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.

Activation of HAND2-FGFR signaling pathway by lncRNA HAND2-AS1 in adenomyosis†.

Heart and neural crest derivatives expressed transcript 2 (HAND2) is a critical mediator of progesterone action in endometrial stromal cells. Silencing of Hand2 expression in mouse uterus leads to an unopposed FGFR-mediated action that causes female mice infertility. To investigate the involvement of HAND2-FGFR signaling in pathogenesis of adenomyosis, immunohistochemistry, in situ hybridization, and quantitative real-time PCR were employed to assess gene expression in the normal endometrium, the paired eutopic endometrium and ectopic lesions obtained from women with adenomyosis. DNA methylation in the regions of HAND2 promoter and the first exon was also monitored in these samples. Our results revealed that HAND2 expression were dramatically reduced, but FGF9 expression and FGFR-ERK1/2-mediated MAPK signaling pathway were enhanced in the eutopic endometrium and ectopic lesions of patients with adenomyosis compared to the normal controls. Interestingly, expression of HAND2-AS1, a long noncoding RNA that resides adjacent to HAND2 in genome, was also reduced in adenomyosis. DNA methylation analysis revealed that the bidirectional promoter between HAND2 and HAND2-AS1, and the first exon of HAND2 gene was heavily methylated in the eutopic endometrium and the ectopic lesions of adenomyosis. To investigate the regulation of gene expression by HAND2-AS1, HAND2-AS1 expression was silenced in human endometrial stromal cells. In contrast to the downregulation of HAND2 in response to HAND2-AS1 silencing, FGF9 expression was augmented significantly. Endometrial stromal cells lacking HAND2-AS1 exhibited enhanced proliferation and migration potentials. Collectively, our studies revealed a new molecular mechanism by which HAND2-AS1 is involved in the pathogenesis of adenomyosis via modulating HAND2-FGFR-mediated signaling.

Genome-wide identification of Kanamycin B binding RNA in Escherichia coli.

BACKGROUND: The aminoglycosides are established antibiotics that inhibit bacterial protein synthesis by binding to ribosomal RNA. Additional non-antibiotic aminoglycoside cellular functions have also been identified through aminoglycoside interactions with cellular RNAs. The full extent, however, of genome-wide aminoglycoside RNA interactions in Escherichia coli has not been determined. Here, we report genome-wide identification and verification of the aminoglycoside Kanamycin B binding to Escherichia coli RNAs. Immobilized Kanamycin B beads in pull-down assays were used for transcriptome-profiling analysis (RNA-seq). RESULTS: Over two hundred Kanamycin B binding RNAs were identified. Functional classification analysis of the RNA sequence related genes revealed a wide range of cellular functions. Small RNA fragments (ncRNA, tRNA and rRNA) or small mRNA was used to verify the binding with Kanamycin B in vitro. Kanamycin B and ibsC mRNA was analysed by chemical probing. CONCLUSIONS: The results will provide biochemical evidence and understanding of potential extra-antibiotic cellular functions of aminoglycosides in Escherichia coli.

The function of twister ribozyme variants in non-LTR retrotransposition in Schistosoma mansoni.

The twister ribozyme is widely distributed over numerous organisms and is especially abundant in Schistosoma mansoni, but has no confirmed biological function. Of the 17 non-LTR retrotransposons known in S. mansoni, none have thus far been associated with ribozymes. Here we report the identification of novel twister variant (T-variant) ribozymes and their function in S. mansoni non-LTR retrotransposition. We show that T-variant ribozymes are located at the 5' end of Perere-3 non-LTR retrotransposons in the S. mansoni genome. T-variant ribozymes were demonstrated to be catalytically active in vitro. In reporter constructs, T-variants were shown to cleave in vivo, and cleavage of T-variants was sufficient for the translation of downstream reporter genes. Our analysis shows that the T-variants and Perere-3 are transcribed together. Target site duplications (TSDs); markers of target-primed reverse transcription (TPRT) and footmarks of retrotransposition, are located adjacent to the T-variant cleavage site and suggest that T-variant cleavage has taken place inS. mansoni. Sequence heterogeneity in the TSDs indicates that Perere-3 retrotransposition is not site-specific. The TSD sequences contribute to the 5' end of the terminal ribozyme helix (P1 stem). Based on these results we conclude that T-variants have a functional role in Perere-3 retrotransposition.

Interactions between the 5' UTR mRNA of the spe2 gene and spermidine regulate translation in S. pombe.

The 5' untranslated regions (5' UTR) of mRNAs play an important role in the eukaryotic translation initiation process. Additional levels of translational regulation may be mediated through interactions between structured mRNAs that can adopt interchangeable secondary or tertiary structures and the regulatory protein/RNA factors or components of the translational apparatus. Here we report a regulatory function of the 5' UTR mRNA of the spe2 gene (SAM decarboxylase) in polyamine metabolism of the fission yeast Schizosaccharomyces pombe Reporter assays, biochemical experiments, and mutational analysis demonstrate that this 5' UTR mRNA of spe2 can bind to spermidine to regulate translation. A tertiary structure transition in the 5' UTR RNA upon spermidine binding is essential for translation regulation. This study provides biochemical evidence for spermidine binding to regulate translation of the spe2 gene through interactions with the 5' UTR mRNA. The identification of such a regulatory RNA that is directly associated with an essential eukaryotic metabolic process suggests that other ligand-binding RNAs may also contribute to eukaryotic gene regulation.

Interactions between SAM and the 5' UTR mRNA of the sam1 gene regulate translation in S. pombe.

The 5' untranslated region (5' UTR) of eukaryotic mRNA plays an important role in translation. Here we report the function of the 5' UTR mRNA of S-adenosylmethionine synthetase (sam1) in translational modulation in the presence of SAM in fission yeast Schizosaccharomyces pombe Reporter assays, binding and chemical probing experiments, and mutational analysis show that the 5' UTR mRNA of sam1 binds to SAM to effect translation. Translational modulation is dependent on a tertiary structure transition in the RNA upon SAM binding. The characterization of such an RNA that is directly associated with an essential metabolic process in eukaryotes provides additional evidence that ligand binding by RNAs plays an important role in eukaryotic gene regulation.

Gastrointestinal events with PARP inhibitors in cancer patients: A meta-analysis of phase II/III randomized controlled trials.

WHAT IS KNOWN AND OBJECTIVE: PARP inhibitors are currently one of the most promising PARP targeted drugs for patients with certain types of cancer. Gastrointestinal (GI) events are common adverse events for all PARP inhibitors. We conducted this meta-analysis of randomized controlled trials (RCTs) to fully investigate the incidence and the relative risk of GI events in cancer patients receiving PARP inhibitors. STUDY DESIGN: Randomized controlled trials in cancer patients treated with PARP inhibitors were retrieved, and the systematic evaluation was conducted. Embase and PubMed/Medline were searched for articles published till July 2020. RESULTS: Twenty-nine RCTs and 9529 patients were included. The present meta-analysis suggests that the use of PARP inhibitors significantly increases the risk of developing all-grade nausea (RR, 1.46; 95% CI, 1.29-1.66; p < .00001), vomiting (RR, 1.39; 95% CI, 1.17-1.64; p = .0001), diarrhoea (RR, 1.14; 95% CI, 1.06-1.23; p = .0003) and decreased appetite (RR, 1.24; 95% CI, 1.14-1.36; p < .00001), but not for constipation. And the use of these agents significantly increased the risk of high-grade nausea (RR, 1.99; 95% CI, 1.44-2.74; p < .0001), vomiting (RR, 1.54; 95% CI, 1.11-2.14; p = .01) and decreased appetite (RR, 2.03; 95% CI, 1.22-3.40; p = .007), except for diarrhoea and constipation. Nausea was the most common GI event for these agents. Patients receiving veliparib were associated with a relatively lower risk of all-grade nausea and vomiting. Patients with ovarian cancer tend to have a higher risk of all-grade nausea and vomiting than those with non-ovarian cancer. The risk of all-grade nausea and vomiting tended to be higher when PARP inhibitors treatment was longer. WHAT IS NEW AND CONCLUSION: PARP inhibitors were associated with a significant increased risk of GI events. Clinicians should be aware of these risks and perform regular monitoring.

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.

Skin Toxicities with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Cancer Patients: A Meta-Analysis of Randomized Controlled Trials.

We fully investigate the skin toxicities of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in cancer patients. The studies about randomized controlled trials in cancer treatment with EGFR-TKIs were retrieved and the systematic evaluation was conducted. The results suggest that EGFR-TKIs significantly increase the risk of skin toxicities including all-grade rash, pruritus, dry skin, and high-grade rash, pruritus. However, the risk of high-grade dry skin did not increase. Rash was the most common toxicity. Physicians should be aware of skin toxicities and should monitor cancer patients when receiving EGFR-TKIs.

Construction of noble-metal-free TiO2 nanobelt/ZnIn2S4 nanosheet heterojunction nanocomposite for highly efficient photocatalytic hydrogen evolution.

A binary nanocomposite composed of two-dimensional (2D) ultrathin ZnIn2S4 nanosheets and one-dimension (1D) TiO2 nanobelts was prepared and applied as a noble-metal-free photocatalyst for hydrogen evolution under solar-light irradiation. The TiO2 nanobelt/ZnIn2S4 nanosheet heterojunction nanocomposites show higher light absorption capacity, larger surface area and higher separation of charge carriers in comparison to pristine TiO2 and ZnIn2S4. As a result, the hydrogen production over the TiO2/ZnIn2S4 nanocomposite with 15 wt% TiO2 can reach up to 348.21 µmol · g-1 · h-1, even without noble metals, which is about 26 and 2.3 times higher than the pristine TiO2 and ZnIn2S4, respectively. Meanwhile, a possible photocatalytic mechanism of TiO2/ZnIn2S4 heterojunction nanocomposites was proposed and corroborated by photoluminescence (PL) spectroscopy and photoelectrochemical (PEC) results. This work paves a way for developing low-cost and high-efficiency noble-metal-free photocatalytic systems for solar-to-hydrogen evolution.

A chlorogenic acid-phospholipid complex ameliorates post-myocardial infarction inflammatory response mediated by mitochondrial reactive oxygen species in SAMP8 mice.

Mitochondrial reactive oxygen species (mtROS) directly stimulate the inflammatory cytokines cascades and participate in age-related changes of cardiovascular diseases. Application of small molecule targeting the mtROS is significant towards development of better therapy to combat inflammatory response after myocardial infarction (MI) in the aging heart. Chlorogenic acid (CGA) is a well-known natural compound while the clinical potential is largely stifled by its poor oral absorption. In the present study, we tested the protective effect of a novel chlorogenic acid-phospholipid complex (CGA-PC) against acute post-MI inflammation in aged senescence accelerated mouse model. 10-month-old SAMP8 mice were treated with CGA-PC (equivalent of CGA 10 or 20 mg/kg body weight) or phospholipid randomly by gavage on a daily basis for 2 weeks. mtROS, lipid peroxidation, H2O2 production and oxygen consumption were evaluated in hearts subjected to ischemia reperfusion (I/R) induced by left anterior descending artery ligation. CGA-PC significantly reduced pro-inflammatory cytokines and myocardial necrosis, accompanied by decreased oxidative stress and mitochondrial respiratory deficits. p-JNK, MnSOD and soluble cytochrome c were up-regulated in the necrotic heart tissue, while CGA-PC treatment increased the expression of MKP-1 and inhibited the downstream activation of JNK. Our study indicated that CGA-PC ameliorated post-MI inflammatory response in aging heart and that it might be a promising candidate for the clinical development of CGA.

Association between the CYP1A2 polymorphisms and risk of cancer: a meta-analysis.

The previously published data on the association between CYP1A2*1C (rs2069514) and CYP1A2*1F (rs762551) polymorphisms and cancer risk have remained controversial. Hence, we performed a meta-analysis to investigate the association between CYP1A2*1F and CYP1A2*1C polymorphisms and cancer risk under different inheritance models. Overall, significant association was observed between CYP1A2*1F and cancer risk when all the eligible studies were pooled into the meta-analysis (dominant model: OR 1.08, 95 % CI 1.02-1.15; heterozygous model: OR 1.06, 95 % CI 1.01-1.12; additive model: OR 1.07, 95 % CI 1.02-1.13). In the further stratified and sensitivity analyses, for CYP1A2*1F polymorphism, significantly increased lung cancer risk and significantly decreased bladder cancer risk were observed in Caucasians. For CYP1A2*1C polymorphism, no significant association was found in overall and all subgroup analyses. In summary, this meta-analysis suggests that CYP1A2*1F polymorphism is associated with lung cancer and bladder cancer risk in Caucasians.

Targeting mitochondrial transcription in fission yeast with ETB, an inhibitor of HSP60, the chaperone that binds to the mitochondrial transcription factor Mtf1.

Mtf1 has been characterized as a mitochondrial transcription factor and is shown to regulat mitochondrial transcription. Mtf1 has an additional function as a transcription factor for the nuclear gene srk1 in fission yeast. Hsp60 has been linked to a variety of important cellular functions such as apoptosis and the immune response. It functions mainly as a molecular chaperone that assists correct protein folding in the mitochondrion. Epolactaene tertiary butyl ester(ETB) is an inhibitor of human Hsp60 that can inhibit Hsp60 chaperone activity. In this study,we report that in fission yeast, Mtf1 binds to Hsp60 in vivo and in vitro, ETB inhibits the binding of Mtf1 and Hsp60, and inhibits mitochondrial transcription but not nuclear transcription of srk1. We propose that Hsp60 may act as a molecular chaperone that folds mitochondrial Mtf1 into a functional form and that ETB inhibits this Hsp60 chaperone activity by disrupting Mtf1 binding to Hsp60 and thus inhibits mitochondrial transcription in fission yeast.

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.

A novel function of the mitochondrial transcription factor Mtf1 in fission yeast; Mtf1 regulates the nuclear transcription of srk1.

In eukaryotic cells, Mtf1 and its homologues function as mitochondrial transcription factors for the mitochondrial RNA polymerase in the mitochondrion. Here we show that in fission yeast Mtf1 exerts a non-mitochondrial function as a nuclear factor that regulates transcription of srk1, which is a kinase involved in the stress response and cell cycle progression. We first found Mtf1 expression in the nucleus. A ChIP-chip approach identified srk1 as a putative Mtf1 target gene. Over expression of Mtf1 induced transcription of the srk1 gene and Mtf1 deletion led to a reduction in transcription of the srk1 gene in vivo. Mtf1 overexpression causes cell elongation in a srk1 dependent manner. Mtf1 overexpression can cause cytoplasmic accumulation of Cdc25. We also provide biochemical evidence that Mtf1 binds to the upstream sequence of srk1. This is the first evidence that a mitochondrial transcription factor Mtf1 can regulate a nuclear gene. Mtf1 may also have a role in cell cycle progression.

Identification and characterization of the mitochondrial RNA polymerase and transcription factor in the fission yeast Schizosaccharomyces pombe.

We have characterized the mitochondrial transcription factor (Mtf1) and RNA polymerase (Rpo41) of Schizosaccharomyces pombe. Deletion mutants show Mtf1 or Rpo41 to be essential for cell growth, cell morphology and mitochondrial membrane potential. Overexpression of Mtf1 and Rpo41 can induce mitochondrial transcription. Mtf1 and Rpo41 can bind and transcribe mitochondrial promoters in vitro and the initiating nucleotides were the same in vivo and in vitro. Mtf1 is required for efficient transcription. We discuss the functional differences between Mtf1 and Rpo41 of S. pombe with Saccharomyces cerevisiae and higher organisms. In contrast to S. cerevisiae, the established model for mitochondrial transcription, S. pombe, a petite-negative yeast, resembles higher organisms that cannot tolerate the loss of mitochondrial function. The S. pombe and human mitochondrial genomes are similar in size and much smaller than that of S. cerevisiae. This is an important first step in the development of S. pombe as an alternative and complementary model system for molecular genetic and biochemical studies of mitochondrial transcription and mitochondrial-nuclear interactions. This is the first systematic study of the cellular function and biochemistry of Rpo41 and Mtf1 in S. pombe.

