Feasibility of Patient Reporting of Symptomatic Adverse Events via the Patient-Reported Outcomes Version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE) in a Chemoradiotherapy Cooperative Group Multicenter Clinical Trial.

PURPOSE: To assess the feasibility of measuring symptomatic adverse events (AEs) in a multicenter clinical trial using the National Cancer Institute's Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE). METHODS AND MATERIALS: Patients enrolled in NRG Oncology's RTOG 1012 (Prophylactic Manuka Honey for Reduction of Chemoradiation Induced Esophagitis-Related Pain during Treatment of Lung Cancer) were asked to self-report 53 PRO-CTCAE items representing 30 symptomatic AEs at 6 time points (baseline; weekly ×4 during treatment; 12 weeks after treatment). Reporting was conducted via wireless tablet computers in clinic waiting areas. Compliance was defined as the proportion of visits when an expected PRO-CTCAE assessment was completed. RESULTS: Among 226 study sites participating in RTOG 1012, 100% completed 35-minute PRO-CTCAE training for clinical research associates (CRAs); 80 sites enrolled patients, of which 34 (43%) required tablet computers to be provided. All 152 patients in RTOG 1012 agreed to self-report using the PRO-CTCAE (median age 66 years; 47% female; 84% white). Median time for CRAs to learn the system was 60 minutes (range, 30-240 minutes), and median time for CRAs to teach a patient to self-report was 10 minutes (range, 2-60 minutes). Compliance was high, particularly during active treatment, when patients self-reported at 86% of expected time points, although compliance was lower after treatment (72%). Common reasons for noncompliance were institutional errors, such as forgetting to provide computers to participants; patients missing clinic visits; Internet connectivity; and patients feeling "too sick." CONCLUSIONS: Most patients enrolled in a multicenter chemoradiotherapy trial were willing and able to self-report symptomatic AEs at visits using tablet computers. Minimal effort was required by local site staff to support this system. The observed causes of missing data may be obviated by allowing patients to self-report electronically between visits, and by using central compliance monitoring. These approaches are being incorporated into ongoing studies.

Transcription of the rpsO-pnp operon of Streptomyces coelicolor involves four temporally regulated, stress responsive promoters.

Primer extension with RNA from an RNase III null mutant of Streptomyces coelicolor M145 and a primer complementary to the polynucleotide phosphorylase gene revealed two major extension products. Two different extension products were observed using RNA from either wild type M145 or the null mutant with a primer complementary to rpsO. Mapping of the 5'-ends of these extension products to the rpsO-pnp intergenic region indicated that all four putative transcription start sites were preceded by possible promoter sequences. These putative promoters were synthesized by the PCR and cloned into pIPP2, a xylE-based streptomycete promoter probe vector. Transfer of the pIPP2 derivatives to S. coelicolor and catechol dioxygenase assays demonstrated that all four cloned fragments had promoter activity in vivo. The activities of the four promoters changed over the course of growth of S. coelicolor and studies in three sigma factor mutant strains demonstrated that three of the promoters were σ(B) dependent. Northern blotting studies showed that the levels of the rpsO-pnp transcripts remained relatively constant over the course of growth of S. coelicolor M145, but that on a molar basis, the levels of the readthrough and pnp transcripts were considerably lower than those of rpsO. PNPase is a cold shock protein in S. coelicolor and the activity of the rpsO-pnp promoters increased during cold shock at 10°, resulting in a two-fold increase in PNPase activity, compared with the activity at 30°.

RNase III is required for actinomycin production in Streptomyces antibioticus.

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain.

RNA-Seq and RNA immunoprecipitation analyses of the transcriptome of Streptomyces coelicolor identify substrates for RNase III.

