Single-cell analysis of Kaposi's sarcoma-associated herpesvirus infection in three-dimensional air-liquid interface culture model.

The oral cavity is the major site for transmission of Kaposi's sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered keratinocyte differentiation and cell death. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host gene expression compared to latent or lytic infected cells. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression.

First in-human evaluation of [1-13C]pyruvate in D2O for hyperpolarized MRI of the brain: A safety and feasibility study.

PURPOSE: To investigate the safety and value of hyperpolarized (HP) MRI of [1-13C]pyruvate in healthy volunteers using deuterium oxide (D2O) as a solvent. METHODS: Healthy volunteers (n = 5), were injected with HP [1-13C]pyruvate dissolved in D2O and imaged with a metabolite-specific 3D dual-echo dynamic EPI sequence at 3T at one site (Site 1). Volunteers were monitored following the procedure to assess safety. Image characteristics, including SNR, were compared to data acquired in a separate cohort using water as a solvent (n = 5) at another site (Site 2). The apparent spin-lattice relaxation time (T1) of [1-13C]pyruvate was determined both in vitro and in vivo from a mono-exponential fit to the image intensity at each time point of our dynamic data. RESULTS: All volunteers completed the study safely and reported no adverse effects. The use of D2O increased the T1 of [1-13C]pyruvate from 66.5 ± 1.6 s to 92.1 ± 5.1 s in vitro, which resulted in an increase in signal by a factor of 1.46 ± 0.03 at the time of injection (90 s after dissolution). The use of D2O also increased the apparent relaxation time of [1-13C]pyruvate by a factor of 1.4 ± 0.2 in vivo. After adjusting for inter-site SNR differences, the use of D2O was shown to increase image SNR by a factor of 2.6 ± 0.2 in humans. CONCLUSIONS: HP [1-13C]pyruvate in D2O is safe for human imaging and provides an increase in T1 and SNR that may improve image quality.

Core implementation strategies for improving cirrhosis care in the Veterans Health Administration.

BACKGROUND AND AIMS: The Veterans Health Administration (VHA) provides care for more than 80,000 veterans with cirrhosis. This longitudinal, multimethod evaluation of a cirrhosis care quality improvement program aimed to (1) identify implementation strategies associated with evidence-based, guideline-concordant cirrhosis care over time, and (2) use qualitative interviews to operationalize strategies for a manualized intervention. APPROACH AND RESULTS: VHA providers were surveyed annually about the use of 73 implementation strategies to improve cirrhosis care in fiscal years 2018 (FY18) and 2019 (FY19). Implementation strategies linked to guideline-concordant cirrhosis care were identified using bivariate statistics and comparative configurational methods. Semistructured interviews were conducted with 12 facilities in the highest quartile of cirrhosis care to specify the successful implementation strategies and their mechanisms of change. A total of 106 VHA facilities (82%) responded at least once over the 2-year period (FY18, n = 63; FY19, n = 100). Facilities reported using a median of 12 (interquartile range [IQR] 20) implementation strategies in FY18 and 10 (IQR 19) in FY19. Of the 73 strategies, 35 (48%) were positively correlated with provision of evidence-based cirrhosis care. Configurational analysis identified multiple strategy pathways directly linked to more guideline-concordant cirrhosis care. Across both methods, a subset of eight strategies was determined to be core to cirrhosis care improvement and specified using qualitative interviews. CONCLUSIONS: In a national cirrhosis care improvement initiative, a multimethod approach identified a core subset of successful implementation strategy combinations. This process of empirically identifying and specifying implementation strategies may be applicable to other implementation challenges in hepatology.

Global epigenomic analysis of KSHV-infected primary effusion lymphoma identifies functional MYC superenhancers and enhancer RNAs.

Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function.

Oncogenic human herpesvirus hijacks proline metabolism for tumorigenesis.

Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.

Using Lean-Facilitation to Improve Quality of Hepatitis C Testing in Primary Care.

