Aberrant DNA N6 -methyladenine incorporation via adenylate kinase 1 is suppressed by ADAL deaminase-dependent 2'-deoxynucleotide pool sanitation.

Intracellular decay of N6 -methyladenine (m6A)-containing RNA potentially induces aberrant N6 -methyl-2'-adenine (6mdA) misincorporation into DNA. Biophysically, misincorporated 6mdA may destabilize the DNA duplex in a manner similar to bona fide methylated 6mdA DNA, thereby affecting DNA replication and transcription. Utilizing heavy stable isotope labeling and ultrasensitive UHPLC-MS/MS assay, we demonstrate that intracellular m6A-RNA decay does not generate free 6mdA species, nor lead to any misincorporated DNA 6mdA in most mammalian cell lines tested, unveiling the existence of a sanitation mechanism that prevents 6mdA misincorporation. Depletion of deaminase ADAL increases the levels of free 6mdA species, concomitant with the presence of DNA-misincorporated 6mdA resulting from intracellular RNA m6A decay, suggesting that ADAL catabolizes 6mdAMP in vivo. Furthermore, we show that the overexpression of adenylate kinase 1 (AK1) promotes 6mdA misincorporation, while AK1 knockdown diminishes 6mdA incorporation, in ADAL-deficient cells. We conclude that ADAL together with other factors (such as MTH1) contributes to 2'-deoxynucleotide pool sanitation in most cells but compromised sanitation (e.g., in NIH3T3 cells) and increased AK1 expression may facilitate aberrant 6mdA incorporation. This sanitation mechanism may provide a framework for the maintenance of the epigenetic 6mdA landscape.

10× single-cell sequencing revealed cellular composition heterogeneity in cardiac myxoma with malignant glandular properties.

Cardiac myxoma is the most common primary cardiac tumor in adults. The histogenesis and cellular composition of myxoma are still unclear. This study aims to reveal the role of myxoma cell components and their gene expression in tumor development. We obtained single living cells by enzymatic digestion of tissues from 4 cases of surgically resected cardiac myxoma. Of course, there was 1 case of glandular myxoma and 3 cases of nonglandular myxoma. Then, 10× single-cell sequencing was performed. We identified 12 types and 11 types of cell populations in glandular myxoma and nonglandular myxoma, respectively. Heterogeneous epithelial cells are the main components of glandular myxoma. The similarities and differences in T cells in both glandular and nonglandular myxoma were analyzed by KEGG and GO. The most important finding was that there was active communication between T cells and epithelial cells. These results clarify the possible tissue occurrence and heterogeneity of cardiac myxoma and provide a theoretical basis and guidance for clinical diagnosis and treatment.

Chlorobenzoquinones Aggravate RSL3-Induced Ferroptosis in ROS-Dependent Manner.

Chlorobenzoquinones (CBQs) are a class of emerging water disinfection byproducts that pose significant risks to public health. In this study, we found that three CBQs (tetrachloro-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, and 2-chloro-1,4-benzoquinone) can significantly aggravate cell death caused by Ras-selective lethal small molecule 3 (RSL3). Further study showed that the cell death caused by CBQs, either alone or in combination with RSL3, was related to iron accumulation and GPX4 inactivation, suggesting the occurrence of ferroptosis. Furthermore, reactive oxygen species are found to play a potential key role in mediating the toxicity of CBQs in CBQs and RSL3-induced ferroptosis. These findings will be helpful in understanding the toxic mechanism of CBQs to mammalian cells.

