Riboswitch control of aminoglycoside antibiotic resistance.

The majority of riboswitches are regulatory RNAs that regulate gene expression by binding small-molecule metabolites. Here we report the discovery of an aminoglycoside-binding riboswitch that is widely distributed among antibiotic-resistant bacterial pathogens. This riboswitch is present in the leader RNA of the resistance genes that encode the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes that confer resistance to aminoglycoside antibiotics through modification of the drugs. We show that expression of the AAC and AAD resistance genes is regulated by aminoglycoside binding to a secondary structure in their 5' leader RNA. Reporter gene expression, direct measurements of drug RNA binding, chemical probing, and UV crosslinking combined with mutational analysis demonstrate that the leader RNA functions as an aminoglycoside-sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycosides antibiotic resistance.

A p21-ATD mouse model for monitoring and eliminating senescent cells and its application in liver regeneration post injury.

Cellular senescence associates with pathological aging and tissue dysfunctions. Studies utilizing mouse models for cell lineage tracings have emphasized the importance of senescence heterogeneity in different organs and cell types. Here, we constructed a p21- (Akaluc - tdTomato - Diphtheria Toxin Receptor [DTR]) (ATD) mouse model to specifically study the undefined mechanism for p21-expressing senescent cells in the aged and liver injury animals. The successful expressions of these genes enabled in vitro flow cytometric sorting, in vivo tracing, and elimination of p21-expressing senescent cells. During the natural aging process, p21-expressing cells were found in various tissues of p21-ATD mice. Eliminating p21-expressing cells in the aged p21-ATD mice recovered their multiple biological functions. p21-ATD/Fah-/- mice, bred from p21-ATD mice and fumarylacetoacetate hydrolase (Fah)-/- mice of liver injury, showed that the majority of their senescent hepatocytes were the phenotype of p21+ rather than p16+. Furthermore, eliminating the p21-expressing hepatocytes significantly promoted the engraftment of grafted hepatocytes and facilitated liver repopulation, resulting in significant recovery from liver injury. Our p21-ATD mouse model serves as an optimal model for studying the pattern and function of p21-expressing senescent cells under the physical and pathological conditions during aging.

Exploring transient global transcriptional changes induced by ascorbic acid revealed via atKAS-seq profiling.

Transcription initiates the formation of single-stranded DNA (ssDNA) regions within the genome, delineating transcription bubbles, a highly dynamic genomic process. Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) utilizing N3-kethoxal has emerged as a potent tool for mapping specific guanine positions in ssDNA on a genome-wide scale. However, the original KAS-seq method required the costly Accel-NGS Methyl-seq DNA library kit. This study introduces an optimized iteration of the KAS-seq technique, referred to as adapter-tagged KAS-seq (atKAS-seq), incorporating an adapter tagging strategy. This modification involves integrating sequencing adapters via complementary strand synthesis using random N9 tagging. Additionally, by harnessing the potential of ascorbic acid (ASC), recognized for inducing global epigenetic changes, we employed the atKAS-seq methodology to elucidate critical pathways influenced by short-term, high-dose ASC treatment. Our findings underscore that atKAS-seq enables rapid and precise analyses of transcription dynamics and enhancer activities concurrently. This method offers a streamlined, cost-efficient, and low-input approach, affirming its utility in probing intricate genomic regulatory mechanisms.

The AaBBX21-AaHY5 module mediates light-regulated artemisinin biosynthesis in Artemisia annua L.

The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood (Artemisia annua L.). Light was previously shown to promote artemisinin production, but the underlying regulatory mechanism remains elusive. In this study, we demonstrate that ELONGATED HYPOCOTYL 5 (HY5), a central transcription factor in the light signaling pathway, cannot promote artemisinin biosynthesis on its own, as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription. Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5. AaBBX21 showed a trichome-specific expression pattern. Additionally, the AaBBX21-AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1, AaMYB108, and AaORA, encoding positive regulators of artemisinin biosynthesis. Moreover, AaHY5 and AaBBX21 physically interacted with the A. annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). In the dark, AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5-AaBBX21 complex, explaining the enhanced production of artemisinin upon light exposure. Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom.

