Jiawei Shoutai Pill promotes decidualization by regulating the SGK1/ENaC pathway in recurrent spontaneous abortion.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. AIM OF STUDY: This study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. MATERIALS AND METHODS: 30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor ß (ERß), and progesterone receptor (PR) in patients' decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERß, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. RESULTS: The decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERß, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERß, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERß, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). CONCLUSIONS: Abnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA.

Inhibitory effect of Lonicera japonica-derived exosomal miR2911 on human papilloma virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Lonicera japonica Thunb. has been used as a traditional medicinal herb in China for thousands of years for its heat-clearing and detoxification effects. In recent years, experimental and clinical studies have shown that some Lonicera japonica-containing Chinese medicine prescriptions have been used to treat intraepithelia neoplasia caused by human papilloma virus (HPV) infection. However, its bioactive molecules and mechanism of action have not been fully explored. AIM OF THE STUDY: In this study, Lonicera japonica-derived exosomes was extracted and exosomal miR2911 was identified. Bioinformatic analysis indicated that miR2911 potentially binds to the sequence of HPV. The mechanism of miR2911 action on HPV and the effect of exosomal miR2911 on HPV-induced cervical cancer cells were investigated. METHODS: The potential targets of miR2911 on the HPV sequence were predicted and confirmed by using RNAhybrid and dual-luciferase reporter assays. Lonicera japonica exosomes were characterized by transmission electronic microscopy and zeta sizer analysis. RT-qPCR was used to measure miR2911 concentration in various tissues and exosomes. Synthetic miR2911 and GFP-E6/E7 plasmids were transfected into HEK293T cells to examine the effect of miR2911 on E6/E7 gene expression. The effects of miR2911 on endogenous E6/E7 mRNA and protein levels were detected in HPV16/18-positive cervical cancer cells by RT-qPCR and Western blotting. The proliferation and apoptosis of CaSki, SiHa and HeLa cells by the treatment of miR2911 or miR2911-containing exosomes were examined by CCK8, colony formation and flow cytometry assays. RESULTS: MiR2911 is found to be enriched in various Lonicera japonica tissues, and is stably present in Lonicera japonica-derived exosomes. It is observed that MiR2911 directly binds to E6 and E7 oncogenes of HPV16/18, leading to the suppression of their protein expression. In addition, the endogenous E6/E7 mRNA and protein levels were significantly decreased by using miR2911 treatment in HPV16/18-positive cervical cancer cells. Furthermore, both miR2911 and miR2911-containing exosomes inhibited cell proliferation of SiHa, CaSki and HeLa cells, meanwhile inducing the cell apoptosis through E6/E7-p53/Caspase3 axis. CONCLUSION: Our findings indicate that miR2911, an active component present in Lonicera japonica exosomes, inhibits proliferation and induces apoptosis of cervical cancer cells by targeting the E6/E7 genes of HPV16/18. Thus, Lonicera japonica-derived exosomal miR2911 has implications for the development of novel therapeutic strategies for the treatment of HPV-associated cervical cancers.

Effects of BXSMD on ESR1 and ESR2 expression in CSD female mice.

ETHNIC PHARMACOLOGICAL RELEVANCE: Due to the rapid pace of modern society, chronic insomnia has become universal phenomenon. In China, Banxia Shumi Decoction (BXSMD) has been used in treating chronic insomnia for thousands of years, but its chemical composition and action mechanism are still unknown. AIM OF THE STUDY: This study aims to explore the chemical composition of BXSMD and its effects on Estrogen receptor 1 (ESR1) and Estrogen receptor 2 (ESR2) in mice with chronic sleep deprivation (CSD). MATERIALS AND METHODS: UHPLC-Q-Orbitrap-MS/MS was applied in determining the chemical composition of BXSMD. After 21-day sleep deprivation (SD) in platform water environment, CSD mice model was prepared. By conducting open field test, 24-h autonomic diurnal and nocturnal activity of mice in each group was detected. ELISA was employed to measure the contents of 5-HT, DA, NE, GABA, Glu, and MT. With RT-PCR, Western blot (WB), and immunohistochemistry (IHC), mRNA and protein expressions of ESR1 and ESR2 in the hypothalamus and hippocampus were tested. RESULTS: BXSMD included ferulic acid, kaverol, daidzein, apigenin, berberine, adenosine, aesculin, vanillin, naringin, and glycine, which might constitute the material basis forthe improvement of chronic insomnia. With BXSMD, the total moving distance and the rest time in dark period of CSD mice were shortened, while its rest time in light period was increased. Besides, BXSMD enhanced the contents of 5-HT, GABA, and MT in CSD mice, and decreased the contents of Glu, NE, and DA. BXSMD elevated the mRNA of Esr1 and Esr2, and elevated the protein expressions of ESR1 and ESR2 in the hypothalamus and hippocampus of CSD mice. CONCLUSIONS: BXSMD contains various chemical components for sleep-wake regulation, with the mechanism of stimulating estrogen signaling pathway by regulating the expressions of ESR1 and ESR2, ultimately realizing the regulation to sleep-wake disorder caused by CSD.