Ferredoxin 1 regulates granulosa cell apoptosis and autophagy in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), a common reproductive endocrine disorder in women of reproductive age, causes anovulatory infertility. Increased apoptosis of granulosa cells has been identified as one of the key factors contributing to abnormal follicular development. Ferredoxin 1 (FDX1) encodes a small ferredoxin that is involved in the reduction in mitochondrial cytochromes and the synthesis of various steroid hormones and has the potential to influence the function of granulosa cells. In the present study, we aimed to determine the relationship between FDX1 and follicular granulosa cell function. To this end, we investigated the difference between FDX1 expression in the granulosa cells of 50 patients with PCOS and that of the controls. Furthermore, we sought to elucidate the role and mechanism of FDX1 in PCOS granulosa cells by establishing a mouse PCOS model with dehydroepiandrosterone and KGN (a steroidogenic human granulosa cell-like tumor cell line). The results indicated significant up-regulation of FDX1 in the granulosa cells after androgen stimulation. Knockdown of FDX1 promoted the proliferation of KGN and inhibited apoptosis. Moreover, FDX1 could regulate autophagy by influencing the autophagy proteins ATG3 and ATG7. Our results demonstrated that FDX1 plays a critical role in female folliculogenesis by mediating apoptosis, autophagy, and proliferation. Therefore, FDX1 may be a potential prognostic factor for female infertility.

Necrosulfonamide Selectively Induces DNA Double-Strand Breaks in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that causes endless pain for patients and accounts for thousands of deaths worldwide. The development of an effective AML treatment is a topic of ongoing interest. Here, we demonstrated that a pyroptosis inhibitor necrosulfonamide (NSA) can selectively induce highly toxic double-strand breaks and kill AML cells. Mechanistically, reactive oxygen species (ROS) were the key effectors mediating the toxicity of NSA. These results probably indicate that NSA is a novel candidate for the treatment of AML.

Immunoassay of Small Molecule Mediated by a Triply Functional DNA.

Benefiting from specific target recognition by antibodies, the immunoassay is one of the widely used assays for the detection of biologically and environmentally important small molecules in broad fields. It can be challenge to isolate small molecules from their antibody complex in an immobilization-free immunoassay with separation for the detection of small-molecule targets. Here we present an immunoassay mediated by a triply functional DNA probe. A DNA strand is dually labeled with a fluorophore and the target small molecule. This DNA probe integrates three functions, including specific binding to the antibody, signal reporting for sensitive fluorescence detection, and carrying negative charges to facilitate capillary electrophoresis (CE) separation. The binding of the probe to an antibody brings many negative charges in the complex and causes significant changes in mass-to-charge ratios, so the antibody-probe complex can be well separated from the unbound probe in CE analysis. A simple immunoassay is achieved by target competition with this DNA probe for antibody binding in CE coupled to ultrasensitive laser-induced fluorescence (LIF) detection. To show a proof of concept, we detected two model small-molecule targets, digoxin, a therapeutic drug, and ochratoxin A (OTA), an important mycotoxin for food safety. In addition, the use of two DNA probes with distinguished migration times in CE allowed the simultaneous detection of OTA and digoxin. This immunoassay provides new opportunities for wide applications.

One-pot intramolecular cyclization of 5-hydroxymethylcytosine for sequencing DNA hydroxymethylation at single-base resolution.

Here we establish a one-pot reaction to directly convert the DNA base 5-hydroxymethylcytosine (5hmC) to an intramolecular cyclization nucleobase, which loses both protons of the exocyclic N4-amino group and thus is recognized as thymine (T) by DNA polymerase. Based on this 5hmC-specific reaction, a prospective bisulfite-free strategy for 5hmC sequencing is proposed. This is also the first example to show modified DNA labeling in non-water solvent-dominant media for DNA sequencing.

Development of Human Lung Induction Models for Air Pollutants' Toxicity Assessment.

There is an urgent need for reliable and effective models to study air pollution health effects on human lungs. Here, we report the utilization of human pluripotent stem cell (hPSC) induction models for human lung progenitor cells (hLPs) and alveolar type 2 epithelial cell-like cells (ATLs) for the toxicity assessment of benzo(a)pyrene, nano-carbon black, and nano-SiO2, as common air pollutants. We induced hPSCs to generate ATLs, which recapitulated key features of human lung type 2 alveolar epithelial cells, and tested the induction models for cellular uptake of nanoparticles and toxicity evaluations. Our findings reveal internalization of nano-carbon black, dose-dependent uptake of nano-SiO2, and interference with surfactant secretion in ATLs exposed to benzo(a)pyrene/nano-SiO2. Thus, hLP and ATL induction models could facilitate the evaluation of environmental pollutants potentially affecting the lungs. In conclusion, this is one of the first studies that managed to adopt hPSC pulmonary induction models in toxicology studies.

