A dose-response study on functional and transcriptomic effects of FSH on ex vivo mouse folliculogenesis.

Follicle-stimulating hormone (FSH) binds to its membrane receptor (FSHR) in granulosa cells to activate various signal transduction pathways and drive the gonadotropin-dependent phase of folliculogenesis. Poor female reproductive outcomes can result from both FSH insufficiency owing to genetic or non-genetic factors and FSH excess as encountered with ovarian stimulation in assisted reproductive technology (ART), but the underlying molecular mechanisms remain elusive. Herein, we conducted single-follicle and single-oocyte RNA sequencing analysis along with other approaches in an ex vivo mouse folliculogenesis and oogenesis system to investigate the effects of different concentrations of FSH on key follicular events. Our study revealed that a minimum FSH threshold is required for follicle maturation into the high estradiol-secreting preovulatory stage, and such threshold is moderately variable among individual follicles between 5-10 mIU/mL. FSH at 5, 10, 20, and 30 mIU/mL induced distinct expression patterns of follicle maturation-related genes, follicular transcriptomics, and follicular cAMP levels. RNA-seq analysis identified FSH-stimulated activation of G proteins and downstream canonical and novel signaling pathways that may critically regulate follicle maturation, including the cAMP/PKA/CREB, PI3K-AKT/FOXO1, and glycolysis pathways. High FSH at 20 and 30 mIU/mL resulted in non-canonical FSH responses including premature luteinization, high production of androgen and proinflammatory factors, and reduced expression of energy metabolism-related genes in oocytes. Together, this study improves our understanding of gonadotropin-dependent folliculogenesis and provides crucial insights into how high doses of FSH used in ART may impact follicular health, oocyte quality, pregnancy outcome, and systemic health.

Periprocedural intravenous heparin in patients with acute ischemic stroke treated with endovascular thrombectomy after intravenous thrombolysis.

OBJECTIVE: The benefit-to-risk ratio of periprocedural heparin in patients treated with endovascular thrombectomy (EVT) after intravenous thrombolysis (IVT) remains unclear. This study aimed to evaluate the potential effects of periprocedural heparin on clinical outcomes of EVT after IVT. METHODS: The authors retrospectively analyzed patients from multicenter studies treated with EVT after IVT in the anterior circulation. The endpoints were unfavorable outcome (defined as modified Rankin Scale score ≥ 3 at 90 days), 90-day mortality, symptomatic intracranial hemorrhage (SICH), successful recanalization, and early neurological deterioration. Patients were divided into two groups based on whether they were treated with heparin (heparin-treated group) or not (untreated group), and the efficacy and safety outcomes were compared using multivariable logistic regression models and propensity score-matching methods. RESULTS: Among the 322 included patients (mean age 67.4 years, 54.3% male), 32% of patients received periprocedural heparin. In multivariable analyses, the administration of periprocedural heparin was a significant predictor for unfavorable outcome (OR 2.821, 95% CI 1.15-7.326; p = 0.027), SICH (OR 24.925, 95% CI 2.363-780.262; p = 0.025), and early neurological deterioration (OR 5.344, 95% CI 1.299-28.040; p = 0.029). Regarding successful recanalization and death, no significant differences between the groups were found after propensity score matching. CONCLUSIONS: The results showed that periprocedural heparin is associated with an increased risk of unfavorable outcomes and SICH in patients treated with EVT after IVT. Further studies are warranted to evaluate the utility and safety of periprocedural heparin.

Analyzing prehospital delays in recurrent acute ischemic stroke: Insights from interpretable machine learning.

