Intragenic suppressors unravel the role of the SCREAM ACT-like domain for bHLH partner selectivity in stomatal development.

Multicellular organisms develop specialized cell types to achieve complex functions of tissues and organs. The basic helix-loop-helix (bHLH) proteins act as master regulatory transcription factors of such specialized cell types. Plant stomata are cellular valves in the aerial epidermis for efficient gas exchange and water control. Stomatal differentiation is governed by sequential actions of three lineage-specific bHLH proteins, SPEECHLESS (SPCH), MUTE, and FAMA, specifying initiation and proliferation, commitment, and terminal differentiation, respectively. A broadly expressed bHLH, SCREAM (SCRM), heterodimerizes with SPCH/MUTE/FAMA and drives stomatal differentiation via switching its partners. Yet nothing is known about its heterodimerization properties or partner preference. Here, we report the role of the SCRM C-terminal ACT-like (ACTL) domain for heterodimerization selectivity. Our intragenic suppressor screen of a dominant scrm-D mutant identified the ACTL domain as a mutation hotspot. Removal of this domain or loss of its structural integrity abolishes heterodimerization with MUTE, but not with SPCH or FAMA, and selectively abrogates the MUTE direct target gene expression. Consequently, the scrm-D ACTL mutants confer massive clusters of arrested stomatal precursor cells that cannot commit to differentiation when redundancy is removed. Structural and biophysical studies further show that SPCH, MUTE, and FAMA also possess the C-terminal ACTL domain, and that ACTL•ACTL heterodimerization is sufficient for partner selectivity. Our work elucidates a role for the SCRM ACTL domain in the MUTE-governed proliferation-differentiation switch and suggests mechanistic insight into the biological function of the ACTL domain, a module uniquely associated with plant bHLH proteins, as a heterodimeric partner selectivity interface.

Loss of splicing factor IK impairs normal skeletal muscle development.

BACKGROUND: IK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo. RESULTS: The ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells. CONCLUSION: This study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

Negative regulation of NOD1 mediated angiogenesis by PPARγ-regulated miR-125a.

Infection with pathogens activates the endothelial cell and its sustained activation may result in impaired endothelial function. Endothelial dysfunction contributes to the pathologic angiogenesis that is characteristic of infection-induced inflammatory pathway activation. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is a protein receptor which recognizes bacterial molecules and stimulates an immune reaction in various cells; however, the underlying molecular mechanisms in the regulation of inflammation-triggered angiogenesis are not fully understood. Here we report that peroxisome proliferator-activated receptor gamma (PPARγ)-mediated miR-125a serves as an important regulator of NOD1 agonist-mediated angiogenesis in endothelial cells by directly targeting NOD1. Treatment of human umbilical vein endothelial cells with natural PPARγ ligand, 15-Deoxy-Delta12,14-prostaglandin J2, led to inhibition of NOD1 expression; contrarily, protein levels of NOD1 were significantly increased by PPARγ knockdown. We report that PPARγ regulation of NOD1 expression is a novel microRNA-mediated regulation in endothelial cells. MiR-125a expression was markedly decreased in human umbilical vein endothelial cells subjected to PPARγ knockdown while 15-Deoxy-Delta12,14-prostaglandin J2 treatment increased the level of miR-125a. In addition, NOD1 is closely regulated by miR-125a, which directly targets the 3' untranslated region of NOD1. Moreover, both overexpression of miR-125a and PPARγ activation led to inhibition of NOD1 agonist-induced tube formation in endothelial cells. Finally, NOD1 agonist increased the formation of cranial and subintestinal vessel plexus in zebrafish, and this effect was abrogated by concurrent PPARγ activation. Overall, these findings identify a PPARγ-miR-125a-NOD1 signaling axis in endothelial cells that is critical in the regulation of inflammation-mediated angiogenesis.

Bone subtraction 3D CT venography for the evaluation of cerebral veins and venous sinuses: imaging techniques, normal variations, and pathologic findings.