Antidepressant effect of Perilla frutescens essential oil through monoamine neurotransmitters and BDNF/TrkB signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine posits that affect-mind ill-being is the primary cause of depression, with Qi movement stagnation as its pathogenesis. As such, clinical treatment for depression should prioritize regulating Qi and relieving depressive symptoms. The pharmacological properties of traditional Chinese medicine indicate that Perilla frutescens may have potential therapeutic effects on depression and other neuropsychiatric diseases due to its ability to regulate Qi and alleviate depressive symptoms. Although previous studies have reported the antidepressant effects of Perilla frutescens, the mechanism underlying PFEO inhalation-mediated antidepressant effect remains unclear. AIM OF THE STUDY: The aim of this investigation is to elucidate the antidepressant mechanisms of PFEO by examining its effects on monoamine neurotransmitters and the BDNF/TrkB signaling pathway. MATERIALS AND METHODS: The CUMS rat model of depression was established, and the depressive state of the animals was assessed through sucrose preference and forced swim tests. ELISA assays were conducted to determine monoamine neurotransmitter levels in the hippocampus and cerebral cortex of rats. Immunohistochemistry, western blotting, and RT-PCR experiments were employed to investigate the BDNF/TrkB signaling pathway's regulation of depression via PFEO inhalation. RESULTS: It has been observed that inhalation administration of PFEO can significantly enhance the preference for sugar water in CUMS rats and reduce their immobility time during forced swimming. Additionally, there was an increase in the levels of monoamine transmitters in both the hippocampus and cerebral cortex of these rats. Furthermore, there was an upregulation in the expression levels of BDNF and TrkB positive cells as well as BDNF and TrkB proteins within both regions, along with increased BDNF mRNA and TrkB mRNA expression levels. CONCLUSION: The antidepressant effect of PFEO via inhalation administration is speculated to be mediated through the monoamine neurotransmitters and BDNF/TrkB signaling pathway.

Sensing intracellular signatures with synthetic mRNAs.

The bottom-up assembly of biological components in synthetic biology has contributed to a better understanding of natural phenomena and the development of new technologies for practical applications. Over the past few decades, basic RNA research has unveiled the regulatory roles of RNAs underlying gene regulatory networks; while advances in RNA biology, in turn, have highlighted the potential of a wide variety of RNA elements as building blocks to construct artificial systems. In particular, synthetic mRNA-based translational regulators, which respond to signals in cells and regulate the production of encoded output proteins, are gaining attention with the recent rise of mRNA therapeutics. In this Review, we discuss recent progress in RNA synthetic biology, mainly focusing on emerging technologies for sensing intracellular protein and RNA molecules and controlling translation.

Extracellular RNAs released by plant-associated fungi: from fundamental mechanisms to biotechnological applications.

Extracellular RNAs are an emerging research topic in fungal-plant interactions. Fungal plant pathogens and symbionts release small RNAs that enter host cells to manipulate plant physiology and immunity. This communication via extracellular RNAs between fungi and plants is bidirectional. On the one hand, plants release RNAs encapsulated inside extracellular vesicles as a defense response as well as for intercellular and inter-organismal communication. On the other hand, recent reports suggest that also full-length mRNAs are transported within fungal EVs into plants, and these fungal mRNAs might get translated inside host cells. In this review article, we summarize the current views and fundamental concepts of extracellular RNAs released by plant-associated fungi, and we discuss new strategies to apply extracellular RNAs in crop protection against fungal pathogens. KEY POINTS: • Extracellular RNAs are an emerging topic in plant-fungal communication. • Fungi utilize RNAs to manipulate host plants for colonization. • Extracellular RNAs can be engineered to protect plants against fungal pathogens.

