Single nucleus transcriptomics, pharmacokinetics, and pharmacodynamics of combined CDK4/6 and mTOR inhibition in a phase 0/1 trial of recurrent high-grade glioma.

Outcomes for adult patients with a high-grade glioma continue to be dismal and new treatment paradigms are urgently needed. To optimize the opportunity for discovery, we performed a phase 0/1 dose-escalation clinical trial that investigated tumor pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics following combined ribociclib (CDK4/6 inhibitor) and everolimus (mTOR inhibitor) treatment in recurrent high-grade glioma. Patients with a recurrent high-grade glioma (n = 24) harboring 1) CDKN2A / B deletion or CDK4 / 6 amplification, 2) PTEN loss or PIK3CA mutations, and 3) wild-type retinoblastoma protein (Rb) were enrolled. Patients received neoadjuvant ribociclib and everolimus treatment and no dose-limiting toxicities were observed. The median unbound ribociclib concentrations in Gadolinium non-enhancing tumor regions were 170 nM (range, 65 - 1770 nM) and 634 nM (range, 68 - 2345 nM) in patients receiving 5 days treatment at the daily dose of 400 and 600 mg, respectively. Unbound everolimus concentrations were below the limit of detection (< 0.1 nM) in both enhancing and non-enhancing tumor regions at all dose levels. We identified a significant decrease in MIB1 positive cells suggesting ribociclib-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) based comparisons of 17 IDH-wild-type on-trial recurrences to 31 IDH-wild-type standard of care treated recurrences data demonstrated a significantly lower fraction of cycling and neural progenitor-like (NPC-like) malignant cell populations. We validated the CDK4/6 inhibitor-directed malignant cell state shifts using three patient-derived cell lines. The presented clinical trial highlights the value of integrating pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics to assess treatment effects in phase 0/1 surgical tissues, including malignant cell state shifts. ClinicalTrials.gov identifier: NCT03834740 .

Predicting outcomes of phase III oncology trials with Bayesian mediation modeling of tumor response.

Pivotal cancer trials often fail to yield evidence in support of new therapies thought to offer promising alternatives to standards-of-care. Conducting randomized controlled trials in oncology tends to be considerably more expensive than studies of other diseases with comparable sample size. Moreover, phase III trial design often takes place with a paucity of survival data for experimental therapies. Experts have explained the failures on the basis of design flaws which produce studies with unrealistic expectations. This article presents a framework for predicting outcomes of phase III oncology trials using Bayesian mediation models. Predictions, which arise from interim analyses, derive from multivariate modeling of the relationships among treatment, tumor response, and their conjoint effects on survival. Acting as a safeguard against inaccurate pre-trial design assumptions, the methodology may better facilitate rapid closure of negative studies. Additionally the models can be used to inform re-estimations of sample size for under-powered trials that demonstrate survival benefit via tumor response mediation. The methods are applied to predict the outcomes of two colorectal cancer studies. Simulation is used to evaluate and compare models in the absence versus presence of reliable surrogate markers of survival.

A binding protein regulates myosin-7a dimerization and actin bundle assembly.

Myosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex's processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.

Pharmacokinetic/pharmacodynamic data extrapolation models for improved pediatric efficacy and toxicity estimation, with application to secondary hyperparathyroidism.

In most drug development settings, the regulatory approval process is accompanied by extensive studies performed to understand the drug's pharmacokinetic (PK) and pharmacodynamic (PD) properties. In this article, we attempt to utilize the rich PK/PD data to inform the borrowing of information from adults during pediatric drug development. In pediatric settings, it is especially crucial that we are parsimonious with the patients recruited for experimentation. We illustrate our approaches in the context of clinical trials of cinacalcet for treating secondary hyperparathyroidism in pediatric and adult patients with chronic kidney disease, where we model both parathyroid hormone (efficacy endpoint) and corrected calcium levels (safety endpoint). We use population PK/PD modeling of the cinacalcet data to quantitatively assess the similarity between adults and children, and use this information in various hierarchical Bayesian adult borrowing rules whose statistical properties can then be evaluated. In particular, we simulate the bias and mean square error performance of our approaches in settings where borrowing is and is not warranted to inform guidelines for the future use of our methods.

Cigarette Packs With URLs Leading to Tobacco Company Websites: Content Analysis.