OBJECTIVE: This study investigates prehospital delays in recurrent Acute Ischemic Stroke (AIS) patients, aiming to identify key factors contributing to these delays to inform effective interventions. METHODS: A retrospective cohort analysis of 1419 AIS patients in Shenzhen from December 2021 to August 2023 was performed. The study applied the Extreme Gradient Boosting (XGBoost) algorithm and SHapley Additive exPlanations (SHAP) for identifying determinants of delay. RESULTS: Living with others and lack of stroke knowledge emerged as significant risk factors for delayed hospital presentation in recurrent AIS patients. Key features impacting delay times included residential status, awareness of stroke symptoms, presence of conscious disturbance, diabetes mellitus awareness, physical weakness, mode of hospital presentation, type of stroke, and presence of coronary artery disease. CONCLUSION: Prehospital delays are similarly prevalent among both recurrent and first-time AIS patients, highlighting a pronounced knowledge gap in the former group. This discovery underscores the urgent need for enhanced stroke education and management. PRACTICE IMPLICATION: The similarity in prehospital delay patterns between recurrent and first-time AIS patients emphasizes the necessity for public health initiatives and tailored educational programs. These strategies aim to improve stroke response times and outcomes for all patients.

The impact of ovarian stimulation on the human endometrial microenvironment.

STUDY QUESTION: How does ovarian stimulation (OS), which is used to mature multiple oocytes for ART procedures, impact the principal cellular compartments and transcriptome of the human endometrium in the periovulatory and mid-secretory phases? SUMMARY ANSWER: During the mid-secretory window of implantation, OS alters the abundance of endometrial immune cells, whereas during the periovulatory period, OS substantially changes the endometrial transcriptome and impacts both endometrial glandular and immune cells. WHAT IS KNOWN ALREADY: Pregnancies conceived in an OS cycle are at risk of complications reflective of abnormal placentation and placental function. OS can alter endometrial gene expression and immune cell populations. How OS impacts the glandular, stromal, immune, and vascular compartments of the endometrium, in the periovulatory period as compared to the window of implantation, is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study carried out between 2020 and 2022 included 25 subjects undergoing OS and 25 subjects in natural menstrual cycles. Endometrial biopsies were performed in the proliferative, periovulatory, and mid-secretory phases. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were processed to determine serum estradiol and progesterone levels. Both the endometrial transcriptome and the principal cellular compartments of the endometrium, including glands, stroma, immune, and vasculature, were evaluated by examining endometrial dating, differential gene expression, protein expression, cell populations, and the three-dimensional structure in endometrial tissue. Mann-Whitney U tests, unpaired t-tests or one-way ANOVA and pairwise multiple comparison tests were used to statistically evaluate differences. MAIN RESULTS AND THE ROLE OF CHANCE: In the periovulatory period, OS induced high levels of differential gene expression, glandular-stromal dyssynchrony, and an increase in both glandular epithelial volume and the frequency of endometrial monocytes/macrophages. In the window of implantation during the mid-secretory phase, OS induced changes in endometrial immune cells, with a greater frequency of B cells and a lower frequency of CD4 effector T cells. LARGE SCALE DATA: The data underlying this article have been uploaded to the Genome Expression Omnibus/National Center for Biotechnology Information with accession number GSE220044. LIMITATIONS, REASONS FOR CAUTION: A limited number of subjects were included in this study, although the subjects within each group, natural cycle or OS, were homogenous in their clinical characteristics. The number of subjects utilized was sufficient to identify significant differences; however, with a larger number of subjects and additional power, we may detect additional differences. Another limitation of the study is that proliferative phase biopsies were collected in natural cycles, but not in OS cycles. Given that the OS cycle subjects did not have known endometrial factor infertility, and the comparisons involved subjects who had a similar and robust response to stimulation, the findings are generalizable to women with a normal response to OS. WIDER IMPLICATIONS OF THE FINDINGS: OS substantially altered the periovulatory phase endometrium, with fewer transcriptomic and cell type-specific changes in the mid-secretory phase. Our findings show that after OS, the endometrial microenvironment in the window of implantation possesses many more similarities to that of a natural cycle than does the periovulatory endometrium. Further investigation of the immune compartment and the functional significance of this cellular compartment under OS conditions is warranted. STUDY FUNDING/COMPETING INTERESTS: Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (R01AI148695 to A.M.B. and N.C.D.), Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD109152 to R.A.), and the March of Dimes (5-FY20-209 to R.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or March of Dimes. All authors declare no conflict of interest.

Healthcare-Seeking Delays in Acute Ischemic Stroke Patients: The Influence of Gender, Immigrant Status, and Educational Background.

