TGFBR3 supports anoikis through suppressing ATF4 signaling.

Epithelial morphogenesis and oncogenic transformation can cause loss of cell adhesion, and detached cells are eliminated by anoikis. Here, we reveal that transforming growth factor ß receptor 3 (TGFBR3) acts as an anoikis mediator through the coordination of activating transcription factor 4 (ATF4). In breast cancer tissues, TGFBR3 is progressively lost, but elevated TGFBR3 is associated with a histologic subtype characterized by cellular adhesion defects. Dissecting the impact of extracellular matrix (ECM) deprivation, we demonstrate that ECM loss promotes TGFBR3 expression, which in turn causes differentiation of cell aggregates, conferring a low-adhesion phenotype, and drives the intrinsic apoptotic pathway. We demonstrate that inhibition of TGFBR3 impairs epithelial anoikis by activating ATF4 signaling. These preclinical findings provide a rationale for therapeutic inhibition of ATF4 in the subgroup of breast cancer patients with low TGFBR3 expression.

Novel tests for evaluating two ROC curves under paired samples.

Disease prevention is important and can be accomplished by developing diagnostic tests. The receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) are used to assess the accuracy of diagnostic tests. The assessment for the superiority between evaluating two diagnostic tests is needed when comparing two diagnostic tests. Existing tests are constructed by comparing two AUCs under the paired samples. Nevertheless, it is problematic when two ROC curves are crossing. This article proposes a test that takes into account the possible correlation between pairs. Simulations are conducted to evaluate the feasibility of the test.

Joint model of multiple longitudinal measures and a binary outcome: An application to predict orthostatic hypertension for subacute stroke patients.

Stroke patients with orthostatic hypertensive responses that are one of the blood pressure regulation problems can easily fall down while doing rehabilitation, which may result in prolonged hospitalization and delayed treatment and recovery. This may result in increasing the medical cost and burden. In turn, developing a diagnostic test for the orthostatic hypertension (OH) is clinically important for patients who are suffering from stroke. Clinically, a patient needs to have a tilt testing that requires measuring the change of blood pressures and heart rate at all angles to determine whether a stroke patient has OH. It takes lots of time and effort to perform the test. Assuming there exist measurement errors when obtaining the blood pressures and heart rate at all angles, this paper proposes using multiple mixed-effect models to obtain the true trajectories of these measurements, which take into account the measurement error and the possible correlation among multiple measurements, and a logistic regression uses these true trajectories at a given time and other fixed-effect covariates as predictors to predict the status of OH. The joint likelihood function is derived to estimate parameters and the area under the receiver operating characteristics curve is used to estimate the predictive power of the model. Monte Carlo simulations are performed to evaluate the feasibility of the proposed methods. Also, the proposed model is implemented in the real data and provides an acceptable predictive power.

Identifying single-cell molecular programs by stochastic profiling.

Cells in tissues can be morphologically indistinguishable yet show molecular expression patterns that are remarkably heterogeneous. Here we describe an approach to comprehensively identify co-regulated, heterogeneously expressed genes among cells that otherwise appear identical. The technique, called stochastic profiling, involves repeated, random selection of very small cell populations via laser-capture microdissection followed by a customized single-cell amplification procedure and transcriptional profiling. Fluctuations in the resulting gene-expression measurements are then analyzed statistically to identify transcripts that are heterogeneously coexpressed. We stochastically profiled matrix-attached human epithelial cells in a three-dimensional culture model of mammary-acinar morphogenesis. Of 4,557 transcripts, we identified 547 genes with strong cell-to-cell expression differences. Clustering of this heterogeneous subset revealed several molecular 'programs' implicated in protein biosynthesis, oxidative-stress responses and NF-kappaB signaling, which we independently confirmed by RNA fluorescence in situ hybridization. Thus, stochastic profiling can reveal single-cell heterogeneities without the need to measure expression in individual cells.

Misspecification of a binary dependent variable in the logistic model controlling for the repeated longitudinal measures.

Many medical applications are interested to know the disease status. The disease status can be related to multiple serial measurements. Nevertheless, owing to various reasons, the binary outcome can be measured incorrectly. The estimators derived from the misspecified outcome can be biased. This paper derives the complete data likelihood function to incorporate both the multiple serial measurements and the misspecified outcome. Owing to the latent variables, EM algorithm is used to derive the maximum-likelihood estimators. Monte Carlo simulations are conducted to compare the impact of misspecification on the estimates. A retrospective data for the recurrence of atrial fibrillation is used to illustrate the usage of the proposed model.