BACKGROUND: Lean management has been successfully employed in healthcare to improve outcomes and efficiencies. Facilitation is increasingly being used to support evidence-based practice uptake in healthcare. However, while both Lean and Facilitation are used in healthcare quality improvement, limited research has explored their integration and the sustainability of their combined effects. OBJECTIVE: To improve hepatitis C virus (HCV) screening rates among persons born between 1945 and 1965 through the design and evaluation of a multi-modal Lean-Facilitation intervention (LFI) for Department of Veterans Affairs primary care community clinics. DESIGN: We conducted a mixed methods quasi-experimental evaluation in eight clinics, guided by the integrated Promoting Action on Research Implementation in Health Services framework. PARTICIPANTS: We engaged regional and local leadership (N = 9), implemented our LFI with clinicians and staff (N = 68), and conducted summative interviews with participants (N = 13). INTERVENTION: The LFI included six implementation strategies: (1) external facilitation, (2) stakeholder engagement, (3) champion activation, (4) rapid process improvement sessions, (5) Plan-Do-Study-Act cycles, and (6) audit-feedback. MEASURES: The primary outcome was rate of new HCV screening among previously untested patients with a primary care visit. Using interrupted time series, we analyzed intervention and time effects on HCV testing rates, and administered organizational readiness surveys, conducted summative qualitative interviews, and tracked facilitation events. RESULTS: The LFI was associated with significant, immediate, and sustained increases in HCV testing. No change was detected at matched comparison clinics. Staff accepted the LFI and the philosophy of "bottom-up" solution development yet had mixed feedback on its appropriateness and feasibility. Enablers of implementation and early sustainment included lower satisfaction with baseline HCV testing processes and staff culture, while later sustainment was related to implementation climate support, measurement, and evaluation. CONCLUSIONS: High-reach and relatively low effort, but persistent intervention led to significant improvement in guideline-concordant HCV testing rates which were sustained. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02936648.

Mapping the road to elimination: a 5-year evaluation of implementation strategies associated with hepatitis C treatment in the veterans health administration.

BACKGROUND: While few countries and healthcare systems are on track to meet the World Health Organization's hepatitis C virus (HCV) elimination goals, the US Veterans Health Administration (VHA) has been a leader in these efforts. We aimed to determine which implementation strategies were associated with successful national viral elimination implementation within the VHA. METHODS: We conducted a five-year, longitudinal cohort study of the VHA Hepatic Innovation Team (HIT) Collaborative between October 2015 and September 2019. Participants from 130 VHA medical centers treating HCV were sent annual electronic surveys about their use of 73 implementation strategies, organized into nine clusters as described by the Expert Recommendations for Implementing Change taxonomy. Descriptive and nonparametric analyses assessed strategy use over time, strategy attribution to the HIT, and strategy associations with site HCV treatment volume and rate of adoption, following the Theory of Diffusion of Innovations. RESULTS: Between 58 and 109 medical centers provided responses in each year, including 127 (98%) responding at least once, and 54 (42%) responding in all four implementation years. A median of 13-27 strategies were endorsed per year, and 8-36 individual strategies were significantly associated with treatment volume per year. Data warehousing, tailoring, and patient-facing strategies were most commonly endorsed. One strategy-"identify early adopters to learn from their experiences"-was significantly associated with HCV treatment volume in each year. Peak implementation year was associated with revising professional roles, providing local technical assistance, using data warehousing (i.e., dashboard population management), and identifying and preparing champions. Many of the strategies were driven by a national learning collaborative, which was instrumental in successful HCV elimination. CONCLUSIONS: VHA's tremendous success in rapidly treating nearly all Veterans with HCV can provide a roadmap for other HCV elimination initiatives.

Exploring the landscape of sustainability performance management systems in U.S. local governments.

