Endometrial Proliferative Phase-Centered View of Transcriptome Dynamics across the Menstrual Cycle.

The endometrium, the inner mucosal lining of the uterus, undergoes complex molecular and cellular changes across the menstrual cycle in preparation for embryo implantation. Transcriptome-wide analyses have mainly been utilized to study endometrial receptivity, the prerequisite for successful implantation, with most studies, so far, comparing the endometrial transcriptomes between (i) secretory and proliferative endometrium or (ii) mid-secretory and early secretory endometrium. In the current study, we provide a complete transcriptome description of the endometrium across the entire menstrual cycle and, for the first time, comprehensively characterize the proliferative phase of the endometrium. Our temporal transcriptome analysis includes five time points including the mid-proliferative, late proliferative (peri-ovulatory phase), early secretory, mid-secretory, and late secretory phases. Thus, we unveil exhaustively the transitions between the consecutive proliferative and secretory phases, highlighting their unique gene expression profiles and possible distinct biological functions. The transcriptome analysis reveals many differentially expressed genes (DEGs) across the menstrual cycle, most of which are phase-specific. As an example of coordinated gene activity, the expression profile of histone-encoding genes within the HIST cluster on chromosome 6 shows an increase in cluster activity during the late proliferative and a decline during the mid-secretory phase. Moreover, numerous DEGs are shared among all phases. In conclusion, in the current study, we delineate the endometrial proliferative phase-centered view of transcriptome dynamics across the menstrual cycle. Our data analysis highlights significant transcriptomic and functional changes occurring during the late proliferative phase-an essential transition point from the proliferative phase to the secretory phase. Future studies should explore how the biology of the late proliferative phase endometrium impacts the achievement of mid-secretory endometrial receptivity or contributes to molecular aberrations leading to embryo implantation failure.

Electrospun Fibers of Biocompatible and Biodegradable Polyesters, Poly(Ethylene Oxide) and Beeswax with Anti-Bacterial and Anti-Fungal Activities.

Fibrous materials composed of core-sheath fibers from poly(ethylene oxide) (PEO), beeswax (BW) and 5-nitro-8-hydroxyquinoline (NQ) were prepared via the self-organization of PEO and BW during the single-spinneret electrospinning of a homogeneous blend solution of the partners. Additionally, the application of the same approach enabled the preparation of fibrous materials composed of core-double sheath fibers from PEO, poly(L-lactide) (PLA) and NQ or 5-chloro-7-iodo-8-hydroxyquinoline (CQ), as well as from PEO, poly(ε-caprolactone) (PCL) and NQ. The consecutive selective extraction of BW and of the polyester with hexane and tetrahydrofuran, respectively, evidenced that core-double sheath fibers from PEO/polyester/BW/drug consisted of a PEO core, a polyester inner sheath and a BW outer sheath. In order to evaluate the possibility of the application of fibrous materials from PEO/BW/NQ, PEO/PLA/BW/NQ, PEO/PCL/BW/NQ and PEO/PLA/BW/CQ for plant protection, microbiological studies were performed using both phytopathogenic microorganisms (Pseudomonas corrugata, Fusarium graminearum and Fusarium avenaceum) and beneficial microorganisms (Pseudomonas chlororaphis, Bacillus amyloliquefaciens and Trichoderma asperellum). It was found that the fibrous materials had anti-bacterial and anti-fungal activity against both phytopathogenic and beneficial microorganisms. This is the first report on the activity of fibrous materials loaded with 8-hydroxyquinoline derivatives not only against phytopathogenic but also against beneficial microorganisms that are of importance in agriculture.

Electrospun Polymer Materials with Fungicidal Activity: A Review.

In recent years, there has been special interest in innovative technologies such as polymer melt or solution electrospinning, electrospraying, centrifugal electrospinning, coaxial electrospinning, and others. Applying these electrokinetic methods, micro- or nanofibrous materials with high specific surface area, high porosity, and various designs for diverse applications could be created. By using these techniques it is possible to obtain fibrous materials from both synthetic and natural biocompatible and biodegradable polymers, harmless to the environment. Incorporation of low-molecular substances with biological activity (e.g., antimicrobial, antifungal) is easily feasible. Moreover, biocontrol agents, able to suppress the development and growth of plant pathogens, have been embedded in the fibrous materials as well. The application of such nanotechnologies for the creation of plant protection products is an extremely promising new direction. This review emphasizes the recent progress in the development of electrospun fungicidal dressings and their potential to be applied in modern agriculture.

