Heterozygous Knockout of ARID4B Using CRISPR/Cas9 Attenuates Some Aggressive Phenotypes in a Breast Cancer Cell Line.

Breast cancer is one of the leading causes of death in women around the world. Over time, many genes and mutations that are associated with the development of this disease have been identified. However, the specific role of many genes has not yet been fully elucidated. Higher ARID4B expression has been identified as a risk factor for diverse cancer types. Silencing experiments also showed that ARID4B is associated with developing cancer-associated characteristics. However, no transcriptomic studies have shown the overall cellular effect of loss of function in breast cancer in humans. This study addresses the impact of loss-of-function mutations in breast cancer MCF-7 cells. Using the CRISPR/Cas9 system, we generated mutations that caused heterozygous truncated proteins, isolating three monoclonal lines carrying insertions and deletions in ARID4B. We observed reduced proliferation and migration in in vitro experiments. In addition, from RNA-seq assays, a differential expression analysis shows known and novel deregulated cancer-associate pathways in mutated cells supporting the impact of ARID4B. For example, we found the AKT-PI3K pathway to be altered at the transcript level but through different genes than those reported for ARID4B. Our transcriptomic results also suggest new insights into the role of ARID4B in aggressiveness by the epithelial-to-mesenchymal transition and TGF-ß pathways and in metabolism through cholesterol and mevalonate pathways. We also performed exome sequencing to show that no off-target effects were apparent. In conclusion, the ARID4B gene is associated with some aggressive phenotypes in breast cancer cells.

The retinoic acid response is a minor component of the cardiac phenotype in H9c2 myoblast differentiation.

The H9c2 myoblast cell line, isolated from the left ventricular tissue of rat, is currently used in vitro as a mimetic for skeletal and cardiac muscle due to its biochemical, morphological, and electrical/hormonal signaling properties. During culture, H9c2 cells acquire a myotube phenotype, where a critical component is the inclusion of retinoic acid (RA). The results from some authors on H9c2 suggested that thousands of genes respond to RA stimuli, while others report hundreds of genes responding to RA over different cell types. In this article, using a more appropriate experimental design, we first confirm the H9c2 cardiac phenotype with and without RA and report transcriptomic and physiological changes regarding calcium handling, bioenergetics, and other biological concepts. Interestingly, of the 2360 genes showing a transcriptional change, 622 genes were statistically associated with the RA response. Of these genes, only 305 were RA-specific, and the rest also showed a culture-time component. Thus, the major expression changes (from 74 to 87%) were indeed due to culture conditions over time. Unexpectedly, only a few components of the retinol pathway in KEGG responded to RA. Our results show the role of RA in the H9c2 cultures impacting the interpretation using H9c2 as an in vitro model.

Dynamic landscape of chromatin accessibility and transcriptomic changes during differentiation of human embryonic stem cells into dopaminergic neurons.

Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. Studies on dopaminergic differentiation have identified coding genes, but the relationship with non-coding genes or chromatin accessibility remains elusive. Using RNA-Seq and ATAC-Seq we profiled differentially expressed transcripts and open chromatin regions during early dopaminergic neuron differentiation. Hierarchical clustering of differentially expressed genes, resulted in 6 groups with unique characteristics. Surprisingly, the abundance of long non-coding RNAs (lncRNAs) was high in the most downregulated transcripts, and depicted positive correlations with target mRNAs. We observed that open chromatin regions decrease upon differentiation. Enrichment analyses of accessibility depict an association between open chromatin regions and specific functional pathways and gene-sets. A bioinformatic search for motifs allowed us to identify transcription factors and structural nuclear proteins that potentially regulate dopaminergic differentiation. Interestingly, we also found changes in protein and mRNA abundance of the CCCTC-binding factor, CTCF, which participates in genome organization and gene expression. Furthermore, assays demonstrated co-localization of CTCF with Polycomb-repressed chromatin marked by H3K27me3 in pluripotent cells, progressively decreasing in neural precursor cells and differentiated neurons. Our work provides a unique resource of transcription factors and regulatory elements, potentially involved in the acquisition of human dopaminergic neuron cell identity.

