Genesis Of Cells Research Articles

Abstract 365: The impact of the RASSF1C-PIWIL1-piRNA pathway on DNA methylation

Abstract Introduction: Epigenomic regulation is becoming critically important in human cancers as epigenetically regulated genes can be used as biomarkers for diagnosis, prognosis, molecular classification of tumors, and predicting response to therapies. Hence, identification of epigenetic biomarkers for specific cancers is highly desirable for development of precision medicine tools. Recently we have identified a novel RASSF1C-PIWIL1-piRNA pathway that appears to promote lung cell proliferation and migration. PIWI-like proteins interact with piRNAs to form complexes that regulate gene expression at the transcriptional (epigenetic control) and translational (mRNA silencing) levels, leading to stimulation of cell stem renewal and proliferation. We have shown that RASSF1C modulates the PIWIL1-piRNA gene axis, suggesting the hypothesis that the RASSF1C-PIWI-piRNA pathway could potentially contribute to lung cancer stem cell development and progression, in part, through modulation of gene methylation of both oncogenic and tumor suppressor genes. Thus, we wanted to validate this hypothesis using a non-small cell lung cancer (NSCLC) cell model to determine if we can identify candidate genes targeted by the RASSF1C-PIWIL1-piRNA pathway through a gene methylation mechanism. Methods: We conducted a pilot study on the impact of over-expressing RASSF1C and knocking down RASSF1C and P1WIL1 expression on global gene DNA methylation in the NSCLC cell line H1299. DNA from cells over-expressing RASSF1C, RASSF1C-knockdown, PIWIL1-knockdwn, and control cells was used for the methylation study using the Reduced Representation Bisulfite Sequencing (RRBS) method. Results: Candidate Differentially Methylated Regions (DMR) were identified by comparing DNA methylation profiles of experimental and control cells. We found that over-expression of RASSF1C and knocking down RASSF1C and PIWIL1 modulated DNA methylation of genomic regions; and statistically significant candidate genes residing in hypo- and hyper-methylated regions in lung cancer cells were identified, including the Dual-specificity phosphatase 4 gene (DUSP4) that functions as a tumor suppressor. Conclusion: The RASSF1C-PIWI-piRNA pathway may play a critical role in epigenome regulation and genesis of lung cancer stem cells through modulation of key genes such as DUSP4. We found that RASSSF1C over-expression increases, while knock-down of RASSF1C or PIWIL1 decreases methylation/expression of the DUSP4 gene perhaps providing a potential mechanism through which the RASSF1C-PIWIL1-piRNA pathway could promote lung cancer stem cell development and progression. DUSP4 is an interesting target gene because knockdown of DUSP4 has recently been shown to enhance cancer stem cell formation. Citation Format: Yousef G. Amaar, Mark E. Reeves. The impact of the RASSF1C-PIWIL1-piRNA pathway on DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 365.

A New Thermo‐, pH‐ and CO2‐Responsive Fluorescent Four‐Arm Star Polymer with Aggregation‐Induced Emission for Long‐Term Cellular Tracing

AbstractDevelopment of fluorescent bioprobes for long‐term cell tracking is of great importance to monitor the processes of genesis, development, invasion, and metastasis of cancerous cells. Herein, a new multistimuli‐responsive star polymer of tetraphenylethene‐graft‐tetra‐poly[N‐[2‐(diethylamino)‐ethyl]acrylamide] (TPE‐tetraPDEAEAM) with aggregation‐induced emission (AIE) effect for tracing live cells over a long period of time is synthesized by atom transfer radical polymerization using TPE derivative as initiator. TPE‐tetraPDEAEAM gives both lower critical solution temperature and fluorescence responses to the stimulus of the temperature, pH, and CO2 by combining the thermoresponsive and pH/CO2‐responsive moieties of the diethylamino and acrylamide groups. The AIE‐active TPE‐tetraPDEAEAM has the advantages of very low cytotoxicity, efficient cellular uptake, and strong fluorescence of polymer‐treated cells, which ensure its good performance in long‐term cell tracing. This facile tracking of HeLa cells for as long as nine passages exhibits superior performance in long‐term cell tracing as compared with some commercial cell tracing probes.

Cut loose and run: The complex role of ADAM proteases during neural crest cell development.

ADAM metalloproteases have been shown to play critical roles during development. In this review, we will describe functional evidence that implicates ADAM proteins during the genesis, migration and differentiation of neural crest cells. We will restrict our analysis to the transmembrane ADAMs as other reviews have addressed the role of extracellular metalloproteases (Christian et al. [2013] Critical Reviews in Biochemistry and Molecular Biology 48:544-560). This review will describe advances that have been obtained mainly through the use of two vertebrate model systems, the frog, and avian embryos. The role of the principal substrates of ADAMs, the cadherins, has been extensively described in other reviews, most recently in (Cousin [1997] Mechanisms of Development 148:79-88; Taneyhill and Schiffmacher [2017] Genesis, 55). The function of ADAMs in the migration of other cell types, including the immune system, wound healing and cancer has been described previously in (Dreymueller et al. [2017] Mediators of Inflammation 2017: 9621724). Our goal is to illustrate both the importance of ADAMs in controlling neural crest behavior and how neural crest cells have helped us understand the molecular interactions, substrates, and functions of ADAM proteins in vivo.

Low omega-6/omega-3 ratio in a maternal protein-deficient diet promotes histone-3 changes in progeny neural cells and favors leukemia inhibitory factor genetranscription

Omega-3 (n-3) fatty acids modulate epigenetic changes critical to genesis and differentiation of neural cells. Conversely, maternal protein-malnutrition can negatively modify these changes. This study investigated whether a low n-6/n-3 ratio in a maternal diet could favor histone-3 (H3) modifications, gene transcription and differentiation in the offspring neural cells even under protein-deficiency. Female rats fed a control (Ct), or 3 types of multideficient diets differing in protein levels or linoleic/alpha-linolenic fatty acid ratios (RBD, RBD-C, RBD-SO) from 30 days prior to mating and during pregnancy. Cerebral cortex tissue and cortical cultures of progeny embryonic neurons and postnatal astrocytes were analyzed. H3K9 acetylation and H3K27 or H3K4 di-methylation levels were assessed by flow cytometry and/or immunocytochemistry. In astrocyte cultures and cortical tissue, the GFAP protein levels were assessed. Glial derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) gene expression were evaluated in the cortical tissue. GFAP levels were similar in astrocytes of Ct, RBD and RBD-C, but 65% lower in RBD-SO group. Higher levels of H3K9Ac were found in the neurons and H3K4Me2 in the astrocytes of the RBD group. No intergroup difference in the cortical GDNF mRNA expression or the H3K27Me2 levels in astrocytes was detected. LIF mRNA levels were higher in the RDB (P=.002) or RBD-C (P=.004) groups than in the control. The findings indicate the importance of dietary n-3 availability for the brain, even under a protein-deficient condition, inducing Histone modifications and increasing LIF gene transcription, involved in neural cell differentiation and reactivity.

Chapter 2 - Embryology

With the growing recognition of the extent and prevalence of human cerebellar disorders, an understanding of developmental programs that build the mature cerebellum is necessary. In this chapter we present an overview of the basic epochs and key molecular regulators of the developmental programs of cerebellar development. These include early patterning of the cerebellar territory, the genesis of cerebellar cells from multiple spatially distinct germinal zones, and the extensive migration and coordinated cellular rearrangements that result in the formation of the exquisitely foliated and laminated mature cerebellum. This knowledge base is founded on extensive analysis of animal models, particularly mice, due in large part to the ease of genetic manipulation of this important model organism. Since cerebellar structure and function are largely conserved across species, mouse cerebellar development is highly relevant to humans and has led to important insights into the developmental pathogenesis of human cerebellar disorders. Human fetal cerebellar development remains largely undescribed; however, several human-specific developmental features are known which are relevant to human disease and underline the importance of ongoing human fetal research.

Differential Roles of Glycogen Synthase Kinase 3 Subtypes Alpha and Beta in Cortical Development.

Glycogen synthase kinases 3 (GSK3) α and β are expressed in the nervous system, and disruption of GSK3 signaling has been implicated in a wide range of neurodevelopmental and psychiatric disorders. Although several studies have established a role of GSK3 signaling in the nervous system, much less is known about isoform-specific functions. Here, we have examined the role of GSK3α and GSK3β in the developing neocortex by performing in utero electroporation with specific small interfering RNAs targeting each isoform. We found that depletion of either GSK3α or GSK3β commonly promoted the proliferation of neural progenitor cells in the ventricular zone, but at later stages, knocking down of each isoform resulted in distinct outcomes. In particular, the transformation of radial progenitors to intermediate progenitor cells was promoted in GSK3α-depleted cells, but markedly prevented in GSK3β-depleted cells. Moreover, knocking down of GSK3β but not GSK3α prevented the generation of upper-layer Cux1+ neurons. Consistent with the distinct outcomes, protein levels of c-Myc and β-catenin, well-known substrates of GSK3, were differentially affected by depletion of GSK3α and GSK3β. Together, these results suggest that GSK3α and GSK3β might play distinct roles in the genesis and differentiation of neuronal lineage cells during neocortex development by differential regulation of downstream signaling pathways.