Galectin-3 mediates pulmonary vascular endothelial cell dynamics via TRPC1/4 under acute hypoxia.

Galectin-3 (Gal-3) has been implicated in various biological functions, yet little is known about its role in regulating the dynamics of pulmonary vascular endothelial cells. Gal-3 was shown to be increased in hypoxic model rats by sequencing analysis. We exposed pulmonary vessel endothelial cells (PVECs) to hypoxia or Gal-3 stimulation, following which cell apoptosis and autophagy were measured with the relevant methods. The results demonstrated that hypoxia elevated nuclear factor-κB (NF-κB) activity and Gal-3 expression. Gla-3 decreased the expression of Bcl-2, Alix, Beclin-1, Atg5, and LC3A/B. The messenger RNA and protein levels of transient receptor potential channel 1/4 (TRPC1/4) and calpain were reduced after Gal-3 treatment. Gal-3 also activated protein kinase B/glycogen synthase kinase-3 ß/mammalian target of rapamycin signaling pathways in PVECs. These results suggest that a hypoxia-mediated increase in Gal-3 promotes apoptosis and inhibits autophagy by inhibiting the TRPC1/4 pathway and activating the protein kinase B/glycogen synthase kinase-3 ß/mammalian target of rapamycin signaling pathway in PVECs. Furthermore, these results may provide us with a new direction to explore the pathogenesis of pulmonary artery hypertension.

Revealing the pathogenic changes of PAH based on multiomics characteristics.

BACKGROUND: Pulmonary artery hypertension (PAH), which is characterized by an increase in pulmonary circulation blood pressure, is a fatal disease, and its pathogenesis remains unclear. METHODS: In this study, RNA sequencing (RNA-seq), tandem mass tags (TMT) and reduced representation bisulfite sequencing (RRBS) were performed to detect the levels of mRNA, protein, and DNA methylation in pulmonary arteries (PAs), respectively. To screen the possible pathways and proteins related to PAH, pathway enrichment analysis and protein-protein interaction (PPI) network analysis were performed. For selected genes, differential expression levels were confirmed at both the transcriptional and translational levels by real-time PCR and Western blot analyses, respectively. RESULTS: A total of 362 differentially expressed genes (|Fold-change| > 1.5 and p < 0.05), 811 differentially expressed proteins (|Fold-change| > 1.2 and p < 0.05) and 76,562 differentially methylated regions (1000 bp slide windows, 500 bp overlap, p < 0.05, and |Fold-change| > 1.2) were identified when the PAH group (n = 15) was compared with the control group (n = 15). Through an integrated analysis of the characteristics of the three omic analyses, a multiomics table was constructed. Additionally, pathway enrichment analysis showed that the differentially expressed proteins were significantly enriched in five Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways and ten Gene Ontology (GO) terms for the PAH group compared with the control group. Moreover, protein-protein interaction (PPI) networks were constructed to identify hub genes. Finally, according to the genes identified in the PPI and the protein expression fold-change, nine key genes and their associated proteins were verified by real-time PCR and Western blot analyses, including Col4a1, Itga5, Col2a1, Gstt1, Gstm3, Thbd, Mgst2, Kng1 and Fgg. CONCLUSIONS: This study conducted multiomic characteristic profiling to identify genes that contribute to the hypoxia-induced PAH model, identifying new avenues for basic PAH research.

Galectin-3- Mediated Transdifferentiation of Pulmonary Artery Endothelial Cells Contributes to Hypoxic Pulmonary Vascular Remodeling.