Eco-Friendly Hybrid PLLA/Chitosan/Trichoderma asperellum Nanomaterials as Biocontrol Dressings against Esca Disease in Grapevines.

Fungi constitute the largest number of plant pathogens and are responsible for a range of serious plant diseases. Phaeomoniella chlamydospora (P. chlamydospora) and Phaeoacremonium aleophilum (P. aleophilum) are the main fungal pathogens causing esca disease in grapevines. On the other hand, there are beneficial microorganisms such as Trichoderma spp., which are able to control the growth of many phytopathogens. In the present study, innovative, eco-friendly hybrid nanomaterials were created by electrospinning PLLA, followed by the formation of a film of chitosan/Trichoderma asperellum (T. asperellum) spores on the fibers. The polymer carrier used in this study plays an active role in ensuring the viability of the biological agent during storage and, when placed in contact with moisture, ensures the agent's normal development. Oligochitosan, as well as low molecular weight and high molecular weight chitosan, were used. The effects of chitosan molecular weight on the dynamic viscosity of chitosan solutions, film formation, mechanical properties, spore incorporation and growth were studied. The morphology of the prepared nanomaterials, and the presence of a film based on the formation of chitosan/T. asperellum spores on the PLLA fibers, were examined using scanning electron microscopy (SEM). The surface chemical compositions of the fibrous materials were studied using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The mechanical properties of the obtained materials were also tested. The microbiological screening that was performed revealed that the eco-friendly hybrid nanomaterials incorporated with the beneficial microorganism, T. asperellum, to hamper the growth of the pathogenic P. chlamydospora and P. aleophilum fungi. The suppression rate depended on the viscosity of the chitosan solution used for the film formation. The use of oligochitosan resulted in the most effective infection of the material with T. asperellum spores. The environmentally friendly hybrid nanomaterials obtained in this study-in which the bioagent was embedded-are promising bioactive dressings for protecting grapevines against esca disease.

Tobacco Plant Growth-Promoting and Antifungal Activities of Three Endophytic Yeast Strains.

In this research, the biosynthetic and biocontrol potential of endophytic yeast to improve the growth and development of tobacco has been elucidated. Three yeast strains were enriched and isolated from different plant tissues. Partial sequence analysis of ITS5-5.8-ITS4 region of the nuclear ribosomal DNA with universal primers identified YD5, YE1, and YSW1 as Saccharomyces cerevisiae (S. cerevisiae), Zygosaccharomyces bailii (Z. bailii), and Saccharomyces kudriavzevii (S. kudriavzevii), respectively. When cultivated in a medium supplemented with 0.1% L-tryptophan, isolated yeast strains produced indole-3-acetic acid (IAA). The capacities of those strains to improve the mobility of phosphorus and synthesize siderophores has been proven. Their antimicrobial activities against several Solanaceae plant pathogenic fungi (Alternaria solani pathovar. tobacco, Rhizoctonia solani, and Fusarium solani pathovar. phaseoli) were determined. S. cerevisiae YD5, Z. bailii YE1, and S. kudriavzevii YSW1 inhibited the growth of all tested pathogens. Yeast strains were tested for endophytic colonization of tobacco by two different inoculation methods: soil drench (SD) and leaf spraying (LS). To establish colonization in the various tissues of tested tobacco (Nicotiana tabaccum L.) plants, samples were taken on the seventh, fourteenth, and twenty-first days after treatment (DAT), and explants were inoculated on yeast malt agar (YMA). Both techniques of inoculation showed a high frequency of colonization from 83.33% to 100%. To determine the effectiveness of the microbial endophytes, their effect on some physiological processes in the plant were analyzed, such as photosynthesis, stomatal conductivity, and transpiration intensity. The effect of single and double treatment with yeast inoculum on the development and biochemical parameters of tobacco was reported. Plants have the ability of structural and functional adaptation to stress effects of different natures. All treated plants had a higher content of photosynthetic pigments compared to the control. Photosynthesis is probably more intense, and growth stimulation has been observed. The chlorophyll a/b ratio remained similar, and the total chlorophyll/carotenoid ratio slightly increased as a result of elevated chlorophyll levels. The most significant stimulating effect was recorded in tobacco plants treated by foliar spraying with Z. bailii YE1 and S. cerevisiae YD5. In contrast, S. kudriavzevii YSW1 had a better effect when applied as a soil drench. Thus, S. cerevisiae YD5, Z. bailii YE1, and S. kudriavzevii YSW1 have a high potential to be used as a biocontrol agents in organic agriculture.

