A Flexible, Pooled CRISPR Library for Drug Development Screens.

Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging and require carefully and appropriately adapted screening approaches. In particular, negative selection (or sensitivity) screening, often the most experimentally desirable modality of screening, has remained a challenge in drug discovery. Here we assess whether our new, modular genome-wide pooled CRISPR library can improve negative selection CRISPR screening and add utility throughout the drug development pipeline. Our pooled library is split into three parts, allowing it to be scaled to accommodate the experimental challenges encountered during drug development, such as target identification using unlimited cell numbers compared with target identification studies for cell populations where cell numbers are limiting. To test our new library, we chose to look for drug-gene interactions using a well-described small molecule inhibitor targeting poly(ADP-ribose) polymerase 1 (PARP1), and in particular to identify genes which sensitise cells to this drug. We simulate hit identification and performance using each library partition and support these findings through orthogonal drug combination cell panel screening. We also compare our data with a recently published CRISPR sensitivity dataset obtained using the same PARP1 inhibitor. Overall, our data indicate that generating a comprehensive CRISPR knockout screening library where the number of guides can be scaled to suit the biological question being addressed allows a library to have multiple uses throughout the drug development pipeline, and that initial validation of hits can be achieved through high-throughput cell panels screens where clinical grade chemical or biological matter exist.

Bikini versus traditional incision direct anterior approach: is there any difference in soft tissue damage?

INTRODUCTION: The direct anterior approach for total hip replacements has reported advantages of improved early function and muscle preservation. In an effort to improve healing and cosmesis, a change in the orientation of the incision has been proposed. Traditionally, the skin incision is in-line with the tensor fasciae latae muscle belly. The bikini incision is orthogonal to this orientation. The hypothesis was that muscle damage would be increased by using the bikini incision. METHODS: A traditional or bikini incision was performed on 18 cadaveric hips. On each of the 9 specimens, the traditional incision was performed on 1 side, and a bikini incision on the contralateral hip, with an even distribution of right or left side. Blinded anatomists performed the hip dissections, and assessed for muscle damage as well as for damage to the lateral femoral cutaneous nerve. RESULTS: No difference in muscle damage was identified in the tensor fasciae latae between muscle groups. Muscle damage was very minimal to the gluteus medius and minimus. Damage to the lateral femoral cutaneous nerve occurred equally for both the bikini and traditional skin incisions. CONCLUSIONS: The bikini incision for the direct anterior approach to the hip can be performed safely, with no increase in muscle damage or damage to the lateral femoral cutaneous nerve compared to the traditional incision.

A cost analysis of single-stage bilateral versus two-stage direct anterior total hip arthroplasty.

PURPOSE: The potential cost savings of single-stage bilateral total hip arthroplasty (THA) are unclear, and the risks associated with it are not well defined. We sought to compare the costs and perioperative complications of single-stage bilateral THA via the direct anterior approach (DAA) to a two-stage bilateral protocol. METHODS: We retrospectively reviewed patients who underwent a single- stage bilateral DAA THA and compared them to a two-stage THA group. We conducted a cost analysis from both the hospital perspective and the Ministry of Health (MOH) perspective. RESULTS: 24 patients were included in this study. The 2 groups were similar in age (58.9 vs 63.9 yrs), height (169.2 vs 170.9 cm), weight (80.2 vs 78.6 kg), BMI (27.9 vs 26.3 kg/m2), ASA score (2.2 vs 2.2), and CCI score (2.3 vs 2.9). The mean cost per patient from the hospital perspective for the single-stage group was $10,728.13 (SD = 621.46) compared to $12,670.63 (SD = 519.72) for the two-stage group (Mean Difference = $1,942.50, 95% CI = $1,457.49 to $2,427.51, p<0.001). Similarly, from the MOH perspective, the cost for the single-stage group was $12,552.34 (SD = 644.93) compared to $14,740.58 (SD = 598.07) for the two-stage group (Mean Difference = $2,188.24, 95% CI = $1,661.67 to $2,714.81, p<0.001). There were no significant differences in complication rate between groups. The largest percent of total cost savings from a hospital perspective was attributed to cost of operating room staff and OR set-up (55%). CONCLUSIONS: Our results suggest that single-stage bilateral DAA THA results in significant cost savings compared to two-stage DAA THA.

Long-term outcomes for Kienböck's disease.

