A rapid bioluminescence assay for measuring ​myeloperoxidase activity in human plasma

Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l(−1) MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders

Short-term studies underestimate 30-generation changes in a butterfly metapopulation

Most studies of rare and endangered species are based on work carried out within one generation, or over one to a few generations of the study organism. We report the results of a study that spans 30 generations (years) of the entire natural range of a butterfly race that is endemic to 35 km2 of north Wales, UK. Short-term studies (surveys in single years and dynamics over 4 years) of this system led to the prediction that the regional distribution would be quite stable, and that colonization and extinction dynamics would be relatively unimportant. However, a longer-term study revealed unexpectedly high levels of population turnover (local extinction and colonization), affecting 18 out of the 20 patches that were occupied at any time during the period. Modelling the system (using the 'incidence function model' (IFM) for metapopulations) also showed higher levels of colonization and extinction with increasing duration of the study. The longer-term dynamics observed in this system can be compared, at a metapopulation level, with the increased levels of variation observed with increasing time that have been observed in single populations. Long-term changes may arise from local changes in the environment that make individual patches more or less suitable for the butterfly, or from unusual colonization or extinction events that take metapopulations into alternative states. One implication is that metapopulation and population viability analyses based on studies that cover only a few animal or plant generations may underestimate extinction threats

The ARF Tumor Suppressor Regulates Bone Remodeling and Osteosarcoma Development in Mice

The ARF tumor suppressor regulates p53 as well as basic developmental processes independent of p53, including osteoclast activation, by controlling ribosomal biogenesis. Here we provide evidence that ARF is a master regulator of bone remodeling and osteosarcoma (OS) development in mice. Arf-/- mice displayed increased osteoblast (OB) and osteoclast (OC) activity with a significant net increase in trabecular bone volume. The long bones of Arf-/- mice had increased expression of OB genes while Arf-/- OB showed enhanced differentiation in vitro. Mice transgenic for the Tax oncogene develop lymphocytic tumors with associated osteolytic lesions, while Tax+Arf-/- mice uniformly developed spontaneous OS by 7 months of age. Tax+Arf-/- tumors were well differentiated OS characterized by an abundance of new bone with OC recruitment, expressed OB markers and displayed intact levels of p53 mRNA and reduced Rb transcript levels. Cell lines established from OS recapitulated characteristics of the primary tumor, including the expression of mature OB markers and ability to form mineralized tumors when transplanted. Loss of heterozygosity in OS tumors arising in Tax+Arf+/- mice emphasized the necessity of ARF-loss in OS development. Hypothesizing that inhibition of ARF-regulated bone remodeling would repress development of OS, we demonstrated that treatment of Tax+Arf-/- mice with zoledronic acid, a bisphosphonate inhibitor of OC activity and repressor of bone turnover, prevented or delayed the onset of OS. These data describe a novel role for ARF as a regulator of bone remodeling through effects on both OB and OC. Finally, these data underscore the potential of targeting bone remodeling as adjuvant therapy or in patients with genetic predispositions to prevent the development of OS

Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer

INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is diagnosed in approximately 15% of all human breast cancer (BrCa) patients. Currently, no targeted therapies exist for this subtype of BrCa and prognosis remains poor. Our laboratory has previously identified a proliferation/DNA repair/cell cycle gene signature (Tag signature) that is characteristic of human TNBC. We hypothesize that targeting the dysregulated biological networks in the Tag gene signature will lead to the identification of improved combination therapies for TNBC. METHODS: Cross-species genomic analysis was used to identify human breast cancer cell lines that express the Tag signature. Knock-down of the up-regulated genes in the Tag signature by siRNA identified several genes that are critical for TNBC cell growth. Small molecule inhibitors to two of these genes were analyzed, alone and in combination, for their effects on cell proliferation, cell cycle, and apoptosis in vitro and tumor growth in vivo. Synergy between the two drugs was analyzed by the Chou-Talalay method. RESULTS: A custom siRNA screen was used to identify targets within the Tag signature that are critical for growth of TNBC cells. Ribonucleotide reductase 1 and 2 (RRM1 and 2) and checkpoint kinase 1 (CHK1) were found to be critical targets for TNBC cell survival. Combination therapy, to simultaneously attenuate cell cycle checkpoint control through inhibition of CHK1 while inducing DNA damage with gemcitabine, improved therapeutic efficacy in vitro and in xenograft models of TNBC. CONCLUSIONS: This combination therapy may have translational value for patients with TNBC and improve therapeutic response for this aggressive form of breast cancer

Synthetic Lethality of Chk1 Inhibition Combined with p53 and/or p21 Loss During a DNA Damage Response in Normal and Tumor Cells

