Rapid assessment of 3-dimensional intra-tumor heterogeneity through cycling temperature capillary electrophoresis.

OBJECTIVE: Tumors are heterogeneous three-dimensional masses populated by numerous cell types, including distinct sub-clones of cancerous cells. Various sub-clones within the same tumor mass may respond differently to cancer treatment, and intra-tumor heterogeneity contributes to acquired therapeutic resistance. Thus, one tissue biopsy will in most cases not be representative of the entire genetic landscape of a tumor mass. In this study, we aimed to establish an easily accessible, low cost method to address intra-tumor heterogeneity in three dimensions, for a limited number of DNA alterations. RESULTS: This study includes analyses of the three-dimensional (3D) distribution of DNA mutations in human colon cancer and mouse mammary gland tumor tissue samples. We used laser capture microdissection for the unbiased collection of tissue in several XY-planes throughout the tumor masses. Cycling temperature capillary electrophoresis was used to determine mutant allele frequency. High-resolution distribution maps of KRAS and Trp53 mutations were generated for each XY-plane in human and mouse tumor samples, respectively. To provide a holistic interpretation of the mutation distribution, we generated interactive 3D heatmaps giving an easily interpretable understanding of the spatial distribution of the analyzed mutations. The method described herein provides an accessible way of describing intra-tumor heterogeneity for a limited number of mutations.

PML Regulates the Epidermal Differentiation Complex and Skin Morphogenesis during Mouse Embryogenesis.

The promyelocytic leukemia (PML) protein is an essential component of nuclear compartments called PML bodies. This protein participates in several cellular processes, including growth control, senescence, apoptosis, and differentiation. Previous studies have suggested that PML regulates gene expression at a subset of loci through a function in chromatin remodeling. Here we have studied global gene expression patterns in mouse embryonic skin derived from Pml depleted and wild type mouse embryos. Differential gene expression analysis at different developmental stages revealed a key role of PML in regulating genes involved in epidermal stratification. In particular, we observed dysregulation of the late cornified envelope gene cluster, which is a sub-region of the epidermal differentiation complex. In agreement with these data, PML body numbers are elevated in basal keratinocytes during embryogenesis, and we observed reduced epidermal thickness and defective hair follicle development in PML depleted mouse embryos.

Coordinated collective migration and asymmetric cell division in confluent human keratinocytes without wounding.

Epithelial sheet spreading is a fundamental cellular process that must be coordinated with cell division and differentiation to restore tissue integrity. Here we use consecutive serum deprivation and re-stimulation to reconstruct biphasic collective migration and proliferation in cultured sheets of human keratinocytes. In this system, a burst of long-range coordinated locomotion is rapidly generated throughout the cell sheet in the absence of wound edges. Migrating cohorts reach correlation lengths of several millimeters and display dependencies on epidermal growth factor receptor-mediated signaling, self-propelled polarized migration, and a G1/G0 cell cycle environment. The migration phase is temporally and spatially aligned with polarized cell divisions characterized by pre-mitotic nuclear migration to the cell front and asymmetric partitioning of nuclear promyelocytic leukemia bodies and lysosomes to opposite daughter cells. This study investigates underlying mechanisms contributing to the stark contrast between cells in a static quiescent state compared to the long-range coordinated collective migration seen in contact with blood serum.

Does Fear Increase Search Effort in More Numerate People? An Experimental Study Investigating Information Acquisition in a Decision From Experience Task.

The aim of this study was to investigate the effect of numeracy and the emotion of fear on the decision-making process. While previous research demonstrated that these factors are independently related to search effort, search policy and choice in a decision from experience task, less is known about how their interaction contributes to processing information under uncertainty. We attempted to address this problem and to fill this gap. In the present study, we hypothesized that more numerate people would sample more information about a decision problem and that the effect of fear would depend on the source of this emotion: whether it is integral (i.e., relevant) or incidental (i.e., irrelevant) to a decision problem. Additionally, we tested how these factors predict choices. We addressed these hypotheses in a series of two experiments. In each experiment, we used a sampling paradigm to measure search effort, search policy and choice in nine binary problems included in a decision from experience task. In Experiment 1, before the sampling task we elicited incidental fear by asking participants to recall fearful events from their life. In Experiment 2, integral fear was elicited by asking participants to make choices concerning medical treatment. Decision problems and their payoff distributions were the same in the two experiments and across each condition. In both experiments, we assessed objective statistical numeracy and controlled for a change in the current emotional state. We found that more numerate people sampled more information about a decision problem and switched less frequently between alternatives. Incidental fear marginally predicted search effort. Integral fear led to larger sample sizes, but only among more numerate people. Neither numeracy nor fear were related to the number of choices that maximized expected values. However, across two experiments sample sizes predicted the number of choices that maximized experienced mean returns. The findings suggest that people with higher numeracy may be more sensitive to integral emotions; this may result in more effortful sampling of relevant information leading to choices maximizing experienced returns.

