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Trophoblast derived extracellular vesicles specifically alter the transcriptome of endometrial cells and may constitute a critical component of embryo-maternal communication
Reproductive Biology and Endocrinology volume 19, Article number: 115 (2021)
The period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation—known as “window of implantation”—is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication.
To investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95–2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing.
Trophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors’ signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs.
Our study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.
Embryo implantation is a crucial step in human reproduction where the embryo initiates physical contact with maternal tissue. The embryo first loosely attaches itself to the endometrial epithelial lining and then proceeds to invade the uterine stromal cell layer . The endometrial epithelium and the underlying stromal cells go through remarkable alterations to facilitate the process of implantation. Most of the physiological and biochemical changes happening in the vicinity of embryo attachment site are localized to the implantation site only, giving rise to the theory that the embryo itself directs the changes required for implantation in the endometrium [2, 3]. Subsequently, the mechanisms of endometrial regulation arising from the embryo have emerged as a subject of intense scientific investigation.
One of the hypotheses being investigated in this regard is the theory of embryo-maternal cross-talk which posits that the embryo and the endometrium undergo a complex set of “negotiations” prior to and during apposition, the first step of implantation, in the time period known as the “window of implantation” (WOI) [4, 5]. The cross-talk prepares both the embryo and the endometrium for a successful implantation by inducing specific biochemical alterations in the epithelial cells and the underlying uterine architecture [6, 7]. Inadequate intercellular dialog would result in implantation failure, which is also the main cause of unsuccessful attempts in assisted reproduction [8,9,10]. Evidence of embryo-maternal cross-talk is reported by a number of studies focusing on different modes of signalling. For example, hormonal regulation of endometrial tissues is a well-known phenomenon [11,12,13], There is also evidence of cytokine based regulation of endometrial tissue during the WOI [14,15,16,17] and direct protein–protein embryo-maternal interactions . However, as embryo implantation is a temporally and spatially complex process, it likely involves several other mechanisms that are less known.
One of the alternative methods of communication under consideration is extracellular vesicle (EV) mediated communications. EVs are a heterogeneous group of membrane bound small (diameter of 20 – 1000 nm) spherical biological structures known to be secreted by every known cell type. Although they were initially considered ‘cellular garbage’ of little interest, in the recent years the concept of EVs as means of intercellular communication has gained traction.
EV-mediated communication may involve diverse mechanisms. They carry an extensive surface proteome and possess a large surface to volume ratio. Mediated by these surface bound molecules, EVs take part in a multitude of molecular interactions with the target cell membrane. These exchanges form a connection between the EV source and the target cell accomplishing a plethora of physiological functions [19,20,21]. Another well-known method of EV based communication concerns the EV cargo. EVs are known to carry a diverse cargo of RNAs, lipids and proteins which are protected from the destructive enzymesin the extracellular space [22,23,24]. EVs are taken up by target cells via various mechanisms such as macropinocytosis, phagocytosis, and lipid raft–mediated internalization . The cargo molecules are then released into the target cell’s cytoplasm. Various instances of intercellular communication, including embryo-maternal communication, are reported to involve EVs [26,27,28,29].
In an earlier study employing an in vitro model of embryo-maternalcommunication, we showed that labelled RNA can be transferred remotely from trophoblast cells to endometrial cells via EVs . In the current study, using the same in vitro system, we investigated the hypothesis that embryo-derived EVs are capable of specifically altering the transcriptomic profiles of endometrial cells. We analysed the RNA cargo of trophoblast EVs and EVs from a cell line of a non-reproductive origin (HEK-293, kidney origin). Co-incubation with trophoblast derived EVs induced substantial changes to the transcriptomic profile of endometrial cells, while no such alterations were observed after HEK EV co-incubation with endometrial cells. These data support the hypothesis that trophoblast EVs specifically interact with endometrial cells and may affect the gene expression in endometrial cells partially through their molecular cargo.
Because of the well-known ethical dilemmas of using human embryos for experimentation, we have used an in vitro model of the pre-implantation microenvironment in the current study. Human choriocarcinoma cell line JAr in 3D spheroidal form was used as an analogue for the trophoblast cells and the RL95-2 cell line was used as an analogue for the mid-secretary/WOI receptive endometrium.
Cell culture and spheroid formation
The human endometrial adenosquamous carcinoma cell line (RL95-2) was obtained from American Type Culture Collection (ATCC CRL-1671, Teddington, UK). RL95-2 was cultured in Dulbecco’s Modified Eagles Medium (DMEM 12-604F, Lonza, Verviers, Belgium) supplemented with 1% Penicillin/Streptomycin (P/S, Gibco™ 15,140,122, Bleiswijk, Netherlands), 5 μg/ml Insulin (human recombinant insulin, Gibco, Invitrogen, Denmark), 1% L-glutamine (Sigma, 59202C, Saint Louis, USA) and 10% fetal bovine serum (FBS, Gibco™, 10,500,064) at 37 °C in 5% CO2 conditions.
The human choriocarcinoma cell line (JAr) from the first trimester trophoblasts was acquired from ATCC® (HTB-144 ™, Teddington, UK). JAr cells were cultured in a T75 flask in RPMI 1640 media (Gibco, Scotland) supplemented with 10% FBS, 1% L-glutamine and 1% P/S at 5% CO2 in 37 °C. At confluency, JAr cells were washed with Dulbecco’s phosphate-buffered saline without Ca+2 and Mg+2 (DPBS, Verviers, Belgium), harvested using trypsin–EDTA (Gibco® Trypsin, New York, USA) and pelleted by centrifugation at 250 g for 5 min. 1 × 106 cells/ml were cultured in 5 ml of supplemented RPMI 1640 medium in 60 mm Petri dishes at 5% CO2 in 37 °C. The cells were kept on a gyratory shaker (Biosan PSU-2 T, Riga, Latvia), set at 295 rotations per min (rpm) for 18 h. The multicellular spheroids were used to mimic trophoblast cells in vitro.
The human embryo kidney (HEK) 293 T cell line (acquired from ATCC®, CRL-3216™, Teddington, UK) was cultured in DMEM supplemented with 10% of heat-inactivated FBS (Gibco) and 1% L-glutamine (Sigma). All cells were grown in T75 flasks at 37 °C in 5% CO2conditions. The media was changed every second day until confluence of the cells. One million cells were counted with a haemocytometer and cultured overnight on a gyratory shaker to form multicellular spheroids as described above.
Preparation of EV depleted medium
EV depleted FBS was produced using the ultrafiltration method described by Kornilov et al. in 2018 . Briefly, the FBS was filtered using Amicon ultra-15 centrifugal filters (100 kDa, MERCK KGAA, Darmstadt, Germany) at 3,000 g for 55 min. This method removed 90% of the nanoparticles from the FBS. The filtered FBS was used as a 10% supplementation for all the cell type specific complete culture media described above to prepare the EV depleted complete media.
EVs purification and characterization
EVs were harvested from conditioned media of spheroid culture of both the JAr and HEK293 cell lines. Conditioned media was centrifuged at 400 g for 10 min. The supernatant was again centrifuged at 4,000 g for 10 min and thereafter at 20,000 g for 15 min to get rid of cell debris and apoptotic bodies. To isolate EVs, conditioned media was concentrated to 500 µl with Amicon® Ultra-15 centrifugal filter devices (10 kDa cut-off). RNase inhibitor (1u/µl, Recombinant RNasin®, Promega corp., 2800, Woods Hollow Road, Madison, WI) was added to conditioned media to protect EVs’ RNA during the isolation process. EVs were isolated using size exclusion chromatography (SEC). A cross linked 4% agarose matrix of 90 µm beads were used (Sepharose 4 fast flow™, GE HealthCare Bio-Sciences AB, Uppsala, Sweden) in a 10 cm column. Fractions 7–10 (fraction size 1 ml) were collected. Fractions were concentrated using Amicon® Ultra-15 centrifugal filter devices (10 kDa cut-off). Isolated EVs were characterized following the protocols described elsewhere in details [26, 31]. Briefly, EVs were quantified by nano-particle tracking analysis using ZetaView (Particle Metrix GmbH, Inning am Ammersee, Germany). Surface proteome of the isolated EVs were analysed using western blot for standard EV markers, CD63, CD81 and CD9. Morphology of the EVs was observed using transmission electron microscopy. EV characterization methods were published in our earlier study .
Whole RNA extraction and quality control
