lhCLIP reveals the in vivo RNA-RNA interactions recognized by hnRNPK.

RNA-RNA interactions play a crucial role in regulating gene expression and various biological processes, but identifying these interactions on a transcriptomic scale remains a challenge. To address this, we have developed a new biochemical technique called pCp-biotin labelled RNA hybrid and ultraviolet crosslinking and immunoprecipitation (lhCLIP) that enables the transcriptome-wide identification of intra- and intermolecular RNA-RNA interactions mediated by a specific RNA-binding protein (RBP). Using lhCLIP, we have uncovered a diverse landscape of intermolecular RNA interactions recognized by hnRNPK in human cells, involving all major classes of noncoding RNAs (ncRNAs) and mRNA. Notably, hnRNPK selectively binds with snRNA U4, U11, and U12, and shapes the secondary structure of these snRNAs, which may impact RNA splicing. Our study demonstrates the potential of lhCLIP as a user-friendly and widely applicable method for discovering RNA-RNA interactions mediated by a particular protein of interest and provides a valuable tool for further investigating the role of RBPs in gene expression and biological processes.

Saponins of Panax japonicus ameliorates cardiac aging phenotype in aging rats by enhancing basal autophagy through AMPK/mTOR/ULK1 pathway.

Heart disease is a significant health concern for elderly individuals, with heart aging being the primary cause. Recent studies have shown that autophagy can play a protective role in preventing cardiac aging. Our previous research confirmed that Chikusetsu saponin IVa, a fundamental component of Saponins of Panax japonics (SPJ), can enhance basic autophagy levels in cardiomyocyte of isoproterenol induced cardiac fibrosis mice. However, it remains unclear whether SPJ possesses a protective effect on cardiac dysfunction during the natural aging process. Rats were randomly divided into four groups: adult control group (6 months old), aging group (24 months old), aging group treated with 10 mg/kg SPJ, and aging group treated with 30 mg/kg SPJ. The heart function, blood pressure, and heart mass index (HMI) were measured. Hematoxylin and eosin staining (H&E) and Wheat Germ Agglutinin (WGA) staining were used to observe the changes in morphology, while Masson staining was used to examine collagen deposition in the rat hearts and CD45 immunohistochemistry was conducted to examine the macrophage infiltration in heart tissues. TUNEL kit was used to detect apoptosis level of cardiomyocyte, and western blot was used to evaluate autophagy-related proteins as well as AMPK/mTOR/ULK1 pathway-related markers. SPJ treatment improved the cardiac function, reduced HMI, attenuated myocardial fiber disorder, inhibited inflammatory cell infiltration, and decreased collagen deposition and cardiomyocyte apoptosis in aging rats. Additionally, SPJ treatment decreased the expression of aging-related proteins and restored the expression of autophagy-related markers. SPJ activated autophagy through the activation of AMPK, which in turn increased the phosphorylation of ULK1(Ser555), while inhibited the phosphorylation of mTOR and ULK1(Ser757). Our study demonstrates that SPJ improves the cardiac function of aging rats by enhancing basal autophagy through the AMPK/mTOR/ULK1 pathway. These results offer a theoretical foundation and empirical evidence to support the clinical advancement of SPJ in enhancing age-related cardiac dysfunction.

Dynamic reprogramming of H3K9me3 at hominoid-specific retrotransposons during human preimplantation development.

Reprogramming of H3K9me3-dependent heterochromatin is required for early development. How H3K9me3 is involved in early human development remains, however, largely unclear. Here, we resolve the temporal landscape of H3K9me3 during human preimplantation development and its regulation for diverse hominoid-specific retrotransposons. At the 8-cell stage, H3K9me3 reprogramming at hominoid-specific retrotransposons termed SINE-VNTR-Alu (SVA) facilitates interaction between certain promoters and SVA-derived enhancers, promoting the zygotic genome activation. In trophectoderm, de novo H3K9me3 domains prevent pluripotent transcription factors from binding to hominoid-specific retrotransposons-derived regulatory elements for inner cell mass (ICM)-specific genes. H3K9me3 re-establishment at SVA elements in the ICM is associated with higher transcription of DNA repair genes, when compared with naive human pluripotent stem cells. Our data demonstrate that species-specific reorganization of H3K9me3-dependent heterochromatin at hominoid-specific retrotransposons plays important roles during early human development, shedding light on how the epigenetic regulation for early development has evolved in mammals.

The X box binding protein 1/C/EBP homologous protein pathway induces apoptosis of endothelial cells under hyperglycemia.

