is to provide novel information through Next Generation Sequencing (NGS) for the characterization of viral and bacterial RNA cargo of human sperm cells from healthy fertile donors. For this, RNA-seq raw data of poly(A) RNA from 12 sperm samples from fertile donors were aligned to microbiome databases using the GAIA software. Species of viruses and bacteria were quantified in Operational Taxonomic Units (OTU) and filtered by minimal expression level (>1% OTU in at least one sample). Mean expression values (and their standard deviation) of each species were estimated. A Hierarchical Cluster Analysis (HCA) and a Principal Component Analysis (PCA) were performed to detect common microbiome patterns among samples. Sixteen microbiome species, families, domains, and orders surpassed the established expression threshold. Of the 16 categories, nine corresponded to viruses (23.07% OTU) and seven to bacteria (2.77% OTU), among which the Herperviriales order and Escherichia coli were the most abundant, respectively. HCA and PCA displayed four clusters of samples with a differentiated microbiome fingerprint. This work represents a pilot study into the viruses and bacteria that make up the human sperm microbiome. Despite the high variability observed, some patterns of similarity among individuals were identified. Further NGS studies under standardized methodological procedures are necessary to achieve a deep knowledge of the semen microbiome and its implications in male fertility.

This study assessed the potential effect of a co-feed liquid whey-integrated diet on the fecal microbiota of 14 crossbred pigs. The experimental design was as follows: seven pigs were in the control group, fed with a control feed, and seven were in the experimental group, fed with the same control feed supplemented daily with liquid whey. The collection of fecal samples was conducted on each animal before the dietary treatment (T0) and one (T1), and two (T2) months after the beginning of the co-feed integration. In addition, blood samples were collected from each pig at the same time points in order to evaluate the physiological parameters. Taxonomic analysis showed a bacterial community dominated by Firmicutes , Bacteroidetes , Spirochaetes , and Proteobacteria phyla that populated the crossbred pig feces. The diversity metrics suggested that the co-feed supplementation affected some alpha diversity indexes of the fecal microbiota. In addition, the differential abundance analysis at the genus level revealed significant differences for various genera, suggesting that the liquid whey supplementation potentially influenced a part of the bacterial community over time. Spearman's correlations revealed that the differential abundant genera identified are positively or negatively correlated with the physiological parameters.

Objective: The transcriptional landscape of Klinefelter syndromeduring early embryogenesis remains elusive. This study aimed to evaluate the impact of X chromosome overdosage in 47,XXY males induced pluripotent stem cells (iPSCs) obtained from patients with different genomic backgrounds and ethnicities., Design and Method: We derived and characterized 15 iPSC lines from four Saudi 47,XXY KS patients and one Saudi 46,XY male. We performed a comparative transcriptional analysis using the Saudi KS-iPSCs and a cohort of European and North American KS-iPSCs., Results: We identified a panel of X-linked and autosomal genes commonly dysregulated in Saudi and European/North American KS-iPSCs vs 46,XY controls. Our findings demonstrate that seven PAR1 and nine non-PAR escape genes are consistently dysregulated and mostly display comparable transcriptional levels in both groups. Finally, we focused on genes commonly dysregulated in both iPSC cohorts and identified several gene-ontology categories highly relevant to KS physiopathology, including aberrant cardiac muscle contractility, skeletal muscle defects, abnormal synaptic transmission, and behavioral alterations., Conclusions: Our results indicate that a transcriptomic signature of X chromosome overdosage in KS is potentially attributable to a subset of X-linked genes sensitive to sex chromosome dosage and escaping X inactivation, regardless of the geographical area of origin, ethnicity, and genetic makeup.

