Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ~7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ~25 min as follows: (i) DNA immobilization ~6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ~6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light.     

The Wnt signaling pathway has tumor suppressor properties in retinoblastoma

Retinoblastoma is a pediatric retinal tumor caused by mutational inactivation of the tumor suppressor pRb. Additional genetic changes, as yet unidentified, are believed to be required for tumor initiation. Mutations in the Wnt signaling pathway have been implicated in the pathogenesis of many cancers. Multiple Wnt pathway genes are expressed in the retina and the pRb and Wnt pathways interact biochemically, raising the possibility that alterations in the Wnt pathway contribute to retinoblastoma. Our studies showed that Wnt signaling activation significantly decreased the viability of retinoblastoma cell lines by inducing cell cycle arrest, which was associated with upregulated p53. Furthermore, immunolocalization of the Wnt signaling mediator beta-catenin in human and mouse retinoblastoma tissue indicated that canonical Wnt signaling is suppressed in tumors in vivo. These studies are consistent with the Wnt pathway acting as a tumor suppressor in retinoblastoma and suggest that loss of Wnt signaling is tumorigenic in the retina.     

U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity

Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content.     

Breast cancer cell-derived matrix supports vascular morphogenesis

The extracellular matrix (ECM), important for maintaining tissue homeostasis, is abnormally expressed in mammary tumors and additionally plays a crucial role in angiogenesis. We hypothesize that breast cancer cells (BCCs) deposit ECM that supports unique patterns of vascular morphogenesis of endothelial cells (ECs). Evaluation of ECM expression revealed that a nontumorigenic cell line (MCF10A), a tumorigenic cell line (MCF7), and a metastatic cell line (MDA-MB-231) express collagens I and IV, fibronectin, and laminin, with tenascin-C limited to MCF10A and MCF7. The amount of ECM deposited by BCCs was found to be higher in MCF10A compared with MCF7 and MDA231, with all ECM differing in their gross structure but similar in mean fiber diameter. Nonetheless, deposition of ECM from BCC lines was overall difficult to detect and insufficient to support capillary-like structure (CLS) formation of ECs. Therefore, a coculture approach was undertaken in which individual BCC lines were cocultured with fibroblasts. Variation in abundance of deposited ECM, deposition of ECM proteins, such as absent collagen I deposition from MDA231-fibroblast cocultures, and fibril organization was found. Deposited ECM from fibroblasts and each coculture supported rapid CLS formation of ECs. Evaluation of capillary properties revealed that CLS grown on ECM deposited from MDA231-fibroblast cocultures possessed significantly larger lumen diameters, occupied the greatest percentage of area, expressed the highest levels of von Willebrand factor, and expressed the greatest amount of E-selectin, which was upregulated independent of exposure to TNF-α. To our knowledge, this is the first study to report tumor cell ECM-mediated differences in vascular capillary features, and thus offers the framework for future investigations interrogating the role of the tumor ECM in supporting vascular morphogenesis.     

Parkin and Mitofusins Reciprocally Regulate Mitophagy and Mitochondrial Spheroid Formation

Mitochondrial homeostasis via mitochondrial dynamics and quality control is crucial to normal cellular functions. Mitophagy (mitochondria removed by autophagy) stimulated by a mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), requires Parkin, but it is not clear why Parkin is crucial to this process. We found that in the absence of Parkin, carbonyl cyanide m-chlorophenylhydrazone induced the formation of mitochondrial spheroids. Mitochondrial spheroid formation is also induced in vivo in the liver by acetaminophen overdose, a condition causing severe oxidative mitochondrial damages and liver injury. Mitochondrial spheroids could undergo a maturation process by interactions with acidic compartments. The formation of this new structure required reactive oxygen species and mitofusins. Parkin suppressed these mitochondrial dynamics by promoting mitofusin degradation. Consistently, genetic deletion of mitofusins without concomitant expression of Parkin was sufficient to prevent mitochondrial spheroid formation and resumed mitophagy. Mitochondrial spheroid formation and mitophagy could represent different strategies of mitochondrial homeostatic response to oxidative stress and are reciprocally regulated by mitofusins and Parkin.     

Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.     

Developmental and genetic components explain enhanced pulmonary volumes of female peruvian Quechua

High altitude natives have enlarged vital capacities and residual volumes (RV). Because pulmonary volumes are an indication of functionally relevant traits, such as diffusion capacity, the understanding of the factors (genetic/developmental) that influence lung volumes provides insight into the adaptive responses of highlanders. In order to test for the effect of growth and development at high altitude on lung volumes, we obtained forced vital capacities (FVC), RV, and total lung capacities (TLC) for a sample of 65 Peruvian females of mostly Quechua origins (18-34 years) who were sub-divided into two well-matched groups: 1) sea-level born and raised females (BSL, n = 34) from Lima, Peru (150 m), and 2) high-altitude born and raised females (BHA, n = 31) from Cerro de Pasco, Peru (4,338 m). To determine Quechua origins, Native American ancestry proportion (NAAP) for each individual was assessed using a panel of 70 ancestry informative markers. NAAP was similar between groups (BSL = 91.71%; BHA = 89.93%; P = 0.240), and the analysis confirmed predominantly Quechua origins. After adjusting for body size and NAAP, BHA females had significantly higher FVC (3.79 ± 0.06 l; P < 0.001), RV (0.98 ± 0.03 l; P < 0.001) and TLC (4.80 ± 0.07 l; P < 0.001) compared to BSL females (FVC = 3.33 ± 0.05 l; RV = 0.69 ± 0.03 l; TLC = 4.02 ± 0.06 l). NAAP was not associated with FVC (P = 0.352) or TLC (P = 0.506). However, NAAP was positively associated with RV (P = 0.004). In summary, results indicate that developmental exposure to high altitude in females constitutes an important factor for all lung volumes, whereas both genetic and developmental factors seem to be important for RV.     

ATP depletion triggers acute myeloid leukemia differentiation through an ATR/Chk1 protein-dependent and p53 protein-independent pathway

Despite advances in oncology drug development, most commonly used cancer therapeutics exhibit serious adverse effects. Often the toxicities of chemotherapeutics are due to the induction of significant DNA damage that is necessary for their ability to kill cancer cells. In some clinical situations, the direct induction of significant cytotoxicity is not a requirement to meet clinical goals. For example, differentiation, growth arrest, and/or senescence is a valuable outcome in some cases. In fact, in the case of acute myeloid leukemia (AML), the use of the differentiation agent all-trans-retinoic acid (ATRA) has revolutionized the therapy for a subset of leukemia patients and led to a dramatic survival improvement. Remarkably, this therapeutic approach is possible even in many elderly patients, who would not be able to tolerate therapy with traditional cytotoxic chemotherapy. Because of the success of ATRA, there is widespread interest in identifying differentiation strategies that may be effective for the 90-95% of AML patients who do not clinically respond to ATRA. Utilizing an AML differentiation agent that is in development, we found that AML differentiation can be induced through ATP depletion and the subsequent activation of DNA damage signaling through an ATR/Chk1-dependent and p53-independent pathway. This study not only reveals mechanisms of AML differentiation but also suggests that further investigation is warranted to investigate the potential clinical use of low dose chemotherapeutics to induce differentiation instead of cytotoxicity. This therapeutic approach may be of particular benefit to patients, such as elderly AML patients, who often cannot tolerate traditional AML chemotherapy.