BACKGROUND: Tobacco companies include on the packaging of their products URLs directing consumers to websites that contain protobacco messages. Online media tend to be underregulated and provide the industry with an opportunity to present users with protobacco communication. OBJECTIVE: The objective of our study was to document the content of websites that were advertised on tobacco packs in 14 low- and middle-income countries. METHODS: We purchased tobacco packs from 14 low- and middle-income countries in 2013 and examined them for the presence of URLs. We visited unique URLs on multiple occasions between October 1, 2016 and August 9, 2017. We developed a coding checklist and used it to conduct a content analysis of active corporate websites to identify types of protobacco communication. The coding checklist included the presence of regulatory controls and warnings, engagement strategies, marketing appeals (eg, description of product popularity, luxury/quality, taste), corporate social responsibility programs, and image management. We coded brand websites separately and also described social media and other website types. RESULTS: We identified 89 unique URLs, of which 54 were active during the search period. We assessed 26 corporate websites, 21 brand websites, 2 nontobacco websites, and 5 social media pages. We excluded 2 corporate websites and 14 brand websites due to limited accessible content or incomplete content. Corporate social responsibility was discussed on all corporate websites, and marketing appeals were also common. Corporate websites were also more likely to include more nonspecific (12/24, 50%) than specific (7/24, 29%) health warnings. Promotions (6/7, 86%) and sociability appeals (3/7, 43%) were common on brand websites. The small number of social media webpages in our sample used gendered marketing. CONCLUSIONS: URLs appearing on tobacco packs direct consumers to websites where users are exposed to marketing that highlights the "positive" contributions of tobacco companies on corporate websites, and extensive promotions and marketing appeals on brand websites and social media pages. It is essential that marketing regulations become more comprehensive and ban all protobacco communication, a policy that is in line with articles 5.3 and 13 of the World Health Organization Framework Convention on Tobacco Control. For countries that already ban internet tobacco advertising, enforcement efforts should be strengthened. Tobacco companies' use of URLs on packs may also be compelling for plain packaging advocacy, where all branding is removed from the pack and large graphic health warning labels are the only communication on the tobacco packaging. Future research should consider including tobacco websites in marketing surveillance.

A database of geopositioned Middle East Respiratory Syndrome Coronavirus occurrences.

As a World Health Organization Research and Development Blueprint priority pathogen, there is a need to better understand the geographic distribution of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and its potential to infect mammals and humans. This database documents cases of MERS-CoV globally, with specific attention paid to zoonotic transmission. An initial literature search was conducted in PubMed, Web of Science, and Scopus; after screening articles according to the inclusion/exclusion criteria, a total of 208 sources were selected for extraction and geo-positioning. Each MERS-CoV occurrence was assigned one of the following classifications based upon published contextual information: index, unspecified, secondary, mammal, environmental, or imported. In total, this database is comprised of 861 unique geo-positioned MERS-CoV occurrences. The purpose of this article is to share a collated MERS-CoV database and extraction protocol that can be utilized in future mapping efforts for both MERS-CoV and other infectious diseases. More broadly, it may also provide useful data for the development of targeted MERS-CoV surveillance, which would prove invaluable in preventing future zoonotic spillover.

Adaptive optics improves multiphoton super-resolution imaging.

We improve multiphoton structured illumination microscopy using a nonlinear guide star to determine optical aberrations and a deformable mirror to correct them. We demonstrate our method on bead phantoms, cells in collagen gels, nematode larvae and embryos, Drosophila brain, and zebrafish embryos. Peak intensity is increased (up to 40-fold) and resolution recovered (up to 176 ± 10 nm laterally, 729 ± 39 nm axially) at depths ∼250 µm from the coverslip surface.

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue.

Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 µm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.

The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles.

The endocycle is a developmentally programmed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. In Drosophila, the endocycle is driven by the oscillations of Cyclin E/Cdk2 activity. How the periodicity of Cyclin E/Cdk2 activity is achieved during endocycles is poorly understood. Here, we demonstrate that the p21(cip1)/p27(kip1)/p57(kip2)-like cyclin-dependent kinase inhibitor (CKI), Dacapo (Dap), promotes replication licensing during Drosophila endocycles by reinforcing low Cdk activity during the endocycle Gap-phase. In dap mutants, cells in the endocycle have reduced levels of the licensing factor Double Parked/Cdt1 (Dup/Cdt1), as well as decreased levels of chromatin-bound minichromosome maintenance (MCM2-7) complex. Moreover, mutations in dup/cdt1 dominantly enhance the dap phenotype in several polyploid cell types. Consistent with a reduced ability to complete genomic replication, dap mutants accumulate increased levels of DNA damage during the endocycle S-phase. Finally, genetic interaction studies suggest that dap functions to promote replication licensing in a subset of Drosophila mitotic cycles.

twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis.

Cyclins regulate progression through the cell cycle. Control of cyclin levels is essential in Drosophila oogenesis for the four synchronous divisions that generate the 16 cell germ line cyst and for ensuring that one cell in each cyst, the oocyte, is arrested in meiosis, while the remaining fifteen cells become polyploid nurse cells. Changes in cyclin levels could be achieved by regulating transcription, translation or protein stability. The proteasome limits cyclin protein levels in the Drosophila ovary, but the mechanisms regulating RNA turnover or translation remain largely unclear. Here, we report the identification of twin, a homolog of the yeast CCR4 deadenylase. We show that twin is important for the number and synchrony of cyst divisions and oocyte fate. Consistent with the deadenylase activity of CCR4 in yeast, our data suggest that Twin controls germ line cyst development by regulating poly(A) tail lengths of several targets including Cyclin A (CycA) RNA. twin mutants exhibit very low expression of Bag-of-marbles (Bam), a regulator of cyst division, indicating that Twin/Ccr4 activity is necessary for wild-type Bam expression. Lowering the levels of CycA or increasing the levels of Bam suppresses the defects we observe in twin ovaries, implicating CycA and Bam as downstream effectors of Twin. We propose that Twin/Ccr4 functions during early oogenesis to coordinate cyst division, oocyte fate specification and egg chamber maturation.

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I.

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. We demonstrate a role for the p21(CIP)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. Our data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts.