PCBP1 regulates LIFR through FAM3C to maintain breast cancer stem cell self-renewal and invasiveness.

The poly(rC) binding protein 1 gene (PCBP1) encodes the heterogeneous nuclear ribonucleoprotein E1 (hnRNPE1), a nucleic acid-binding protein that plays a tumor-suppressive role in the mammary epithelium by regulating phenotypic plasticity and cell fate. Following the loss of PCBP1 function, the FAM3C gene (encoding the Interleukin-like EMT inducer, or "ILEI" protein) and the leukemia inhibitory factor receptor (LIFR) gene are upregulated. Interaction between FAM3C and LIFR in the extracellular space induces phosphorylation of signal transducer and activator of transcription 3 (pSTAT3). Overexpression and/or hyperactivity of STAT3 has been detected in 40% of breast cancer cases and is associated with a poor prognosis. Herein, we characterize feed-forward regulation of LIFR expression in response to FAM3C/LIFR/STAT3 signaling in mammary epithelial cells. We show that PCBP1 upregulates LIFR transcription through activity at the LIFR promoter, and that FAM3C participates in transcriptional regulation of LIFR. Additionally, our bioinformatic analysis reveals a signature of transcriptional regulation associated with FAM3C/LIFR interaction and identifies the TWIST1 transcription factor as a downstream effector that participates in the maintenance of LIFR expression. Finally, we characterize the effect of LIFR expression in cell-based experiments that demonstrate the promotion of invasion, migration, and self-renewal of breast cancer stem cells (BCSCs), consistent with previous studies linking LIFR expression to tumor initiation and metastasis in mammary epithelial cells.

Uncovering the molecular mechanisms of Fructus Choerospondiatis against coronary heart disease using network pharmacology analysis and experimental pharmacology.

Fructus Choerospondiatis (FC), a Mongolian medicine, was mainly used in Mongolian medical theory for the treatment of coronary heart disease (CHD). Nonetheless, the main components and mechanisms of action of FC in the treatment of coronary artery disease have not been studied clearly. AIM OF THE STUDY: The aim of this study is to identify the components of FC and analyze the pathways affected by the targets of these components to probe into the potential mechanisms of action of FC on coronary heart disease. MATERIALS AND METHODS: Identification of compounds in FC employing high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS) method, then further investigate the network pharmacology and molecular docking to obtain potential targets and elucidate the potential mechanism of action of FC in the therapy of CHD. Experimental validation was established to verify the mechanism of FC in vitro. RESULTS: 21 FC components were identified and 65 overlapping targets were gained. In addition, these ingredients regulated AMPK and PPAR signaling pathway by 65 target genes including IL6, AKT1 and PPARg, etc. Molecular docking displayed that the binding ability of the key target PPARg to FC components turned out to be better. Experimental validation proved that FC treatment decreased the expression of PPARg (p < 0.05) compare with model group, which may be involved in the PPAR signaling pathway. CONCLUSIONS: This study was the first to elucidate the mechanism of action of components of FC for the treatment of CHD using network pharmacology. It alleviated CHD by inhibiting the expression of PPARg to attenuate hypoxia/reoxygenation injury, and the results give a basis for elucidating the molecular mechanism of action of FC for the treatment of coronary heart disease.

MicroRNA-593-5p contributes to cell death following exposure to 1-methyl-4-phenylpyridinium by targeting PTEN-induced putative kinase 1.

Neurodegenerative diseases are characterized by a decline in neuronal function and structure, leading to neuronal death. Understanding the molecular mechanisms of neuronal death is crucial for developing therapeutics. MiRs are small noncoding RNAs that regulate gene expression by degrading target mRNAs or inhibiting translation. MiR dysregulation has been linked to many neurodegenerative diseases, but the underlying mechanisms are not well understood. As mitochondrial dysfunction is one of the common molecular mechanisms leading to neuronal death in many neurodegenerative diseases, here we studied miRs that modulate neuronal death caused by 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of complex I in mitochondria. We identified miR-593-5p, levels of which were increased in SH-SY5Y human neuronal cells, after exposure to MPP+. We found that intracellular Ca2+, but not of reactive oxygen species, mediated this miR-593-5p increase. Furthermore, we found the increase in miR-593-5p was due to enhanced stability, not increased transcription or miR processing. Importantly, we show the increase in miR-593-5p contributed to MPP+-induced cell death. Our data revealed that miR-593-5p inhibits a signaling pathway involving PTEN-induced putative kinase 1 (PINK1) and Parkin, two proteins responsible for the removal of damaged mitochondria from cells, by targeting the coding sequence of PINK1 mRNA. Our findings suggest that miR-593-5p contributes to neuronal death resulting from MPP+ toxicity, in part, by impeding the PINK1/Parkin-mediated pathway that facilitates the clearance of damaged mitochondria. Taken together, our observations highlight the potential significance of inhibiting miR-593-5p as a therapeutic approach for neurodegenerative diseases.

