Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms

Can We Improve Screening Costs in Asymptomatic Metal on Metal Total Hip Arthroplasties?

Introduction: Metal on Metal (MoM) total hip arthroplasty (THA) has been largely abandoned in the United States secondary to high failure rates. Many of the failures are attributed to adverse local tissue reactions (ALTR). Therefore, patients that have a MoM THA are routinely screened by checking serum metal ion levels every two years, as was recommended by the FDA. However, there is limited data on the costs of current screening protocols.   Materials and Methods: 318 consecutive patients who underwent a MoM THA at a single institution were retrospectively enrolled. The average follow-up was 8.2 years. Clinical data, metal ion levels, revision and reoperation rates were prospectively collected. The costs of clinical screening for this patient population was calculated and compared to the cost of an annual screening protocol.   Results: 12 patients had either an elevated Co or Cr level (>4.5 ppb). Eight patients were revised secondary to ALTR. The total cost of screening during the study was $612,250. Additionally, if annual screening had been performed, total screening costs would be approximately$1,719,200.   Discussion: Eight patients in the following study were revised secondary to ALTR with a total cost of screening of $612,500. These costs are substantially less than the cost of annual screening ($1,719,200). Due to the considerable costs of screening asymptomatic MoM THA patients, we recommend both optimizing the frequency of screening and evaluating the specific risk of the implant being screened

Moving beyond the front line: a 20-year retrospective cohort study of career trajectories from the Indigenous Health Program at the University of Queensland

This report examines critical success factors for enabling Aboriginal and Torres Strait Islander leadership across the health system as demonstrated by alumni of the University of Queensland (UQ) Indigenous Health Program (IHP) (1994–2005) who today work in various leadership roles throughout the country.\ua0It determines the enablers of professional success of these health leaders in various facets of the health system and investigates the impact of active participation in the community of Aboriginal and Torres Strait Islander health professionals over the course of a career. Through this analysis, the report further theorises the confluence of community, subjectivity, self-determination and health

Is Utilizing a Modular Stemmed Tibial Component in Obese Patients Undergoing Primary Total Knee Replacement Cost-Effective?

Introduction: There has been recent enthusiasm for the use of modular stemmed tibial components in obese (BMI ≥35kg/m2) patients undergoing primary total knee arthroplasty (TKA). This has been mainly driven by studies demonstrating statistically significant increases in the rates of aseptic tibial loosening (ATL) in this patient population. However, to our knowledge, no study has specifically evaluated the cost effectiveness of this current recommendation. Methods: The following study was performed utilizing previously obtained data on the incidence of ATL in obese patients undergoing primary TKA. This data was then utilized to create a cost calculator that can evaluate the price point at which the use of a stemmed tibial component in all obese patients would be less than or equal to the costs of revision surgery if a stemmed implant was not utilized. Results: Utilizing historical data with a revision rate of 4% for aseptic loosening of the tibia on obese patients, a cost calculator was developed. The cost calculator requires the input of expected or known incidence of ATL utilizing a stem extension and the expected or known costs of revision for ATL. Conclusion: The following cost calculator quickly determines a price point at which the use of a tibial stem offsets the costs of revision surgery. While this study may not provide an exact cost-effectiveness of modular stem fixation due to model limitations, it will hopefully initiate the discussion for providing more cost-effective individualized care for this patient population

High Failure Rates of Concomitant Periprosthetic Joint Infection and Extensor Mechanism Disruption

Background Patients presenting with both chronic periprosthetic joint infection (PJI) and extensor mechanism disruption (EMD) pose a significant challenge. As there is little in the literature regarding outcomes of patients with concomitant PJI and EMD, we performed a multicenter study to evaluate the outcomes. Methods Sixty patients with concomitant diagnoses of PJI and EMD were evaluated from 5 institutions. Patient demographics, presentation type, surgical management, and outcomes including recurrent infections, final surgery, and ambulatory status were documented. Results Fifty-three of 60 patients had an attempted extensor mechanism reconstruction/repair (EMR) of which 12 (23%) were successful, averaging 3.5 (range, 2-7) intervening surgeries. Forty-one patients (77%) were considered failures with recurrence of infection as most common failure (80%); 26 ended in fusion, 10 in above knee amputation, 3 with chronic resection arthroplasty, and 2 with chronic spacers/EMD. Seven patients had no attempt at EMR but proceeded directly to fusion (n = 6) or amputation (n = 1). There was no statistical difference between groups that had success or failure of EMR in age, American Society of Anesthesiologists Physical Status Classification System, or body mass index. Conclusion Our study demonstrates that concomitant EMD and PJI is a dreaded combination with poor outcomes regardless of treatment. Eradication of infection and reconstruction of the extensor mechanism often require numerous surgeries and despite great effort often end in failure. Consideration of early fusion or amputation may be preferable in some patients to avoid the morbidity and mortality of repeated surgeries

The Impact of Implementing a Test, Treat and Retain HIV Prevention Strategy in Atlanta among Black Men Who Have Sex with Men with a History of Incarceration: A Mathematical Model

Background Annually, 10 million adults transition through prisons or jails in the United States (US) and the prevalence of HIV among entrants is three times higher than that for the country as a whole. We assessed the potential impact of increasing HIV Testing/Treatment/Retention (HIV-TTR) in the community and within the criminal justice system (CJS) facilities, coupled with sexual risk behavior change, focusing on black men-who-have-sex-with-men, 15–54 years, in Atlanta, USA. Methods We modeled the effect of a HIV-TTR strategy on the estimated cumulative number of new (acquired) infections and mortality, and on the HIV prevalence at the end of ten years. We additionally assessed the effect of increasing condom use in all settings. Results In the Status Quo scenario, at the end of 10 years, the cumulative number of new infections in the community, jail and prison was, respectively, 9246, 77 and 154 cases; HIV prevalence was 10815, 69 and 152 cases, respectively; and the cumulative number of deaths was 2585, 18 and 34 cases, respectively. By increasing HIV-TTR coverage, the cumulative number of new infections could decrease by 15% in the community, 19% in jail, and 8% in prison; HIV prevalence could decrease by 8%, 9% and 7%, respectively; mortality could decrease by 20%, 39% and 18%, respectively. Based on the model results, we have shown that limited use and access to condoms have contributed to the HIV incidence and prevalence in all settings. Conclusions Aggressive implementation of a CJS-focused HIV-TTR strategy has the potential to interrupt HIV transmission and reduce mortality, with benefit to the community at large. To maximize the impact of these interventions, retention in treatment, including during the period after jail and prison release, and increased condom use was vital for decreasing the burden of the HIV epidemic in all settings

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer‐related mortality. Despite significant advances made in the treatment of other cancers, current chemotherapies offer little survival benefit in this disease. Pancreaticoduodenectomy offers patients the possibility of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found that LOX expression is upregulated in metastatic tumors from Pdx1‐Cre KrasG12D/+ Trp53R172H/+ (KPC) mice and that inhibition of LOX in these mice suppressed metastasis. Mechanistically, LOX inhibition suppressed both migration and invasion of KPC cells. LOX inhibition also synergized with gemcitabine to kill tumors and significantly prolonged tumor‐free survival in KPC mice with early‐stage tumors. This was associated with stromal alterations, including increased vasculature and decreased fibrillar collagen, and increased infiltration of macrophages and neutrophils into tumors. Therefore, LOX inhibition is able to reverse many of the features that make PDAC inherently refractory to conventional therapies and targeting LOX could improve outcome in surgically resectable disease