DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long noncoding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo disease models. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, which indicates mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease.

Elevated miR-200a and miR-141 inhibit endocrine gland-derived vascular endothelial growth factor expression and ciliogenesis in preeclampsia.

KEY POINTS: Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is a critical factor that facilitates trophoblast invasion in placenta. Plasma miR-141 and miR-200a levels were elevated, while EG-VEGF was decreased in peripheral blood and placenta of preeclamptic patients. Furthermore, numbers of cilia in the placenta from preeclamptic women were significantly decreased. Elevated miR-141 and miR-200a inhibited the expression of EG-VEGF, downstream extracellular signal-regulated kinase (ERK)/matrix metalloproteinase 9 signalling and cilia formation, thus leading to defective trophoblast invasion. The growth of the primary cilium, which transduced ERK signalling upon EG-VEGF induction for proper trophoblast invasion, was also inhibited by miR-141 and miR-200a upregulation. ABSTRACT: Preeclampsia is a severe gestational complication, and inadequate trophoblast invasion during placental development is an important pathoaetiology. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is a critical factor that facilitates trophoblast invasion in placenta. By binding to the primary cilium, EG-VEGF initiates the signalling cascade for proper embryo implantation and placental development. The miR-200 family was predicted to target the EG-VEGF 5'-untranslated region, and its specific binding site was confirmed using a dual luciferase and a co-transfection assay. In the peripheral blood and placenta of preeclamptic patients, EG-VEGF showed significantly lower expression, whereas plasma miR-141 and miR-200a had higher expression compared with the controls. The biological significance of miR-141 and miR-200a was verified using an overexpression method in a trophoblast cell line (HTR-8/SVneo). Elevated miR-141 and miR-200a inhibited the expression of EG-VEGF, matrix metalloproteinase 9 (MMP9) and downstream extracellular signal-regulated kinase (ERK) signalling, thus leading to defective trophoblast invasion. Additionally, the growth of the primary cilium, which transduces ERK/MMP9 signalling upon EG-VEGF induction, was inhibited by miR-141 and miR-200a upregulation. Furthermore, the number of cilia in the human placenta of preeclamptic women was significantly decreased compared to normal placenta. In conclusion, the study uncovers the clinical correlations among the miR-200 family, EG-VEGF and the primary cilium in preeclampsia and the underlying molecular mechanisms. The results indicate that miR-141 and miR-200a directly targeted EG-VEGF, suppressed primary cilia formation and inhibited trophoblast invasion. Thus, miR-141 and miR-200a could be explored as promising miRNA biomarkers and therapeutic targets in preeclampsia.

NLRP7 Is Involved in the Differentiation of the Decidual Macrophages.

Macrophage polarization, regulated appropriately, may play important roles in successful pregnancy. In the face of the vital roles of decidua macrophages in pregnancy, it is insufficient to recognize the trigger of macrophage differentiation and polarization. We aimed to explore the link between the NLRP7 gene and macrophage polarization in human deciduas. Here, we enrolled the endometrial tissues from eight pregnant women in the first trimester. We found that NLRP7 was abundant in endometrial tissues and that NLRP7 was expressed in decidual macrophages of the first-trimester pregnancy. NLRP7 was predominately expressed in the decidual M2 macrophages, as compared with the M1 macrophages. Furthermore, our results suggest that NLRP7 is associated with decidual macrophage differentiation. NLRP7 over-expression suppresses the expression of M1 markers and enhances the expression of the M2 markers. Considering that NLRP7 relates to decidualization and macrophage differentiation, we propose that NLRP7 is a primate-specific multitasking gene to maintain endometrial hemostasis and reproductive success. This finding may pave the way for therapies of pathological pregnancies.