Multifunctional magnetic nanocarriers for delivery of siRNA and shRNA plasmid to mammalian cells: Characterization, adsorption and release behaviors.

Nucleic acids are promising candidates for treating various diseases. Nucleic acid is negatively charged and hydrophilic; therefore, it is not efficiently taken up by cells. Successful gene therapy requires the development of carriers for efficient delivery of gene-expressing DNA plasmid and small interfering RNA (siRNA) duplex. In this study, we developed MNP-CA-PEI, a citric acid (CA)-modified magnetic nanoparticle (MNP) cross-linked with polyethyleneimine (PEI), using carbonyldiimidazole as the crosslinker. The physical properties of MNP-CA-PEI (particle size, morphologies, surface coating, surface potentials, magnetic hystereses, superparamagnetic behaviors, and infrared spectra) were systematically characterized by transmission electron microscopy imaging, dynamic light scattering, thermogravimetric analysis, superconducting quantum interference device, and Fourier transform infrared spectroscopy. The adsorption isotherm and kinetics were determined by the Langmuir model, the Freundlich model, a pseudo-first-order equation, and a pseudo-second-order equation. MNP-CA-PEI could form polyelectrolyte complexes with negatively charged nucleic acids, enabling the efficient delivery of nucleic acids into cells. Using MNP-CA-PEI nanoparticles, we magnetically triggered the intracellular delivery of green fluorescence protein (GFP)-expressing DNA plasmid, plasmid-expressing short hairpin RNA (shRNA) against GFP, or siRNA targeting GFP into different cell lines. Nucleic acid/MNP-CA-PEI displayed the enhanced cellular uptake of GFP-expressing DNA plasmid, and it improved the silencing efficiency of shRNA and siRNA, determined by fluorescence imaging. Gene knockdowns mediated by shRNA and siRNA were also confirmed by a quantitative real-time polymerase chain reaction. MNP-CA-PEI delivered nucleic acids into cytosol through caveolae-mediated endocytosis. This study introduces a new MNP functionalization that can be used for the magnetically driven intracellular delivery of nucleic acids.

Tetraethylenepentamine-Coated ß Cyclodextrin Nanoparticles for Dual DNA and siRNA Delivery.

Nucleic acid reagents, including plasmid-encoded genes and small interfering RNA (siRNA), are promising tools for validating gene function and for the development of therapeutic agents. Native ß-cyclodextrins (BCDs) have limited efficiency in gene delivery due to their instable complexes with nucleic acid. We hypothesized that cationic BCD nanoparticles could be an efficient carrier for both DNA and siRNA. Tetraethylenepentamine-coated ß-cyclodextrin (TEPA-BCD) nanoparticles were synthesized, characterized, and evaluated for targeted cell delivery of plasmid DNA and siRNA. The cationic TEPA coating provided ideal zeta potential and effective nucleic acid binding ability. When transfecting plasmid encoding green fluorescent protein (GFP) by TEPA-BCD, excellent GFP expression could be achieved in multiple cell lines. In addition, siRNA transfected by TEPA-BCD suppressed target GFP gene expression. We showed that TEPA-BCD internalization was mediated by energy-dependent endocytosis via both clathrin-dependent and caveolin-dependent endocytic pathways. TEPA-BCD nanoparticles provide an effective means of nucleic acid delivery and can act as potential carriers in future pharmaceutical application.

Systematic quantification of negative feedback mechanisms in the extracellular signal-regulated kinase (ERK) signaling network.

