Functional rescue of dystrophin deficiency in mice caused by frameshift mutations using Campylobacter jejuni Cas9

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout (KO) mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the eGFP gene in the tibialis anterior muscle of the Dmd KO mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out-of-frame to in-frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9 has great potential for the treatment of DMD and other neuromuscular diseases

Hepatic retransplantation in cholestatic liver disease: Impact of the interval to retransplantation on survival and resource utilization

The aim of our study was to quantitatively assess the impact of hepatic retransplantation on patient and graft survival and resource utilization. We studied patients undergoing hepatic retransplantation among 447 transplant recipients with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) at 3 transplantation centers. Cox proportional hazards regression analysis was used for survival analysis. Measures of resource utilization included the duration of hospitalization, length of stay in the intensive care unit, and the duration of transplantation surgery. Forty-six (10.3%) patients received 2 or more grafts during the follow-up period (median, 2.8 years). Patients who underwent retransplantation had a 3.8-fold increase in the risk of death compared with those without retransplantation (P < .01). Retransplantation after an interval of greater than 30 days from the primary graft was associated with a 6.7-fold increase in the risk of death (P < .01). The survival following retransplantations performed 30 days or earlier was similar to primary transplantations. Resource utilization was higher in patients who underwent multiple consecutive transplantations, even after adjustment for the number of grafts during the hospitalization. Among cholestatic liver disease patients, poor survival following hepatic retransplantation is attributed to late retransplantations, namely those performed more than 30 days after the initial transplantation. While efforts must be made to improve the outcome following retransplantation, a more critical evaluation may be warranted for late retransplantation candidates

Microbicides 2008 conference: From discovery to advocacy

Recently revised statistics show the number of individuals living with HIV at over 33 million worldwide, with 68% being in sub-Saharan Africa. Current HIV prevention methods, such as condom use, monogamy and abstinence, are not always feasible. The need for improved HIV preventative technologies remains urgent. Of these, microbicides represent a promising female-initiated preventative method. Microbicides are designed to be applied vaginally to prevent HIV and STI acquisition. Research is also being undertaken to assess the safety of the product during rectal application

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases. Koo et al. demonstrate that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. This study provides the therapeutic utility of CjCas9 for the treatment of Duchenne muscular dystrophy and other neuromuscular diseases

Normal cognition in transgenic BRI2-Aβ mice

BACKGROUND: Recent research in Alzheimer’s disease (AD) field has been focused on the potential role of the amyloid-β protein that is derived from the transmembrane amyloid precursor protein (APP) in directly mediating cognitive impairment in AD. Transgenic mouse models overexpressing APP develop robust AD-like amyloid pathology in the brain and show various levels of cognitive decline. In the present study, we examined the cognition of the BRI2-Aβ transgenic mouse model in which secreted extracellular Aβ1-40, Aβ1-42 or both Aβ1-40/Aβ1-42 peptides are generated from the BRI-Aβ fusion proteins encoded by the transgenes. BRI2-Aβ mice produce high levels of Aβ peptides and BRI2-Aβ1-42 mice develop amyloid pathology that is similar to the pathology observed in mutant human APP transgenic models. RESULTS: Using established behavioral tests that reveal deficits in APP transgenic models, BRI2-Aβ1-42 mice showed completely intact cognitive performance at ages both pre and post amyloid plaque formation. BRI2-Aβ mice producing Aβ1-40 or both peptides were also cognitively intact. CONCLUSIONS: These data indicate that high levels of Aβ1-40 or Aβ1-42, or both produced in the absence of APP overexpression do not reproduce memory deficits observed in APP transgenic mouse models. This outcome is supportive of recent data suggesting that APP processing derivatives or the overexpression of full length APP may contribute to cognitive decline in APP transgenic mouse models. Alternatively, Aβ aggregates may impact cognition by a mechanism that is not fully recapitulated in these BRI2-Aβ mouse models