The Light- and Jasmonic Acid-Induced AaMYB108-like Positive Regulates the Initiation of Glandular Secretory Trichome in Artemisia annua L.

The plant Artemisia annua L. is famous for producing "artemisinin", which is an essential component in the treatment of malaria. The glandular secretory trichomes (GSTs) on the leaves of A. annua secrete and store artemisinin. Previous research has demonstrated that raising GST density can effectively raise artemisinin content. However, the molecular mechanism of GST initiation is not fully understood yet. In this study, we identified an MYB transcription factor, the AaMYB108-like, which is co-induced by light and jasmonic acid, and positively regulates glandular secretory trichome initiation in A. annua. Overexpression of the AaMYB108-like gene in A. annua increased GST density and enhanced the artemisinin content, whereas anti-sense of the AaMYB108-like gene resulted in the reduction in GST density and artemisinin content. Further experiments demonstrated that the AaMYB108-like gene could form a complex with AaHD8 to promote the expression of downstream AaHD1, resulting in the initiation of GST. Taken together, the AaMYB108-like gene is a positive regulator induced by light and jasmonic acid for GST initiation in A. annua.

16-membered ring macrolides and erythromycin induce ermB expression by different mechanisms.

BACKGROUND: Ribosome stalling on ermBL at the tenth codon (Asp) and mRNA stabilization are believed to be mechanisms by which erythromycin (Ery) induces ermB expression. Expression of ermB is also induced by 16-membered ring macrolides (tylosin, josamycin and spiramycin), but the mechanism underlying this induction is unknown. METHODS: We introduced premature termination codons, alanine-scanning mutagenesis and amino acid mutations in ermBL and ermBL2. RESULTS: In this paper, we demonstrated that 16-membered ring macrolides can induce ermB expression but not ermC expression. The truncated mutants of the ermB-coding sequence indicate that the regulatory regions of ermB whose expression is induced by Ery and 16-membered ring macrolides are different. We proved that translation of the N-terminal region of ermBL is key for the induction of ermB expression by Ery, spiramycin (Spi) and tylosin (Tyl). We also demonstrated that ermBL2 is critical for the induction of ermB expression by erythromycin but not by 16-membered ring macrolides. CONCLUSIONS: The translation of ermBL and the RNA sequence of the C-terminus of ermBL are critical for the induction of ermB expression by Spi and Tyl.

Genome-Wide Identification, Classification and Expression Analysis of the MYB Transcription Factor Family in Petunia.

A lot of researches have been focused on the evolution and function of MYB transcription factors (TFs). For revealing the formation of petunia flower color diversity, MYB gene family in petunia was identified and analyzed. In this study, a total of 155 MYB genes, including 40 1R-MYBs, 106 R2R3-MYBs, 7 R1R2R3-MYBs and 2 4R-MYBs, have been identified in the Petunia axillaris genome. Most R2R3 genes contain three exons and two introns, whereas the number of PaMYB introns varies from 0 to 12. The R2R3-MYB members could be divided into 28 subgroups. Analysis of gene structure and protein motifs revealed that members within the same subgroup presented similar exon/intron and motif organization, further supporting the results of phylogenetic analysis. Genes in subgroup 10, 11 and 21 were mainly expressed in petal, not in vegetative tissues. Genes in subgroup 9, 19, 25 and 27 expressed in all tissues, but the expression patterns of each gene were different. According to the promoter analysis, five R2R3-MYB and two MYB-related genes contained MBSI cis-element, which was involved in flavonoid biosynthetic regulation. PaMYB100/DPL has been reported to positively regulate to pigmentation. However, although PaMYB82, PaMYB68 and Pa1RMYB36 contained MBSI cis-element, their function in flavonoid biosynthesis has not been revealed. Consistent with existing knowledge, PaMYBs in subgroup 11 had similar function to AtMYBs in subgroup 6, genes in which played an important role in anthocyanin biosynthesis. In addition, PaMYB1 and PaMYB40 belonged to P9 (S7) and were potentially involved in regulation of flavonoid synthesis in petunia vegetative organs. This work provides a comprehensive understanding of the MYB gene family in petunia and lays a significant foundation for future studies on the function and evolution of MYB genes in petunia.

