Circadian Rhythm Disruption Promotes Lung Tumorigenesis

Circadian rhythms are 24-hr oscillations that control a variety of biological processes in living systems, including two hallmarks of cancer, cell division and metabolism. Circadian rhythm disruption by shift work is associated with greater risk for cancer development and poor prognosis, suggesting a putative tumor-suppressive role for circadian rhythm homeostasis. Using a genetically engineered mouse model of lung adenocarcinoma, we have characterized the effects of circadian rhythm disruption on lung tumorigenesis. We demonstrate that both physiologic perturbation (jet lag) and genetic mutation of the central circadian clock components decreased survival and promoted lung tumor growth and progression. The core circadian genes Per2 and Bmal1 were shown to have cell-autonomous tumor-suppressive roles in transformation and lung tumor progression. Loss of the central clock components led to increased c-Myc expression, enhanced proliferation, and metabolic dysregulation. Our findings demonstrate that both systemic and somatic disruption of circadian rhythms contribute to cancer progression

Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8[superscript +]T-cells, and not CD4[superscript +]T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8[superscript +]T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8[superscript +]T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8[superscript +]T-cells, and decoupling of antigen uptake from B-cell activation.Kathy and Curt Marble Cancer Research Fund (Frontier Research Programme Grant)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32CA180586

Effect of temperature and moisture on high pressure lipid/oil extraction from microalgae

Commercially viable carbon–neutral biodiesel production from microalgae has potential for replacing depleting petroleum diesel. The process of biodiesel production from microalgae involves harvesting, drying and extraction of lipids which are energy- and cost-intensive processes. The development of effective large-scale lipid extraction processes which overcome the complexity of microalgae cell structure is considered one of the most vital requirements for commercial production. Thus the aim of this work was to investigate suitable extraction methods with optimised conditions to progress opportunities for sustainable microalgal biodiesel production. In this study, the green microalgal species consortium, Tarong polyculture was used to investigate lipid extraction with hexane (solvent) under high pressure and variable temperature and biomass moisture conditions using an Accelerated Solvent Extraction (ASE) method. The performance of high pressure solvent extraction was examined over a range of different process and sample conditions (dry biomass to water ratios (DBWRs): 100%, 75%, 50% and 25% and temperatures from 70 to 120 °C, process time 5–15 min). Maximum total lipid yields were achieved at 50% and 75% sample dryness at temperatures of 90–120 °C. We show that individual fatty acids (Palmitic acid C16:0; Stearic acid C18:0; Oleic acid C18:1; Linolenic acid C18:3) extraction optima are influenced by temperature and sample dryness, consequently affecting microalgal biodiesel quality parameters. Higher heating values and kinematic viscosity were compliant with biodiesel quality standards under all extraction conditions used. Our results indicate that biodiesel quality can be positively manipulated by selecting process extraction conditions that favour extraction of saturated and mono-unsaturated fatty acids over optimal extraction conditions for polyunsaturated fatty acids, yielding positive effects on cetane number and iodine values. Exceeding biodiesel standards for these two parameters opens blending opportunities with biodiesels that fall outside the minimal cetane and maximal iodine values

A Conditional System to Specifically Link Disruption of Protein-Coding Function with Reporter Expression in Mice

Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes. Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements. We present a strategy that couples Cre/loxP-mediated conditional gene disruption with faithful GFP reporter expression in mice in which Cre-mediated stable inversion of a splice acceptor-GFP-splice donor cassette concurrently disrupts protein production and creates a GFP fusion product. Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements. To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus

Genetic and Clonal Dissection of Murine Small Cell Lung Carcinoma Progression by Genome Sequencing

SummarySmall cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs

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Delivery to human immune cells.

<p><b>A)</b> Human T cells and MDDCs were tested for delivery of cascade blue labeled 3kDa dextran, fluorescein labeled 70kDa dextran, and APC labeled IgG1. The representative histograms for a 30–4 (T cells) and 10–7 (MDDCs) device (left) and replicates across device designs (right) are displayed. <b>B)</b> SiRNA mediated knockdown of CD4 and DC-SIGN protein levels in CD4⁺ T cells and MDDCs respectively. <b>C)</b> Knockdown of CD4 expression in human regulatory T cells in response to treatment by a 30–4 device. Dead cells were excluded for delivery or knockdown analysis. <b>D)</b> Comparison of device performance in T cells to nucleofection by Amaxa. Protein expression 72hrs after siRNA delivery and cell viability after treatment are shown. <b>E)</b> Intracellular staining for the p24 antigen was used as an indicator of HIV infection level in treated human CD4⁺ T cells 24hrs after infection. In these studies, vif and/or gag, siRNA was delivered 24hrs prior to infection while CD4 siRNA was delivered 48hrs prior to infection. <b>F)</b> Median fluorescence intensity of the p24 antigen stain across repeats (min. N = 4) of the experimental conditions. Data are represented as mean + 1 standard error.</p