Larval behaviour, dispersal and population connectivity in the deep sea.

Ecosystem connectivity is an essential consideration for marine spatial planning of competing interests in the deep sea. Immobile, adult communities are connected through freely floating larvae, depending on new recruits for their health and to adapt to external pressures. We hypothesize that the vertical swimming ability of deep-sea larvae, before they permanently settle at the bottom, is one way larvae can control dispersal. We test this hypothesis with more than [Formula: see text] simulated particles with a range of active swimming behaviours embedded within the currents of a high-resolution ocean model. Despite much stronger horizontal ocean currents, vertical swimming of simulated larvae can have an order of magnitude impact on dispersal. These strong relationships between larval dispersal, pathways, and active swimming demonstrate that lack of data on larval behaviour traits is a serious impediment to modelling deep-sea ecosystem connectivity; this uncertainty greatly limits our ability to develop ecologically coherent marine protected area networks.

Measurements of Aerial Spore Load by qPCR Facilitates Lettuce Downy Mildew Risk Advisement.

The lettuce downy mildew pathogen, Bremia lactucae, is an obligate oomycete that causes extensive produce losses. Initial chlorotic symptoms that severely reduce the market value of the produce are followed by the appearance of white, downy sporulation on the abaxial side of the leaves. These spores become airborne and disseminate the pathogen. Controlling lettuce downy mildew has relied on repeated fungicide applications to prevent outbreaks. However, in addition to direct economic costs, heterogeneity and rapid adaptation of this pathogen to repeatedly applied fungicides has led to the development of fungicide-insensitivity in the pathogen. We deployed a quantitative PCR assay-based detection method using a species-specific DNA target for B. lactucae coupled with a spore trap system to measure airborne B. lactucae spore loads within three commercial fields that each contained experimental plots, designated EXP1 to EXP3. Based upon these measurements, when the spore load in the air reached a critical level (8.548 sporangia per m3 air), we advised whether or not to apply fungicides on a weekly basis within EXP1 to EXP3. This approach saved three sprays in EXP1, and one spray each in EXP2 and EXP3 without a significant increase in disease incidence. The reduction in fungicide applications to manage downy mildew can decrease lettuce production costs while slowing the development of fungicide resistance in B. lactucae by eliminating unnecessary fungicide applications.

Ocean sprawl facilitates dispersal and connectivity of protected species.

Highly connected networks generally improve resilience in complex systems. We present a novel application of this paradigm and investigated the potential for anthropogenic structures in the ocean to enhance connectivity of a protected species threatened by human pressures and climate change. Biophysical dispersal models of a protected coral species simulated potential connectivity between oil and gas installations across the North Sea but also metapopulation outcomes for naturally occurring corals downstream. Network analyses illustrated how just a single generation of virtual larvae released from these installations could create a highly connected anthropogenic system, with larvae becoming competent to settle over a range of natural deep-sea, shelf and fjord coral ecosystems including a marine protected area. These results provide the first study showing that a system of anthropogenic structures can have international conservation significance by creating ecologically connected networks and by acting as stepping stones for cross-border interconnection to natural populations.

Sensitivity of marine protected area network connectivity to atmospheric variability.

International efforts are underway to establish well-connected systems of marine protected areas (MPAs) covering at least 10% of the ocean by 2020. But the nature and dynamics of ocean ecosystem connectivity are poorly understood, with unresolved effects of climate variability. We used 40-year runs of a particle tracking model to examine the sensitivity of an MPA network for habitat-forming cold-water corals in the northeast Atlantic to changes in larval dispersal driven by atmospheric cycles and larval behaviour. Trajectories of Lophelia pertusa larvae were strongly correlated to the North Atlantic Oscillation (NAO), the dominant pattern of interannual atmospheric circulation variability over the northeast Atlantic. Variability in trajectories significantly altered network connectivity and source-sink dynamics, with positive phase NAO conditions producing a well-connected but asymmetrical network connected from west to east. Negative phase NAO produced reduced connectivity, but notably some larvae tracked westward-flowing currents towards coral populations on the mid-Atlantic ridge. Graph theoretical metrics demonstrate critical roles played by seamounts and offshore banks in larval supply and maintaining connectivity across the network. Larval longevity and behaviour mediated dispersal and connectivity, with shorter lived and passive larvae associated with reduced connectivity. We conclude that the existing MPA network is vulnerable to atmospheric-driven changes in ocean circulation.

