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 Trial XXXX (XXXX) were asked to self-report 53 PRO-CTCAE items representing 30 symptomatic AEs at 6 time points (baseline; weekly x4 during treatment; 12-weeks post-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 Trial XXXX, 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 Trial XXXX agreed to self-report using the PRO-CTCAE (median age 66; 47% female; 84% white). Median time for CRAs to learn the system was 60 minutes (range 30–240), and median time for CRAs to teach a patient to self-report was 10 minutes (range 2–60). Compliance was high, particularly during active treatment when patients self-reported at 86% of expected time points, although compliance was lower post-treatment (72%). Common reasons for non-compliance 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 adverse events 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 employing central compliance monitoring. These approaches are being incorporated into ongoing studies.

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 proteincoding 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.T he double-strand-specific endonuclease RNase III is found in bacteria and eukaryotes (12,25,33). In addition to its general role in RNA processing, RNase III is involved in the regulation of gene expression in Escherichia coli and other bacteria. Thus, RNase III autoregulates its own expression in E. coli and is also involved in the regulation of the expression of the polynucleotide phosphorylase (PNPase) gene, pnp (3, 40). There are a number of other examples of the regulation of gene expression by RNase III in E. coli and other bacteria (reviewed in references 12, 25, and 33).Streptomyces are Gram-positive soil bacteria notable for their ability to sporulate and for the production of antibiotics (6,7,11). Members of the genus Streptomyces produce nearly 70% of all antibiotics used in clinical and veterinary medicine worldwide (5). Much is known about the regulation of antibiotic production in the paradigm for the study of antibiotic production in streptomycetes, Streptomyces coelicolor (6, 7). Of particular relevance to the present report are the studies of Champness and coworkers (1, 2) on the absB locus of S. coelicolor. absB was shown to encode a homolog of E. coli RNase III, and the function of the absB product as a double-strand-specific endoribonuclease has been subsequently verified (1, 2, 10). Mutations in the absB locus reduce or abolish the production of all four of the antibiotics normally synthesized by S. coelicolor (2). Moreover, Acet...

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.The alarmone, ppGpp (GDP; 3Ј-diphosphate) regulates gene expression in bacteria. In the classical stringent response to amino acid starvation in Escherichia coli, ppGpp is synthesized on idling ribosomes by the product of the relA gene, a (p)ppGpp synthetase (6). ppGpp then inhibits the synthesis of ribosomal and transfer RNAs, decreasing the levels of ribosome and tRNA synthesis when rates of protein synthesis decrease (reviewed in references 3, 6, 31, and 35). ppGpp can also activate transcription in E. coli. E. coli mutants lacking ppGpp are auxotrophic for a number of amino acids, and ppGpp has been shown to activate transcription of the operons for those amino acids (3,6,31,35). Recent studies indicate that ppGpp interacts with RNA polymerase in concert with the effector protein DksA, and this interaction results in promoterspecific inhibition of transcription, in the case of stable RNA promoters, or the activation of transcription in the case of the promoters for amino acid biosynthesis (11,12,18). It has also been argued that ppGpp regulates growth rate in E. coli (2, 19), although there is other evidence suggesting that it is not essential to this process (15). Microarray studies indicate that the expression of several hundred genes is affected by changes in ppGpp levels in E. coli (13). In other systems, ppGpp is involved in sporulation, stress survival, and virulence (1, 14, 17).The stringent response also occurs in the soil-dwelling actinomycete, Streptomyces (36). Streptomyces species contain homologs of relA (8,22,28), and ppGpp levels increase in response to amino acid starvation in Streptomyces, as in E. coli (36). Of particular interest is the observation that ppGpp serves as both a negative and a positive regulator of antibiotic production in Streptomyces species. Thus,...

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 IIIdependent 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.Polynucleotide phosphorylase (PNPase) is a 3Ј-5Ј-exoribonuclease that functions in the phosphorolytic degradation of RNA molecules in bacteria and in eukaryotic organelles (14,21). In Escherichia coli and other bacteria, PNPase plays an important role in the degradation of mRNAs. Thus, endonucleolytic cleavage of RNA molecules generates 3Ј ends that are substrates for the action of PNPase and RNase II, an exonuclease that functions hydrolytically (8,12,28). PNPase plays another role in E. coli, at least under some circumstances. As is the case in eukaryotes, the 3Ј ends of at least some RNA molecules in bacteria are polyadenylated (31, 32). Polyadenylation facilitates the degradation of RNAs in bacteria (22,24,27). While the major enzyme responsible for RNA polyadenylation in E. coli is poly(A) polymerase I (PAP I [7]), mutants of E. coli lacking PAP I still retain the ability to polyadenylate RNAs (26), indicating that there is at least one other polyadenylating enzyme in those cells. Mohanty and Kushner have presented evidence indicating that the second PAP in E. coli is none other than PNPase (25). They argue that under appropriate conditions in vivo, PNPase can serve to degrade RNAs or synthesize poly(A) tails and that this enzyme is responsible for the G, C, and U residues that are found at low frequency in the poly(A) tails of RNAs from wild-type E. coli (25).PNPase structure, function, and expression have been studied extensively in species of the soil-dwelling, antibiotic-pro...

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 halflives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain. RNase III is a double-strand-specific endonuclease that is found in bacteria and eukaryotes (1-3). In addition to its general role in RNA processing, RNase III is involved in the regulation of gene expression in Escherichia coli and other bacteria. Thus, RNase III autoregulates its own expression in E. coli by cleaving an untranslated leader in its mRNA. This cleavage leads to an increased rate of decay of the RNase III mRNA, downregulating expression of the RNase III gene, rnc (4-6). RNase III is also involved in the regulation of the expression of the polynucleotide phosphorylase (PNPase) gene, pnp, in bacteria. RNase III cleaves a stem-loop structure in the 5= leader of the pnp transcripts in E. coli. The 3= ends produced by this cleavage then serve as targets for polyadenylation by poly(A) polymerase I, and the polyadenylated 3= ends are degraded by PNPase itself (7,8). Thus, RNase III cleavage, polyadenylation, and PNPase action on its own mRNA lead to instability of that mRNA, resulting in decreased synthesis of PNPase (7-9). There are a number of other examples of the regulation of gene expression by RNase III in E. coli and other bacteria (reviewed in references 1, 2, and 3).RNase III also plays an important role in the regulation of antibiotic synthesis in Streptomyces coelicolor. Some years ago, Champness and coworkers identified an S. coelicolor locus, which they designated absB (10). The synthesis of all four antibiotics normally produced by S. coelicolor was severely reduced in an absB mutant, C120. Specifically, they reported that the production of actinorhodin (encoded by the act gene cluster) by the C120 mutant was reduced to only 2% of the normally observed level and that the level of undecylprodigiosin (encoded by the red gene cluster) was only 15% of that normally observed (10). Production of the calcium-dependent antibiotic and of methylenomycin was also reduced. Champness and coworkers subsequently demonstrated that the absB locus encoded a homolog of RNase III, the double-strand-specific endoribonuclease discussed above, and the C120 absB mutant was shown to contain a point mutation that resulted in a change of leucine to proline in the R...