Whole RNA was extracted from cells and EVs by TRIzol Reagent and isopropanol precipitation (TRIzol® reagent; Invitrogen). To increase the efficiency of RNA extraction, 20 µg glycogen (UltraPure™ Glycogen, Cat. no. 10814–010, Thermo Fisher Scientific, Bleiswijk, Netherlands) was added to the lysis buffer per sample. The RNA pellet was washed three times by 75% ethanol. Quality and quantity of the extracted RNA samples were analyzed by Bioanalyzer Automated Electrophoresis instrument (Agilent technologies, Santa Clara, CA) using Agilent RNA 6000 Pico kit (Agilent technologies).
cDNA Library preparation and mRNA sequencing
RNA sequencing libraries were generated using multiplexing capacity of Smart-seq2 methodology with slight modifications . Instead of single cells, 20 ng of total RNA was used for cDNA synthesis and 10 cycles of PCR for pre-amplification. KAPA HiFi DNA polymerase was replaced with Phusion High-Fidelity DNA Polymerase (Thermo Scientific) compatible with the original protocol. 2 μL of diluted cDNA was applied to dual-index library preparation using Illumina Nextera XT DNA Sample Preparation Kit (FC-131–1024). AMPure XP beads (Beckman Coulter) were used for all clean-up steps and for size selection (200–700 bp). All samples were pooled into single library by equal concentration and sequenced on Illumina NextSeq500 using High Output Flow Cell v2.5 (single-end, 75 bp).
Processing, alignment, and quantification of RNAsequencing(RNAseq) reads
The quality of raw reads was assessed using FASTQC v0.11.8 . Trimmomatic v0.39 was used for read trimming and removal of adaptor sequences, using the following parameters: LEADING:20, SLIDINGWINDOW:4:15, ILLUMINACLIP: *:1:30:15 and MINLEN:25.
Reads were aligned to the hg38 human reference genome. The alignment was performed using HISAT2  with default parameters and with the inclusion of splice site information derived from the corresponding Ensembl H. sapiens annotation file (GRCh38.97). The sequencing of EV RNA samples yielded relatively low percentage of reads mapped to the genome. For HEK293 EVs, on average 3.08% of 6,820,518 total alignments were successfully assigned. In case of JAr EVs, 4.48% of 4,672,213 total alignments were successfully assigned on average. For RL95-2 cells treated with JAr EVs, 5,747,968 reads were aligned on average and 32.39% of which were successfully assigned to the genome. Average number of aligned reads and percentage of successfully assigned reads were 5,282,088 (56.84%) in case of untreated RL95-2 cells and 4,974,678 (54.94%) for RL 95–2 cells treated with HEK293 EVs. Gene-level read counts were obtained using featureCounts  with default parameters, using the Ensembl H. sapiens annotation file (GRCh38.97) for genomic feature annotations. Genes with at least 10 counts for all the samples in at least one of the experimental groups were retained in the analysis for subsequent differential expression testing.
Differential gene expression analysis
Differential expression (DE) analysis was carried out in R version 3.6.1 using the edgeR package version 3.26.8 . Tagwise dispersion estimates were obtained based on the trended dispersions, and statistical comparisons were performed using a generalized linear model followed by likelihood ratio tests, also accounting for the experiment batch. We considered the differential expression of geneswith a false discovery rate (FDR) ≤ 0.05 to be statistically significant.
Gene set enrichment analysis (GSEA), and pathway over-representation analysis was conducted using the ReactomePA package  and Reactome Pathway database annotations . GSEA was used for full gene lists obtained from DE analysis that were ranked by -log10p × log2FC, where p denotes unadjusted p-values and FC the fold-change.
Principal components were calculated using prcomp function from the Stats package and visualized using the ggplot2 package . The pheatmap package  was used for heatmap visualization with hierarchical clustering based on Euclidean distance.
RNA extraction for miRNA sequencing
The EVs (1 × 108 EVs extracted from 10 ml of conditioned medium)were sorted into 100 µl of RLT buffer (Qiagen) and proceeded for RNA extraction. Briefly, 100 µl of RLT buffer with sorted EVs was mixed with 2 µl of pellet paint (Merck Millipore), vortexed briefly, 19 µl of 3 M Sodium Acetate (pH 5.5) and 300 µl of 100% ethanol was added and vortexed briefly and incubated at + 4 °C overnight. The contents were then centrifuged at 16,000 g for 15 min at 4 °C and the supernatant wascarefully discarded without disturbing the pellet. The pellet was washed twice with fresh 1 ml of 80% ethanol and air dried. The pellet was resuspended in 10 µl of RNase free water and stored at -80 °C till further use.
Small RNA library construction and data analysis
The small RNA transcriptome library was constructed for the EV’s from both the JAr and HEK293 cell lines,as described in the protocol published by Faridani et. al. [40, 41], using 3 µl of extracted whole RNA from EV’s and HEK293cells. The amplified libraries were then purified using AMPure XP beads with 1:1 ratio of sample to beads as per the manufsacturer’s protocol and eluted in 10 µl of RNase free water. DNA quantification was done using Qubit HS DNA analysis (Thermo Scientific) and the DNA library quality control was performed on Bioanalyzer 2100 station (Agilent). 5 ng of DNA from each sample was pooled and sequenced 1 × 100 bp using Illumina NovaSeq platform (National Genomics Infrastructure, SciLifeLab, Sweden).
The initial data analysis was performed on the Partek Flow bioinformatics software (Partek Inc, USA). Briefly, all the fastq files were screened and the contaminating reads from the mitochondrial DNA and ribosomal DNA were removed. Theunique molecular identifiers (UMI’s) were removed from the sequences and appended to the read names for later analysis. Adapters and poly(A) sequences were removed from the reads and the trimmed reads were aligned to human genome Hg38 using Bowtie 2 aligner with a seed length of 10 and seed mismatch of 1nt. Post alignment the UMIs were deduplicated and the reads were quantified to Hg38 miRBase mature miRNAs database, version 22.
Identification of putative JAr-specific miRNAs and their putative targets in RL95 cells
To identify putative JAr EV-specific miRNAs, we examined miRNA alignment counts from three small RNAseq libraries derived from JAr EVs (total genome-mapped reads: 1,359,431) alongside three derived from HEK293EVs (total genome-mapped reads: 1,912,942). The dataset was filtered to retain miRNAs which were detected in at least 2/3 libraries of either of JAr or HEK293EVs. We subsequently counted the number of miRNAs which were detected above raw counts thresholds of 1, 3, 5, and 10 in the required number of samples. For downstream analysis, miRNAs were considered specific to JAr EVs if they were represented by at least five counts in 2/3 JAr EV libraries while not being detected at all in any of the HEK293EV libraries.
We obtained a list of all predicted target transcripts from miRDB . These were filtered to retain only high-confidence targets (those with a target score of ≥ 90). Using the R package AnnotationDbi , REFSEQ transcript IDs were converted to ENSEMBL gene IDs to obtain the list of predicted miR targets at the gene level. We were thus able to identify putative miRNA targets in the RL95-2 gene expression dataset by matching the ENSEMBL IDs. We subsequently counted the number of putative targets within the RL95-2 gene expression dataset that were downregulated, upregulated, and non-DE for each miRNA.
Focusing on downregulated putative targets, we then sought to ascertain whether the abundance of a given miRNA from EVs corresponded with the extent of repression of downregulated targets in RL95-2. We obtained the mean counts per million (CPM) value for each miRNA in JAr EVs and the mean log2FC of downregulated putative target genes for each miRNA. We then performed a weighted Pearson’s correlation using the R package ‘weights’ (https://cran.r-project.org/web/packages/weights/index.html),whereby each miRNA was weighted according to the number of downregulated targets (Fig. 1).
Investigating the RNA cargo of extracellular vesicles
JAr and HEK293 cells were cultured and spheroids were formed according to the methods and conditions described above. Approximately 1 × 105 spheroids were prepared from each cell type. Once the spheroids were fully formed, they were transferred into 60 mm dishes containing 5 ml EV depleted culture media (5,000 spheroids per dish). Spheroids were incubated in a slow rotating gyratory shaker for 24 h to stop the spheroids from losing the structural cohesion. After incubation, conditioned media (approximately 100 ml) were collected and EVs were isolated. After removing the EVs used for supplementation, remaining EVs (approximately 1 × 1012 EVs per each sample) were subjected to RNA extraction and mRNA sequencing was performed. Samples were prepared in three different days for EV supplementation and mRNAseq. Samples used for miRNA sequencing were prepared separately and a minimum of 1 × 107EVs were used for miRNA sequencing for each sample in three biological replicates for each group.
Determining the effects of JAr and HEK293 cell derived EVs on RL95-2 cellular transcriptome
Endometrial analogue (RL95-2) cells were cultured in 12 well plates until 80% confluency using the culture methods and conditions described above. At the desired confluency, growth media was removedand 1 × 108 EVs derived from trophoblast analogue (JAr) and non-reproductive cellular spheroid (HEK293) cells, were added to the RL95-2 cell monolayer separately in an EV-depleted supplementation media. Controls were prepared using untreated RL95-2 cells cultured in EV-depleted media. Cells were incubated for 24 h. After incubation, the media was removed and cellular RNA was collected for sequencing. The experiment was performed on three different days to prepare the three biological replicates.