Fetuin-A Inhibits Placental Cell Growth and Ciliogenesis in Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a type of unbalanced glucose tolerance that occurs during pregnancy, which affects approximately 10% of pregnancies worldwide. Fetuin-A is associated with insulin resistance, and the concentration of circulating fetuin-A increases in women with GDM, however, the role of fetuin-A in the placenta remains unclear. In this study, we enrolled placental samples from twenty pregnant women with GDM and twenty non-GDM pregnant women and found that the abundance of fetuin-A was upregulated in terms of mRNA and protein levels. Fetuin-A inhibited placental cell growth by inducing apoptosis and inhibiting S phase entry. Irregular alignment of mitotic chromosomes and aberrant mitotic spindle poles were observed. In addition, centrosome amplification was induced by fetuin-A treatment, and these amplified centrosomes nucleated microtubules with disorganized microtubule arrays in placental cells. Furthermore, fetuin-A inhibited autophagy, and thus blocked the growth of the primary cilium, a cellular antenna that regulates placenta development and differentiation. Thus, our study uncovered the novel function of fetuin-A in regulating placental cell growth and ciliogenesis.

Differential miR-346 and miR-582-3p Expression in Association with Selected Maternal and Fetal Complications.

Several miRNAs are expressed in human gestational tissue, and some have been shown to be associated with placental dysfunction and complicated pregnancy outcomes. To investigate the roles of miR-346 and miR-582-3p in adverse obstetric events, we analyzed these 2 miRNAs in three samples (maternal blood, umbilical cord blood and placenta) obtained from pregnant women in four groups, including healthy control (n = 60), preeclampsia (n = 31), preterm delivery (n = 29) and small for gestational age (n = 19) patients. The expression levels of miR-346 and miR-582-3p in all included adverse obstetric outcome groups were significantly higher in the maternal plasma samples but lower in the placenta samples (all p value < 0.05). In addition, the miR-346 expression levels in fetal cord blood were also significantly lower in all of the included adverse obstetric outcome groups (all p < 0.05). Multivariate analysis of the three specimens after adjusting for maternal age and gestational age at delivery gave the same results. In conclusion, aberrant miR-346 and miR-582-3p expression level in pregnancy was associated with multiple maternal and fetal complications. Their differential expression in maternal blood, umbilical cord blood and placenta could be potential biomarkers or therapeutic targets for adverse obstetric outcomes.

Aspirin alleviates fibronectin-induced preeclampsia phenotypes in a mouse model and reverses fibronectin-mediated trophoblast invasiveness under hypoxia by regulating ciliogenesis and Akt and MAPK signaling.

The placenta experiences a low-oxygen stage during early pregnancy. Aspirin is an effective preventative treatment for preeclampsia if applied early in pregnancy. Elevation of fibronectin (FN) level has been reported to be associated with preeclampsia; however, the role of FN in the physiological hypoxic phase and whether aspirin exerts its effect on FN at this hypoxic stage remain unknown. We determined pregnancy outcomes by injecting saline or recombinant FN protein into C57BL/6 pregnant mice and one group of FN-injected mice was fed aspirin. The effects of FN, the underlying pathways on trophoblast biology, and cilia formation under hypoxia were investigated in FN-pretreated or FN-knockdown HTR-8/SVneo cells in a hypoxic chamber (0.1 % O2). Preeclampsia-like phenotypes, including blood pressure elevation and proteinuria, developed in FN-injected pregnant mice. The fetal weight of FN-injected mice was significantly lower than that of non-FN-injected mice (p < 0.005). Trophoblast FN expression was upregulated under hypoxia, which could be suppressed by aspirin treatment. FN inhibited trophoblast invasion and migration under hypoxia, and this inhibitory effect occurred through downregulating ZEB1/2, MMP 9 and the Akt and MAPK signaling pathways. Ciliogenesis of trophoblasts was stimulated under hypoxia but was inhibited by FN treatment. Aspirin was shown to reverse the FN-mediated inhibitory effect on trophoblast invasion/migration and ciliogenesis. In conclusion, FN overexpression induces preeclampsia-like symptoms and impairs fetal growth in mice. Aspirin may exert its suppressive effect on FN upregulation and FN-mediated cell function in the hypoxic stage of pregnancy and therefore provides a preventative effect on preeclampsia development.

