A novel method for RNA extraction from FFPE samples reveals significant differences in biomarker expression between orthotopic and subcutaneous pancreatic cancer patient-derived xenografts.

Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies

Designer TGFβ Superfamily Ligands with Diversified Functionality

Transforming Growth Factor – beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values

Ordered three-fold symmetric graphene oxide/buckled graphene/graphene heterostructures on MgO(111) by carbon molecular beam epitaxy

Theory and experiment demonstrate the direct growth of a graphene oxide/buckled graphene/graphene heterostructure on an incommensurate MgO(111) substrate. X-ray photoelectron spectroscopy, electron energy loss, Auger electron spectroscopy, low energy electron diffraction, Raman spectroscopy and first-principles density functional theory (DFT) calculations all demonstrate that carbon molecular beam epitaxy on either a hydroxylated MgO(111) single crystal or a heavily twinned thin film surface at 850 K yields an initial C layer of highly ordered graphene oxide with C_(3v) symmetry. A 5 × 5 unit cell of carbon, with one missing atom, forms on a 4 × 4 unit cell of MgO, with the three C atoms surrounding the C vacancy surface forming covalent C–O bonds to substrate oxide sites. This leads to a bowed graphene-oxide with slightly modified D and G Raman lines and a calculated band gap of 0.36 eV. Continued C growth results in the second layer of graphene that is stacked AB with respect to the first layer and buckled conformably with the first layer while maintaining C_(3v) symmetry, lattice spacing and azimuthal orientation with the first layer. Carbon growth beyond the second layer yields graphene in azimuthal registry with the first two C layers, but with graphene-characteristic lattice spacing and π → π* loss feature. This 3rd layer is also p-type, as indicated by the 5.6 eV energy loss feature. The significant sp^3 character and C_(3v) symmetry of such heterostructures suggest that spin–orbit coupling is enabled, with implications for spintronics and other device applications

Leaf colour as a signal of chemical defence to insect herbivores in wild cabbage (Brassica Oleracea)

Leaf colour has been proposed to signal levels of host defence to insect herbivores, but we lack data on herbivory, leaf colour and levels of defence for wild host populations necessary to test this hypothesis. Such a test requires measurements of leaf spectra as they would be sensed by herbivore visual systems, as well as simultaneous measurements of chemical defences and herbivore responses to leaf colour in natural host-herbivore populations. In a large-scale field survey of wild cabbage (Brassica oleracea) populations, we show that variation in leaf colour and brightness, measured according to herbivore spectral sensitivities, predicts both levels of chemical defences (glucosinolates) and abundance of specialist lepidopteran (Pieris rapae) and hemipteran (Brevicoryne brassicae) herbivores. In subsequent experiments, P. rapae larvae achieved faster growth and greater pupal mass when feeding on plants with bluer leaves, which contained lower levels of aliphatic glucosinolates. Glucosinolate-mediated effects on larval performance may thus contribute to the association between P. rapae herbivory and leaf colour observed in the field. However, preference tests found no evidence that adult butterflies selected host plants based on leaf coloration. In the field, B. brassicae abundance varied with leaf brightness but greenhouse experiments were unable to identify any effects of brightness on aphid preference or performance. Our findings suggest that although leaf colour reflects both levels of host defences and herbivore abundance in the field, the ability of herbivores to respond to colour signals may be limited, even in species where performance is correlated with leaf colour

Immune Activation Reduces Sperm Quality in the Great Tit

Mounting an immune response against pathogens incurs costs to organisms by its effects on important life-history traits, such as reproductive investment and survival. As shown recently, immune activation produces large amounts of reactive species and is suggested to induce oxidative stress. Sperm are highly susceptible to oxidative stress, which can negatively impact sperm function and ultimately male fertilizing efficiency. Here we address the question as to whether mounting an immune response affects sperm quality through the damaging effects of oxidative stress. It has been demonstrated recently in birds that carotenoid-based ornaments can be reliable signals of a male's ability to protect sperm from oxidative damage. In a full-factorial design, we immune-challenged great tit males while simultaneously increasing their vitamin E availability, and assessed the effect on sperm quality and oxidative damage. We conducted this experiment in a natural population and tested the males' response to the experimental treatment in relation to their carotenoid-based breast coloration, a condition-dependent trait. Immune activation induced a steeper decline in sperm swimming velocity, thus highlighting the potential costs of an induced immune response on sperm competitive ability and fertilizing efficiency. We found sperm oxidative damage to be negatively correlated with sperm swimming velocity. However, blood resistance to a free-radical attack (a measure of somatic antioxidant capacity) as well as plasma and sperm levels of oxidative damage (lipid peroxidation) remained unaffected, thus suggesting that the observed effect did not arise through oxidative stress. Towards the end of their breeding cycle, swimming velocity of sperm of more intensely colored males was higher, which has important implications for the evolution of mate choice and multiple mating in females because females may accrue both direct and indirect benefits by mating with males having better quality sperm

