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Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor 1 – 6 . This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses 7 , 8 . However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of ‘orphan’ CpG islands 9 . In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA 10 , which prevents activation of the MDA5 receptor 11 . We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment. Inverted-repeat Alu elements are the main source of drug-induced immunogenic double-stranded RNAs, which are destabilized by the RNA deaminase ADAR1, thereby limiting activation of the immune response.

The non-coding RNAs (ncRNA) are RNA transcripts with different sizes, structures and biological functions that do not encode functional proteins. RNA G-quadruplexes (rG4s) have been found in small and long ncRNAs. The existence of an equilibrium between rG4 and stem−loop structures in ncRNAs and its effect on biological processes remains unexplored. For example, deviation from the stem−loop leads to deregulated mature miRNA levels, demonstrating that miRNA biogenesis can be modulated by ions or small molecules. In light of this, we report several examples of rG4s in certain types of ncRNAs, and the implications of G4 stabilization using small molecules, also known as G4 ligands, in the regulation of gene expression, miRNA biogenesis, and miRNA−mRNA interactions. Until now, different G4 ligands scaffolds were synthesized for these targets. The regulatory role of the above-mentioned rG4s in ncRNAs can be used as novel therapeutic approaches for adjusting miRNA levels.

Acidic soils, where aluminum (Al) toxicity is a major agricultural constraint, are globally widespread and are prevalent in developing countries. In sorghum, the root citrate transporter SbMATE confers Al tolerance by protecting root apices from toxic Al3+, but can exhibit reduced expression when introgressed into different lines. We show that allele-specific SbMATE transactivation occurs and is caused by factors located away from SbMATE. Using expression-QTL mapping and expression genome-wide association mapping, we establish that SbMATE transcription is controlled in a bipartite fashion, primarily in cis but also in trans. Multiallelic promoter trans-activation and ChIP analyses demonstrated that intermolecular effects on SbMATE expression arise from a WRKY and a zinc finger-DHHC transcription factor (TF) that bind to and trans-activate the SbMATE promoter. A haplotype analysis in sorghum RILs indicates that the TFs influence SbMATE expression and Al tolerance. Variation in SbMATE expression likely results from changes in tandemly repeated cis sequences flanking a transposable element (a miniature inverted repeat transposable element) insertion in the SbMATE promoter, which are recognized by the Al3+-responsive TFs. According to our model, repeat expansion in Al-tolerant genotypes increases TF recruitment and, hence, SbMATE expression, which is, in turn, lower in Al-sensitive genetic backgrounds as a result of lower TF expression and fewer binding sites. We thus show that even dominant cis regulation of an agronomically important gene can be subjected to precise intermolecular fine-tuning. These concerted cis/trans interactions, which allow the plant to sense and respond to environmental cues, such as Al3+ toxicity, can now be used to increase yields and food security on acidic soils.

Clustered, regularly interspaced short palindromic repeats (CRISPR) provide bacteria and archaea with sequence-specific, acquired defense against plasmids and phage. Because mobile elements constitute up to 25% of the genome of multidrug-resistant (MDR) enterococci, it was of interest to examine the codistribution of CRISPR and acquired antibiotic resistance in enterococcal lineages. A database was built from 16 Enterococcus faecalis draft genome sequences to identify commonalities and polymorphisms in the location and content of CRISPR loci. With this data set, we were able to detect identities between CRISPR spacers and sequences from mobile elements, including pheromone-responsive plasmids and phage, suggesting that CRISPR regulates the flux of these elements through the E. faecalis species. Based on conserved locations of CRISPR and CRISPR- cas loci and the discovery of a new CRISPR locus with associated functional genes, CRISPR3- cas , we screened additional E. faecalis strains for CRISPR content, including isolates predating the use of antibiotics. We found a highly significant inverse correlation between the presence of a CRISPR- cas locus and acquired antibiotic resistance in E. faecalis , and examination of an additional eight E. faecium genomes yielded similar results for that species. A mechanism for CRISPR- cas loss in E. faecalis was identified. The inverse relationship between CRISPR- cas and antibiotic resistance suggests that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense. IMPORTANCE For many bacteria, including the opportunistically pathogenic enterococci, antibiotic resistance is mediated by acquisition of new DNA and is frequently encoded on mobile DNA elements such as plasmids and transposons. Certain enterococcal lineages have recently emerged that are characterized by abundant mobile DNA, including numerous viruses (phage), and plasmids and transposons encoding multiple antibiotic resistances. These lineages cause hospital infection outbreaks around the world. The striking influx of mobile DNA into these lineages is in contrast to what would be expected if a self (genome)-defense system was present. Clustered, regularly interspaced short palindromic repeat (CRISPR) defense is a recently discovered mechanism of prokaryotic self-defense that provides a type of acquired immunity. Here, we find that antibiotic resistance and possession of complete CRISPR loci are inversely related and that members of recently emerged high-risk enterococcal lineages lack complete CRISPR loci. Our results suggest that antibiotic therapy inadvertently selects for enterococci with compromised genome defense. For many bacteria, including the opportunistically pathogenic enterococci, antibiotic resistance is mediated by acquisition of new DNA and is frequently encoded on mobile DNA elements such as plasmids and transposons. Certain enterococcal lineages have recently emerged that are characterized by abundant mobile DNA, including numerous viruses (phage), and plasmids and transposons encoding multiple antibiotic resistances. These lineages cause hospital infection outbreaks around the world. The striking influx of mobile DNA into these lineages is in contrast to what would be expected if a self (genome)-defense system was present. Clustered, regularly interspaced short palindromic repeat (CRISPR) defense is a recently discovered mechanism of prokaryotic self-defense that provides a type of acquired immunity. Here, we find that antibiotic resistance and possession of complete CRISPR loci are inversely related and that members of recently emerged high-risk enterococcal lineages lack complete CRISPR loci. Our results suggest that antibiotic therapy inadvertently selects for enterococci with compromised genome defense.