TrypTag.org: from images to discoveries using genome-wide protein localisation in Trypanosoma brucei.

TrypTag was a 4-year project to tag the N- and C-termini of almost all Trypanosoma brucei proteins with a fluorescent protein and record the subcellular localisation through images and manual annotation. We highlight the new routes to cell biological discovery this transformative resource is enabling for parasitologists and cell biologists.

Fluorescent toys 'n' tools lighting the way in fungal research.

Although largely overlooked compared to bacterial infections, fungal infections pose a significant threat to the health of humans and other organisms. Many pathogenic fungi, especially Candida species, are extremely versatile and flexible in adapting to various host niches and stressful situations. This leads to high pathogenicity and increasing resistance to existing drugs. Due to the high level of conservation between fungi and mammalian cells, it is hard to find fungus-specific drug targets for novel therapy development. In this respect, it is vital to understand how these fungi function on a molecular, cellular as well as organismal level. Fluorescence imaging allows for detailed analysis of molecular mechanisms, cellular structures and interactions on different levels. In this manuscript, we provide researchers with an elaborate and contemporary overview of fluorescence techniques that can be used to study fungal pathogens. We focus on the available fluorescent labelling techniques and guide our readers through the different relevant applications of fluorescent imaging, from subcellular events to multispecies interactions and diagnostics. As well as cautioning researchers for potential challenges and obstacles, we offer hands-on tips and tricks for efficient experimentation and share our expert-view on future developments and possible improvements.

Unlike Its Paralog LEDGF/p75, HRP-2 Is Dispensable for MLL-R Leukemogenesis but Important for Leukemic Cell Survival.

HDGF-related protein 2 (HRP-2) is a member of the Hepatoma-Derived Growth Factor-related protein family that harbors the structured PWWP and Integrase Binding Domain, known to associate with methylated histone tails or cellular and viral proteins, respectively. Interestingly, HRP-2 is a paralog of Lens Epithelium Derived Growth Factor p75 (LEDGF/p75), which is essential for MLL-rearranged (MLL-r) leukemia but dispensable for hematopoiesis. Sequel to these findings, we investigated the role of HRP-2 in hematopoiesis and MLL-r leukemia. Protein interactions were investigated by co-immunoprecipitation and validated using recombinant proteins in NMR. A systemic knockout mouse model was used to study normal hematopoiesis and MLL-ENL transformation upon the different HRP-2 genotypes. The role of HRP-2 in MLL-r and other leukemic, human cell lines was evaluated by lentiviral-mediated miRNA targeting HRP-2. We demonstrate that MLL and HRP-2 interact through a conserved interface, although this interaction proved less dependent on menin than the MLL-LEDGF/p75 interaction. The systemic HRP-2 knockout mice only revealed an increase in neutrophils in the peripheral blood, whereas the depletion of HRP-2 in leukemic cell lines and transformed primary murine cells resulted in reduced colony formation independently of MLL-rearrangements. In contrast, primary murine HRP-2 knockout cells were efficiently transformed by the MLL-ENL fusion, indicating that HRP-2, unlike LEDGF/p75, is dispensable for the transformation of MLL-ENL leukemogenesis but important for leukemic cell survival.

Understanding new molecular and cell biology findings based on progressive scientific practices and interconnected activities in undergraduate students.

