ABSTRACT
For several decades, Mfd has been studied as the bacterial transcription-coupled repair factor. However, recent observations indicate that this factor influences cell functions beyond DNA repair. Our lab recently described a role for Mfd in disulfide stress that was independent of its function in nucleotide excision repair and base excision repair. Because reports showed that Mfd influenced transcription of single genes, we investigated the global differences in transcription in wild-type and mfd mutant growth-limited cells in the presence and absence of diamide. Surprisingly, we found 1,997 genes differentially expressed in Mfd- cells in the absence of diamide. Using gene knockouts, we investigated the effect of genetic interactions between Mfd and the genes in its regulon on the response to disulfide stress. Interestingly, we found that Mfd interactions were complex and identified additive, epistatic, and suppressor effects in the response to disulfide stress. Pathway enrichment analysis of our RNASeq assay indicated that major biological functions, including translation, endospore formation, pyrimidine metabolism, and motility, were affected by the loss of Mfd. Further, our RNASeq findings correlated with phenotypic changes in growth in minimal media, motility, and sensitivity to antibiotics that target the cell envelope, transcription, and DNA replication. Our results suggest that Mfd has profound effects on the modulation of the transcriptome and on bacterial physiology, particularly in cells experiencing nutritional and oxidative stress.
ABSTRACT
BACKGROUND: Despite well-described analytical effects of autoantibodies against cardiac troponin (cTn) I on experimental assays, no study has systematically examined their impact on cTn assays in clinical use. We determined the effects of endogenous antibodies on 5 different cTnI assays and a cTnT assay. METHODS: cTn was measured by 6 methods: Siemens hs-cTnI Centaur, Siemens hs-cTnI Vista, Abbott hs-cTnI Architect, Beckman hs-cTnI Access, Beckman cTnI Access, and Roche hs-cTnT Elecsys. Measurements were repeated on 5 assays (all except Siemens hs-cTnI Vista) following immunoglobulin depletion by incubation with protein A. Low recovery of cTnI (<40%) following immunoglobulin depletion was considered positive for macro-cTnI. Protein A findings were validated by gel filtration chromatography and polyethylene glycol precipitation. RESULTS: In a sample of 223 specimens selected from a community laboratory that uses the Siemens hs-cTnI Centaur assay and from which cTn was requested, 76% of samples demonstrated increased cTnI (median, 88 ng/L; interquartile range, 62-204 ng/L). Macro-cTnI was observed in 123 (55%) of the 223 specimens. Comparisons of cTnI assays markedly improved once patients with macro-cTnI were removed. Passing-Bablok regression analysis between hs-cTnI assays demonstrated different slopes for patients with and without macro-cTnI. In patients with macro-cTnI, 89 (72%) showed no effect on the recovery of cTnT, whereas 34 (28%) had reduced recovery of cTnT. The proportion of results above the manufacturers' 99th percentile varied with the cTn assay and macro-cTnI status. CONCLUSION: We suggest that the observed discrepancy between hs-cTnI assays may be attributed in part to the presence of macro-cTnI.