TNF is an important mediator in numerous inflammatory diseases, e.g., in inflammatory bowel diseases (IBDs). In IBD, acute increases in TNF production can lead to disease flares. Glucocorticoids (GCs), which are steroids that bind and activate the glucocorticoid receptor (GR), are able to protect animals and humans against acute TNF-induced inflammatory symptoms. Mice with a poor transcriptional response of GR dimer-dependent target genes were studied in a model of TNF-induced lethal inflammation. In contrast to the GRWT/WT mice, these GRdim/dim mice displayed a substantial increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GRdim/dim mice had a strong IFN-stimulated gene (ISG) signature, along with STAT1 upregulation and phosphorylation. This ISG signature was gut specific and, based on our studies with antibiotics, depended on the gut microbiota. GR dimers directly bound to short DNA sequences in the STAT1 promoter known as inverted repeat negative GRE (IR-nGRE) elements. Poor control of STAT1 in GRdim/dim mice led to failure to repress ISG genes, resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay among gut microbiota, IFNs, necroptosis, and GR in both the basal response to acute inflammatory challenges and pharmacological intervention by GCs.

Tightly regulated promoters are essential for numerous biological applications, where strong inducibility, portability, and scalability are desirable. Current systems are often incompatible with large-scale fermentations due to high inducer costs and strict media requirements. Here, we describe the bottom-up engineering of ‘Jungle Express’, an expression system that enables efficient gene regulation in diverse proteobacteria. This system is guided by EilR, a multidrug-binding repressor with high affinity to its optimized operator and cationic dyes that act as powerful inducers at negligible costs. In E. coli , the engineered promoters exhibit minimal basal transcription and are inducible over four orders of magnitude by 1 µM crystal violet, reaching expression levels exceeding those of the strongest current bacterial systems. Further, we provide molecular insights into specific interactions of EilR with its operator and with two inducers. The versatility of Jungle Express opens the way for tightly controlled and efficient gene expression that is not restricted to host organism, substrate, or scale. Tightly regulated promoters with strong inducibility and scalability are highly desirable for biological applications. Here the authors describe ‘Jungle Express’, a EilR repressor-based broad host system activated by cationic dyes.

The increasing rate of antimicrobial-resistant Neisseria gonorrhoeae poses a considerable public health threat due to the difficulty in treating gonococcal infections. This study examined antimicrobial resistance (AMR) to drugs recommended for gonorrhea treatment between 2015 and 2017, and the AMR determinants and genetic compositions of plasmids in 3 gonococcal strains with high-level penicillin resistance. We collected 117 N. gonorrhoeae isolates from patients with gonococcal infections who attended Siriraj Hospital, Bangkok, Thailand, between 2015 and 2017. Minimum inhibitory concentrations (MICs) of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, cefixime, and ceftriaxone were determined by the agar dilution method. PCR amplification and sequencing of 23S rRNA and mtrR (a negative regulator of MtrCDE efflux pump) were performed. Whole genomes of 3 PPNG strains with high-level penicillin resistance (MIC ≥ 128 μg/ml) were sequenced using Illumina and Nanopore sequencing platforms. The proportions of N. gonorrhoeae isolates with resistance were 84.6% for penicillin, 91.5% for tetracycline, and 96.6% for ciprofloxacin. All isolates were susceptible to spectinomycin, azithromycin, cefixime, and ceftriaxone. An adenine deletion within a 13 bp inverted repeat sequence in the mtrR promoter and an H105Y mutation in the mtrR coding region were found in the N. gonorrhoeae isolate with the highest azithromycin MIC value (1 μg/ml). Three high-level penicillin-resistant isolates contained nonmosaic type II penA and had mutations in penB and the mtrR coding region. All isolates with high-level penicillin resistance carried the conjugative plasmids with or without the Dutch type tetM determinant, the beta-lactamase plasmid (Rio/Toronto), and the cryptic plasmid. The gonococcal population in Thailand showed high susceptibility to ceftriaxone and azithromycin, current dual therapy recommended for gonorrhea treatment. As elevated MIC of azithromycin has been observed in 1 strain of N. gonorrhoeae, expanded and enhanced surveillance of antimicrobial susceptibility and study of genetic resistance determinants are essential to improve treatment guidelines.

