Discovery of Aurovertin B as a Potent Metastasis Inhibitor against Triple-Negative Breast Cancer: Elucidating the Complex Role of the ATF3-DUSP1 Axis.

Triple-negative breast cancer (TNBC) is characterized by high mortality rates primarily due to its propensity for metastasis. Addressing this challenge necessitates the development of effective antimetastatic therapies. This study aimed to identify natural compounds with potential antimetastatic properties mainly based on the high-throughput phenotypic screening system. This system, utilizing luciferase reporter gene assays combined with scratch wound assays, evaluates compounds based on their influence on the epithelial-mesenchymal transition (EMT) marker E-cadherin. Through this approach, aurovertin B (AVB) was revealed to have significant antimetastatic capability. Notably, AVB exhibited substantial metastasis suppression in many TNBC cell lines, including MDA-MB-231, HCC1937 and 4T1. Also, its remarkable antimetastatic activity was demonstrated in vivo via the orthotopic breast cancer mouse model. Further exploration revealed a pronounced association between AVB-induced upregulation of DUSP1 (dual-specificity phosphatase 1) and its inhibitory effect on TNBC metastasis. Additionally, microarray analysis conducted to elucidate the underlying mechanism of the AVB-DUSP1 interaction identified ATF3 (activating transcription factor 3) as a critical transcription factor instrumental in DUSP1 transcriptional activation. This discovery, coupled with observations of enhanced ATF3-DUSP1 expression and consequent reduction in TNBC metastatic foci in response to AVB, provides novel insights into the molecular mechanisms driving metastasis in TNBC. Significance Statement We construct a high-throughput phenotypic screening system utilizing EMT marker E-cadherin promoter luciferase reporter gene combined with scratch wound assays. Aurovertin B was revealed to possess significant antimetastatic activity through this approach, which was further demonstrated via in vivo and in vitro experiments. The discovery of the regulatory role of the ATF3-DUSP1 pathway enriches our understanding of TNBC metastasis mechanism and suggests the potential of ATF3 and DUSP1 as biomarkers for diagnosing TNBC metastasis.

A new method to estimate the histological stage of primary biliary cholangitis.

OBJECTIVE: To analyze the diagnostic efficacy of the periportal hypoechoic band (PHB) in the histological stage of patients with primary biliary cholangitis (PBC). METHODS: We prospectively included 77 cases of PBC pathologically or clinically confirmed, and high-frequency ultrasound (HFUS) measurements of the PHB were performed in all included patients. Ludwig staging system of histopathology was used as the gold standard. RESULTS: The width of the PHB was positively correlated with histological staging (r = 0.844, p < 0.001). By area under the receiving operating characteristic curve (AUROC), the best cutoff value for PHB for advanced stage (≥ stage 3) was 2.4 mm (AUROC: 0.934; 95%CI: 0.841-0.981) and 0.93 for sensitivity, and 0.91 for specificity, the concordance rates of PHB vs. liver biopsy was 90.3%. The correct rate for early-stage PBC was 87.9% and for the progressive stage was 93.1%. After multi-factor regression analysis, the PHB (OR = 1.331, CI = 1.105-1.603, p = 0.003) and total bilirubin (OR = 1.156, CI = 1.041-1.285, p = 0.007) were independent influencing factors for progressive PBC. CONCLUSIONS: Measurement of the PHB to assess advanced PBC is a simple and effective method. This method may complement current methods for the histological staging assessment of patients with PBC. REGISTRATION: Clinical trial registration: ChiCTR 2000032053, 2020/04/19. CLINICAL RELEVANCE STATEMENT: The measurement of periportal hypoechoic band (PHB) provides a simple and easy assessment of the degree of disease progression in patients with PBC and provides an important clinical reference in predicting the histological staging of PBC from an ultrasound perspective. KEY POINTS: • The PHB is correlated with histological staging in the patient with PBC. • The area under the ROC curves of PHB for detecting advanced stage (≥ stage 3) were 0.934 and 0.93 for sensitivity, and 0.91 for specificity, the concordance rates of PHB vs. liver biopsy was 90.3%. The application of PHB can better assess the advanced PBC. • Measurement of the PHB to assess advanced PBC is a simple and effective method that can significantly reduce the need for liver biopsy.

