DNA repair endonuclease ERCC1�CXPF as a novel therapeutic target to overcome chemoresistance in cancer therapy

The ERCC1–XPF complex is a structure-specific endonuclease essential for the repair of DNA damage by the nucleotide excision repair pathway. It is also involved in other key cellular processes, including DNA interstrand crosslink (ICL) repair and DNA double-strand break (DSB) repair. New evidence has recently emerged, increasing our understanding of its requirement in these additional roles. In this review, we focus on the protein–protein and protein–DNA interactions made by the ERCC1 and XPF proteins and discuss how these coordinate ERCC1–XPF in its various roles. In a number of different cancers, high expression of ERCC1 has been linked to a poor response to platinum-based chemotherapy. We discuss prospects for the development of DNA repair inhibitors that target the activity, stability or protein interactions of the ERCC1–XPF complex as a novel therapeutic strategy to overcome chemoresistance.     

First case of highly pathogenic H5N1 avian influenza virus in Spain

Background  

In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8-seroprevalence in sheep and goats.     

Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors

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The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H⁺ with K⁺ and, to lesser extent, Na⁺. Here, we investigated the response to NaCl supply and K⁺ deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K⁺ the opposite was found. Analysis of mineral composition showed a higher K⁺ content in roots, shoots and xylem sap of transgenic plants and no differences in Na⁺ content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na⁺/H⁺ and, above all, K⁺/H⁺ transport activity in root intracellular membrane vesicles. Under K⁺ limiting conditions, transgenic plants enhanced root expression of the high‐affinity K⁺ uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K⁺ depletion rates and half cytosolic K⁺ activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K⁺ and lower cytosolic K⁺ activity than untransformed plants. These results indicate the fundamental role of K⁺ homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

Introduction

Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method

We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results

Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions

Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.     

Metabolic distribution of radioactivity in Sprague-Dawley rats and B6C3F1 mice exposed to 1,3-[2,3-14C]-butadiene by whole body exposure

The uptake of 1,3-[2,3-(14)C]-butadiene and its disposition, measured as radioactivity in urine, faeces, exhaled volatiles and CO(2) during and following 6 h whole body exposure to 20 ppm butadiene has been investigated in male Sprague-Dawley rats and B6C3F1 mice. Whilst there were similarities between the two species, the uptake and metabolic distribution of butadiene were somewhat different for rats and mice. The major differences observed were in the urinary excretion of radioactivity and in the exhalation of 14C-CO(2). After 42 h from the start of exposure, 51.1% of radioactivity was eliminated in rat urine compared with 39.5% for mouse urine. 34.9% of the recovered radioactivity was exhaled by rats as 14C-CO(2), compared with 48.7% by mice. Excretion of radioactivity in faeces was similar for both species (3.8% for rats and 3.4% for mice). The tissue concentrations of 14C-butadiene equivalents measured in liver, testes, lung and blood of exposed mice were 0.493, 0460, 0.457, and 1.626 nmol/g tissue, respectively. The values for the corresponding rat tissues were 0.869, 0.329, 0.457, and 1.626 nmol butadiene equivalents/g tissue, respectively. For rats, 6.2% of recovered radioactivity (0.288 nmol butadiene equivalents/g tissue) was retained in carcasses whereas for mice the amount was 3.6% (0.334 nmol butadiene equivalents/g tissue). There were also some significant differences between the metabolic conversion of 1,3-[2,3-(14)C]-butadiene and excretion by mice following the 20 ppm whole body exposure compared to previously reported data for nose-only exposure to 200 ppm butadiene [Richardson et al., Toxicol. Sci. 49 (1999) 186]. The main difference between the high- and low-exposure studies was in the exhalation of 14C-CO(2). At the 200 ppm exposure, 40% of the radioactivity was exhaled as 14C-CO(2) by rats whereas 6% was measured by this route for mice. The proportional conversion of butadiene to CO(2) by mice was significantly greater at the low exposure concentration compared with that reported for the higher concentration. This shift was not observed for rats. The difference between species could be caused by a saturation of metabolism in mice between 20 and 200 ppm for the pathways leading to CO(2). Restraint or error in collection of CO(2) in the 200 ppm study could also be factors.     

Determination of protein fold class from Raman or Raman optical activity spectra using random forests

Knowledge of the fold class of a protein is valuable because fold class gives an indication of protein function and evolution. Fold class can be accurately determined from a crystal structure or NMR structure, though these methods are expensive, time-consuming, and inapplicable to all proteins. In contrast, vibrational spectra [infra-red, Raman, or Raman optical activity (ROA)] are rapidly obtained for proteins under wide range of biological molecules under diverse experimental and physiological conditions. Here, we show that the fold class of a protein can be determined from Raman or ROA spectra by converting a spectrum into data of 10 cm⁻¹ bin widths and applying the random forest machine learning algorithm. Spectral data from 605 and 1785 cm⁻¹ were analyzed, as well as the amide I, II, and III regions in isolation and in combination. ROA amide II and III data gave the best performance, with 33 of 44 proteins assigned to one of the correct four top-level structural classification of proteins (SCOP) fold class (all α, all β, α and β, and disordered). The method also shows which spectral regions are most valuable in assigning fold class.     

Vibrational communication and mating behaviour of Dichelops melacanthus (Hemiptera: Pentatomidae) recorded from loudspeaker membranes and plants

Vibrational communication is important for successful mating in various stink bugs species. The vibrational signals from males and females of Dichelops melacanthus Dallas (Hemiptera: Pentatomidae) are recorded from a nonresonant substrate (i.e. a loudspeaker membrane) to characterize the temporal and spectral properties of these vibrational signals, as well as on a resonant substrate (i.e. bean plants) to obtain information about how these signals are altered when they are transmitted through the plants. On the loudspeaker membrane, D. melacanthus males and females emit only one male or one female song, respectively. However, when the insects are placed on bean leaves, a more complex repertoire is recorded, with three different songs for each sex. The first female and male songs appear to have calling functions and the third male and female songs are emitted during courtship. The second female and male songs are emitted after the first song, although their functions in mating behaviour are not clear. The identified repertoire is similar to those of other Neotropical stink bugs, starting with songs 1 and 2 and developing into song 3. Frequency modulation is observed in the female songs recorded from the loudspeaker membrane and the plants. The signals recorded from plants present higher harmonic peaks compared with the signals recorded from the loudspeaker membrane. The presence of species and sex‐specific songs during mating confirms the important role of vibrational communication in mate location and recognition. The temporal and spectral characteristic signals are influenced by the substrate used to record the songs emitted by D. melacanthus.