Fibronectin, DHPS and SLC3A2 Signaling Cooperate to Control Tumor Spheroid Growth, Subcellular eIF5A1/2 Distribution and CDK4/6 Inhibitor Resistance.

Extracellular matrix (ECM) protein expression/deposition within and stiffening of the breast cancer microenvironment facilitates disease progression and correlates with poor patient survival. However, the mechanisms by which ECM components control tumorigenic behaviors and responses to therapeutic intervention remain poorly understood. Fibronectin (FN) is a major ECM protein controlling multiple processes. In this regard, we previously reported that DHPS-dependent hypusination of eIF5A1/2 is necessary for fibronectin-mediated breast cancer metastasis and epithelial to mesenchymal transition (EMT). Here, we explored the clinical significance of an interactome generated using hypusination pathway components and markers of intratumoral heterogeneity. Solute carrier 3A2 (SLC3A2 or CD98hc) stood out as an indicator of poor overall survival among patients with basal-like breast cancers that express elevated levels of DHPS. We subsequently discovered that blockade of DHPS or SLC3A2 reduced triple negative breast cancer (TNBC) spheroid growth. Interestingly, spheroids stimulated with exogenous fibronectin were less sensitive to inhibition of either DHPS or SLC3A2 - an effect that could be abrogated by dual DHPS/SLC3A2 blockade. We further discovered that a subset of TNBC cells responded to fibronectin by increasing cytoplasmic localization of eIF5A1/2. Notably, these fibronectin-induced subcellular localization phenotypes correlated with a G0/G1 cell cycle arrest. Fibronectin-treated TNBC cells responded to dual DHPS/SLC3A2 blockade by shifting eIF5A1/2 localization back to a nucleus-dominant state, suppressing proliferation and further arresting cells in the G2/M phase of the cell cycle. Finally, we observed that dual DHPS/SLC3A2 inhibition increased the sensitivity of both Rb-negative and -positive TNBC cells to the CDK4/6 inhibitor palbociclib. Taken together, these data identify a previously unrecognized mechanism through which extracellular fibronectin controls cancer cell tumorigenicity by modulating subcellular eIF5A1/2 localization and provides prognostic/therapeutic utility for targeting the cooperative DHPS/SLC3A2 signaling axis to improve breast cancer treatment responses.

Evaluating the Indonesia Free Pasung Movement: Understanding continuing use of restraint of the mentally ill in rural Java.

Forcible restraint and confinement of persons suffering from mental illness occurs throughout the world, including in Indonesia. Since 2010, when Gerakan Bebas Pasung (GBP) or the Indonesian Freedom from Forcible Restraint (Pasung) of Mentally Ill Persons movement was launched, national policy has been published to eradicate Pasung in Indonesia by improving the mental healthcare system. This article analyses this policy, specifically the National Mental Health Legislation (2014) and the Ministry of Health Regulation Tackling Forcible Restraint of People with Mental Illness (2017), and evaluates their current state of implementation through a local, in-depth case study. Using mental health institution mapping, two sets of semi-structured qualitative interviews with government officials and healthcare workers, and participant observation in a facility practicing Pasung, we identify the extent to which the 2017 regulation has been implemented in Winong village and discuss current efforts and persistent obstacles to eradicating Pasung. We suggest that despite reforms and the new treatment facility in our case study, the continuing use of Pasung is due to a combination of access to care issues and a widely held explanatory model of mental illness characterized by strong curative beliefs that, when disappointed, lead to a sense of threat and hopelessness.

A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers.

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akthigh/p-gH2AXlow, MCL1high/p-gH2AXlow and GRP78high/VIMhigh breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis.

Sternopygus macrurus electric organ transcriptome and cell size exhibit insensitivity to short-term electrical inactivity.

Electrical activity is an important regulator of cellular function and gene expression in electrically excitable cell types. In the weakly electric teleost fish Sternopygus macrurus, electrocytes, i.e., the current-producing cells of the electric organ, derive from a striated muscle lineage. Mature electrocytes are larger than muscle fibers, do not contain sarcomeres, and are driven continuously at frequencies higher than those exerted on muscle cells. Previous work showed that the removal of electrical activity by spinal cord transection (ST) for two and five weeks led to an upregulation of some sarcomeric proteins and a decrease in electrocyte size. To test whether changes in gene transcription preceded these phenotypic changes, we determined the sensitivity of electrocyte gene expression to electrical inactivity periods of two and five days after ST. Whole tissue gene expression profiles using deep RNA sequencing showed minimal alterations in the levels of myogenic transcription factor and sarcomeric transcripts after either ST period. Moreover, while analysis of differentially expressed genes showed a transient upregulation of genes associated with proteolytic mechanisms at two days and an increase in mRNA levels of cytoskeletal genes at five days after electrical silencing, electrocyte size was not affected. Electrical inactivity also resulted in the downregulation of genes that were classified into enriched clusters associated with functions of axon migration and synapse structure. Overall, these data demonstrate that unlike tissues in the myogenic lineage in other vertebrate species, regulation of gene transcription and cell size in the muscle-like electrocytes of S. macrurus is highly insensitive to short-term electrical inactivity. Moreover, together with data obtained from control and long-term ST studies, the present data suggest that neural input might influence post-transcriptional processes to affect the mature electrocyte phenotype.