Formation of a stable RNase Y-RicT (YaaT) complex requires RicA (YmcA) and RicF (YlbF).

In Bacillus subtilis, the RicT (YaaT), RicA (YmcA), and RicF (YlbF) proteins, which form a stable ternary complex, are needed together with RNase Y (Rny) to cleave and thereby stabilize several key transcripts encoding enzymes of intermediary metabolism. We show here that RicT, but not RicA or RicF, forms a stable complex with Rny and that this association requires the presence of RicA and RicF. We propose that RicT is handed off from the ternary complex to Rny. We show further that the two iron-sulfur clusters carried by the ternary Ric complex are required for the formation of the stable RicT-Rny complex. We demonstrate that proteins of the degradosome-like network of B. subtilis, which also interact with Rny, are dispensable for processing of the gapA operon. Thus, Rny participates in distinct RNA-related processes, determined by its binding partners, and a RicT-Rny complex is likely the functional entity for gapA mRNA maturation. IMPORTANCE The action of nucleases on RNA is universal and essential for all forms of life and includes processing steps that lead to the mature and functional forms of certain transcripts. In Bacillus subtilis, it has been shown that key transcripts for energy-producing steps of glycolysis, for nitrogen assimilation, and for oxidative phosphorylation, all of them crucial processes of intermediary metabolism, are cleaved at specific locations, resulting in mRNA stabilization. The proteins required for these cleavages in B. subtilis [Rny (RNase Y), RicA (YmcA), RicF (YlbF), and RicT (YaaT)] are broadly conserved among the firmicutes, including several important pathogens, hinting that regulatory mechanisms they control may also be conserved. Several aspects of these regulatory events have been explored: phenotypes associated with the absence of these proteins have been described, the impact of these absences on the transcriptome has been documented, and there has been significant exploration of the biochemistry and structural biology of Rny and the Ric proteins. The present study further advances our understanding of the association of Ric proteins and Rny and shows that a complex of Rny with RicT is probably the entity that carries out mRNA maturation.

C-C Motif Chemokine Receptor 3-Mediated Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Signaling: Promising Targets for Human Airway Epithelial Mucin 5AC Induction by Eotaxin-2 and Eotaxin-3.

INTRODUCTION: Eotaxin-2 and -3 of the C-C chemokine subfamily function as potent chemoattractant factors for eosinophil recruitment and various immune responses in allergic and inflammatory airway diseases. Mucin 5AC (MUC5AC), a major gel-forming secretory mucin, is overexpressed in airway inflammation. However, the association between mucin secretion and eotaxin-2/3 expression in the upper and lower airway epithelial cells has not been fully elucidated. Therefore, in this study, we investigated the effects of eotaxin-2/3 on MUC5AC expression and its potential signaling mediators. METHODS: We analyzed the effects of eotaxin-2 and -3 on NCI-H292 human airway epithelial cells and primary human nasal epithelial cells (HNEpCs) via reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. Along with immunoblot analyses with specific inhibitors and small interfering RNA (siRNA), we explored the signaling pathway involved in MUC5AC expression following eotaxin-2/3 treatment. RESULTS: In HCI-H292 cells, eotaxin-2/3 activated the mRNA expression and protein production of MUC5AC. A specific inhibitor of C-C motif chemokine receptor 3 (CCR3), SB328437, suppressed eotaxin-2/3-induced MUC5AC expression at both the mRNA and protein levels. Eotaxin-2/3 induced the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38, whereas pretreatment with a CCR3 inhibitor significantly attenuated this effect. Induction of MUC5AC expression with eotaxin-2/3 was decreased by U0126 and SB203580, specific inhibitors of ERK1/2 and p38 mitogen-activated protein kinase (MAPK), respectively. In addition, cell transfection with ERK1/2 and p38 siRNAs inhibited eotaxin-2/3-induced MUC5AC expression. Moreover, specific inhibitors (SB328437, U0126, and SB203580) attenuated eotaxin-2/3-induced MUC5AC expression in HNEpCs. CONCLUSION: Our results imply that CCR3-mediated ERK1/2 and p38 MAPK are involved in the signal transduction of eotaxin-2/3-induced MUC5AC overexpression.

