Comprehensive analysis of m6A methylome alterations after azacytidine plus venetoclax treatment for acute myeloid leukemia by nanopore sequencing.

N6 adenosine methylation (m6A), one of the most prevalent internal modifications on mammalian RNAs, regulates RNA transcription, stabilization, and splicing. Growing evidence has focused on the functional role of m6A regulators on acute myeloid leukemia (AML). However, the global m6A levels after azacytidine (AZA) plus venetoclax (VEN) treatment in AML patients remain unclear. In our present study, bone marrow (BM) sample pairs (including pre-treatment [AML] and post-treatment [complete remission (CR)] samples) were harvested from three AML patients who had achieved CR after AZA plus VEN treatment for Nanopore direct RNA sequencing. Notably, the amount of m6A sites and the m6A levels in CR BMs was significantly lower than those in the AML BMs. Such a significant reduction in the m6A levels was also detected in AZA-treated HL-60 cells. Thirteen genes with decreased m6A and expression levels were identified, among which three genes (HPRT1, SNRPC, and ANP32B) were closely related to the prognosis of AML. Finally, we speculated the mechanism via which m6A modifications affected the mRNA stability of these three genes. In conclusion, we illustrated for the first time the global landscape of m6A levels in AZA plus VEN treated AML (CR) patients and revealed that AZA had a significant demethylation effect at the RNA level in AML patients. In addition, we identified new biomarkers for AZA plus VEN-treated AML via Nanopore sequencing technology in RNA epigenetics.

Structural and functional prediction, evaluation, and validation in the post-sequencing era.

The surge of genome sequencing data has underlined substantial genetic variants of uncertain significance (VUS). The decryption of VUS discovered by sequencing poses a major challenge in the post-sequencing era. Although experimental assays have progressed in classifying VUS, only a tiny fraction of the human genes have been explored experimentally. Thus, it is urgently needed to generate state-of-the-art functional predictors of VUS in silico. Artificial intelligence (AI) is an invaluable tool to assist in the identification of VUS with high efficiency and accuracy. An increasing number of studies indicate that AI has brought an exciting acceleration in the interpretation of VUS, and our group has already used AI to develop protein structure-based prediction models. In this review, we provide an overview of the previous research on AI-based prediction of missense variants, and elucidate the challenges and opportunities for protein structure-based variant prediction in the post-sequencing era.

Small extrachromosomal circular DNAs as biomarkers for multi-cancer diagnosis and monitoring.

BACKGROUND: Small extrachromosomal circular DNAs (eccDNAs) have the potential to be cancer biomarkers. However, the formation mechanisms and functions of small eccDNAs selected in carcinogenesis are not clear, and whether the small eccDNA profile in the plasma of cancer patients represents that in cancer tissues remains to be elucidated. METHODS: A novel sequencing workflow based on the nanopore sequencing platform was used to sequence naturally existing full-length small eccDNAs in tissues and plasma collected from 25 cancer patients (including prostate cancer, hepatocellular carcinoma and colorectal cancer), and from an independent validation cohort (including 7 cancer plasma and 14 healthy plasma). RESULTS: Compared with those in non-cancer tissues, small eccDNAs detected in cancer tissues had a significantly larger number and size (P = 0.040 and 2.2e-16, respectively), along with more even distribution and different formation mechanisms. Although small eccDNAs had different general characteristics and genomic annotation between cancer tissues and the paired plasma, they had similar formation mechanisms and cancer-related functions. Small eccDNAs originated from some specific genes had great multi-cancer diagnostic value in tissues (AUC ≥ 0.8) and plasma (AUC > 0.9), especially increasing the accuracy of multi-cancer prediction of CEA/CA19-9 levels. The high multi-cancer diagnostic value of small eccDNAs originated from ALK&ETV6 could be extrapolated from tissues (AUC = 0.804) to plasma and showed high positive predictive value (100%) and negative predictive value (82.35%) in a validation cohort. CONCLUSIONS: As independent and stable circular DNA molecules, small eccDNAs in both tissues and plasma can be used as ideal biomarkers for cost-effective multi-cancer diagnosis and monitoring.