Neuron-derived exosomes mediate sevoflurane-induced neurotoxicity in neonatal mice via transferring lncRNA Gas5 and promoting M1 polarization of microglia.

Sevoflurane exposure during rapid brain development induces neuronal apoptosis and causes memory and cognitive deficits in neonatal mice. Exosomes that transfer genetic materials including long non-coding RNAs (lncRNAs) between cells play a critical role in intercellular communication. However, the lncRNAs found in exosomes derived from neurons treated with sevoflurane and their potential role in promoting neurotoxicity remain unknown. In this study, we investigated the role of cross-talk of newborn mouse neurons with microglial cells in sevoflurane-induced neurotoxicity. Mouse hippocampal neuronal HT22 cells were exposed to sevoflurane, and then co-cultured with BV2 microglial cells. We showed that sevoflurane treatment markedly increased the expression of the lncRNA growth arrest-specific 5 (Gas5) in neuron-derived extracellular vesicles, which inhibited neuronal proliferation and induced neuronal apoptosis by promoting M1 polarization of microglia and the release of inflammatory cytokines. We further revealed that the exosomal lncRNA Gas5 significantly upregulated Foxo3 as a competitive endogenous RNA of miR-212-3p in BV2 cells, and activated the NF-κB pathway to promote M1 microglial polarization and the secretion of inflammatory cytokines, thereby exacerbating neuronal damage. In neonatal mice, intracranial injection of the exosomes derived from sevoflurane-treated neurons into the bilateral hippocampi significantly increased the proportion of M1 microglia, inhibited neuronal proliferation and promoted apoptosis, ultimately leading to neurotoxicity. Similar results were observed in vitro in BV2 cells treated with the CM from HT22 cells after sevoflurane exposure. We conclude that sevoflurane induces the transfer of lncRNA Gas5-containing exosomes from neurons, which in turn regulates the M1 polarization of microglia and contributes to neurotoxicity. Thus, modulating the expression of lncRNA Gas5 or the secretion of exosomes could be a strategy for addressing sevoflurane-induced neurotoxicity.

Effect of Remimazolam Supplementation on Propofol Requirements During Hysteroscopy: A Double-Blind, Dose-Response Study.

BACKGROUND: Propofol is commonly used for procedural sedation but may increase side effects in a dose-dependent manner. Remimazolam, an ultrashort-acting benzodiazepine, has been approved for procedural sedation but may delay awakening. This study tested the hypothesis that remimazolam as a supplement reduces effect-site propofol concentration (Ceprop) required to suppress response to cervical dilation in patients undergoing hysteroscopy. METHODS: One hundred and fifty patients who were scheduled for hysteroscopy were randomized to receive 0, 0.05, 0.1, 0.15, or 0.2 mg·kg-1 intravenous remimazolam, followed by a bolus of sufentanil 0.15 µg⋅kg-1, and a target-controlled propofol infusion. The initial target Ceprop was 3.5 µg·mL-1 and was increased or decreased in subsequent patients by steps of 0.5 µg·mL-1 according to whether there was loss of response to cervical dilation in the previous patient. We used up-down sequential analysis to determine values of Ceprop that suppressed response to cervical dilation in 50% of patients (EC50). RESULTS: The EC50 of propofol for suppressing response to cervical dilation was lower in patients given 0.1 mg·kg-1 (2.08 [95% confidence interval, CI, 1.88-2.28] µg·mL-1), 0.15 mg⋅kg-1 (1.83 [1.56-2.10] µg·mL-1), and 0.2 mg⋅kg-1 (1.43 [1.27-1.58] µg·mL-1) remimazolam than those given 0 mg⋅kg-1 (3.67 [3.49-3.86] µg·mL-1) or 0.05 mg⋅kg-1 (3.47 [3.28-3.67] µg·mL-1) remimazolam (all were P < .005). Remimazolam at doses of 0.1, 0.15, and 0.2 mg·kg-1 decreased EC50 of propofol by 43.3% (95% CI, 41.3%-45.5%), 50.3% (48.0%-52.8%), and 61.2% (58.7%-63.8%), respectively, from baseline (remimazolam 0 mg⋅kg-1). Propofol consumption was lower in patients given 0.1 mg⋅kg-1 (4.15 [3.51-5.44] mg·kg-1), 0.15 mg⋅kg-1 (3.54 [3.16-4.46] mg·kg-1), and 0.2 mg⋅kg-1 (2.74 [1.73-4.01] mg·kg-1) remimazolam than those given 0 mg⋅kg-1 (6.09 [4.99-7.35] mg·kg-1) remimazolam (all were P < .005). Time to anesthesia emergence did not differ significantly among the 5 groups. CONCLUSIONS: For women undergoing hysteroscopic procedures, remimazolam at doses from 0.1 to 0.2 mg·kg-1 reduced the EC50 of propofol inhibiting response to cervical dilation and the total propofol requirement. Whether the combination could improve perioperative outcomes deserves further investigation.