Visualization of PML nuclear import complexes reveals FG-repeat nucleoporins at cargo retrieval sites.

Selective nuclear import in eukaryotic cells involves sequential interactions between nuclear import receptors and phenylalanine-glycine (FG)-repeat nucleoporins. Traditionally, binding of cargoes to import receptors is perceived as a nuclear pore complex independent event, while interactions between import complexes and nucleoporins are thought to take place at the nuclear pores. However, studies have shown that nucleoporins are mobile and not static within the nuclear pores, suggesting that they may become engaged in nuclear import before nuclear pore entry. Here we have studied post-mitotic nuclear import of the tumor suppressor protein PML. Since this protein forms nuclear compartments called PML bodies that persist during mitosis, the assembly of putative PML import complexes can be visualized on the surface of these protein aggregates as the cell progress from an import inactive state in mitosis to an import active state in G1. We show that these post-mitotic cytoplasmic PML bodies incorporate a multitude of peripheral nucleoporins, but not scaffold or nuclear basket nucleoporins, in a manner that depends on FG-repeats, the KPNB1 import receptor, and the PML nuclear localization signal. The study suggests that nucleoporins have the ability to target certain nuclear cargo proteins in a nuclear pore-uncoupled state, before nuclear pore entry.

Do endometriomas induce an inflammatory reaction in nearby follicles?

STUDY QUESTION: Do endometriomas induce an inflammatory reaction with increased cytokine concentrations in nearby follicles and thereby affect follicular development during controlled ovarian stimulation for in vitro fertilization (IVF)? SUMMARY ANSWER: With most endometriomas, there is no evidence of increased cytokine concentrations in the ipsilateral leading follicle. Infrequently, the concentration of inflammatory cytokines is increased in the follicular fluid (FF) and associated with diminished ovarian response. WHAT IS KNOWN ALREADY: The link between peritoneal endometriosis, inflammation and infertility is well established; however, the association between intraovarian inflammation and endometrioma is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 117 infertile women undergoing IVF in a tertiary infertility clinic at Oslo University Hospital Rikshospitalet, Norway, during the period May 2009 to September 2011. PARTICIPANTS, SETTING, METHODS: There were 47 patients with unilateral endometrioma and 17 patients with bilateral endometrioma, while the 53 control patients had unexplained or male factor infertility. Concentrations of IL-1ß, IL-6, IL-8, IL-10, IL-12 and TNF-α were measured in serum and in the fluid of the largest pre-ovulatory follicles from each ovary of each participant. MAIN RESULTS AND THE ROLE OF CHANCE: Cytokine levels in the follicular fluid from the two ovaries in women with unilateral endometriomas were comparable, and were not significantly altered compared with that of control groups with male factor infertility, unexplained infertility or bilateral endometriomas. Compared with serum levels, the follicular fluid levels of IL-8 and IL-6 were higher, suggesting a local production or recruitment. The follicular fluid IL-8 level varied considerably and showed an inverse relationship with IL-12, IL-10 and TNF-∝, suggesting a complex interaction between various immune cells. A small group of patients (n = 3) had increased levels of all follicular fluid cytokines combined with moderately to slightly elevated serum levels and these patients had a significantly lower ovarian response. LIMITATIONS, REASONS FOR CAUTION: For ethical reasons, the endometriomas were diagnosed indirectly by ultrasound rather than by histology. WIDER IMPLICATIONS OF THE FINDINGS: This paper reveals that endometriomas seldom induce inflammation in nearby follicles during IVF; therefore, routine cystectomy prior to IVF may not be necessary. Cytokine levels in the follicular fluid, nonetheless, show distinctive patterns and increased levels may be linked to reduced ovarian response independent of the cause of infertility.