A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila.

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.

Artificial intelligence-based recognition for variant pathogenicity of BRCA1 using AlphaFold2-predicted structures.

With the surge of the high-throughput sequencing technologies, many genetic variants have been identified in the past decade. The vast majority of these variants are defined as variants of uncertain significance (VUS), as their significance to the function or health of an organism is not known. It is urgently needed to develop intelligent models for the clinical interpretation of VUS. State-of-the-art artificial intelligence (AI)-based variant effect predictors only learn features from primary amino acid sequences, leaving out information about the most important three-dimensional structure that is more related to its function. Methods: We proposed a deep convolutional neural network model named variant effect recognition network for BRCA1 (vERnet-B) to recognize the clinical pathogenicity of missense single-nucleotide variants in the BRCT domain of BRCA1. vERnet-B learned features associated with the pathogenicity from the tertiary protein structures of variants predicted by AlphaFold2. Results: After performing a series of validation and analyses on vERnet-B, we discovered that it exhibited significant advances over previous works. Recognizing the phenotypic consequences of VUS is one of the most daunting challenges in genetic informatics; however, we achieved 85% accuracy in recognizing disease BRCA1 variants with an ideal balance of false-positive and true-positive detection rates. vERnet-B correctly recognized the pathogenicity of variant A1708E, which was poorly predicted by AlphaFold2 as previously described. The vERnet-B web server is freely available from URL: http://ai-lab.bjrz.org.cn/vERnet. Conclusions: We applied protein tertiary structures to successfully recognize the pathogenic missense SNVs, which were difficult to be addressed by classical approaches based on sequences. Our work demonstrated that AlphaFold2-predicted structures were expected to be used for rich feature learning and revealed unique insights into the clinical interpretation of VUS in disease-related genes, using vERnet-B as a discovery tool.

Genotoxicity of organic contaminants in the soil: A review based on bibliometric analysis and methodological progress.

Organic contaminants (OCs) are ubiquitous in the environment, posing severe threats to human health and ecological balance. In particular, OCs and their metabolites could interact with genetic materials to induce genotoxicity, which has attracted considerable attention. In this review, bibliometric analysis was executed to analyze the publications on the genotoxicity of OCs in soil from 1992 to 2021. The result indicated that significant contributions were made by China and the United States in this field and the research hotspots were biological risks, damage mechanisms, and testing methods. Based on this, in this review, we summarized the manifestations and influencing factors of genotoxicity of OCs to soil organisms, the main damage mechanisms, and the most commonly utilized testing methods. OCs can induce genotoxicity and the hierarchical response of soil organisms, which could be influenced by the physicochemical properties of OCs and the properties of soil. Specific mechanisms of genotoxicity can be classified into DNA damage, epigenetic toxicity, and chromosomal aberrations. OCs with different molecular weights lead to genetic material damage by inducing the generation of ROS or forming adducts with DNA, respectively. The micronucleus test and the comet test are the most commonly used testing methods. Moreover, this review also pointed out that future studies should focus on the relationships between bioaccessibilities and genotoxicities, transcriptional regulatory factors, and potential metabolites of OCs to elaborate on the biological risks and mechanisms of genotoxicity from an overall perspective.

Nitric oxide inhibits endothelial cell apoptosis by inhibiting cysteine-dependent SOD1 monomerization.

Endothelial cell apoptosis is an important pathophysiology in many cardiovascular diseases. The gasotransmitter nitric oxide (NO) is known to regulate cell survival and apoptosis. However, the mechanism underlying the effect of NO remains unclear. In this research, by targeting cytosolic copper/zinc superoxide dismutase (SOD1) monomerization, we aimed to explore how NO inhibited endothelial cell apoptosis. We showed that treatment with the NO synthase (NOS) inhibitor nomega-nitro-l-arginine methyl ester hydrochloride (L-NAME) significantly decreased the endogenous NO content of endothelial cells, facilitated the formation of SOD1 monomers, inhibited dismutase activity, and promoted reactive oxygen species (ROS) accumulation in human umbilical vein endothelial cells (HUVECs); by contrast, supplementation with the NO donor sodium nitroprusside (SNP) upregulated NO content, prevented the formation of SOD1 monomers, enhanced dismutase activity, and reduced ROS accumulation in L-NAME-treated HUVECs. Mechanistically, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), a specific reducer of cysteine thiol, increased SOD1 monomer formation, thus preventing the NO-induced increase in dismutase activity and the decrease in ROS. Furthermore, SNP inhibited HUVEC apoptosis caused by the decrease in endogenous NO, whereas TCEP abolished this protective effect of SNP. In summary, our data reveal that NO protects endothelial cells against apoptosis by inhibiting cysteine-dependent SOD1 monomerization to enhance SOD1 activity and inhibit oxidative stress.

Endogenous SO2-dependent Smad3 redox modification controls vascular remodeling.

Sulfur dioxide (SO2) has emerged as a physiological relevant signaling molecule that plays a prominent role in regulating vascular functions. However, molecular mechanisms whereby SO2 influences its upper-stream targets have been elusive. Here we show that SO2 may mediate conversion of hydrogen peroxide (H2O2) to a more potent oxidant, peroxymonosulfite, providing a pathway for activation of H2O2 to convert the thiol group of protein cysteine residues to a sulfenic acid group, aka cysteine sulfenylation. By using site-centric chemoproteomics, we quantified >1000 sulfenylation events in vascular smooth muscle cells in response to exogenous SO2. Notably, ~42% of these sulfenylated cysteines are dynamically regulated by SO2, among which is cysteine-64 of Smad3 (Mothers against decapentaplegic homolog 3), a key transcriptional modulator of transforming growth factor ß signaling. Sulfenylation of Smad3 at cysteine-64 inhibits its DNA binding activity, while mutation of this site attenuates the protective effects of SO2 on angiotensin II-induced vascular remodeling and hypertension. Taken together, our findings highlight the important role of SO2 in vascular pathophysiology through a redox-dependent mechanism.

Protease-activated receptor 4 activation increases the expression of calcitonin gene-related peptide mRNA and protein in dorsal root ganglion neurons.

Accumulating evidence demonstrates that nociceptor activation evokes a rapid change in mRNA and protein levels of calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons. Although the colocalization of CGRP and protease-activated receptor-4 (PAR4), a potent modulator of pain processing and inflammation, was detected in DRG neurons, the role of PAR4 activation in the expression of CGRP has not been investigated. In the present study, the expression of CGRP and activation (phosphorylation) of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in rat DRG neurons were measured by immunofluorescence, real-time PCR, and Western blotting after AYPGKF-NH2 (selective PAR4-activating peptide; PAR4-AP) intraplantar injection or treatment of cultured DRG neurons. The expression of CGRP in cultured DRG neurons was also assessed after treatment with AYPGKF-NH2 with preaddition of PD98059 (an inhibitor for ERK1/2 pathway). Results showed that PAR4-AP intraplantar injection or treatment of cultured DRG neurons evoked significant increases in DRG cells displaying CGRP immunoreactivity and cytoplasmic and nuclear staining for phospho-ERK1/2 (p-ERK1/2). Percentages of total DRG neurons expressing both CGRP and PAR4 or p-ERK1/2 also increased significantly at 2 hr after PAR4-AP treatment. Real-time PCR and Western blotting showed that PAR4-AP treatment significantly increased expression of CGRP mRNA and protein levels in DRG neurons. The PAR4 activation-evoked CGRP expression both at mRNA and at protein levels was significantly inhibited after p-ERK1/2 was inhibited by PD98059. These results provide evidence that activation of PAR4 upregulates the expression of CGRP mRNA and protein levels in DRG neurons via the p-ERK1/2 signal pathway.

Interleukin-1ß increased the expression of protease-activated receptor 4 mRNA and protein in dorsal root ganglion neurons.

Protease-activated receptor-4 (PAR4) is localized in primary sensory neurons and is believed to implicate in the modulation of nociceptive mechanisms. The pro-inflammatory cytokine interleukin-1ß (IL-1ß) is involved in the generation of hyperalgesia in pathological states such as neuropathy and inflammation. Previous studies have shown that IL-1ß enhances the expression of PAR4 in many cell types but the effect of this cytokine on primary sensory neuron PAR4 expression is less clear. In the present study, we evaluated in rat dorsal root ganglion (DRG) neurons the influence of IL-1ß on PAR4 mRNA and protein levels after IL-1ß intraplantar injection into the hind-paw or treatment of cultured DRG neurons. The expression of PAR4 in cultured DRG neurons was also assessed after treatment with IL-1ß with pre-addition of phorbol-12-myristate 13-acetate (PMA, a PKC activator) or chelerythrine chloride (a PKC inhibitor). We found that IL-1ß intraplantar injection into the hind-paw or long-term exposure of cultured DRG neurons to IL-1ß significantly increased the proportion of DRG neurons expressing PAR4 immunoreactivity. Real-time PCR and western blotting showed that IL-1ß treatment also significantly elevated PAR4 mRNA and protein levels in DRG neurons. This IL-1ß effect was enhanced in DRG neurons when DRG cultures were pre-treatment with the PMA. But pre-incubation with chelerythrine chloride strongly inhibited the IL-1ß-induced increase of PAR4 mRNA and protein levels. These results demonstrate that the expression of PAR4 mRNA and protein induced by IL-1ß is PKC signaling pathway dependent.

Capsaicin up-regulates protease-activated receptor-4 mRNA and protein in primary cultured dorsal root ganglion neurons.

Previous study has shown that there is a functional link between the transient receptor potential vanilloid type 1 (TRPV1) receptor and protease-activated receptor-4 (PAR4) in modulation of inflammation and pain. Capsaicin activation of TRPV1 is involved in enhancement of the expression of TRPV1 in mRNA and protein in dorsal root ganglion (DRG) in vivo. Whether capsaicin could influence expression of PAR4 in primary sensory neurons remains unknown. In the present study, expression of PAR4 in cultured rat DRG neurons was observed using immunofluorescence, real-time PCR and Western blots to examine whether increases in PAR4 mRNA and protein levels are induced by capsaicin treatment with or without pre-treatment of forskolin, a cyclic AMP/protein kinase A (cAMP/PKA) activator or PKA inhibitor fragment 14-22 (PKI14-22), a PKA inhibitor. Capsaicin treatment of cultured DRG neurons significantly increased the expression of PAR4 in mRNA and protein levels. The percentage of PAR4-, TRPV1-immunoreactive neurons and their co-localization in cultured DRG neurons increased significantly in the presence of capsaicin as compared with that in the absence of capsaicin. Compared with capsaicin-only group, pre-incubation with forskolin strongly enhanced the capsaicin-induced increase of PAR4 in mRNA and protein levels. Consistent with the involvement of PKA in the modulation of PAR4 expression, this evoked expression both at mRNA and protein levels was significantly inhibited after PKA was inhibited by pre-incubation with PKI14-22. Taken together, these results provide evidence that TRPV1 activation significantly increases the expression of PAR4 mRNA and protein levels in primary cultures of DRG neurons after capsaicin incubation. Effects of capsaicin on PAR4 expression appear to be mediated by cAMP/PKA signal pathways in DRG neurons.