Venous endothelial cell apoptosis can be induced by endoplasmic reticulum (ER) stress, thus serving an important role in the formation of deep venous thrombosis. X-box binding protein 1 (XBP1) is a protein associated with ER. The present study aimed to explore the function of XBP1/C/EBP homologous protein (CHOP) pathway in the process of endothelial cell apoptosis under hyperglycemia. Small interfering (si)RNAs targeting XBP1 and CHOP were designed to downregulate the expression of XBP1 and CHOP in human umbilical vein endothelial cell, respectively. Flow cytometry was used to determine cell apoptosis. The expression of XBP1, glucose-regulated protein 78 (GRP78), CHOP, Puma, cleaved caspase-3 and Cytochrome c was evaluated by western blotting. There were seven groups of cells that were used in the present study: i) Control (5.5 mM D-glucose); ii) hypertonic (hypertonic control, 27.8 mM mannitol and 5.5 mM D-glucose); iii) 16.7 mM D-glucose; iv) 33.3 mM D-glucose; v) 33.3 mM + NC (33.3 mM D-glucose incubated with NC); vi) 33.3 mM + si-XBP1 (33.3 mM D-glucose incubated with siRNA against XBP1); and vii) 33.3 mM + si-CHOP (33.3 mM D-glucose incubated with siRNA against CHOP). Compared with the control, the apoptosis rate of human umbilical vein endothelial cells (HUVECs) increased greatly with the increase in the concentration of D-glucose. Compared with the 33.3 mM D-glucose group, the HUVECs incubated with 33.3 mM D-glucose and si-XBP1 or 33.3 mM D-glucose and si-CHOP demonstrated a significantly lower apoptosis rate. Compared with the control, XBP1, GRP78, CHOP, Puma, cleaved caspase-3 and cytochrome c were significantly upregulated in the hypertonic, 16.7 mM D-glucose, 33.3 mM D-glucose and 33.3 mM + negative control (NC) groups. Compared with the 33.3 mM D-glucose group, the expression levels of XBP1, GRP78, CHOP, Puma, cleaved caspase-3 and cytochrome c in the 33.3 mM + si-XBP1 or 33.3 mM + si-CHOP groups significantly decreased. High dosage of glucose induced endothelial cell apoptosis by promoting the expression of apoptotic proteins by activating endoplasmic reticulum stress. XBP1/CHOP may be a potential target for the treatment of deep vein thrombosis as one of the key pathways regulating ERS by regulating apoptosis of endothelial cells.

Chemical-induced chromatin remodeling reprograms mouse ESCs to totipotent-like stem cells.

Totipotent cells have more robust developmental potency than any other cell types, giving rise to both embryonic and extraembryonic tissues. Stable totipotent cell cultures and deciphering the principles of totipotency regulation would be invaluable to understand cell plasticity and lineage segregation in early development. Our approach of remodeling the pericentromeric heterochromatin and re-establishing the totipotency-specific broad H3K4me3 domains promotes the pluri-to-totipotency transition. Our protocol establishes a closer match of mouse 2-cell (2C) embryos than any other 2C-like cells. These totipotent-like stem cells (TLSCs) are stable in culture and possess unique molecular features of the mouse 2C embryo. Functionally, TLSCs are competent for germline transmission and give rise to both embryonic and extraembryonic lineages at high frequency. Therefore, TLSCs represent a highly valuable cell type for studies of totipotency and embryology.

Hippocampal CD39/ENTPD1 promotes mouse depression-like behavior through hydrolyzing extracellular ATP.

Emerging evidence implicates that low levels of ATP in the extracellular space may contribute to the pathophysiology of major depressive disorder (MDD). The concentration of extracellular ATP is regulated by its hydrolase ectonucleotide tri(di)phosphohydrolase (ENTPD). However, the role of ENTPD in depression remains poorly understood. Here we examine the role of CD39 (known as ENTPD1) in mouse depression-like behavior induced by chronic social defeat stress (CSDS). We demonstrate that CSDS enhances the expression and activity of CD39 in hippocampus. The CD39 functional analog apyrase also induces depression-like behavior, which can be ameliorated by ATP replenishment. Pharmacological inhibition and genetic silencing of CD39 has an antidepressant-like effect via increasing hippocampal extracellular ATP concentration, accompanied with an increase in hippocampal neurogenesis and dendritic spine numbers in defeated mice. These results suggest that hippocampal CD39 contributes to CSDS-induced depression-like behavior via hydrolyzing extracellular ATP, indicating that CD39 may be a promising new target for the treatment of depression.

