piggyBac Transposon Somatic Mutagenesis with an Activated Reporter and Tracker (PB-SMART) for Genetic Screens in Mice

Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease

Ultra-low threshold CW Triply Resonant OPO in the near infrared using Periodically Poled Lithium Niobate

We have operated a CW triply resonant OPO using a PPLN crystal pumped by a Nd:YAG laser at 1.06 micron and generating signal and idler modes in the 2-2.3 micron range. The OPO was operated stably in single mode operation over large periods of time with a pump threshold as low as 500 microwatts.Comment: 7 pages, 5 figures, submitted to JEOS

Frontiers in Pigment Cell and Melanoma Research

We identify emerging frontiers in clinical and basic research of melanocyte biology and its associated biomedical disciplines. We describe challenges and opportunities in clinical and basic research of normal and diseased melanocytes that impact current approaches to research in melanoma and the dermatological sciences. We focus on four themes: (1) clinical melanoma research, (2) basic melanoma research, (3) clinical dermatology, and (4) basic pigment cell research, with the goal of outlining current highlights, challenges, and frontiers associated with pigmentation and melanocyte biology. Significantly, this document encapsulates important advances in melanocyte and melanoma research including emerging frontiers in melanoma immunotherapy, medical and surgical oncology, dermatology, vitiligo, albinism, genomics and systems biology, epidemiology, pigment biophysics and chemistry, and evolution

A Role for ATF2 in Regulating MITF and Melanoma Development

The transcription factor ATF2 has been shown to attenuate melanoma susceptibility to apoptosis and to promote its ability to form tumors in xenograft models. To directly assess ATF2's role in melanoma development, we crossed a mouse melanoma model (Nras(Q61K)::Ink4a⁻/⁻) with mice expressing a transcriptionally inactive form of ATF2 in melanocytes. In contrast to 7/21 of the Nras(Q61K)::Ink4a⁻/⁻ mice, only 1/21 mice expressing mutant ATF2 in melanocytes developed melanoma. Gene expression profiling identified higher MITF expression in primary melanocytes expressing transcriptionally inactive ATF2. MITF downregulation by ATF2 was confirmed in the skin of Atf2⁻/⁻ mice, in primary human melanocytes, and in 50% of human melanoma cell lines. Inhibition of MITF transcription by MITF was shown to be mediated by ATF2-JunB-dependent suppression of SOX10 transcription. Remarkably, oncogenic BRAF (V600E)-dependent focus formation of melanocytes on soft agar was inhibited by ATF2 knockdown and partially rescued upon shMITF co-expression. On melanoma tissue microarrays, a high nuclear ATF2 to MITF ratio in primary specimens was associated with metastatic disease and poor prognosis. Our findings establish the importance of transcriptionally active ATF2 in melanoma development through fine-tuning of MITF expression

A 750 mW, continuous-wave, solid-state laser source at 313 nm for cooling and manipulating trapped 9Be+ ions

We present a solid-state laser system that generates 750 mW of continuous-wave single-frequency output at 313 nm. Sum-frequency generation with fiber lasers at 1550 nm and 1051 nm produces up to 2 W at 626 nm. This visible light is then converted to UV by cavity-enhanced second-harmonic generation. The laser output can be tuned over a 495 GHz range, which includes the 9Be+ laser cooling and repumping transitions. This is the first report of a narrow-linewidth laser system with sufficient power to perform fault-tolerant quantum-gate operations with trapped 9Be+ ions by use of stimulated Raman transitions.Comment: 9 pages, 4 figure

Workshop on challenges, insights, and future directions for mouse and humanized models in cancer immunology and immunotherapy: a report from the associated programs of the 2016 annual meeting for the Society for Immunotherapy of cancer

Understanding how murine models can elucidate the mechanisms underlying antitumor immune responses and advance immune-based drug development is essential to advancing the field of cancer immunotherapy. The Society for Immunotherapy of Cancer (SITC) convened a workshop titled, “Challenges, Insights, and Future Directions for Mouse and Humanized Models in Cancer Immunology and Immunotherapy” as part of the SITC 31st Annual Meeting and Associated Programs on November 10, 2016 in National Harbor, MD. The workshop focused on key issues in optimizing models for cancer immunotherapy research, with discussions on the strengths and weaknesses of current models, approaches to improve the predictive value of mouse models, and advances in cancer modeling that are anticipated in the near future. This full-day program provided an introduction to the most common immunocompetent and humanized models used in cancer immunology and immunotherapy research, and addressed the use of models to evaluate immune-targeting therapies. Here, we summarize the workshop presentations and subsequent panel discussion

