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.
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•Physiologic disruption of circadian rhythms accelerates lung cancer•Genetic loss of Per2 or Bmal1 promotes lung tumorigenesis•Cell-autonomous loss of circadian genes enhances transformation and growth•Circadian rhythm disruption leads to increased c-Myc levels and metabolic reprogramming
Papagiannakopoulos et al. demonstrate that both physiologic and genetic circadian rhythm disruption accelerates lung tumorigenesis in mice, pointing to a tumor cell-autonomous, tumor-suppressive role of the circadian machinery. Mechanistically, circadian rhythm disruption leads to increased c-Myc levels, enhanced proliferation, and metabolic reprogramming.
Small 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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•Murine SCLCs acquire few point mutations in the absence of tobacco mutagens•Pten is recurrently mutated, and engineered deletion accelerates tumor progression•Mycl1 amplifications play an early, central role in mSCLC tumorigenesis•Analysis of related primary and metastatic mSCLC suggests complex clonal evolution
Comprehensive genomic analysis in a mouse model of small-cell lung carcinoma delineates metastatic progression in this tumor type, showing that selection for specific cancer mutations can drive large genomic rearrangements and that distant metastases likely arise from a common metastatic seeding step involving the lymph nodes.
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(+)T-cells, and not CD4(+)T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8(+)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(+)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(+)T-cells, and decoupling of antigen uptake from B-cell activation.
Intracellular delivery of biomolecules, such as proteins and siRNAs, into primary immune cells, especially resting lymphocytes, is a challenge. Here we describe the design and testing of microfluidic ...intracellular delivery systems that cause temporary membrane disruption by rapid mechanical deformation of human and mouse immune cells. Dextran, antibody and siRNA delivery performance is measured in multiple immune cell types and the approach's potential to engineer cell function is demonstrated in HIV infection studies.
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.
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•Genetic system couples gene disruption with GFP expression•System preserves function of all nonprotein-coding elements after recombination•System allows tracking of cells harboring targeted protein deficiency
Current gene-targeting approaches typically involve simultaneous ablation of all elements within targeted protein-coding genes, thus potentially disrupting protein coding and important noncoding functions. Chiou et al. now present a strategy coupling Cre/loxP-mediated gene disruption with faithful GFP reporter expression in mice. Importantly, Cre-mediated cassette inversion maintains microRNA-mediated regulation of the GFP reporter as well as the expression of nonprotein-coding elements. The authors tested this potentially generalizable strategy by targeting the Hmga2 locus.
CD8
T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8
T cell ...responses, therapeutic approaches to generate Ag-specific CD8
T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8
T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ∼1000-fold more potent at eliciting CD8
T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8
T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8
T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8
T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8
T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.
Receptors for advanced glycation end-products (RAGE) are cell-surface receptors expressed by pulmonary tissue that influence alveolar type (AT) II-ATI transition required for normal alveolar ...formation. However, the precise contribution of RAGE in interactions between pulmonary epithelium and splanchnic mesenchyme during lung organogenesis remains uncertain. To test the hypothesis that RAGE misexpression adversely affects lung morphogenesis, conditional transgenic mice were generated that overexpress RAGE. Mice that overexpress RAGE throughout embryogenesis experienced 100% mortality and significant lung hypoplasia coincident with large, vacuous areas in the periphery when compared with normal airway and alveolar architecture observed in control mouse lungs. Flow cytometry and immunohistochemistry employing cell-specific markers for distal (forkhead box protein A2) and respiratory (thyroid transcription factor-1) epithelium, ATII cells (pro-surfactant protein-C), and ATI cells (T1-α) demonstrated anomalies in key epithelial cell populations resulting from RAGE up-regulation. These results reveal that precise regulation of RAGE expression is required during lung formation. Furthermore, abundant RAGE results in profound alterations in epithelial cell differentiation that culminate in severe respiratory distress and perinatal lethality.
Modic changes (MC) are signs of vertebral pathology visible on magnetic resonance (MR) images that have been associated with low back pain (LBP) and disc degeneration in people. Multiple breeds of ...dogs also develop MCs and coincident back pain. However, the association between breed, MC, and spinal pathologies has yet to be fully elucidated. This study aimed to identify the prevalence of MC that occur spontaneously in the lumbar vertebral column of dogs diagnosed with intervertebral disc disease (IVDD) and examine their association with demographic criteria and the disc width index (DWI).
Medical records and lumbar vertebral column MR images were examined from 104 dogs (831 intervertebral disc spaces and adjacent vertebrae), which were divided into three groups: chondrodystrophic dogs (CD;
=54) and non-chondrodystrophic dogs (NCD;
=30) with IVDD as the primary diagnosis, and control dogs (
=20) with other spinal diseases as their primary diagnosis.
Increasing age and a diagnosis of IVDD were significantly associated with MC in dogs (
< 0.001 and
= 0.0062, respectively). In CD dogs with IVDD, Type 2 MC were most prevalent, whereas, in NCD dogs, Type 3 MC were the most prevalent type. Type 2 MC were distributed nearly equally across the lumbar vertebral column, while Type 3 MC were primarily detected at the level of L7-S1.
This study demonstrated that MC developed spontaneously in dogs, are common in dogs diagnosed with IVDD, and the type observed varies by breed. Further research is needed to understand the pathogenesis of MC; however, the increased presence of Type 2 MC in CD dogs, similar to what is found in people with disc degeneration, suggests that CD dogs could serve as models for MC in people.
•Microalgae biomass was extracted at high pressure.•Moisture and temperature has significant effect on extraction performance.•Fatty acid composition changes with extraction condition.
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 5–15min). 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.
Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650)
1
. AARSs are grouped into two ...broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650)
1
. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia–c and IIa–c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes—(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.
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