Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01, via the CD44 pathway

PURPOSE: FKBPL and its peptide derivative, AD-01, have already demonstrated tumour growth inhibition and CD44 dependent anti-angiogenic activity. Here we explore the ability of AD-01 to target CD44 positive breast cancer stem cells (BCSCs). EXPERIMENTAL DESIGN: Mammosphere assays and flow cytometry were utilized to analyse the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anti-cancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples and xenografts. Delays in tumour initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays, qPCR and immunofluorescence. RESULTS: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere forming efficiency (MFE) and ESA+/CD44+/CD24- or ALDH+ cell subpopulations in vitro and tumour initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism appears to be due to AD-01-mediated BCSC differentiation demonstrated by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4 and Sox2, were also significantly reduced. Furthermore, we demonstrated additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signalling. CONCLUSIONS: AD-01 has dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies , somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.

Interaction of pollen-specific actin-depolymerizing factor with actin

We have examined the interaction of recombinant lily pollen ADF, LIADF1, with actin and found that whilst it bound both G- and F-actin, it had a much smaller effect on the polymerization and depolymerization rate constants than the maize vegetative ADF, ZmADF3. An antiserum specific to pollen ADF, antipADF, was raised and used to localize pollen ADF in daffodil - a plant in which massive reorganizations of the actin cytoskeleton have been seen to occur as pollen enters and exits dormancy. We show, for the first time, an ADF decorating F-actin in cells that did not result from artificial increase in ADF concentration. In dehydrated pollen this ADF:actin array is replaced by actin:ADF rodlets and aggregates of actin, which presumably act as a storage form of actin during dormancy. In germinated pollen ADF has no specific localization, except when an adhesion is made at the tip where actin and ADF now co-localize. These activities of pollen ADF are discussed with reference to the activities of ZmADF3 and other members of the ADF/cofilin group of proteins.

A Trip from a Tube to a Chip Applied Micro and Nanotechnology in Biotechnology, Veterinary and Life Sciences

More than 200 known diseases are transmitted via foods or food products. In the United States, food-borne diseases are responsible for 76 million cases of illness, 32,500 cases of hospitalisation and 5000 cases of death yearly. The ongoing increase in worldwide trade in livestock, food, and food products in combination with increase in human mobility (business- and leisure travel, emigration etc.) will increase the risk of emergence and spreading of such pathogens. There is therefore an urgent need for development of rapid, efficient and reliable methods for detection and identification of such pathogens.

Microchipfabrication has had a major impact on electronics and is expected to have an equally pronounced effect on life sciences. By combining micro-fluidics with micromechanics, micro-optics, and microelectronics, systems can be realized to perform complete chemical or biochemical analyses. These socalled ’Lab-on-a-Chip’ will completely change the face of laboratories in the future where smaller, fully automated devices will be able to perform assays faster, more accurately, and at a lower cost than equipment of today. A general introduction of food safety and applied micro-nanotechnology in life sciences will be given. In addition, examples of DNA micro arrays, micro fabricated integrated PCR chips and total integrated lab-on-achip systems from different National and EU research projects being carried out at the Laboratory of Applied Micro- Nanotechnology (LAMINATE) group at the National Veterinary Institute (DTU-Vet) Technical University of Denmark and the BioLabchip group at the Department of Micro and Nanotechnology (DTU-Nanotech), Technical University of Denmark (DTU), Ikerlan-IK4 (Spain) and other 16 partners from different European countries will be presented.

The anatomy of Last Glacial Maximum climate variations in south Westland, New Zealand, derived from pollen records