Predicting fluid responsiveness in spontaneously breathing parturients undergoing caesarean section via carotid artery blood flow and velocity time integral measured by carotid ultrasound: a prospective cohort study.

BACKGROUND: Present evidence suggests that the Doppler ultrasonographic indices, such as carotid artery blood flow (CABF) and velocity time integral (VTI), had the ability to predict fluid responsiveness in non-obstetric patients. The purpose of this study was to assess their capacity to predict fluid responsiveness in spontaneous breathing parturients undergoing caesarean section and to determine the effect of detecting and management of hypovolemia (fluid responsiveness) on the incidence of hypotension after anaesthesia. METHODS: A total of 72 full term singleton parturients undergoing elective caesarean section were enrolled in this study. CABF, VTI, and hemodynamic parameters were recorded before and after fluid challenge and assessed by carotid artery ultrasonography. Fluid responsiveness was defined as an increase in stroke volume index (SVI) of 15% or more after the fluid challenge. RESULTS: Thirty-one (43%) patients were fluid responders. The area under the ROC curve to predict fluid responsiveness for CABF and VTI were 0.803 (95% CI, 0.701-0.905) and 0.821 (95% CI, 0.720-0.922). The optimal cut-off values of CABF and VTI for fluid responsiveness was 175.9 ml/min (sensitivity of 74.0%; specificity of 78.0%) and 8.7 cm/s (sensitivity of 67.0%; specificity of 90.0%). The grey zone for CABF and VTI were 114.2-175.9 ml/min and 6.8-8.7 cm/s. The incidence of hypotension after the combined spinal-epidural anaesthesia (CSEA) was significantly higher in the Responders group 25.8% (8/31) than in the Non-Responders group 17.1(7/41) (P < 0.001). The total incidence of hypotension after CSEA of the two groups was 20.8% (15/72). CONCLUSIONS: Ultrasound evaluation of CABF and VTI seem to be the feasible parameters to predict fluid responsiveness in parturients undergoing elective caesarean section and detecting and management of hypovolemia (fluid responsiveness) could significantly decrease incidence of hypotension after anaesthesia. TRIAL REGISTRATION: The trial was registered at the Chinese Clinical Trial Registry (ChiCTR) ( www.chictr.org ), registration number was ChiCTR1900022327 (The website link: https://www.chictr.org.cn/showproj.html?proj=37271 ) and the date of trial registration was in April 5, 2019. This study was performed in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee of Women's Hospital, Zhejiang University School of Medicine (20,180,120).

Infrared and Visual Image Fusion Based on a Local-Extrema-Driven Image Filter.

The objective of infrared and visual image fusion is to amalgamate the salient and complementary features of the infrared and visual images into a singular informative image. To accomplish this, we introduce a novel local-extrema-driven image filter designed to effectively smooth images by reconstructing pixel intensities based on their local extrema. This filter is iteratively applied to the input infrared and visual images, extracting multiple scales of bright and dark feature maps from the differences between continuously filtered images. Subsequently, the bright and dark feature maps of the infrared and visual images at each scale are fused using elementwise-maximum and elementwise-minimum strategies, respectively. The two base images, representing the final-scale smoothed images of the infrared and visual images, are fused using a novel structural similarity- and intensity-based strategy. Finally, our fusion image can be straightforwardly produced by combining the fused bright feature map, dark feature map, and base image together. Rigorous experimentation conducted on the widely used TNO dataset underscores the superiority of our method in fusing infrared and visual images. Our approach consistently performs on par or surpasses eleven state-of-the-art image-fusion methods, showcasing compelling results in both qualitative and quantitative assessments.