Mechanism, function and modulation of oncogenic cripto signaling

Cripto is a multifunctional protein with key roles during embryonic development and tumorigenesis. Cripto's oncogenic functions have been linked to its regulation of TGF-[beta] ligand signaling via the Smad2/3 pathway and its growth factor activity via the MAPK/PI3K pathways. Notably, Cripto is required for signaling by certain TGF- [beta] superfamily members such as Nodal, but also antagonizes others such as activin and TGF-[beta]. The opposing effects of Cripto on Nodal and activin signaling seem contradictory, however, since these closely related ligands utilize the same receptors. Chapter one addresses this apparent paradox by demonstrating that Cripto forms analogous receptor complexes with Nodal and activin and functions as a non-competitive activin antagonist. The results show that activin-A and Nodal elicit similar maximal signaling responses in the presence of Cripto that are substantially lower than that of activin-A in the absence of Cripto. In addition, biochemical data reveal complexes containing activin-A, Cripto and both receptor types and show that the assembly of such complexes is competitively inhibited by Nodal. Furthermore, both Nodal and activin-A share the same binding residues on ActRII. Finally, data presented here demonstrate that the ALK4 ECD has distinct and separable binding sites for activin-A and Cripto. Importantly, ALK4 ECD mutants with disrupted activin-A binding can bind and block Cripto effects on both activin-A and Nodal signaling. We recently demonstrated that Cripto forms a cell surface complex with glucose-regulated protein-78 (GRP78). Chapter two provides evidence that shRNA knockdown or immunoneutralization of GRP78 inhibits the ability of Cripto to function as a Nodal co-receptor, an antagonist of activin and TGF-[beta] signaling and an activator of MAPK/PI3K pathways. Disrupting the Cripto/GRP78 complex also prevents Cripto from increasing cellular proliferation, downregulating E- Cadherin, decreasing cell adhesion and promoting mitogenic activin-A and Nodal signaling. The results indicate that GRP78 is a critical mediator of Cripto signaling in human tumor, mammary epithelial and embryonic stem cells and suggest that the Cripto/GRP78 complex plays key roles during normal developmental processes and tumorigenesis. Collectively, the work presented in this dissertation represents seminal advances in understanding the mechanism and function of Cripto-mediated signalin

Mechanism, function and modulation of oncogenic cripto signaling

Cripto is a multifunctional protein with key roles during embryonic development and tumorigenesis. Cripto's oncogenic functions have been linked to its regulation of TGF-[beta] ligand signaling via the Smad2/3 pathway and its growth factor activity via the MAPK/PI3K pathways. Notably, Cripto is required for signaling by certain TGF- [beta] superfamily members such as Nodal, but also antagonizes others such as activin and TGF-[beta]. The opposing effects of Cripto on Nodal and activin signaling seem contradictory, however, since these closely related ligands utilize the same receptors. Chapter one addresses this apparent paradox by demonstrating that Cripto forms analogous receptor complexes with Nodal and activin and functions as a non-competitive activin antagonist. The results show that activin-A and Nodal elicit similar maximal signaling responses in the presence of Cripto that are substantially lower than that of activin-A in the absence of Cripto. In addition, biochemical data reveal complexes containing activin-A, Cripto and both receptor types and show that the assembly of such complexes is competitively inhibited by Nodal. Furthermore, both Nodal and activin-A share the same binding residues on ActRII. Finally, data presented here demonstrate that the ALK4 ECD has distinct and separable binding sites for activin-A and Cripto. Importantly, ALK4 ECD mutants with disrupted activin-A binding can bind and block Cripto effects on both activin-A and Nodal signaling. We recently demonstrated that Cripto forms a cell surface complex with glucose-regulated protein-78 (GRP78). Chapter two provides evidence that shRNA knockdown or immunoneutralization of GRP78 inhibits the ability of Cripto to function as a Nodal co-receptor, an antagonist of activin and TGF-[beta] signaling and an activator of MAPK/PI3K pathways. Disrupting the Cripto/GRP78 complex also prevents Cripto from increasing cellular proliferation, downregulating E- Cadherin, decreasing cell adhesion and promoting mitogenic activin-A and Nodal signaling. The results indicate that GRP78 is a critical mediator of Cripto signaling in human tumor, mammary epithelial and embryonic stem cells and suggest that the Cripto/GRP78 complex plays key roles during normal developmental processes and tumorigenesis. Collectively, the work presented in this dissertation represents seminal advances in understanding the mechanism and function of Cripto-mediated signalin

Innate colour preferences of a hawkmoth depend on visual context

Solitary insects that feed on floral nectar must use innate knowledge to find their first flower. While innate preferences for flower colours are often described as fixed, species-specific traits, the nature and persistence of these preferences have been debated, particularly in relation to ontogenetic processes such as learning. Here we present evidence for a strong context-dependence of innate colour preferences in the crepuscular hawkmoth Manduca sexta. Contrary to expectations, our results show that innate colour biases shift with changes in the visual environment, namely illuminance and background. This finding reveals that innate responses might emerge from a contextual integration of sensory inputs involved in object class recognition rather than from the deterministic matching of such inputs with a fixed internal representation