JAr cell derived EVs induced significantly differentiated gene expression in RL95-2 cells while HEK293 cell derived EVs failed to induce a similar effect
JAr cell spheroid-derived EVs and HEK293 cell spheroid-derived EVs were supplemented to RL95-2 cell monolayers separately and incubated for 24 h. Control samples were prepared using untreated RL95-2 cells. After incubation, the cellular RNA was extracted and sequenced for mRNA expression. Differential expression was calculated with reference to untreated control (R). The expression profile of RL95-2 cells treated with JAr spheroid derived EVs (RJ) was clearly different from the untreated control (R) and the RL95-2 cells treated with HEK293 spheroid-derived EVs (RH) (Fig. 2A). One of the RH samples was excluded from this analysis as an outlier (supplementary Figure 1). Nevertheless, it is apparent that the untreated RL95-2 cells and RL95-2 cells treated with HEK293 EVs are clustered relatively closely together, indicating that there was little or no effect on RL95-2 cells from HEK293 derived EVs. Differential expression (DE) analysis of the RJ and R group yielded 1166 upregulated and 588 downregulated genes in RJ group compared to the untreated RL95-2 cells.Similar analysis comparing R and RH group did not yield any significant differential expression. The similarity between the R and RH groups is abundantly clear based on the expression levels of aforementioned DEGs (Fig. 2B). The results of DE analysis suggest that JAr spheroid derived EVs can induce significant changes in RL95-2 transcriptome while the HEK293 derived EVs lack that capability.
Gene set enrichment analysis of JAr EV-targeted genes in RL95-2
The most significantly enriched pathways in RL95-2 cells supplemented with JAr EVs are listed in the Table 1.
Two major pathways, ECM organization (R-HSA-1474244) and signalling by GPCR (R-HSA-372790) were significantly affected by JAr EVs effects on RL95-2 cells. Other significantly affected pathways are events of the two major pathways, for instance, the pathway GPCR downstream signalling (R-HSA-388396) is one of the two first level events of the signalling by GPCR pathway. Other significantly enriched pathways indicated in the table are events of ECM organization (R-HSA-1474244) pathway, such as collagen formation (R-HSA-1474290). The net positive Normalized Enrichment Score (NES) values of these pathways indicate that the genes involved in them were mostly upregulated in RL95-2 cells supplemented with JAr EVs, suggesting that the pathways themselves could also have been induced as the result of EV supplementation.
JAr spheroid derived EVs carry distinct mRNA cargo compared to HEK293 cell derived EVs
EVs were isolated from JAr and HEK293 spheroid conditioned media using size exclusion chromatography. EV RNA was extracted and the mRNA cargo of the EVs were sequenced. Enrichment of mRNA was calculated by contrasting the abundance of JAr EV mRNA to the abundance of HEK293 EV mRNA using edgeR as previously described. The population of RNA fragments aligning to known genes was substantially different between the JAr EV RNA and HEK293 EV RNA (Fig. 3A). 400 genes were found to be significantly enriched among the EV RNA fragments at logFC > 1and FDR ≤ 0.05 in JAr EV while 501 mRNA were significantly depleted at logFC < 1 and FDR ≤ 0.05 compared to HEK293 EV (Fig. 3B). Based on these data, the mRNA cargo appears to be significantly dependent on the type of cells producing the EVs.
JAr spheroid derived EVs carry distinct miRNA cargo compared to HEK293 cell derived EVs
JAr EVs were also distinguishable from HEK293EVs according to their miRNA content. The miRNA filtering criteria used for the analysis influenced both the total number of miRNAs detected in either of the two EV types examined (Fig. 4A) and the number of miRNAs which were unique to either JAr or HEK293 EVs (Fig. 4B). When considering a read count threshold of five which had to be met in 2/3 libraries within a given group (JAr or HEK293), 11 microRNAs were detected only in JAr EVs while only two were detected in HEK293 EVs. These 11 microRNAs were subsequently retained for further analysis of their target genes. miRNA abundance in JAr EVs correlates with fold change of downregulated target genes in RL95 cells.
For the 11 JAr-specific miRNAs, a total of 1,188 high-confidence putative gene targets were identified from miRDB database and applying a target score cut-off of 90. Of these, 744 were present within the RL95 gene expression dataset. Only a small proportion of these genes were differentially expressed, with 53 of them downregulated and 68 of them upregulated.Although more putative targets were upregulatedthan downregulated,putative miRNA targets constituted a higher percentage of total downregulated genes (9%) compared to both total upregulated(5.8%) and total non-differentially expressed genes (6.4%). Furthermore, six out of the eleven miRNAs had a greater number of targets which were downregulated than upregulated, while only four had a greater number of upregulated than downregulated targets (Fig. 5A). hsa-miR-524-5p had the largest number of putative targets represented in the expression dataset, the 26 downregulated targets of which constituted 4.4% of the total downregulated genes.
Although the number of miRNAs examined was low, the mean log2FC of downregulated target genes displayed a moderate negative correlation with the abundance of a given miRNA in JAr EVs (weighted Pearson’s correlation, r = -0.65, p = 0.041; Fig. 5B). The most abundant JAr-specific miRNA was has-miR-1323, the downregulated targets of which had the lowest log2FC of all miRNAs except for hsa-miR-526b-5p, which had only one downregulated target.The negative correlation was found to be influenced both by the target score cut-off and FDR cut-off used to detect downregulated genes, with no such correlations evident at more relaxed criteria (supplementary Figure 2).
We also examined whether any downregulated genes constituted high-confidence predicted targets of multiple miRNAs. In this regard, we found only twodownregulated genes that were the putative targets of at least three JAr-specific miRNAs: ATF2 (predicted target of hsa-miR-524-5p, hsa-miR-520a-5p, and hsa-miR-525-5p) and SPTSSA (predicted target of hsa-miR-524-5p, hsa-miR-526b-5p, and hsa-miR-1323), respectively.
Target prediction analysis was not carried out for the transcriptome of RL95-2 cells treated with HEK293 derived EVs because there was no significant differential expression observed.
EVs are known to transport various molecules between cells and alter the biochemical and physiological state of the target cells [44,45,46]. Despite the increasing popularity and the ever increasing knowledge of EV-mediated intercellular communication , the specificity of the effect induced by EVs on target cells and the mechanism of EV-mediated communication is not yet well understood. In the current study we endeavoured to study the effect of JAr spheroid derived EVs (an analogue for pre-implantation embryo) and HEK293 spheroid derived EVs (a cell line of non-reproductive origin) on RL95-2 cells, analogue of WOI-status endometrium.
JAr EVs induced substantial alterations to the RL95-2 cells’ transcriptome. Interestingly, EVs derived from a non-reproductive cell line, HEK293, failed to induce a similar profile of differential expression in the RL95-2 cells (Fig. 2), thus demonstrating that trophoblast-like-cell derived EVs are uniquely capable of altering the transcriptome of endometrial cells.The specificity of EVs is a topic of intense scientific discourse . Some reports [49, 50] posit that EVs are highly targeted and will only act on a specific type of cell or a tissue. However, other reports [51,52,53] suggest that EVs are indiscriminately uptaken by any type of the cells. In the current study we have demonstrated that trophoblast-like cell derived EVs exhibit functional specificity. Studies on functional specificity of EVs are conducted, to a large degree, in the field of cancer metastasis where EVs derived from cancerous cells are reported to regulate the reaction of target cells in metastasis [54, 55]. To the best of our knowledge, this is the first report of functional specificity of EVs in the context of embryo-maternal communication. This compelling effect could be attributed to either the selective uptake of JAr-EVs, when compared to HEK293 derived EVs by RL 95–2 cells or to the differences between the EV cargo of JAr and HEK293 EVs. Since in this study the JAr/RL95-2 model was used to mimic the pre-implantation uterine microenvironment, we could deduce that the transcriptomic changes induced by EVs are not random, but specific to embryo derived EVs and have functional significance for embryo-maternal communication.
The probable functional importance of the JAr EVs’ effect on RL95-2 cells is more apparent when considering the pathways affected by the DEGs (Table 1). For example, the majority of the events of the extracellular matrix (ECM) organization pathway (R-HSA-1474244) were tagged by the genes upregulated in the RL95-2 cells treated with JAr EVs. ECM remodelling is a critical morphological and biochemical alteration the endometrium undergoes in preparation for implantation. It promotes and stabilizes the embryo adhesion while protects the underlying stromal cell layer from over invasion by the extravillous trophoblasts [56,57,58]. Major components of the pathways, such as laminin interactions (R-HSA-3000157) [59, 60], integrin cell surface interactions (R-HSA-216083) [61,62,63,64,65,66], and non-integrin membrane-ECM interactions (R-HSA-3000171)  are all known to be implicated in endometrial modifications in the WOI.
