Mutations in AcrR and RNA Polymerase Confer High-Level Resistance to Psoralen-UVA Irradiation.

DNA interstrand cross-links, such as those formed by psoralen-UVA irradiation, are highly toxic lesions in both humans and bacteria, with a single lesion being lethal in Escherichia coli. Despite the lack of effective repair, human cancers and bacteria can develop resistance to cross-linking treatments, although the mechanisms of resistance remain poorly defined. Here, we subjected E. coli to repeated psoralen-UVA exposure to isolate three independently derived strains that were >10,000-fold more resistant to this treatment than the parental strain. Analysis of these strains identified gain-of-function mutations in the transcriptional regulator AcrR and the alpha subunit of RNA polymerase that together could account for the resistance of these strains. Resistance conferred by the AcrR mutation is mediated at least in part through the regulation of the AcrAB-TolC efflux pump. Resistance via mutations in the alpha subunit of RNA polymerase occurs through a still-uncharacterized mechanism that has an additive effect with mutations in AcrR. Both acrR and rpoA mutations reduced cross-link formation in vivo. We discuss potential mechanisms in relation to the ability to repair and survive interstrand DNA cross-links. IMPORTANCE Psoralen DNA interstrand cross-links are highly toxic lesions with antimicrobial and anticancer properties. Despite the lack of effective mechanisms for repair, cells can become resistant to cross-linking agents through mechanisms that remain poorly defined. We derived resistant mutants and identified that two gain-of-function mutations in AcrR and the alpha subunit of RNA polymerase confer high levels of resistance to E. coli treated with psoralen-UVA. Resistance conferred by AcrR mutations occurs through regulation of the AcrAB-TolC efflux pump, has an additive effect with RNA polymerase mutations, acts by reducing the formation of cross-links in vivo, and reveals a novel mechanism by which these environmentally and clinically important agents are processed by the cell.

Extracellular Vesicle Proteins and MicroRNAs as Biomarkers for Traumatic Brain Injury.

Traumatic brain injury (TBI) is a heterogeneous condition, associated with diverse etiologies, clinical presentations and degrees of severity, and may result in chronic neurobehavioral sequelae. The field of TBI biomarkers is rapidly evolving to address the many facets of TBI pathology and improve its clinical management. Recent years have witnessed a marked increase in the number of publications and interest in the role of extracellular vesicles (EVs), which include exosomes, cell signaling, immune responses, and as biomarkers in a number of pathologies. Exosomes have a well-defined lipid bilayer with surface markers that reflect the cell of origin and an aqueous core that contains a variety of biological material including proteins (e.g., cytokines and growth factors) and nucleic acids (e.g., microRNAs). The presence of proteins associated with neurodegenerative changes such as amyloid-ß, α-synuclein and phosphorylated tau in exosomes suggests a role in the initiation and propagation of neurological diseases. However, mechanisms of cell communication involving exosomes in the brain and their role in TBI pathology are poorly understood. Exosomes are promising TBI biomarkers as they can cross the blood-brain barrier and can be isolated from peripheral fluids, including serum, saliva, sweat, and urine. Exosomal content is protected from enzymatic degradation by exosome membranes and reflects the internal environment of their cell of origin, offering insights into tissue-specific pathological processes. Challenges in the clinical use of exosomal cargo as biomarkers include difficulty in isolating pure exosomes, variable yields of the isolation processes, quantification of vesicles, and lack of specificity of exosomal markers. Moreover, there is no consensus regarding nomenclature and characteristics of EV subtypes. In this review, we discuss current technical limitations and challenges of using exosomes and other EVs as blood-based biomarkers, highlighting their potential as diagnostic and prognostic tools in TBI.

Limited Capacity or Involvement of Excision Repair, Double-Strand Breaks, or Translesion Synthesis for Psoralen Cross-Link Repair in Escherichia coli.

DNA interstrand cross-links are complex lesions that covalently bind complementary strands of DNA and whose mechanism of repair remains poorly understood. In Escherichia coli, several gene products have been proposed to be involved in cross-link repair based on the hypersensitivity of mutants to cross-linking agents. However, cross-linking agents induce several forms of DNA damage, making it challenging to attribute mutant hypersensitivity specifically to interstrand cross-links. To address this, we compared the survival of UVA-irradiated repair mutants in the presence of 8-methoxypsoralen-which forms interstrand cross-links and monoadducts-to that of angelicin-a congener forming only monoadducts. We show that incision by nucleotide excision repair is not required for resistance to interstrand cross-links. In addition, neither RecN nor DNA polymerases II, IV, or V is required for interstrand cross-link survival, arguing against models that involve critical roles for double-strand break repair or translesion synthesis in the repair process. Finally, estimates based on Southern analysis of DNA fragments in alkali agarose gels indicate that lethality occurs in wild-type cells at doses producing as few as one to two interstrand cross-links per genome. These observations suggest that E. coli may lack an efficient repair mechanism for this form of damage.