Electrospun 5-Chloro-7-iodo-8-hydroxyquinoline (Clioquinol)-Containing Poly(3-hydroxybutyrate)/Polyvinylpyrrolidone Antifungal Materials Prospective as Active Dressings against Esca.

Esca is a grapevine disease known for centuries which pertains to the group of so-called vine trunk diseases. Phaeomoniella chlamydospora (P. chlamydospora) and Phaeoacremonium aleophilum (P. aleophilum) are the two main fungal pathogens associated with esca. Novel fibrous materials with antifungal properties based on poly(3-hydroxybutyrate) (PHB), polyvinylpyrrolidone (PVP) and 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol, CQ) were developed. One-pot electrospinning ("in" strategy) or electrospinning in conjunction with electrospraying ("on" strategy) were applied to obtain the materials. The materials' morphology and their surface chemical composition were examined using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). CQ incorporated in the bulk of the fibers or in PVP particles deposited on the fibers was in the amorphous phase, which was confirmed by differential scanning calorimetry (DSC) and X-ray diffraction analysis (XRD). The in vitro release of CQ depended on the composition of the electrospun materials and on their design. The performed microbiological screening revealed that, unlike the non-loaded mats, the fibrous mats loaded with CQ were effective in inhibiting the growth of the pathogenic P. chlamydospora and P. aleophilum fungi. Therefore, the created materials are promising as active dressings for grapevine protection against esca.

The Dynamics of miR-449a/c Expression during Uterine Cycles Are Associated with Endometrial Development.

The human endometrium is a highly dynamic tissue. Increasing evidence has shown that microRNAs (miRs) play essential roles in human endometrium development. Our previous assay, based on small RNA-sequencing (sRNA-seq) indicated the complexity and dynamics of numerous sequence variants of miRs (isomiRs) that can act together to control genes of functional relevance to the receptive endometrium (RE). Here, we used a greater average depth of sRNA-seq to detect poorly expressed small RNAs. The sequencing data confirmed the up-regulation of miR-449c and uncovered other members of the miR-449 family up-regulated in RE-among them miR-449a, as well as several isoforms of both miR-449a and miR-449c, while the third family member, miR-449b, was not identified. Stem-looped RT-qPCR analysis of miR expression at four-time points of the endometrial cycle verified the increased expression of the miR-449a/c family members in RE, among which the 5' isoform of miR-449c-miR-449c.1 was the most strongly up-regulated. Moreover, we found in a case study that the expression of miR-449c.1 and its precursor correlated with the histological assessment of the endometrial phase and patient age. We believe this study will promote the clinical investigation and application of the miR-449 family in the diagnosis and prognosis of human reproductive diseases.

Coupling miR/isomiR and mRNA Expression Signatures Unveils New Molecular Layers of Endometrial Receptivity.

