Shear shock wave injury in vivo: High frame-rate ultrasound observation and histological assessment.

Using high frame-rate ultrasound and ¡1µm sensitive motion tracking we previously showed that shear waves at the surface of ex vivo and in situ brains develop into shear shock waves deep inside the brain, with destructive local accelerations. However post-mortem tissue cannot develop injuries and has different viscoelastodynamic behavior from in vivo tissue. Here we present the ultrasonic measurement of the high-rate shear shock biomechanics in the in vivo porcine brain, and histological assessment of the resulting axonal pathology. A new biomechanical model of brain injury was developed consisting of a perforated mylar surface attached to the brain and vibrated using an electromechanical shaker. Using a custom sequence with 8 interleaved wide beam emissions, brain imaging and motion tracking were performed at 2900 images/s. Shear shock waves were observed for the first time in vivo wherein the shock acceleration was measured to be 2.6 times larger than the surface acceleration ( 95g vs. 36g). Histopathology showed axonal damage in the impacted side of the brain from the brain surface, accompanied by a local shock-front acceleration of >70g. This shows that axonal injury occurs deep in the brain even though the shear excitation was at the brain surface, and the acceleration measurements support the hypothesis that shear shock waves are responsible for deep traumatic brain injuries.

Measuring How Public Health Stakeholders Seek to Influence Alcohol Premises Licensing in England and Scotland: The Public Health Engagement In Alcohol Licensing (PHIAL) Measure.

OBJECTIVE: In the United Kingdom, some public health teams (PHTs) routinely engage with local alcohol premises licensing systems, through which licenses to sell alcohol are granted. We aimed to categorize PHT efforts and to develop and apply a measure of their efforts over time. METHOD: Preliminary categories of PHT activity were developed based on prior literature and were used to guide data collection with PHTs in 39 local government areas (27 in England; 12 in Scotland), sampled purposively. Relevant activity from April 2012 to March 2019 was identified through structured interviews (N = 62), documentation analysis, and follow-up checks, and a grading system was developed. The measure was refined based on expert consultation and used to grade relevant PHT activity for the 39 areas in 6-month periods. RESULTS: The Public Health engagement In Alcohol Licensing (PHIAL) Measure includes 19 activities in six categories: (a) staffing; (b) reviewing license applications; (c) responding to license applications; (d) data usage; (e) influencing licensing stakeholders or policy; and (f) public involvement. PHIAL scores for each area demonstrate fluctuation in type and level of activity between and within areas over time. Participating PHTs in Scotland were more active on average, particularly on senior leadership, policy development, and working with the public. In England, activity to influence license applications before decision was more common, and a clear increase in activity is apparent from 2014 onward. CONCLUSIONS: The novel PHIAL Measure successfully assessed diverse and fluctuating PHT engagement in alcohol licensing systems over time and will have practice, policy, and research applications.

Geospatial analysis of reported activity locations to identify sites for tuberculosis screening.

Mobile screening units can help close tuberculosis case detection gaps. Placing screening units where people at high risk for undiagnosed tuberculosis preferentially spend time could make screening more resource-effective. We conducted a case-control study in Lima, Peru to identify locations where people with tuberculosis were more likely to spend time than community controls. We surveyed participants about activity locations over the past 6 months. We used density-based clustering to assess how patient and control activity locations differed, and logistic regression to compare location-based exposures. We included 109 tuberculosis patients and 79 controls. In density-based clustering analysis, the two groups had similar patterns of living locations, but their work locations clustered in distinct areas. Both groups were similarly likely to use public transit, but patients predominantly used buses and were less likely to use rapid transit (adjusted odds ratio [aOR] 0.31, 95% confidence interval [CI] 0.10-0.96) or taxis (aOR 0.42, 95% CI 0.21-0.85). Patients were more likely to have spent time in prison (aOR 11.55, 95% CI 1.48-90.13). Placing mobile screening units at bus terminals serving locations where tuberculosis patients have worked and within and around prisons could help reach people with undiagnosed tuberculosis.

Identifying barriers and facilitators to implementation of community-based tuberculosis active case finding with mobile X-ray units in Lima, Peru: a RE-AIM evaluation.