OBJECTIVE: Varying anatomic characteristics and clinical and radiologic manifestations are diagnostic challenges in the evaluation of the cerebral vein and of venous sinus diseases. The purpose of this article is to introduce bone subtraction CT venography and review normal variations and diseases involving the cerebral veins and venous sinuses. CONCLUSION: Knowledge of the normal variations and pathologic findings will be helpful for the accurate diagnosis of diseases involving the cerebral venous system. Bone subtraction CT venography offers complete 3D visualization of the cerebral venous system and can be useful for the evaluation of the cerebral vein and venous sinus diseases.

Rational design of pH-responsive near-infrared spirocyclic cyanines: the effects of substituents and the external environment.

pH-responsive spirocyclic cyanine dyes were designed and synthesized. The equilibrium constant for cyclization (pKcycl) could be rationally controlled by changing the nucleophilic moiety and the side chains. Encapsulation in polymeric micelles inhibited the H-aggregation of the dye, and the pKcycl could be shifted according to the amphiphilic polymer employed.

Complete chloroplast genome sequence of the liverwort, Porella grandiloba Lindb. (Porellaceae).

Porella grandiloba Lindb. is a liverwort species of Porellaceae, primarily distributed in East Asia. Here, we determined the complete chloroplast (cp) genome sequence of P. grandiloba. The complete cp genome was 121,433 bp in length with a typical quadripartite structure consisting of a large single-copy region (83,039 bp), a small single-copy region (19,586 bp), and two copies of inverted repeat regions (9,404 bp, each). Genome annotation predicted 131 genes, including 84 protein-coding, 36 tRNA, and eight rRNA genes. The maximum likelihood tree indicated that P. grandiloba was sister to P. perrottetiana, which species formed a clade with Radula japonica (Radulaceae).

Global analysis of cell behavior and protein dynamics reveals region-specific roles for Shroom3 and N-cadherin during neural tube closure.

Failures of neural tube closure are common and serious birth defects, yet we have a poor understanding of the interaction of genetics and cell biology during neural tube closure. Additionally, mutations that cause neural tube defects (NTDs) tend to affect anterior or posterior regions of the neural tube but rarely both, indicating a regional specificity to NTD genetics. To better understand the regional specificity of cell behaviors during neural tube closure, we analyzed the dynamic localization of actin and N-cadherin via high-resolution tissue-level time-lapse microscopy during Xenopus neural tube closure. To investigate the regionality of gene function, we generated mosaic mutations in shroom3, a key regulator or neural tube closure. This new analytical approach elucidates several differences between cell behaviors during cranial/anterior and spinal/posterior neural tube closure, provides mechanistic insight into the function of shroom3, and demonstrates the ability of tissue-level imaging and analysis to generate cell biological mechanistic insights into neural tube closure.

Dynamic chromatin accessibility deploys heterotypic cis/trans-acting factors driving stomatal cell-fate commitment.

Chromatin architecture and transcription factor (TF) binding underpin cell-fate specification during development, but their mutual regulatory relationships remain unclear. Here we report an atlas of dynamic chromatin landscapes during stomatal cell-lineage progression, in which sequential cell-state transitions are governed by lineage-specific bHLH TFs. Major reprogramming of chromatin accessibility occurs at the proliferation-to-differentiation transition. We discover novel co-cis regulatory elements (CREs) signifying the early precursor stage, BBR/BPC (GAGA) and bHLH (E-box) motifs, where master-regulatory bHLH TFs, SPEECHLESS and MUTE, consecutively bind to initiate and terminate the proliferative state, respectively. BPC TFs complex with MUTE to repress SPEECHLESS expression through a local deposition of repressive histone marks. We elucidate the mechanism by which cell-state-specific heterotypic TF complexes facilitate cell-fate commitment by recruiting chromatin modifiers via key co-CREs.

Dutomycin Induces Autophagy and Apoptosis by Targeting the Serine Protease Inhibitor SERPINB6.