Embryo implantation depends on endometrial receptivity (ER). To achieve ER, the preparation of the uterine lining requires controlled priming by ovarian hormones and the expression of numerous genes in the endometrial tissue. microRNAs (miRs) have emerged as critical genetic regulators of ER in fertility and of the diseases that are associated with infertility. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes can produce canonical miRs and variants-isomiRs. Here, we describe miR/isomiR expression dynamics across the four time points of natural chorionic gonadotropin (hCG)-administered cycles. Sequencing of the small RNAs (sRNA-seq) revealed that the most significant expression changes during the transition from the pre-receptive to the receptive phase occurred in the isomiR families of miR-125a, miR-125b, miR-10a, miR-10b, miR-449c, miR-92a, miR-92b, and miR-99a. Pairing the analysis of the differentially expressed (DE) miRs/isomiRs and their predicted DE mRNA targets uncovered 280 negatively correlating pairs. In the receptive endometrium, the 5'3'-isomiRs of miR-449c, which were among the most highly up-regulated isomiRs, showed a negative correlation with their target, transcription factor (TF) MYCN, which was down-regulated. Joint analysis of the miR/isomiR and TF expression identified several regulatory interactions. Based on these data, a regulatory TF-miR/isomiR gene-target circuit including let7g-5p and miR-345; the isomiR families of miR-10a, miR-10b, miR-92a, and miR-449c; and MYCN and TWIST1 was proposed to play a key role in the establishment of ER. Our work uncovers the complexity and dynamics of the endometrial isomiRs that can act cooperatively with miRs to control the functionally important genes that are critical to ER. Further studies of miR/isomiR expression patterns that are paired with those of their target mRNAs may provide a more in-depth picture of the endometrial pathologies that are associated with implantation failure.

Electrospun Polymer-Fungicide Nanocomposites for Grapevine Protection.

Nowadays, diseases in plants are a worldwide problem. Fungi represent the largest number of plant pathogens and are responsible for a range of serious plant diseases. Esca is a grapevine disease caused mainly by fungal pathogens Phaeomoniella chlamydospora (P. chlamydospora) and Phaeoacremonium aleophilum (P. aleophilum). The currently proposed methods to fight esca are not curative. In this study, polymer composites based on biodegradable polymer containing chemical fungicides with antifungal activity were successfully prepared by electrospinning. The obtained materials were hydrophobic with good mechanical properties. In vitro studies demonstrated that the fungicide release was higher from PLLA/K5N8Q fibrous mats (ca. 72% for 50 h) compared to the released drug amount from PLLA/5-Cl8Q materials (ca. 52% for 50 h), which is due to the better water-solubility of the salt. The antifungal activity of the fibrous materials against P. chlamydospora and P. aleophilum was studied as well. The incorporation of the fungicide in the biodegradable fibers resulted in the inhibition of fungal growth. The obtained materials are perspective candidates for the protection of vines from the penetration and growth of fungal pathogens.

Electrospun Eco-Friendly Materials Based on Poly(3-hydroxybutyrate) (PHB) and TiO2 with Antifungal Activity Prospective for Esca Treatment.

Esca is a type of grapevine trunk disease that severely affects vine yield and longevity. Phaeomoniella chlamydospora (P. chlamydospora) is one of the main fungi associated with esca. The aim of the present study was to obtain eco-friendly materials with potential antifungal activity against P. chlamydospora based on biodegradable and biocompatible poly(3-hydroxybutyrate) (PHB), nanosized TiO2-anatase (nanoTiO2), and chitosan oligomers (COS) by conjunction of electrospinning and electrospraying. One-pot electrospinning of a suspension of nanosized TiO2 nanoparticles in PHB solution resulted in materials in which TiO2 was incorporated within the fibers (design type "in"). Simultaneous electrospinning of PHB solution and electrospraying of the dispersion of nanosized TiO2 in COS solution enabled the preparation of materials consisting of PHB fibers on which TiO2 was deposited on the fibers' surface (design type "on"). Several methods including scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction analysis (XRD), thermogravimetric analyses (TGA) and water contact angle were utilized to characterize the obtained materials. The incorporation of nanoTiO2 in the PHB fibers, as well as nanoTiO2 deposition onto the surface of the PHB fibers resulted in increased roughness and hydrophobicity of the obtained composite fibrous materials. Moreover, TiO2-on-PHB fibrous material exhibited complete inhibition of fungal growth of P. chlamydospora. Therefore, the obtained eco-friendly fibrous materials based on PHB and nanoTiO2 are promising candidates for protection against esca in agriculture.

Electrospun 5-chloro-8-hydroxyquinoline-Loaded Cellulose Acetate/Polyethylene Glycol Antifungal Membranes Against Esca.