Municipalities around the globe are increasingly adopting and integrating sustainability goals into their service functions. As with many public programs, local sustainability initiatives benefit from regular and systematic assessment aimed at determining the extent to which efforts are achieving their performance goals. However, there currently is little understanding of how local governments are collecting and tracking data on their sustainability-related performance and to what extent they integrate its lessons into their sustainability management practice. This study helps fill this gap by investigating U.S. local governments' sustainability performance management systems and provides empirical evidence on some of their key institutional and system characteristics. Our findings indicate that a significant number of U.S. local governments are engaged in sustainability performance assessment, but they vary in their ability to establish appropriate indicators as well as supporting mechanisms, indicating significant room for improvement in local sustainability performance management.

Strategy Configurations Directly Linked to Higher Hepatitis C Virus Treatment Starts: An Applied Use of Configurational Comparative Methods.

BACKGROUND: The Department of Veterans Affairs (VA) cares for more patients with hepatitis C virus (HCV) than any other US health care system. We tracked the implementation strategies that VA sites used to implement highly effective new treatments for HCV with the aim of uncovering how combinations of implementation strategies influenced the uptake of the HCV treatment innovation. We applied Configurational Comparative Methods (CCMs) to uncover causal dependencies and identify difference-making strategy configurations, and to distinguish higher from lower HCV treating sites. METHODS: We surveyed providers to assess VA sites' use of 73 implementation strategies to promote HCV treatment in the fiscal year 2015. CCMs were used to identify strategy configurations that uniquely distinguished higher HCV from lower HCV treating sites. RESULTS: From the 73 possible implementation strategies, CCMs identified 5 distinct strategy configurations, or "solution paths." These were comprised of 10 individual strategies that collectively explained 80% of the sites with higher HCV treatment starts with 100% consistency. Using any one of the following 5 solution paths was sufficient to produce higher treatment starts: (1) technical assistance; (2) engaging in a learning collaborative AND designating leaders; (3) site visits AND outreach to patients to promote uptake and adherence; (4) developing resource sharing agreements AND an implementation blueprint; OR (5) creating new clinical teams AND sharing quality improvement knowledge with other sites AND engaging patients. There was equifinality in that the presence of any one of the 5 solution paths was sufficient for higher treatment starts. CONCLUSIONS: Five strategy configurations distinguished higher HCV from lower HCV treating sites with 100% consistency. CCMs represent a methodological advancement that can help inform high-yield implementation strategy selection and increase the efficiency and effectiveness of future implementation efforts.

Discovery of Selective SIRT2 Inhibitors as Therapeutic Agents in B-Cell Lymphoma and Other Malignancies.

Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 µM, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells.

Mechanisms of Transformation of Bulk Aluminum-Lithium Alloys to Aluminum Metal-Organic Nanowires.

Fabrication and applications of lightweight, high load-bearing, thermally stable composite materials would benefit greatly from leveraging the high mechanical strength of ceramic nanowires (NWs) over conventional particles or micrometer-scale fibers. However, conventional synthesis routes to produce NWs are rather expensive. Recently we discovered a novel method to directly convert certain bulk bimetallic alloys to metal-organic NWs at ambient temperature and pressure. This method was demonstrated by a facile transformation of polycrystalline aluminum-lithium (AlLi) alloy particles to aluminum alkoxide NWs, which can be further transformed to mechanically robust aluminum oxide (Al2O3) NWs. However, the transformation mechanisms have not been clearly understood. Here, we conducted advanced materials characterization (via electron microscopy and nuclear magnetic resonance spectroscopies) and chemo-mechanical modeling to elucidate key physical and chemical mechanisms responsible for NWs formation. We further demonstrated that the content of Li metal in the AlLi alloy could be reduced to about 4 wt % without compromising the success of the NWs synthesis. This new mechanistic understanding may open new avenues for large-scale, low-cost manufacturing of NWs and nanofibers for a broad range of composites and flexible ceramic membranes.

Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice.

BACKGROUND & AIMS: It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing. METHODS: We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be up-regulated after tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines. RESULTS: We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation. CONCLUSIONS: Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

Early emergence of de novo EGFR T790M gatekeeper mutations during erlotinib treatment in PC9 non-small cell lung cancer cells.