OBJECTIVES: Identify barriers and facilitators to integrating community tuberculosis screening with mobile X-ray units into a health system. METHODS: Reach, effectiveness, adoption, implementation and maintenance evaluation. SETTING: 3-district region of Lima, Peru. PARTICIPANTS: 63 899 people attended the mobile units from 7 February 2019 to 6 February 2020. INTERVENTIONS: Participants were screened by chest radiography, which was scored for abnormality by computer-aided detection. People with abnormal X-rays were evaluated clinically and by GeneXpert MTB/RIF (Xpert) sputum testing. People diagnosed with tuberculosis at the mobile unit were accompanied to health facilities for treatment initiation. PRIMARY AND SECONDARY OUTCOME MEASURES: Reach was defined as the percentage of the population of the three-district region that attended the mobile units. Effectiveness was defined as the change in tuberculosis case notifications over a historical baseline. Key implementation fidelity indicators were the percentages of people who had chest radiography performed, were evaluated clinically, had sputum samples collected, had valid Xpert results and initiated treatment. RESULTS: The intervention reached 6% of the target population and was associated with an 11% (95% CI 6 to 16) increase in quarterly case notifications, adjusting for the increasing trend in notifications over the previous 3 years. Implementation indicators for screening, sputum collection and Xpert testing procedures all exceeded 85%. Only 82% of people diagnosed with tuberculosis at the mobile units received treatment; people with negative or trace Xpert results were less likely to receive treatment. Suboptimal treatment initiation was driven by health facility doctors' lack of familiarity with Xpert and lack of confidence in diagnoses made at the mobile unit. CONCLUSION: Mobile X-ray units were a feasible and effective strategy to extend tuberculosis diagnostic services into communities and improve early case detection. Effective deployment however requires advance coordination among stakeholders and targeted provider training to ensure that people diagnosed with tuberculosis by new modalities receive prompt treatment.

Exploring the impact of public health teams on alcohol premises licensing in England and Scotland (ExILEnS): procotol for a mixed methods natural experiment evaluation.

BACKGROUND: Recent regulatory changes in the system by which premises are licensed to sell alcohol, have given health representatives a formal role in the process in England and Scotland. The degree to which local public health teams engage with this process varies by locality in both nations, which have different licensing regimes. This study aims to critically assess the impact on alcohol-related harms - and mechanisms - of public health stakeholders' engagement in alcohol premises licensing from 2012 to 2018, comparing local areas with differing types and intensities of engagement, and examining practice in Scotland and England. METHODS: The study will recruit 20 local authority areas where public health stakeholders have actively engaged with the alcohol premises licensing system (the 'intervention') and match them to a group of 20 lower activity areas using genetic matching. Four work packages are included: (1) Structured interviews and documentary analysis will examine the type and level of intervention activity from 2012 to 2018, creating a novel composite measure of the intensity of such activity and will assess the local licensing system and potential confounding activities over the same period. In-depth interviews with public health, licensing, police and others will explore perceived mechanisms of change, acceptability, and impact. (2) Using longitudinal growth models and time series analyses, the study will evaluate the impact of high and low levels of activity on alcohol-related harms using routine data from baseline 2009 to 2018. (3) Intervention costs, estimated National Health Service cost savings and health gains will be evaluated using the Sheffield Alcohol Policy Model to estimate impact on alcohol consumption and health inequalities. (4) The study will engage public health teams to create a new theory of change for public health involvement in the licensing process using our data. We will share findings with local, national and international stakeholders. DISCUSSION: This interdisciplinary study examines, for the first time, whether and how public health stakeholders' involvement in alcohol licensing impacts on alcohol harms. Using mixed methods and drawing on complex systems thinking, it will make an important contribution to an expanding literature evaluating interventions not suited to traditional epidemiological research.

CDK4/6 Inhibition on Glucose and Pancreatic Beta Cell Homeostasis in Young and Aged Rats.

Genetic deletion of cyclin-dependent kinase 4 (Cdk4) is associated with pancreatic beta cell loss and glucose dysregulation in rodents. Palbociclib, one of the first selective CDK4/6 inhibitors approved for the treatment of advanced breast cancer, is currently being investigated as an adjuvant treatment in patients with early-stage breast cancer and in a variety of cancers covering a wide-range of patient populations. Hence, longer chronic toxicity studies were necessary to further examine its safety profile. The effects of different doses and duration of palbociclib administration on glucose and beta cell homeostasis in young (two months) versus aged (12 months) rats was compared. Glucose dysregulation, due to pancreatic beta cell degeneration, was observed in young rats administered the highest dose of palbociclib for 6 months. Abnormal pancreatic islet histology and activation of the endoplasmic reticulum stress response in beta cells were detected after shorter administration with high-dose palbociclib in young rats. To test the hypothesis that palbociclib-associated inhibition of beta cell proliferation will more profoundly affect younger animals that have not achieved replicative quiescence, we administered high-dose palbociclib to aged rats for 6 months. In contrast to the young rats, despite equivalent exposures to palbociclib, no evidence of impaired glucose tolerance, hypoinsulinemia, beta cell vacuolization, or beta cell loss was seen in aged rats. Palbociclib administration induces beta cell failure in young but not aged rats.Implications: Although adult humans receiving palbociclib have not displayed detectable adverse effects on glucose metabolism, the risk of beta cell failure in children remains unexplored. Mol Cancer Res; 15(11); 1531-41. ©2017 AACR.

PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations.

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.

Comparison of dynamic contrast-enhanced MR, ultrasound and optical imaging modalities to evaluate the antiangiogenic effect of PF-03084014 and sunitinib.

Noninvasive imaging has been widely applied for monitoring antiangiogenesis therapy in cancer drug discovery. In this report, we used different imaging modalities including high-frequency ultrasound (HFUS), dynamic contrast enhanced-MR (DCE-MR), and fluorescence molecular tomography (FMT) imaging systems to monitor the changes in the tumor vascular properties after treatment with γ-secretase inhibitor PF-03084014. Sunitinib was tested in parallel for comparison. In the MDA-MB-231Luc model, we demonstrated that antiangiogenesis was one of the contributing mechanisms for the therapeutic effect of PF-03084014. By immunohistochemistry and FITC-lectin perfusion assays, we showed that the vascular defects upon treatment with PF-03084014 were associated with Notch pathway modulation, evidenced by a decrease in the HES1 protein and by the changes in VEGFR2 and HIF1α levels, which indicates down-stream effects. Using a 3D power Doppler scanning method, ultrasound imaging showed that the% vascularity in the MDA-MB-231Luc tumor decreased significantly at 4 and 7 days after the treatment with PF-03084014. A decrease in the tumor vessel function was also observed through contrast-enhanced ultrasound imaging with microbubble injection. These findings were consistent with the PF-03084014-induced functional vessel changes measured by suppressing the K(trans) values using DCE-MRI. In contrast, the FMT imaging with the AngioSence 680EX failed to detect any treatment-associated tumor vascular changes. Sunitinib demonstrated an outcome similar to PF-03084014 in the tested imaging modalities. In summary, ultrasound and DCE-MR imaging successfully provided longitudinal measurement of the phenotypic and functional changes in tumor vasculature after treatment with PF-03084014 and sunitinib.

Pan-FGFR inhibition leads to blockade of FGF23 signaling, soft tissue mineralization, and cardiovascular dysfunction.

The fibroblast growth factor receptors (FGFR) play a major role in angiogenesis and are desirable targets for the development of therapeutics. Groups of Wistar Han rats were dosed orally once daily for 4 days with a small molecule pan-FGFR inhibitor (5mg/kg) or once daily for 6 days with a small molecule MEK inhibitor (3mg/kg). Serum phosphorous and FGF23 levels increased in all rats during the course of the study. Histologically, rats dosed with either drug exhibited multifocal, multiorgan soft tissue mineralization. Expression levels of the sodium phosphate transporter Npt2a and the vitamin D-metabolizing enzymes Cyp24a1 and Cyp27b1 were modulated in kidneys of animals dosed with the pan-FGFR inhibitor. Both inhibitors decreased ERK phosphorylation in the kidneys and inhibited FGF23-induced ERK phosphorylation in vitro in a dose-dependent manner. A separate cardiovascular outcome study was performed to monitor hemodynamics and cardiac structure and function of telemetered rats dosed with either the pan-FGFR inhibitor or MEK inhibitor for 3 days. Both compounds increased blood pressure (~+ 17 mmHg), decreased heart rate (~-75 bpm), and modulated echocardiography parameters. Our data suggest that inhibition of FGFR signaling following administration of either pan-FGFR inhibitor or MEK inhibitor interferes with the FGF23 pathway, predisposing animals to hyperphosphatemia and a tumoral calcinosis-like syndrome in rodents.

Dose-dependent effects of small-molecule antagonists on the genomic landscape of androgen receptor binding.

BACKGROUND: The androgen receptor plays a critical role throughout the progression of prostate cancer and is an important drug target for this disease. While chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) is becoming an essential tool for studying transcription and chromatin modification factors, it has rarely been employed in the context of drug discovery. RESULTS: Here we report changes in the genome-wide AR binding landscape due to dose-dependent inhibition by drug-like small molecules using ChIP-Seq. Integration of sequence analysis, transcriptome profiling, cell viability assays and xenograft tumor growth inhibition studies enabled us to establish a direct cistrome-activity relationship for two novel potent AR antagonists. By selectively occupying the strongest binding sites, AR signaling remains active even when androgen levels are low, as is characteristic of first-line androgen ablation therapy. Coupled cistrome and transcriptome profiling upon small molecule antagonism led to the identification of a core set of AR direct effector genes that are most likely to mediate the activities of targeted agents: unbiased pathway mapping revealed that AR is a key modulator of steroid metabolism by forming a tightly controlled feedback loop with other nuclear receptor family members and this oncogenic effect can be relieved by antagonist treatment. Furthermore, we found that AR also has an extensive role in negative gene regulation, with estrogen (related) receptor likely mediating its function as a transcriptional repressor. CONCLUSIONS: Our study provides a global and dynamic view of AR's regulatory program upon antagonism, which may serve as a molecular basis for deciphering and developing AR therapeutics.

