The virtual ecologist approach: simulating data and observers

Ecologists carry a well‐stocked toolbox with a great variety of sampling methods, statistical analyses and modelling tools, and new methods are constantly appearing. Evaluation and optimisation of these methods is crucial to guide methodological choices. Simulating error‐free data or taking high‐quality data to qualify methods is common practice. Here, we emphasise the methodology of the ‘virtual ecologist’ (VE) approach where simulated data and observer models are used to mimic real species and how they are ‘virtually’ observed. This virtual data is then subjected to statistical analyses and modelling, and the results are evaluated against the ‘true’ simulated data. The VE approach is an intuitive and powerful evaluation framework that allows a quality assessment of sampling protocols, analyses and modelling tools. It works under controlled conditions as well as under consideration of confounding factors such as animal movement and biased observer behaviour. In this review, we promote the approach as a rigorous research tool, and demonstrate its capabilities and practical relevance. We explore past uses of VE in different ecological research fields, where it mainly has been used to test and improve sampling regimes as well as for testing and comparing models, for example species distribution models. We discuss its benefits as well as potential limitations, and provide some practical considerations for designing VE studies. Finally, research fields are identified for which the approach could be useful in the future. We conclude that VE could foster the integration of theoretical and empirical work and stimulate work that goes far beyond sampling methods, leading to new questions, theories, and better mechanistic understanding of ecological systems.     

Modeling the Expansion of an Introduced Tree Disease

Pine wilt disease is caused by the introduced pinewood nematode, Bursaphelenchus xylophilus, for which the vector is the pine sawyer beetle, Monochamus alternatus. Native Japanese pines, black pine (Pinus thunbergii) and red pine (P. densiflora), are extremely sensitive to the nematode's infection, and the parasite has been expanding nationwide in the last few decades, despite intensive control efforts. To understand the parasite's range expansion in Japan, we modeled the dynamics of the pines and the beetle that disperses the nematode, using an integro-difference equation in a one-dimensional space. Based on field data collected in Japan, we investigated the dependence of the parasite's rate of range expansion on the eradication rate of the beetle, the initial pine density, and the beetle dispersal ability. Our model predicts several results. (1) The Allee Effect operates on beetle reproduction, and consequently the parasite cannot invade a pine stand, once the beetle density decreases below a threshold. (2) The distribution of the dispersal distance of the beetles critically affects the expansion rate of the disease. As the fraction of the beetles that travel over long distance increases from zero, the range expansion accelerates sharply. (3) However, too frequent long-range dispersal results in a failure of the parasite invasion due to the Allee Effect, suggesting the importance of correctly assessing the beetle's mobility to predict the speed of range expansion of the parasite. (4) As the eradication rate is increased, the range expansion speed decreases gradually at first and suddenly drops to zero at a specific value of the eradication rate. This revised version was published online in July 2006 with corrections to the Cover Date.     

STAT3 Regulates MMP3 in Heme-Induced Endothelial Cell Apoptosis

Background

We have previously reported that free Heme generated during experimental cerebral malaria (ECM) in mice, is central to the pathogenesis of fatal ECM. Heme-induced up-regulation of STAT3 and CXCL10 promotes whereas up-regulation of HO-1 prevents brain tissue damage in ECM. We have previously demonstrated that Heme is involved in the induction of apoptosis in vascular endothelial cells. In the present study, we further tested the hypothesis that Heme reduces blood-brain barrier integrity during ECM by induction of apoptosis of brain vascular endothelial cells through STAT3 and its target gene matrix metalloproteinase three (MMP3) signaling.

Methods

Genes associated with the JAK/STAT3 signaling pathway induced upon stimulation by Heme treatment, were assessed using real time RT² Profile PCR arrays. A human MMP3 promoter was cloned into a luciferase reporter plasmid, pMMP3, and its activity was examined following exposure to Heme treatment by a luciferase reporter gene assay. In order to determine whether activated nuclear protein STAT3 binds to the MMP3 promoter and regulates MMP3 gene, we conducted a ChIP analysis using Heme-treated and untreated human brain microvascular endothelial cells (HBVEC), and determined mRNA and protein expression levels of MMP3 using qRT-PCR and Western blot. Apoptosis in HBVEC treated with Heme was evaluated by MTT and TUNEL assay.

Results

The results show that (1) Heme activates a variety of JAK/STAT3 downstream pathways in HBVEC. STAT3 targeted genes such as MMP3 and C/EBPb (Apoptosis-related genes), are up regulated in HBVEC treated with Heme. (2) Heme-induced HBVEC apoptosis via activation of STAT3 as well as its downstream signaling molecule MMP3 and upregulation of CXCL10 and HO-1 expressions. (3) Phosphorylated STAT3 binds to the MMP3 promoter in HBVEC cells, STAT3 transcribed MMP3 and induced MMP3 protein expression in HBVEC cells.

Conclusions

Activated STAT3 binds to the MMP3 promoter region and regulates MMP3 in Heme-induced endothelial cell apoptosis.     

Genetic Mechanisms in Apc-Mediated Mammary Tumorigenesis

Many components of Wnt/β-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre–mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of β-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; Apc^CKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre–induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre–mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC.     

Context-dependent activation of p53 target genes and induction of apoptosis by actinomycin D in aerodigestive tract cancers

Actinomycin D (ActD) was the first anticancer antibiotic approved for the management of human cancers. However, the notorious toxicity profile limits its widespread application in cancers, including cancers of the aerodigestive tract. Recent studies show that combining low-dose ActD with existing chemotherapies could potentially protect normal cells from the toxicity of chemotherapy drugs through p53 activation (cyclotherapy). An understanding of ActD’s effect on p53 signaling is critical for the meaningful application of ActD in cyclotherapy-based combinations. This study evaluated the anti-tumor efficacy and mechanism of action of ActD in aerodigestive tract cancers. We found that ActD strongly inhibited the growth of a panel of aerodigestive tract cancer cell lines and induced efficient apoptosis, although the sensitivity varies among cell lines. The IC₅₀ values of ActD spanned between 0.021 and 2.96?nM. Mechanistic studies revealed that ActD increased the expression of total and phosphorylated p53 (ser15) in a time- and dose-dependent manner. Moreover, ActD-induced apoptosis is dependent on p53 in cells expressing wild-type p53 and that ActD induced context-dependent differential expression of downstream targets p21 and PUMA without significant effects on p27. In the final analysis, this study revealed that p53-p21 is the predominant pathway activated by low-dose ActD, supporting further development of ActD in cyclotherapy.

     

Calcium carbonate-methylene blue nanohybrids for photodynamic therapy and ultrasound imaging

Photodynamic therapy plays an important role in cancer treatment. In this work, methylene blue (MB)-embedded calcium carbonate nanorods (CaCO₃-MB NRs) have been synthesized for pH-responsive photodynamic therapy and ultrasound imaging. The morphology of CaCO₃-MB NRs can be controlled by modulating the concentration of Na₂CO₃ aqueous solution. The generation of effective reactive oxygen species (ROS) were confirmed by 1,3-diphenylisobenzofuran (DPBF) probe. Both photodynamic therapy performance and echogenic performance of CaCO₃-MB NRs were investigated to confirm the feasibility of CaCO₃-MB nanohybrids for ultrasound image-guided photodynamic therapy.