Esca is one of the earliest described diseases in grapevines and causes trunk damage and the sudden wilting of the entire plant; it is caused mainly by the species Phaeomoniella chlamydospora (P. chlamydospora) and Phaeoacremonium aleophilum (P. aleophilum). In practice, there are no known curative approaches for fighting esca directly, which is a huge problem for preserving vineyards. Micro- and nanofibrous membranes from cellulose acetate (CA) and cellulose acetate/polyethylene glycol (CA/PEG) containing 5-chloro-8-hydroxyquinolinol (5-Cl8Q) were successfully prepared by electrospinning. The surface morphologies and optical and mechanical properties of the membranes were characterized by using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-Vis), water contact angle measurements and mechanical tests. It was found that the bioactive compound release was facilitated by PEG. The antifungal activities of the obtained materials against P. chlamydospora and P. aleophilum were studied. We have demonstrated that 5-Cl8Q is an efficient and sustainable antifungal agent against P. chlamydospora and P. aleophilum. Moreover, for the first time, the present study reveals the possibility of using electrospun polymer membranes containing 5-Cl8Q which impede the penetration and growth of P. chlamydospora and P. aleophilum. Thus, the obtained fibrous materials can be suitable candidates for plant protection against diverse fungi.

Transposon-associated polymorphisms of stress-responsive gene promoters in selected accessions of Arabidopsis thaliana.

Genetic diversity caused by transposable element movement can play an important role in plant adaptation to local environments. Regarding genes, transposon-induced alleles were mostly related to gene bodies and a few of them to promoter regions. In this study, promoter regions of 9 stress-related genes were searched for transposable element insertions in 12 natural accessions of Arabidopsis thaliana. The promoter screening was performed via PCR amplification with primers designed to flank transposable element insertions in the promoter regions of the reference accession Col-0. Transposable element-associated insertion/deletion (indel) polymorphisms were identified in 7 of the 12 promoter loci across studied accessions that can be developed further as molecular markers. The transposable element absence in the promoter regions of orthologous genes in A. lyrata indicated that the insertion of these transposable elements in A. thaliana lineage had occurred after its divergence from A. lyrata. Sequence analysis of the promoter regions of CML41 (Calmodulin-like protein 41) and CHAP (chaperone protein dnaJ-related) confirmed the indel polymorphic sites in four accessions - Col-0, Wassilewskija, Shahdara, and Pirin. The observed indel polymorphism of the CHAP promoter region was associated with specific gene expression profiles in the different accessions grown at a normal and elevated temperature in a plant growth chamber. The collected data can be a starting point for gene expression profiling studies under conditions resembling the natural habitats of accessions.

HPLC-HRMS method for fast phytochelatins determination in plants. Application to analysis of Clinopodium vulgare L.

An optimized analytical method based on C8 core-shell reverse phase chromatographic separation and high resolution mass spectral (HRMS) detection is developed for a fast analysis of unbound phytochelatins (PCs) in plants. Its application to analysis of Clinopodium vulgare L. is demonstrated where proper PCs liberating and preservation conditions were employed using dithiotreitol in the extraction step. A baseline separation of glutathione (GSH) and phytochelatins from 2 to 5 (PC2-PC5) for 3 min was achieved at conventional HPLC backpressure, with detection limits from 3 ppt (for GSH) to 2.5 ppb (for PC5). It is shown, that the use of HRMS with tandem mass spectral (MS/MS) capabilities permits additional wide range screening ability for iso-phytochelatins and PC similar compounds, based on exact mass and fragment spectra in a post acquisition manner.

High-temperature effect on genes engaged in DNA methylation and affected by DNA methylation in Arabidopsis.