BACKGROUND/AIMS: Vascular muscularity is a key event in vessel remodeling during pulmonary artery hypertension (PAH). Endothelial-mesenchymal transdifferentiation (EndMT) has been increasingly reported to play a role in disease occurrence. Galectin-3, a carbohydrate-binding protein regulates cell proliferation, differentiation, migration and neovascularization. However, whether galectin-3 controls endothelial cell transdifferentiation during the development of PAH is unknown. METHODS: Rats were exposed to normoxic or hypoxic conditions (fraction of inspired O2 0.10) for 21 d to establish PAH models. Hemodynamic changes were evaluated through surgery of the right jugular vein and ultrasound biomicroscopy inviVue. And vessel pathological alterations were detected by H&E staining. Galectin-3 (Gal-3)-induced pulmonary artery endothelium cell (PAEC) dynamic alterations were measured by MTT assays, Cell immunofluorescence, Flow cytometry, Real-time PCR and Western blot. RESULTS: Our study demonstrated that Gal-3 was expressed in hypoxic pulmonary vascular adventitia and intima. The increased Gal-3 expression was responsible for hypoxic vessel remodeling and PAH development in vivo. Gal-3 was found to inhibit cell proliferation and apoptosis in cultured endothelial cells. Meanwhile endothelial cell morphology was altered and exhibited smooth muscle-like cell features as demonstrated by the expression of α-SMA after Gal-3 treatment. Gal-3 activated Jagged1/Notch1 pathways and induced MyoD and SRF. When MyoD or SRF were silenced with siRNAs, Gal-3-initiated transdifferentiation in endothelial cells was blocked as indicated by a lack of α-SMA. CONCLUSION: These results suggest that Gal-3 induces PAECs to acquire an α-SMA phenotype via a transdifferentiation process which depends on the activation of Jagged1/Notch1 pathways that mediate MyoD and SRF expression.

Elevated 8-oxo-7,8-dihydro-2'-deoxyguanosine in genome of T24 bladder cancer cells induced by halobenzoquinones.

Halobenzoquinones (HBQs) are an emerging class of halogenated disinfection byproducts (DBPs) in drinking water, which raised public concerns due to potential carcinogenic effects to human bladder. Our previous work demonstrated that HBQs and hydrogen peroxide (H2O2) together generated oxidative DNA damage via a metal-independent and intercalation-enhanced oxidation mechanism in vitro. This study further investigated the efficiency of various HBQs to induce oxidative DNA damage in T24 bladder cancer cells. Compared with T24 cells without treatment (3.1 lesions per 106 dG), the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) significantly increased by 1.4, 3.2, 8.8, and 9.2 times after treatment with tetrabromo-1,4-benzoquinone (TBBQ), terachloro-1,4-benzoquinone (TCBQ), 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) and 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ) for 24hr, respectively. Interestingly, we found that the oxidative potency of HBQs in T24 cells (2,5-DCBQ≈2,6-DCBQ>TCBQ>TBBQ) is inconsistent with that of in vitro dsDNA oxidation (TCBQ>TBBQ>2,5-DCBQ>2,6-DCBQ), suggesting HBQs induce oxidative lesions in cellular genomic DNA probably involved with a complex mechanism.

Recent advances in high-performance liquid chromatography tandem mass spectrometry techniques for analysis of DNA damage and epigenetic modifications.

Environmental exposure would cause DNA damage and epigenetic modification changes, potentially resulting in physiological dysfunction, thereby triggering diseases and even cancer. DNA damage and epigenetic modifications are thus promising biomarkers for environmental exposures and disease states. Benefiting from its high sensitivity and accuracy, high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is considered the "gold standard technique" for investigating epigenetic DNA modifications. This review summarizes the recent advancements of UHPLC-MS/MS-based technologies for DNA damage and epigenetic modifications analysis, mainly focusing on the innovative methods developed for UHPLC-MS/MS-related pretreatment technologies containing efficient genomic DNA digestion and effective removal of the inorganic salt matrix, and the new strategies for improving detection sensitivity of liquid chromatography-mass spectrometry. Moreover, we also summarized the novel hyphenated techniques of the advanced UHPLC-MS/MS coupled with other separation and analysis methods for the measurement of DNA damage and epigenetic modification changes in special regions and fragments of chromosomes.

Adenosine Deaminase-Like Gene-Carried Lentivirus Toolkit for Identification of DNA N6-Methyladenine Origins.

