AAV5 delivery of CRISPR-Cas9 supports effective genome editing in mouse lung airway.

Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. The NIH Somatic Cell Genome Editing (SCGE) Consortium is developing safe and effective methods for genome editing in disease tissues. Methods developed by consortium members are independently validated by the SCGE small animal testing center to establish rigor and reproducibility. We have developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. After intratracheal injection of the AAV serotype 5 (AAV5)-packaged S. pyogenes Cas9 (SpCas9) and single guide RNAs (sgRNAs), we observed ∼19%-26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. This highly effective AAV delivery platform will facilitate the study of therapeutic genome editing in the lung and other tissue types.

UBR1 Promotes Sex-Dependent ACE2 Ubiquitination in Hypertension.

Background: Angiotensin (Ang)-II impairs the function of the antihypertensive enzyme ACE2 by promoting its internalization, ubiquitination and degradation thus contributing to hypertension. However, few ACE2 ubiquitination partners have been identified and their role in hypertension remains unknown. Methods: Proteomics and bioinformatic analysis were used to identify ACE2 ubiquitination partners in the brain, heart, and kidney from Ang-II-infused C57BL6/J mice from both sexes and validated the interaction between UBR1 and ACE2 in cells. Central and peripheral UBR1 knockdown was then performed in male mice to investigate its role in the maintenance of hypertension. Results: Proteomics analysis from hypothalamus identified UBR1 as a potential E3 ligase promoting ACE2 ubiquitination. Enhanced UBR1 expression, associated with ACE2 reduction, was confirmed in various tissues from hypertensive male mice and human samples. Treatment of endothelial and smooth muscle cells with testosterone, but not 17ß-estradiol, confirmed a sex-specific regulation of UBR1. In vivo silencing of UBR1 using chronic administration of small interference RNA resulted in the restoration of ACE2 levels in hypertensive males. A transient decrease in blood pressure following intracerebroventricular, but not systemic, infusion was also observed. Interestingly, UBR1 knockdown increased the brain activation of Nedd4-2, an E3 ligase promoting ACE2 ubiquitination and reduced expression of SGK1, the kinase inactivating Nedd4-2. Conclusions: These data demonstrate that UBR1 is a novel ubiquitin ligase targeting ACE2 in hypertension. UBR1 and Nedd4-2 E3 ligases appear to work synergistically to ubiquitinate ACE2. Targeting of these ubiquitin ligases may represent a novel strategy to restore ACE2 compensatory activity in hypertension.

Interoperability of 300 watt and 150 watt xenon arc solar simulators in sun protection factor and in UVA protection factor clinical testing.

BACKGROUND: The purpose of these clinical trials was to determine if 300 W and 150 W xenon arc solar simulators (SSs) deliver the same sun protection factor (SPF) and UVA protection factor (PFA). METHODS: First, the SPF of the P7 control standard and of the P2 control standard was determined, testing 20 subjects using the method described in the Food and Drug Administration (FDA) Final Monograph and using 150 W and 300 W SSs. In the second clinical trial, the PFA of the Japanese Cosmetic Industry Association (JCIA) control standard and of the P2 control standard was determined, testing 10 subjects using the method described in the JCIA Technical Bulletin and using 150 W and 300 W SSs. RESULTS: The SPF values for P7 control standard determined using the 150 W and 300 W SSs were 4.54 ± 0.35 and 4.61 ± 0.32, respectively. The SPF values for P2 control standard determined using the 150 W and 300 W SSs were 17.0 ± 0.9 and 16.7 ± 0.9, respectively. The resultant PFA values for JCIA control standard determined using the 150 W and 300 W SSs were 4.06 ± 0.70 and 4.06 ± 0.70, respectively. The resultant PFA values for P2 control standard determined using the 150 W and 300 W SSs were 3.28 ± 0.25 and 3.44 ± 0.39, respectively. CONCLUSION: As the values are essentially identical for SPF and for PFA, the 150 W and 300 W SSs can be used interchangeably for SPF and PFA determinations.

Concurrent Validity of The Expanded Cutting Alignment Scoring Tool (E-CAST).