Properties of skeletal muscle in the teleost Sternopygus macrurus are unaffected by short-term electrical inactivity.

Skeletal muscle is distinguished from other tissues on the basis of its shape, biochemistry, and physiological function. Based on mammalian studies, fiber size, fiber types, and gene expression profiles are regulated, in part, by the electrical activity exerted by the nervous system. To address whether similar adaptations to changes in electrical activity in skeletal muscle occur in teleosts, we studied these phenotypic properties of ventral muscle in the electric fish Sternopygus macrurus following 2 and 5 days of electrical inactivation by spinal transection. Our data show that morphological and biochemical properties of skeletal muscle remained largely unchanged after these treatments. Specifically, the distribution of type I and type II muscle fibers and the cross-sectional areas of these fiber types observed in control fish remained unaltered after each spinal transection survival period. This response to electrical inactivation was generally reflected at the transcript level in real-time PCR and RNA-seq data by showing little effect on the transcript levels of genes associated with muscle fiber type differentiation and plasticity, the sarcomere complex, and pathways implicated in the regulation of muscle fiber size. Data from this first study characterizing the acute influence of neural activity on muscle mass and sarcomere gene expression in a teleost are discussed in the context of comparative studies in mammalian model systems and vertebrate species from different lineages.

The myogenic electric organ of Sternopygus macrurus: a non-contractile tissue with a skeletal muscle transcriptome.

In most electric fish species, the electric organ (EO) derives from striated muscle cells that suppress many muscle properties. In the gymnotiform Sternopygus macrurus, mature electrocytes, the current-producing cells of the EO, do not contain sarcomeres, yet they continue to make some cytoskeletal and sarcomeric proteins and the muscle transcription factors (MTFs) that induce their expression. In order to more comprehensively examine the transcriptional regulation of genes associated with the formation and maintenance of the contractile sarcomere complex, results from expression analysis using qRT-PCR were informed by deep RNA sequencing of transcriptomes and miRNA compositions of muscle and EO tissues from adult S. macrurus. Our data show that: (1) components associated with the homeostasis of the sarcomere and sarcomere-sarcolemma linkage were transcribed in EO at levels similar to those in muscle; (2) MTF families associated with activation of the skeletal muscle program were not differentially expressed between these tissues; and (3) a set of microRNAs that are implicated in regulation of the muscle phenotype are enriched in EO. These data support the development of a unique and highly specialized non-contractile electrogenic cell that emerges from a striated phenotype and further differentiates with little modification in its transcript composition. This comprehensive analysis of parallel mRNA and miRNA profiles is not only a foundation for functional studies aimed at identifying mechanisms underlying the transcription-independent myogenic program in S. macrurus EO, but also has important implications to many vertebrate cell types that independently activate or suppress specific features of the skeletal muscle program.

Unique patterns of transcript and miRNA expression in the South American strong voltage electric eel (Electrophorus electricus).

BACKGROUND: With its unique ability to produce high-voltage electric discharges in excess of 600 volts, the South American strong voltage electric eel (Electrophorus electricus) has played an important role in the history of science. Remarkably little is understood about the molecular nature of its electric organs. RESULTS: We present an in-depth analysis of the genome of E. electricus, including the transcriptomes of eight mature tissues: brain, spinal cord, kidney, heart, skeletal muscle, Sachs' electric organ, main electric organ, and Hunter's electric organ. A gene set enrichment analysis based on gene ontology reveals enriched functions in all three electric organs related to transmembrane transport, androgen binding, and signaling. This study also represents the first analysis of miRNA in electric fish. It identified a number of miRNAs displaying electric organ-specific expression patterns, including one novel miRNA highly over-expressed in all three electric organs of E. electricus. All three electric organ tissues also express three conserved miRNAs that have been reported to inhibit muscle development in mammals, suggesting that miRNA-dependent regulation of gene expression might play an important role in specifying an electric organ identity from its muscle precursor. These miRNA data were supported using another complete miRNA profile from muscle and electric organ tissues of a second gymnotiform species. CONCLUSIONS: Our work on the E. electricus genome and eight tissue-specific gene expression profiles will greatly facilitate future research on determining the coding and regulatory sequences that specify the function, development, and evolution of electric organs. Moreover, these data and future studies will be informed by the first comprehensive analysis of miRNA expression in an electric fish presented here.