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

A novel approach for next-generation sequencing of circulating tumor cells.

BACKGROUND: Next-generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low-input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching. METHODS: To address this, we have developed a novel approach combining tumor cell isolation from preserved blood with Repli-G WGA and Illumina TruSeq Amplicon Cancer Panel-based NGS. We purified cell pools ranging from 10 to 1000 cells from three different cell lines, and quantitatively demonstrate comparable quality of DNA extracted from preserved versus unpreserved samples. RESULTS: Preservation and WGA were compatible with the generation of high-quality libraries. Known point mutations and gene amplification were detected for libraries that had been prepared from amplified DNA from preserved blood. CONCLUSION: These spiking experiments provide proof of concept of a clinically applicable workflow for real-time monitoring of patient tumor using noninvasive liquid biopsies.

Accuracy of Rain Forecasts for Use in Scheduling Late Blight Management Tactics in the Columbia Basin of Washington and Oregon.

Accuracy of prediction was analyzed for 17- and 30-day rain forecasts at two locations in the Columbia Basin to determine whether forecasts were sufficiently accurate to be included as a model component to schedule fungicide applications for potato late blight. Accuracy was partitioned into specificity (percentage of forecasted nonrainfall events classified correctly) and sensitivity (percentage of forecasted daily rainfall events classified correctly). An adjusted sensitivity, which included the forecasted rain day plus the next 2 days, was also used to give a wider target than only 1 day for evaluating accuracy of forecasted rain events. For 17-day forecasts, specificity during the seasonal test period was ≥70% from mid-June through September and specificity over the days of the forecast was >70% for the first 8 days at both locations both years. Adjusted sensitivity over days of the forecast was initially >80% and then decreased as forecasts increased from 7 to 17 days for 17-day forecasts at both locations and years. Sensitivity and adjusted sensitivity during the seasonal test period were both positively correlated with the number of rainy days while specificity was negatively correlated. Adjusted sensitivity was considerably higher for May (month with highest incidence of rain) than July (month with lowest incidence of rain) at both locations. For 30-day forecasts, specificity during the test period was >75% in July and August and adjusted sensitivity ranged from 60 to 100% for time periods occurring in May and June during both sample seasons. Specificity was generally above 80% as days of the forecast increased and adjusted sensitivity varied greatly over days of the forecasts, with extremes between 0 and 100% at both locations and years for the 30-day forecasts. Specificity of 17- and 30-day rain forecasts and adjusted sensitivity of 17-day rain forecasts have utility in scheduling late blight fungicides in the Columbia Basin.

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential.

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice.

The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45(-)/CD11b(-)/CD31(-)/MIC1-1C3(+)/CD133(+)/CD26(-), at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creER(T2)-R26R(YFP) mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.

Foxa1 and Foxa2 maintain the metabolic and secretory features of the mature beta-cell.

Foxa1 and Foxa2 play both redundant and distinct roles in early pancreas development. We demonstrate here that inducible ablation of both transcription factors in mature mouse beta-cells leads to impaired glucose homeostasis and insulin secretion. The defects in both glucose-stimulated insulin secretion and intracellular calcium oscillation are more pronounced than those in beta-cells lacking only Foxa2. Unexpectedly, in contrast to the severe reduction of beta-cell-enriched factors contributing to metabolic and secretory pathways, expression of a large number of genes that are involved in neural differentiation and function is significantly elevated. We further demonstrate that expression of carbohydrate response element-binding protein (ChREBP or Mlxipl), an important transcriptional regulator of carbohydrate metabolism, is significantly affected in compound Foxa1/a2 mutant beta-cells. ChREBP expression is directly controlled by Foxa1 and Foxa2 in both the fetal endocrine pancreas as well as mature islets. These data demonstrate that Foxa1 and Foxa2 play crucial roles in the development and maintenance of beta-cell-specific secretory and metabolic pathways.

Genome-wide analysis of histone modifications in human pancreatic islets.