Integron-Derived Aminoglycoside-Sensing Riboswitches Control Aminoglycoside Acetyltransferase Resistance Gene Expression.

Class 1 integrons accumulate antibiotic resistance genes by site-specific recombination at aatI-1 sites. Captured genes are transcribed from a promoter located within the integron; for class 1 integrons, the first gene to be transcribed and translated normally encodes an aminoglycoside antibiotic resistance protein (either an acetyltransferase [AAC] or adenyltransferase [AAD]). The leader RNA from the Pseudomonas fluorescens class 1 integron contains an aminoglycoside-sensing riboswitch RNA that controls the expression of the downstream aminoglycoside resistance gene. Here, we explore the relationship between integron-dependent DNA recombination and potential aminoglycoside-sensing riboswitch products of recombination derived from a series of aminoglycoside-resistant clinical strains. Sequence analysis of the clinical strains identified a series of sequence variants that were associated with class I integron-derived aminoglycoside-resistant (both aac and aad) recombinants. For the aac recombinants, representative sequences showed up to 6-fold aminoglycoside-dependent regulation of reporter gene expression. Microscale thermophoresis (MST) confirmed RNA binding. Covariance analysis generated a secondary-structure model for the RNA that is an independent verification of previous models that were derived from mutagenesis and chemical probing data and that was similar to that of the P. fluorescens riboswitch RNA. The aminoglycosides were among the first antibiotics to be used clinically, and the data suggest that in an aminoglycoside-rich environment, functional riboswitch recombinants were selected during integron-mediated recombination to regulate aminoglycoside resistance. The incorporation of a functional aminoglycoside-sensing riboswitch by integron recombination confers a selective advantage for the expression of resistance genes of diverse origins.

Ascorbic Acid Reprograms Epigenome and Epitranscriptome by Reducing Fe(III) in the Catalytic Cycle of Dioxygenases.

Ascorbic acid (ASC) has been reported to stimulate DNA iterative oxidase ten-eleven translocation (TET) enzymes, Jumonji C-domain-containing histone demethylases, and potentially RNA m6A demethylases FTO and ALKBH5 as a cofactor. Although ascorbic acid has been widely investigated in reprogramming DNA and histone methylation status in vitro, in cultured cells and mouse models, its specific role in the catalytic cycle of dioxygenases remains enigmatic. Here, we systematically investigated the stimulation of ASC toward TET2, ALKBH3, histone demethylases, and FTO. We find that ASC reprograms epitranscriptome by erasing the hypermethylated m6A sites in mRNA. Biochemistry and electron spin resonance assays demonstrate that ASC enters the active pocket of dioxygenases and reduces Fe(III), either incorporated upon protein synthesis or generated upon rebounding the hydroxyl radical during oxidation, into Fe(II). Finally, we propose a remedied model for the catalytic cycle of dioxygenases by adding in the essential cofactor, ASC, which refreshes and regenerates inactive dioxygenase through recycling Fe(III) into Fe(II) in a dynamic "hit-and-run" manner.

New Therapeutic Targets and Drugs for Schizophrenia Beyond Dopamine D2 Receptor Antagonists.