Post-replicative DNA N6-methyladenine (pr6mdA) can form via bona fide methylase-catalyzed adenine methylation, playing a pivotal role in embryonic development and other biological processes. Surprisingly, pre-methylated adenine can be erroneously incorporated into DNA as misincorporated N6-methyladenine (i6mdA) via DNA polymerase-mediated replication. Despite pr6mdA and i6mdA sharing identical chemical structures, their biological functions diverge significantly, presenting a substantial challenge in distinguishing between the two. Here, for the first-time, it is exploited that the adenosine deaminase-like (Adal) protein and a corresponding activity-null mutant to construct an Adal lentivirus toolkit. With this newly designed toolkit, both pr6mdA and i6mdA can be identified and quantified simultaneously. The presence of 6mdA in the bone marrow cells of mice is shown, with its levels serving as indicators for growth with age, probably reflecting the cellular stress-caused changes in RNA decay, nucleotide pool sanitation, and transcription. Collectively, a powerful toolkit to advance understanding of both pr6mdA and i6mdA is demonstrated.

The suppressive role of p38 MAPK in cellular vacuole formation.

Vacuolization of the cytoplasm is one of the dramatic and frequently observed phenomena in various cell types. Cellular vacuoles occur spontaneously or via a wide range of inductive stimuli, but the molecular mechanism involved in this process remains largely unknown. In this study, we investigated the role of the p38 and JNK pathways in the formation of cytoplasmic vacuoles. We found that p38 and JNK agonist anisomycin abolishes spontaneous cytoplasmic vacuolization of HepG2 cells through p38 activation, but not through JNK activation. Importantly, blocking the activity of p38 or suppression the expression of p38 elicits cytoplasmic vacuoles formation in various cancer cells. Furthermore, cytoplasmic vacuoles induced by p38 blocking are derived from the perinuclear region. These observations provide direct evidence for a role of p38 signaling in regulating the formation of cytoplasmic vacuoles.

c-Met function requires N-linked glycosylation modification of pro-Met.

c-Met, the receptor for hepatocyte growth factor (HGF), is cell surface tyrosine kinase that controls cancer cell growth, survival, invasion, and metastasis. Post-translational modification, such as glycosylation, plays an essential role in regulating the function of cell surface molecules. Whether glycosylation modification regulates the enzymatic properties of c-Met is unknown. In this study, we investigated the effect of glycosylation on the function of c-Met. We found that c-Met is an N-linked glycosylated protein. Both pro-Met and p145Met (the ß subunit of mature c-Met) have N-linked glycosylation. Glycosylation inhibitor studies revealed that the N-glycosylation modification of p145Met is from pro-Met, but not due to the further modification of pro-Met. Importantly, blocking the N-glycosylation targets pro-Met to cytoplasm and initiates its phosphorylation independent of HGF engagement. Nonglycosylated pro-Met activates c-Met downstream pathways to a certain extent to compensate for the degradation of p145Met induced by glycosylation blocking-mediated endoplasmic reticulum (ER) stress.

[Phosphorylation of eukaryotic initiation factor 2-alpha inhibits cisplatin-mediated apoptosis of hepatocellular carcinoma cells].

OBJECTIVE: To investigate whether the phosphorylation (functionally inhibitive) of eukaryotic initiation factor 2-alpha (eIF2-a) affects the molecular mechanism of cisplatin-induced cellular apoptosis in human hepatocellular carcinoma (HCC). METHODS: The human HCC cultured cell lines SMMC-7221 and HepG2 were treated with cisplatin alone (controls; 24 h) or in combination with pre-transfection of a dominant-negative eIF2-a mutant (eIF2aS51A) or pre-exposure to an eIF2-a-specific phosphatase inhibitor (salubrinal) to decrease or increase the phosphorylation level, respectively. Changes in expression of apoptosis markers were quantitatively and qualitatively assessed by flow cytometry and western blot analysis. The significance of differences among groups was assessed by analysis of variance testing and of differences between groups was assessed by t-test. RESULTS: Cisplatin treatment induced the appropriate functional-inhibitive phosphorylation of eIF2-a on serine 51. Cisplatin treatment (10 mg/ml) induced significant apoptosis in the eIF2aS51A pre-transfected SMMC-7721 (control: 21.7 +/- 1.5% vs. 50.7 +/- 2.1%, t = 19.454, P less than 0.05) and HepG2 (21.0 +/- 1.0% vs. 57.3 +/- 2.1%, t = 27.250, P less than 0.05). Salubrinal pre-treatment significantly inhibited the cisplatin (15 mg/ml)-induced apoptosis in SMMC-7721 (control: 50.3 +/- 2.5% vs. 16.3 +/- 2.1%, t = 18.031, P less than 0.05) and HepG2 (42.0 +/- 2.6% vs. 12.0 +/- 2.0%, t = 15.667, P less than 0.05). CONCLUSION: Phosphorylation of eIF2-a may act to inhibit cisplatin-induced apoptosis of HCC.