Cell responses are actuated by tightly controlled signal transduction pathways. Although the concept of an integrated signaling network replete with interpathway cross-talk and feedback regulation is broadly appreciated, kinetic data of the type needed to characterize such interactions in conjunction with mathematical models are lacking. In mammalian cells, the Ras/ERK pathway controls cell proliferation and other responses stimulated by growth factors, and several cross-talk and feedback mechanisms affecting its activation have been identified. In this work, we take a systematic approach to parse the magnitudes of multiple regulatory mechanisms that attenuate ERK activation through canonical (Ras-dependent) and non-canonical (PI3K-dependent) pathways. In addition to regulation of receptor and ligand levels, we consider three layers of ERK-dependent feedback: desensitization of Ras activation, negative regulation of MEK kinase (e.g. Raf) activities, and up-regulation of dual-specificity ERK phosphatases. Our results establish the second of these as the dominant mode of ERK self-regulation in mouse fibroblasts. We further demonstrate that kinetic models of signaling networks, trained on a sufficient diversity of quantitative data, can be reasonably comprehensive, accurate, and predictive in the dynamical sense.

Metabolic Stress Adaptations Underlie Mammary Gland Morphogenesis and Breast Cancer Progression.

Breast cancers display dynamic reprogrammed metabolic activities as cancers develop from premalignant lesions to primary tumors, and then metastasize. Numerous advances focus on how tumors develop pro-proliferative metabolic signaling that differs them from adjacent, non-transformed epithelial tissues. This leads to targetable oncogene-driven liabilities among breast cancer subtypes. Other advances demonstrate how microenvironments trigger stress-response at single-cell resolution. Microenvironmental heterogeneities give rise to cell regulatory states in cancer cell spheroids in three-dimensional cultures and at stratified terminal end buds during mammary gland morphogenesis, where stress and survival signaling juxtapose. The cell-state specificity in stress signaling networks recapture metabolic evolution during cancer progression. Understanding lineage-specific metabolic phenotypes in experimental models is useful for gaining a deeper understanding of subtype-selective breast cancer metabolism.

PI3K-dependent cross-talk interactions converge with Ras as quantifiable inputs integrated by Erk.

Although it is appreciated that canonical signal-transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross-talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross-talk mechanisms in the platelet-derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3-kinase (PI3K) and Ras/extracellular signal-regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross-talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K-dependent and -independent modes of Ras/Erk activation.

Encapsulating curcumin in ethylene diamine-ß-cyclodextrin nanoparticle improves topical cornea delivery.

Curcumin is a powerful scavenger of reactive oxygen species and could prevent the corneal cells from oxidative damage. However, the clinical efficacy of curcumin is limited by its low aqueous solubility and stability, leading to poor bioavailability. ß-cyclodextrin, with a hydrophilic surface and a hydrophobic cavity and self-assembling properties, can form inclusion complexes with lipophilic drugs such as curcumin for ocular delivery. We synthesized ethylene diamine (EDA)-modified ß-cyclodextrin and prepared the curcumin complexation using the solvent evaporation method. The EDA-ß-cyclodextrin provided a better thermodynamic stability and higher complex yield for curcumin complexes, compared to ß-cyclodextrin, which were demonstrated on the analysis of their van't Hoff plots and phase solubility diagrams. We characterized EDA-ß-cyclodextrin curcumin nanoparticles and determined that the EDA modified ß-cyclodextrin is a more suitable carrier than parental ß-cyclodextrin, using FT-IR, XRD, TEM, and analyses of solubility and storage stability. In addition, the curcumin-EDA-ß-cyclodextrin nanoparticles had better in vitro corneal penetration and 3 -h cumulative flux in a porcine cornea experiment, and displayed an improved biocompatibility, confirmed by the histological examination of porcine corneas and cell viability of bovine corneal epithelial cells. These results together revealed a role of EDA modification in the ß-cyclodextrin carrier, including the improvement of curcumin complex formation, thermodynamic properties, cytotoxicity, and the in vitro corneal penetration. The EDA-ß-cyclodextrin inclusion can provide curcumin a higher degree of aqueous solubility and corneal permeability.

Sporadic activation of an oxidative stress-dependent NRF2-p53 signaling network in breast epithelial spheroids and premalignancies.