Contraception determinants in youths of Sierra Leone are largely behavioral

Background: Sexual initiation occurs early in Sierra Leone. This study aims to analyze the determinants of condom and/or contraceptive use among a representative sample of young persons (10 to 24 years) in Sierra Leone. Methods: This is a secondary analysis of data from a study conducted to monitor the implementation of a UNFPA package of interventions directed to improve SRH in young people of Sierra Leone. This assessment was conducted in 2016 at the end of the Ebola outbreak. In consequence, determinants linked to healthy lifestyle behaviors and UNFPA interventions were explored in addition to the usual determinants: socio demographic and sexual lifestyle. This study is a household quantitative survey with open ended questions used to illustrate and complete the analysis. Results: A total of 1409 young people were interviewed: of these, 216 boys and 381 girls were sexually active. Those who were pregnant or wished for pregnancy were excluded, leaving 194 boys and 268 girls for the analysis of determinants. The proportion of young people using neither condom nor other contraception at their last sexual intercourse in the whole sample was 40.5% and there was no statistically significant difference between boys and girls (42.3 vs 39.2; P = 0.504). Determinants were assessed and, after multivariable analysis, results differed between boys and girls and showed the importance of behavioral aspects. Four determinants were common to boys and girls: literacy, distance, negotiation capacity and hand washing. However, the distance factor for girls was to the health facility and for boys it was to school. Three more determinants remained in the boy's model: sleeping under a bednet, number of sexual partners and knowledge of contraceptive methods. Opinions about condoms and contraception revealed important barriers; opposition to contraceptive use was the main reason for non-use for both boys and girls, while lack of access was an important reason for boys. Conclusion: There is a need to reach out to the 40% of young people who are sexually active and neither pregnant nor with pregnancy desire, and are not using condom or contraception

Quantitative transcription factor binding kinetics at the single-molecule level

We have investigated the binding interaction between the bacteriophage lambda repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large, step-wise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated and dissociated from individually labeled single molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants, ka and kd respectively. We resolved in detail how ka and kd varied as a function of 3 control parameters, the DNA length L, the CI dimer concentration, and the binding affinity. Our results show that although interaction with non-operator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking non-operator DNA.Comment: 34 pages, 10 figures, accepted by Biophysical Journa

Effect of a consumer-focused website for low back pain on health literacy, treatment choices, and clinical outcomes: randomized controlled trial

BACKGROUND: The internet is used for information related to health conditions, including low back pain (LBP), but most LBP websites provide inaccurate information. Few studies have investigated the effectiveness of internet resources in changing health literacy or treatment choices. OBJECTIVE: This study aims to evaluate the effectiveness of the MyBackPain website compared with unguided internet use on health literacy, choice of treatments, and clinical outcomes in people with LBP. METHODS: This was a pragmatic, web-based, participant- and assessor-blinded randomized trial of individuals with LBP stratified by duration. Participants were randomly allocated to have access to the evidence-based MyBackPain website, which was designed with input from consumers and expert consensus or unguided internet use. The coprimary outcomes were two dimensions of the Health Literacy Questionnaire (dimension 2: “having sufficient information to manage my health;” dimension 3: “actively managing my health;” converted to scores 1-100) at 3 months. Secondary outcomes included additional Health Literacy Questionnaire dimensions, quality of treatment choices, and clinical outcomes. RESULTS: A total of 453 participants were recruited, and 321 (70.9%) completed the primary outcomes. Access to MyBackPain was not superior to unguided internet use on primary outcomes (dimension 2: mean difference −0.87 units, 95% CI −3.56 to 1.82; dimension 3: mean difference −0.41 units, 95% CI −2.78 to 1.96). Between-group differences in other secondary outcomes had inconsistent directions and were unlikely to be clinically important, although a small improvement of unclear importance in the quality of stated treatment choices at 1 month was found (mean difference 0.93 units, 95% CI 0.03 to 1.84). CONCLUSIONS: MyBackPain was not superior to unguided internet use for health literacy, but data suggest some short-term improvement in treatment choices. Future research should investigate if greater interactivity and engagement with the website may enhance its impact. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12617001292369; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=372926 INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1136/bmjopen-2018-02751