Herpetic ocular manifestations in a patient with GATA2 deficiency

Specific genetic deficiencies are a rare cause that should be included in the diagnostic algorithm of disseminated herpetic lesions. The aim of this article is to describe the ocular herpetic manifestations in a rare genetic disorder called GATA2 deficiency. We present the clinical case of a 26-year-old male with dendritic ulcers in his cornea, marrow aplasia and idiopathic chronic lymphedema. He was diagnosed with GATA2 deficiency. GATA2 gene is critical for the genesis and function of hematopoietic stem cells. Its deficiency can cause myelodysplastic syndromes, congenital lymphedema and severe viral infections. Our patient presented these three manifestations, added to a deletion in 20q12 that confirmed the suspicion of GATA2 mutation. A bone marrow transplant was suggested as definitive treatment. The corneal herpetic epithelial lesion was analogous to a regular hepetic keratitis with none stromal keratitis.

Abstract 120: A study of in vitro and in vivo effects of a novel peptide and rhenium compounds on prostate cancer

Abstract Emerging evidence suggests that acquisition of the epithelial-to-mesenchymal transition (EMT), a process that resembles the genesis of cancer stem-like cells, contributes to tumor aggressiveness and is mediated by deregulated expression of microRNAs (miRNAs), such as miR-200 and let-7 family. Loss of miR-200 expression results in the over-expression of Lin28B, which is prevalent in human Prostate Cancer( PCa) . Lin28B is also known to block the processing of another miRNA (pre-let-7 and pri-let-7), resulting in decreased mature let-7, thereby leads to increased Suz12 and EZH2 expression, which are important components of the polycomb repressive complex 2 (PRC2). Thus, over- expression of Lin28B and loss of miR-200 and let-7 appear to be responsible for PCa aggressiveness. A group of novel rhenium compounds have shown promising anti-cancer properties in various cancer cell lines tested in our laboratories. Our current investigations show that re-expression of miR-200b, miR-200c, and let-7 could be achieved by treating cells with our newly developed rhenium compounds by down-regulating the expression of Lin28B and EZH2. Based on our preliminary results, we hypothesize that over-expression of Lin28B leads to the acquisition of invasive and metastatic characteristics in PCa cells (EMT-phenotype cells) via down-regulation of miR-200b and miR-200c, resulting in increased expression of Suz12, ZEB1, and ZEB2. We also hypothesize that over-expression of Lin28B represses the maturation of let-7 family, leading to increased expression of EZH2, and these processes can be attenuated by treatment of cells with novel rhenium compounds in vitro. Our experiments were done on prostate cancer cell lines derived from both Caucasian(CA) and African American (AA)patients and also biopsy samples obtained from both CA and AA patients at Henry Ford Hospital and Karmanos Cancer Center of Wayne State University, Detroit, MI. We confirmed using gene expression studies by micro array and Real-Time PCR and then data analysis by Ingenuity software systemthe validity of our hypothesis and continued our research by testing one of these rhenium compounds-RPR1 on prostate cancer model of nude mice at Greenbaum Cancer Center(GCC) of University of Maryland at Baltimore. In vitro studies by Real -Time PCR, Flow Cytometry, Western Blotting ,Smart-Flare technology, cell death and proliferation assays along with inhibition of spheroid forming assays showed efficacy of these rhenium compounds as anti cancer agents. In vivo studies also showed decrease of tumor volume and mass ,no toxicity, thus efficacy of these novel drugs. ACKNOWLEDGEMENT: Supported by NIH- 3R01CA164318-03S1 and NIH-T-34-GM100831, a NSF-VESTEM award and NSF-LSAMP award . Citation Format: Hirendra N. Banerjee, Sasha Hodge, William Kahan, Santosh Mandal, David Weber, Rena Lapidus, Fazlul Sarkar, Somiranjan Ghosh. A study of in vitro and in vivo effects of a novel peptide and rhenium compounds on prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 120. doi:10.1158/1538-7445.AM2017-120

Development of Mural Cells: From In Vivo Understanding to In Vitro Recapitulation

Mural cells are indispensable for the development and maintenance of healthy mature vasculature, valuable for vascular therapies and as developmental models. However, their functional plasticity, developmental diversity, and multitude of differentiation pathways complicate in vitro generation. Fortunately, there is a vast pool of untapped knowledge from in vivo studies that can guide in vitro engineering. This review highlights the in vivo genesis of mural cells from progenitor populations to recruitment pathways to maturation and identity with an emphasis on how this knowledge is applicable to in vitro models of stem cell differentiation.