Ultrasound imaging findings in primary biliary cholangitis.

BACKGROUND: Our study aimed to analyze the characteristics of ultrasound images corresponding to each histological stage of primary biliary cholangitis (PBC). METHODS: We prospectively analyzed 75 confirmed cases of PBC and used liver biopsy as the gold standard to determine the disease stage. RESULTS: The typical ultrasound images of patients with PBC were characterized by a thickening of the portal vein wall (PVW) and periportal hypoechoic band (PHB) width with increasing histological stages, and significant increases in the left hepatic lobe diameter (LHLD) in stage II (by 64.0%) and stage III (by 69.2%). PHB width (r = 0.857, p < 0.001), PVW thickness (r = 0.488, p < 0.001), and spleen area (r = 0.8774, p < 0.001) were positively correlated with the histological stage. Significant changes were noted in the liver surface, echo texture, and edge between different stages. The areas under the receiver operating characteristic curve of composite indicators were 0.965 for predicting progressive PBC(≥ stage 2), and 0.926 for predicting advanced PBC(≥ stage 3). CONCLUSIONS: The ultrasound imaging characteristics of patients with PBC varied according to the histological staging. LHLD, PVW thickness, and PHB width were significantly correlated with the histological stage. A combination of high- and low-frequency ultrasound imaging can provide relevant cues regarding the degree of PBC progression and important clinical reference values. The application of all the ultrasound image findings as the composite indicators can better predict progressive and advanced PBC, providing important clinical reference values.

A Portable Sensing Platform Using an Upconversion-Based Nanosensor for Visual Quantitative Monitoring of Mesna.

Mesna is an important regional antidote for protecting the urinary system of chemotherapy patients, which requires monitoring its level in real time to ensure the curative effect. The fluorescence method is a powerful tool in real-time detection with the advantages of fast response and visualization. However, the background interference limits its application in biological sensing. Here, we developed a portable sensing platform using an upconversion-based nanosensor for visual quantitative monitoring of mesna in real-time/on-site conditions. The nanosensor was constructed by upconversion nanoparticles (UCNPs) and ethyl violet (EV), in which the UCNPs emitted red and green light, while EV quenched the green light due to the inner filter effect (IFE). The reaction of mesna with EV caused its fading and broke the IFE process, leading to the recovery of green light. By the fluorescence and colorimetric chromaticity variations, the nanosensor achieved a dual-readout detection for mesna with low limits of detection (LODs) of 26 and 48 nM, respectively. Furthermore, a highly compatible sensing platform was fabricated for facile determination of mesna with an LOD of 56 nM, realizing visual quantitative monitoring of the mesna level to ensure the curative effect and providing a new strategy for point-of-care testing of drugs in clinical settings.

Chromaticity Evolutionary Detection of Food Contaminant Semicarbazide through an Upconversion Luminescence-Based Nanosensor.

Semicarbazide (SEM) is a widespread carcinogenic and neurotoxic food contaminant, originating from the metabolite of antibiotic nitrofurazone, which is used in aquaculture, or thermal decomposition byproduct of a flour blowing agent azodicarbonamide. Although optical detection technologies are powerful tools considering the advantages of fast response and visualization detection, there are few optical nanosensors for highly sensitive and visual assays of SEM due to no luminescence response and UV absorbance of SEM. Herein, an upconversion luminescence (UCL)-based nanosensor was designed for visual detection of SEM with high sensitivity and good selectivity. The nanosensor was constructed by combining upconversion nanoparticles (UCNPs) and phosphomolybdic acid (PMA), which was used as the specific recognition element of SEM. The developed nanosensor exhibited selective absorbance enhancement and UCL quenching behavior with the addition of SEM based on the inner filter effect (IFE). Since the change in absorbance translated into an exponential change in the luminescence, the sensitivity of the nanosensor was greatly improved. The nanosensor realized a highly sensitive and visual response to SEM in the linear range of 0.5-16 µM with a low limit of detection of 58 nM. Moreover, satisfactory recovery values ranging from 90 to 112% in spiked real samples indicated the practical applicability of the nanosensor. The nanosensor designed here provides a sensitive and convenient sensing strategy for visual detection of hazardous substances and is expected to develop the upconversion sensing application in food safety.

