The Crystallization and Melting Behavior of Neodymium-Based Butadiene Rubber Blends.

This study investigates the influence of poly(butadiene-isoprene) copolymer rubber (BIR) and TDAE oil on the crystallization and melting behavior of neodymium-based butadiene rubber (Nd-BR). The study demonstrates that the melting points of Nd-BR and its blends decrease with lower crystallization temperatures. Below the critical crystallization temperature (Tc,c), the melting behavior shows dual peaks in distinct temperature ranges, which are attributed to different spherulitic sizes. The addition of BIR or TDAE oil lowers the Tc,c, with TDAE oil exerting a more substantial effect. These diluents mainly influence the nucleation temperature and crystallinity level of Nd-BR while having a minimal effect on the crystallization mechanism. A master curve, which overlaps for various samples, is developed by correlating the peak melting temperature (Tm,peak) with the Tc. This curve facilitates a quantitative assessment of the effects of BIR and TDAE oil on Nd-BR, highlighting the greater influence of TDAE oil on the crystalline structure compared with BIR at equivalent mass fractions. By applying the Lorentz equation and multi-peak fitting, a relationship between the melting points and crystallization temperatures is established, enabling the calculation of the equilibrium melting points (Tm0) for different samples. The findings show a reduction in the Tm0 due to the diluents; specifically, the Tm0 is approximately 0 °C for pure Nd-BR, and it decreases to -4.579 °C and -6.579 °C for samples with 50 PHR TDAE oil and 60 wt.% BIR, respectively.

Study on the Crystallization Behavior of Neodymium Rare-Earth Butadiene Rubber Blends and Its Effect on Dynamic Mechanical Properties.

Utilizing neodymium-based butadiene rubber as a baseline, this study examines the effect of eco-friendly aromatic TDAE oil, fillers, and crosslinking reactions on neodymium-based rare-earth butadiene rubber (Nd-BR) crystallization behavior. The findings suggest that TDAE oil hinders crystallization, resulting in decreased crystallization temperatures and heightened activation energies (Ea). The crystallization activation energies for 20 parts per hundreds of rubber (PHR) and 37.5 PHR oil stand at -116.8 kJ/mol and -48.1 kJ/mol, respectively, surpassing the -264.3 kJ/mol of the unadulterated rubber. Fillers act as nucleating agents, hastening crystallization, which in turn elevates crystallization temperatures and diminishes Ea. In samples containing 20 PHR and 37.5 PHR oil, the incorporation of carbon black and silica brought the Ea down to -224.9 kJ/mol and -239.1 kJ/mol, respectively. Crosslinking considerably restricts molecular motion and crystallization potential. In the examined conditions, butadiene rubber containing 37.5 PHR oil displayed no crystallization following crosslinking, albeit crystallization was discernible with filler inclusion. Simultaneously, the crystallinity level sharply declined, manifesting cold crystallization behavior. The crosslinking process elevates Ea, while the equilibrium melting point (Tm0) noticeably diminishes. For instance, the Tm0 of pure Nd-BR is approximately -0.135 °C. When blended with carbon black and silica, the Tm0 values are -3.13 °C and -5.23 °C, respectively. After vulcanization, these values decrease to -21.6 °C and -10.16 °C. Evaluating the isothermal crystallization kinetics of diverse materials via the Avrami equation revealed that both the oil and crosslinking process can bring about a decrease in n values, with the Avrami index n for various samples oscillating between 1.5 and 2.5. Assessing the dynamic mechanical attributes of different specimens reveals that Nd-BR crystallization notably curtails its glass transition, marked by a modulus shift in the transition domain and a decrement in loss factor. The modulus in the rubbery state also witnesses a substantial augmentation.

Comparative effectiveness of prophylactic strategies for preeclampsia: a network meta-analysis of randomized controlled trials.