Structural basis for high-order complex of SARNP and DDX39B to facilitate mRNP assembly.

mRNA in eukaryotic cells is packaged into highly compacted ribonucleoprotein particles (mRNPs) in the nucleus and exported to the cytoplasm for translation. mRNP packaging and export require the evolutionarily conserved transcription-export (TREX) complex. TREX facilitates loading of various RNA-binding proteins on mRNA through the action of its DDX39B subunit. SARNP (Tho1 [transcriptional defect of Hpr1 by overexpression 1] in yeast) is shown to interact with DDX39B and affect mRNA export. The molecular mechanism of how SARNP recognizes DDX39B and functions in mRNP assembly is unclear. Here, we determine the crystal structure of a Tho1/DDX39B/RNA complex, revealing a multivalent interaction mediated by tandem DDX39B interacting motifs in SARNP/Tho1. The high-order complex of SARNP and DDX39B is evolutionarily conserved, and human SARNP can engage with five DDX39B molecules. RNA sequencing (RNA-seq) from SARNP knockdown cells shows the most affected RNAs in export are GC rich. Our work suggests the role of the high-order SARNP/DDX39B/RNA complex in mRNP assembly and export.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate toxicity of the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine in human placental villous trophoblast BeWo cells.

Trichloroethylene (TCE) is a known human carcinogen with toxicity attributed to its metabolism. S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is a metabolite of TCE formed downstream in TCE glutathione (GSH) conjugation and is upstream of several toxic metabolites. Despite knowledge that DCVC stimulates reactive oxygen species (ROS) generation and apoptosis in placental cells, the extent to which these outcomes are attributable to DCVC metabolism is unknown. The current study used N-acetyl-L-cysteine (NAC) at 5 mM and aminooxyacetic acid (AOAA) at 1 mM as pharmacological modifiers of DCVC metabolism to investigate DCVC toxicity at concentrations of 5-50 µM in the human placental trophoblast BeWo cell model capable of forskolin-stimulated syncytialization. Exposures of unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells were studied. NAC pre/co-treatment with DCVC either failed to inhibit or exacerbated DCVC-induced H2O2 abundance, PRDX2 mRNA expression, and BCL2 mRNA expression. Although NAC increased mRNA expression of CYP3A4, which would be consistent with increased generation of the toxic metabolite N-acetyl-DCVC sulfoxide (NAcDCVCS), a CYP3A4 inhibitor ketoconazole did not significantly alter BeWo cell responses. Moreover, AOAA failed to inhibit cysteine conjugate ß-lyase (CCBL), which bioactivates DCVC, and did not affect the percentage of nuclei condensed or fragmented, a measure of apoptosis, in all BeWo cell models. However, syncytialized cells had higher CCBL activity compared to unsyncytialized cells, suggesting that the former may be more sensitive to DCVC toxicity. Together, although neither NAC nor AOAA mitigated DCVC toxicity, differences in CCBL activity and potentially CYP3A4 expression dictated the differential toxicity derived from DCVC.

Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation.

Cleavage and polyadenylation (CPA) is responsible for 3' end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets CPSF-73, is the first known CPA inhibitor (CPAi) in mammalian cells. Here we show that JTE-607 perturbs gene expression through both transcriptional readthrough and alternative polyadenylation (APA). Sensitive genes are associated with features similar to those previously identified for PCF11 knockdown, underscoring a unified transcriptomic signature of CPAi. The degree of inhibition of an APA site by JTE-607 correlates with its usage level and, consistently, cells with elevated CPA activities, such as those with induced overexpression of FIP1, display greater transcriptomic disturbances when treated with JTE-607. Moreover, JTE-607 causes S phase crisis and is hence synergistic with inhibitors of DNA damage repair pathways. Together, our data reveal CPA activity and proliferation rate as determinants of CPAi-mediated cell death, raising the possibility of using CPAi as an adjunct therapy to suppress certain cancers.

Comparison of Endoplasmic Reticulum Stress Messenger Ribonucleic Acid Expression Between Chronic Otitis Media With and Without Cholesteatoma.