A review of current methods for assessing hemostasis in vivo and introduction to a potential alternative approach.

A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents in preclinical animal models and in patients with inherited bleeding disorders, such as von Willebrand disease (VWD) and hemophilia A, or with acquired bleeding disorders such as those resulting from medications or disease processes. In this review, we discuss current methods for assessing hemostasis in vivo and the associated challenges. We also present ARFI-Monitored Hemostatic Challenge; a new, potentially alternate method for in vivo hemostasis monitoring that is in development by our group.

[(18)F]FLT-PET imaging does not always "light up" proliferating tumor cells.

PURPOSE: [(18)F]FLT (3'-Fluoro-3' deoxythymidine)-PET imaging was proposed as a tool for measuring in vivo tumor cell proliferation. The aim of this article was to validate the use of [(18)F]FLT-PET imaging for measuring xenograft proliferation and subsequent monitoring of targeted therapy. EXPERIMENTAL DESIGN: In exponentially growing xenografts, factors that could impact the outcome of [(18)F]FLT-PET imaging, such as nucleoside transporters, thymidine kinase 1, the relative contribution of DNA salvage pathway, and the ratio of FLT to thymidine, were evaluated. The [(18)F]FLT tracer avidity was compared with other proliferation markers. RESULTS: In a panel of proliferating xenografts, [(18)F]FLT or [(3)H]thymidine tracer avidity failed to reflect the tumor growth rate across different tumor types, despite the high expressions of Ki67 and TK1. When FLT was injected at the same dose level as used in the preclinical [(18)F]FLT-PET imaging, the plasma exposure ratio of FLT to thymidine was approximately 1:200. Thymidine levels in different tumor types seemed to be variable and exhibited an inverse relationship with the FLT tracer avidity. In contrast, high-dose administration of bromdeoxyuridine (BrdUrd; 50 mg/kg) yielded a plasma exposure of more than 4-fold higher than thymidine and leads to a strong correlation between the BrdUrd uptake and the tumor proliferation rate. In FLT tracer-avid models, [(18)F]FLT-PET imaging as a surrogate biomarker predicted the therapeutic response of CDK4/6 inhibitor PD-0332991. CONCLUSIONS: Tumor thymidine level is one of the factors that impact the correlation between [(18)F]FLT uptake and tumor cell proliferation. With careful validation, [(18)F]FLT-PET imaging can be used to monitor antiproliferative therapies in tracer-avid malignancies.

Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays.

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase 1.

Checkpoints are present in all phases of the cell cycle and are regarded as the gatekeepers maintaining the integrity of the genome. Many conventional agents used to treat cancer impart damage to the genome and activate cell cycle checkpoints. Many tumors are defective in the tumor suppressor p53 and therefore lack a functional G(1) checkpoint. In these tumors, however, the S-G(2) checkpoints remain intact and, in response to DNA damage, arrest cell cycle progression allowing time for DNA repair. Checkpoint kinase 1 (Chk1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G(2)-phase checkpoints. Inhibiting Chk1 represents a therapeutic strategy for creating a "synthetic lethal" response by overriding the last checkpoint defense of tumor cells against the lethal damage induced by DNA-directed chemotherapeutic agents. Chk1 inhibition is consistent with emerging targeted therapies aiming to exploit molecular differences between normal and cancer cells. Adding a Chk1 inhibitor to DNA-damaging cytotoxic therapy selectively targets tumors with intrinsic checkpoint defects while minimizing toxicity in checkpoint-competent normal cells. PF-00477736 was identified as a potent, selective ATP-competitive small-molecule inhibitor that inhibits Chk1 with a K(i) of 0.49 nM. PF-00477736 abrogates cell cycle arrest induced by DNA damage and enhances cytotoxicity of clinically important chemotherapeutic agents, including gemcitabine and carboplatin. In xenografts, PF-00477736 enhanced the antitumor activity of gemcitabine in a dose-dependent manner. PF-00477736 combinations were well tolerated with no exacerbation of side effects commonly associated with cytotoxic agents.