Background: Green feed diet in ruminants exerts a beneficial effect on rumen metabolism and enhances the content of milk nutraceutical quality. At present, a comprehensive analysis focused on the identification of genes, and therefore, biological processes modulated by the green feed in buffalo rumen has never been reported. We performed RNA-sequencing in the rumen of buffaloes fed a total mixed ration (TMR) + the inclusion of 30% of ryegrass green feed (treated) or TMR (control), and identified differentially expressed genes (DEGs) using EdgeR and NOISeq tools., Results: We found 155 DEGs using EdgeR (p-values < 0.05) and 61 DEGs using NOISeq (prob ≥0.8), 30 of which are shared. The rt-qPCR validation suggested a higher reliability of EdgeR results as compared with NOISeq data, in our biological context. Gene Ontology analysis of DEGs identified using EdgeR revealed that green feed modulates biological processes relevant for the rumen physiology and, then, health and well-being of buffaloes, such as lipid metabolism, response to the oxidative stress, immune response, and muscle structure and function. Accordingly, we found: (i) up-regulation of HSD17B13, LOC102410803 (or PSAT1) and HYKK, and down-regulation of CDO1, SELENBP1 and PEMT, encoding factors involved in energy, lipid and amino acid metabolism; (ii) enhanced expression of SIM2 and TRIM14, whose products are implicated in the immune response and defense against infections, and reduced expression of LOC112585166 (or SAAL1), ROR2, SMOC2, and S100A11, encoding pro-inflammatory factors; (iii) up-regulation of NUDT18, DNAJA4 and HSF4, whose products counteract stressful conditions, and down-regulation of LOC102396388 (or UGT1A9) and LOC102413340 (or MRP4/ABCC4), encoding detoxifying factors; (iv) increased expression of KCNK10, CACNG4, and ATP2B4, encoding proteins modulating Ca 2+ homeostasis, and reduced expression of the cytoskeleton-related MYH11 and DES., Conclusion: Although statistically unpowered, this study suggests that green feed modulates the expression of genes involved in biological processes relevant for rumen functionality and physiology, and thus, for welfare and quality production in Italian Mediterranean dairy buffaloes. These findings, that need to be further confirmed through the validation of additional DEGs, allow to speculate a role of green feed in the production of nutraceutical molecules, whose levels might be enhanced also in milk., (© 2023. The Author(s).)

Until a few years ago, many studies focused on the transcriptomic response to single stresses. However, tomato cultivations are often constrained by a wide range of biotic and abiotic stress that can occur singularly or in combination, and several genes can be involved in the defensive mechanism response. Therefore, we analyzed and compared the transcriptomic responses of resistant and susceptible genotypes to seven biotic stresses ( Cladosporium fulvum , Phytophthora infestans , Pseudomonas syringae , Ralstonia solanacearum , Sclerotinia sclerotiorum , Tomato spotted wilt virus (TSWV) and Tuta absoluta ) and five abiotic stresses (drought, salinity, low temperatures, and oxidative stress) to identify genes involved in response to multiple stressors. With this approach, we found genes encoding for TFs, phytohormones, or participating in signaling and cell wall metabolic processes, participating in defense against various biotic and abiotic stress. Moreover, a total of 1474 DEGs were commonly found between biotic and abiotic stress. Among these, 67 DEGs were involved in response to at least four different stresses. In particular, we found RLKs, MAPKs, Fasciclin-like arabinogalactans (FLAs), glycosyltransferases, genes involved in the auxin, ET, and JA pathways, MYBs, bZIPs, WRKYs and ERFs genes. Detected genes responsive to multiple stress might be further investigated with biotechnological approaches to effectively improve plant tolerance in the field.

The utilization of dairy by-products as animal feed, especially in swine production, is a strategy to provide functional ingredients to improve gut health. This study explored the potential effect of a liquid whey-supplemented diet on the fecal microbiota of eleven pigs belonging to the Nero Siciliano breed. Five pigs were assigned to the control group and fed with a standard formulation feed, whereas six pigs were assigned to the experimental group and fed with the same feed supplemented with liquid whey. Fecal samples were collected from each individual before the experimental diet (T0), and one (T1) and two (T2) months after the beginning of the co-feed supplementation. Taxonomic analysis, based on the V3-V4 region of the bacterial 16S rRNA, showed that pig feces were populated by a complex microbial community with a remarkable abundance of Firmicutes , Bacteroidetes , and Spirochaetes phyla and Prevotella , Lactobacillus , Clostridium , and Treponema genera. Alpha and beta diversity values suggested that the experimental diet did not significantly affect the overall fecal microbiota diversity. However, analysis of abundance at different time points revealed significant variation in several bacterial genera, suggesting that the experimental diet potentially affected some genera of the microbial community.

Microbiomes have been the focus of a substantial research effort in the last decades. The composition of microbial populations is normally determined by comparing DNA sequences sampled from those populations with the sequences stored in genomic databases. Therefore, the amount of information available in databanks should be expected to constrain the accuracy of microbiome analyses. Albeit normally ignored in microbiome studies, this constraint could severely compromise the reliability of microbiome data. To test this hypothesis, we generated virtual bacterial populations that exhibit the ecological structure of real-world microbiomes. Confronting the analyses of virtual microbiomes with their original composition revealed critical issues in the current approach to characterizing microbiomes, issues that were empirically confirmed by analyzing the microbiome of Galleria mellonella larvae. To reduce the uncertainty of microbiome data, the effort in the field must be channeled towards significantly increasing the amount of available genomic information and optimizing the use of this information., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Serrano-Antón et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Xiphias gladius is an important fishing resource. The Mediterranean stock is affected by overfishing and is declining. In this light, the aim of this study was to evaluate the cross-talk among metabolism, stress response, immune system and reproduction in immature and mature females, coupling histological and transcriptomic approaches. The transcriptome of livers from 3 immature and 3 mature females was analyzed using the Artificial Intelligence RNA-Seq. For the histological analysis, ovary and liver samples were collected from 50 specimens caught during the reproductive season in the Mediterranean Sea. A total of 750 genes were differentially expressed between the livers. The gene ontologtabey analysis showed 91 upregulated and 161 downregulated biological process GO terms. Instead, the KEGG enrichment analysis revealed 15 enriched pathways. Furthermore, the binding occurring between estrogen receptors and aryl hydrocarbon receptor nuclear translocator, upregulated in mature females, could be liable for the inhibition of detoxification pathway. Indeed, at the histological level, mature females showed a higher density and number of melanomacrophage centers, biomarkers of stress. The present findings reveal the cross-talk among response to environmental stressors, metabolism and reproduction, highlighting that mature females invest a lot of energy in reproduction instead of immune response and detoxification.

Crop wild relatives (CWRs) are recognized as the best potential source of traits for crop improvement. However, successful crop improvement using CWR relies on identifying variation in genes controlling desired traits in plant germplasms and subsequently incorporating them into cultivars. Epigenetic diversity may provide an additional layer of variation within CWR and can contribute novel epialleles for key traits for crop improvement. There is emerging evidence that epigenetic variants of functional and/or agronomic importance exist in CWR gene pools. This provides a rationale for the conservation of epigenotypes of interest, thus contributing to agrobiodiversity preservation through conservation and (epi)genetic monitoring. Concepts and techniques of classical and modern breeding should consider integrating recent progress in epigenetics, initially by identifying their association with phenotypic variations and then by assessing their heritability and stability in subsequent generations. New tools available for epigenomic analysis offer the opportunity to capture epigenetic variation and integrate it into advanced (epi)breeding programmes. Advances in -omics have provided new insights into the sources and inheritance of epigenetic variation and enabled the efficient introduction of epi-traits from CWR into crops using epigenetic molecular markers, such as epiQTLs., (© 2022. The Author(s).)

Background: The role of the pulmonary microbiome in sarcoidosis is unknown. The objectives of this study were the following: (1) examine whether the pulmonary fungal and bacterial microbiota differed in patients with sarcoidosis compared with controls; (2) examine whether there was an association between the microbiota and levels of the antimicrobial peptides (AMPs) in protected bronchoalveolar lavage (PBAL)., Methods: Thirty-five sarcoidosis patients and 35 healthy controls underwent bronchoscopy and were sampled with oral wash (OW), protected BAL (PBAL), and left protected sterile brushes (LPSB). The fungal ITS1 region and the V3V4 region of the bacterial 16S rRNA gene were sequenced. Bioinformatic analyses were performed with QIIME 2. The AMPs secretory leucocyte protease inhibitor (SLPI) and human beta defensins 1 and 2 (hBD-1 and hBD-2), were measured in PBAL by enzyme-linked immunosorbent assay (ELISA)., Results: Aspergillus dominated the PBAL samples in sarcoidosis. Differences in bacterial taxonomy were minor. There was no significant difference in fungal alpha diversity between sarcoidosis and controls, but the bacterial alpha diversity in sarcoidosis was significantly lower in OW (p = 0.047) and PBAL (p = 0.03) compared with controls. The beta diversity for sarcoidosis compared with controls differed for both fungi and bacteria. AMP levels were significantly lower in sarcoidosis compared to controls (SLPI and hBD-1: p < 0.01). No significant correlations were found between alpha diversity and AMPs., Conclusions: The pulmonary fungal and bacterial microbiota in sarcoidosis differed from in controls. Lower antimicrobial peptides levels were seen in sarcoidosis, indicating an interaction between the microbiota and the innate immune system. Whether this dysbiosis represents a pathogenic mechanism in sarcoidosis needs to be confirmed in experimental studies. Video Abstract., (© 2022. The Author(s).)

Plastic degradation by biological systems with re-utilization of the by-products could be a future solution to the global threat of plastic waste accumulation. Here, we report that the saliva of Galleria mellonella larvae (wax worms) is capable of oxidizing and depolymerizing polyethylene (PE), one of the most produced and sturdy polyolefin-derived plastics. This effect is achieved after a few hours' exposure at room temperature under physiological conditions (neutral pH). The wax worm saliva can overcome the bottleneck step in PE biodegradation, namely the initial oxidation step. Within the saliva, we identify two enzymes, belonging to the phenol oxidase family, that can reproduce the same effect. To the best of our knowledge, these enzymes are the first animal enzymes with this capability, opening the way to potential solutions for plastic waste management through bio-recycling/up-cycling., (© 2022. The Author(s).)

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.

The histone demethylase KDM1A is a multi-faceted regulator of vital developmental processes, including mesodermal and cardiac tube formation during gastrulation. However, it is unknown whether the fine-tuning of KDM1A splicing isoforms, already shown to regulate neuronal maturation, is crucial for the specification and maintenance of cell identity during cardiogenesis. Here, we discovered a temporal modulation of ubKDM1A and KDM1A+2a during human and mice fetal cardiac development and evaluated their impact on the regulation of cardiac differentiation. We revealed a severely impaired cardiac differentiation in KDM1A -/- hESCs that can be rescued by re-expressing ubKDM1A or catalytically impaired ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. Conversely, KDM1A+2a -/- hESCs give rise to functional cardiac cells, displaying increased beating amplitude and frequency and enhanced expression of critical cardiogenic markers. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants, independent of their enzymatic activity, during hESC differentiation into cardiac cells., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Background: Few studies have examined the stability of the pulmonary mycobiome. We report longitudinal changes in the oral and pulmonary mycobiome of participants with and without COPD in a large-scale bronchoscopy study (MicroCOPD)., Methods: Repeated sampling was performed in 30 participants with and 21 without COPD. We collected an oral wash (OW) and a bronchoalveolar lavage (BAL) sample from each participant at two time points. The internal transcribed spacer 1 region of the ribosomal RNA gene cluster was PCR amplified and sequenced on an Illumina HiSeq sequencer. Differences in taxonomy, alpha diversity, and beta diversity between the two time points were compared, and we examined the effect of intercurrent antibiotic use., Results: Sample pairs were dominated by Candida. We observed less stability in the pulmonary taxonomy compared to the oral taxonomy, additionally emphasised by a higher Yue-Clayton measure in BAL compared to OW (0.69 vs 0.22). No apparent effect was visually seen on taxonomy from intercurrent antibiotic use or participant category. We found no systematic variation in alpha diversity by time either in BAL (p-value 0.16) or in OW (p-value 0.97), and no obvious clusters on bronchoscopy number in PCoA plots. Pairwise distance analyses showed that OW samples from repeated sampling appeared more stable compared to BAL samples using the Bray-Curtis distance metric (p-value 0.0012), but not for Jaccard., Conclusion: Results from the current study propose that the pulmonary mycobiome is less stable than the oral mycobiome, and neither COPD diagnosis nor intercurrent antibiotic use seemed to influence the stability., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Einar M. H. Martinsen, Elise O. Leiten, Gunnar Husebø, and Solveig Tangedal declare no conflict of interest. Walter Sanseverino and Andreu Paytuví-Gallart are employed at Sequentia Biotech SL. Tomas M. L. Eagan reports lecture fees from Boehringer and AstraZeneca, and grants from GlaxoSmithKline outside the submitted work. Harald G. Wiker reports being head of the educational programme for medicine at the University of Bergen. Kristel S. Knudsen reports lecture fees from Boehringer Ingelheim and Roche. Rune Nielsen reports grants from Boehringer Ingelheim, GlaxoSmithKline, AstraZeneca, and the Timber Merchant Delphins Endowment, in addition to lecture fees from GlaxoSmithKline and AstraZeneca. Rune Nielsen also reports being unpaid member of The Norwegian Respiratory Society, unpaid GOLD national delegate, unpaid ERS national delegate, and paid member of the Reference group for new Norwegian guidelines for COPD (The Norwegian Directorate of Health). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Pepper is a major crop species of the Solanaceae family, largely appreciated for its high nutritional and healthy contribution to human diets. In the Mediterranean basin, the favorable pedoclimatic conditions enhanced the selection of several diversified landraces cultivated pepper (Capsicum annuum), for whom Italy can be considered a main pole of diversification. Hence, a survey of traditional C. annuum genetic resources is essential for deep understanding of such diversity and for applications in genomics assisted breeding. Here, we report whole-genome resequencing analyses of two sweet and two pungent genotypes highly diffused in South Italy and representative of the variability for shape, colour and nutritional properties., Results: The four genomes were reconstructed at a chromosomal scale using a reference-guided approach, based on a dataset of 2.6 billion paired-end reads, corresponding to 20× genome coverage and a mapping rate above 99% for a final genomes size of approximately 3 Gb. After five iterations of variant calling, a total of 29,258,818 single nucleotide polymorphisms (SNPs) and 1,879,112 InDels, were identified. Substantial differences were observed among the four genomes based on geographical origin, with chromosomes 9 and 11 showing more polymorphisms in the accessions with higher fruit weight and absence of pungency. Among the identified variants, a small private indel (T - > TA) shared between sweet and big fruits accessions induces a frameshift with the generation of a new stop codon in a gene annotated as extensin, whereas two private SNPs within hot types were identified in 1-aminocyclopropane-1-carboxylate oxidase (ACO), a key gene involved in fruit ripening. The estimation of repetitive elements highlights a preponderant presence of Long Terminal Repeats (LTRs), the majority of which belonged to Gypsy superfamily. By comparing the four genomes with publicly available references including 'CM334' and Zunla-1 highlight the presence of 49,475 shared gene families., Conclusions: The new genomic sequences aim to enrich the whole genome information of pepper local varieties, providing a valuable tool for precision gene mapping, marker discovery, comparative studies. Such knowledge widens the frontiers to understand the selection history of Italian pepper landraces toward the recognition of specificity local agri-food products marks., (© 2022. The Author(s).)