Autophagy plays an important role in maintaining tumor cell progression and survival in response to metabolic stress. Thus, the regulation of autophagy can be used as a strategy for anticancer therapy. Here, we report dutomycin (DTM) as a novel autophagy enhancer that eventually induces apoptosis due to excessive autophagy. Also, human serine protease inhibitor B6 (SERPINB6) was identified as a target protein of DTM, and its novel function which is involved in autophagy was studied for the first time. We show that DTM directly binds SERPINB6 and then activates intracellular serine proteases, resulting in autophagy induction. Inhibitory effects of DTM on the function of SERPINB6 were confirmed through enzyme- and cell-based approaches, and SERPINB6 was validated as a target protein using siRNA-mediated knockdown and an overexpression test. In a zebrafish xenograft model, DTM showed a significant decrease in tumor area. Furthermore, the present findings will be expected to contribute to the expansion of novel basic knowledge about the correlation of cancer and autophagy by promoting active further research on SERPINB6, which was not previously considered the subject of cancer biology.

Cancer cell­specific anticancer effects of Coptis chinensis on gefitinib­resistant lung cancer cells are mediated through the suppression of Mcl­1 and Bcl­2.

The epidermal growth factor receptor (EGFR)­tyrosine kinase inhibitor (TKI), gefitinib, is an effective therapeutic drug used in the treatment of non­small cell lung cancers (NSCLCs) harboring EGFR mutations. However, acquired resistance significantly limits the efficacy of EGFR­TKIs and consequently, the current chemotherapeutic strategies for NSCLCs. It is, therefore, necessary to overcome this resistance. In the present study, the anticancer potential of natural extracts of Coptis chinensis (ECC) against gefitinib­resistant (GR) NSCLC cells were investigated in vitro and in vivo. ECC inhibited the viability, migration and invasion, and effectively induced the apoptosis of GR cells. These effects were associated with the suppression of EGFR/AKT signaling and the expression of anti­apoptotic proteins, Mcl­1 and Bcl­2, which were overexpressed in GR NSCLC cells. Combination treatment with ECC and gefitinib enhanced the sensitivity of GR cells to gefitinib in vitro, but not in vivo. However, ECC increased the survival of individual zebrafish without affecting the anticancer effect to cancer cells in vivo, which indicated a specific cytotoxic effect of ECC on cancer cells, but not on normal cells; this is an important property for the development of novel anticancer drugs. On the whole, the findings of the present study indicate the potential of ECC for use in the treatment of NSCLC, particularly in combination with EGFR­TKI therapy, in EGFR­TKI­resistant cancers.

Cancer-derived exosomes trigger endothelial to mesenchymal transition followed by the induction of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) play a pivotal role in tumor growth, but very little has been known about its characteristics and origin. Recently, cancer-derived exosome has been suggested to transdifferentiate CAFs, by a new mechanism of endothelial to mesenchymal transition (EndMT), initiating angiogenic processes and triggering metastatic evolution. However, an enabling tool in vitro is yet to be developed to investigate complicated procedures of the EndMT and the transdifferentiation under reconstituted tumor microenvironment. Here we proposed an in vitro microfluidic model which enables to monitor a synergetic effect of complex tumor microenvironment in situ, including extracellular matrix (ECM), interstitial flow and environmental exosomes. The number of CAFs differentiated from human umbilical vein endothelial cells (HUVECs) increased with melanoma-derived exosomes, presenting apparent morphological and molecular changes with pronounced motility. Mesenchymal stem cell (MSC)-derived exosomes were found to suppress EndMT, induce angiogenesis and maintain vascular homeostasis, while cancer-derived exosomes promoted EndMT. Capabilities of the new microfluidic model exist in precise regulation of the complex tumor microenvironment and therefore successful reconstitution of 3D microvasculature niches, enabling in situ investigation of EndMT procedure between various cell types. STATEMENT OF SIGNIFICANCE: This study presents an in vitro 3D EndMT model to understand the progress of the CAF generation by recapitulating the 3D tumor microenvironment in a microfluidic device. Both cancer-derived exosomes and interstitial fluid flow synergetically played a pivotal role in the EndMT and consequent formation of CAFs through a collagen-based ECM. Our approach also enabled the demonstration of a homeostatic capability of MSC-derived exosomes, ultimately leading to the recovery of CAFs back to endothelial cells. The in vitro 3D EndMT model can serve as a powerful tool to validate exosomal components that could be further developed to anti-cancer drugs.