Purpose: Timely medical attention is crucial for patients with Acute Ischemic Stroke (AIS), as delays can significantly impact therapeutic outcomes. These delays are influenced by a combination of socio-cultural, educational, and clinical factors. Patients and Methods: An in-depth analysis was conducted to assess the prevalence and median duration of healthcare-seeking delays in AIS patients. The study specifically investigated the independent impacts of sociocultural and clinical determinants on these delays, with a focus on immigrant status, gender disparities, and educational levels. Multivariate regression analysis was employed to identify these independent effects while controlling for potential confounding factors. Results: Among 1419 AIS patients, 82.52% (n = 1171) experienced delays exceeding 2 hours from symptom onset of symptoms to hospital arrival. The median delay was 12.3 hours. Immigrant populations encountering longer delays compared to native groups. Younger males (<45 years) and elderly females were more prone to delay in healthcare-seeking. Identified independent risk factors for delay included male gender (OR = 1.65 [95% CI:1.14-2.48]), self-acknowledged diabetes (OR = 2.50 [95% CI:1.21-5.17]), small vessel (OR = 2.07 [95% CI:1.27-3.36]), and wake stroke (OR = 7.04 [95% CI:3.69-13.44]). Educational background (high school and above), GCS score with 3-8 points (OR = 0.52 [95% CI:0.09-0.69]), understanding stroke-related knowledge (OR = 0.26 [95% CI:0.09-0.44]), conscious disturbance (OR = 0.25 [95% CI:0.10-0.62]) and limb weakness (OR=0.21[95% CI:0.21-0.49]) are protective factors for timely treatment. Conclusion: Immigrant populations experienced longer delays from symptom onset to hospital arrival. The crucial roles of education and knowledge about stroke underscore the need for enhanced health literacy campaigns and public awareness, with a targeted focus on younger males and elderly females.

Lymphocyte to Monocyte Ratio is Independently Associated with Futile Recanalization in Acute Ischemic Stroke After Endovascular Therapy.

Background and Purpose: Acute ischemic stroke (AIS) caused by large artery occlusion (LAO) poses considerable risks in terms of mortality and disability. Endovascular treatment (EVT) has emerged as a primary intervention for this condition. However, the occurrence of futile recanalization (FR) following EVT remains common, necessitating the identification of predictive markers for treatment outcomes. Although the lymphocyte to monocyte ratio (LMR) has been linked to various diseases, its association with FR after EVT in AIS patients has not been investigated. Methods: An analysis was conducted on patients with AIS who underwent EVT within 24 hours of symptom onset. The success of reperfusion was evaluated using the modified Thrombolysis in Cerebral Infarction (mTICI) scale, with patients achieving an mTICI score of ≥2b being included in the study. Various clinical, radiological, and laboratory variables, including lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR), were collected. Logistic regression analysis was used to determine factors associated with FR, and receiver operating characteristic (ROC) analysis was performed to assess the predictive value of LMR. Results: Among the cohort of 101 patients, it was observed that 52.4% experienced FR. Upon admission, lower levels of lymphocyte-to-monocyte ratio (LMR) were found to be associated with older age, higher baseline NIHSS scores, lower ASPECTS, and poorer mRS scores at 90 days. Both univariate and multivariate logistic regression analyses indicated that low LMR independently predicted FR, with an adjusted odds ratio of 0.64 (95% CI = 0.412-0.984, p = 0.042). ROC analysis further demonstrated that LMR had an area under the curve (AUC) of 0.789 for predicting FR. Conclusion: This study establishes the potential value of the lymphocyte-to-monocyte ratio (LMR) as a prognostic marker for predicting FR in patients with AIS undergoing EVT. Decreased LMR levels are associated with unfavorable clinical outcomes.

Mesenchymal stem cell secretome alters gene expression and upregulates motility of human endometrial stromal cells.