Klinefelter syndrome (KS) is the most prevalent aneuploidy in males and is characterized by a 47,XXY karyotype. Less frequently, higher grade sex chromosome aneuploidies (HGAs) can also occur. Here, using a paradigmatic cohort of KS and HGA induced pluripotent stem cells (iPSCs) carrying 49,XXXXY, 48,XXXY, and 47,XXY karyotypes, we identified the genes within the pseudoautosomal region 1 (PAR1) as the most susceptible to dosage-dependent transcriptional dysregulation and therefore potentially responsible for the progressively worsening phenotype in higher grade X aneuploidies. By contrast, the biallelically expressed non-PAR escape genes displayed high interclonal and interpatient variability in iPSCs and differentiated derivatives, suggesting that these genes could be associated with variable KS traits. By interrogating KS and HGA iPSCs at the single-cell resolution we showed that PAR1 and non-PAR escape genes are not only resilient to the X-inactive specific transcript (XIST)-mediated inactivation but also that their transcriptional regulation is disjointed from the absolute XIST expression level. Finally, we explored the transcriptional effects of X chromosome overdosage on autosomes and identified the nuclear respiratory factor 1 (NRF1) as a key regulator of the zinc finger protein X-linked (ZFX). Our study provides the first evidence of an X-dosage-sensitive autosomal transcription factor regulating an X-linked gene in low- and high-grade X aneuploidies., Competing Interests: AS and RA were employed by the company Sequentia Biotech SL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Astro, Alowaysi, Fiacco, Saera-Vila, Cardona-Londoño, Aiese Cigliano and Adamo.)

