The USP46 complex deubiquitylates LRP6 to promote Wnt/ß-catenin signaling.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells.

Introduction: Ex vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. Methods: We compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)-IL2RG donor following double-strand DNA break at the endogenous IL2RG locus. Results and discussion: In vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI-IL2RG HSPCs with efficient T cell development following TI-IL2RG in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before (in vitro) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.

Patient satisfaction in military medicine: model refinement and assessment of continuity of care effects.

The Military Health System (MHS) identified patient-centered care and satisfied beneficiaries as main objectives in their 2006 Strategic Plan. The objective of this study was to expand upon the previous MHS model to determine predictors of patient satisfaction behaviors based upon associated attitudes and beliefs in addition to determining the predictive qualities presented by continuity of care. A sample of 90,318 patient responses from 2002 to 2004 was drawn from the Customer Satisfaction Survey database. Hierarchical multiple linear regression analyses were conducted to assess the uniquely predictive effects of the independent variables on the outcome variable. Results indicated the constructs from previous studies of satisfaction within the MHS using an attitude model were consistent when tested in a behavioral model. Hypothesis tests also indicated continuity of care demonstrated uniquely predictive qualities suggesting inclusion in the model. The final satisfaction with visit model yielded F(29, 90, 288) = 7062.37, p < 0.01 with R2 = 0.694.

Loco-regional administration of nanomedicines for the treatment of lung cancer.

Lung cancer poses one of the most significant challenges to modern medicine, killing thousands every year. Current therapy involves surgical resection supplemented with chemotherapy and radiotherapy due to high rates of relapse. Shortcomings of currently available chemotherapy protocols include unacceptably high levels of systemic toxicity and low accumulation of drug at the tumor site. Loco-regional delivery of nanocarriers loaded with anticancer agents has the potential to significantly increase efficacy, while minimizing systemic toxicity to anticancer agents. Local drug administration at the tumor site using nanoparticulate drug delivery systems can reduce systemic toxicities observed with intravenously administered anticancer drugs. In addition, this approach presents an opportunity for sustained delivery of anticancer drug over an extended period of time. Herein, the progress in the development of locally administered nanomedicines for the treatment of lung cancer is reviewed. Administration by inhalation, intratumoral injection and means of direct in situ application are discussed, the benefits and drawbacks of each modality are explored.