We present pollen records from three sites in south Westland, New Zealand, that document past vegetation and inferred climate change between approximately 30,000 and 15,000 cal. yr BP. Detailed radiocarbon dating of the enclosing sediments at one of those sites, Galway tarn, provides a more robust chronology for the structure and timing of climate-induced vegetation change than has previously been possible in this region. The Kawakawa/Oruanui tephra, a key isochronous marker, affords a precise stratigraphic link across all three pollen records, while other tie points are provided by key pollen-stratigraphic changes which appear to be synchronous across all three sites. Collectively, the records show three episodes in which grassland, interpreted as indicating mostly cold subalpine to alpine conditions, was prevalent in lowland south Westland, separated by phases dominated by subalpine shrubs and montane-lowland trees, indicating milder interstadial conditions. Dating, expressed as a Bayesian-estimated single 'best' age followed in parentheses by younger/older bounds of the 95% confidence modelled age range, indicates that a cold stadial episode, whose onset was marked by replacement of woodland by grassland, occurred between 28,730 (29,390-28,500) and 25,470 (26,090-25,270) cal. yr BP (years before AD, 1950), prior to the deposition of the Kawakawa/Oruanui tephra. Milder interstadial conditions prevailed between 25,470 (26,090-25,270) and 24,400 (24,840-24,120) cal. yr BP and between 22,630 (22,930-22,340) and 21,980 (22,210-21,580) cal. yr BP, separated by a return to cold stadial conditions between 24,400 and 22,630 cal. yr BP. A final episode of grass-dominated vegetation, indicating cold stadial conditions, occurred from 21,980 (22,210-21,580) to 18,490 (18,670-17,950) cal. yr BP. The decline in grass pollen, indicating progressive climate amelioration, was well advanced by 17,370 (17,730-17,110) cal. yr BP, indicating that the onset of the termination in south Westland occurred sometime between ca 18,490 and ca 17,370 cal. yr BP. A similar general pattern of stadials and interstadials is seen, to varying degrees of resolution but generally with lesser chronological control, in many other paleoclimate proxy records from the New Zealand region. This highly resolved chronology of vegetation changes from southwestern New Zealand contributes to the examination of past climate variations in the southwest Pacific region. The stadial and interstadial episodes defined by south Westland pollen records represent notable climate variability during the latter part of the Last Glaciation. Similar climatic patterns recorded farther afield, for example from Antarctica and the Southern Ocean, imply that climate variations during the latter part of the Last Glaciation and the transition to the Holocene interglacial were inter-regionally extensive in the Southern Hemisphere and thus important to understand in detail and to place into a global context. © 2013 Elsevier Ltd. All rights reserved.

The role of lipid peroxidation products and oxidative stress in activation of the canonical wingless-type MMTV integration site (WNT) pathway in a rat model of diabetic retinopathy

Our recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.

Activation of the Wnt pathway plays a pathogenic role in diabetic retinopathy in humans and animal models

Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of beta-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of low-density lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts, were also elevated in the DR models. The high glucose-induced activation of beta-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target.

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal-Regulated Kinase/β-Catenin Signaling Pathway

Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown.

Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia

AtsR is a membrane-bound hybrid sensor kinase of Burkholderia cenocepacia that negatively regulates quorum sensing and virulence factors such as biofilm production, type 6-secretion and protease secretion. Here, we elucidate the mechanism of AtsR phosphorelay by site-directed mutagenesis of predicted histidine and aspartic acid phosphoacceptor residues. We demonstrate by in vitro phosphorylation that histidine-245 and aspartic acid-536 are conserved sites of phosphorylation in AtsR, and we also identify the cytosolic response regulator AtsT (BCAM0381) as a key component of the AtsR phosphorelay pathway. Monitoring the function of AtsR and its derivatives in vivo by measuring extracellular protease activity and swarming motility confirmed the in vitro phosphorylation results. Together, we find that the AtsR receiver domain plays a fine-tuning role in determining the levels of phosphotransfer from its sensor kinase domain to the AtsT response regulator.

Harnessing the complexity of gene expression data from cancer: from single gene to structural pathway methods

High-dimensional gene expression data provide a rich source of information because they capture the expression level of genes in dynamic states that reflect the biological functioning of a cell. For this reason, such data are suitable to reveal systems related properties inside a cell, e.g., in order to elucidate molecular mechanisms of complex diseases like breast or prostate cancer. However, this is not only strongly dependent on the sample size and the correlation structure of a data set, but also on the statistical hypotheses tested. Many different approaches have been developed over the years to analyze gene expression data to (I) identify changes in single genes, (II) identify changes in gene sets or pathways, and (III) identify changes in the correlation structure in pathways. In this paper, we review statistical methods for all three types of approaches, including subtypes, in the context of cancer data and provide links to software implementations and tools and address also the general problem of multiple hypotheses testing. Further, we provide recommendations for the selection of such analysis methods.

A composite pollen-based stratotype for inter-regional evaluation of climatic events in New Zealand over the past 30,000 years (NZ-INTIMATE project)