In this study we investigated the transcriptome and epigenome dynamics of the tomato fruit during post-harvest in a landrace belonging to a group of tomatoes (Solanum lycopersicum L.) collectively known as "Piennolo del Vesuvio", all characterized by a long shelf-life. Expression of protein-coding genes and microRNAs as well as DNA methylation patterns and histone modifications were analysed in distinct post-harvest phases. Multi-omics data integration contributed to the elucidation of the molecular mechanisms underlying processes leading to long shelf-life. We unveiled global changes in transcriptome and epigenome. DNA methylation increased and the repressive histone mark H3K27me3 was lost as the fruit progressed from red ripe to 150 days post-harvest. Thousands of genes were differentially expressed, about half of which were potentially epi-regulated as they were engaged in at least one epi-mark change in addition to being microRNA targets in ~5% of cases. Down-regulation of the ripening regulator MADS-RIN and of genes involved in ethylene response and cell wall degradation was consistent with the delayed fruit softening. Large-scale epigenome reprogramming that occurred in the fruit during post-harvest likely contributed to delayed fruit senescence., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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&#95;Page., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Papiliotrema terrestris strain LS28 is a biocontrol agent selected for its antagonistic activity against several plant pathogens both in the field and postharvest. The availability of a genome sequencing sets the foundation for the identification of the genetic mechanisms of its antagonistic activity. The genome size is 21.29 Mbp with a G+C content of 58.65%, and genome annotation predicts 8,626 protein-encoding genes. Phylogenetic analysis based on whole-genome data confirms that P. terrestris is a Tremellomycetes more closely related to Papiliotrema flavescens than Papiliotrema laurentii., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Thraustochytrids are marine protists that naturally accumulate triacylglycerol with long chains of polyunsaturated fatty acids, such as ω3-docosahexaenoic acid (DHA). They represent a sustainable response to the increasing demand for these "essential" fatty acids (FAs). Following an attempt to transform a strain of Aurantiochytrium limacinum , we serendipitously isolated a clone that did not incorporate any recombinant DNA but contained two to three times more DHA than the original strain. Metabolic analyses indicated a deficit in FA catabolism. However, whole transcriptome analysis did not show down-regulation of genes involved in FA catabolism. Genome sequencing revealed extensive DNA deletion in one allele encoding a putative peroxisomal adenylate transporter. Phylogenetic analyses and yeast complementation experiments confirmed the gene as a peroxisomal adenylate nucleotide transporter ( AlANT1 ), homologous to yeast ScANT1 and plant peroxisomal adenylate nucleotide carrier AtPNC genes. In yeast and plants, a deletion of the peroxisomal adenylate transporter inhibits FA breakdown and induces FA accumulation, a phenotype similar to that described here. In response to this metabolic event, several compensatory mechanisms were observed. In particular, genes involved in FA biosynthesis were upregulated, also contributing to the high FA accumulation. These results support AlANT1 as a promising target for enhancing DHA production in Thraustochytrids.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Spinach (Spinacia oleracea L.) is a member of the Caryophyllales family, a basal eudicot asterid that consists of sugar beet (Beta vulgaris L. subsp. vulgaris), quinoa (Chenopodium quinoa Willd.), and amaranth (Amaranthus hypochondriacus L.). With the introduction of baby leaf types, spinach has become a staple food in many homes. Production issues focus on yield, nitrogen-use efficiency and resistance to downy mildew (Peronospora effusa). Although genomes are available for the above species, a chromosome-level assembly exists only for quinoa, allowing for proper annotation and structural analyses to enhance crop improvement. We independently assembled and annotated genomes of the cultivar Viroflay using short-read strategy (Illumina) and long-read strategies (Pacific Biosciences) to develop a chromosome-level, genetically anchored assembly for spinach. Scaffold N50 for the Illumina assembly was 389 kb, whereas that for Pacific BioSciences was 4.43 Mb, representing 911 Mb (93% of the genome) in 221 scaffolds, 80% of which are anchored and oriented on a sequence-based genetic map, also described within this work. The two assemblies were 99.5% collinear. Independent annotation of the two assemblies with the same comprehensive transcriptome dataset show that the quality of the assembly directly affects the annotation with significantly more genes predicted (26,862 vs. 34,877) in the long-read assembly. Analysis of resistance genes confirms a bias in resistant gene motifs more typical of monocots. Evolutionary analysis indicates that Spinacia is a paleohexaploid with a whole-genome triplication followed by extensive gene rearrangements identified in this work. Diversity analysis of 75 lines indicate that variation in genes is ample for hypothesis-driven, genomic-assisted breeding enabled by this work., (© 2021 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Transcriptome analyses using high-throughput methodologies allow a deeper understanding of biological functions in different cell types/tissues. The present study provides an RNA-seq profiling of human sperm mRNAs and lncRNAs (messenger and long non-coding RNAs) in a well-characterized population of fertile individuals. Sperm RNA was extracted from twelve ejaculate samples under strict quality controls. Poly(A)-transcripts were sequenced and aligned to the human genome. mRNAs and lncRNAs were classified according to their mean expression values (FPKM: Fragments Per Kilobase of transcript per Million mapped reads) and integrity. Gene Ontology analysis of the Expressed and Highly Expressed mRNAs showed an involvement in diverse reproduction processes, while the Ubiquitously Expressed and Highly Stable mRNAs were mainly involved in spermatogenesis. Transcription factor enrichment analyses revealed that the Highly Expressed and Ubiquitously Expressed sperm mRNAs were primarily regulated by zinc-fingers and spermatogenesis-related proteins. Regarding the Expressed lncRNAs, only one-third of their potential targets corresponded to Expressed mRNAs and were enriched in cell-cycle regulation processes. The remaining two-thirds were absent in sperm and were enriched in embryogenesis-related processes. A significant amount of post-testicular sperm mRNAs and lncRNAs was also detected. Even though our study is solely directed to the poly-A fraction of sperm transcripts, results indicate that both sperm mRNAs and lncRNAs constitute a footprint of previous spermatogenesis events and are configured to affect the first stages of embryo development., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2020 John Wiley & Sons, Ltd.)