Interaction between granulosa-lutein cells and monocytes regulates secretion of angiogenic factors in vitro.

BACKGROUND: Leukocyte infiltration and angiogenesis in the forming corpus luteum are prerequisites for normal ovarian function and may also underlie disorders like ovarian hyperstimulation syndrome. We examined whether ovarian angiogenesis could be affected by an interaction between granulosa-lutein (GL) cells and leukocytes. METHODS AND RESULTS: We found that GL cells isolated from the follicular fluid synthesize and secrete the chemokine interleukin-8 (IL-8), which activates IL-8-receptor-specific Ca(2+) and p38 mitogen-activated protein kinase signalling in monocytes and induces a directed migration of these cells towards the chemical gradient. Monocytes were found to further enhance IL-8 release, which suggests that these cells promote a massive leukocyte infiltration of the forming corpus luteum. A possible utility of leukocyte infiltration is the modulation of angiogenesis. We found that GL cells induce migration and capillary tube formation by endothelial cells in vitro. Furthermore, monocytes altered the profile of angiogenic factors released by GL cells, which supports the theory that an interaction between GL cells and leukocytes regulates ovarian angiogenesis. In addition, we found a correlation between increased secretion of pro-angiogenic cytokines and number of oocytes collected during IVF, which suggests that ovarian angiogenesis is related to the clinical response during ovarian stimulation. CONCLUSIONS: An intricate communication may exist between infiltrating leukocytes and ovarian GL cells during the formation of corpus luteum, affecting neo-vascularization of the luteal tissue.

Differential release of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human granulosa-lutein cells and ovarian leukocytes.

Tissue reorganization during ovulation and corpus luteum formation involves a coordinated action of matrix metalloproteinases (MMPs) and tissue MMP inhibitors (TIMPs). In this study we investigated the cellular source of ovarian MMPs and TIMPs. Cells isolated from the preovulatory human follicle were cultured after immunobead depletion of CD45-expressing cells, which allowed differential assessment of leukocyte and granulosa-lutein cell fractions. Secretion of MMP-9 by follicular fluid-derived cells was associated with the presence of leukocytes. Granulosa-lutein cells synthesized low levels of MMP-9 but failed to secrete this enzyme that presumably accumulated in the cytoplasm, indicated by an increased MMP-9 expression of luteinized cells in sectioned midluteal phase corpora lutea. Synthesis and secretion of TIMP by follicular fluid-derived cells was associated with granulosa-lutein cells. TIMPs derived by granulosa-lutein cells failed to inhibit MMP-related pericellular proteolysis. The findings support a two-cell model of periovulatory MMP/TIMP release, in which leukocytes secrete MMPs and granulosa-lutein cells release TIMP, suggesting that there exists an intriguing interaction among cells that intertwingle during ovulation and corpus luteum formation.

Cellular interaction regulates interleukin-8 secretion by granulosa-lutein cells and monocytes/macrophages.

PROBLEM: Peri-ovulatory migration of leukocytes towards the follicle plays an important role during corpus luteum formation. In this study, we examined the secretion of the neutrophil chemoattractant interleukin (IL)-8 by ovarian GL cells and the role of monocytes in IL-8 secretion. METHOD OF STUDY: Granulosa-lutein cells were isolated from the pre-ovulatory follicle. After depletion of contaminating leukocytes, GL cells were co-cultured with the myelo-monocytic cell line THP-1. Intracellular IL-8 accumulation, IL-8 secretion, and chemotactic activity of cell culture media were examined. RESULTS: Intracellular IL-8 was predominantly localized in the endoplasmatic reticulum-Golgi both in GL cells and in THP-1 cells. In co-cultured cells, intracellular IL-8-specific immunofluorescence and IL-8 secretion were increased compared with either GL cells or THP-1 cells that were cultured alone. Conditioned cell culture media from GL cells and THP-1 cells induced directed cell migration by neutrophils. CONCLUSION: Human GL cells constitutively synthesize IL-8. An increased IL-8 secretion by co-cultured GL cells and THP-1 cells suggest that GL cells and monocytes mutually induce chemokine secretion. An initial interaction between GL cells and ovarian leukocytes may therefore contribute to an increased chemokine release and leukocyte recruitment to the forming corpus luteum.