Schizophrenia is a disease with a complex pathological mechanism that is influenced by multiple genes. The study of its pathogenesis is dominated by the dopamine hypothesis, as well as other hypotheses such as the 5-hydroxytryptamine hypothesis, glutamate hypothesis, immune-inflammatory hypothesis, gene expression abnormality hypothesis, and neurodevelopmental abnormality hypothesis. The first generation of antipsychotics was developed based on dopaminergic receptor antagonism, which blocks dopamine D2 receptors in the brain to exert antipsychotic effects. The second generation of antipsychotics acts by dual blockade of 5-hydroxytryptamine and dopamine receptors. From the third generation of antipsychotics onwards, the therapeutic targets for antipsychotic schizophrenia expanded beyond D2 receptor blockade to explore D2 receptor partial agonism and the antipsychotic effects of new targets such as D3, 5-HT1A, 5-HT7, and mGlu2/3 receptors. The main advantages of the second and third generation antipsychotics over first-generation antipsychotics are the reduction of side effects and the improvement of negative symptoms, and even though third-generation antipsychotics do not directly block D2 receptors, the modulation of the dopamine transmitter system is still an important part of their antipsychotic process. According to recent research, several receptors, including 5-hydroxytryptamine, glutamate, γ-aminobutyric acid, acetylcholine receptors and norepinephrine, play a role in the development of schizophrenia. Therefore, the focus of developing new antipsychotic drugs has shifted towards agonism or inhibition of these receptors. Specifically, the development of NMDARs stimulants, GABA receptor agonists, mGlu receptor modulators, cholinergic receptor modulators, 5-HT2C receptor agonists and alpha-2 receptor modulators has become the main direction. Animal experiments have confirmed the antipsychotic effects of these drugs, but their pharmacokinetics and clinical applicability still require further exploration. Research on alternative targets for antipsychotic drugs, beyond the dopamine D2 receptor, has expanded the potential treatment options for schizophrenia and gives an important way to address the challenge of refractory schizophrenia. This article aims to provide a comprehensive overview of the research on therapeutic targets and medications for schizophrenia, offering valuable insights for both treatment and further research in this field.

Riboswitch control of induction of aminoglycoside resistance acetyl and adenyl-transferases.

The acquisition of antibiotic resistance by human pathogens poses a significant threat to public health. The mechanisms that control the proliferation and expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are a historically important class of antibiotics that were introduced in the 1940s. Aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug or enzymatic modification of the target rRNA through methylation or through the overexpression of efflux pumps. In our recent paper, we reported that expression of the aminoglycoside resistance genes encoding the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes was controlled by an aminoglycoside-sensing riboswitch RNA. This riboswitch is embedded in the leader RNA of the aac/aad genes and is associated with the integron cassette system. The leader RNA can sense and bind specific aminoglycosides such that the binding causes a structural transition in the leader RNA, which leads to the induction of aminoglycoside antibiotic resistance. Specific aminoglycosides induce reporter gene expression mediated by the leader RNA. Aminoglycoside RNA binding was measured directly and, aminoglycoside-induced changes in RNA structure monitored by chemical probing. UV cross-linking and mutational analysis identified potential aminoglycoside binding sites on the RNA.

Brusatol isolated from Brucea javanica (L.) Merr. induces apoptotic death of insect cell lines.

Brucea javanica (L.) Merr. is a medicine plant distributed widely throughout Asia where its bitter fruits have been used traditionally in medicine for treating various ailments and controlling some pests. In recent years, concerns over the potential impact of synthetic pesticides on human health and environment have now become more pressing to develop environmentally friendly pesticides. In this paper, brusatol, a quassinoid, was isolated from the fruit of B. javanica, and identified using X-ray crystallographic analysis. Results showed that brusatol has potent contact toxicity (LD50, 2.91 µg/larva, 72 h) and anfieedant activity (AFC50, 17.4 mg/L, 48 h) against the third-instar larvae of Spodoptera exigua. Brusatol demonstrated cytotoxic effects to the tested insect cell lines, IOZCAS-Spex-II and Sf21, in a time- and dose-dependent manner. After brusatol treatment, apoptotic cell death with the DNA fragmentation, activation of caspase-3 and release of cytochrome c was preliminarily observed in both IOZCAS-Spex-II and Sf21. These results indicated the existence of apoptotic death with the mitochondrial-dependent pathway induced by brusatol in Sf21 and IOZCAS-Spex-II cell lines. Our studies will provide important knowledge to understand mechanisms of action of brusatol and to develop brusatol and its derivatives as insecticides.