USP22 controls multiple signaling pathways that are essential for vasculature formation in the mouse placenta.

USP22, a component of the SAGA complex, is overexpressed in highly aggressive cancers, but the normal functions of this deubiquitinase are not well defined. We determined that loss of USP22 in mice results in embryonic lethality due to defects in extra-embryonic placental tissues and failure to establish proper vascular interactions with the maternal circulatory system. These phenotypes arise from abnormal gene expression patterns that reflect defective kinase signaling, including TGFß and several receptor tyrosine kinase pathways. USP22 deletion in endothelial cells and pericytes that are induced from embryonic stem cells also hinders these signaling cascades, with detrimental effects on cell survival and differentiation as well as on the ability to form vessels. Our findings provide new insights into the functions of USP22 during development that may offer clues to its role in disease states.

DNAzyme signal amplification based on Au@Ag core-shell nanorods for highly sensitive SERS sensing miRNA-21.

Here, we developed a surface-enhanced Raman scattering (SERS) sensor based on functionalized Au@Ag core-shell nanorods (Au@Ag NRs) and cascade DNAzyme amplifier (CSA) for sensitive and accurate determination of microRNA-21 (miRNA-21). The as-prepared SERS nanoprobes were composed of a thiol-modification hairpin probe (HP2)-functionalized Au@Ag NRs and hairpin DNAzyme (HP1-Dz). Compared with original gold nanorods, the silver shell caused an enhancement of plasmonic properties, resulting in a significant enhancement of Raman signals. In the presence of target miRNAs, the hairpin construction of HP1-Dz changed due to DNA/RNA hybridization; subsequently, the DNAzyme-catalyzed cleaving process changed, and the Raman signals of the SERS nanoprobes gradually "turned off" with time elapse because of the dissociation of the Raman reporter from the surface of Au@Ag NRs. Hence, based on this principle, the proposed SERS sensor exhibited good linearity in the range 0.5 fM to 10 nM for miRNA-21 detection with a detection limit (LOD) of 0.5 fM. The proposed SERS platform has potential application in quantitative and precise detection of miRNA-21 in human serum.

Corrected flow time and respirophasic variation in blood flow peak velocity of radial artery predict fluid responsiveness in gynecological surgical patients with mechanical ventilation.

BACKGROUND: Recent evidence suggests that ultrasound measurements of carotid and brachial artery corrected flow time (FTc) and respirophasic variation in blood flow peak velocity (ΔVpeak) are valuable for predicting fluid responsiveness in mechanical ventilated patients. We performed the study to reveal the performance of ultrasonic measurements of radial artery FTc and ΔVpeak for predicting fluid responsiveness in mechanical ventilated patients undergoing gynecological surgery. METHODS: A total of eighty mechanical ventilated patients were enrolled. Radial artery FTc and ΔVpeak, and non-invasive pulse pressure variation (PPV) were measured before and after fluid challenge. Fluid responsiveness was defined as an increase in stroke volume index (SVI) of 15% or more after the fluid challenge. Multivariate logistic regression analyses and receiver operating characteristic (ROC) curve were used to screen multivariate predictors of fluid responsiveness and identify the predictive abilitie of non-invasive PPV, ΔVpeak and FTc on fluid responsiveness. RESULTS: Forty-four (55%) patients were fluid responders. Multivariate logistic regression analysis showed that radial artery FTc, ΔVpeak, and non-invasive PPV were the independent predictors of fluid responsiveness, with odds ratios of 1.152 [95% confidence interval (CI) 1.045 to 1.270], 0.581 (95% CI 0.403 to 0.839), and 0.361 (95% CI, 0.193 to 0.676), respectively. The area under the ROC curve of fluid responsiveness predicted by FTC was 0.802 (95% CI, 0.706-0.898), and ΔVpeak was 0.812 (95% CI, 0.091-0.286), which were comparable with non-invasive PPV (0.846, 95%CI, 0.070-0.238). The optimal cut-off values of FTc for fluid responsiveness was 336.6 ms (sensitivity of 75.3%; specificity of 75.9%), ΔVpeak was 14.2% (sensitivity of 88.2%; specificity of 67.9%). The grey zone for FTc was 313.5-336.6 ms and included 40 (50%) of the patients, ΔVpeak was 12.2-16.5% and included 37(46%) of the patients. CONCLUSIONS: Ultrasound measurement of radial artery FTc and ΔVpeak are the feasible and reliable methods for predicting fluid responsiveness in mechanically ventilated patients. TRIAL REGISTRATION: The trial was registered at the Chinese Clinical Trial Registry (ChiCTR)(www.chictr.org), registration number ChiCTR2000040941.