Transcriptomic and cellular analyses of CRISPR/Cas9-mediated edition of FASN show inhibition of aggressive characteristics in breast cancer cells.

Several genes are significantly mutated in breast cancer but only a small percentage of mutations are well-known to contribute to cancer development. FASN is involved in de novo lipogenesis and the regulation of ERα signaling. However, the effect of genetic mutations affecting FASN in breast cancer has not thoroughly studied. Therefore, we used the CRISPR/Cas9 system to edit the FASN locus in MCF-7 cells and evaluated its biological effect. We obtained four clones carrying mutations and frameshifts in the acyl-transferase domain of FASN. We found that clones had reduced proliferation, migration, viability, and showed alterations in cell cycle profiles. RNA-Seq analysis demonstrates that a lack of fully functional FASN may have a more significant role in proliferation-related genes than in lipid metabolism. We conclude that functional knockouts in FASN contributes to decrease the proliferation and migration of breast cancer cells contrary to point mutations in breast cancer patients.

Anaplastic Large Cell Lymphoma in Central America: A Report From the Central American Association of Pediatric Hematology Oncology (AHOPCA).

BACKGROUND: Although anaplastic large cell lymphoma (ALCL) is curable in high-income countries (HIC), data from low- and middle-income countries (LMIC) are lacking. We therefore conducted a retrospective study of the Central American Association of Pediatric Hematology Oncology (AHOPCA) experience in treating ALCL. PROCEDURE: We included all patients age <18 years newly diagnosed with ALCL treated between 2000 and 2013 in seven AHOPCA institutions. Retrospective data were extracted from the Pediatric Oncology Network Database. RESULTS: Thirty-one patients met inclusion criteria. Twenty-five (81%) had advanced disease (stages III and IV), six (19%) were treated on the APO (doxorubicin, prednisone, vincristine) regimen, 15 (49%) on multi-agent chemotherapy designed for T-cell lineage malignancies (GuatALCL protocol), and 10 (32%) on BFM-based treatment regimens. Five-year overall event-free survival and overall survival were, respectively, 67.1 ± 8.6% and 66.7 ± 8.7%. All 10 events occurred in patients treated on BFM-based treatment regimens or the GuatALCL protocol, none on APO treatment: two patients experienced relapse, six treatment related mortality (TRM), and two abandonment. CONCLUSIONS: Treatment of ALCL in countries with limited resources is feasible with similar outcomes as in HIC, though the causes of treatment failure differ. Less intensive regimens may be preferable in order to decrease TRM and improve outcomes. Prospective clinical trials determining the ideal treatment for LMIC children with ALCL are necessary.

Linear fibroblast alignment on sinusoidal wave micropatterns.

Micrometer and nanometer grooved surfaces have been determined to influence cellular orientation, morphology, and migration through contact guidance. Cells typically elongate along the direction of an underlying groove and often migrate with guidance provided by constraints of the pattern. This phenomenon has been studied primarily using linear grooves, post, or well patterns. We investigated the behavior of mouse embryonic fibroblasts on non-linear, sinusoidal wave grooves created via electron beam lithography on a polymethyl methacrylate (PMMA) substrate that was spin-coated onto a positively charged glass surface. Three different wave patterns, with varying wavelengths and amplitudes, and two different line patterns were created. Cell orientation and adhesion was examined after 4, 24, and 48 h after cell seeding. Attachment strength was studied via subjecting cells on substrates to centrifugal force following a 24-h incubation period. For all wave patterns studied, it was noted that cells did not reside within the groove, rather they were observed to cross over each groove, residing both inside and outside of each wave pattern, aligning linearly along the long axis of the pattern. For the linear patterns, we observed that cells tended to reside within the grooves, consistent with previous observations. The ability to add texture to a surface to manipulate cell adhesion strength and growth with only localized attachment, maintaining free space in curvilinear microtopography underlying the cell, may be a useful addition for tissue engineering and the fabrication of novel biomedical devices.

Nanowell-trapped charged ligand-bearing nanoparticle surfaces: a novel method of enhancing flow-resistant cell adhesion.