Impact of Antenatal SARS-CoV-2 Exposure on SARS-CoV-2 Neutralization Potency.

A pregnancy booster dose significantly reduces the risk and severity of COVID-19, and it is widely recommended. A prospective cohort study was conducted to compare the transplacental passage of maternal antibodies from vaccination or infection during three trimesters against both the vaccine-targeted Wuhan strain and the Omicron strain of SARS-CoV-2. Maternal-infant dyads from vaccinated mothers were collected between 6 June 2022 and 20 September 2022. We analyzed 38 maternal-infant dyads from mothers who had been infected with COVID-19 and 37 from mothers without any previous infection. Pregnant women who received their last COVID-19 vaccine dose in the third trimester exhibited the highest anti-spike protein antibody levels and neutralizing potency against both the Wuhan strain and Omicron BA.2 variant in their maternal and cord plasma. Both second- and third-trimester vaccination could lead to a higher level of neutralization against the Wuhan and Omicron strains. COVID-19 infection had a negative effect on the transplacental transfer ratio of SARS-CoV-2 antibodies. A booster dose during the second or third trimester is encouraged for the maximum transplacental transfer of humoral protection against COVID-19 for infants.

Upregulation of miR-20b-5p inhibits trophoblast invasion by blocking autophagy in recurrent miscarriage.

Recurrent miscarriage is defined as more than three pregnancy failures occurring before 20 weeks of gestation. Poor differentiation of the endometrial stroma or defective trophoblast cell invasion at the maternal-fetal interface leads to recurrent miscarriages. Several miRNAs, including miR-20b-5p, are aberrantly regulated in recurrent miscarriages; however, the underlying molecular mechanisms remain unclear. Primary cilia are antenna-like organelles that coordinate signaling during development and differentiation. Defective primary cilia formation leads to complications, such as recurrent miscarriage or preeclampsia. Here, we demonstrated that miR-20b-5p inhibited trophoblast cell invasion by blocking primary cilia formation. Mechanistically, miR-20b-5p targeted and inhibited ATG16L1 and ATG7 expression, thereby blocking autophagy. Defective autophagy reduced primary cilia formation and stopped ERK activation, which is a crucial signaling pathway for trophoblast invasion. Aspirin is used to prevent recurrent miscarriages in clinical settings. Treatment with aspirin inhibited miR-20b-5p levels, thus restoring primary cilia formation and trophoblast invasion. Thus, our findings uncovered the molecular mechanism by which miR-20b-5p suppressed primary cilia formation and trophoblast invasion by reducing the expression of ATG16L1 and ATG7. Moreover, we found that the defective phenotypes could be rescued by aspirin in recurrent miscarriages.

Nipsnap1-A regulatory factor required for long-term maintenance of non-shivering thermogenesis.

OBJECTIVE: The activation of non-shivering thermogenesis (NST) has strong potential to combat obesity and metabolic disease. The activation of NST however is extremely temporal and the mechanisms surrounding how the benefits of NST are sustained once fully activated, remain unexplored. The objective of this study is to investigate the role of 4-Nitrophenylphosphatase Domain and Non-Neuronal SNAP25-Like 1 (Nipsnap1) in NST maintenance, which is a critical regulator identified in this study. METHODS: The expression of Nipsnap1 was profiled by immunoblotting and RT-qPCR. We generated Nipsnap1 knockout mice (N1-KO) and investigated the function of Nipsnap1 in NST maintenance and whole-body metabolism using whole body respirometry analyses. We evaluate the metabolic regulatory role of Nipsnap1 using cellular and mitochondrial respiration assay. RESULTS: Here, we show Nipsnap1 as a critical regulator of long-term thermogenic maintenance in brown adipose tissue (BAT). Nipsnap1 localizes to the mitochondrial matrix and increases its transcript and protein levels in response to both chronic cold and ß3 adrenergic signaling. We demonstrated that these mice are unable to sustain activated energy expenditure and have significantly lower body temperature in the face of an extended cold challenge. Furthermore, when mice are exposed to the pharmacological ß3 agonist CL 316, 243, the N1-KO mice exhibit significant hyperphagia and altered energy balance. Mechanistically, we demonstrate that Nipsnap1 integrates with lipid metabolism and BAT-specific ablation of Nipsnap1 leads to severe defects in beta-oxidation capacity when exposed to a cold environmental challenge. CONCLUSION: Our findings identify Nipsnap1 as a potent regulator of long-term NST maintenance in BAT.