In brief: Endometrial stromal cell motility is fundamental to regeneration and repair of this tissue and crucial for successful reproduction. This paper shows a role for the mesenchymal stem cell (MSC) secretome in enhancing endometrial stromal cell motility. Abstract: Cyclic regeneration and repair of the endometrium are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, mechanisms remain unclear. This study tested the hypothesis that the BM-MSC and UC-MSC secretomes upregulate human endometrial stromal cell (HESC) proliferation, migration, and invasion and activate pathways to increase HESC motility. BM-MSCs were purchased from ATCC and cultured from the BM aspirate of three healthy female donors. UC-MSCs were cultured from umbilical cords of two healthy male term infants. Using indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system, we demonstrated that co-culture of HESCs with BM-MSCs or UC-MSCs from all donors significantly increased HESC migration and invasion, whereas effects on HESC proliferation varied among BM-MSC and UC-MSC donors. Analysis of gene expression by mRNA sequencing and RT-qPCR showed that expression of CCL2 and HGF was upregulated in HESCs that had been cocultured with BM-MSCs or UC-MSCs. Validation studies revealed that exposure to recombinant CCL2 for 48 h significantly increased HESC migration and invasion. Increased HESC motility by the BM-MSC and UC-MSC secretome appears to be mediated in part by upregulated HESC CCL2 expression. Our data support the potential for leveraging MSC secretome as a novel cell-free therapy to treat disorders of endometrial regeneration.

Expression of glucocorticoid and androgen receptors in bone marrow-derived hematopoietic and nonhematopoietic murine endometrial cells.

OBJECTIVE: To determine whether bone marrow (BM)-derived cells engrafting the murine endometrium express the glucocorticoid receptor (GR) and androgen receptor (AR). Recent data demonstrate that BM is a long-term source of multiple hematopoietic and nonhematopoietic endometrial cell types. Important roles for glucocorticoids and androgens in regulating endometrial functions, including decidualization and early embryo attachment/invasion, have very recently emerged. Whether endometrial cells of BM origin express glucocorticoid or ARs has not been previously studied. DESIGN: Animal study. SETTING: Basic science laboratory. ANIMAL(S): Wild-type C57BL/6J male mice expressing enhanced green fluorescent protein (GFP) and syngeneic wild-type C57BL/6J female mice aged 6-9 weeks. INTERVENTION(S): Murine bone marrow transplant. MAIN OUTCOME MEASURE(S): Bone marrow cells were harvested from adult wild-type C57BL/6 mice and subjected to flow cytometry to identify the percentage of hematopoietic and nonhematopoietic cells expressing GR or AR. Uterine tissue sections from lethally irradiated syngeneic adult female C57BL/6 mice that had been recipients of BM transplants from adult male transgenic donor mice ubiquitously expressing GFP were studied. Immunohistochemistry was performed in the uterine tissue sections of the recipient mice at 5, 9, and 12 months after transplant using specific anti-GR, anti-AR, anti-GFP, anti-CD45 (pan leukocyte marker), and anti-F4/80 (murine macrophage marker) primary antibodies. Confocal laser microscopy was used to localize and quantitate BM-derived (GFP+) cell types in the endometrial stromal and epithelial compartments and determine whether BM-derived cell types in the murine endometrium express GR or AR. RESULT(S): Hematopoietic cells comprised 93.6%-96.6% of all cells in the BM, of which 98.1% ± 0.2% expressed GR and 92.2% ± 4.4% expressed AR. Nonhematopoietic cells comprised 0.4%-1.3% of BM, of which 52.8% ± 5.9% expressed GR and 48.9% ± 3.4% expressed AR. After BM transplant, the proportion of cells originating from BM in the endometrial stromal compartment increased over time, reaching 13.5% ± 2.3% at 12 months after transplant. In the epithelial compartments, <1% of the cells were of BM origin at 12 months after transplant. Most (60%-72%) GR+ and/or AR+ BM-derived cells in the stroma were hematopoietic (CD45+) cells, of which 37%-51% were macrophages. Nonetheless, 28%-33% of GR+ cells, and 28%-40% of AR+ BM-derived cells, were nonhematopoietic (CD45-) stromal cells of BM origin. CONCLUSION(S): A substantial number of BM-derived cells express GR and AR, suggesting a role for these cells in both glucocorticoid-regulated and androgen-regulated endometrial functions, such as proliferation and/or decidualization.