The pathway of signalling by GPCRs (R-HSA-372790) was also found to be significantly enriched by DEGs. GPCRs are the largest family of transmembrane receptors accounting for 4% of the coding regions of the human genome . They are known to bind a highly diverse set of ligands that perform biological functions ranging from sight and olfactory senses to immune regulation [69, 70]. They also act as receptors for a number of ligands known to alter the endometrial microenvironment during the WOI , such as hCG , prostaglandin E2 [73, 74], cytokines [75,76,77] and progesterone via its membrane bound receptor [78,79,80]. Downstream signalling of GPCR pathway (R-HSA-388396) is also significantly enriched by the DEGs. These downstream pathways are secondary messengers that modify the endometrial morphology to facilitate implantation. For example, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) which regulates cell growth and survival, is reported to be involved in endometrial cellular migration, which is crucial in embryo attachment and invasion [81,82,83,84]. Evidence from the top pathways resulting from GSEA leads to the notion that the transcriptomic changes induced by JAr EVs on RL95-2 cells are not only directly modifying the endometrium for imminent implantation, but also modifying and priming the endometrial membrane receptors for further reception of embryonic signals, such as hormones and cytokines.
We investigated the RNA contents of the EV populations derived from JAr and HEK293 cells to collect data about the mechanism of EV induced transcriptomic changes. Identification of the RNA cargo of the two types of EVs by sequencing mRNA and miRNA was the first step of the investigation. There were significant (FDR ≤ 0.05) differences between mRNA found in JAr and HEK293 EVs indicating that the two EV populations are distinct from each other (Fig. 3). However, we were not able to confirm any connection between the observed transcriptomic alterations and the mRNA cargo of EVs.
In addition to mRNA, JAr EVs differed from HEK293 EVs in their microRNA composition. miRNAs are regulators of gene expression which uses multiple mechanisms to inhibit, destabilize and cleave transcripts [85,86,87]. There are about 1115 miRNAs identified and characterized which target about 60% of the human genes [88,89,90]. It is well-documented that miRNAs play important roles in cell-to-cell communication [91,92,93] and numerous studies have further demonstrated the role of EVs in transporting miRNAs to facilitate intercellular signalling. When applying a reasonable counts threshold (5 counts in 2/3 libraries of either JAr or HEK293), we identified eleven microRNAs in JAr EVs which were not detected in HEK293 EVs. Interestingly, while substantially more JAr-specific miRNAs could be detected while applying lower counts thresholds, relaxing the counts threshold did not substantially influence the number of HEK293-specific miRNAs in EVs, suggesting that the majority of miRNAs present in HEK293 EVs are indeed also present in JAr EVs. We reasoned that the 11 microRNAs could be considered to be JAr-specific and were retained for downstream target analysis. Although the ability to detect differences between the miRNA profiles of different EV types is highly influenced by technical factors such as read depth and filtering criteria [94,95,96,97,98], multiple reports nevertheless corroborate the presence of contrasting miRNA profiles in populations of EVs isolated from different cell types and biological fluids [23, 99, 100]. These differences, if present, could aid in interpreting the observed EV induced effects on RL95-2 cellular transcriptome.
Although we hypothesized that some gene expression changes may be putatively linked with miRNAs present in JAr EVs, the finding that the majority of gene expression changes constituted upregulation suggests that canonical miRNA-induced silencing is not the primary mode of action by which JAr EVs modulate gene expression changes in target cells. Indeed, we believe that the 11 microRNAs identified as JAr-specific had high-confidence putative targets among both downregulated and upregulated genes in RL95-2 cells. Moreover, in the absence of any miRNA-induced silencing, we would expect the relative proportions of downregulated and upregulated putative targets to reflect the relative proportions of overall up- and down-regulation of genes, which was not the case in our results. Indeed, most JAr-specific miRNAs had more high-confidence predicted targets that were downregulated than upregulated, and miRNA targets constituted a higher proportion of downregulated genes compared to either upregulated or non-DE genes. Furthermore, the log2FC of downregulated putative targets negatively correlated with the abundance of 11 miRNAs detected in JAr EVs, with the putative targets of the most abundant JAr-specific miRNA (hsa-miR-1323) showing the strongest decrease in mean log2FC. Collectively, these observations suggest that at least some of the downregulation may have been a direct result of canonical miRNA silencing. We also note that, as we co-incubated EVs with RL95-2 cells for 24 h, we cannot exclude the possibility that at least for some of the increased expression levels were caused by the secondary effects of EV-derived miRNAs.
The majority of the observed DEG’s cannot be explained as a direct effect resulting from either mRNA or miRNA transported to the recipient cells via EVs. Considering the relatively small copy number of RNAs transported in EVs, attributing the substantial degree of DE to direct effects of EV RNA on target cells would be illogical [101, 102]. However, based on the results of the current study, the role played by EVs, and specifically JAr-derived EVs as the mediator of the effect is clear, even though the molecular mechanisms underlying the majority of the transcriptomic changes remain elusive. It can be speculated that other types of regulatory mechanisms such as biomolecules bound to the EV surface, other non-coding RNAs, transcription regulation enzymes or cytokines carried in the EVs could play a role in the observed effects on endometrial transcriptome.
The phenomenon of functional specificity of EVs was clearly demonstrated in our experiments. However, it remains unclear to what extent the observed differences in RNA cargo between the two EV types (JAr and HEK293) can account for the transcriptomic changes observed in the target cells. It might be possible to reveal a clearer picture of this form of intercellular communication if this effect would be measured in detailed series of time points.Also, future studies would be required to explore the issue of EVs’ functional specificity via other avenues of investigations, such as the degree of uptake specificity exhibited by the RL95-2 cells towards trophoblast-like-cell and non-trophoblast spheroid derived EVs,the surface proteome differences between the two types of EVs which could play a role in membrane-based intercellular signalling, differences in EV cargo other than regulatory RNA, the mechanisms employed by the target cells to interpret the signals delivered by EVs and the target specificity of trophoblast spheroid derived EVs in terms of target cells of reproductive and non-reproductive origin.
We have shown that trophoblast-like-cell derived EVs were capable of inducing unique transcriptomic alterations in target endometrial cells. These changes were only induced by trophoblast-like-cell derived EVs, implying functional specificity of these EVs.Some of the observed changes, such as those associated with extracellular matrix remodelling and GPCR mediated signalling, may be indicative of functional components of embryo-maternal communication in implantation.This effect was unique to the trophoblast-like-cell derived EVs compared to EVs from a non-reproductive source, which may be at least partly due to the distinct RNA cargo of the EVs from the two sources. However, as the RNA composition of JAr EVs could be putatively linked with only a small proportion of gene expression changes, future studies should aim to explore the role of different molecular pathwaysunderlying EV-mediated transcriptomic changes, as well as further elucidate their functional role in embryo-maternal communication.
Availability of data and materials
The sequencing data has been uploaded to the NCBI SRA repository (www.ncbi.nlm.nih.gov/sra) under the accession number PRJNA692831.
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This work was supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No 668989 (TransGeno) and COMBIVET ERA Chair grant agreement no. 857418.A.S. was supported by grants from the Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (grant EU48695); the European Commission Horizon 2020 research and innovation programme under grant agreement 692065 (project WIDENLIFE); MSCA-RISE-2015 project MOMENDO (grant no 691058), and University of Tartu base funding.
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Supplementary Figure S1: Data pertaining to the outliers observed in the RNAseq analysis.
Supplementary Figure S2: Data pertaining to the variation of the correlation between mean log2FC of downregulated target genes with the abundance of a given miRNA in JAr EVs in different levels of target prediction criteria.
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Godakumara, K., Ord, J., Lättekivi, F. et al. Trophoblast derived extracellular vesicles specifically alter the transcriptome of endometrial cells and may constitute a critical component of embryo-maternal communication. Reprod Biol Endocrinol 19, 115 (2021). https://doi.org/10.1186/s12958-021-00801-5
- Embryo-maternal communication
- Extracellular vesicles
- miRNA signalling


