Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution

Infertility; Molecular medicineInfertilidad; Medicina molecularInfertilitat; Medicina molecularAsherman’s Syndrome is characterized by intrauterine adhesions or scarring, which cause infertility, menstrual abnormalities, and recurrent pregnancy loss. The pathophysiology of this syndrome remains unknown, with treatment restricted to recurrent surgical removal of intrauterine scarring, which has limited success. Here, we decode the Asherman’s Syndrome endometrial cell niche by analyzing data from over 200,000 cells with single-cell RNA-sequencing in patients with this condition and through in vitro analyses of Asherman’s Syndrome patient-derived endometrial organoids. Our endometrial atlas highlights the loss of the endometrial epithelium, alterations to epithelial differentiation signaling pathways such as Wnt and Notch, and the appearance of characteristic epithelium expressing secretory leukocyte protease inhibitor during the window of implantation. We describe syndrome-associated alterations in cell-to-cell communication and gene expression profiles that support a dysfunctional pro-fibrotic, pro-inflammatory, and anti-angiogenic environment.This study was jointly supported by Human Uterus Cell Atlas Project from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 874867, PROMETEO/2018/161 from the Valencia Government, IDI-20201142 CDTI from the Spanish Government and Carlos Simon Foundation, Spain. X.S. and E.F. were partially supported by IDI-20201142 CDTI from the Spanish Government. B.R. was supported by the H2020-funded project Human Uterus Cell Atlas (HUTER) (2020/2021) (Grant Agreement 874867). R.P. was supported by an Industrial Doctorate grant (DIN2020-011069) from the Spanish Ministry of Science and Innovation (MICINN). N.V. was supported by PROMETEO/2018/161. J.G.F. was supported by a PFIS grant [FI19/00159]. J.L. was supported by INVEST/2022/478 program. A.S. was supported by Estonian Research Council (PRG1076) and Horizon 2020 innovation grant (ERIN, grant no. EU952516). I.M. was supported by an FIS project grant [PI21/00235]. F.V. was supported by an FIS project grant [PI21/00528]. Other data that support the findings of this study are available from Asherman Therapy SL. Restrictions apply to data access with data used under license for the current clinical study and are not publicly available. Data are, however, available from the authors upon reasonable request and with permission of the Vall Hebron Ethical Committee

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC

Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus

[EN] Vaccinia virus capping enzyme is a heterodimer of D1 (844 aa) and D12 (287 aa) polypeptides that executes all three steps in m(7)GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1.D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB-fold domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1.D12 structure complements and rationalizes four decades of biochemical studies of this enzyme, which was the first capping enzyme to be purified and characterized, and provides new insights into the origins of the capping systems of other large DNA viruses.We are grateful for access to platforms of the Grenoble Partnership for Structural Biology, especially the High Throughput Crystallization (HTX) laboratory of the European Molecular Biology Laboratory (EMBL) for robotic crystallization. We thank the staff of the European Synchrotron Radiation Facility (ESRF)-EMBL Joint Structural Biology Group for help with data collection on beamlines BM14, ID14-4, ID14-3, and ID23-1. We acknowledge the help of Dr. Heinz Gut in setting up the cross-crystal averaging. This work was supported by National Institutes of Health grant GM42498 (to S.S.). S.S. is an American Cancer Society Research ProfessorKyrieleis, OJP.; Chang, J.; La Peña Del Rivero, MD.; Shuman, S.; Cusack, S. (2014). Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus. Structure. 22(3):452-465. https://doi.org/10.1016/j.str.2013.12.014S45246522

Synthesis and Characterization of Hydrophilic High Glycolic Acid–Poly(dl-Lactic-co-Glycolic Acid)/ Polycaprolactam/Polyvinyl Alcohol Blends and Their Biomedical Application as a Ureteral Material

Synthesis of low molecular weight poly­(dl-lactic-co-glycolic acid), homopolymer poly lactic acid (PLA), and formulation of a hydrophilic blend with polycaprolactam and polyvinyl alcohol is reported. The surface properties and the morphology of the blends were characterized with a goniometer and scanning electron microscopy (SEM). The mechanical property was analyzed with a tensile strength analyzer. The composition of these blends was verified by Fourier transform infrared (FTIR), ¹H, and ¹³C NMR spectroscopy. The biocompatibility and hemocompatibility and the extent of salt encrustation and adhesion of virulent bacterial strains on the blends were also investigated. All the blends were biocompatible and hemocompatible. Adhesion of virulent E. coli was high with blood plasma protein pretreated blends than P. mirabilis. The pattern of encrustation of Ca, Mg, and P was similar on all the blends with calcium being the predominant encrustant. After 3 weeks, 3–10% weight loss was observed with maximum weight loss observed in the poly­(lactic-co-glycolic acid) 10:90 based blend. The PLA based blend dominates all the other blends in all the aspects tested

Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution

Abstract Asherman’s Syndrome is characterized by intrauterine adhesions or scarring, which cause infertility, menstrual abnormalities, and recurrent pregnancy loss. The pathophysiology of this syndrome remains unknown, with treatment restricted to recurrent surgical removal of intrauterine scarring, which has limited success. Here, we decode the Asherman’s Syndrome endometrial cell niche by analyzing data from over 200,000 cells with single-cell RNA-sequencing in patients with this condition and through in vitro analyses of Asherman’s Syndrome patient-derived endometrial organoids. Our endometrial atlas highlights the loss of the endometrial epithelium, alterations to epithelial differentiation signaling pathways such as Wnt and Notch, and the appearance of characteristic epithelium expressing secretory leukocyte protease inhibitor during the window of implantation. We describe syndrome-associated alterations in cell-to-cell communication and gene expression profiles that support a dysfunctional pro-fibrotic, pro-inflammatory, and anti-angiogenic environment

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

<div><p>Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to <i>mature</i> taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) <i>P0-Cre/R26-tdTomato (RFP)</i> to label NC, NC derived Schwann cells and derivatives; (2) <i>Dermo1-Cre/RFP</i> to label mesenchymal cells and derivatives; and (3) <i>Vimentin-CreER/mGFP</i> to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both <i>P0-Cre/RFP</i> and <i>Dermo1-Cre/RFP</i> labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, <i>Vimentin-CreER</i> labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.</p></div

<i>P0-Cre</i> labeled type I, II, III taste bud cells.

<p>In lingual and palatal taste buds of adult <i>P0-Cre/RFP</i> mice, RFP⁺ signals were co-localized with markers for specific taste cell types (white arrowheads), i.e., NTPDaseII for type I cells (A), α-Gustducin for type II cells (B) and SNAP25 for type III cells (C). White dotted lines demarcate the epithelium from underlying connective tissue. Scale bar: 20 μm for all images (single plane laser-scanning confocal).</p

<i>Dermo1-Cre</i> labeled all three types (I, II, III) of taste bud cells in adult mice.

<p>RFP⁺ signals were co-localized with markers for specific taste cell types (white arrowheads), i.e., NTPDaseII for type I cells, α-Gustducin for type II cells and SNAP25 for type III cells in the lingual (A, B, C) and palatal (D) taste buds. White dotted lines demarcate the epithelium from underlying connective tissue. Scale bar: 20 μm for all images (single plane laser-scanning confocal).</p

A-D: Single-plane laser-scanning confocal photomicrographs illustrate the distribution of RFP⁺ cells in mature taste buds in young adult (8 week) <i>P0-Cre/RFP</i> mice.

<p>Taste bud cells in lingual fungiform (A), foliate (B), circumvallate (C) papillae and soft palate (D) were labeled by immunoreactivity of a pan-taste cell marker Keratin 8 (Krt8, green). Tissue sections were counterstained with DAPI (blue) to stain the nuclei of all cells. White dotted lines demarcate the epithelium from underlying connective tissue with short arrows pointing to connective tissue. Green dots in A and D bracket taste buds. <i>P0-Cre</i> driven RFP⁺ cells were abundantly distributed in taste buds and underlying lamina propria of the tongue and soft palate. No RFP⁺ cells were seen in the surrounding epithelium (arrowheads) of taste buds. Scale bars: 20 μm for all images. <u><b>E</b></u>: Histogram shows the average (x̄±SD, n = 3) of RFP⁺Krt8⁺ as a proportion of total Krt8⁺ taste bud cell profiles in fungiform, foliate and circumvallate papillae in 8-week-old mice. <u><b>F</b></u>: Data from 3 mice for each stage (2, 4, 8 and 16 week) are represented as box plot of median±percentile. The diamond within each box represents the average (n = 3) of RFP⁺Krt8⁺ double labeled versus total Krt8⁺ taste bud cell profiles in fungiform papillae at different stages.</p