Gamma Secretase Inhibitors as Potential Therapeutic Targets for Notch Signaling in Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer with few effective therapeutics. The Notch signaling pathway is evolutionarily conserved with oncogenic properties, but it has not been well studied in uLMS. The purpose of our study was to determine expression of Notch family genes and proteins and to investigate the therapeutic effect of γ-secretase inhibitors (GSIs), indirect inhibitors of Notch signaling, in uLMS. We determined expression of Notch genes and proteins in benign uterine smooth muscle tissue, fibroids, and uLMS samples by immunostaining and in two uLMS cell lines, SK-UT-1B (uterine primary) and SK-LMS-1 (vulvar metastasis) by RT-PCR, Western blot and immunostaining. We exposed our cell lines to GSIs, DAPT and MK-0752, and measured expression of HES1, a downstream effector of Notch. Notch proteins were differentially expressed in uLMS. Expression of NOTCH3 and NOTCH4 was higher in uLMS samples than in benign uterine smooth muscle and fibroids. Expression of NOTCH4 was higher in SK-LMS-1 compared to SK-UT-1B. Exposure of SK-UT-1B and SK-LMS-1 to DAPT and MK-0752 decreased expression of HES1 and decreased uLMS cell viability in a dose- and time-dependent manner that was unique to each GSI. Our findings suggest that GSIs are potential therapeutics for uLMS, albeit with limited efficacy.

Implications for preeclampsia: hypoxia-induced Notch promotes trophoblast migration.

In the first trimester of human pregnancy, low oxygen tension or hypoxia is essential for proper placentation and placenta function. Low oxygen levels and activation of signaling pathways have been implicated as critical mediators in the promotion of trophoblast differentiation, migration, and invasion with inappropriate changes in oxygen tension and aberrant Notch signaling both individually reported as causative to abnormal placentation. Despite crosstalk between hypoxia and Notch signaling in multiple cell types, the relationship between hypoxia and Notch in first trimester trophoblast function is not understood. To determine how a low oxygen environment impacts Notch signaling and cellular motility, we utilized the human first trimester trophoblast cell line, HTR-8/SVneo. Gene set enrichment and ontology analyses identified pathways involved in angiogenesis, Notch and cellular migration as upregulated in HTR-8/SVneo cells exposed to hypoxic conditions. DAPT, a γ-secretase inhibitor that inhibits Notch activation, was used to interrogate the crosstalk between Notch and hypoxia pathways in HTR-8/SVneo cells. We found that hypoxia requires Notch activation to mediate HTR-8/SVneo cell migration, but not invasion. To determine if our in vitro findings were associated with preeclampsia, we analyzed the second trimester chorionic villous sampling (CVS) samples and third trimester placentas. We found a significant decrease in expression of migration and invasion genes in CVS from preeclamptic pregnancies and significantly lower levels of JAG1 in placentas from pregnancies with early-onset preeclampsia with severe features. Our data support a role for Notch in mediating hypoxia-induced trophoblast migration, which may contribute to preeclampsia development.

Current Status of Endovascular Treatment for Acute Large Vessel Occlusion in China: A Real-World Nationwide Registry.