First report on rhinoceros from the late Neogene Qin Basin of Shanxi, China.

Dihoplus is a rhinoceros distributed across East Asia and Europe from the Late Miocene to Pliocene. This study describes a new skull from the Qin Basin in Shanxi Province, China, referred to as Dihoplus ringstroemi, which has long been debated in taxonomic identity. This skull confirms that D. ringstroemi is an independent species and reveals the presence of the upper incisor and variations in the degree of constriction of the two lingual cusps of upper cheek teeth. In addition, the new skull indicates that the Qin Basin has a late Neogene sediment and fauna comparable to that of the Yushe Basin.

Integrating network pharmacology and experimental verification to explore the pharmacological mechanisms of asparagus against polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder in women of reproductive age that still lacks effective treatment. Inflammation is one of the important features of PCOS. Asparagus (ASP) has anti-inflammatory, antioxidant, and anti-aging pharmacological effects, and its anti-tumor effects have been demonstrated in a variety of tumors. However, the role and mechanism of ASP in PCOS remain unclear. METHODS: The active components of ASP and the key therapeutic targets for PCOS were obtained by network pharmacology. Molecular docking was used to simulate the binding of PRKCA to the active components of ASP. The effects of ASP on inflammatory and oxidative stress pathways in PCOS, and the regulation of PRKCA were examined by KGN, a human derived granulosa cell line. PCOS mouse model validated the results of in vivo experiments. RESULTS: Network pharmacology identified 9 major active ingredients of ASP with 73 therapeutic targets for PCOS. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment yielded 101 PCOS-related signaling pathways. The hub gene PRKCA was obtained after taking the gene intersection of the top 4 pathways. Molecular docking showed the binding of PRKCA to the 7 active components in ASP. In vitro and in vivo experiments showed that ASP alleviated the course of PCOS through antioxidant, anti-inflammatory effects. ASP can partially restore the low expression of PRKCA in the PCOS models. CONCLUSION: The therapeutic effect of ASP on PCOS is mainly achieved by targeting PRKCA through the 7 active components of ASP. Mechanistically, ASP alleviated the course of PCOS through antioxidant, anti-inflammatory effects, and PRKCA was its potential target.

Activation of PKR/eIF2α signaling cascade is associated with dihydrotestosterone-induced cell cycle arrest and apoptosis in human liver cells.

Androgen receptor (AR) signaling plays an important role in the development and progression of several liver diseases, including hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). Dihydrotestosterone (DHT) is the active metabolite of the major circulating androgen, testosterone. In this study, we investigated the effect of DHT on human liver cells. We found that DHT not only induces cell cycle arrest but also initiates apoptosis in androgen-sensitive liver cells, such as SMMC-7721 and L02. Importantly, DHT/AR induces the activation of RNA-dependent protein kinase (PKR)/eukaryotic initiation factor-2 alpha (eIF2α) cascades in androgen-sensitive liver cells. PKR/eIF2α activation-induced growth arrest and DNA damage-inducible gene 153 (GADD153) and heat shock protein 27 (Hsp27) expression contribute to cell cycle arrest in response to DHT. It is notable that DHT administration results in androgen-sensitive liver cells apoptosis, at least in part, through PKR/eIF2α/GADD153 cascades. These results suggest that the androgen/AR pathway plays a pivotal role in liver cell growth and apoptosis regulating, whose deregulation might be involved in the pathogenesis of liver diseases.