Metformin reduces hepatocarcinogenesis by inducing downregulation of Cyp26a1 and CD8+ T cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly heterogeneous cancer with major challenges in both prevention and therapy. Metformin, adenosine monophosphate-activated protein kinase (AMPK) activator, has been suggested to reduce the incidence of HCC when used for patients with diabetes in preclinical and clinical studies. However, the possible effects of metformin and their mechanisms of action in non-diabetic HCC have not been adequately investigated. METHODS: Fah-/-  mice were used to construct a liver-injury-induced non-diabetic HCC model for exploring hepatocarcinogenesis and therapeutic potential of metformin. Changes in relevant tumour and biochemical indicators were measured. Bulk and single-cell RNA-sequencing analyses were performed to validate the crucial role of proinflammatory/pro-tumour CD8+ T cells. In vitro and in vivo experiments were performed to confirm Cyp26a1-related antitumour mechanisms of metformin. RESULTS: RNA-sequencing analysis showed that chronic liver injury led to significant changes in AMPK-, glucose- and retinol metabolism-related pathways in Fah-/- mice. Metformin prevented the formation of non-diabetic HCC in Fah-/- mice with chronic liver injury. Cyp26a1 ddexpression in hepatocytes was significantly suppressed after metformin treatment. Moreover, downregulation of Cyp26a1 occurred in conjunction with increased levels of all-trans-retinoic acid (atRA), which is involved in the activation of metformin-suppressed hepatocarcinogenesis in Fah-/- mice. In contrast, both CD8+  T-cell infiltration and proinflammatory/pro-tumour cytokines in the liver were significantly upregulated in Fah-/- mice during chronic liver injury, which was notably reversed by either metformin or atRA treatment. Regarding mechanisms, metformin regulated the decrease in Cyp26a1 enzyme expression and increased atRA expression via the AMPK/STAT3/Gadd45ß/JNK/c-Jun pathway. CONCLUSIONS: Metformin inhibits non-diabetic HCC by upregulating atRA levels and downregulating CD8+ T cells. This is the first reporting that the traditional drug metformin regulates the metabolite atRA via the Cyp26a1-involved pathway. The present study provides a potential application of metformin and atRA in non-diabetic HCC.

Translational Attenuation Mechanism of ErmB Induction by Erythromycin Is Dependent on Two Leader Peptides.

Ribosome stalling on ermBL at the tenth codon (Asp) is believed to be a major mechanism of ermB induction by erythromycin (Ery). In this study, we demonstrated that the mechanism of ermB induction by Ery depends not only on ermBL expression but also on previously unreported ermBL2 expression. Introducing premature termination codons in ermBL, we proved that translation of the N-terminal region of ermBL is the key component for ermB induced by Ery, whereas translation of the C-terminal region of ermBL did not affect Ery-induced ermB. Mutation of the tenth codon (Asp10) of ermBL with other amino acids showed that the degree of induction in vivo was not completely consistent with the data from the in vitro toe printing assay. Alanine-scanning mutagenesis of ermBL demonstrated that both N-terminal residues (R7-K11) and the latter part of ermBL (K20-K27) are critical for Ery induction of ermB. The frameshifting reporter plasmid showed that a new leader peptide, ermBL2, exists in the ermB regulatory region. Further, introducing premature termination mutation and alanine-scanning mutagenesis of ermBL2 demonstrated that the N-terminus of ermBL2 is essential for induction by Ery. Therefore, the detailed function of ermBL2 requires further study.

Bruceine a.

The title compound, C(26)H(34)O(11), known as bruceine A, is a natural quassinoid extracted from the dried fruits of Brucea javanica. Its structure consists of five fused rings including an oxygen-containing heterocyclic ring and a lactone ring. Two intra-molecular O-H⋯O links help to establish the mol-ecular conformation. In the crystal, O-H⋯O hydrogen bonds connect the mol-ecules.

Aminoglycoside riboswitch control of the expression of integron associated aminoglycoside resistance adenyltransferases.