BACKGROUND AND PURPOSE: The benefit of endovascular treatment (EVT) for large vessel occlusion in clinical practice in developing countries like China needs to be confirmed. The aim of the study was to determine whether the benefit of EVT for acute ischemic stroke in randomized trials could be generalized to clinical practice in Chinese population. METHODS: We conducted a prospective registry of EVT at 111 centers in China. Patients with acute ischemic stroke caused by imaging-confirmed intracranial large vessel occlusion and receiving EVT were included. The primary outcome was functional independence at 90 days defined as a modified Rankin Scale score of 0 to 2. Outcomes of specific subgroups in the anterior circulation were reported and logistic regression was performed to predict the primary outcome. RESULTS: Among the 1793 enrolled patients, 1396 (77.9%) had anterior circulation large vessel occlusion (median age, 66 [56-73] years) and 397 (22.1%) had posterior circulation large vessel occlusion (median age, 64 [55-72] years). Functional independence at 90 days was reached in 45% and 44% in anterior and posterior circulation groups, respectively. For anterior circulation population, underlying intracranial atherosclerotic disease was identified in 29% of patients, with higher functional independence at 90 days (52% versus 44%; P=0.0122) than patients without intracranial atherosclerotic disease. In the anterior circulation population, after adjusting for baseline characteristics, procedure details, and early outcomes, the independent predictors for functional independence at 90 days were age <66 years (odds ratio [OR], 1.733 [95% CI, 1.213-2.476]), time from onset to puncture >6 hours (OR, 1.536 [95% CI, 1.065-2.216]), local anesthesia (OR, 2.194 [95% CI, 1.325-3.633]), final modified Thrombolysis in Cerebral Infarction 2b/3 (OR, 2.052 [95% CI, 1.085-3.878]), puncture-to-reperfusion time ≤1.5 hours (OR, 1.628 [95% CI, 1.098-2.413]), and National Institutes of Health Stroke Scale score 24 hours after the procedure <11 (OR, 9.126 [95% CI, 6.222-13.385]). CONCLUSIONS: Despite distinct characteristics in the Chinese population, favorable outcome of EVT can be achieved in clinical practice in China. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03370939.

Connexin-43 reduction prevents muscle defects in a mouse model of manifesting Duchenne muscular dystrophy female carriers.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder that affects males. However, 8% of female carriers are symptomatic and underrepresented in research due to the lack of animal models. We generated a symptomatic mouse model of DMD carriers via injection of mdx (murine DMD) embryonic stem cells (ESCs) into wild-type (WT) blastocysts (mdx/WT chimera). mdx/WT chimeras developed cardiomyopathic features and dystrophic skeletal muscle phenotypes including elevated mononuclear invasion, central nucleation, fibrosis and declined forelimb grip strength. The disease was accompanied by connexin-43 (Cx43) aberrantly enhanced in both cardiac and skeletal muscles and remodeled in the heart. Genetic reduction of Cx43-copy number in mdx/WT-Cx43(+/-) chimeras protected them from both cardiac and skeletal muscle fiber damage. In dystrophic skeletal muscle, Cx43 expression was not seen in the fibers but in adjacent F4/80+ mononuclear cells. Ethidium Bromide uptake in purified F4/80+/CD11b+ mdx macrophages revealed functional activity of Cx43, which was inhibited by administration of Gap19 peptide mimetic, a Cx43 hemichannel-specific inhibitor. Thus, we suggest that Cx43 reduction in symptomatic DMD carrier mice leads to prevention of Cx43 remodeling in the heart and prevention of aberrant Cx43 hemichannel activity in the skeletal muscle macrophages neighboring Cx43 non-expressing fibers.

Prevention of connexin-43 remodeling protects against Duchenne muscular dystrophy cardiomyopathy.

Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested as playing a role in the development of cardiac disease in the mdx mouse model of Duchenne muscular dystrophy (DMD); however, a mechanistic understanding of this association is lacking. Here, we identified a reduction of phosphorylation of Cx43 serines S325/S328/S330 in human and mouse DMD hearts. We hypothesized that hypophosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knockin mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or nonphosphorylatable alanines (mdxS3A). The mdxS3E, but not mdxS3A, mice were resistant to Cx43 remodeling, with a corresponding reduction of Cx43 hemichannel activity. MdxS3E cardiomyocytes displayed improved intracellular Ca2+ signaling and a reduction of NADPH oxidase 2 (NOX2)/ROS production. Furthermore, mdxS3E mice were protected against inducible arrhythmias, related lethality, and the development of cardiomyopathy. Inhibition of microtubule polymerization by colchicine reduced both NOX2/ROS and oxidized CaMKII, increased S325/S328/S330 phosphorylation, and prevented Cx43 remodeling in mdx hearts. Together, these results demonstrate a mechanism of dystrophic Cx43 remodeling and suggest that targeting Cx43 may be a therapeutic strategy for preventing heart dysfunction and arrhythmias in DMD patients.