Clinical outcomes of nirmatrelvir-ritonavir use in pregnant women during the Omicron wave of the coronavirus disease 2019 pandemic.

BACKGROUND: Paxlovid is an oral drug composed of nirmatrelvir and ritonavir that has been demonstrated to be effective in decreasing the risk of severe coronavirus disease 2019 (COVID-19). Here, we report the use of paxlovid in pregnant women with COVID-19. METHODS: Pregnant women attending a tertiary referral hospital in Taiwan from 29 April to 30 July 2022 were enrolled in the study. We compared baseline characteristics, clinical manifestations, and adverse events between paxlovid-treated women and those without paxlovid use. Maternal and neonatal outcomes were analysed in women who delivered during the study period. RESULTS: A total of 30 paxlovid-treated pregnant women and 55 women without paxlovid use were included in the analysis. The mean duration of COVID-19-associated symptoms in the paxlovid-treated women was shorter than that in the control group (10.10 days versus 15.59 days, p = 0.04). No severe adverse events due to paxlovid use were observed. Dysgeusia and diarrhoea were the most common adverse effects. Thirteen paxlovid-treated and 28 untreated women delivered during the study period. More pregnant women in the paxlovid group who delivered during the study period underwent caesarean delivery compared to the group without antiviral treatment (10 of 13 [76.92%] versus 12 of 28 [42.86%], p = 0.042), and insignificantly more newborns were born small for gestational age in the paxlovid group compared to the control group (3 of 13 [23.08%] versus 1 of 28 [3.57%], p = 0.086). CONCLUSION: Our study showed that paxlovid was effective and safe for pregnant women during the Omicron wave of the COVID-19 pandemic. A higher proportion of caesarean delivery rates was observed among paxlovid-treated women. Long-term follow-up of pregnant women exposed to paxlovid and their offspring is needed.

Ado-Mediated Depletion of Taurine Impairs Mitochondrial Respiratory Capacity and Alters the Chromatin Landscape of Inguinal Adipose Tissue.

Non-shivering thermogenesis (NST) has strong potential to combat obesity; however, a safe molecular approach to activate this process has not yet been identified. The sulfur amino acid taurine has the ability to safely activate NST and confer protection against obesity and metabolic disease in both mice and humans, but the mechanism of this action is unknown. In this study, we discover that a suite of taurine biosynthetic enzymes, especially that of cysteamine dioxygenase (ADO), significantly increases in response to ß3 adrenergic signaling in inguinal adipose tissue (IWAT) in order to increase intracellular concentrations of taurine. We further show that ADO is critical for thermogenic mitochondrial respiratory function as its ablation in adipocytes significantly reduces taurine levels, which leads to declines in mitochondrial oxygen consumption rates. Finally, we demonstrate via assay for transposase-accessible chromatin with sequencing (ATAC-seq) that taurine supplementation in beige adipocytes has the ability to remodel the chromatin landscape to increase the chromatin accessibility and transcription of genes, such as glucose-6-phosphate isomerase 1 (Gpi1), which are critical for NST. Taken together, our studies highlight a potential mechanism for taurine in the activation of NST that can be leveraged toward the treatment of obesity and metabolic disease.

Setting up a specialized maternity unit in a tertiary hospital: An oasis for pregnant women with COVID-19 during the pandemic.