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted repeat-lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form two or three intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +8 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro . Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea , causal agent of gray mold disease in fruits, vegetables, and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides a structural framework for design of novel, potent antifungal peptides for agricultural use.

A total of 65 spore-forming mercury-resistant bacteria were isolated from natural environments worldwide in order to understand the acquisition of additional genes by and dissemination of mercury resistance transposons across related Bacilli genera by horizontal gene movement. PCR amplification using a single primer complementary to the inverted repeat sequence of TnMERI1-like transposons showed that 12 of 65 isolates had a transposon-like structure. There were four types of amplified fragments: Tn5084, Tn5085, TndMER3 (a newly identified deleted transposon-like fragment) and Tn6294 (a newly identified transposon). TndMER3 is a 3.5-kb sequence that carries a merRETPA operon with no merB or transposase genes. It is related to the mer operon of Bacillus licheniformis strain FA6-12 from Russia. DNA homology analysis shows that Tn6294 is an 8.5-kb sequence that is possibly derived from TndMER3 by integration of a TnMERI1-type transposase and resolvase genes and in addition the merR2 and merB1 genes. Bacteria harboring Tn6294 exhibited broad-spectrum mercury resistance to organomercurial compounds, although Tn6294 had only merB1 and did not have the merB2 and merB3 sequences for organomercurial lyases found in Tn5084 of B. cereus strain RC607. Strains with Tn6294 encode mercuric reductase (MerA) of less than 600 amino acids in length with a single N-terminal mercury-binding domain, whereas MerA encoded by strains MB1 and RC607 has two tandem domains. Thus, TndMER3 and Tn6294 are shorter prototypes for TnMERI1-like transposons. Identification of Tn6294 in Bacillus sp. from Taiwan and in Paenibacillus sp. from Antarctica indicates the wide horizontal dissemination of TnMERI1-like transposons across bacterial species and geographical barriers. The broad-spectrum mercury resistance transposon Tn6294 (the prototype of TnMERI1-like transposons) was derived from TndMER3 by integration of transposase and mer genes and horizontally disseminated in Bacilli species worldwide.

To investigate the presence of metallo-β-lactamase (MBL) genes and the genetic environment of the New Delhi metallo-β-lactamase gene bla(NDM-1) in bacteria of food animal origin. Gram-negative bacteria with low susceptibility to imipenem (MIC>8 µg/mL) were isolated from swab samples collected from 15 animal farms and one slaughterhouse in eastern China. These bacteria were selected for phenotypic and molecular detection of known MBL genes and antimicrobial susceptibility testing. For the bla(NDM-1) positive isolate, conjugation and transformation experiments were carried out to assess plasmid transfer. Southern blotting was conducted to localize the bla(NDM-1) genes, and DNA sequencing was performed to determine the sequences of bla(NDM-1) and the flanking genes. In total, nine gram-negative bacteria of four different species presented a MBL phenotype. bla(NDM-1) was identified on a mobile plasmid named pAL-01 in an Acinetobacter lwoffii isolate of chicken origin. Transfer of pAL-01 from this isolate to E. coli J53 and JM109 resulted in resistance to multiple β-lactams. Sequence analysis revealed that the bla(NDM-1) gene is attached to an intact insertion element ISAba125, whose right inverted repeat (IR-R) overlaps with the promoter sequence of bla(NDM-1). Thus, insertion of ISAba125 likely enhances the expression of bla(NDM-1). The identification of a bla(NDM-1)- carrying strain of A. lwoffii in chickens suggests the potential for zoonotic transmission of bla(NDM-1) and has important implications for food safety.