BACKGROUND: We evaluated and compared the role of endoplasmic reticulum stress in chronic otitis media with cholesteatoma and chronic otitis media without cholesteatoma. METHODS: The messenger ribonucleic acid expression of endoplasmic reticulum stress was measured and compared between chronic otitis media with cholesteatoma and chronic otitis media without cholesteatoma according to the presence or absence of bacteria, type of hearing loss, ossicle destruction, and facial canal dehiscence. RESULTS: The expression of immunoglobulin heavy chain-binding protein messenger ribonucleic acid was higher in the chronic otitis media without cholesteatoma group than in the chronic otitis media with cholesteatoma group, and Protein kinase RNA (PKR)-like endoplasmic reticulum kinase and activating transcription factor 6 messenger ribonucleic acid expression were higher in the chronic otitis media with cholesteatoma group than in the chronic otitis media without cholesteatoma group. CONCLUSION: Endoplasmic reticulum stress messenger ribonucleic acids were expressed in both chronic otitis media with cholesteatoma and chronic otitis media without cholesteatoma. The levels of expression of endoplasmic reticulum stress messenger ribonucleic acids differed according to clinical features, suggesting that different endoplasmic reticulum stress pathways are involved in the pathophysiology of different types of chronic otitis media.

Translation initiation at AUG and non-AUG triplets in plants.

In plants and other eukaryotes, precise selection of translation initiation site (TIS) on mRNAs shapes the proteome in response to cellular events or environmental cues. The canonical translation of mRNAs initiates at a 5' proximal AUG codon in a favorable context. However, the coding and non-coding regions of plant genomes contain numerous unannotated alternative AUG and non-AUG TISs. Determining how and why these unexpected and prevalent TISs are activated in plants has emerged as an exciting research area. In this review, we focus on the selection of plant TISs and highlight studies that revealed previously unannotated TISs used in vivo via comparative genomics and genome-wide profiling of ribosome positioning and protein N-terminal ends. The biological signatures of non-AUG TIS-initiated open reading frames (ORFs) in plants are also discussed. We describe what is understood about cis-regulatory RNA elements and trans-acting eukaryotic initiation factors (eIFs) in the site selection for translation initiation by featuring the findings in plants along with supporting findings in non-plant species. The prevalent, unannotated TISs provide a hidden reservoir of ORFs that likely help reshape plant proteomes in response to developmental or environmental cues. These findings underscore the importance of understanding the mechanistic basis of TIS selection to functionally annotate plant genomes, especially for crops with large genomes.

FBN1 knockout promotes cervical artery dissection by inducing N-glycosylation alternation of extracellular matrix proteins in rat VSMCs.

FBN1 mutation promotes the degeneration of microfibril structures and extracellular matrix (ECM) integrity in the tunica media of the aorta in Marfan syndrome. However, whether FBN1 modulates cervical artery dissection (CAD) development and the potential molecular mechanisms of abnormal FBN1 in CAD remains elusive. In this study, FBN1 deficiency participated in the development of CAD and influenced the proliferation, apoptosis, and migration of vascular smooth muscle cells. FBN1 knockout induced alternations in mRNA levels of the transcriptome, protein expression of the proteome, and abundance of N-glycosylation of the N-glycoproteome. Comprehensive analysis of multiple omics showed up-regulation in mRNA levels of ECM proteins; yet, both the ECM protein levels and relative abundance of N-glycosylation were decreased. Moreover, we performed in vivo experiments to confirm the altered glycosylation of proteins in vascular smooth muscle cells. In conclusion, FBN1 deletion in vascular smooth muscle cells can result in altered N-glycosylation of ECM protein, which were critical for the stability of ECM and the process of CAD. This may open the way for a novel therapeutic strategy to treat people with CAD.

Global gene expression analysis reveals a subtle effect of DEHP in human granulosa cell line HGrC1.

