Gene expression caused by alkylating agents and cis-diamminedichloroplatinum(II) in Escherichia coli

Previous work has demonstrated heterogeneous effects of methylating agents on induction of DNA damage inducible genes in Escherichia coli. These studies employed E. coli mutants that have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents. These mutants were selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methylmethanesulfonate or N-methyl-N\u27-nitro-N-nitrosoguanidine. The current report extends these findings by analyzing gene expression caused by mechlorethamine, chloroethylnitrosoureas and cis-diamminedichloroplatinum(II) (cis-DDP). The results demonstrate heterogeneous effects by these agents on gene expression. While 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea induces alkA, other nitrosoureas, mechlorethamine, and cis-DDP do not cause expression of this gene. Further, while all nitrosoureas caused expression of aidC, mechlorethamine and cis-DDP did not. Lastly, cis-DDP caused marked expression of a sulA fusion mutant while not inducing any of the other E. coli fusion mutants

A pragmatic patient-reported outcome strategy for rare disease clinical trials: application of the EORTC item library to myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia.

BackgroundNovel, pragmatic, patient-centered strategies are needed to ensure fit-for-purpose patient-reported outcomes (PRO) instruments in clinical trial research for rare diseases such as myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and chronic myelomonocytic leukemia (CMML). The objective of the current study was to select supplemental items to add to the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life-Core 30 (QLQ-C30) to ensure content coverage of all important clinical concepts in patients with higher-risk (HR) MDS, low-blast count (LB) AML, and CMML, thus, improving the instrument's ability to detect clinically meaningful treatment benefit for this context of use.MethodsOur mixed methods approach comprised literature review, clinician consultation (n = 3), and qualitative and quantitative analysis of two stages of patient interview data (n = 14, n = 18) to select library bank items to supplement a generic cancer PRO, the EORTC QLQ-C30.ResultsUnique symptom (n = 54) and impact (n = 72) concepts were organized into conceptual frameworks of treatment benefit, compared with EORTC QLQ-C30 items and conceptual gaps identified. Supplemental items (n = 13) addressing those gaps were selected from the EORTC Item Library and tested with patients. Supplemental item endorsement frequencies met World Health Organization Quality of Life criteria, suggesting good targeting and relevance for this sample. However, three supplemental items were confirmed as problematic based upon cognitive debriefing results, and expert clinical consultations. Ultimately, 10 supplemental items (n = 7 symptom; n = 3 impact) were selected for the MDS/AML/CMML context.ConclusionSupplemental items were selected to enhance the conceptual coverage of the EORTC QLQ-C30 in the areas of fatigue, shortness of breath, and functioning

Lessons Learned From the United States Ocean Observatories Initiative

The Ocean Observatories Initiative (OOI) is a United States National Science Foundation-funded major research facility that provides continuous observations of the ocean and seafloor from coastal and open ocean locations in the Atlantic and Pacific. Multiple cycles of OOI infrastructure deployment, recovery, and refurbishment have occurred since operations began in 2014. This heterogeneous ocean observing infrastructure with multidisciplinary sampling in important but challenging locations has provided new scientific and engineering insights into the operation of a sustained ocean observing system. This paper summarizes the challenges, successes, and failures experienced to date and shares recommendations on best practices that will be of benefit to the global ocean observing community

“Charity Begins at Home”: Informal Caring Barriers to Formal Volunteering Among Older People

Formal volunteering is an important economic and social activity. In many countries, prevalence of volunteering is decreasing overall, including among older people who constitute a major volunteering resource. This qualitative study explored reasons for non-volunteering among seniors, with a focus on those who attribute their non-volunteering to their existing helping commitments. Forty-nine Australian interviewees aged 60 + years described a range of social, psychological, and temporal factors that resulted in their prioritization of informal rather than formal volunteering activities. These factors are mapped onto a theoretical framework matrix, with social identity and social capital theories appearing to possess the most explanatory power. The findings suggest that programs designed to encourage formal volunteering among older people need to be implemented in a manner that recognizes that members of this group can hold many other responsibilities that limit their ability to participate, especially those assisting in the care of multiple generations

Mutagenesis and repair of DNA damage caused by nitrogen mustard, N,N\u27-bis(2-chloroethyl)-N-nitrosourea (BCNU), streptozotocin, and mitomycin C in E. coli

Cytotoxicity and mutagenesis by streptozotocin, BCNU, nitrogen mustard, and mitomycin C were evaluated in E. coli mutants deficient in SOS repair, SOS-mediated mutagenesis, the adaptive response, and mutants that engage in aberrant mismatch repair. The results demonstrate that premutagenic lesions are caused by nitrogen mustard, BCNU and streptozotocin that are not repaired by ada or recognized by umuDC. Further, recA mutants were hypomutable after exposure to nitrogen mustard, BCNU, and streptozotocin compared to wild type. With the exception of the monofunctional nitrosourea, streptozotocin, both recA and uvrA gene products contribute to the repair of DNA damage caused by the alkylating agents tested. In the case of streptozotocin, although recA mutants were more sensitive than wild type, uvrA mutants were not. Moreover, while ada and alkA E. coli mutants showed increased sensitivity to streptozotocin, they were not more sensitive to the other alkylating agents evaluated

Studies on mutagenesis and repair induced by platinum analogs

Mutagenesis and cytotoxicity were studied in Escherichia coli by iproplatin and carboplatin, two analogs of cisplatin (CDDP) currently undergoing clinical trial. As with CDDP, mutagenesis by these agents was mediated by the umuDC gene product. In contrast to CDDP, however, mismatch repair did not substantially contribute to survival of cells after exposure to these agents since dam-3 E. coli were not more sensitive than wild type E. coli. UvrA- E. coli, however were more sensitive to these analogs demonstrating that as with CDDP, uvr endonuclease-mediated excision contributes to the repair of DNA damage induced by platinum compounds

Gene expression in E. coli after treatment with streptozotocin

Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N\u27-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants

Characterization of acquired resistance to cis-diamminedichloroplatinum (II) in BE human colon carcinoma cells

To study mechanisms underlying resistance to cis-diamminedichloroplatinum (II) (cis-DDP) we have induced resistance to this agent in BE human colon carcinoma cells. A 5-fold increase in the IC50 of resistant compared to sensitive cells was noted as analyzed by the inhibition of cellular growth. Up to a 4-fold reduction in interstrand cross-link formation by cis-DDP in resistant compared to sensitive cells was present as measured by alkaline elution. No significant differences were detectable either in the extent of DNA platination as analyzed by atomic absorption spectroscopy or in the induction of cis-DDP DNA adducts as evaluated by an enzyme-linked immunosorbent assay employing antiserum that detects intrastrand cross-links formed by cis-DDP. Further, no differences in the kinetics of excision of DNA interstrand cross-links, cis-DDP DNA adducts, or total platinum in DNA were present. Levels of glutathione, however, were increased about threefold in resistant compared to sensitive cells. Loss of resistance was associated with increased interstrand cross-link formation and declines in glutathione levels. Our results are consistent with a critical role of glutathione in preventing platinum monoadduct rearrangements resulting in lower levels of interstrand cross-links and resistance to cis-DDP in resistant BE cells