The proliferation of antibiotic resistance has its origins in horizontal gene transfer. The class 1 integrons mediate gene transfer by assimilating antibiotic-resistance genes through site-specific recombination. For the class 1 integrons the first assimilated gene normally encodes an aminoglycoside antibiotic resistance protein which is either an aminoglycoside acetyltransferase (AAC), nucleotidyltransferase - (ANT), or adenyl transferase (AAD). An aminoglycoside-sensing riboswitch RNA in the leader RNA of AAC/AAD that controls the expression of aminoglycoside resistance genes has been previously described. Here we explore the relationship between the recombinant products of integron recombination and a series of candidate riboswitch RNAs in the 5' UTR of aad (aminoglycoside adenyltransferases) genes. The RNA sequences from the 5' UTR of the aad genes from pathogenic strains that are the products of site-specific DNA recombination by class 1 integrons were investigated. Reporter assays, MicroScale Thermophoresis (MST) and covariance analysis revealed that a functional aminoglycoside-sensing riboswitch was selected at the DNA level through integron-mediated site-specific recombination. This study explains the close association between integron recombination and the aminoglycoside-sensing riboswitch RNA.

IL22RA1/STAT3 Signaling Promotes Stemness and Tumorigenicity in Pancreatic Cancer.

Chronic inflammation is a feature of pancreatic cancer, but little is known about how immune cells or immune cell-related signals affect pancreatic cancer stemness and development. Our previous work showed that IL22/IL22RA1 plays a vital role in acute and chronic pancreatitis progression by mediating cross-talk between immune cells and acinar cells or stellate cells, respectively. Here, we find IL22RA1 is highly but heterogeneously expressed in pancreatic cancer cells, with high expression associated with poor prognosis of patients with pancreatic cancer. The IL22RA1hi population from pancreatic cancer harbored higher stemness potential and tumorigenicity. Notably, IL22 promoted pancreatic cancer stemness via IL22RA1/STAT3 signaling, establishing the mechanism of regulation of cancer stemness by microenvironmental factors. Moreover, STAT3 was indispensable for the maintenance of IL22RA1hi cells. Overall, these findings provide a therapeutic strategy for patients with PDAC with high expression of IL22RA1.Significance: IL22RA1/STAT3 signaling enhances stemness and tumorigenicity in pancreatic cancer. Cancer Res; 78(12); 3293-305. ©2018 AACR.

Characterization of DNA topoisomerase-1 in Spodoptera exigua for toxicity evaluation of camptothecin and hydoxy-camptothecin.

Camptothecin (CPT), a plant alkaloid originally isolated from the native Chinese tree, Camptotheca acuminate, exerts the toxic effect by targeting eukaryotic DNA topoisomerase 1 (DNA Topo1). Besides as potent anti-cancer agents, CPT and its derivatives are now being explored as potential pesticides for insect control. In this study, we assessed their toxicity to an insect homolog, the Topo1 protein from beet armyworms (Spodoptera exigua Hübner), a worldwide pest of many important crops. The S. exigua Topo1 gene contains an ORF of 2790 base pairs that is predicted to encode a polypeptide of 930 amino acids. The deduced polypeptide exhibits polymorphism at residue sites V420, L530, A653 and T729 (numbered according to human Topo1) among insect species, which are predicted to confer sensitivity to CPT. The DNA relaxation activity of this protein was subsequently examined using a truncated form that contained the residues 337-930 and was expressed in bacteria BL21 cells. The purified protein retained the ability to relax double-stranded DNA and was susceptible to CPT and its derivative hydroxy-camptothecin (HCPT) in a dose-dependent manner. The same inhibitory effect was also found on the native Topo1 extracted from IOZCAS-Spex-II cells, a cell line established from beet armyworms. Additionally, CPT and HCPT treatment reduced the steady accumulation of Topo1 protein despite the increased mRNA expression in response to the treatment. Our studies provide information of the S. exigua Topo1 gene and its amino acid polymorphism in insects and uncover some clues about potential mechanisms of CPT toxicity against insect pests. These results also are useful for development of more effective Topo1-targeted CPT insecticides in the future.