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Refugee access to health care during COVID-19 should not be an afterthought
The racial and ethnic disparities in health care and unequal health burdens of refugee populations put them at disproportionate health risks from COVID-19.
Rohingya refugees are pictured in the Kutupalong camp in Bangladesh in 2018. A recent study projected the potential impacts and burden of COVID-19 on Rohingya refugees from Myanmar, where a single introduction of the virus in the Kutupalong-Balukhali Expansion Site with 600,000 people would lead to up to 370 people infected within the first month and up to 589,000 people infected in 12 months. U.K. Department for International Development photograph by Russell Watkins

domain: health


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Our family leaves on an extended holiday trip in just over 2 weeks and I keep wondering how I’m going to get everything done. So, here are a few tips I’ve created to help myself maintain balance during this busy time of year – I hope you find them helpful also.
1. Prioritze your schedule to include activities most important to you. Say no to everything else and to obligations someone else can manage.
For me, that includes attending my son’s Christmas party at school, special church services, a holiday piano performance in our hometown, a few Christmas parties, and various other events. However, it doesn’t include ladies bunko night, the symphony performance, or lunch with each of my girlfriends to exchange gifts – there simply isn’t time for all that.
2. Start each day with a spiritual act – prayer, devotional, Bible reading, listening to songs of praise, etc. to center your mind and soul for the day.
When we start our day with God in control, it allows for a God-centered day instead of a man-centered one.
3. Don’t allow someone else power over your emotions (i.e. ex-spouses, children/stepchildren).
Commit to staying in control of your emotions instead of allowing someone else to take that power from you. Walk away from volatile emotions or heated conversations. Engage in communication via e-mail or texting if necessary.
4. Stay faithful to healthy eating patterns and a regular exercise routine.
Get up earlier than usual if you need to, but don’t skimp on exercise and sensible eating. You will feel better and manage your demanding schedule more competently if you maintain healthy habits through the season.
5. Break down consuming tasks into chunk-size actions that can be completed a little at a time.
For instance, I easily become overwhelmed when I think about shopping for our five children in addition to parents, siblings, nieces, nephews, etc. However, if I choose one child to focus on until I’m finished and then move to the next child or a parent or whatever, the task seems less daunting.
6. Commit to making each day positive.
We have so much to be thankful for and if we choose to focus on the positives in our life, we will manage our schedule with greater ease. If we have a bump in the road one day, we can choose to pick ourselves up and keep moving forward instead of allowing negative thoughts to set in.
7. Read Thriving at the Holidays: A Stepparent’s Guide to Success – Unwrapping the Gift of Peace (an easy-to-read e-book) to find additional tips on maintaing balance and creating a peaceful season.
There they are – 7 tips for finding balance during holiday chaos.
Do you another tip to add? Would you please share it with us?