The global diabetes epidemic poses a major challenge. Epigenetic events contribute to the etiology of diabetes; however, the lack of epigenomic analysis has limited the elucidation of the mechanistic basis for this link. To determine the epigenetic architecture of human pancreatic islets we mapped the genome-wide locations of four histone marks: three associated with gene activation-H3K4me1, H3K4me2, and H3K4me3-and one associated with gene repression, H3K27me3. Interestingly, the promoters of the highly transcribed insulin and glucagon genes are occupied only sparsely by H3K4me2 and H3K4me3. Globally, we identified important relationships between promoter structure, histone modification, and gene expression. We demonstrated co-occurrences of histone modifications including bivalent marks in mature islets. Furthermore, we found a set of promoters that is differentially modified between islets and other cell types. We also use our histone marks to determine which of the known diabetes-associated single-nucleotide polymorphisms are likely to be part of regulatory elements. Our global map of histone marks will serve as an important resource for understanding the epigenetic basis of type 2 diabetes.

Evidence for limited genetic compartmentalization of HIV-1 between lung and blood.

BACKGROUND: HIV-1 is frequently detected in the lungs of infected individuals and is likely important in the development of pulmonary opportunistic infections. The unique environment of the lung, rich in alveolar macrophages and with specialized local immune responses, may contribute to differential evolution or selection of HIV-1. METHODOLOGY AND FINDINGS: We characterized HIV-1 in the lung in relation to contemporaneous viral populations in the blood. The C2-V5 region of HIV-1 env was sequenced from paired lung (induced sputum or bronchoalveolar lavage) and blood (plasma RNA and proviral DNA from sorted or unsorted PBMC) from 18 subjects. Compartmentalization between tissue pairs was assessed using 5 established tree or distance-based methods, including permutation tests to determine statistical significance. We found statistical evidence of compartmentalization between lung and blood in 10/18 subjects, although lung and blood sequences were intermingled on phylogenetic trees in all subjects. The subject showing the greatest compartmentalization contained many nearly identical sequences in BAL sample, suggesting clonal expansion may contribute to reduced viral diversity in the lung in some cases. However, HIV-1 sequences in lung were not more homogeneous overall, nor were we able to find a lung-specific genotype associated with macrophage tropism in V3. In all four subjects in whom predicted X4 genotypes were found in blood, predicted X4 genotypes were also found in lung. CONCLUSIONS: Our results support a picture of continuous migration of HIV-1 between circulating blood and lung tissue, with perhaps a very limited degree of localized evolution or clonal replication.

The transcriptional response of the islet to pregnancy in mice.

The inability of the ss-cell to meet the demand for insulin brought about by insulin resistance leads to type 2 diabetes. In adults, ss-cell replication is one of the mechanisms thought to cause the expansion of ss-cell mass. Efforts to treat diabetes require knowledge of the pathways that drive facultative ss-cell proliferation in vivo. A robust physiological stimulus of ss-cell expansion is pregnancy and identifying the mechanisms underlying this stimulus may provide therapeutic leads for the treatment of type 2 diabetes. The peak in ss-cell proliferation during pregnancy occurs on d 14.5 of gestation in mice. Using advanced genomic approaches, we globally characterize the gene expression signature of pancreatic islets on d 14.5 of gestation during pregnancy. We identify a total of 1907 genes as differentially expressed in the islet during pregnancy. The islet's ability to compensate for relative insulin deficiency during metabolic stress is associated with the induction of both proliferative and survival pathways. A comparison of the genes induced in three different models of islet expansion suggests that diverse mechanisms can be recruited to expand islet mass. The identification of many novel genes involved in islet expansion during pregnancy provides an important resource for diabetes researchers to further investigate how these factors contribute to the maintenance of not only islet mass, but ultimately ss-cell mass.

Male genital tract compartmentalization of human immunodeficiency virus type 1 (HIV).

We present phylogenetic evidence supporting viral compartmentalization between the blood (peripheral blood mononuclear cells or plasma) and multiple genitourinary sites in HIV-infected men. Four of the five subjects evaluated demonstrated compartmentalization of viral sequences between urogenital tract specimens (tissue or fluid) and at least one blood category. HIV sequence migration from blood to urogenital tract was detected in four of five men, with migration from urogenital tract to blood in the fifth, and cross migration between both compartments noted in one man. These observations add 5 additional cases to the 27 total reported cases in which male urogenital tract compartmentalization has been studied, investigate surgical samples/specimens that have not been evaluated previously, and provide further evidence for restricted flow of HIV between the blood and the genital tract. As such, our study findings are important for understanding the long-term response to antiretroviral therapy, the design of vaccines, and the sexual transmission of HIV.