Cloning and Functional Characterization of a Novel ß-GRP Gene From Melanotus cribricollis.

In this study, a novel ß-1,3-glucan recognition protein gene (ß-GRP) was identified from Melanotus cribricollis, and its potential role in the immune response was investigated. The full length of ß-GRP cDNA (Accession Number: MT941530) was 1644 bp, encoding a protein composed of 428 amino acids. The theoretical molecular weight and the isoelectric point were 51.53 kDa and 6.17, respectively. The amino acid sequence of ß-GRP from M. cribricollis was closely related to that of. ß-GRP-like from Photinus pyralis, and was predicted to contain conserved GH16 domain without glucanase active site. The results of real-time quantitative PCR showed that fungal infection of Metarhizium pingshaense may significantly upregulated the expression level of ß-GRP gene. The RNAi suppression of ß-GRP gene expression significantly increased the corrected cumulative mortality. Meanwhile, antimicrobial peptide genes defensin and lysozyme were significantly downregulated after interference of ß-GRP. Taken together, these results suggest that ß-GRP of M. cribricollis probably participates in the host immune system by mediating the expression of antimicrobial peptides. This study provides comprehensive insights into the innate immune system of insect larvae.

Portable Smartphone Platform Based on a Single Dual-Emissive Ratiometric Fluorescent Probe for Visual Detection of Isopropanol in Exhaled Breath.

The components in the exhaled breath have been confirmed to be related to certain diseases, especially studies have shown that isopropanol (IPA) might be closely associated with illnesses such as lung cancer, and are considered as a biomarker. Herein, we designed a portable smartphone platform based on a chemically synthesized ratiometric fluorescent probe for real-time/on-site, sensitive, and quantitative visual detection of IPA in exhaled breath. The fluorescent probe was fabricated by a nicotinamide adenine dinucleotide (NAD+) functional modified onto fluorescent internal standard red carbon dots (RCDs). Whereas, IPA can convert NAD+ into reduced nicotinamide adenine dinucleotide (NADH) through an enzymatic reaction of secondary alcohol dehydrogenase (S-ADH). The electron transfer from IPA to NAD+ emitted a blue emission of NADH, which displayed consecutive color changes from red to light blue. Under optimum conditions, the fluorescent probe shows sensitive responses to IPA with a detection limit as low as 4.45 nM. Moreover, combined with the smartphone color recognizer application (APP), the ratio of fluorescence intensity response was recorded on a blue channel (B)/red channel (R), which has been employed for the visual quantitative determination of IPA with a detection limit of 8.34 nM and a recovery rate of 90.65-110.09% (RSD ≤ 4.83). The method reported here provides a convenient pathway for real-time/on-site and visual detection of IPA in exhaled air and is expected to extend the application of investigation of potential volatile biomarkers for preliminary monitoring and clinical diagnosis.

The liver steatosis severity and lipid characteristics in primary biliary cholangitis.