The emergence of the T790M gatekeeper mutation in the Epidermal Growth Factor Receptor (EGFR) gene is an important mechanism that can lead to the acquired resistance to EGFR-targeted tyrosine kinase inhibitors such as erlotinib or gefitinib. These drugs have been used in treating a subset of non-small cell lung cancer (NSCLC) patients harboring EGFR activating mutations. Here we investigated the paths leading to the acquisition of the T790M mutation by establishing an erlotinib resistant PC9 cell model harboring ectopically introduced EGFR cDNA. We detected the emergence of T790M mutation within the EGFR cDNA in a subset of erlotinib resistant PC9 cell models through Sanger sequencing and droplet digital PCR-based methods, demonstrating that T790M mutation can emerge via de novo events following treatment with erlotinib. In addition, we show that the de novo T790M bearing erlotinib resistant PC9 cells are sensitive to the 3rd generation EGFR-targeted drug, WZ4002. Furthermore, GFP-based competition cell proliferation assays reveal that PC9 cells ectopically expressing EGFR mutant become more rapidly resistant to erlotinib than parental PC9 cells through the acquisition of the T790M mutation. Taken together, we believe that our findings expand upon the previous notion of evolutionary paths of T790M development, providing an important clue to designing a therapeutic strategy to overcome drug resistance.

To Control Site-Specific Skin Gene Expression, Autocrine Mimics Paracrine Canonical Wnt Signaling and Is Activated Ectopically in Skin Disease.

Despite similar components, the heterogeneity of skin characteristics across the human body is enormous. It is classically believed that site-specific fibroblasts in the dermis control postnatal skin identity by modulating the behavior of the surface-overlying keratinocytes in the epidermis. To begin testing this hypothesis, we characterized the gene expression differences between volar (ventral; palmoplantar) and nonvolar (dorsal) human skin. We show that KERATIN 9 (KRT9) is the most uniquely enriched transcript in volar skin, consistent with its etiology in genetic diseases of the palms and soles. In addition, ectopic KRT9 expression is selectively activated by volar fibroblasts. However, KRT9 expression occurs in the absence of all fibroblasts, although not to the maximal levels induced by fibroblasts. Through gain-of-function and loss-of-function experiments, we demonstrate that the mechanism is through overlapping paracrine or autocrine canonical WNT-ß-catenin signaling in each respective context. Finally, as an in vivo example of ectopic expression of KRT9 independent of volar fibroblasts, we demonstrate that in the human skin disease lichen simplex chronicus, WNT5a and KRT9 are robustly activated outside of volar sites. These results highlight the complexities of site-specific gene expression and its disruption in skin disease.

Who Gets Testosterone? Patient Characteristics Associated with Testosterone Prescribing in the Veteran Affairs System: a Cross-Sectional Study.

BACKGROUND: There has been concern about the growing off-label use of testosterone. Understanding the context within which testosterone is prescribed may contribute to interventions to improve prescribing. OBJECTIVE: To evaluate patient characteristics associated with receipt of testosterone. DESIGN: Cross-sectional. SETTING: A national cohort of male patients, who had received at least one outpatient prescription within the Veterans Affairs (VA) system during Fiscal Year 2008- Fiscal Year 2012. PARTICIPANTS: The study sample consisted of 682,915 non-HIV male patients, of whom 132,764 had received testosterone and a random 10% sample, 550,151, had not. MAIN MEASURES: Conditions and medications associated with testosterone prescription. KEY RESULTS: Only 6.3% of men who received testosterone from the VA during the study period had a disorder of the testis, pituitary or hypothalamus associated with male hypogonadism. Among patients without a diagnosed disorder of hypogonadism, the use of opioids and obesity were the strongest predictors of testosterone prescription. Patients receiving >100 mg/equivalents of oral morphine daily (adjusted odds ratio = 5.75, p < 0.001) and those with body mass index (BMI) >40 kg/m2 (adjusted odds ratio = 3.01, p < 0.001) were more likely to receive testosterone than non-opioid users and men with BMI <25 kg/m2. Certain demographics (age 40-54, White race), comorbid conditions (sleep apnea, depression, and diabetes), and medications (antidepressants, systemic corticosteroids) also predicted a higher likelihood of testosterone receipt, all with an adjusted odds ratio less than 2 (p < 0.001). CONCLUSIONS: In the VA, 93.7% of men receiving testosterone did not have a diagnosed condition of the testes, pituitary, or hypothalamus. The strongest predictors of testosterone receipt (e.g., obesity, receipt of opioids), which though are associated with unapproved, off-label use, may be valid reasons for therapy. Interventions should aim to increase the proportion of testosterone recipients who have a valid indication.

