Single-cell transcriptomics : a high-resolution avenue for plant functional genomics

Plant function is the result of the concerted action of single cells in different tissues. Advances in RNA-seq technologies and tissue processing allow us now to capture transcriptional changes at single-cell resolution. The incredible potential of single-cell RNA-seq lies in the novel ability to study and exploit regulatory processes in complex tissues based on the behaviour of single cells. Importantly, the independence from reporter lines allows the analysis of any given tissue in any plant. While there are challenges associated with the handling and analysis of complex datasets, the opportunities are unique to generate knowledge of tissue functions in unprecedented detail and to facilitate the application of such information by mapping cellular functions and interactions in a plant cell atlas. [Abstract copyright: Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

Deficiency in clonogenic endometrial mesenchymal stem cells in obese women with reproductive failure – a pilot study

The mechanisms of obesity associated reproductive complications remain poorly understood. Endometrial mesenchymal stem-cells are critical for cyclic renewal and uterine function. Recently, W5C5+ cells, with high clonogenicity, capable of producing endometrial stroma in vivo, have been described. We sought to investigate the abundance and cloning efficiency of W5C5+ and W5C5− endometrial cells in relation to Body Mass Index, age and reproductive outcome. Design W5C5+ and W5C5− cells were purified from mid-luteal endometrial biopsies (n = 54) by magnetic bead separation and subjected to in vitro colony-forming assays. Results First trimester pregnancy losses were significantly higher in obese subjects (n = 12) compared to overweight (n = 20) and subjects with normal Body Mass Index (n = 22) (P0.05). Conclusions Our observations suggest that the regenerative capacity and plasticity of the endometrium of obese women is suboptimal, which in turn may account for the increased risk of reproductive complications associated with obesity

Success after failure : the role of endometrial stem cells in recurrent miscarriage

Endometrial stem-like cells, including mesenchymal stem cells (MSCs) and epithelial progenitor cells, are essential for cyclic regeneration of the endometrium following menstrual shedding. Emerging evidence indicates that endometrial MSCs (eMSCs) constitute a dynamic population of cells that enables the endometrium to adapt in response to a failed pregnancy. Recurrent miscarriage is associated with relative depletion of endometrial eMSCs, which not only curtails the intrinsic ability of the endometrium to adapt to reproductive failure but also compromises endometrial decidualization, an obligatory transformation process for embryo implantation. These novel findings should pave the way for more effective screening of women at risk of pregnancy failure prior to conception

Recurrent pregnancy loss is associated with a pro-senescent decidual response during the peri-implantation window

During the implantation window, the endometrium becomes poised to transition to a pregnant state, a process driven by differentiation of stromal cells into decidual cells (DC). Perturbations in this process, termed decidualization, leads to breakdown of the feto-maternal interface and miscarriage, but the underlying mechanisms are poorly understood. Here, we reconstructed the decidual pathway at single-cell level in vitro and demonstrate that stromal cells first mount an acute stress response before emerging as DC or senescent DC (snDC). In the absence of immune cell-mediated clearance of snDC, secondary senescence transforms DC into progesterone-resistant cells that abundantly express extracellular matrix remodelling factors. Additional single-cell analysis of midluteal endometrium identified DIO2 and SCARA5 as marker genes of a diverging decidual response in vivo. Finally, we report a conspicuous link between a pro-senescent decidual response in peri-implantation endometrium and recurrent pregnancy loss, suggesting that pre-pregnancy screening and intervention may reduce the burden of miscarriage

Analysis of chromatin accessibility in decidualizing human endometrial stromal cells

Spontaneous decidualization of the endometrium in response to progesterone signaling is confined to menstruating species, including humans and other higher primates. During this process, endometrial stromal cells (EnSCs) differentiate into specialized decidual cells that control embryo implantation. We subjected undifferentiated and decidualizing human EnSCs to an assay for transposase accessible chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility upon decidualization was strongly associated with differential gene expression. Analysis of 1533 opening and closing chromatin regions revealed over-representation of DNA binding motifs for known decidual transcription factors (TFs) and identified putative new regulators. ATAC-seq footprint analysis provided evidence of TF binding at specific motifs. One of the largest footprints involved the most enriched motif-basic leucine zipper-as part of a triple motif that also comprised the estrogen receptor and Pax domain binding sites. Without exception, triple motifs were located within Alu elements, which suggests a role for this primate-specific transposable element (TE) in the evolution of decidual genes. Although other TEs were generally under-represented in open chromatin of undifferentiated EnSCs, several classes contributed to the regulatory DNA landscape that underpins decidual gene expression

Progesterone-dependent induction of phospholipase C-related catalytically inactive protein 1 (PRIP-1) in decidualizing human endometrial stromal cells

Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca2+ release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors

Impact of sitagliptin on endometrial mesenchymal stem-like progenitor cells : a randomised, double-blind placebo-controlled feasibility trial