OBJECTIVE: Preeclampsia is a common disease during pregnancy that leads to fetal and maternal adverse events. Few head-to-head clinical trials are currently comparing the effectiveness of prophylactic strategies for preeclampsia. In this network meta-analysis, we aimed to compare the efficacy of prophylactic strategies for preventing preeclampsia in pregnant women at risk. DATA SOURCES: Articles published in or before September 2021 from PubMed, Embase, Web of Science, Cochrane Library, and ClinicalTrials.gov, references of key articles, and previous meta-analyses were manually searched. STUDY ELIGIBILITY CRITERIA: Randomized controlled trials comparing prophylactic strategies preventing preeclampsia with each other or with negative controls were included. METHODS: Two reviewers independently extracted data, assessed the risk of bias, and assessed evidence certainty. The efficacy of prophylactic strategies was estimated by frequentist and Bayesian network meta-analysis models. The primary composite outcome was preeclampsia/ pregnancy-induced hypertension. RESULTS: In total, 130 trials with a total of 112,916 patients were included to assess 13 prophylactic strategies. Low-molecular-weight heparin (0.60; 95% confidence interval, 0.42-0.87), vitamin D supplementation (0.65; 95% confidence interval, 0.45-0.95), and exercise (0.68; 95% confidence interval, 0.50-0.92) were as efficacious as calcium supplementation (0.71; 95% confidence interval, 0.62-0.82) and aspirin (0.79; 95% confidence interval, 0.72-0.86) in preventing preeclampsia/pregnancy-induced hypertension, with a P score ranking of 85%, 79%, 76%, 74%, and 61%, respectively. In the head-to-head comparison, no differences were found between these effective prophylactic strategies for preventing preeclampsia and pregnancy-induced hypertension, except with regard to exercise, which tended to be superior to aspirin and calcium supplementation in preventing pregnancy-induced hypertension. Furthermore, the prophylactic effects of aspirin and calcium supplementation were robust across subgroups. However, the prophylactic effects of low-molecular-weight heparin, exercise, and vitamin D supplementation on preeclampsia and pregnancy-induced hypertension varied with different risk populations, dosages, areas, etc. The certainty of the evidence was moderate to very low. CONCLUSION: Low-molecular-weight heparin, vitamin D supplementation, exercise, calcium supplementation, and aspirin reduce the risk of preeclampsia/pregnancy-induced hypertension. No significant differences between effective prophylactic strategies were found in preventing preeclampsia. These findings raise the necessity to reevaluate the prophylactic effects of low-molecular-weight heparin, vitamin D supplementation, and exercise on preeclampsia.

Different transcriptional profiles of human embryonic stem cells grown in a feeder-free culture system and on human foreskin fibroblast feeder layers.

Feeder cells provide an optimal microenvironment for the propagation of human embryonic stem cells (hESCs) by supplying currently known or unknown factors. However, the hESCs grown on feeder cells are not suitable for the purpose of clinical application because of the risk of contamination. In recent years, the feeder-free culture method has been developed to eliminate contamination, but some studies show that hESCs exhibit poor growth patterns in a feeder-free culture system. Regarding this phenomenon, we speculate that some genes related to hESC propagation were differently expressed in hESCs grown on feeder cells. To test this hypothesis, 3 hESC lines (NF4, NF5 and P096) were efficiently expanded in a feeder-free culture system or on human foreskin fibroblast (HFF) cells. The different gene expression patterns of hESCs in these 2 conditions were analyzed through microarrays. The results revealed that the hESCs cultured in both conditions maintained the expression of stemness markers and the ability to spontaneously differentiate into the 3 germ layers. The analysis of gene expression profiles revealed that 23 lncRNA and 15 genes were significantly differentially expressed in these two culture conditions. Furthermore, GO analyses showed that these genes were involved in such biological processes as growth factor stimuli, cell growth, and stem cell maintenance. To summarize, our study demonstrated that the hESCs grown on the HFF showed different gene expression patterns compared to those grown in a feeder-free culture system, suggesting that these differently expressed lncRNAs and genes played important roles in maintaining hESC propagation.

Protocol to derive human trophoblast stem cells directly from primed pluripotent stem cells.

Human trophoblast stem cells (hTSCs) are useful for studying human placenta development and diseases, but primed human pluripotent stem cells (hPSCs) routinely cultured in most laboratories do not support hTSC derivation. Here, we present a protocol to derive hTSCs directly from primed hPSCs. This approach, containing two strategies either with or without bone morphogenetic protein 4 (BMP4), provides a simple and accessible tool for deriving hTSCs to study placenta development and disease modeling without ethical limitations or reprogramming process. For complete details on the use and execution of this protocol, please refer to Wei et al. (2021).