Background: The Expanded Cutting Alignment Scoring Tool (E-CAST) has been previously shown to be reliable when assessing lower extremity alignment during a 45-degree sidestep cut, however, the validity of this tool remains unknown. The purpose of this study was to assess the concurrent validity of the E-CAST by comparing visually identified movement errors from two-dimensional (2D) video with three-dimensional (3D) biomechanical variables collected using motion capture. Study Design: Cross Sectional. Methods: Sixty female athletes (age 14.1 ± 1.5 years) who regularly participated in cutting/pivoting sports performed a sidestep cut with 2D video and 3D motion capture simultaneously recording. One clinician scored the 2D videos for each limb using the E-CAST criteria. Joint angles and moments captured in 3D were computed for the trunk and knee. Receiver operating characteristic (ROC) curve analyses were performed to determine the accuracy of each E-CAST item and to provide cut-off points for risk factor identification. Results: ROC analyses identified a cut-off point for all biomechanical variables with sensitivity and specificity ranging from 70-85% and 55-89%, respectively. Across items, the area under the curve ranged from 0.67 to 0.91. Conclusion: The E-CAST performed with acceptable to outstanding area under the curve values for all variables except static knee valgus. Level of evidence: 3b.

No Difference in Two-Dimensional Kinematic Assessment of a 45-Degree Sidestep Cut Compared to Qualitative Assessment.

Background and Purpose: The Expanded Cutting Alignment Scoring Tool (E-CAST) is a two-dimensional qualitative scoring system that has demonstrated moderate inter-rater and good intra-rater reliability for the assessment of trunk and lower extremity alignment during a 45-degree sidestep cut. The primary purpose of this study was to examine the reliability of the quantitative version of the E-CAST among physical therapists and to compare the reliability of the quantitative E-CAST to the original qualitative E-CAST. The hypothesis was that the quantitative version of the E-CAST would demonstrate greater inter-rater and intra-rater reliability compared to the qualitative E-CAST. Study Design: Observational cohort, repeated measures reliability study. Methods: Twenty-five healthy female athletes (age 13.8±1.4 years) performed three sidestep cuts with two-dimensional video capturing frontal and sagittal views. Two physical therapist raters independently scored a single trial using both views on two separate occasions. Based on the E-CAST criteria, select kinematic measurements were extracted using a motion analysis phone application. Intraclass correlation coefficients and 95% confident intervals were calculated for the total score, and kappa coefficients were calculated per kinematic variable. Correlations were converted to z-scores and compared to the six original criteria for significance (α<0.05). Results: Cumulative intra- and inter-rater reliability were both good (ICC=0.821, 95% CI 0.687-0.898 and ICC=0.752, 95% CI 0.565-0.859). Cumulative intra-rater kappa coefficients ranged from moderate to almost perfect, and cumulative inter-rater kappa coefficients ranged from slight to good. No significant differences were observed between the quantitative and qualitative criteria for either inter- or intra-rater reliability (Zobs(intra)= -0.38, p=0.352 and Zobs(inter)= -0.30, p=0.382). Conclusion: The quantitative E-CAST is a reliable tool to assess trunk and lower extremity alignment during a 45-degree sidestep cut. No significant differences were observed in reliability of the quantitative versus qualitative assessment. Level of evidence: 3b.

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector.

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Sand resistance of sunscreens.

Like water resistance in sunscreens, sand resistance in sunscreens is the ability of the sunscreen to retain its effectiveness while undergoing sand treatment. The influence of the type of sand on the sand resistance of sunscreens has not been described. The sand resistance of a control standard sunscreen, P2, and data on three grades of Quickrete commercial grade sand, #1961, #1962, and #1152, are described. These sands represent a fine sand, a medium sand, and an all-purpose sand. Using the methodology described in the 2007 proposed amendment of the Final Monograph (1) with one exception, we obtained an SPF of 16.5 (1.6) for the control standard, compared to the expected SPF of 16.3 (3.4). After a five-minute treatment of sand #1961, #1962, or #1151, the SPF of the control standard was 18.3 (1.6), 18.4 (2.0), and 17.5 (2.2), respectively. Thus, all three sands exhibited a similar sand-resistance response. Thus, there was no significant difference in the average SPF with and without sand. The medium grade sand, Quickrete commercial grade #1962, was preferred for sand-resistance testing because the fine sand was difficult to remove from the subject's backs and the coarse sand was unpleasant to the subjects.