Assuring cell adhesion to an underlying biomaterial surface is vital in implant device design and tissue engineering, particularly under circumstances where cells are subjected to potential detachment from overriding fluid flow. Cell-substrate adhesion is a highly regulated process involving the interplay of mechanical properties, surface topographic features, electrostatic charge, and biochemical mechanisms. At the nanoscale level, the physical properties of the underlying substrate are of particular importance in cell adhesion. Conventionally, natural, pro-adhesive, and often thrombogenic, protein biomaterials are frequently utilized to facilitate adhesion. In the present study, nanofabrication techniques are utilized to enhance the biological functionality of a synthetic polymer surface, polymethymethacrylate, with respect to cell adhesion. Specifically we examine the effect on cell adhesion of combining: 1. optimized surface texturing, 2. electrostatic charge and 3. cell adhesive ligands, uniquely assembled on the substrata surface, as an ensemble of nanoparticles trapped in nanowells. Our results reveal that the ensemble strategy leads to enhanced, more than simply additive, endothelial cell adhesion under both static and flow conditions. This strategy may be of particular utility for enhancing flow-resistant endothelialization of blood-contacting surfaces of cardiovascular devices subjected to flow-mediated shear.

Microfluidic immunosensor with integrated liquid core waveguides for sensitive Mie scattering detection of avian influenza antigens in a real biological matrix.

This work presents the use of integrated, liquid core, optical waveguides for measuring immunoagglutination-induced light scattering in a microfluidic device, towards rapid and sensitive detection of avian influenza (AI) viral antigens in a real biological matrix (chicken feces). Mie scattering simulations were performed and tested to optimize the scattering efficiency of the device through proper scatter angle waveguide geometry. The detection limit is demonstrated to be 1 pg mL(-1) in both clean buffer and real biological matrix. This low detection limit is made possible through on-chip diffusional mixing of AI target antigens and high acid content microparticle assay reagents, coupled with real-time monitoring of immunoagglutination-induced forward Mie scattering via high refractive index liquid core optical waveguides in close proximity (100 µm) to the sample chamber. The detection time for the assay is <2 min. This device could easily be modified to detect trace levels of any biological molecules that antibodies are available for, moving towards a robust platform for point-of-care disease diagnostics.

FRET detection of Octamer-4 on a protein nanoarray made by size-dependent self-assembly.

An alternative approach for fabricating a protein array at nanoscale is suggested with a capability of characterization and/or localization of multiple components on a nanoarray. Fluorescent micro- and nanobeads each conjugated with different antibodies are assembled by size-dependent self-assembly (SDSA) onto nanometer wells that were created on a polymethyl methacrylate (PMMA) substrate by electron beam lithography (EBL). Antibody-conjugated beads of different diameters are added serially and electrostatically attached to corresponding wells through electrostatic attraction between the charged beads (confirmed by zeta potential analysis) and exposed p-doped silicon substrate underneath the PMMA layer. This SDSA method is enhanced by vibrated-wire-guide manipulation of droplets on the PMMA surface containing nanometer wells. Saturation rates of antibody-conjugated beads to the nanometer patterns are up to 97% under one component and 58-70% under two components nanoarrays. High-density arrays (up to 40,000 wells) could be fabricated, which can also be multi-component. Target detection utilizes fluorescence resonance energy transfer (FRET) from fluorescent beads to fluorescent-tagged secondary antibodies to Octamer-4 (Oct4), which eliminates the need for multiple steps of rinsing. The 100 nm green beads are covalently conjugated with anti-Oct4 to capture Oct4 peptides (39 kDa); where the secondary anti-Oct4 and F(ab)(2) fragment of anti-gIgG tagged with phycoerythrin are then added to function as an indicator of Oct4 detection. FRET signals are detected through confocal microscopes, and further confirmed by Fluorolog3 spectrofluorometer. The success rates of detecting Oct4 are 32% and 14% of the beads in right place under one and two component nanoarrays, respectively. Ratiometric FRET is used to quantify the amount of Oct4 peptides per each bead, which is estimated about 2 molecules per bead.