Nonhuman genetics. Genomic basis for the convergent evolution of electric organs.

Little is known about the genetic basis of convergent traits that originate repeatedly over broad taxonomic scales. The myogenic electric organ has evolved six times in fishes to produce electric fields used in communication, navigation, predation, or defense. We have examined the genomic basis of the convergent anatomical and physiological origins of these organs by assembling the genome of the electric eel (Electrophorus electricus) and sequencing electric organ and skeletal muscle transcriptomes from three lineages that have independently evolved electric organs. Our results indicate that, despite millions of years of evolution and large differences in the morphology of electric organ cells, independent lineages have leveraged similar transcription factors and developmental and cellular pathways in the evolution of electric organs.

Mechanisms of muscle gene regulation in the electric organ of Sternopygus macrurus.

Animals perform a remarkable diversity of movements through the coordinated mechanical contraction of skeletal muscle. This capacity for a wide range of movements is due to the presence of muscle cells with a very plastic phenotype that display many different biochemical, physiological and morphological properties. What factors influence the maintenance and plasticity of differentiated muscle fibers is a fundamental question in muscle biology. We have exploited the remarkable potential of skeletal muscle cells of the gymnotiform electric fish Sternopygus macrurus to trans-differentiate into electrocytes, the non-contractile electrogenic cells of the electric organ (EO), to investigate the mechanisms that regulate the skeletal muscle phenotype. In S. macrurus, mature electrocytes possess a phenotype that is intermediate between muscle and non-muscle cells. How some genes coding for muscle-specific proteins are downregulated while others are maintained, and novel genes are upregulated, is an intriguing problem in the control of skeletal muscle and EO phenotype. To date, the intracellular and extracellular factors that generate and maintain distinct patterns of gene expression in muscle and EO have not been defined. Expression studies in S. macrurus have started to shed light on the role that transcriptional and post-transcriptional events play in regulating specific muscle protein systems and the muscle phenotype of the EO. In addition, these findings also represent an important step toward identifying mechanisms that affect the maintenance and plasticity of the muscle cell phenotype for the evolution of highly specialized non-contractile tissues.

Conserved and differential gene interactions in dynamical biological systems.

MOTIVATION: While biological systems operated from a common genome can be conserved in various ways, they can also manifest highly diverse dynamics and functions. This is because the same set of genes can interact differentially across specific molecular contexts. For example, differential gene interactions give rise to various stages of morphogenesis during cerebellar development. However, after over a decade of efforts toward reverse engineering biological networks from high-throughput omic data, gene networks of most organisms remain sketchy. This hindrance has motivated us to develop comparative modeling to highlight conserved and differential gene interactions across experimental conditions, without reconstructing complete gene networks first. RESULTS: We established a comparative dynamical system modeling (CDSM) approach to identify conserved and differential interactions across molecular contexts. In CDSM, interactions are represented by ordinary differential equations and compared across conditions through statistical heterogeneity and homogeneity tests. CDSM demonstrated a consistent superiority over differential correlation and reconstruct-then-compare in simulation studies. We exploited CDSM to elucidate gene interactions important for cellular processes poorly understood during mouse cerebellar development. We generated hypotheses on 66 differential genetic interactions involved in expansion of the external granule layer. These interactions are implicated in cell cycle, differentiation, apoptosis and morphogenesis. Additional 1639 differential interactions among gene clusters were also identified when we compared gene interactions during the presence of Rhombic lip versus the presence of distinct internal granule layer. Moreover, compared with differential correlation and reconstruct-then-compare, CDSM makes fewer assumptions on data and thus is applicable to a wider range of biological assays. AVAILABILITY: Source code in C++ and R is available for non-commercial organizations upon request from the corresponding author. The cerebellum gene expression dataset used in this article is available upon request from the Goldowitz lab (dang@cmmt.ubc.ca, http://grits.dglab.org/). CONTACT: joemsong@cs.nmsu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

S. macrurus myogenic regulatory factors (MRFs) induce mammalian skeletal muscle differentiation; evidence for functional conservation of MRFs.