Results of a Regional Effort to Improve Warfarin Management.

BACKGROUND: Improved anticoagulation control with warfarin reduces adverse events and represents a target for quality improvement. No previous study has described an effort to improve anticoagulation control across a health system. OBJECTIVE: To describe the results of an effort to improve anticoagulation control in the New England region of the Veterans Health Administration (VA). METHODS: Our intervention encompassed 8 VA sites managing warfarin for more than 5000 patients in New England (Veterans Integrated Service Network 1 [VISN 1]). We provided sites with a system to measure processes of care, along with targeted audit and feedback. We focused on processes of care associated with site-level anticoagulation control, including prompt follow-up after out-of-range international normalized ratio (INR) values, minimizing loss to follow-up, and use of guideline-concordant INR target ranges. We used a difference-in-differences (DID) model to examine changes in anticoagulation control, measured as percentage time in therapeutic range (TTR), as well as process measures and compared VISN 1 sites with 116 VA sites located outside VISN 1. RESULTS: VISN 1 sites improved on TTR, our main indicator of quality, from 66.4% to 69.2%, whereas sites outside VISN 1 improved from 65.9% to 66.4% (DID 2.3%, P < 0.001). Improvement in TTR correlated strongly with the extent of improvement on process-of-care measures, which varied widely across VISN 1 sites. CONCLUSIONS: A regional quality improvement initiative, using performance measurement with audit and feedback, improved TTR by 2.3% more than control sites, which is a clinically important difference. Improving relevant processes of care can improve outcomes for patients receiving warfarin.

Suppressor mutations suggest a surface on PAT-4 (Integrin-linked Kinase) that interacts with UNC-112 (Kindlin).

Caenorhabditis elegans striated muscle cells attach to basement membrane and transmit the force of muscle contraction through integrin adhesion complexes. The cytoplasmic tail of ß-integrin (PAT-3) is associated with a conserved four-protein complex that includes UNC-112 (kindlin), PAT-4 (integrin-linked kinase), PAT-6 (α-parvin/actopaxin), and UNC-97 (PINCH). The proper localization of UNC-112 to muscle integrin adhesion sites requires PAT-4. A recent report (Qadota, H., Moerman, D. G., and Benian, G. M. (2012) A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (kindlin) to integrin adhesion sites. J. Biol. Chem. 287, 28537-28551) suggests a possible molecular mechanism for this requirement: that UNC-112 exists in closed inactive and open active conformations, and conversion to the open active form is promoted by binding to PAT-4 (ILK). Previously, we also reported identification of a single missense mutation in UNC-112, D382V, which abolishes both binding to PAT-4 and normal localization to integrin adhesion sites in vivo. In this report, we describe isolation and characterization of PAT-4 missense mutations that permit binding with UNC-112 D382V and place nine affected residues on a homology model of PAT-4. These nine residues cluster in two regions on the surface of PAT-4, do not overlap the likely binding surface for PAT-6 (α-parvin), and therefore may reside along the interaction surface of PAT-4 for UNC-112 (kindlin). We also show that one of these PAT-4 mutations restores the ability of UNC-112 D382V to localize to integrin adhesions and participate in complex formation.

Balancing collaborative and independent practice roles in clinical pharmacy: a qualitative research study.