BACKGROUND: Patients with primary biliary cholangitis (PBC) often have comorbid dyslipidemia, and determining the degree of hepatic steatosis can help predict the risk of cardiovascular events in PBC patients. The aim of our study was to analyze the characteristics of lipid distribution and the degree of hepatic steatosis in PBC. METHODS: We retrospectively analyzed 479 cases of PBC, chronic hepatitis B (CHB), chronic hepatitis C (CHC), non-alcoholic fatty liver disease (NAFLD), and healthy subjects (Normal) diagnosed by liver biopsy or definitive clinical diagnosis. Controlled attenuation parameter (CAP) values were applied to assess the degree of steatosis of the liver, and lipid levels were also compared in the five cohorts. RESULTS: We found that among the five groups of subjects, the PBC group had the lowest CAP values (P < 0.001), and the high-density lipoprotein cholesterol (HDL-C) level in the PBC group was higher than normal, CHC and CHB group (P = 0.004, P = 0.033, P < 0.001, respectively).In the multivariate linear analysis, only BMI (ß = 1.280, P = 0.028), ALP (ß = - 0.064, P = 0.012), TBA (ß = - 0.126, P = 0.020), TG (ß = 12.520, P = 0.000), HDL-C (ß = - 11.338, P = 0.001) and LDL-C (ß = 7.012, P = 0.002) were independent predictors of CAP. CONCLUSIONS: Among PBC, CHB, CHC, NAFLD and healthy subjects, PBC had the lowest degree of hepatic steatosis and higher HDL-C levels, all of which were found to be protective factors against atherosclerosis and cardiovascular risk and would provide a valuable reference for the risk of developing cardiovascular events in PBC patients.

DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells.

DNMT3L (DNMT3-like), a member of the DNMT3 family, has no DNA methyltransferase activity but regulates de novo DNA methylation. While biochemical studies show that DNMT3L is capable of interacting with both DNMT3A and DNMT3B and stimulating their enzymatic activities, genetic evidence suggests that DNMT3L is essential for DNMT3A-mediated de novo methylation in germ cells but is dispensable for de novo methylation during embryogenesis, which is mainly mediated by DNMT3B. How DNMT3L regulates DNA methylation and what determines its functional specificity are not well understood. Here we show that DNMT3L-deficient mouse embryonic stem cells (mESCs) exhibit downregulation of DNMT3A, especially DNMT3A2, the predominant DNMT3A isoform in mESCs. DNA methylation analysis of DNMT3L-deficient mESCs reveals hypomethylation at many DNMT3A target regions. These results confirm that DNMT3L is a positive regulator of DNA methylation, contrary to a previous report that, in mESCs, DNMT3L regulates DNA methylation positively or negatively, depending on genomic regions. Mechanistically, DNMT3L forms a complex with DNMT3A2 and prevents DNMT3A2 from being degraded. Restoring the DNMT3A protein level in DNMT3L-deficient mESCs partially recovers DNA methylation. Thus, our work uncovers a role for DNMT3L in maintaining DNMT3A stability, which contributes to the effect of DNMT3L on DNMT3A-dependent DNA methylation.

PRMT1 loss sensitizes cells to PRMT5 inhibition.

PRMT5 is an arginine methyltransferase that accounts for the vast majority of the symmetric methylation in cells. PRMT5 exerts its function when complexed with MEP50/WDR77. This activity is often elevated in cancer cells and correlates with poor prognosis, making PRMT5 a therapeutic target. To investigate the PRMT5 signaling pathway and to identify genes whose loss-of-function sensitizes cancer cells to PRMT5 inhibition, we performed a CRISPR/Cas9 genetic screen in the presence of a PRMT5 inhibitor. We identified known components of the PRMT5 writer/reader pathway including PRMT5 itself, MEP50/WDR77, PPP4C, SMNDC1 and SRSF3. Interestingly, loss of PRMT1, the major asymmetric arginine methyltransferase, also sensitizes cells to PRMT5 inhibition. We investigated the interplay between PRMT5 and PRMT1, and found that combinatorial inhibitor treatment of small cell lung cancer and pancreatic cancer cell models have a synergistic effect. Furthermore, MTAP-deleted cells, which harbor an attenuated PRMT5-MEP50 signaling pathway, are generally more sensitive to PRMT1 inhibition. Together, these findings demonstrate that there is a degree of redundancy between the PRMT5 and PRMT1 pathways, even though these two enzymes deposit different types of arginine methylation marks. Targeting this redundancy provides a vulnerability for tumors carrying a co-deletion of MTAP and the adjacent CDKN2A tumor suppressor gene.