domain: psychology


Social media has swiftly become an essential staple in every marketing strategy. Having the ability to connect with thousands of people daily in a responsive, engaging manner brings added life and personality to your brand in a way that traditional marketing lacked. It is estimated that by 2019, there will be approximately 2.77 billion social network users across the entire globe making up approximately 37% of the world’s entire population. With numbers like these, it seems inevitable that businesses would choose to either incorporate part of their marketing efforts or shift their entire focus to digital marketing in favour of a medium that is placed in front of potential customer’s eyes numerous times a day.
When it comes to marketing your product, business or service successfully online, there are two main avenues you can go down – Social Media Marketing and Social Media Advertising. As with any important business decision, it is imperative that you understand each in detail, including the pros and cons in order to decipher the correct plan and strategy for your company.
Before we delve further and determine what goes into them, lets first determine what we are referring to with each of these terms in this article.
Social Media Marketing
In this article, Social Media Marketing essentially refers to organic social media activity. So, the use of social media to promote your business without the addition of paid advertising (e.g Facebook Ads or PPC for example). This could be through engaging blog posts, creative infographics or in-house videos that provide your potential customers with a deeper look into your business.
Social Media Advertising
Social Media Advertising includes Facebook, Twitter, LinkedIn and other channels and refers to all types of paid social media promotion that can be found either built into each platform or available through external companies. A common example of this would be the ‘Promoted’ posts you see pop up on your own personal Facebook or Instagram feed – these are all paid promotions that companies have actioned to achieve a specific goal.
When it comes to constructing the most effective social media strategy for your business, it is vital that you understand how both advertising and marketing on these platforms can benefit your business. Let’s take a look at each option individually and establish how they can be used to achieve your overall goals.
Social Media Marketing Explained
Social media marketing is the process of promoting your business, product or service to your followers and potential customers in an organic manner. In an ordinary case, this would involve minimal to no financial investment on your side and sees the highest success from the spread and production of engaging content. For businesses looking to implement successful digital strategies into their businesses, this should be at the heart of any businesses social media goals as it is important to remember that these platforms were originally produced as a digital communication option for friends and family. Although it is now commonplace to see businesses active and engaging with followers online, remember that the majority of social users aren’t actively seeking out purchase opportunities when they use them on a daily basis. However, according to the Social Media Examiner, 66% of marketers see lead generation benefits with social media, making it a highly viable route to go down as a part of your business goals.
Organic marketing enables you to create an engaging community hub for your followers that brings together like-minded individuals into one digital space. Through the creation of content such as videos, blog posts or influencer activity, you are able to build an online world where your potential customers can learn more about your brand and decide whether they resonate with the message and ethos you portray. With 95% of online adults between 18-34 being likely to follow a brand via social networking), connecting with potential customers online has never been so important and working this into your existing plan can see highly beneficial results. Organic marketing allows you to do this in a natural and non-invasive manner where you are able to integrate yourselves into the everyday life of your followers and become the obvious choice when it comes to their purchasing decisions.
The essence of social media marketing is your content strategy and by creating a sound and strategic plan, you are able to both minimise costs and ensure continual traffic. Inclusive of all kinds of content such as influencer outreach (which may incur an initial cost but should pull continual traffic to your platform long into the future), photo creation, blogs or videos, a content strategy indicates the potential results that you envision from the promotion of and engagement with this information. Posts with visuals including photos and videos have been proven time and time again to generate higher engagement and activity from followers so the right photos or videos have the ability to capture your audience and bring them inside your world.
In addition, well-constructed and produced organic content has the potential to offer continual value to your target audience, customers and followers, therefore producing a steady stream of traffic to both your social media platforms and external sites such as your company blog. Produce a well-written and resonating blog post that perfectly explains your brand’s positioning on a specific subject and over the months, you will continue to see traffic flowing through in a beneficial manner. Differing from advertising in its longevity, successful marketing on social media can ensure a continual flow of new followers and potential customers into your businesses eyesight.
It is important to understand that successful organic marketing content ensures a presence for your business or brand in search engine results. Running alongside high SEO rankings, engaging organic content shows Google that your website is trustworthy and that is relevant to the searching needs of its users. By creating an engaging blog post that answers some of your company’s most frequently asked questions, for example, you have produced an engaging piece of content that you can confidently acquire traffic from using the knowledge you already have about your customers. Combining relevant content with a topic that is sure to generate repeated traffic will help different search engines pick up on your website and return it as a viable result option on more than one occasion.
Social Media Advertising Explained
If we take a look on the other side of the fence, Social Media Advertising is the paid promotion of your posts, content or promotions. Unlike organic, this form of business advertising has been shown to generate higher and more successful leads and conversions– mostly due to its accuracy. The powerful thing about social media and platforms such as Facebook is that they store and offer a wealth of knowledge about all of their users. Accessible to businesses of all shapes and sizes, Facebook Insights can supply you with information from which of your followers has just got engaged through to which ones are interested in dirt bike racing, for example. Using this information, you are better able to decipher the passions of your target audience and use these to create content that appeals to their needs and wants. Where traditional advertising focused on reader/viewer numbers and a publication’s desired demographic, social media advertising affords us the opportunity to drill down into the exact customer base that we currently have and produce tailored promotions specifically for them.
Paid advertising, as the name suggests, requires investment to see the results and in general, the bigger the investment, the higher the return. For example, by digging into the analytics and insights provided by your chosen platform, you could produce a specific promotion for customers who have shown an interest in the previous activity you have pushed. Through platforms such as Facebook, you are then able to create a fully personalised advertisement with your chosen tagline, images and URL link (where applicable) and ask Facebook to publish this post to the feeds of your target audience. The production of completely personalised and specific promotions improves your chances of seeing successful results and therefore moves you closer to your end goals.
In general, social media advertising is used when a specific message needs to be pushed out in a timely manner. So, for example, if you have a seasonal promotion running or a limited edition item out of stock, paid posts help to ensure that this information is at the forefront of your follower’s minds at your chosen time. The focus is generally on acquisition and conversion so your success will ordinarily be measured in traffic to your website/blog/e-commerce site, no. of conversions seen during this period or the completion of a specific action chosen in-house (such as signing up to a new course or email list).
Businesses that see success with social media advertising are able to rehash existing promotions to suit audiences on different platforms and demographics. Keeping their content new and fresh eliminates the chance of looking spam-like and makes the inclusion of your business in a users feed feel much more natural. It is important to remember that users of social media are generally savvy and understand the paid promotional aspect to some degree. As these platforms continue to function in a social manner, ensuring that your posts don’t come across as hard selling is essential so having the ability to alter and adapt posts to suit different audiences affords you a practical solution. For those businesses looking to retarget past customers or even those looking to find similar users to their own customers, these promotional avenues offer the ability to capture groups of audiences in a manner that traditional advertising could not with the near promise of quick wins when done right.
It is worth noting that, in general, social media advertising is used as a short-term growth plan. Well tailored and targeted activity will generate successful results for businesses of all sizes, however once the promoted post period comes to an end, businesses can expect to see a dip in traffic and activity as a result. This isn’t to say that the followers you managed to engage with have lost interest, but rather than your post is no longer being pushed directly on to their feeds. If the platform’s algorithms and ranking systems deem your content unrelated to specific users, you’ll no longer find yourself in front of their eyes in the same manner. However, advertising through social media offers the opportunity to capture the imagination and interest of potential customers before encouraging them to delve further into the organic content you have produced. In this manner, advertising can be highly beneficial to all aspects of your business – from your content strategy through to your conversion rate.
For a business to see true successes on social media, it is important to consider both social media advertising and social media marketing in your plans. Where advertising will afford you those quick wins and high exposure a business needs to elevate above their competition, marketing allows you to build a strong and secure hub of loyalty that supports repeat customers and true brand advocates. The percentage split between the two depends largely on the nature of your business and the goals you have set yourself in an existing strategy. For small businesses, advertising can be the boost they need to capture the first batch of customers and entice them back for more. In the long run, successful organic activity or social media marketing is the holy grail, bringing followers back again and again while ensuring a stream of consistently engaged purchasers.
Marketing any business on social media has been shown to return considerable benefits in more ways than just follower numbers or likes. Before embarking on any set course of marketing activity, it is important to understand your overreaching business goals fully and determine how you expect to achieve them in the most cost and time-efficient manner. And, with social media, it is important to remember the pros and cons of both social media marketing (organic) and social media advertising (paid) and how they can be used collaboratively to achieve the outcome you expect to see.
If you need any help with Facebook advertising in London or throughout the UK, please get in touch.