Genomic Variance and Transcriptional Comparisons Reveal the Mechanisms of Leaf Color Affecting Palatability and Stressed Defense in Tea Plant.

Leaves are one of the most important organs of plants, and yet, the association between leaf color and consumable traits remains largely unclear. Tea leaves are an ideal study system with which to investigate the mechanism of how leaf coloration affects palatability, since tea is made from the leaves of the crop Camellia sinensis. Our genomic resequencing analysis of a tea cultivar ZiJuan (ZJ) with purple leaves and altered flavor revealed genetic variants when compared with the green-leaf, wild type cultivar YunKang(YK). RNA-Seq based transcriptomic comparisons of the bud and two youngest leaves in ZJ and YK identified 93%, 9% and 5% expressed genes that were shared in YK- and ZJ-specific cultivars, respectively. A comparison of both transcript abundance and particular metabolites revealed that the high expression of gene UFGT for anthocyanin biosynthesis is responsible for purple coloration, which competes with the intermediates for catechin-like flavanol biosynthesis. Genes with differential expression are enriched in response to stress, heat and defense, and are casually correlated with the environmental stress of ZJ plant origin in the Himalayas. In addition, the highly expressed C4H and LDOX genes for synthesizing flavanol precursors, ZJ-specific CLH1 for degrading chlorophyll, alternatively spliced C4H and FDR and low photosynthesis also contributed to the altered color and flavor of ZJ. Thus, our study provides a better molecular understanding of the effect of purple coloration on leaf flavor, and helps to guide future engineering improvement of palatability.

Comparative genome-wide characterization leading to simple sequence repeat marker development for Nicotiana.

BACKGROUND: Simple sequence repeats (SSRs) are tandem repeats of DNA that have been used to develop robust genetic markers. These molecular markers are powerful tools for basic and applied studies such as molecular breeding. In the model plants in Nicotiana genus e.g. N. benthamiana, a comprehensive assessment of SSR content has become possible now because several Nicotiana genomes have been sequenced. We conducted a genome-wide SSR characterization and marker development across seven Nicotiana genomes. RESULTS: Here, we initially characterized 2,483,032 SSRs (repeat units of 1-10 bp) from seven genomic sequences of Nicotiana and developed SSR markers using the GMATA® software package. Of investigated repeat units, mono-, di- and tri-nucleotide SSRs account for 98% of all SSRs in Nicotiana. More complex SSR motifs, although rare, are highly variable between Nicotiana genomes. A total of 1,224,048 non-redundant Nicotiana (NIX) markers were developed, of which 99.98% are novel. An efficient and uniform genotyping protocol for NIX markers was developed and validated. We created a web-based database of NIX marker information including amplicon sizes of alleles in each genome for downloading and online analysis. CONCLUSIONS: The present work constitutes the first deep characterization of SSRs in seven genomes of Nicotiana, and the development of NIX markers for these SSRs. Our online marker database and an efficient genotyping protocol facilitate the application of these markers. The NIX markers greatly expand Nicotiana marker resources, thus providing a useful tool for future research and breeding. We demonstrate a novel protocol for SSR marker development and utilization at the whole genome scale that can be applied to any lineage of organisms. The Tobacco Markers & Primers Database (TMPD) is available at http://biodb.sdau.edu.cn/tmpd/index.html.

Hematopoietic Id Deletion Triggers Endomyocardial Fibrotic and Vascular Defects in the Adult Heart.