Preeclampsia associated changes in volume density of fetoplacental vessels in Chinese women and mouse model of preeclampsia.

INTRODUCTION: Preeclampsia (PE) is associated with abnormal placental vascular structure. However, the volume density of fetoplacental vessels in PE remains unclear. Additionally, manually annotated CT angiography, which is widely used to analyze placental vessels, has issues regarding inaccuracy. Thus, computer-aided CT angiography for analyzing the volume density of fetoplacental vessels would facilitate the understanding of PE pathogenesis. METHODS: We performed computer-aided CT angiography to compare differences in placentas among 17 women with PE and 34 normotensive women. The contrast ratio in CT angiography was significantly enhanced using a three-dimensional (3-D) Hessian matrix algorithm. The PE-like mouse model was established by administration of 125 mg/kg/day NG-nitro-l-arginine methyl ester (l-NAME) for 10 days. The presence of endothelial marker CD31 was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The expression of angiogenic factors (PlGF, VEGFA, and sFlt1) in placentas was detected using qRT-PCR and western blotting. RESULTS: The volume density in fetoplacental vessels and CD31 expression were significantly reduced in women with PE and l-NAME-induced mice. Additionally, the downregulation of angiogenic factors (PlGF/VEGFA) and upregulation of an anti-angiogenic factor (sFlt1) were determined in a mouse model. DISCUSSION: Contrast-enhanced CT angiography with the aid of a 3-D Hessian matrix algorithm was performed. PE significantly affects the formation of vascular vessels, resulting in a lower volume density of fetoplacental vessels in humans and mice. This may be explained by the abnormal release of angiogenic factors during PE.

Coordination of EZH2 and SOX2 specifies human neural fate decision.

Polycomb repressive complexes (PRCs) are essential in mouse gastrulation and specify neural ectoderm in human embryonic stem cells (hESCs), but the underlying molecular basis remains unclear. Here in this study, by employing an array of different approaches, such as gene knock-out, RNA-seq, ChIP-seq, et al., we uncover that EZH2, an important PRC factor, specifies the normal neural fate decision through repressing the competing meso/endoderm program. EZH2-/- hESCs show an aberrant re-activation of meso/endoderm genes during neural induction. At the molecular level, EZH2 represses meso/endoderm genes while SOX2 activates the neural genes to coordinately specify the normal neural fate. Moreover, EZH2 also supports the proliferation of human neural progenitor cells (NPCs) through repressing the aberrant expression of meso/endoderm program during culture. Together, our findings uncover the coordination of epigenetic regulators such as EZH2 and lineage factors like SOX2 in normal neural fate decision.

ACE2 Is Expressed in Immune Cells That Infiltrate the Placenta in Infection-Associated Preterm Birth.

COVID-19 is associated with increased incidence of preterm birth (PTB). We assessed pathways by which SARS-CoV-2 could access the placenta. Placentae, from PTB with or without chorioamnionitis (ChA), or from term pregnancies (n = 12/13/group) were collected. Peripheral blood was collected from healthy pregnant women (n = 6). Second trimester placental explants (16-20 weeks, n = 5/group) were treated with lipopolysaccharide (LPS, to mimic bacterial infection) and ACE2, CCL2, IL-6/8 and TNFα mRNA was assessed. ChA-placentae exhibited increased ACE2 and CCL2 mRNA expression (p < 0.05). LPS increased cytokine and ACE2 mRNA in placental explants. Placental ACE2 protein localized to syncytiotrophoblast, fetal endothelium, extravillous trophoblast and in immune cells-subsets (M1/M2 macrophage and neutrophils) within the villous stroma. Significantly increased numbers of M1 macrophage and neutrophils were present in the ChA-placenta (p < 0.001). Subsets of peripheral immune cells from pregnant women express the ACE2 mRNA and protein. A greater fraction of granulocytes was positive for ACE2 protein expression compared to lymphocytes or monocytes. These data suggest that in pregnancies complicated by ChA, ACE2 positive immune cells in the maternal circulation have the potential to traffic SARS-CoV-2 virus to the placenta and increase the risk of vertical transmission to the placenta/fetus.

Efficient derivation of human trophoblast stem cells from primed pluripotent stem cells.