BACKGROUND: Clinical pharmacists (CPs) with a scope of practice operate as direct care providers and health care team members. Research often focuses on one role or the other; little is understood about the dynamic relationship between roles in practice settings. OBJECTIVE: To identify the challenges CPs face in balancing dual roles as direct care providers and health care team members and the implications for CP effectiveness and quality of care. METHODS: Pharmacists were interviewed with a primary purpose of informing an implementation effort. Besides the implementation, there were emergent themes regarding the challenges posed for CPs in negotiating dual roles. This study is, therefore, a secondary analysis of semistructured interviews and direct observation of 48 CPs, addressing this phenomenon. Interview data were entered into NVivo 10 and systematically analyzed using an emergent thematic coding strategy. RESULTS: Pharmacists describe role ambiguity, where they perform as direct providers or team members simultaneously or in quick succession. They note the existence of a "transaction cost," where switching causes loss of momentum or disruption of work flow. Additionally, pharmacists feel that fellow providers lack an understanding of what they do and that CP contributions are not evaluated accurately by other health professionals. CONCLUSION: It is a challenge for CPs to balance the distinct roles of serving as collaborators and primary providers. Frequent role switching is not conducive to optimal work efficiency or patient care. Our findings suggest concrete steps that medical centers can take to improve both CP worklife and quality of patient care.

The interplay of contextual elements in implementation: an ethnographic case study.

BACKGROUND: Contextual elements have significant impact on uptake of health care innovations. While existing conceptual frameworks in implementation science suggest contextual elements interact with each other, little research has described how this might look in practice. To bridge this gap, this study identifies the interconnected patterns among contextual elements that influence uptake of an anticoagulation clinic improvement initiative. METHODS: We completed 51 semi-structured interviews and ethnographic observations across five case study sites involved in an evidence-based practice (EBP) quality improvement initiative. We analyzed data in NVivo 10 using an a priori approach based on the Promoting Action on Research Implementation in Health Services (PARIHS) model and an emergent thematic analysis. RESULTS: Key contextual elements, such as leadership, teamwork, and communication, interacted with each other in contributing to site-level uptake of the EBP, often yielding results that could not be predicted by looking at just one of these elements alone. Sites with context conducive to change in these areas predictably had high uptake, while sites with uniformly weak contextual elements had low uptake. Most sites presented a mixed picture, with contextual elements being strongly supportive of change in some areas and weak or moderate in others. In some cases, we found that sites with strong context in at least one area only needed to have adequate context in other areas to yield high uptake. At other sites, weak context in just one area had the potential to contribute to low uptake, despite countervailing strengths. Even a site with positive views of EBPs could not succeed when context was weak. CONCLUSION: Interrelationships among different contextual elements can act as barriers to uptake at some sites and as facilitators at others. Accounting for interconnections among elements enables PARIHS to more fully describe the determinants of successful implementation as they operate in real-world settings.

Colon cancer-derived oncogenic EGFR G724S mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab.

BACKGROUND: Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradoxically, anti-EGFR antibodies are more effective in colon and head and neck cancers where EGFR mutations occur less frequently. In colorectal cancer, anti-EGFR antibodies are routinely used as second-line therapy of KRAS wild-type tumors. However, detailed mechanisms and genomic predictors for pharmacological response to these antibodies in colon cancer remain unclear. FINDINGS: We describe a case of colorectal adenocarcinoma, which was found to harbor a kinase domain mutation, G724S, in EGFR through whole genome sequencing. We show that G724S mutant EGFR is oncogenic and that it differs from classic lung cancer derived EGFR mutants in that it is cetuximab responsive in vitro, yet relatively insensitive to small molecule kinase inhibitors. Through biochemical and cellular pharmacologic studies, we have determined that cells harboring the colon cancer-derived G719S and G724S mutants are responsive to cetuximab therapy in vitro and found that the requirement for asymmetric dimerization of these mutant EGFR to promote cellular transformation may explain their greater inhibition by cetuximab than small-molecule kinase inhibitors. CONCLUSION: The colon-cancer derived G719S and G724S mutants are oncogenic and sensitive in vitro to cetuximab. These data suggest that patients with these mutations may benefit from the use of anti-EGFR antibodies as part of the first-line therapy.