Breast and mammary epithelial cells experience different local environments during tissue development and tumorigenesis. Microenvironmental heterogeneity gives rise to distinct cell regulatory states whose identity and importance are just beginning to be appreciated. Cellular states diversify when clonal three-dimensional (3D) spheroids are cultured in basement membrane, and one such state is associated with stress tolerance and poor response to anticancer therapeutics. Here, we found that this state was jointly coordinated by the NRF2 and p53 pathways, which were costabilized by spontaneous oxidative stress within 3D cultures. Inhibition of NRF2 or p53 individually disrupted some of the transcripts defining the regulatory state but did not yield a notable phenotype in nontransformed breast epithelial cells. In contrast, combined perturbation prevented 3D growth in an oxidative stress-dependent manner. By integrating systems models of NRF2 and p53 signaling in a single oxidative stress network, we recapitulated these observations and made predictions about oxidative stress profiles during 3D growth. NRF2 and p53 signaling were similarly coordinated in normal breast epithelial tissue and hormone-negative ductal carcinoma in situ lesions but were uncoupled in triple-negative breast cancer (TNBC), a subtype in which p53 is usually mutated. Using the integrated model, we correlated the extent of this uncoupling in TNBC cell lines with the importance of NRF2 in the 3D growth of these cell lines and their predicted handling of oxidative stress. Our results point to an oxidative stress tolerance network that is important for single cells during glandular development and the early stages of breast cancer.

An efficient method for the N-bromosuccinimide catalyzed synthesis of indolyl-nitroalkanes.

An efficient and practical method for the synthesis of indolyl-nitroalkane derivatives catalyzed by N-bromosuccinimide is described. The generality of this method was demonstrated by synthesizing an array of diverse 3-substituted indole derivatives by the reaction of different beta-nitrostyrenes with various substituted indoles. Simple reaction conditions accompanied by good yields of indolyl-nitroalkanes are the merits of this methodology.

Biocompatible quantum dot-antibody conjugate for cell imaging, targeting and fluorometric immunoassay: crosslinking, characterization and applications.

Quantum dots (QDs) are important fluorescent probes that offer great promise for bio-imaging research due to their superior optical properties. However, QDs for live cell imaging and the tracking of cells need more investigation to simplify processing procedures, improving labeling efficiency, and reducing chronic toxicity. In this study, QDs were functionalized with bovine serum albumin (BSA) via a chemical linker. Anti-human immunoglobulin antibodies were oxidized by sodium periodate to create reactive aldehyde groups for a spontaneous reaction with the amine groups of BSA-modified QDs. An antibody-labeled QD bioconjugate was characterized using agarose gel electrophoresis, dynamic light scattering, and zeta potential. Using fluorescence spectroscopy, we found that the fluorescence of QDs was retained after multiple conjugation steps. The cell-labeling function of the QD bioconjugate was confirmed using an image analyzer and confocal microscopy. The QD bioconjugate specifically targeted human immunoglobulin on the membrane surface of recombinant cells. In addition, the QD bioconjugate applied in fluorometric immunoassay was effective for the quantitative analysis of human immunoglobulin in an enzyme-linked immunosorbent assay. The developed QD bioconjugate may offer a promising platform to develop biocompatible tools to label cells and quantify antibodies in the immunoassay.

Genetic dissection of seedling vigour in a diverse panel from the 3,000 Rice (Oryza sativa L.) Genome Project.

Seedling vigour (SV) is important for direct seeding rice (Oryza sativa L.), especially in a paddy-direct seeding system, but the genetic mechanisms behind the related traits remain largely unknown. Here, we used 744 germplasms, having at least two subsets, for the detection of quantitative trait loci (QTLs) affecting the SV-related traits tiller number, plant height, and aboveground dry weight at three sampling stages, 27, 34, and 41 d after sowing. A joint map based on GAPIT and mrMLM produced a satisfying balance between type I and II errors. In total, 42 QTL regions, containing 18 (42.9%) previously reported overlapping QTL regions and 24 new ones, responsible for SV were detected throughout the genome. Four QTL regions, qSV1a, qSV3e, qSV4c, and qSV7c, were delimited and harboured quantitative trait nucleotides that are responsible for SV-related traits. Favourable haplotype mining for the candidate genes within these four regions, as well as the early SV gene OsGA20ox1, was performed, and the favourable haplotypes were presented with donors from the 3,000 Rice Genome Project. This work provides new information and materials for the future molecular breeding of direct seeding rice, especially in paddy-direct seeding cultivation systems.

Efficient TCT-catalyzed synthesis of 1,5-benzodiazepine derivatives under mild conditions.

2,4,6-Trichloro-1,3,5-triazine (TCT) efficiently catalyzed the condensation reactions between 1,2-diamines and various enolizable ketones to afford 1,5-benzodiazepines in good to excellent yields. Simple and mild reaction conditions, the use of a cheap catalyst and easy workup and isolation are notable features of this method.