Background: The fungal part of the pulmonary microbiome (mycobiome) is understudied. We report the composition of the oral and pulmonary mycobiome in participants with COPD compared to controls in a large-scale single-centre bronchoscopy study (MicroCOPD)., Methods: Oral wash and bronchoalveolar lavage (BAL) was collected from 93 participants with COPD and 100 controls. Fungal DNA was extracted before sequencing of the internal transcribed spacer 1 (ITS1) region of the fungal ribosomal RNA gene cluster. Taxonomic barplots were generated, and we compared taxonomic composition, Shannon index, and beta diversity between study groups, and by use of inhaled steroids., Results: The oral and pulmonary mycobiomes from controls and participants with COPD were dominated by Candida, and there were more Candida in oral samples compared to BAL for both study groups. Malassezia and Sarocladium were also frequently found in pulmonary samples. No consistent differences were found between study groups in terms of differential abundance/distribution. Alpha and beta diversity did not differ between study groups in pulmonary samples, but beta diversity varied with sample type. The mycobiomes did not seem to be affected by use of inhaled steroids., Conclusion: Oral and pulmonary samples differed in taxonomic composition and diversity, possibly indicating the existence of a pulmonary mycobiome., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Einar M. H. Martinsen, Elise O. Leiten, Ingvild Haaland, Gunnar R. Husebø, Kristel S. Knudsen, and Christine Drengenes declare no conflict of interest. Walter Sanseverino and Andreu Paytuví-Gallart are employed at Sequentia Biotech SL. Rune Nielsen reports grants from the Endowment of Timber Merchant A. Delphin and Wife (The Norwegian Medical Association) and grants from GlaxoSmithKline during the conduct of the study, and grants and personal fees from AstraZeneca, grants and personal fees from GlaxoSmithKline, grants and personal fees from Boehringer Ingelheim, and grants from Novartis outside the submitted work. Tomas M. L. Eagan reports grants from Helse Vest (Western Norway Regional Health Authority) during the conduct of the study, and personal fees from Boehringer Ingelheim outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Isolation of nuclei tagged in specific cell types (INTACT) is a method developed to isolate cell-type-specific nuclei that are tagged through in vivo biotin labeling of a nuclear targeting fusion (NTF) protein. In our work, INTACT was used to capture nuclei of meiocytes and to generate a meiotic transcriptome in Arabidopsis . Using the promoter of AtDMC1 recombinase to label meiotic nuclei, we generated transgenic plants carrying AtDMC1:NTF along with biotin ligase enzyme ( BirA ) under the constitutive ACTIN2 ( ACT2 ) promoter. AtDMC1 -driven expression of biotin-labeled NTF allowed us to collect nuclei of meiocytes by streptavidin-coated magnetic beads. The nuclear meiotic transcriptome was obtained by RNA-seq using low-quantity input RNA. Transcripts grouped into different categories according to their expression levels were investigated by gene ontology enrichment analysis (GOEA). The most enriched GO term "DNA demethylation" in mid/high-expression classes suggests that this biological process is particularly relevant to meiosis onset. The majority of genes with established roles in meiosis were distributed in the classes of mid/high and high expression. Meiotic transcriptome was compared with public available transcriptomes from other tissues in Arabidopsis. Bioinformatics analysis by expression network identified a core of more than 1,500 genes related to meiosis landmarks., Competing Interests: RAC was employed by company Sequentia Biotech SL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Barra, Termolino, Aiese Cigliano, Cremona, Paparo, Lanzillo, Consiglio and Conicella.)