Human trophoblast stem cells (hTSCs) provide a valuable model to study placental development and function. While primary hTSCs have been derived from embryos/early placenta, and transdifferentiated hTSCs from naïve human pluripotent stem cells (hPSCs), the generation of hTSCs from primed PSCs is problematic. We report the successful generation of TSCs from primed hPSCs and show that BMP4 substantially enhances this process. TSCs derived from primed hPSCs are similar to blastocyst-derived hTSCs in terms of morphology, proliferation, differentiation potential, and gene expression. We define the chromatin accessibility dynamics and histone modifications (H3K4me3/H3K27me3) that specify hPSC-derived TSCs. Consistent with low density of H3K27me3 in primed hPSC-derived hTSCs, we show that knockout of H3K27 methyltransferases (EZH1/2) increases the efficiency of hTSC derivation from primed hPSCs. Efficient derivation of hTSCs from primed hPSCs provides a simple and powerful model to understand human trophoblast development, including the pathogenesis of trophoblast-related disorders, by generating disease-specific hTSCs.

Establishment of a CRISPR/Cas9-mediated ANP32A homozygous knockout human embryonic stem cell line.

ANP32A is a member of acidic leucine-rich nuclear phosphoprotein 32 family, which is involved in diverse biochemical processes, including chromatin modification and remodeling. Here, we established the CRISPR/Cas9-mediated ANP32A homozygous knockout human embryonic stem cell (ESC) line to investigate the roles of ANP32A in pluripotency maintenance and differentiation process of human ESCs. This cell line shows the normal karyotype and typical stem cell morphology, in accordance with high expression of pluripotent genes and the differentiation potential in vitro. Consequently, the ANP32A knockout cell line provides a promising approach for investigating the roles of ANP32A in human ESC cell fate decisions.

Generation of two RNF2 homozygous knockout human embryonic stem cell lines by CRISPR/Cas9 system.

Human RNF2 (RING1B) gene is a critical epigenetic modification factor for embryonic development, pluripotency and differentiation of embryonic stem cells (ESCs). To further gain insights into the role of RNF2 in cell fate decisions of human ESCs, here we generated two RNF2 homozygous knockout human ESC lines by CRISPR/Cas9 genome editing technology. These cell lines maintained a normal karyotype and typical undifferentiated state in terms of morphology, pluripotent gene expression, and had differentiation potential in vivo. These cell lines provide good cell resources to explore the role of RNF2 gene in embryonic development and lineage differentiation in vitro.

Deciphering a distinct regulatory network of TEAD4, CDX2 and GATA3 in humans for trophoblast transition from embryonic stem cells.

The placenta is an essential organ for the fetus, but its regulatory mechanism for formation of functional trophoblast lineage remains elusive in humans. Although widely known in mice, TEAD4 and its downstream targets CDX2 and GATA3 have not been determined in human models. In this work, we used a human model of trophoblast transition from BAP (BMP4, A83-01 and PD173074)-treated human embryonic stem cells (hESCs) and performed multiple gain- and loss-of-function tests of TEAD4, CDX2 or GATA3 to study their roles during this process. Although hESCs with TEAD4 deletion maintain pluripotency, their trophoblast transition potentials are attenuated. This impaired trophoblast transition could be rescued by separately overexpressing TEAD4, CDX2 or GATA3. Furthermore, trophoblast transition from hESCs is also attenuated by knockout of CDX2 but remains unaffected with deletion of GATA3. However, CDX2-overexpressed hESCs maintain pluripotency, whereas overexpression of GATA3 in hESCs leads to spontaneous differentiation including trophoblast lineage. In brief, our findings using a human model of trophoblast transition from BAP-treated hESCs reveal transcription roles of TEAD4, CDX2 and GATA in humans that are different from those in mice. We hope that this evidence can aid in understanding the distinct transcriptional network regulating trophoblast development in humans.

TNF-related apoptosis-inducing ligand (TRAIL) promotes trophoblast cell invasion via miR-146a-EGFR/CXCR4 axis: A novel mechanism for preeclampsia?