SMAC mimetic Debio 1143 synergizes with taxanes, topoisomerase inhibitors and bromodomain inhibitors to impede growth of lung adenocarcinoma cells

Targeting anti-apoptotic proteins can sensitize tumor cells to conventional chemotherapies or other targeted agents. Antagonizing the Inhibitor of Apoptosis Proteins (IAPs) with mimetics of the pro-apoptotic protein SMAC is one such approach. We used sensitization compound screening to uncover possible agents with the potential to further sensitize lung adenocarcinoma cells to the SMAC mimetic Debio 1143. Several compounds in combination with Debio 1143, including taxanes, topoisomerase inhibitors, and bromodomain inhibitors, super-additively inhibited growth and clonogenicity of lung adenocarcinoma cells. Co-treatment with Debio 1143 and the bromodomain inhibitor JQ1 suppresses the expression of c-IAP1, c-IAP2, and XIAP. Non-canonical NF-κB signaling is also activated following Debio 1143 treatment, and Debio 1143 induces the formation of the ripoptosome in Debio 1143-sensitive cell lines. Sensitivity to Debio 1143 and JQ1 co-treatment was associated with baseline caspase-8 expression. In vivo treatment of lung adenocarcinoma xenografts with Debio 1143 in combination with JQ1 or docetaxel reduced tumor volume more than either single agent alone. As Debio 1143-containing combinations effectively inhibited both in vitro and in vivo growth of lung adenocarcinoma cells, these data provide a rationale for Debio 1143 combinations currently being evaluated in ongoing clinical trials and suggest potential utility of other combinations identified here

Revision of the Melanocytic Pathology Assessment Tool and Hierarchy for Diagnosis Classification Schema for Melanocytic Lesions: A Consensus Statement

IMPORTANCE A standardized pathology classification system for melanocytic lesions is needed to aid both pathologists and clinicians in cataloging currently existing diverse terminologies and in the diagnosis and treatment of patients. The Melanocytic Pathology Assessment Tool and Hierarchy for Diagnosis (MPATH-Dx) has been developed for this purpose. OBJECTIVE To revise the MPATH-Dx version 1.0 classification tool, using feedback from dermatopathologists participating in the National Institutes of Health-funded Reducing Errors in Melanocytic Interpretations (REMI) Study and from members of the International Melanoma Pathology Study Group (IMPSG). EVIDENCE REVIEW Practicing dermatopathologists recruited from 40 US states participated in the 2-year REMI study and provided feedback on the MPATH-Dx version 1.0 tool. Independently, member dermatopathologists participating in an IMPSG workshop dedicated to the MPATH-Dx schema provided additional input for refining the MPATH-Dx tool. A reference panel of 3 dermatopathologists, the original authors of the MPATH-Dx version 1.0 tool, integrated all feedback into an updated and refined MPATH-Dx version 2.0. FINDINGS The new MPATH-Dx version 2.0 schema simplifies the original 5-class hierarchy into 4 classes to improve diagnostic concordance and to provide more explicit guidance in the treatment of patients. This new version also has clearly defined histopathological criteria for classification of classes I and II lesions; has specific provisions for the most frequently encountered low-cumulative sun damage pathway of melanoma progression, as well as other, less common World Health Organization pathways to melanoma; provides guidance for classifying intermediate class II tumors vs melanoma; and recognizes a subset of pT1a melanomas with very low risk and possible eventual reclassification as neoplasms lacking criteria for melanoma. CONCLUSIONS AND RELEVANCE The implementation of the newly revised MPATH-Dx version 2.0 schema into clinical practice is anticipated to provide a robust tool and adjunct for standardized diagnostic reporting of melanocytic lesions and management of patients to the benefit of both health care practitioners and patients