Drugging evolution of antibiotic resistance at a regulatory network hub.

Evolution of antibiotic resistance is a world health crisis, fueled by new mutations. Drugs to slow mutagenesis could, as cotherapies, prolong the shelf-life of antibiotics, yet evolution-slowing drugs and drug targets have been underexplored and ineffective. Here, we used a network-based strategy to identify drugs that block hubs of fluoroquinolone antibiotic-induced mutagenesis. We identify a U.S. Food and Drug Administration- and European Medicines Agency-approved drug, dequalinium chloride (DEQ), that inhibits activation of the Escherichia coli general stress response, which promotes ciprofloxacin-induced (stress-induced) mutagenic DNA break repair. We uncover the step in the pathway inhibited: activation of the upstream "stringent" starvation stress response, and find that DEQ slows evolution without favoring proliferation of DEQ-resistant mutants. Furthermore, we demonstrate stress-induced mutagenesis during mouse infections and its inhibition by DEQ. Our work provides a proof-of-concept strategy for drugs to slow evolution in bacteria and generally.

ppGpp and RNA-polymerase backtracking guide antibiotic-induced mutable gambler cells.

Antibiotic resistance is a global health threat and often results from new mutations. Antibiotics can induce mutations via mechanisms activated by stress responses, which both reveal environmental cues of mutagenesis and are weak links in mutagenesis networks. Network inhibition could slow the evolution of resistance during antibiotic therapies. Despite its pivotal importance, few identities and fewer functions of stress responses in mutagenesis are clear. Here, we identify the Escherichia coli stringent starvation response in fluoroquinolone-antibiotic ciprofloxacin-induced mutagenesis. Binding of response-activator ppGpp to RNA polymerase (RNAP) at two sites leads to an antibiotic-induced mutable gambler-cell subpopulation. Each activates a stress response required for mutagenic DNA-break repair: surprisingly, ppGpp-site-1-RNAP triggers the DNA-damage response, and ppGpp-site-2-RNAP induces σS-response activity. We propose that RNAP regulates DNA-damage processing in transcribed regions. The data demonstrate a critical node in ciprofloxacin-induced mutagenesis, imply RNAP-regulation of DNA-break repair, and identify promising targets for resistance-resisting drugs.

Stress-Induced Mutagenesis, Gambler Cells, and Stealth Targeting Antibiotic-Induced Evolution.

Mechanisms of evolution and evolution of antibiotic resistance are both fundamental and world health problems. Stress-induced mutagenesis defines mechanisms of mutagenesis upregulated by stress responses, which drive adaptation when cells are maladapted to their environments-when stressed. Work in mutagenesis induced by antibiotics had produced tantalizing clues but not coherent mechanisms. We review recent advances in antibiotic-induced mutagenesis that integrate how reactive oxygen species (ROS), the SOS and general stress responses, and multichromosome cells orchestrate a stress response-induced switch from high-fidelity to mutagenic repair of DNA breaks. Moreover, while sibling cells stay stable, a mutable "gambler" cell subpopulation is induced by differentially generated ROS, which signal the general stress response. We discuss other evolvable subpopulations and consider diverse evolution-promoting molecules as potential targets for drugs to slow evolution of antibiotic resistance, cross-resistance, and immune evasion. An FDA-approved drug exemplifies "stealth" evolution-slowing drugs that avoid selecting resistance to themselves or antibiotics.

Two mechanisms of chromosome fragility at replication-termination sites in bacteria.

Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

Gamblers: An Antibiotic-Induced Evolvable Cell Subpopulation Differentiated by Reactive-Oxygen-Induced General Stress Response.

Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (σS) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small σS-response-"on" subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents σS induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a "gambler" cell subpopulation promote resistance evolution without risking most cells.

Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.

Fluorescent fusions of the N protein of phage Mu label DNA damage in living cells.

The N protein of phage Mu was indicated from studies in Escherichia coli to hold linear Mu chromosomes in a circular conformation by non-covalent association, and thus suggested potentially to bind DNA double-stranded ends. Because of its role in association with linear Mu DNA, we tested whether fluorescent-protein fusions to N might provide a useful tool for labeling DNA damage including double-strand break (DSB) ends in single cells. We compared N-GFP with a biochemically well documented DSB-end binding protein, the Gam protein of phage Mu, also fused to GFP. We find that N-GFP produced in live E. coli forms foci in response to DNA damage induced by radiomimetic drug phleomycin, indicating that it labels damaged DNA. N-GFP also labels specific DSBs created enzymatically by I-SceI double-strand endonuclease, and by X-rays, with the numbers of foci corresponding with the numbers of DSBs generated, indicating DSB labeling. However, whereas N-GFP forms about half as many foci as GamGFP with phleomycin, its labeling of I-SceI- and X-ray-induced DSBs is far less efficient than that of GamGFP. The data imply that N-GFP binds and labels DNA damage including DSBs, but may additionally label phleomycin-induced non-DSB damage, with which DSB-specific GamGFP does not interact. The data indicate that N-GFP labels DNA damage, and may be useful for general, not DSB-specific, DNA-damage detection.

Legonaridin, a new member of linaridin RiPP from a Ghanaian Streptomyces isolate.