INTRODUCTION: Preeclampsia (PE) is associated with failure of uterovascular transformation due to impaired trophoblast invasion. Previously, TNF-related apoptosis-inducing ligand (TRAIL) has been controversially reported to correlate with PE, but whether it regulates trophoblast invasion yet to be defined. METHODS: We treated HTR8/SVneo cells with sTRAIL at concentrations of 0.1 ng/mL, 1 ng/mL and 10 ng/mL for a 72-h time course and compared cell proliferation, apoptosis and invasion ability by CCK8 assay, flow cytometry analysis and Matrigel cell invasion assay, respectively. The expressions levels of miR-146a, CXCR4, EGFR and MMP2 were quantified by qRT-PCR and Western blot. Moreover, HTR8/SVneo cells were transfected with miR-146a mimics and miR-146a inhibitor, followed by analyzing invasion ability and gene expressions of CXCR4, EGFR and MMP2. Finally, both serum and placental samples from preeclamptic and gestational week-matched normotensive women were collected and assessed for the expression levels of TRAIL by ELISA assay and immunochemistry staining. RESULTS: sTRAIL treatment of HTR8/SVneo cells resulted in no change in proliferation or apoptosis, but dose- and time-dependently enhanced invasion. TRAIL downregulated the expressions of miR-146a and upregulated CXCR4, EGFR and MMP2. Transfection of miR-146a resulted in the inhibition of invasion and downregulation of CXCR4, EGFR and MMP2. Lastly, the expression levels of TRAIL decreased in both serum and placenta of preeclamptic pregnancies and correlated with the disease severity. DISCUSSION: TRAIL regulated miR-146a-CXCR/EGFR axis to promote the invasion of trophoblast like cells. Its deceased levels in preeclamptic sera and placenta, suggest that low levels of TRAIL might contribute to the pathogenesis of preeclampsia.

JMJD3 and UTX determine fidelity and lineage specification of human neural progenitor cells.

Neurogenesis, a highly orchestrated process, entails the transition from a pluripotent to neural state and involves neural progenitor cells (NPCs) and neuronal/glial subtypes. However, the precise epigenetic mechanisms underlying fate decision remain poorly understood. Here, we delete KDM6s (JMJD3 and/or UTX), the H3K27me3 demethylases, in human embryonic stem cells (hESCs) and show that their deletion does not impede NPC generation from hESCs. However, KDM6-deficient NPCs exhibit poor proliferation and a failure to differentiate into neurons and glia. Mechanistically, both JMJD3 and UTX are found to be enriched in gene loci essential for neural development in hNPCs, and KDM6 impairment leads to H3K27me3 accumulation and blockade of DNA accessibility at these genes. Interestingly, forced expression of neuron-specific chromatin remodelling BAF (nBAF) rescues the neuron/glia defect in KDM6-deficient NPCs despite H3K27me3 accumulation. Our findings uncover the differential requirement of KDM6s in specifying NPCs and neurons/glia and highlight the contribution of individual epigenetic regulators in fate decisions in a human development model.

Decidual small extracellular vesicles induce trophoblast invasion by upregulating N-cadherin.

Small extracellular vesicles (sEVs) are important mediators of cell-to-cell communication involved in the successful establishment of a pregnancy. Human decidual stromal cells play a key role in regulating trophoblast invasion. Nevertheless, the regulatory functions of decidual stromal cells-derived sEVs in human trophoblast cells are still unclear. In this study, primary human decidual stromal cells were isolated, and immortalized human endometrial stromal cell line (HESCs) were decidualized into human decidual stromal cells (HDSCs) using hormonal cocktail containing medroxy progesterone 17-acetate (MPA), estrogen and cAMP analog. HDSC-sEVs were isolated from both primary human decidual stromal cells and immortal HDSCs, respectively, and identified by transmission electron microscopy and western blotting. EV uptake assay indicated that HDSC-sEVs could be uptaken by trophoblast cells. HDSC-sEVs could increase the invasiveness and the expression level of N-cadherin of trophoblast cells with elevated phosphorylation of SMAD2 and SMAD3 in the cells. Silencing of N-cadherin could block cell invasion induced by HDSC-sEVs, while knockdown of SMAD2 and SMAD3 could inhibit the upregulation of N-cadherin in trophoblast cells. Taken together, our results suggested a regulatory effect of HDSC-sEVs in the invasion of trophoblast cells, and HDSC-sEVs may be important mediators of trophoblasts during embryo implantation and placentation.