OBJECTIVE: The COVID-19 pandemic has had an enormous impact on society and the medical environment in Taiwan in 2022. As pregnant women with COVID-19 are at higher risk for multiple complications, Taiwan needs a COVID-19 specialized maternity unit to improve the quality of maternal and neonatal care. MATERIALS AND METHODS: We share our experience with specialized maternity unit for pregnant women with COVID-19 at the National Cheng Kung University Hospital, where we can have careful evaluation, safe birth, and comprehensive postpartum care. RESULTS: Our COVID-19 specialized maternity unit enrolled 253 pregnant women with COVID-19, 90 (35.6%) pregnant women were admitted to the specialized maternity unit, and 71 (28.1%) pregnant women gave birth during hospitalization in two months. All pregnant women recovery well and real-time polymerase chain reaction tests on all infants were negative for COVID-19. CONCLUSION: A specialized maternity unit can provide pregnant women with a safe birth environment, immediate maternity care, and high medical quality. It can also help health workers in non-specialized maternity units deal with COVID-19-related psychological stress. Therefore, setting up one specialized maternity unit in the city during the pandemic should be guardedly considered.

Platelet-derived growth factor-AA promotes placental choriocarcinoma JAR cell proliferation via primary cilia.

OBJECTIVE: During early pregnancy, the proliferation placental cells is crucial for proper implantation and formation of maternal-fetal circulation. Platelet-derived growth factor-AA (PDGF-AA) has been detected in placenta during early pregnancy; however, the role of PDGF-AA in placental cell growth has not been studied extensively. Primary cilium, a centrosome-based cellular protrusion, is an signaling hub for regulating development and differentiation. Importantly, the receptor of PDGF-AA (Pdgfr-α) is detected in the primary cilium and primary cilia-mediated PDGF-AA signaling regulates development and differentiation. Here we would like to investigate whether PDGF-AA regulates placental cell growth and whether primary cilia play roles in this process. MATERIALS AND METHODS: Human placental choriocarcinoma JAR cells were treated with PDGF-AA followed by examining cell growth. Primary cilia and subcellular localization of Pdgfr-α were observed by immunofluorescence staining. Manipulation of primary cilia was performed by treating cells with roscovitine or by transfecting cells with siRNA against IFT88. RESULTS: Here we showed that PDGF-AA induced JAR cell proliferation. In addition, JAR cells grew primary cilia where Pdgfr-α was detected. More importantly, pharmacological inhibition of primary cilia formation or depletion of cilia-related gene, IFT88, alleviated PDGF-AA induced JAR cell proliferation. CONCLUSION: Thus, our study show that PDGF-AA facilitates human placental choriocarcinomaJARcell growth via primary cilia.

Aspirin Inhibits Fibronectin Expression and Reverses Fibronectin-Mediated Cell Invasiveness by Activating Akt Signaling in Preeclampsia.

Preeclampsia is a severe gestational hypertensive disorder that may lead to maternal multiple organ dysfunction and adverse fetal outcomes. Aspirin provides a protective effect by reducing the risk of preeclampsia; however, its mechanism of action is unclear. Fibronectin (FN) is a key factor in cell motility and is associated with preeclampsia. Here, we demonstrated that cellular FN expression was elevated in the placenta of preeclamptic patients. The functional roles of plasma and cellular FN in trophoblasts were investigated by treating HTR-8/SVneo cells with exogenous recombinant human FN protein (rhFN) and siRNA, respectively. Trophoblast migration and invasion were inhibited by rhFN and facilitated by FN knockdown. Moreover, rhFN activated ERK and Akt signaling in trophoblasts, and FN-suppressed cell motility was rescued by ERK and/or Akt inhibitors. In this study, aspirin suppressed trophoblast cellular FN expression and reversed FN-mediated cell functions, including cell migration, invasion, and ERK/Akt signal changes. Taken together, the results of this study revealed the effects of FN on trophoblast motility and signaling; aspirin inhibits FN expression and reverses FN-mediated trophoblast biology. These results provide a drug mechanism for disease prevention and a target for preeclampsia intervention.