Our review of paleoclimate information for New Zealand pertaining to the past 30,000 years has identified a general sequence of climatic events, spanning the onset of cold conditions marking the final phase of the Last Glaciation, through to the emergence to full interglacial conditions in the early Holocene. In order to facilitate more detailed assessments of climate variability and any leads or lags in the timing of climate changes across the region, a composite stratotype is proposed for New Zealand. The stratotype is based on terrestrial stratigraphic records and is intended to provide a standard reference for the intercomparison and evaluation of climate proxy records. We nominate a specific stratigraphic type record for each climatic event, using either natural exposure or drill core stratigraphic sections. Type records were selected on thebasis of having very good numerical age control and a clear proxy record. In all cases the main proxy of the type record is subfossil pollen. The type record for the period from ca 30 to ca 18 calendar kiloyears BP (cal. ka BP) is designated in lake-bed sediments from a small morainic kettle lake (Galway tarn) in western South Island. The Galway tarn type record spans a period of full glacial conditions (Last Glacial Coldest Period, LGCP) within the Otira Glaciation, and includes three cold stadials separated by two cool interstadials. The type record for the emergence from glacial conditions following the termination of the Last Glaciation (post-Termination amelioration) is in a core of lake sediments from a maar (Pukaki volcanic crater) in Auckland, northern North Island, and spans from ca 18 to 15.64±0.41 cal. ka BP. The type record for the Lateglacial period is an exposure of interbedded peat and mud at montane Kaipo bog, eastern North Island. In this high-resolution type record, an initial mild period was succeeded at 13.74±0.13 cal. ka BP by a cooler period, which after 12.55±0.14 cal. ka BP gave way to a progressive ascent to full interglacial conditions that were achieved by 11.88±0.18 cal. ka BP. Although a type section is not formally designated for the Holocene Interglacial (11.88±0.18 cal. ka BP to the present day), the sedimentary record of Lake Maratoto on the Waikato lowlands, northwestern North Island, is identified as a prospective type section pending the integration and updating of existing stratigraphic and proxy datasets, and age models. The type records are interconnected by one or more dated tephra layers, the ages of which are derived from Bayesian depositional modelling and OxCal-based calibrations using the IntCal09 dataset. Along with the type sections and the Lake Maratoto record, important, well-dated terrestrial reference records are provided for each climate event. Climate proxies from these reference records include pollen flora, stable isotopes from speleothems, beetle and chironomid fauna, and glacier moraines. The regional composite stratotype provides a benchmark against which to compare other records and proxies. Based on the composite stratotype, we provide an updated climate event stratigraphic classification for the New Zealand region. © 2013 Elsevier Ltd.

Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3-and viral-induced expression of type i interferons

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression. © 2012 Nature America, Inc. All rights reserved.

A high resolution 15,600-year pollen and microcharcoal record from the Cederberg Mountains, South Africa

The Cederberg Mountains (Western Cape Province, South Africa) are located within the Fynbos Biome, which exhibits some of the highest levels of species richness and endemism in the world. The region's post-glacial vegetation history, however, remains largely unknown. Presented here are high resolution pollen and microcharcoal records spanning the last 15,600 years obtained from the De Rif rock hyrax midden from the Driehoek Valley of the central Cederberg. In this region, previous pollen studies have shown muted variability in vegetation community composition during periods of globally marked climatic variability (e.g. the last glacial-interglacial transition). In our record, however, significant changes in vegetation composition are apparent. Most notably, they indicate a shift from ericaceous/restioid fynbos (present from 15,600 to 13,300 cal yr BP) to a brief, but prominent, development of proteoid fynbos at the beginning of the Holocene around 11,200 cal yr BP. This vegetation shift is associated with increased moisture at the site, and coincides with reduced fire frequency as indicated by the microcharcoal record. At 10,400 cal yr BP, there is a marked reduction in Protea-type pollen, which is replaced by thicket, characterised by Dodonaea, which became the dominant arboreal pollen type. This shift was likely the result of a long relatively fire-free period coupled with warmer and wetter climates spanning much of the early Holocene. A brief but marked decrease in water availability around 8500-8000 cal yr BP resulted in the strong decrease of Dodonaea pollen. The vegetation of the mid- to late Holocene is characterised by the increased occurrence of Asteraceae and succulent taxa, suggesting substantially drier conditions. These data give unprecedented insight into the vegetation dynamics across a period of substantial, rapid climate change, and while they confirm the presence of fynbos elements throughout the last 15,600 years, the results highlight significant fluctuations in the vegetation that were triggered by changes in both climate and fire regimes. (C) 2013 Elsevier B.V. All rights reserved.

Fabrication and optical properties of large-scale arrays of gold nanocavities based on rod-in-a-tube coaxials

Centimeter sized arrays of gold coaxial rod-in-a tube cavities have been fabricated using anodized aluminum oxide as a template. The etching process used to create the cavities enables the production of extremely small gaps between tube and rod, on the order of 5 nm, smaller than those created by standard fabrication techniques. Normal incidence spectroscopy reveals two extinction peaks in the visible and near infrared wavelength range associated with resonant plasmonic modes excited in the structure. Numerical simulations show that the modes are associated with in-phase and out-of-phase hybridization of transverse dipolar excitations in the nanorod and in the tube.