Background: Recurrent pregnancy loss (RPL) is associated with the loss of endometrial mesenchymal stem-like progenitor cells (eMSC). DPP4 inhibitors may increase homing and engraftment of bone marrow-derived cells to sites of tissue injury. Here, we evaluated the effect of the DPP4 inhibitor sitagliptin on eMSC in women with RPL, determined the impact on endometrial decidualization, and assessed the feasibility of a full-scale clinical trial. Methods: A double-blind, randomised, placebo-controlled feasibility trial on women aged 18 to 42 years with a history of 3 or more miscarriages, regular menstrual cycles, and no contraindications to sitagliptin. Thirty-eight subjects were randomised to either 100 mg sitagliptin daily for 3 consecutive cycles or identical placebo capsules. Computer generated, permuted block randomisation was used to allocate treatment packs. Colony forming unit (CFU) assays were used to quantify eMSC in midluteal endometrial biopsies. The primary outcome measure was CFU counts. Secondary outcome measures were endometrial thickness, study acceptability, and first pregnancy outcome within 12 months following the study. Tissue samples were subjected to explorative investigations. Findings: CFU counts following sitagliptin were higher compared to placebo only when adjusted for baseline CFU counts and age (RR: 1.52, 95% CI: 1.32–1.75, P<0.01). The change in CFU count was 1.68 in the sitagliptin group and 1.08 in the placebo group. Trial recruitment, acceptability, and drug compliance were high. There were no serious adverse events. Explorative investigations showed that sitagliptin inhibits the expression of DIO2, a marker gene of senescent decidual cells. Interpretation: Sitagliptin increases eMSCs and decreases decidual senescence. A large-scale clinical trial evaluating the impact of preconception sitagliptin treatment on pregnancy outcome in RPL is feasible and warranted. Funding: Tommy's Baby Charity. Clinical trial registration: EU Clinical Trials Register no. 2016-001120-54

Expression of the Mxra8 receptor promotes alphavirus infection and pathogenesis in mice and Drosophila

Mxra8 is a recently described receptor for multiple alphaviruses, including Chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), and O\u27nyong nyong (ONNV) viruses. To determine its role in pathogenesis, we generated mice with mutant Mxra8 alleles: an 8-nucleotide deletion that produces a truncated, soluble form (Mxra

The clock protein period 2 synchronizes mitotic expansion and decidual transformation of human endometrial stromal cells

Implantation requires coordinated interactions between the conceptus and surrounding decidual cells, but the involvement of clock genes in this process is incompletely understood. Circadian oscillations are predicated on transcriptional-translational feedback loops, which balance the activities of the transcriptional activators CLOCK (circadian locomotor output cycles kaput) and brain muscle arnt-like 1 and repressors encoded by PER (Period) and Cryptochrome genes. We show that loss of PER2 expression silences circadian oscillations in decidualizing human endometrial stromal cells (HESCs). Down-regulation occurred between 12 and 24 hours following differentiation and coincided with reduced CLOCK binding to a noncanonical E-box enhancer in the PER2 promoter. RNA sequencing revealed that premature inhibition of PER2 by small interfering RNA knockdown leads to a grossly disorganized decidual response. Gene ontology analysis highlighted a preponderance of cell cycle regulators among the 1121 genes perturbed upon PER2 knockdown. Congruently, PER2 inhibition abrogated mitotic expansion of differentiating HESCs by inducing cell cycle block at G2/M. Analysis of 70 midluteal endometrial biopsies revealed an inverse correlation between PER2 transcript levels and the number of miscarriages in women suffering reproductive failure (Spearman rank test, ρ = -0.3260; P = 0.0046). Thus, PER2 synchronizes endometrial proliferation with initiation of aperiodic decidual gene expression; uncoupling of these events may cause recurrent pregnancy loss.-Muter, J., Lucas, E. S., Chan, Y.-W., Brighton, P. J., Moore, J. D., Lacey, L., Quenby, S., Lam, E. W.-F., Brosens, J. J. The clock protein period 2 synchronizes mitotic expansion and decidual transformation of human endometrial stromal cells

Cancer survivors' experience with telehealth: A systematic review and thematic synthesis

Background: Net survival rates of cancer are increasing worldwide, placing a strain on health service provision. There is a drive to transfer the care of cancer survivors—individuals living with and beyond cancer—to the community and encourage them to play an active role in their own care. Telehealth, the use of technology in remote exchange of data and communication between patients and health care professionals (HCPs), is an important contributor to this evolving model of care. Telehealth interventions are “complex,” and understanding patient experiences of them is important in evaluating their impact. However, a wider view of patient experience is lacking as qualitative studies detailing cancer survivor engagement with telehealth are yet to be synthesized. Objective: To systematically identify, appraise, and synthesize qualitative research evidence on the experiences of adult cancer survivors participating in telehealth interventions, to characterize the patient experience of telehealth interventions for this group. Methods: Medline (PubMed), PsychINFO, Cumulative Index for Nursing and Allied Health Professionals (CINAHL), Embase, and Cochrane Central Register of Controlled Trials were searched on August 14, 2015, and March 8, 2016, for English-language papers published between 2006 and 2016. Inclusion criteria were as follows: adult cancer survivors aged 18 years and over, cancer diagnosis, experience of participating in a telehealth intervention (defined as remote communication or remote monitoring with an HCP delivered by telephone, Internet, or hand-held or mobile technology), and reporting qualitative data including verbatim quotes. An adapted Critical Appraisal Skill Programme (CASP) checklist for qualitative research was used to assess paper quality. The results section of each included article was coded line by line, and all papers underwent inductive analysis, involving comparison, reexamination, and grouping of codes to develop descriptive themes. Analytical themes were developed through an iterative process of reflection on, and interpretation of, the descriptive themes within and across studies. Results: Across the 22 included papers, 3 analytical themes emerged, each with 3 descriptive subthemes: (1) influence of telehealth on the disrupted lives of cancer survivors (convenience, independence, and burden); (2) personalized care across physical distance (time, space, and the human factor); and (3) remote reassurance—a safety net of health care professional connection (active connection, passive connection, and slipping through the net). Telehealth interventions represent a convenient approach, which can potentially minimize treatment burden and disruption to cancer survivors’ lives. Telehealth interventions can facilitate an experience of personalized care and reassurance for those living with and beyond cancer; however, it is important to consider individual factors when tailoring interventions to ensure engagement promotes benefit rather than burden. Conclusions: Telehealth interventions can provide cancer survivors with independence and reassurance. Future telehealth interventions need to be developed iteratively in collaboration with a broad range of cancer survivors to maximize engagement and benefit