Nowadays Molecular Cell Biology (MCB) must be taught as science is practiced. Even though there are several approaches based on scientific practices, a key aspect is to define the purpose of each of these teaching strategies and, most importantly, their implementation. Our goal was to train students to acquire, understand, and communicate new scientific knowledge in the field. The main feature of our new teaching methodology was progressive training in scientific practices associated with a back-and-forward interplay between activities and assessments. The methodology was implemented over 4 years, in students attending the MCB course of the undergraduate degree in Biological Sciences. In the first two modules, the students were prepared to comprehend MCB concepts and techniques and to experience activities based on scientific practices. In the third module, the students analyzed a primary paper in-depth. They were assessed by midterm exams based on a primary paper, written laboratory reports, and the oral presentation of a scientific paper. Our teaching proposal was evaluated through the students' academic performance and by their opinion on the teaching methodology. Most students were satisfied since they improved their acquisition of concepts, their interpretation and integration of scientific knowledge, and developed skills to communicate scientific knowledge in writing and orally. The novelty of transversal interconnections and progressive training in scientific practices provides students with skills in acquiring and understanding new scientific information, even beyond the MCB course.

Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways.

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

Tailored Teaching for Specialized (Para-)medical Students - Experience From Incorporating a Relevant Genetic Disease Throughout a Course of Molecular Cell Biology.

Worldwide, a mandatory course in Molecular Cell Biology is often part of the (para-) medical curricula. Student audiences are regularly not receptive to such relatively theoretical courses and teachers often struggle to convey the necessary information. Here, positive experience is shared on rigorously embedding a genetic disease that severely affects the movement apparatus, fibrodysplasia ossificans progressiva (FOP), in all aspects of a course for an international group of Research Master Human Movement Sciences students. Various molecular cell biological aspects of FOP were systematically implemented in the course, covering genetics, the biochemical consequences of the mutation, signaling pathways that affect bone formation and lectures on how to clone the mutation or cure the mutation. Students were invited to critically think about how to use the theories learned in the course to analyze a research paper. During the practical part of the course, students assisted in novel, cutting edge research on FOP patient derived or control cells. Research findings were reported in a research paper format. By building a Molecular Cell Biology course around an appealing disease, we managed to increase the general motivation of the students for the course as reflected in two specific questions of the course evaluations (p < 0.05). It convincingly taught the relevance of a course of Molecular Cell Biology to students with a primary background in biomechanics and physiotherapy for their paramedical professional life. This approach of embedding an audience-tailored human disease with a known genetic cause into a course can be implemented to many medical curriculum related courses and will increase students' perception of the relevance of a course.

Prothrombin is a binding partner of the human receptor of advanced glycation end products.

The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a γ-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury.

Using anticipated learning outcomes for backward design of a molecular cell biology Course-based Undergraduate Research Experience.

Anticipated learning outcomes (LOs) were defined and used for the backward design of a Course-based Undergraduate Research Experience (CURE). These LOs reflect the inquiry-based nature of CUREs and capture key knowledge and skills inherent to scientific practice and essential in research. The LOs were used to plan a formative and summative assessment strategy to support and evaluate student achievement. A research question was identified that aligned with the learning goals of the course, provided an opportunity for discovery and iteration, and introduced a variety of molecular, cellular, and biochemical techniques. The course is offered to students in the final year of their degree and delivered over a 12-week period with two 3-hr labs each week. These LOs, and the rigorous assessment strategy used to support them, could be adapted to different projects. Likewise, the laboratory exercises are presented as a series of modules highlighting opportunities for adaptation to a variety of schedules.

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex.

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization.

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation.

A recently-discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The nonenzymatic nature of K-PPn means that the degree of this modification depends on both polyP abundance and the amino acids surrounding the modified lysine. K-PPn was originally discovered in budding yeast (Saccharomyces cerevisiae), in which polyP anabolism and catabolism are well-characterized. However, yeast vacuoles accumulate large amounts of polyP, and upon cell lysis, the release of the vacuolar polyP could nonphysiologically cause K-PPn of nuclear and cytosolic targets. Moreover, yeast vacuoles possess two very active endopolyphosphatases, Ppn1 and Ppn2, that could have opposing effects on the extent of K-PPn. Here, we characterized the contribution of vacuolar polyP metabolism to K-PPn of two yeast proteins, Top1 (DNA topoisomerase 1) and Nsr1 (nuclear signal recognition 1). We discovered that whereas Top1-targeting K-PPn is only marginally affected by vacuolar polyP metabolism, Nsr1-targeting K-PPn is highly sensitive to the release of polyP and of endopolyphosphatases from the vacuole. Therefore, to better study K-PPn of cytosolic and nuclear targets, we constructed a yeast strain devoid of vacuolar polyP by targeting the exopolyphosphatase Ppx1 to the vacuole and concomitantly depleting the two endopolyphosphatases (ppn1Δppn2Δ, vt-Ppx1). This strain enabled us to study K-PPn of cytosolic and nuclear targets without the interfering effects of cell lysis on vacuole polyP and of endopolyphosphatases. Furthermore, we also define the fundamental nature of the acidic amino acid residues to the K-PPn target domain.