Di(2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor that exerts anti-steroidogenic effects in human granulosa cells; however, the extent of this effect depends on the concentration of DEHP and granulosa cell models used for exposure. The objective of this study was to identify the effects of low- and high-dose DEHP exposure in human granulosa cells. We exposed human granulosa cell line HGrC1 to 3 nM and 25 µM DEHP for 48 h. The whole genome transcriptome was analyzed using the DNBSEQ sequencing platform and bioinformatics tools. The results revealed that 3 nM DEHP did not affect global gene expression, whereas 25 µM DEHP affected the expression of only nine genes in HGrC1 cells: ABCA1, SREBF1, MYLIP, TUBB3, CENPT, NUPR1, ASS1, PCK2, and CTSD. We confirmed the downregulation of ABCA1 mRNA and SREBP-1 protein (encoded by the SREBF1 gene), both involved in cholesterol homeostasis. Despite these changes, progesterone production remained unaffected in low- and high-dose DEHP-exposed HGrC1 cells. The high concentration of DEHP decreased the levels of ABC1A mRNA and SREBP-1 protein and prevented the upregulation of STAR, a protein involved in progesterone synthesis, in forskolin-stimulated HGrC1 cells; however, the observed changes were not sufficient to alter progesterone production in forskolin-stimulated HGrC1 cells. Overall, this study suggests that acute exposure to low concentration of DEHP does not compromise the function of HGrC1 cells, whereas high concentration causes only subtle effects. The identified nine novel targets of high-dose DEHP require further investigation to determine their role and importance in DEHP-exposed human granulosa cells.

HET0016 inhibits neuronal pyroptosis in the immature brain post-TBI via the p38 MAPK signaling pathway.

Traumatic brain injury (TBI) is a serious health threat worldwide, especially for the younger demographic. Our previous study demonstrated that HET0016 (a specific inhibitor of 20-hydroxyeicosatetraenoic acid synthesis) can decrease the lesion volume in the immature brain post-TBI; however, its mechanism of action and its association with pyroptosis post-TBI are unclear. In this study, we established a controlled cortical impact (CCI) injury rat model (postnatal day 9-10) and observed that increased expression of indicators for pyroptosis, including NLR family pyrin domain containing 3 (NLRP3), caspase-1 and gasdermin D (GSDMD) proteins and interleukin (IL)-18/IL-1ß mRNA during the acute phase of TBI, especially on post-injury day (PID) 1. Additionally, we found that caspase-1 was primarily expressed in the neurons and microglia. HET0016 (1 mg/kg/d, ip, 3 consecutive days since TBI) reduced the lesion volume; neuronal death; expression of NLRP3, caspase-1, and GSDMD; and expression of IL-18/IL-1ß mRNA. Bioinformatics analysis suggested involvement of mitogen-activated protein kinase (MAPK) signaling pathway in the HET0016-mediated neuroprotective role against TBI in the immature brain. Western blot analysis revealed reduced expression of p-p38 MAPK and nuclear factor-kappa B (NF-κB) p65 in the neurons and microglia upon HET0016 treatment in TBI rats. In cultured primary cortical neurons subjected to oxygen-glucose deprivation/re-oxygenation (OGD) + (lipopolysaccharide) LPS, HET0016-induced the reduction of p-p38 MAPK, NLRP3, cleaved-caspase-1, GSDMD, IL-18, and IL-1ß was reversed by co-treatment with p38 MAPK activator as well as NLRP3 agonist. Therefore, we conclude that pyroptosis is involved in neuronal death in the immature brains post-TBI and that HET0016 administration can alleviate neuronal pyroptosis possibly via inhibiting the phosphorylation of p38 MAPK.

Oxidative stress and activation of H2A.X in lung alveolar epithelial cells (A549) by nanoparticulate carbon black.

Fine airborne particulate matter enter the respiratory system, induce oxidative stress and initiate DNA damage. The aim of our study was the estimation of cell viability, oxidative stress, DNA damage, cell cycle alterations and activation of histone H2A.X. Experiments were done on lung alveolar epithelial (A549) cells grown for 24 h with 200 µg mL-1 coarse carbon black (CB), or nanoparticulate CB (NPCB). Neither CB nor glutathione depletion altered cell viability, growth rates, and H2A.X expression while NPCB decreased cell viability, increased oxidative stress and DNA damage. The cell cycle was blocked at G0/G1. NPCB but not CB increased expression and activation of H2A.X at mRNA and protein levels. Co-expression data point to γH2A.X as a major NPCB target, and show the interdependence of γH2A.X and oxidative stress. We conclude, that NPCB increases γ-H2A.X expression in A549 cells at mRNA and protein levels and stimulates H2A.X (Ser139), phosphorylation, associated with oxidative stress, the DNA damage response and G1 cell cycle arrest.

lncRNA CLRN1-AS1 reduces adhesion ability of human trophoblasts via CXCL10/CXCL11.