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OK, so I haven’t watched the BBC version of Pride and Prejudice once, let alone a hundred times. But I still do swoon over Jane Austen’s romances. I mean, really, how could I not?
One of the latest books to meet the needs of Austen-addicted modern women is Shannon Hale’s Austenland. It’s Hale’s first venture into adult fiction from her award-winning young-adult writing, and she does a fine job of addressing more mature readers’ interests without adding “mature content.”
Jane Hayes, the heroine of the novel, gets to go on a three-week Austen-style immersion vacation in England when her rich great-aunt dies and leaves her a paid-for package deal. Having a serious thing for Darcy but no serious history of real, full-bodied, contemporary romances of her own, she realizes she probably has a bit of a problem. Embarrassed, she decides to just go on the vacation and look her demon in the face before she flings it aside to look for men who actually exist today.
On the flight, Jane prepares herself for the experience by familiarizing herself with Regency customs. If she doesn’t play the role absolutely correctly, she could be kicked out. She gets to wear authentic clothes, a bonus, but she has to hand over her modern devices, which she is not pleased about.
Jane fumbles her way through what she considers an embarrassing charade. Sure, she’s always longed deep-down for her real life to be full of gallant men and the quaint customs of the Austen books, but play-acting it for three weeks is just a full-on reminder of how pitiful both her real and fantasy lives are.
The other participants in this charade serve to confuse her as well as any characters. The “gentlemen” visiting the estate are played by well-trained actors who are paid to be attentive, so it’s impossible to experience anything real. And dealing with the other paying guests, some ludicrous and some very authentic, is quite an experience.
Jane’s excursion in “Austenland” mirrors the plots of Austen’s books; a cad deludes and confuses her, and other characters, male and female, aren’t what they seem. The ending, naturally, is happy.
Austenland is an entertaining read, perfect for anyone who just can’t get enough Darcy.
Rated: Mild, for six uses of mild/moderate language usage and some mild sensual references (the author herself said this book would be like a “PG” movie.)

domain: entertainment


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Towards a Sudanese Humanitarian Vision on Violent Extremism
This paper highlights some key theoretical issues relating to violent extremism and their relevance and compatibility with Sudanese reality, and accordingly presents practical proposals and recommendations. By violent extremism, we mean convictions and practices that deliberately target civilians and non-combatants with violence to achieve ideological, religious, political or economic goals. We use the terms violent extremism, terrorism, and brutal violence as synonyms.
Terms and Concepts
Many scholars in this field agree that there is no single comprehensive definition of terrorism, and one expert has counted 109 definitions of the term. This is because the interpretation of the term itself is colored by contentious social and ideological currents and is defined and approached based on these different perspectives.
In general, the writings of Western specialists tend to expand the scope of “terrorism” to the extent that some use the term to stigmatize any political or ideological violence perpetrated by groups or individuals other than the state. While this expansion may be justified in the West, where democratic regimes prevails, allowing for the condemnation of the practice of any violence against them, other specialists have noted that such an expanded use of the term would render resistance to colonialism, fascism and Nazism as “terrorism.” While this is wrong in essence, it also points to the ideological manipulation and use of the term.
The roots of the modern use of the term terrorism go back to the French Revolution, from the Latin word “Terrere”, meaning “horrified-freezes” – a horror that freezes the blood in one’s veins. Initially, it described the methods used by the Jacobins – a radical revolutionary force whose ascendance lasted from June 1793 to July 1794, and is known as the Reign of Terror. It was also used by the philosopher Edmund Burke in 1795 to describe the various forms of intimidation used by the regime in France, i.e. to rule through repression and terror.
Many reject the equation of the terrorism of the French Revolution with current forms of terrorism and present a number of arguments. The most important of these arguments is that the Jacobin violence was an exceptional and temporary, intended to breed a new society that would establish processes for the peaceful transfer of power. Current forms of terrorism, however, are seen as either dark violence intended to revert to a past pattern that is practically impossible to achieve or a violence without a purpose whose only aim is revenge.

domain: sociology


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United Rentals intends to invest more than $900 million in capital in 2007, CEO Wayland Hicks told RER in a recent interview. Although the interview was granted before United Rentals’ recent announcement that the company is exploring strategic alternatives and that Hicks will retire in June, company officials this week told RER that the company’s growth plans have not changed and that United Rentals is going forward with “business as usual.”
Hicks said United Rentals has budgeted about $125 million in growth capex. “Our outlook is based on the expectation that private non-residential construction spending in North America will continue to grow in 2007, although at a more moderate pace than we saw in 2005 and 2006.”
Hicks also said United Rentals plans to open 30 to 35 new branches in 2007, after opening 36 cold starts in 2006. He added that the company still intends to add about $100 million a year in revenue through acquisition.
The expanded text of RER’s interview with Hicks can be read by scrolling down to Trends & Analysis.

domain: news


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CHARLESTON, S.C. — A combination of intralesional steroids, antifungal therapy, and physical therapy may be effective for the treatment of vulvar vestibulitis syndrome, Carolyn Gardella, M.D., reported in a poster at the annual meeting of the Infectious Diseases Society for Obstetrics and Gynecology.
Of 21 women treated with intralesional steroids after failing treatment with topical steroids, 68% had complete resolution of symptoms, and all patients had at least 80% improvement.
Twelve of the 21 women also were treated with yeast suppressive therapy, and 14 underwent physical therapy for levator muscle hypertonus, noted Dr. Gardella of the University of Washington, Seattle.
Of the 12 women who also received yeast suppressive therapy, 10 (83%) had complete resolution of symptoms, but only 4 of the 9 (44%) who did not receive yeast suppressive therapy had symptom resolution, Dr. Gardella noted.
All patients were seen by a single clinician at a vulvovaginal specialty clinic during a 5-month period. A chart review showed that the women were symptomatic for a mean of 27 months before presenting at the clinic.
Eleven of the 21 had yeast by culture despite a lack of clinical evidence of yeast vaginitis, and all had failed treatment with 0.25% desoximetasone ointment applied twice daily for an average of 11 weeks.
The intralesional injections included 4-mg of betamethasone and 4 cc of 0.5% bupivacaine with epinephrine; the women received a mean of nine injections.
A randomized controlled trial of a regimen using intralesional steroids, antifungal treatment, and physical therapy for vulvar vestibulitis syndrome is warranted, Dr. Gardella wrote.
Her poster also indicated that the high prevalence of yeast by culture in the study population supports the “biologic plausibility” of empiric yeast suppressive therapy for women with vulvar vestibulitis syndrome.