Structural basis of specific DNA binding by the transcription factor ZBTB24.

ZBTB24, encoding a protein of the ZBTB family of transcriptional regulators, is one of four known genes-the other three being DNMT3B, CDCA7 and HELLS-that are mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a genetic disorder characterized by DNA hypomethylation and antibody deficiency. The molecular mechanisms by which ZBTB24 regulates gene expression and the biological functions of ZBTB24 are poorly understood. Here, we identified a 12-bp consensus sequence [CT(G/T)CCAGGACCT] occupied by ZBTB24 in the mouse genome. The sequence is present at multiple loci, including the Cdca7 promoter region, and ZBTB24 binding is mostly associated with gene activation. Crystallography and DNA-binding data revealed that the last four of the eight zinc fingers (ZFs) (i.e. ZF5-8) in ZBTB24 confer specificity of DNA binding. Two ICF missense mutations have been identified in the ZBTB24 ZF domain, which alter zinc-binding cysteine residues. We demonstrated that the corresponding C382Y and C407G mutations in mouse ZBTB24 abolish specific DNA binding and fail to induce Cdca7 expression. Our analyses indicate and suggest a structural basis for the sequence specific recognition by a transcription factor centrally important for the pathogenesis of ICF syndrome.

ATF3-Induced Mammary Tumors Exhibit Molecular Features of Human Basal-Like Breast Cancer.

Basal-like breast cancer (BLBC) is an aggressive and deadly subtype of human breast cancer that is highly metastatic, displays stem-cell like features, and has limited treatment options. Therefore, developing and characterizing preclinical mouse models with tumors that resemble BLBC is important for human therapeutic development. ATF3 is a potent oncogene that is aberrantly expressed in most human breast cancers. In the BK5.ATF3 mouse model, overexpression of ATF3 in the basal epithelial cells of the mammary gland produces tumors that are characterized by activation of the Wnt/ß-catenin signaling pathway. Here, we used RNA-Seq and microRNA (miRNA) microarrays to better define the molecular features of BK5.ATF3-derived mammary tumors. These analyses showed that these tumors share many characteristics of human BLBC including reduced expression of Rb1, Esr1, and Pgr and increased expression of Erbb2, Egfr, and the genes encoding keratins 5, 6, and 17. An analysis of miRNA expression revealed reduced levels of Mir145 and Mir143, leading to the upregulation of their target genes including both the pluripotency factors Klf4 and Sox2 as well as the cancer stem-cell-related gene Kras. Finally, we show through knock-down experiments that ATF3 may directly modulate MIR145/143 expression. Taken together, our results indicate that the ATF3 mouse mammary tumor model could provide a powerful model to define the molecular mechanisms leading to BLBC, identify the factors that contribute to its aggressiveness, and, ultimately, discover specific genes and gene networks for therapeutic targeting.

The arginine methyltransferase CARM1 represses p300•ACT•CREMτ activity and is required for spermiogenesis.

CARM1 is a protein arginine methyltransferase (PRMT) that has been firmly implicated in transcriptional regulation. However, the molecular mechanisms by which CARM1 orchestrates transcriptional regulation are not fully understood, especially in a tissue-specific context. We found that Carm1 is highly expressed in the mouse testis and localizes to the nucleus in spermatids, suggesting an important role for Carm1 in spermiogenesis. Using a germline-specific conditional Carm1 knockout mouse model, we found that it is essential for the late stages of haploid germ cell development. Loss of Carm1 led to a low sperm count and deformed sperm heads that can be attributed to defective elongation of round spermatids. RNA-seq analysis of Carm1-null spermatids revealed that the deregulated genes fell into similar categories as those impacted by p300-loss, thus providing a link between Carm1 and p300. Importantly, p300 has long been known to be a major Carm1 substrate. We found that CREMτ, a key testis-specific transcription factor, associates with p300 through its activator, ACT, and that this interaction is negatively regulated by the methylation of p300 by Carm1. Thus, high nuclear Carm1 levels negatively impact the p300•ACT•CREMτ axis during late stages of spermiogenesis.