Usability testing of the Internet program: "Teens Taking Charge: Managing My Transplant Online".

Adolescents with SOT demonstrate high rates of medication non-adherence and higher rates of graft loss compared to all other age groups. Self-management interventions encompass information-based material designed to achieve disease-related learning and changes in the participant's knowledge and skill acquisition, while providing social support. These interventions have had some success in chronic disease populations by reducing symptoms and promoting self-efficacy and empowerment. Using findings from a needs assessment, an Internet-based self-management program, Teens Taking Charge: Managing My Transplant Online, for youth with SOT was developed. This program contains information on transplant, self-management and transition skills, and opportunities for peer support. The purpose of this study was to determine the usability and acceptability of the initial three modules (Medication and Vaccines; Diet after Transplant; and Living with a Transplant Organ) of the online program from the perspectives of youth with SOT. Participants were recruited from SOT clinics at a large pediatric tertiary care center in Canada. Three iterative cycles (seven patients per iteration) of usability testing took place to refine the Web site prototype. Study procedures involved participants finding items from a standardized list of features and talking aloud about issues they encountered, followed by a semi-structured interview to generate feedback about what they liked and disliked about the program. All 21 patients (mean age = 14.9 yr) found the Web site content to be trustworthy, they liked the picture content, and they found the videos of peer experiences to be particularly helpful. Participants had some difficulties finding information within submodules and suggested a more simplistic design with easier navigation. This web-based intervention is appealing to teenagers and may foster improved self-management with their SOT. Nine additional teen and two parent modules are being developed, and the completed Web site will undergo usability testing. In the future, a randomized control trial will determine the feasibility and effectiveness of this online self-management program on adherence, self-efficacy, and transition skills.

Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.

The T7 concatemer junction sequence interferes with expression from a downstream T7 promoter in vivo.

A recently described new signal for transcription termination in vitro by T7 RNA polymerase has now been tested in vivo. This signal, identified during transcription of the cloned human preproparathyroid hormone (PTH) gene, is also found in the phage T7 genome, at the concatemer junction (CJ). We introduced the 17-bp concatemer junction sequence at the ends of a test gene and control gene (both derived from T7 gene 9) in a T7 vector previously used to study effects of rare codons on expression. The CJ elements replaced the original vector's RNase III processing sites, and a new T7 promoter was also introduced to drive the downstream (control) gene. We assayed for test and control gene mRNA and protein by direct labeling with [32P]phosphate and [35S]methionine. The altered vector with CJ sequences (pCT1.1) expressed the upstream test gene, but showed poor expression of the downstream control gene. No discrete T7 mRNA bands could be discerned by direct labeling with 32P. A precursor vector with only the control gene in single copy expressed the protein much better, suggesting that the inhibition of control gene expression in pCT1.1 was a result of the upstream CJ element at the 3' end of the test gene. RT-PCR experiments were consistent with readthrough and possibly pausing at CJ. An RNA folding program predicts a highly stable secondary structure between the upstream CJ element and the control gene's translation start signals. These data support an interpretation that the CJ element is ineffective as a T7 transcription terminator in vivo in this vector, and that structure of the readthrough transcript blocks ribosome access to the downstream translation start. The readthrough transcripts are also likely to be less stable than properly terminated or processed T7 mRNA, because levels of test protein expression in pCT1.1 were reduced compared to original vector, and basal expression was negligible, while the original codon test vector shows substantial basal expression.

Compliance with cyclosporine in adolescent renal transplant recipients.

Inadequate compliance with prescribed medication regimens in children is complex and poorly understood. We measured the extent and pattern of noncompliance with cyclosporine in our adolescent renal transplant population and attempted to determine factors associated with poor compliance. After informed consent, each patient was provided cyclosporine capsules in a medication bottle equipped with an electronic monitoring device (MEMS-4) in the lid. Of the 24 patients eligible, 19 patients (8 female, 11 male) completed the study. Four (21%) patients took less than 80% of the prescribed cyclosporine doses. Five (26%) patients took drug holidays involving > or = consecutive doses. There was a trend towards improved compliance with the evening dose (88.5% vs. 93.4%, P = 0.09) and a downward trend in compliance over the course of the study (P = 0.17). None of the variables tested were found to be associated with noncompliance. Experienced physicians and nurses were able to identify 2 of the 4 individuals who were identified by MEMS as noncompliant. Additionally, 2 of the 4 noncompliance patients demonstrated low cyclosporine trough levels (< 50 ng/ml). Noncompliance with cyclosporine regimens occurs commonly in adolescent renal transplant recipients. Unexpectedly low cyclosporine levels are strongly suggestive of noncompliance, whereas other variables, including prediction by physicians and nurses intimately involved in the care, were not reflective of noncompliance.