Linaridins are rare linear ribosomally-synthesized and post-translationally modified peptides (RiPPs) and only two, cypemycin and SGR-1832, in this family have been identified so far. Legonaridin 1 has been discovered as a new member of linaridins through chemical isolation, peptidogenomics, comprehensive 1- and 2-D NMR and advanced Marfey's analyses from the soil bacterium Streptomyces sp. CT34, an isolate collected from Legon, Ghana. Bioinformatics analysis of the gene cluster suggested that the biosynthesis of legonaridin 1 is different from those of cypemycin and SGR-1832. Consistent with bioinformatics and peptidogenomics analyses, 1 has a total of nine post-modifications, 8 dehydrobutyrine residues and a N,N-dimethylated N-terminus with a carboxylic acid at the C-terminus. Legonaridin 1 is structurally different from the two known linaridins comprising a new subfamily. This is the first time that NMR spectroscopy is used to establish the 2-D structure of a linaridin RiPP.

Draft Genome Sequence of Streptomyces sp. Strain CT34, Isolated from a Ghanaian Soil Sample.

Presented here is a draft genome sequence of Streptomyces sp. strain CT34, which produces a novel ribosomally synthesized and posttranslationally modified peptide (RiPP). Analysis of the deduced open reading frame set identified the putative RiPP biosynthesis gene cluster, as well as other secondary metabolite gene clusters.

The role of C/EBP-α expression in human liver and liver fibrosis and its relationship with autophagy.

AIM: To investigate the expression of CCAAT enhancer binding protein-α (C/EBP-α) in normal human liver and liver fibrosis and its probable association with autophagy. METHODS: Double label immunohistochemistry was used to detect the location of C/EBP-α in hepatocytes and hepatic stellate cells (HSCs). The expression of C/EBP-α, Atg5, and Atg6 was also evaluated by immunohistochemistry in paraffin sections of human liver. HSC-T6 cells were treated with rapamycin and 3-methyladenine (3MA) to induce or inhibit autophagy, and the expression of C/EBP-α protein was detected by Western blotting. RESULTS: Double label immunohistochemistry showed that C/EBP-α was predominantly located in hepatocytes and that its expression was significantly decreased in fibrosis compared with normal liver. Atg5 expression was increased in fibrosis but was located primarily in liver septa and peri-vascular areas, which was consistent with the distribution of HSCs. In contrast, Atg6 was not expressed in normal or fibrotic liver. Treatment of HSC-T6 cells in culture with rapamycin or 3MA decreased or increased C/EBP-α expression, respectively, as shown by Western blotting. CONCLUSION: C/EBP-α was primarily expressed in hepatocytes in normal liver, but its expression decreased significantly in liver fibrosis. Autophagy might play a role in liver fibrosis through its association with C/EBP-α, but this hypothesis warrants further investigation.

[Variation of forest vegetation carbon storage and carbon sequestration rate in Liaoning Province, Northeast China].

The forest vegetation carbon stock and carbon sequestration rate in Liaoning Province, Northeast China, were predicted by using Canadian carbon balance model (CBM-CFS3) combining with the forest resource data. The future spatio-temporal distribution and trends of vegetation carbon storage, carbon density and carbon sequestration rate were projected, based on the two scenarios, i. e. with or without afforestation. The result suggested that the total forest vegetation carbon storage and carbon density in Liaoning Province in 2005 were 133.94 Tg and 25.08 t x hm(-2), respectively. The vegetation carbon storage in Quercus was the biggest, while in Robinia pseudoacacia was the least. Both Larix olgensis and broad-leaved forests had higher vegetation carbon densities than others, and the vegetation carbon densities of Pinus tabuliformis, Quercus and Robinia pseudoacacia were close to each other. The spatial distribution of forest vegetation carbon density in Liaoning Province showed a decrease trend from east to west. In the eastern forest area, the future increase of vegetation carbon density would be smaller than those in the northern forest area, because most of the forests in the former part were matured or over matured, while most of the forests in the later part were young. Under the scenario of no afforestation, the future increment of total forest vegetation carbon stock in Liaoning Province would increase gradually, and the total carbon sequestration rate would decrease, while they would both increase significantly under the afforestation scenario. Therefore, afforestation plays an important role in increasing vegetation carbon storage, carbon density and carbon sequestration rate.

Hb J-Wenchang-Wuming [α11(A9)Lys→Gln (AAG>CAG) (α2 or α1)] compromises neonatal screening for α-thalassemia with the Sebia Capillarys2 electrophoresis system.

We describe a Chinese newborn who was assumed to have α(0)-thalassemia (α(0)-thal) by determining the amount of Hb Bart's (γ4) in the cord blood, but was later shown to have only α(+)-thal. Hb J-Wenchang-Wuming [α11(A9)Lys→Gln (AAG>CAG) (α2 or α1)] was mistaken for Hb Bart's as both hemoglobin (Hb) variants have the same mobility.

A novel case of Hb Phnom Penh: codons 117/118 (+ATC) as a cause of α+ -thalassemia.

We report a novel case of Hb Phnom Penh [α117(GH5)Phe-Ile-α118(H1)Thr (α1)] detected through cord blood screening for hemoglobinopathies. Sequence analyses identified this in-frame mutation at codons 117/118 (+ATC) in exon 3 of the α1-globin gene. This mutation causes a silent α-thalassemia (α-thal).