Criteria for effective design, construction, and gene knockdown by shRNA vectors

BACKGROUND: RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized. RESULTS: To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings. CONCLUSION: The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors

Stakeholder perspectives on addressing adverse events from adjuvant cancer therapy: A qualitative study

Background: With increasing survival rates, a growing population of patients with cancer have received or will receive adjuvant therapy to prevent cancer recurrences. Patients and caregivers will confront the complexities of balancing the preventative benefits of adjuvant therapy with possible near-term or long-term adverse events (AEs). Adjuvant treatment–related AEs (from minimal to severe) can impact therapeutic adherence, quality of life, emotional and physical health, and survival. However, to the authors' knowledge, limited information is available regarding how stakeholders use or desire to use adjuvant-related AE information to inform the care of patients with cancer. Methods: A qualitative, purposeful sampling approach was used to elicit stakeholder feedback via semistructured interviews (24 interviews). Drug development, drug regulatory, clinical, payer, and patient/patient advocacy stakeholders were questioned about the generation, dissemination, and use of adjuvant treatment–related AE information to inform the care of patients with cancer. Transcripts were coded independently by 2 senior health care researchers and reconciled to identify key themes. Results: All stakeholder groups in the current study identified needed improvements in each of the following 4 areas: 1) improving the accessibility and relevance of AE-related information; 2) better integrating and implementing available information regarding AEs for decisions; 3) connecting contemporary cultural and economic value systems to the generation and use of information regarding adjuvant treatment–related AEs; and 4) addressing a lack of alignment and ownership of stakeholder efforts to improve the use of AE information in the adjuvant setting. Conclusions: Despite commonalities in the overall needs identified by the diverse stakeholders in the current study, broad systemic change has been stymied. The current study identified the lack of alignment and the absence of a central “owner” of these diffuse efforts as a previously unrecognized hurdle to realizing the desired systemic improvements. Future initiatives aimed at improving quality of life and outcomes for patients receiving adjuvant therapy through the improved use of AE information must address this challenge through innovative collectives and novel leadership strategies

The Effects of State Psychiatric Hospital Waitlist Policies on Length of Stay and Time to Readmission

This study examined the effects of a waitlist policy for state psychiatric hospitals on length of stay and time to readmission using data from North Carolina for 2004–2010. Cox proportional hazards models tested the hypothesis that patients were discharged “quicker-but-sicker” post-waitlist, as hospitals struggled to manage admission delays and quickly admit waitlisted patients. Results refute this hypothesis, indicating that waitlists were associated with increased length of stay and time to readmission. Further research is needed to evaluate patients’ clinical outcomes directly and to examine the impact of state hospital waitlists in other areas, such as state hospital case mix, local emergency departments, and outpatient mental health agencies

Methylation Analysis of Several Tumour Suppressor Genes Shows a Low Frequency of Methylation of CDKN2A and RARB in Uveal Melanomas

We have investigated the frequency of methylation of several tumour suppressor genes in uveal melanoma. As the loss of one copy of chromosome 3 (monosomy 3), which is found in about half of these tumours, is tightly associated with metastatic disease, a special emphasis was laid on genes located on this chromosome, including the fragile histidine triad (FHIT), von Hippel–Lindau (VHL), β-catenin (CTNNB1), activated leukocyte cell adhesion molecule (ALCAM) and retinoic acid receptor-β2 (RARB) genes. In addition, the methylation patterns of the CpG-rich regions 5′ of the E-cadherin (CDH1), p16/cyclin-dependent kinase inhibitor 2 A (CDKN2A) and retinoblastoma (RB1) genes were analysed by bisulphite genomic sequencing or methylation-specific PCR (MSP). Furthermore, the SNRPN and D15S63 loci, which are located in the imprinted region of chromosome 15, were included in the study. Aberrant methylation was detected in nine of 40 tumours analysed: The imprinted SNRPN and D15S63 loci were hypermethylated in three tumours, all of which retained both copies of chromosome 3. Methylated RARB alleles were detected in three tumours, whereas in three other tumours CDKN2A was found to be methylated. As we did not find RARB and CDKN2A preferentially methylated in tumours with monosomy 3, which is a significant predictor of metastatic disease, we suggest that these genes may play a causative role in the formation of uveal melanoma but not in the development of metastases

A novel Vision Zero leadership training model to support collaboration and strategic action planning

Introduction: While collaboration and cooperation are regarded as foundational to Vision Zero (VZ) and Safe Systems initiatives, there is little guidance on structuring VZ collaboration, conducting collaborative goal setting, and aligning tangible action across organizations. As part of a larger VZ mutual learning model, we developed a VZ Leadership Team Institute to support communities in collaborative VZ strategic planning and goal setting. The purpose of this paper is to describe the development and evaluation of the Institute, which can serve as a foundation for other initiatives seeking to move VZ planning and implementation forward in a collaborative, systems-aware manner.Methods: In June 2021, eight multi-disciplinary teams of 3–6 persons each (n = 42 participants) attended the Institute, representing leaders from communities of various sizes. Surveys were administered pre, immediately post, and 6 months following the Institute. We measured confidence in a range of skills (on a 5-point scale, 1: not confident to 5: very confident). Surveys also measured coalition collaboration pre-Institute and 6 months post-Institute (on a 4-point scale, 1: strongly disagree to 4: strongly agree).Results: The largest increases in confidence from pre- to immediately post-Institute were for collaboratively drafting objectives and actions for VZ goals (pre-mean: 2.6, SD: 0.9 to post-mean: 3.8, SD: 0.9); incorporating equity into goals (pre-mean: 2.8, SD: 1.0 to post-mean: 3.9, SD: 0.8); and knowing how to keep VZ planning and implementation efforts on track (pre-mean: 2.6, SD: 1.0 to post-mean: 3.7, SD: 0.7). For all measures, average confidence in skills decreased from immediately post-Institute to 6 months post-Institute, but remained greater than average scores pre-Institute. Several measures of coalition collaboration maintained high agreement across time, and mean agreement increased for reporting that the future direction of the coalition was clearly communicated to everyone (pre-mean: .6, SD: 0.8; 6 months post-mean: 3.1, SD: 0.4). However, average scores decreased for feeling like the coalition had adequate staffing (pre-mean: 3.0, SD: 0.6; 6 months post-mean: 2.3, SD: 0.5).Discussion: The Institute utilized innovative content, tools, and examples to support VZ coalitions’ collaborative and systems-aware planning and implementation processes. As communities work toward zero transportation deaths and serious injuries, providing effective support models to aid multidisciplinary planning and action around a Safe Systems approach will be important to accelerate progress toward a safer transportation system