Reliability of the Expanded Cutting Alignment Scoring Tool (E-CAST) to Assess Trunk and Limb Alignment During a 45-Degree Side-Step Cut.

Background: Current clinical screening tools assessing risky movements during cutting maneuvers do not adequately address sagittal plane foot and ankle evaluations. The Cutting Alignment Scoring Tool (CAST) is reliable in evaluating frontal plane trunk and lower extremity alignment during a 45-degree side-step cut. The Expanded Cutting Alignment Scoring Tool (E-CAST) includes two new sagittal plane variables, knee flexion and ankle plantarflexion angle. Hypothesis/Purpose: To assess the inter-and intra-rater reliability of the E-CAST to evaluate trunk and lower extremity alignment during a 45-degree side-step cut. Study Design: Repeated Measures. Methods: Participants included 25 healthy females (13.8 ± 1.4 years) regularly participating in cutting or pivoting sports. Participants were recorded performing a side-step cut in frontal and sagittal planes. One trial was randomly selected for analysis. Two physical therapists independently scored each video using the E-CAST on two separate occasions, with randomization and a two-week wash-out between rounds. Observed movement variables were awarded a score of "1", with higher scores representing poorer technique. Intraclass correlation coefficients (ICC) and 95% confident intervals (95% CI) were calculated for the total score, and a kappa coefficient (k) was calculated for each variable. Results: The cumulative intra-rater reliability was good (ICC=0.78, 95% CI 0.59-0.96) and the cumulative inter-rater reliability was moderate (ICC=0.71, 95% CI 0.50-0.91). Intra-rater kappa coefficients ranged from moderate to excellent for all variables (k= 0.50-0.84) and inter-rater kappa coefficients ranged from slight to excellent for all variables (k=0.20-0.90). Conclusion: The addition of two sagittal plane variables resulted in lower inter-rater ICC compared to the CAST (ICC= 0.81, 95% CI 0.64-0.91). The E-CAST is a reliable tool to evaluate trunk and LE alignment during a 45-degree side-step cut, with good intra-rater and moderate inter-rater reliability. Level of Evidence: Level 2, Diagnosis.

Reliability of the Cutting Alignment Scoring Tool (CAST) to Assess Trunk and Limb Alignment During a 45-Degree Side-Step Cut.

BACKGROUND: Three-dimensional (3D) motion analysis is considered the gold standard for evaluating human movement. However, its clinical utility is limited due to cost, operating expertise, and lengthy data processing time. Numerous qualitative scoring systems have been introduced to assess trunk and lower extremity biomechanics during functional tasks. However, the reliability of qualitative scoring systems to evaluate cutting movements is understudied. Purpose/Hypotheses: To assess the inter-rater and intra-rater reliability of the Cutting Alignment Scoring Tool (CAST) among sports medicine providers and to evaluate rater agreement of each component of the CAST. The hypotheses were: 1) there would be good-to-excellent inter-rater and intra-rater reliability among sports medicine providers, 2) there would be good to almost perfect agreement for cut width and trunk lean variables and moderate to good agreement for valgus variables of the CAST. STUDY DESIGN: Repeated Measures. METHODS: Ten videos of a 45-degree side-step cut performed by adolescent athletes were independently rated on two occasions by six raters (2 medical doctors, 2 physical therapists, and 2 athletic trainers). The variables assessed include trunk lean to the opposite direction of the cut, increased cut width, knee valgus at initial load acceptance (static), and knee valgus throughout the task (dynamic). Variables were scored as either present, which were given a score of "1", or not present, which were given a score of "0". Video sequence was randomized in each rating session, and a two-week wash out period was given. RESULTS: The cumulative inter-rater and intra-rater reliabilities were good (ICC: 0.808 and ICC: 0.753). Almost perfect kappa coefficients were recorded for cut width (k=0.949). Moderate kappa coefficients were found for trunk lean (k= 0.632) and fair kappa coefficients were noted for dynamic and static valgus (k=0.462 and k= 0.533 respectively). CONCLUSION: These findings suggest that the CAST is a reliable tool to evaluate trunk and LE alignment during a cutting task by sports medicine providers. LEVEL OF EVIDENCE: Level 2 Diagnosis.