Soft tissue volume of upper arm in predicting small-for-gestational-age fetuses using three-dimensional ultrasound.

PURPOSE: To assess the value of fetal soft tissue volume (STV) of the upper arm in predicting small-for-gestational-age (SGA) fetuses using three-dimensional (3D) ultrasound (US). METHODS: We used 3D US to test the accuracy of fetal STV of the upper arm measurement in predicting SGA in a prospective cross-sectional study. RESULTS: Fetal STV of the upper arm assessed by 3D US can differentiate SGA fetuses from appropriate-for-gestational-age (AGA) fetuses. Using the 5th percentile as the cutoff, the sensitivity of fetal upper arm STV in predicting SGA fetuses was 84.1%, specificity, 93.4%, positive predictive value, 71.1%, negative predictive value, 96.8%, and overall accuracy, 91.9%. In addition, the diagnostic accuracy of fetal arm STV was better than that of the biparietal diameter, abdominal circumference, and femur length. CONCLUSION: Fetal STV of upper arm assessment by 3D US is a novel method to predict SGA fetuses.

Aspirin facilitates trophoblast invasion and epithelial-mesenchymal transition by regulating the miR-200-ZEB1 axis in preeclampsia.

Preeclampsia is a severe gestational hypertensive disorder that occurs after 20 weeks' of gestation. It involves several maternal systems, such as cardiovascular, renal, coagulatory systems, and poses a major threat to the maternal and fetal health. Recent clinical evidence showed that aspirin is an effective preventative treatment for reducing the incidence of premature preeclampsia among high-risk pregnant women, however, the mechanism of drug action is not clear. miR-200 family has been shown to be associated with preeclampsia and upregulated in the plasma and placenta of preeclamptic patients. Here we revealed that miR-200 family inhibited trophoblast invasion and epithelial-mesenchymal transition (EMT) process by stimulating epithelial marker expression (E-cadherin and ZO-1) and repressing mesenchymal marker expression (ZEB1 and TGFß1). Similarly, EMT markers in the placenta of preeclamptic patients showed higher E-cadherin and lower ZEB1 and TGF-ß1 protein expression. Moreover, aspirin was shown to suppress miR-200 family and these miR-200 family-mediated cell functions, including cell invasion and EMT changes, were completely reversed. In conclusion, this study demonstrates the effect of miR-200 family on trophoblast invasion and EMT. For the first time, aspirin was shown to fully reverse miR-200-mediated trophoblast biology and act through the network signaling of TGF-ß1/ZEB1/miR-200. These results provide a plausible mechanism explaining aspirin's effect on preeclampsia prevention and a therapeutic target for disease intervention.

Insight into the strong inhibitory action of salt on activity of neocarzinostatin.

Enediyne anticancer drugs belong to one of the most potent category in inducing DNA damage. We report 85+/-5% inhibition on activity of neocarzinostatin by salt. As high sodium ion concentration is a known tumor cell feature, we explored the dynamic mechanism of inhibition. Using various analytical tools, we examined parameters involved in the four consecutive steps of the drug action, namely, drug releasing from carrier protein, drug-DNA binding, drug activating, and DNA damaging. Neither protein stability, nor drug release rate, was altered by salt. The salt inhibition level was similar in between the protein-bound and unbound enediyne chromophore. Salt did not quench the thiol-induced drug activation. The inhibition was independent of DNA lesion types and irrelevant with thiol structures. Collectively, no salt interaction was found in the releasing, activating, and DNA damaging step of the drug action. However, binding with DNA decreased linearly with salt and corresponded well with the salt-induced inhibition on the drug activity. Salt interference on the affinity of DNA binding was the main and sole cause of the severe salt inhibition. The inhibition factor should be carefully considered for all agents with similar DNA binding mode.