INTRODUCTION: Trophoblast cells play an important role in embryo recognition and localization, as well as placental development during embryo implantation. Dysfunction of trophoblastic cells causes pathological changes that lead to insufficient recognition, positioning, and adhesion during embryo implantation, ultimately leading to embryo development has stopped. METHODS: High-throughput sequencing was used to identify differentially expressed the mRNA and lncRNA in the villi tissue of pregnant women diagnosed with embryo cessation. In vitro implantation cell models, characteristic analysis, and bio information analysis confirmed that CLRN1-AS1 affected the adhesion function of trophoblast cells by influencing the chemokines CXCL10/CXCL11. RESULTS: High throughput sequencing technology was used to identify 438 differentially expressed mRNAs and 41 lncRNAs. The three lncRNAs, namely CLRN1-AS1, USP27X-AS1, and AC104809.4, were screened by the mRNA-lncRNA network. In vitro implantation model suggested that all three lncRNAs could affect the adhesion between trophoblast cells, among which CLRN1-AS1 had the most significant effect. Characteristic analysis and correlation analysis showed that CLRN1-AS1 was closely related to the expression of six adhesion-related genes, LAMA1, FGL2, ITGB2, FBN1, EMP2, and PODN. Cell experiments and re-sequencing confirmed that CLRN1-AS1 could affect the adhesion ability of trophoblast cells to the extracellular matrix, and its process was related to the chemokine CXCL10/CXCL11. DISCUSSION: These results constructed the network of mRNA-lncRNA and enrichment when embryonic development has stopped and found CLRN1-AS1 highly correlated to failure of embryo implantation, and revealed that CLRN1-AS1 modulates the adhesion ability of trophoblast cells to the extracellular matrix via the chemokines CXCL10/CXCL11 during the early stage of embryo implantation.

Cell surface associated protein mucin 15 (MUC15) is elevated in preeclampsia.

BACKGROUND: Mucins are a family of proteins that protect the epithelium. A particular type of mucin, MUC15 is highly expressed in the placenta. This study aimed to characterise MUC15 in preeclampsia and investigate its role in placental stem cell biology. METHODS: MUC15 mRNA and protein were measured in placentas from patients with early onset (<34 weeks' gestation) preeclampsia. Circulating serum MUC15 was measured via ELISA. MUC15 was localised in the placenta using in situ hybridisation. MUC15 mRNA expression was measured across differentiation of human trophoblast stem cells (hTSCs) to syncytiotrophoblast and extravillous trophoblasts. MUC15 was measured after syncytialised hTSCs were cultured in hypoxic (1% O2) and proinflammatory (TNF α, IL-6) conditions. MUC15 secretion was assessed when syncytialised hTSCs were treated with brefeldin A (impairs protein trafficking) and batimastat (inhibits matrix metalloproteinases). RESULTS: MUC15 protein was significantly increased in the placenta (P = 0.0003, n = 32 vs n = 20 controls) and serum (P = 0.016, n = 32 vs n = 22 controls) of patients with preeclampsia, whilst MUC15 mRNA remained unchanged (n = 61 vs n = 18 controls). MUC15 mRNA (P = 0.005) and protein secretion (P = 0.006) increased following differentiation to syncytiotrophoblast cells. In situ hybridisation confirmed MUC15 localised to the syncytiotrophoblast cell within the placenta. Neither hypoxic or inflammatory conditions changed MUC15 mRNA expression or secretion. Brefeldin A treated hTSCs did not alter MUC15 secretion, whilst batimastat reduced MUC15 secretion (P = 0.044). CONCLUSIONS: MUC15 is increased in early onset preeclampsia and is cleaved by matrix metalloproteinases. Increased MUC15 may reflect a protective mechanism associated with placental dysfunction. Further research will aid in confirming this.

Towards in silico CLIP-seq: predicting protein-RNA interaction via sequence-to-signal learning.

We present RBPNet, a novel deep learning method, which predicts CLIP-seq crosslink count distribution from RNA sequence at single-nucleotide resolution. By training on up to a million regions, RBPNet achieves high generalization on eCLIP, iCLIP and miCLIP assays, outperforming state-of-the-art classifiers. RBPNet performs bias correction by modeling the raw signal as a mixture of the protein-specific and background signal. Through model interrogation via Integrated Gradients, RBPNet identifies predictive sub-sequences that correspond to known and novel binding motifs and enables variant-impact scoring via in silico mutagenesis. Together, RBPNet improves imputation of protein-RNA interactions, as well as mechanistic interpretation of predictions.