Inhibitor of DNA binding (Id) proteins play important roles in regulating cardiac development via paracrine signaling. Id1/Id3 knockout mice die at mid-gestation with multiple cardiac defects. Single Id knockout studies have not reported cardiomyopathies. To bypass embryonic lethality we used Tie2CRE-mediated recombination to conditionally delete Id1 against global Id3 ablation (Id cDKOs), which develops adult-onset dilated cardiomyopathy. We confirm upregulation of thrombospondin-1 (TSP1) in Id cDKO hearts. Colocalization studies reveal increased TSP1 expression in the vicinity of endothelial cells and near regions of endocardial fibrosis/disruption. Downstream fibrotic molecules were upregulated. Endocardial capillary density was reduced with evidence of vascular distention. Treatment of Id cDKO cardiac explants with LSKL, a peptide antagonist of TSP1 activation of TGFß, reversed the increased expression of fibrotic molecules. We conducted bone marrow transplant experiments in which we transferred bone marrow cells from Id cDKO mice into lethally irradiated WT mice. The majority of WT recipients of Id cDKO bone marrow cells phenocopied Id cDKO cardiac fibrosis 4 months post-transplantation. Injection of LSKL into adult Id cDKO mice led to downregulation of fibrotic molecules. The results prompt caution when bone marrow transfers from individuals potentially carrying mutations in the Id axis are applied in clinical settings.

Small Fractions of Muscular Dystrophy Embryonic Stem Cells Yield Severe Cardiac and Skeletal Muscle Defects in Adult Mouse Chimeras.

Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.

Combined Id1 and Id3 Deletion Leads to Severe Erythropoietic Disturbances.

The Inhibitor of DNA Binding (Id) proteins play a crucial role in regulating hematopoiesis and are known to interact with E proteins and the bHLH family of transcription factors. Current efforts seek to elucidate the individual roles of Id members in regulating hematopoietic development and specification. However, the nature of their functional redundancies remains elusive since ablation of multiple Id genes is embryonically lethal. We developed a model to test this compensation in the adult. We report that global Id3 ablation with Tie2Cre-mediated conditional ablation of Id1 in both hematopoietic and endothelial cells (Id cDKO) extends viability to 1 year but leads to multi-lineage hematopoietic defects including the emergence of anemia associated with defective erythroid development, a novel phenotype unreported in prior single Id knockout studies. We observe decreased cell counts in the bone marrow and splenomegaly to dimensions beyond what is seen in single Id knockout models. Transcriptional dysregulation of hematopoietic regulators observed in bone marrow cells is also magnified in the spleen. E47 protein levels were elevated in Id cDKO bone marrow cell isolates, but decreased in the erythroid lineage. Chromatin immunoprecipitation (ChIP) studies reveal increased occupancy of E47 and GATA1 at the promoter regions of ß-globin and E2A. Bone marrow transplantation studies highlight the importance of intrinsic Id signals in maintaining hematopoietic homeostasis while revealing a strong extrinsic influence in the development of anemia. Together, these findings demonstrate that loss of Id compensation leads to dysregulation of the hematopoietic transcriptional network and multiple defects in erythropoietic development in adult mice.

Reversible mitochondrial DNA accumulation in nuclei of pluripotent stem cells.

According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

Dystrophin-compromised sarcoglycan-δ-knockout diaphragm requires full wild-type embryonic stem cell reconstitution for correction.

Limb-girdle muscular dystrophy-2F (LGMD-2F) is an incurable degenerative muscle disorder caused by a mutation in the sarcoglycan-δ (SGδ)-encoding gene (SGCD in humans). The lack of SGδ results in the complete disruption of the sarcoglycan complex (SGC) in the skeletal and cardiac muscle within the larger dystrophin-glycoprotein complex (DGC). The long-term consequences of SG ablation on other members of the DGC are currently unknown. We produced mosaic mice through the injection of wild-type (WT) embryonic stem cells (ESCs) into SGδ-knockout (KO) blastocysts. ESC-derived SGδ was supplied to the sarcolemma of 18-month-old chimeric muscle, which resulted in the restoration of the SGC. Despite SGC rescue, and contrary to previous observations obtained with WT/mdx chimeras (a mouse rescue paradigm for Duchenne muscular dystrophy), low levels of ESC incorporation were insufficient to produce histological corrections in SGδ-KO skeletal muscle or heart. The inefficient process of ESC rescue was more evident in the SGδ-KO diaphragm, which had reduced levels of dystrophin and no compensatory utrophin, and needed almost full WT ESC reconstitution for histological improvement. The results suggest that the SGδ-KO mouse model of LGMD is not amenable to ESC treatment.