FADS3 is a Δ14Z sphingoid base desaturase that contributes to gender differences in the human plasma sphingolipidome.

Sphingolipids (SLs) are structurally diverse lipids that are defined by the presence of a long-chain base (LCB) backbone. Typically, LCBs contain a single Δ4E double bond (DB) (mostly d18:1), whereas the dienic LCB sphingadienine (d18:2) contains a second DB at the Δ14Z position. The enzyme introducing the Δ14Z DB is unknown. We analyzed the LCB plasma profile in a gender-, age-, and BMI-matched subgroup of the CoLaus cohort (n = 658). Sphingadienine levels showed a significant association with gender, being on average ∼30% higher in females. A genome-wide association study (GWAS) revealed variants in the fatty acid desaturase 3 (FADS3) gene to be significantly associated with the plasma d18:2/d18:1 ratio (p = -log 7.9). Metabolic labeling assays, FADS3 overexpression and knockdown approaches, and plasma LCB profiling in FADS3-deficient mice confirmed that FADS3 is a bona fide LCB desaturase and required for the introduction of the Δ14Z double bond. Moreover, we showed that FADS3 is required for the conversion of the atypical cytotoxic 1-deoxysphinganine (1-deoxySA, m18:0) to 1-deoxysphingosine (1-deoxySO, m18:1). HEK293 cells overexpressing FADS3 were more resistant to m18:0 toxicity than WT cells. In summary, using a combination of metabolic profiling and GWAS, we identified FADS3 to be essential for forming Δ14Z DB containing LCBs, such as d18:2 and m18:1. Our results unravel FADS3 as a Δ14Z LCB desaturase, thereby disclosing the last missing enzyme of the SL de novo synthesis pathway.

High-resolution genome-wide expression analysis of single myofibers using SMART-Seq.

Skeletal muscle is a heterogeneous tissue. Individual myofibers that make up muscle tissue exhibit variation in their metabolic and contractile properties. Although biochemical and histological assays are available to study myofiber heterogeneity, efficient methods to analyze the whole transcriptome of individual myofibers are lacking. Here, we report on a single-myofiber RNA-sequencing (smfRNA-Seq) approach to analyze the whole transcriptome of individual myofibers by combining single-fiber isolation with Switching Mechanism at 5' end of RNA Template (SMART) technology. Using smfRNA-Seq, we first determined the genes that are expressed in the whole muscle, including in nonmyogenic cells. We also analyzed the differences in the transcriptome of myofibers from young and old mice to validate the effectiveness of this new method. Our results suggest that aging leads to significant changes in the expression of metabolic genes, such as Nos1, and structural genes, such as Myl1, in myofibers. We conclude that smfRNA-Seq is a powerful tool to study developmental, disease-related, and age-related changes in the gene expression profile of skeletal muscle.

Genomic tagging of endogenous human ESCRT-I complex preserves ESCRT-mediated membrane-remodeling functions.

The endosomal sorting complexes required for transport (ESCRT) machinery drives membrane scission for diverse cellular functions that require budding away from the cytosol, including cell division and transmembrane receptor trafficking and degradation. The ESCRT machinery is also hijacked by retroviruses, such as HIV-1, to release virions from infected cells. The crucial roles of the ESCRTs in cellular physiology and viral disease make it imperative to understand the membrane scission mechanism. Current methodological limitations, namely artifacts caused by overexpression of ESCRT subunits, obstruct our understanding of the spatiotemporal organization of the endogenous human ESCRT machinery. Here, we used CRISPR/Cas9-mediated knock-in to tag the critical ESCRT-I component tumor susceptibility 101 (Tsg101) with GFP at its native locus in two widely used human cell types, HeLa epithelial cells and Jurkat T cells. We validated this approach by assessing the function of these knock-in cell lines in cytokinesis, receptor degradation, and virus budding. Using this probe, we measured the incorporation of endogenous Tsg101 in released HIV-1 particles, supporting the notion that the ESCRT machinery initiates virus abscission by scaffolding early-acting ESCRT-I within the head of the budding virus. We anticipate that these validated cell lines will be a valuable tool for interrogating dynamics of the native human ESCRT machinery.