Asperolide A, a marine-derived tetranorditerpenoid, induces G2/M arrest in human NCI-H460 lung carcinoma cells, is mediated by p53-p21 stabilization and modulated by Ras/Raf/MEK/ERK signaling pathway.

Here we first demonstrate that asperolide A, a very recently reported marine-derived tetranorditerpenoid, leads to the inhibition of NCI-H460 lung carcinoma cell proliferation by G2/M arrest with the activation of the Ras/Raf/MEK/ERK signaling and p53-dependent p21 pathway. Treatment with 35 µM asperolide A (2 × IC(50)) resulted in a significant increase in the proportion of G2/M phase cells, about a 2.9-fold increase during 48 h. Immunoblot assays demonstrated time-dependent inhibition of G2/M regulatory proteins. Moreover, asperolide A significantly activated MAP kinases (ERK1/2, JNK and p38 MAP kinase) by phosphorylation, and only the inhibition of ERK activation by PD98059 reversed downregulation of G2/M regulatory proteins CDC2, and suppressed upregulation of p21 and p-p53 levels. Transfection of cells with dominant-negative Ras (RasN17) mutant genes up-regulated asperolide A-induced the decrease of cyclin B1 and CDC2, suppressed Raf, ERK activity and p53-p21 expression, and at last, abolished G2/M arrest. This study indicates that asperolide A-induced G2/M arrest in human NCI-H460 lung carcinoma cells relys on the participation of the Ras/Raf/MEK/ERK signaling pathway in p53-p21 stabilization. An in vivo study with asperolide A illustrated a marked inhibition of tumor growth, and little toxcity compared to Cisplatin therapy. Overall, these findings provide potential effectiveness and a theoretical basis for the therapeutic use of asperolide A in the treatment of malignancies.

Ophiobolin-O reverses adriamycin resistance via cell cycle arrest and apoptosis sensitization in adriamycin-resistant human breast carcinoma (MCF-7/ADR) cells.

Multidrug-resistance is a major obstacle facing cancer chemotherapy. This paper demonstrates that novel compound Ophiobolin-O reverses MCF-7/ADR resistance to adriamycin (ADM). The IC50 of ADM treated MCF-7 cells was 2.02 ± 0.05 µM and 74.00 ± 0.18 µM treated MCF-7/ADR cells, about 37-fold, compared to the former. However, 0.1 µM Ophiobolin-O (less than 20% inhibition concentration) combined with ADM caused the decreased IC50 of ADM to 6.67 ± 0.98 µM, indicating it reversed ADM resistance of MCF-7/ADR cells (11-fold). Furthermore, Ophiobolin-O increased ADM-induced mitochondrial pathway apoptosis and G2/M phase arrest, which is partly due to the elevation level of ROS in MCF-7/ADR cells. As we described in this paper, the reversal effect of Ophiobolin-O may be due to the reduction of resistance-related protein P-Glycoprotein (P-gp, also known as MDR1) through inhibiting the activity of the multidrug resistance 1 (MDR1) gene promoter, which makes MCF-7/ADR cells more sensitive to ADM treatment. Assays in nude mice also showed that the combination of ADM and Ophiobolin-O significantly improved the effect of ADM.

(6,6-Dimethyl-1-phenyl-6,7-di-hydro-5H-pyrrolizin-2-yl)(thio-phen-2-yl)methanone.

In the title compound, C20H19NOS, the pyrrolizine ring is essentially planar (r.m.s. deviation = 0.001 Å) while the fused dihydro-pyrrolizine ring adopts an envelope comformation with the C atom bearing the methyl substituents as the flap. The dihedral angles between the pyrrolizine and the phenyl and thio-phene rings are 34.54 (7) and 44.93 (7)°, respectively. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into infinite zigzag chains parallel to the b-axis direction.