Pannexin-1 channel dysfunction in the medial prefrontal cortex mediates depressive-like behaviors induced by chronic social defeat stress and administration of mefloquine in mice.

Mefloquine (MFQ) is widely used for the treatment of malaria clinically. Apart from antimalarial effect, psychiatric side effects such as depression and anxiety of MFQ have been reported. Interestingly, MFQ is also known as a broad-spectrum pannexin-1 (Panx1) inhibitor. Panx1 is a new gap junction channel in the brain which mediates efflux of adenosine triphosphate (ATP). Although exogenous ATP has been known to produce a potential antidepressant-like effect, little is known about the role of Panx1 in pathophysiology of depression, especially the depression induced by administration of MFQ. Here, we used the chronic social defeat stress (CSDS) model and found a decrease in the expression and function of Panx1 in the medial prefrontal cortex (mPFC) of susceptible mice. Furthermore, pharmacological blockade of Panx1 in the mPFC with carbenoxolone (CBX) (100 mM) or 10Panx (100 µM) was sufficient to induce depressive-like behaviors and increase vulnerability to stress in mice, which were prevented by preconditioning with ATP (25 µM). Finally, systemic and intral-mPFC injection of MFQ both inhibited the activity of Panx1 and induced depressive-like and anxiety behaviors in mice with sub-threshold social defeat stress. Indeed, the behavioral abnormalities induced by MFQ were prevented by preconditioning with ATP in the mPFC. In conclusion, our study demonstrates a role of the Panx1 channel in chronic stress and MFQ-induced depressive-like and anxiety behaviors, which may provide a novel molecular mechanism for psychiatric side effects of MFQ.

Multiple H+ sensors mediate the extracellular acidification-induced [Ca2+]i elevation in cultured rat ventricular cardiomyocytes.

Acidosis has been known to cause "Ca2+ transients", however, the mechanism is still uncertain. Here, we demonstrated that multiple H+ sensors, such as ASICs, TRPV1 and proton-sensing G protein coupled receptors (GPCRs) are involved in extracellular acidification-induced intracellular calcium ([Ca2+]i) elevation. By using calcium imaging measures, we observed that both ASIC and TRPV1 channels inhibitors suppressed the [Ca2+]i elevation induced by extracellular acidosis in cultured rat cardiac myocytes. Then, both channels mRNA and proteins were identified by RT-PCR, western blotting and immunofluorescence. ASIC-like and TRPV1-like currents were induced by extracellular acidification, suggesting that functional ASIC and TRPV1 channels jointly mediated extracellular calcium entry. Furthermore, either pre-exhaustion of sarcoplasmic reticulum (SR) Ca2+ with thapsigargin or IP3 receptor blocker 2-APB or PLC inhibitor U73122 significantly attenuated the elevation of [Ca2+]i, indicating that the intracellular Ca2+ stores and the PLC-IP3 signaling also contributed to the acidosis-induced elevation of [Ca2+]i. By using genetic and pharmacological approaches, we identified that ovarian cancer G protein-coupled receptor 1 (OGR1) might be another main component in acidosis-induced release of [Ca2+]i. These results suggest that multiple H+-sensitive receptors are involved in "Ca2+ transients" induced by acidosis in the heart.

[HPLC-ELSD fingerprint and chemical constituents of Rhizoma Panacis Japonici].

The aim was to establish the chromatographic fingerprint of Rhizoma Panacis Japonici for its quality evaluation. Analysis was performed on a YMC-Pack ODS-AQ column (4.6 mm x 250 mm, 5 microm) eluted with the mobile phases of acetonitrile(A) and 5% acetic acid solution (B) at in gradient a flow rate of 1.0 mL x min(-1). The elution program was as follow: 0-5 min, changed from 20% A to 40% A, 5-20 min, kept 40% A. The temperature of column was 30 degrees C. The temperate of drift tube was 40 degrees C, and the nitrogen pressure was 33 Psi. Ten batches of Rhizoma Panacis japonici were determined. The HPLC-ELSD chromatographic fingerprint of chemical constituents was established from the 10 batches of Rhizoma Panacis japonici and it had 9 characteristic common peaks. The 10 batches of samples were classified into 2 cluster by cluster analysis. Furthermore, five known chemical constuituents were identified after isolation and purification by means of silica gel column chromatography, and semi-preparative high performance liquid chromatography. They were panaxsaponins Re, chikusetsusaponins IV, IVa, V and pjs-2, respectively. This study provided experimental data for comprehensive evaluation of the quality of Rhizoma Panacis japonici.