Hematopoietic PBX-interacting protein is a substrate and an inhibitor of the APC/C-Cdc20 complex and regulates mitosis by stabilizing cyclin B1.

Proper cell division relies on the coordinated regulation between a structural component, the mitotic spindle, and a regulatory component, anaphase-promoting complex/cyclosome (APC/C). Hematopoietic PBX-interacting protein (HPIP) is a microtubule-associated protein that plays a pivotal role in cell proliferation, cell migration, and tumor metastasis. Here, using HEK293T and HeLa cells, along with immunoprecipitation and immunoblotting, live-cell imaging, and protein-stability assays, we report that HPIP expression oscillates throughout the cell cycle and that its depletion delays cell division. We noted that by utilizing its D box and IR domain, HPIP plays a dual role both as a substrate and inhibitor, respectively, of the APC/C complex. We observed that HPIP enhances the G2/M transition of the cell cycle by transiently stabilizing cyclin B1 by preventing APC/C-Cdc20-mediated degradation, thereby ensuring timely mitotic entry. We also uncovered that HPIP associates with the mitotic spindle and that its depletion leads to the formation of multiple mitotic spindles and chromosomal abnormalities, results in defects in cytokinesis, and delays mitotic exit. Our findings uncover HPIP as both a substrate and an inhibitor of APC/C-Cdc20 that maintains the temporal stability of cyclin B1 during the G2/M transition and thereby controls mitosis and cell division.

Transcription factor TFAP2B up-regulates human corneal endothelial cell-specific genes during corneal development and maintenance.

The corneal endothelium, which originates from the neural crest via the periocular mesenchyme (PM), is crucial for maintaining corneal transparency. The development of corneal endothelial cells (CECs) from the neural crest is accompanied by the expression of several transcription factors, but the contribution of some of these transcriptional regulators to CEC development is incompletely understood. Here, we focused on activating enhancer-binding protein 2 (TFAP2, AP-2), a neural crest-expressed transcription factor. Using semiquantitative/quantitative RT-PCR and reporter gene and biochemical assays, we found that, within the AP-2 family, the TFAP2B gene is the only one expressed in human CECs in vivo and that its expression is strongly localized to the peripheral region of the corneal endothelium. Furthermore, the TFAP2B protein was expressed both in vivo and in cultured CECs. During mouse development, TFAP2B expression began in the PM at embryonic day 11.5 and then in CECs during adulthood. siRNA-mediated knockdown of TFAP2B in CECs decreased the expression of the corneal endothelium-specific proteins type VIII collagen α2 (COL8A2) and zona pellucida glycoprotein 4 (ZP4) and suppressed cell proliferation. Of note, we also found that TFAP2B binds to the promoter of the COL8A2 and ZP4 genes. Furthermore, CECs that highly expressed ZP4 also highly expressed both TFAP2B and COL8A2 and showed high cell proliferation. These findings suggest that TFAP2B transcriptionally regulates CEC-specific genes and therefore may be an important transcriptional regulator of corneal endothelial development and homeostasis.

Muscular Dystrophy Model.

Muscular dystrophy (MD) is a group of muscle weakness disease involving in inherited genetic conditions. MD is caused by mutations or alteration in the genes responsible for the structure and functioning of muscles. There are many different types of MD which have a wide range from mild symptoms to severe disability. Some types involve the muscles used for breathing which eventually affect life expectancy. This chapter provides an overview of the MD types, its gene mutations, and the Drosophila MD models. Specifically, the Duchenne muscular dystrophy (DMD), the most common form of MD, will be thoroughly discussed including Dystrophin genes, their isoforms, possible mechanisms, and signaling pathways of pathogenesis.