BACKGROUND: The precise etiology of Kienböck's disease is unclear. Controversy exists regarding the appropriate treatment modality. The present study sought to investigate and compare surgical and nonsurgical treatment outcomes of patients suffering from Kienböck's disease in the province of Newfoundland and Labrador (NL), Canada. METHODS: The present study was a retrospective analysis of 66 patients. The primary outcome was the Disabilities of the Arm, Shoulder, and Hand (DASH) score. Student's t test was used to assess differences in outcomes between treatment groups. One-way ANOVA was used to assess differences in primary outcome in time since first assessed in an effort to examine progression over time. Pearson correlation was used to assess for correlation between primary outcome and age at diagnosis. RESULTS: The average age was 38.6 ± 11.4 (18-70) years; Four patients were excluded due to inaccessible imaging. Of the remaining patients, 44 were treated conservatively, while 18 were treated surgically. The DASH scores for the surgical group were 23.7 ± 24.5 (0.9-82.8) and nonsurgical group were 20.0 ± 20.1 (1.7-81). As expected, the surgical group was mainly comprised of late-stage Kienböck's. When both groups were compared, there was no significant difference in the DASH scores. There were no difference in DASH scores within groups according to time since first diagnosed (<5 years; between 5 and 10 years; and >10 years). A positive correlation was found between age at diagnosis and DASH score (r = 0.42, p = 0.007), despite treatment modality. This finding remained significant after accounting for confounding factors (p = 0.029). CONCLUSION: The DASH score for the surgical group was 23.7 ± 24.5 (0.9-82.8) and nonsurgical group was 20.0 ± 20.1 (1.7-81). No significant difference in DASH scores was found between surgically and nonsurgically treated patients. A positive association was found between the age at diagnosis of Kienböck's and DASH score, which suggests that patients diagnosed and treated later in life tend not to do as well.

Lack of association of ghrelin precursor gene variants and percentage body fat or serum lipid profiles.

Ghrelin has been recognized for its involvement in food intake, control of energy homeostasis, and lipid metabolism. However, the roles of genetic variations in the ghrelin precursor gene (GHRL) on body compositions and serum lipids are not clear in humans. Our study investigated five single-nucleotide polymorphisms (SNPs) within GHRL to determine their relationship with body fat percentage (BF), trunk fat percentage (TF), lower body (legs) fat percentage (LF), and serum lipids in 1,464 subjects, which were recruited from the genetically homogeneous population of Newfoundland and Labrador (NL), Canada. Serum glucose, insulin, total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and triglycerides were determined. Five SNPs are rs35684 (A/G: a transition substitution in exon 1), rs4684677 (A/T: a missense mutation), rs2075356 (C/T: intron), rs26802 (G/T: intron), and rs26311 (A/G: near the 3' untranslated region) of GHRL were genotyped using TaqMan validated or functionally tested SNP genotyping assays. Our study found no significant evidence of an allele or genotype association between any of the variant sites and body compositions or serum lipids. Furthermore, haplotype frequencies were not found to be significantly different between lean and obese subjects. In summary, the results of our study do not support a significant role for genetic variations in GHRL in the differences of body fat and serum lipid profiles in the NL population.

Altered calcium homeostasis is correlated with abnormalities of fasting serum glucose, insulin resistance, and beta-cell function in the Newfoundland population.

Alteration of extracellular calcium concentration may be involved in glucose metabolism in a number of pathways. The present study was designed to investigate the relationship between total serum calcium and 1) fasting serum glucose, 2) insulin, 3) insulin resistance, and 4) beta-cell function in 1,182 healthy subjects from the province of Newfoundland and Labrador, Canada. All variables were log10 transformed, and confounding factors including age, trunk fat percentage, serum phosphorus, magnesium, 25-OH vitamin D, and parathyroid hormone were adjusted before analyses. Significant positive correlations between glucose and insulin resistance with calcium were found in both sexes, whereas an inverse correlation between beta-cell function and calcium was found only in women. Similar results were found in medication-free women and men, as well as in pre- and postmenopausal women. Subjects with low calcium levels had the lowest concentration of glucose and the least insulin resistance, whereas subjects with high calcium levels had the highest concentration of glucose and insulin resistance in women but not in men. This relationship remained after calcium was adjusted for 25-OH vitamin D and parathyroid hormone. Our results suggest that alteration of serum calcium homeostasis is significantly correlated with the abnormality of glucose level, insulin resistance, and beta-cell function.

Comparison of multifrequency bioelectrical impedance analysis with dual-energy X-ray absorptiometry for assessment of percentage body fat in a large, healthy population.

BACKGROUND: Bioelectrical impedance analysis (BIA) is widely used in clinics and research to measure body composition. However, the results of BIA validation with reference methods are contradictory, and few data are available on the influence of adiposity on the measurement of body composition by BIA. OBJECTIVE: The goal was to determine the effects of sex and adiposity on the difference in percentage body fat (%BF) predicted by BIA compared with dual-energy X-ray absorptiometry (DXA). DESIGN: A total of 591 healthy subjects were recruited in Newfoundland and Labrador. %BF was predicted by using BIA and was compared with that measured by DXA. Methods agreement was assessed by Pearson's correlation and Bland and Altman analysis. Differences in %BF among groups based on sex and adiposity were analyzed by using one-factor analysis of variance with Bonferroni correction. RESULTS: Correlations between BIA and DXA were 0.88 for the whole population, 0.78 for men, and 0.85 for women. The mean %BF determined by BIA (32.89 +/- 8.00%) was significantly lower than that measured by DXA (34.72 +/- 8.66%). The cutoffs were sex specific. BIA overestimated %BF by 3.03% and 4.40% when %BF was <15% in men and <25% in women, respectively, and underestimated %BF by 4.32% and 2.71% when %BF was >25% in men and >33% in women, respectively. CONCLUSIONS: BIA is a good alternative for estimating %BF when subjects are within a normal body fat range. BIA tends to overestimate %BF in lean subjects and underestimate %BF in obese subjects.