Joint Exploration of Favorable Haplotypes for Mineral Concentrations in Milled Grains of Rice (Oryza sativa L.).

Grain minerals in rice, especially those in milled grains, are important sources of micro-nutrition elements, such as iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), and selenium (Se), and of toxic heavy metal elements, especially cadmium (Cd), for populations consuming a rice diet. To date, the genetic mechanism underlying grain mineral concentrations (GMCs) in milled grain remains largely unknown. In this report, we adopted a set of 698 germplasms consisting of two subsets [indica/Xian (X-set) and japonica/Geng (G-set)], to detect quantitative trait loci (QTL) affecting GMC traits of Fe, Zn, Cd, Mn, Cu, and Se in milled grains. A total of 47 QTL regions, including 18 loci and 29 clusters (covering 62 Cd loci), responsible for the GMCs in milled grains were detected throughout the genome. A joint exploration of favorable haplotypes of candidate genes was carried out as follows: (1) By comparative mapping, 10 chromosome regions were found to be consistent with our previously detected QTL from linkage mapping. (2) Within eight of these regions on chromosomes 1, 4, 6, 7, and 8, candidate genes were identified in the genome annotation database. (3) A total of 192 candidate genes were then submitted to further haplotype analysis using million-scale single nucleotide polymorphisms (SNPs) from the X-set and the G-set. (4) Finally, 37 genes (19.3%) were found to be significant in the association between the QTL targeting traits and the haplotype variations by pair-wise comparison. (5) The phenotypic values for the haplotypes of each candidate were plotted. Three zinc finger (like) genes within two candidate QTL regions (qFe6-2 and qZn7), and three major GMC traits (Fe, Zn, and Cd) were picked as sample cases, in addition to non-exhausted cross validations, to elucidate this kind of association by trait value plotting. Taken together, our results, especially the 37 genes with favorable haplotype variations, will be useful for rice biofortification molecular breeding.

Application of the nonlinear, double-dynamic Taguchi method to the precision positioning device using combined piezo-VCM actuator.

In this research, the nonlinear, double-dynamic Taguchi method was used as design and analysis methods for a high-precision positioning device using the combined piezo-voice-coil motor (VCM) actuator. An experimental investigation into the effects of two input signals and three control factors were carried out to determine the optimum parametric configuration of the positioning device. The double-dynamic Taguchi method, which permits optimization of several control factors concurrently, is particularly suitable for optimizing the performance of a positioning device with multiple actuators. In this study, matrix experiments were conducted with L9(3(4)) orthogonal arrays (OAs). The two most critical processes for the optimization of positioning device are the identification of the nonlinear ideal function and the combination of the double-dynamic signal factors for the ideal function's response. The driving voltage of the VCM and the waveform amplitude of the PZT actuator are combined into a single quality characteristic to evaluate the positioning response. The application of the double-dynamic Taguchi method, with dynamic signal-to-noise ratio (SNR) and L9(3(4)) OAs, reduced the number of necessary experiments. The analysis of variance (ANOVA) was applied to set the optimum parameters based on the high-precision positioning process.

Tumor-Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous carcinoma in which various tumor-suppressor genes are lost by mutation, deletion, or silencing. Here we report a tumor-suppressive mode of action for growth-differentiation factor 11 (GDF11) and an unusual mechanism of its inactivation in TNBC. GDF11 promotes an epithelial, anti-invasive phenotype in 3D triple-negative cultures and intraductal xenografts by sustaining expression of E-cadherin and inhibitor of differentiation 2 (ID2). Surprisingly, clinical TNBCs retain the GDF11 locus and expression of the protein itself. GDF11 bioactivity is instead lost because of deficiencies in its convertase, proprotein convertase subtilisin/kexin type 5 (PCSK5), causing inactive GDF11 precursor to accumulate intracellularly. PCSK5 reconstitution mobilizes the latent TNBC reservoir of GDF11 in vitro and suppresses triple-negative mammary cancer metastasis to the lung of syngeneic hosts. Intracellular GDF11 retention adds to the concept of tumor-suppressor inactivation and reveals a cell-biological vulnerability for TNBCs lacking therapeutically actionable mutations.