domain: medical


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Menstrual health is an integral part of a woman’s well-being, and the challenges of menstrual cramps and uterine discomfort can be physically and emotionally taxing. Thankfully, nature offers a variety of herbs that have been used for centuries to support menstrual health, alleviate cramps, and promote overall uterine well-being.
In this article, we will explore the power of herbs in addressing menstrual cramps and supporting a healthy menstrual cycle. From soothing herbal teas to nourishing tonics, these natural remedies offer a gentle and effective approach to enhancing women’s reproductive health.
Chamomile (Matricaria chamomilla):
Chamomile, with its delicate white flowers and calming aroma, is a gentle herb renowned for its soothing properties. When it comes to menstrual cramps, chamomile can work wonders by relaxing the uterine muscles and reducing inflammation. Its anti-inflammatory and antispasmodic properties can provide relief from painful menstrual cramps and help ease tension during the menstrual cycle.
Preparation: To prepare chamomile tea, steep dried chamomile flowers in hot water for 5-10 minutes. Strain the tea and enjoy its calming effects.
Vitex (Vitex agnus-castus):
Vitex, also known as chasteberry, is a powerful herb that has been used for centuries to support women’s reproductive health. It acts on the pituitary gland, helping to balance hormone levels and regulate the menstrual cycle. Vitex is particularly beneficial for women experiencing irregular menstrual cycles, premenstrual syndrome (PMS), and menstrual cramps.
Preparation: Vitex is commonly taken in tincture form or as a capsule. Follow the recommended dosage on the product label or consult with a healthcare professional for personalized guidance.
Red Raspberry Leaf (Rubus idaeus):
Red raspberry leaf has long been treasured for its uterine-toning properties. It is rich in essential nutrients, including vitamins C, E, and calcium, which nourish the reproductive system and support overall menstrual health. Red raspberry leaf can help ease menstrual cramps and regulate menstrual flow.
Preparation: To prepare red raspberry leaf tea, steep dried leaves in hot water for 10-15 minutes. Strain the tea and enjoy its nourishing benefits.
Dong Quai (Angelica sinensis):
Dong Quai, often referred to as “female ginseng,” is a traditional Chinese herb known for its hormone-balancing and blood-moving properties. It is widely used to alleviate menstrual cramps, regulate irregular periods, and promote hormonal balance.
Preparation: Dong Quai is available in various forms, including capsules, tablets, and tinctures. Follow the dosage instructions on the product label or consult with a healthcare professional for personalized recommendations.
Cramp Bark (Viburnum opulus):
Cramp bark, as the name suggests, is an excellent herbal ally for relieving cramps and muscle spasms, including those associated with menstrual pain. This herb works by relaxing the smooth muscles of the uterus, providing relief from painful contractions.
Preparation: Cramp bark is commonly used as a tincture. Follow the recommended dosage on the product label or consult with a healthcare professional for appropriate usage.
Menstrual health and uterine support are essential aspects of a woman’s well-being. These five remarkable herbs – chamomile, vitex, red raspberry leaf, dong quai, and cramp bark – offer natural and effective solutions for menstrual cramps and overall reproductive health. By incorporating these herbal allies into your self-care routine, you can embrace the power of nature and find comfort and relief during your menstrual cycle.
As always, it is advisable to consult with a healthcare professional or herbalist to determine the most suitable herbs and formulations for your individual needs. With the gentle support of these botanical treasures, women can enhance their menstrual health and find balance, harmony, and empowerment in their natural cycles.


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Sega Master System / Mark III / Game GearSG-1000 / SC-3000 / SF-7000 / OMV
Home - Forums - Games - Scans - Maps - Cheats - CreditsMusic - Videos - Development - Hacks - Translations - Homebrew
Huge game area, great graphics and a few puzzles thrown in for good measure. Can you do without this one?
See more reviews of Wonder Boy in Monster World
See the main page for Wonder Boy in Monster World

domain: game


Police officers in Winter Haven wrangled a 5-foot gator taking a stroll on a neighborhood street Sunday.
Sgt. Gregory and Officer Ramos wrangled the gator lassoed by Officer Avery on Manolin Boulevard in Winter Haven.
The gator was successfully relocated to the local pond.
Winter Haven Police Department shared the photos on their facebook page on Monday morning.


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At the cost of crop production crops about 35%, and in the structure of sown areas, they account for almost 60%. On the production of grain consumed 20% of all labor costs in crop production.
Grain, as agricultural production, in economic terms has several advantages. It is well kept in dry form is easily transported over long distances, has a high degree of flow ability. All these features of the grain are used in the construction of grain elevators, silos, as well as transport and the creation of public stocks of food and feed.
Agriculture is the starting point for grain production and the initial stage of manufacturing the final product. Grain production employs more than 15% of agricultural workers. Implementation of the grain gives agricultural producers about 18% of revenue.
Contemporary situation in grain farming and the grain market of the country, was the result of many internal and external reasons, the main of which includes adverse macroeconomic conditions of grain farming, associated with the deformation of inter-industry relations, costs of ongoing agrarian reform, heightened disparity in prices for grain and industrial products, a sharp reduction in state support for the grain industry and the lack of active management of the grain market, etc. This has greatly contributed to the destruction and degradation of its productive capacity, low susceptibility to the introduction of scientific and technological progress and strengthening de-intensification of the industry, followed by a nearly ubiquitous ongoing reduction in the area of the grain of the wedge and the volume of grain production, the continued deterioration of its territorial and sector structure and grain quality, steady "compression" of the economy the grain industry, reducing its share of world gross harvest of grain crops.
As we know the market is a system of socio-economic relations in the area of exchange, through which products sell. Market is the link between producer and consumer. The market emerged and exacerbated economic and social problems led to a reduction in grain production. Grain on a daily basis, is a biological liquid of complex composition, containing all the nutrients: proteins, fats, carbohydrates, minerals and vitamins.
Grain needed in the country for food needs of 500 thousand tons over the past 10 years produced 1,279.8 thousand tons in the same time, food grain for bread and pasta production is completely insufficient.
Bashkortostan has a large area of arable land of 4.8 million hectares. More than half of that land is black soil of various kinds with good natural fertility. The country has gained great scientific and practical experience in the production of grain, the volume of which in some years reached 6 million tons and more
With the application of modern intensive technologies and the introduction of higher doses of fertilizers and crop protection agricultural enterprises of the republic with the growth in demand for grain could produce it in large quantities. Note that in some periods the area of grain crops in the country amounted to 2.5-3 million hectares. It is also important that the production of grain - the only major sector of agriculture, which has been and remains profitable through the years and in all areas of its cultivation.
Involvement of agricultural land in the major grain production with science-based crop rotations is an effective system, the basis of all agriculture; as well as the base of the most rational use of agricultural land and renewal of its fertility. Bashkortostan is located in the zone of risky agriculture, but despite that in many areas with insufficient moisture supply, a compressed period of growth and the difficulties during harvest grain production is justified. Therefore, in the future of Bashkortostan has to be one of the major regions of the Russian Federation for the production of marketable grain.
In a market where business activity is carried out at your own risk and under own financial liability, the profitability is a particular importance.
Level of profitability, as we know, is influenced by cost and revenue from sales, and the latter in turn depends on crop yields and prices of products sold.
Assuming the accepted among Russian economists - agrarian notions of growth efficiency with increasing scale of enterprise, we can expect increase of profitability. Profitability of production is considered as an effective index. As a rule, this dependence is illustrated by the grouping of agricultural enterprises on farmland and a number of employees, which demonstrates the systematic (from group to group) increase in profitability.
The indicators of the size of the agricultural enterprise can act as values of key factors of production (land, labor, capital) and the effective indexes (volumes of output, revenue, gross profit, profit). In agriculture, the most commonly used two indicators are: area of farmland and number of employees.
The most important condition for improving the efficiency of the complex stands coordination capacity of processing enterprises with production and revenues on processing of grain raw materials from the economic zone and the production of dietary grain products in quantity and range corresponding to the purchasing power of consumers.
Grain is produced once a year, but it can be stored under certain conditions, a longer time than milk. Therefore, grain producers may hold back production to more favorable terms. Grain is produced in almost all agricultural enterprises of the republic. Processing plants can operate effectively only when the capacity utilization, which is the reduction of production, declines. More stable supply of products can achieve higher purchasing prices for grain in the winter months and lower them in summer. Increase in revenues of raw materials to processing plants traditionally causes falling grain prices and products from it. Conversely, the reduction proceeds of grain processing enterprises prices rise. Consequently, manufacturers and processors of grain can work more efficiently only if their mutual interests meet.
The main reasons for the decline of grain production and reduction of its profitability are:

domain: food