RNase III is a key enzyme in the pathways of RNA degradation and processing in bacteria and has been suggested as a global regulator of antibiotic production in Streptomyces coelicolor. Using RNA-Seq, we have examined the transcriptomes of S. coelicolor M145 and an RNase III (rnc)-null mutant of that strain. RNA preparations with reduced levels of structural RNAs were prepared by subtractive hybridization prior to RNA-Seq analysis. We initially identified 7,800 transcripts of known and putative protein-coding genes in M145 and the null mutant, JSE1880, along with transcripts of 21 rRNA genes and 65 tRNA genes. Approximately 3,100 of the protein-coding transcripts were categorized as low-abundance transcripts. For further analysis, we selected those transcripts of known and putative protein-coding genes whose levels changed by ≥ 2-fold between the two S. coelicolor strains and organized those transcripts into 16 functional categories. We refined our analysis by performing RNA immunoprecipitation of the mRNA preparation from JSE1880 using a mutant RNase III protein that binds to transcripts but does not cleave them. This analysis identified ca. 800 transcripts that were enriched in the RNA immunoprecipitates, including 28 transcripts whose levels also changed by ≥ 2-fold in the RNA-Seq analysis. We compare our results with those obtained by microarray analysis of the S. coelicolor transcriptome and with studies describing the characterization of small noncoding RNAs. We have also used the RNA immunoprecipitation results to identify new substrates for RNase III cleavage.

Expression of a polycistronic messenger RNA involved in antibiotic production in an rnc mutant of Streptomyces coelicolor.

RNase III is a double strand specific endoribonuclease that is involved in the regulation of gene expression in bacteria. In Streptomyces coelicolor, an RNase III (rnc) null mutant manifests decreased ability to synthesize antibiotics, suggesting that RNase III globally regulates antibiotic production in that species. As RNase III is involved in the processing of ribosomal RNAs in S. coelicolor and other bacteria, an alternative explanation for the effects of the rnc mutation on antibiotic production would involve the formation of defective ribosomes in the absence of RNase III. Those ribosomes might be unable to translate the long polycistronic messenger RNAs known to be produced by operons containing genes for antibiotic production. To examine this possibility, we have constructed a reporter plasmid whose insert encodes an operon derived from the actinorhodin cluster of S. coelicolor. We show that an rnc null mutant of S. coelicolor is capable of translating the polycistronic message transcribed from the operon. We show further that RNA species with the mobilities expected for mature 16S and 23S ribosomal RNAs are produced in the rnc mutant even though the mutant contains higher levels of the 30S rRNA precursor than the wild-type strain.

RNase III-dependent expression of the rpsO-pnp operon of Streptomyces coelicolor.

We have examined the expression of the rpsO-pnp operon in an RNase III (rnc) mutant of Streptomyces coelicolor. Western blotting demonstrated that polynucleotide phosphorylase (PNPase) levels increased in the rnc mutant, JSE1880, compared with the parental strain, M145, and this observation was confirmed by polymerization assays. It was observed that rpsO-pnp mRNA levels increased in the rnc mutant by 1.6- to 4-fold compared with M145. This increase was observed in exponential, transition, and stationary phases, and the levels of the readthrough transcript, initiated upstream of rpsO in the rpsO-pnp operon; the pnp transcript, initiated in the rpsO-pnp intergenic region; and the rpsO transcript all increased. The increased levels of these transcripts in JSE1880 reflected increased chemical half-lives for each of the three. We demonstrated further that overexpression of the rpsO-pnp operon led to significantly higher levels of PNPase activity in JSE1880 compared to M145, reflecting the likelihood that PNPase expression is autoregulated in an RNase III-dependent manner in S. coelicolor. To explore further the increase in the level of the pnp transcript initiated in the intergenic region in JSE1880, we utilized that transcript as a substrate in assays employing purified S. coelicolor RNase III. These assays revealed the presence of hitherto-undiscovered sites of RNase III cleavage of the pnp transcript. The position of those sites was determined by primer extension, and they were shown to be situated in the loops of a stem-loop structure.

(p)ppGpp inhibits polynucleotide phosphorylase from streptomyces but not from Escherichia coli and increases the stability of bulk mRNA in Streptomyces coelicolor.

ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in Streptomyces species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli. We have shown that (p)ppGpp inhibits the activities of both Streptomyces PNPases but not the E. coli enzyme. The inhibition kinetics for polymerization using the Streptomyces enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the rpsO-pnp operon of S. coelicolor. We have shown further that the chemical stability of mRNA increases during the stationary phase in S. coelicolor and that induction of a plasmid-borne copy of relA in a relA-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition in vitro may reflect a role of ppGpp in the regulation of antibiotic production in vivo.