Cell cycle checkpoints ensure genome integrity and are frequently compromised in human cancers. A therapeutic strategy being explored takes advantage of checkpoint defects in p53-deficient tumors in order to sensitize them to DNA-damaging agents by eliminating Chk1-mediated checkpoint responses. Using mouse models, we demonstrated that p21 is a key determinant of how cells respond to the combination of DNA damage and Chk1 inhibition (combination therapy) in normal cells as well as in tumors. Loss of p21 sensitized normal cells to the combination therapy much more than did p53 loss and the enhanced lethality was partially blocked by CDK inhibition. In addition, basal pools of p21 (p53 independent) provided p53 null cells with protection from the combination therapy. Our results uncover a novel p53-independent function for p21 in protecting cells from the lethal effects of DNA damage followed by Chk1 inhibition. As p21 levels are low in a significant fraction of colorectal tumors, they are predicted to be particularly sensitive to the combination therapy. Results reported in this study support this prediction

Anticancer Gene Transfer for Cancer Gene Therapy

Gene therapy vectors are among the treatments currently used to treat malignant tumors. Gene therapy vectors use a specific therapeutic transgene that causes death in cancer cells. In early attempts at gene therapy, therapeutic transgenes were driven by non-specific vectors which induced toxicity to normal cells in addition to the cancer cells. Recently, novel cancer specific viral vectors have been developed that target cancer cells leaving normal cells unharmed. Here we review such cancer specific gene therapy systems currently used in the treatment of cancer and discuss the major challenges and future directions in this field

Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems

Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β(+))-emitting radionuclides in small animals and humans. Upon β(+) decay, the initial velocity of high-energy β(+) particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms.To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes (64)Cu and (18)F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705) to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with (99m)Tc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), herein we define the Cerenkov radiation energy transfer (CRET) ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [(18)F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [(18)F]FDG showed CRET ratios in vivo as high as 3.5±0.3.Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals

Type I Interferons Link Viral Infection to Enhanced Epithelial Turnover and Repair

The host immune system functions constantly to maintain chronic commensal and pathogenic organisms in check. The consequences of these immune responses on host physiology are as yet unexplored, and may have long-term implications in health and disease. We show that chronic viral infection increases epithelial turnover in multiple tissues, and the antiviral cytokines type I interferons (IFNs) mediate this response. Using a murine model with persistently elevated type I IFNs in the absence of exogenous viral infection, the Irgm1−/− mouse, we demonstrate that type I IFNs act through nonepithelial cells, including macrophages, to promote increased epithelial turnover and wound repair. Downstream of type I IFN signaling, the highly related IFN-stimulated genes Apolipoprotein L9a and b activate epithelial proliferation through ERK activation. Our findings demonstrate that the host immune response to chronic viral infection has systemic effects on epithelial turnover through a myeloid-epithelial circuit

Capturing threshold responses of marine benthos along gradients of natural and anthropogenic change

1. Ecologists and managers need to understand what types of communities emerge with continued human alterations to ecosystems against a background of natural change. Both natural and anthropogenic drivers are well known to affect organisms’ distributions; however, it often remains unclear where along a range of environmental and anthropogenic gradients important compositional community changes occur. 2. We used a big-data approach, including over 175,000 presence records of benthic genera for the North Sea, to identify environmental (bed shear stress, sediment grain size, temperature) and anthropogenic parameters (trawling effort) driving benthic community composition over a 21-year period. We applied a Gradient Forest analysis, based on Random Forests, to estimate the locations and importance of thresholds where small cumulative increases in the predictors drive a much greater change in genus composition than would be expected from linear effects. 3. Shear stress was the most important predictor of benthic community composition. Trawling effort, temperature gradients and sediment grain size were of intermediate importance. This corroborates that current and wave effects (typically associated with seabed substrate types) are primary determinants of benthic communities. 4. Our results suggest that a genus composition threshold for both infauna and epifaunal benthic communities is crossed when the seafloor is trawled as little as once every 4 years. Higher trawling levels corresponded with gradual compositional change without obvious thresholds, which would be consistent with chronic fishing in the North Sea over the last two centuries having caused persistent, long-term changes in ecosystem structure and functioning. This was corroborated by the large-scale spatial patterns of benthic community composition undergoing limited temporal changes during the 21-year study period. 5. Synthesis and applications. Although well established in theory, threshold effects are poorly validated in the field. We generated new information on multi-organism responses to environmental change at the scale of a continental shelf ecosystem and over a multi-decadal time period. This will help pure and applied scientists better understand the conditions under which community thresholds are crossed and provide environmental managers with empirical evidence that is expected to reduce uncertainty regarding decisions on the protection and sustainable use of the marine environment