Along with its essential role in the maintenance of genome integrity, DNA methylation takes part in regulation of genes which are important for plant development and stress response. In plants, DNA methylation process can be directed by small RNAs in process known as RNA-directed DNA methylation (RdDM) involving two plant-specific RNA polymerases - PolIV and PolV. The aim of the present study was to investigate the effect of heat stress on the expression of genes encoding key players in DNA methylation - DNA methyltransferase (MET1, CMT3, and DRM2), the largest subunits of PoIIV and PolV (NRPD1 and NRPE1 respectively) and the DNA demethylase ROS1. We also examined the high-temperature effect on two protein-coding genes - At3g50770 and At5g43260 whose promoters contain transposon insertions and are affected by DNA-methylation, as well as on the AtSN1, a SINE-like retrotransposon. To assess the involvement of PolIV and PolV in heat stress response, the promoter methylation status and transcript levels of these genes were compared between wild type and double mutant lacking NRPD1 and NRPE1. The results demonstrate coordinated up-regulation of the DRM2, NRPD1 and NRPE1 in response to high temperature and suggest that PolIV and/or PolV might be required for the induction of DRM2 expression under heat stress. The ROS1 expression was confirmed to be suppressed in the mutant lacking active PolIV and PolV that might be a consequence of abolished DNA methylation. The increased expression of At3g50770 in response to elevated temperature correlated with reduced promoter DNA methylation, while the stress response of At5g43260 did not show inverse correlation between promoter methylation and gene expression. Our results also imply that PolIV and/or PolV could regulate gene expression under stress conditions not only through RdDM but also by acting in other regulatory processes.

Insight into small RNA abundance and expression in high- and low-temperature stress response using deep sequencing in Arabidopsis.

Small RNA profiling and assessing its dependence on changing environmental factors have expanded our understanding of the transcriptional and post-transcriptional regulation of plant stress responses. Insufficient data have been documented earlier to depict the profiling of small RNA classes in temperature-associated stress which has a wide implication for climate change biology. In the present study, we report a comparative assessment of the genome-wide profiling of small RNAs in Arabidopsis thaliana using two conditional responses, induced by high- and low-temperature. Genome-wide profiling of small RNAs revealed an abundance of 21 nt small RNAs at low temperature, while high temperature showed an abundance of 21 nt and 24 nt small RNAs. The two temperature treatments altered the expression of a specific subset of mature miRNAs and displayed differential expression of a number of miRNA isoforms (isomiRs). Comparative analysis demonstrated that a large number of protein-coding genes can give rise to differentially expressed small RNAs following temperature shifts. Low temperature caused accumulation of small RNAs, corresponding to the sense strand of a number of cold-responsive genes. In contrast, high temperature stimulated the production of small RNAs of both polarities from genes encoding functionally diverse proteins.

Implementation of a de novo genome-wide computational approach for updating Brachypodium miRNAs.

Plant microRNAs (miRNAs) are single-stranded 20-22 nt small RNAs (sRNA) that are produced from their own genes. We have developed a de novo genome-wide approach for the computational identification of novel plant miRNAs based on the integration of the complete genome sequence with sRNA libraries. It comprises three modules - the clustering module identifies genomic regions that have two closely-located unidirectional sRNA clusters, the mirplan module explores the secondary structure of the genomic regions, and the duplex module predicts miRNA/miRNA* duplexes. We applied our approach to the Brachypodium genome and publicly available sRNA libraries and predicted 102 miRNAs. Our results extend the list of known miRNAs with 58 novel miRNAs and define the genomic loci of all predicted miRNAs. Because this approach considers specific features of plant miRNAs, it can be employed for the analysis of the genome and sRNA libraries generated for plant species to achieve systematic miRNA discovery.

Identification of RNA-dependent DNA-methylation regulated promoters in Arabidopsis.

RNA-dependent DNA methylation (RdDM) is an important regulatory event involved in repressive epigenetic modifications that can trigger transcriptional gene silencing (TGS). The criteria we used to pick out promoter sequences targeted by RdDM in Arabidopsis thaliana were the main RdDM hallmark properties: 24nt siRNAs as inducers of DNA methylation and transposable elements (TE) as one of the major targets of RdDM. Those genes whose promoters comprised overlapping sites for 24nt siRNA hits, TE and DNA methylation (siRNA/TE/Methylation overlapping regions), were defined as candidates that might be silenced by RdDM. On this basis two gene sets were created which include abiotic and biotic stress-responsive genes whose promoters may be silenced by RdDM. The DNA methylation status of the At3g50770 (CML41) promoter - one of the selected candidates, was experimentally assayed, and it showed dependence on the RdDM-associated Polymerase IV and Polymerase V. A publicly available 24nt siRNA-centered database called starPRO was developed that allows users easily to discover whether a particular promoter sequence is related to RdDM-associated features such as 24nt siRNA-target sites, TE, tandem repeats and DNA methylation.