The current-producing cells of the electric organ, i.e., electrocytes, in Sternopygus macrurus derive from skeletal muscle fibers. Mature electrocytes are not contractile, but they do retain some muscle proteins, are multinucleated, and receive cholinergic innervation. Electrocytes express the myogenic regulatory factors (MRFs) MyoD, myogenin, Myf5 and MRF4 despite their incomplete muscle phenotype. Although S. macrurus MRFs share functional domains which are highly conserved and their expression is confined to the myogenic lineage, their capability to induce the muscle phenotype has not been determined. To test the functional conservation of S. macrurus MRFs to transcriptionally activate skeletal muscle gene expression and induce the myogenic program, we transiently over-expressed S. macrurus MyoD (SmMyoD) and myogenin (SmMyoG) in mouse C3H/10T1/2 and NIH3T3 embryonic cells. RT-PCR and immunolabeling studies showed that SmMyoD and SmMyoG can efficiently convert these two cell lines into multinucleated myotubes which expressed differentiated muscle markers. The levels of myogenic induction by SmMyoD and SmMyoG were comparable to those obtained with mouse MRF homologs. Furthermore, SmMyoD and SmMyoG proteins were able to induce mouse MyoD and myogenin in C3H/10T1/2 cells. We conclude that S. macrurus MRFs are functionally conserved as they can transcriptionally activate skeletal muscle gene expression and induce the myogenic program in mammalian non-muscle cells. Hence, these data suggest that the partial muscle phenotype of electrocytes is not likely due to differences in the MRF-dependent transcriptional program between skeletal muscle and electric organ.

Constituents with independence from growth temperature for bacteria using pyrolysis-gas chromatography/differential mobility spectrometry with analysis of variance and principal component analysis.

Four bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus warneri, and Micrococcus luteus, were grown at temperatures of 23, 30, and 37 degrees C and were characterized by pyrolysis-gas chromatography/differential mobility spectrometry (Py-GC/DMS) providing, with replicates, 120 data sets of retention time, compensation voltage, and ion intensity, each for negative and positive polarity. Principal component analysis (PCA) for 96 of these data sets exhibited clusters by temperature of culture growth and not by genus. Analysis of variance was used to isolate the constituents with dependences on growth temperature. When these were subtracted from the data sets, Fisher ratios with PCA resulted in four clusters according to genus at all temperatures for ions in each polarity. Comparable results were obtained from unsupervised PCA with 24 of the original data sets. The ions with taxonomic features were reconstructed into 3D plots of retention time, compensation voltage, and Fisher ratio and were matched, through GC-mass spectrometry (MS), with chemical standards attributed to the thermal decomposition of proteins and lipid A. Results for negative ions provided simpler data sets than from positive ions, as anticipated from selectivity of gas phase ion-molecule reactions in air at ambient pressure.

Analysis of bacteria by pyrolysis gas chromatography-differential mobility spectrometry and isolation of chemical components with a dependence on growth temperature.

Pyrolysis gas chromatography-differential mobility spectrometry (py-GC-DMS) analysis of E. coli, P. aeruginosa, S. warneri and M. luteus, grown at temperatures of 23, 30, and 37 degrees C, provided data sets of ion intensity, retention time, and compensation voltage for principal component analysis. Misaligned chromatographic axes were treated using piecewise alignment, the impact on the degree of class separation (DCS) of clusters was minor. The DCS, however, was improved between 21 to 527% by analysis of variance with Fisher ratios to remove chemical components independent of growth temperature. The temperature dependent components comprised 84% of all peaks in the py-GC-DMS analysis of E. coli and were attributed to the pyrolytic decomposition of proteins rather than lipids, as anticipated. Components were also isolated in other bacteria at differing amounts: 41% for M. luteus, 14% for P. aeruginosa, and 4% for S. warneri, and differing patterns suggested characteristic dependence on temperature of growth for these bacteria. These components are anticipated to have masses from 100 to 200 Da by inference from differential mobility spectra.

Analysis of bacterial strains with pyrolysis-gas chromatography/differential mobility spectrometry.

Eight vegetative bacterial strains and two spores were characterized by pyrolysis-gas chromatography with differential mobility spectrometry (py-GC/DMS) yielding topographic plots of ion intensity, retention time, and compensation voltage simultaneously for ions in positive and negative polarity. Biomarkers were found in the pyrolysate at characteristic retention times and compensation voltages and were confirmed by standard addition with GC/MS analyses providing discrimination between Gram negative and Gram positive bacterial types, but no recognition of individual strains within the Gram negative bacteria. Principal component analysis was applied using two dimensional data sets of ion intensity versus retention time at five compensation voltages including the reactant ion peaks all in positive and negative ion polarity. Clustering was observed with compensation voltage (CV) chromatograms associated with ion separation in the DMS detector and little or no clustering was observed with the reactant ion peaks or CV chromatograms where ion separation is poor. Consistent clustering of Gram positive B. odysseyi and Gram negative E. coli in both positive and negative polarities with the reactant ion peak chromatograms and key CV chromatograms suggests common but unknown common chemical compositions in the pyrolysate.