Comparison of Dinoprostone and Oxytocin for the Induction of Labor in Late-Term Pregnancy and the Rate of Cesarean Section: A Retrospective Study in Ten Centers in South China.

BACKGROUND Dinoprostone is the recommended primary option for induction of labor (IOL) in late-term pregnancies (LTPs). However, oxytocin is used in developing and rural areas, and studies have supported similar effectiveness for oxytocin and dinoprostone in reducing the rate of cesarean delivery of LTPs with a Bishop's score of between 4-6. This study aimed to compare dinoprostone and oxytocin for IOL in LTPs and the rate of cesarean section in ten centers in South China. MATERIAL AND METHODS A retrospective study included 1,408 women with LTP, with subgroups including a Bishop's score of 0-3 and 4-6. Rates of cesarean delivery were compared between women given vaginal dinoprostone and intravenous oxytocin for IOL. Secondary outcomes included the duration of labor, and maternal and fetal complications. RESULTS Comparison between women who received oxytocin (N=365) and dinoprostone (N=1,043) showed significantly lower rates of cesarean delivery with dinoprostone, but no significant difference between the subgroups with Bishop's scores of 0-3 and 4-6. The interval between induction to labor and duration of the active phase of labor were significantly reduced in the dinoprostone group with a Bishop's score of between 4-6. CONCLUSIONS For LTPs with a Bishop's score of 0-3, dinoprostone was superior to oxytocin for IOL with a lower rate of cesarean delivery, but both agents had a similar outcome for women with a Bishop's score of 4-6. These findings may have implications for the choice of agent used in IOL when dinoprostone is unavailable.

Tuning FOXD3 expression dose-dependently balances human embryonic stem cells between pluripotency and meso-endoderm fates.

Forkhead box D3 (FOXD3) is a key transcription factor maintaining pluripotency in mouse embryonic stem cells (ESCs). Yet to date studies on its role in human ESCs are quite limited. In this study, we report that deletion of FOXD3 in human ESCs results in loss of pluripotency and spontaneous differentiation toward meso-endoderm. Ectopic overexpression of FOXD3 in hESCs leads to two different phenotypes: Human ESCs expressing high levels of FOXD3 undergo spontaneous meso-endoderm differentiation, whereas those with lower levels of FOXD3 maintain pluripotency. Next we deleted endogenous FOXD3 in the low ectopic expression model and find that addition of exogenous FOXD3 at a low level could rescue FOXD3-deficiency phenotype in hESCs. In summary, our findings suggest that FOXD3 dose-dependently regulates the balance of human ESCs between pluripotency and meso-endoderm fates, which adds to our understanding of the role of FOXD3 in humans.

Decreased expression of fibroblast growth factor 13 in early-onset preeclampsia is associated with the increased trophoblast permeability.

OBJECTIVE: Intracellular protein fibroblast growth factor 13 (FGF13) is highly expressed in human placenta, although its biological function remains unexplored. The aims of this study were to investigate the expression of FGF13 in placentae with early-onset preeclampsia (PE) and the associated mechanisms in the pathophysiology of PE. METHODS: The expression levels of FGF13 in placentae obtained from patients with early-onset PE and normal pregnancies were assessed using immunofluorescent staining, Western blot assays and quantitative PCR. We knocked down FGF13 in trophoblast cell lines BeWo and HTR8/SVneo, and analyzed cell permeability. Clinical trophoblast cell-cell junctions were identified by cytokeratin 7 (CK7) immunofluorescent staining of human placental sections. The expressions of FGF13 were manipulated in BeWo and HTR8/SVneo cell lines, and the expressions of E-cadherin were quantified by reverse transcription followed by quantitative PCR, Western blot assays and immunofluorescent staining. The expressions of FGF13 and E-cadherin were further confirmed in the isolated human primary trophoblasts. RESULTS: Downregulation of FGF13 along with trophoblast disarrangement were found in human placentae with early-onset PE. In trophoblast cell lines decreased FGF13 expression resulted in increased cell permeability and decreased E-cadherin expression. The FGF13 insufficiency-mediated loss of E-cadherin was further confirmed in the human villous trophoblasts isolated from PE patients. CONCLUSION: FGF13 was downregulated in human placentae with early-onset PE. FGF13 played an important role in maintaining placental trophoblast permeability via the modulation of E-cadherin.