Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and β-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression

Far-Red and Near-Infrared Seminaphthofluorophores for Targeted Pancreatic Cancer Imaging

Molecular probes that selectively highlight pancreatic cancer (PC) tissue have the potential to improve pancreatic ductal adenocarcinoma (PDAC) margin assessment through the selective highlighting of individual PC cells. Herein, we report a simple and unique family of systematically modified red and near-infrared fluorescent probes that exhibit a field-effect-derived redshift. Two of thirteen probes distributed to the normal mouse pancreas following systemic administration. One selectively accumulated in genetically modified mouse models of PDAC. The probe exhibited intracellular accumulation and enabled visualization of four levels of the structure, including the whole organ, resected tissue, individual cells, and subcellular organelles. In contrast to the small-molecule probes reported previously, it possesses an inherent affinity toward PDAC cells and thus does not require conjugation to any targeting agent. The fluorescent probe can thus promote new strategies not only for precision image-guided surgery, but also for PC detection, monitoring of therapeutic outcomes, and basic research

Greenhouse gas emissions in coffee grown with differing input levels under conventional and organic management

Coffee plays a key role in sustaining millions of livelihoods around the world. Understanding GHG emissions from coffee supply chains is important in evaluating options for climate change mitigation within the sector. We use data from two long-term coffee agroforestry experiments in Costa Rica and Nicaragua to calculate carbon footprints (CF) for coffee and identify emission hotspots within different management systems, levels of inputs and shade types. Management system and input level were the main cause of variation in CFs. Carbon footprints for 1 kg of fresh coffee cherries were between 0.26 and 0.67 kgCO2e for conventional and 0.12 and 0.52 kgCO2e for organic management systems. The main contributor to GHG emissions for all management systems was the inputs of organic and inorganic nitrogen. Nitrous oxide emissions from pruning inputs contributed between 7% and 42 % of CFs. However, these estimates were strongly influenced by the choice of emission factors

Sink or source—The potential of coffee agroforestry systems to sequester atmospheric CO2 into soil organic carbon

Current carbon accounting methodologies often assume interactions between above-ground and below-ground carbon, without considering effects of land management. We used data from two long-term coffee agroforestry experiments in Costa Rica and Nicaragua to assess the effect on total soil organic carbon (SOC) stocks of (i) organic versus conventional management, (ii) higher versus moderate agronomic inputs, (iii) tree shade types. During the first nine years of coffee establishment total 0–40 cm depth SOC stocks decreased by 12.4% in Costa Rica and 0.13% in Nicaragua. Change in SOC differed consistently amongst soil layers: at 0–10 cm SOC stocks increased by 2.14 and 1.26 Mg C ha−1 in Costa Rica and Nicaragua respectively; however much greater reduction occurred at 20–40 cm (9.65 and 2.85 Mg C ha−1 respectively). Organic management caused a greater increase in 0–10 cm SOC but did not influence its reduction at depth. Effects of shade type were smaller, though heavily pruned legume shade trees produced a greater increase in 0–10 cm SOC than unpruned timber trees. No significant differences in SOC stocks were found between shaded and unshaded systems at any depth and SOC was poorly correlated with above-ground biomass stocks highlighting poor validity of “expansion factors” currently used to estimate SOC. SOC stock changes were significantly negatively correlated with initial SOC stock per plot, providing evidence that during establishment of these woody-plant-dominated agricultural systems SOC stocks tend to converge towards a new equilibrium as a function of the change in the quantity and distribution of organic inputs. Therefore it cannot be assumed that tree-based agricultural systems necessarily lead to increases in soil C stocks. While high inputs of organic fertiliser/tree pruning mulch increased surface-layer SOC stocks, this did not affect stocks in deeper soil, where decreases generally exceeded any gains in surface soil. Therefore site- and system-specific sampling is essential to draw meaningful conclusions for climate change mitigation strategies

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

The cloning and sequence characterization of cytochrome P-450 homologous sequences

Thesis (B.S.) in Chemistry -- University of Illinois at Urbana-Champaign, 1986.Bibliography: leaf 22.U of I OnlyTheses restricted to UIUC community onl

Integrin Alpha Subunit Expression and Cytoplasmic Domain Function

181 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.The \beta\sb1 integrins are a family of $\alpha\beta$ heterodimeric cell surface receptors which mediate cell adhesion to molecules of the extracellular matrix (ECM), and also span the membrane and associate with the cytoskeleton via their cytoplasmic domains. By mediating the interaction of the cellular cytoskeleton with the ECM, the \beta\sb1 integrins are central to issues of cell migration, proliferation, differentiation, cytoskeletal organization, cell morphology, and the maintenance of tissue architecture and integrity. The focus of this work has been to investigate the expression and function of individual chicken \beta\sb1 integrin family members. First, the integrins were affinity purified from various adult chicken tissues to reveal the diversity and differential expression of the chicken \beta\sb1 integrins. The purified receptors were then used to immunize mice and generate monoclonal antibodies (mAbs) against the $\alpha$ subunits which distinguish each receptor. Antibodies were generated against two $\alpha$ subunits (i.e. two receptors), and were used to identify these $\alpha$ subunits as homologs of the human \alpha\sb5 subunit (a fibronectin receptor) and human \alpha\sb6 subunit (a laminin receptor). Also, the cross-reaction with chicken $\alpha$ subunits of antisera specific for the human $\alpha$ subunit cytoplasmic domains revealed conserved sequences, and presumably a conserved function, within each $\alpha$ subunit cytoplasmic domain. Immuno-cytochemistry studies using the anti-\alpha\sb5 and anti-\alpha\sb6 mAbs revealed: (1) the cell-specific expression of the receptors; (2) the changes in receptor expression during embryonic development; and (3) the localization of the receptors in specific membrane-cytoskeletal junctions. The observed junctional specificity of the receptors suggested a unique recognition between cytoskeletal molecules at each membrane-cytoskeleton junction and the integrin $\alpha$ subunit cytoplasmic domains. We have initiated mutagenesis studies to dissect the function(s) within the $\alpha$ subunit cytoplasmic domains. In addition, we have generated antibodies against an apparently novel integrin-associated cytoskeletal protein complex. These antibodies have permitted investigations of the composition and molecular associations at specific integrin-cytoskeleton junctions, and lead to the identification and characterization of several novel cytoskeletal molecules which are components of these junctions.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Cell-matrix interactions in mammary gland development and breast cancer.

The mammary gland is an organ that at once gives life to the young, but at the same time poses one of the greatest threats to the mother. Understanding how the tissue develops and functions is of pressing importance in determining how its control mechanisms break down in breast cancer. Here we argue that the interactions between mammary epithelial cells and their extracellular matrix (ECM) are crucial in the development and function of the tissue. Current strategies for treating breast cancer take advantage of our knowledge of the endocrine regulation of breast development, and the emerging role of stromal–epithelial interactions (Fig. 1). Focusing, in addition, on the microenvironmental influences that arise from cell–matrix interactions will open new opportunities for therapeutic intervention. We suggest that ultimately a three-pronged approach targeting endocrine, growth factor, and cell-matrix interactions will provide the best chance of curing the disease