OncomicroRNAs-Mediated Tumorigenesis: Implication in Cancer Diagnosis and Targeted Therapy.

MicroRNAs (miRNAs) control the expression of approximately 60% of protein-coding genes and regulate cell metabolism, proliferation, differentiation, and apoptosis. Notably, aberrant expression of miRNAs contributes to several diseases including cancer. Accumulating evidence indicates that miRNAs play important roles in EMT, genesis of cancer stem cells, cancer metabolism and carcinogenesis. Aberrant expression of miRNAs triggers tumor initiation, progression and poor prognosis of cancer patients. Accordingly, oncogenic miRNAs have emerged as diagnostic biomarkers and targets for novel anti-cancer drug discovery. However, the mechanisms of miRNAs contriving tumorigenesis are not completely understood. This review aims to clarify the identification of tumorspecific miRNAs, verification of oncogenic miRNA signatures, and dynamic study of oncogenic miRNAs in cancer initiation and development. Despite sound progress in miRNA-mediated anticancer therapy, several barriers like drug stability, immunogenicity, off-target effects and toxicities still remain. We hope our review could stimulate the further study of miRNAs in cancer research field, which may lead to new insights into the mechanisms of carcinogenesis and create new avenues for targeted cancer therapy.

Dual Shp2 and Pten Deficiencies Promote Non-alcoholic Steatohepatitis and Genesis of Liver Tumor-Initiating Cells

The complexity of liver tumorigenesis is underscored by the recently observed anti-oncogenic effects of oncoproteins, although the mechanisms are unclear. Shp2/Ptpn11 is a proto-oncogene in hematopoietic cells and antagonizes the effect of tumor suppressor Pten in leukemogenesis. In contrast, we show here cooperative functions of Shp2 and Pten in suppressing hepatocarcinogenesis. Ablating both Shp2 and Pten in hepatocytes induced early-onset non-alcoholic steatohepatitis (NASH) and promoted genesis of liver tumor-initiating cells likely due to augmented cJun expression/activation and elevated ROS and inflammation in the hepatic microenvironment. Inhibiting cJun partially suppressed NASH-driven liver tumorigenesis without improving NASH. SHP2 and PTEN deficiencies were detected in liver cancer patients with poor prognosis. These data depict a mechanism of hepato-oncogenesis and suggest a potential therapeutic strategy.

Abstract SY08-02: Normal and neoplastic stem cells

Abstract The transition from single cell species to multicellular organisms led to organ and tissue development. The development and maintenance of organs/tissues in multicellular organisms has resulted in the emergence of specialized cells that self-renew as well as differentiate; these rare cells are stem cells. Following embryonic development, most of our tissues and organs are continuously regenerated from tissue/organ specific stem cells. The principal property that distinguishes such stem cells from their daughter cells is self-renewal; when stem cells divide they give rise to stem cells (by self-renewal) and progenitors (by differentiation). In most tissues only the primitive stem cells self-renew. Even within a particular tissue, stem cells may be diverse, and subject to stem cell competition for their niche. We have documented both germline stem cell and somatic stem cell competition in species as diverse as colonial protochordates and humans. Stem cell isolation and transplantation is the basis for regenerative medicine. Self-renewal is dangerous, and therefore strictly regulated. Poorly regulated self-renewal can lead to the genesis of cancer stem cells, the only self-renewing cells in the cancerous tumor. The Weissman lab has followed the progression from hematopoietic stem cells (HSCs) to myelogenous leukemias. They have found that the developing cancer clones progress at the stage of HSCs, until they become fully malignant. At this point, the “leukemia” stem cell is at a stage of a downstream oligolineage or multilineage progenitor that has evaded programmed cell death and programmed cell removal, while acquiring or keeping self-renewal. In the case of chronic myeloid leukemia, bcr-abl+ HSC clones outcompete normal HSCs in the chronic phase. The transition from the chronic phase to myeloid blast crisis results in the leukemia stem cells appearing in the granulocyte-macrophage progenitor (GMP) stage. While there are many ways to defeat programmed cell death and senescence, there appears to be one dominant method to avoid programmed cell removal—the expression of the cell surface “don't eat me” protein CD47, the ligand for macrophage SIRPα. All cancers tested express CD47 to overcome expression of “eat me” signals such as calreticulin. Antibodies that block the CD47-SIRPα interaction enable phagocytosis and killing of the tumor cells in vitro and in vivo. All tested human solid tumors and lymphomas/leukemias/myelomas express CD47 and are susceptible to phagocytosis in the presence of anti-CD47 blocking antibodies. Citation Format: Irving L. Weissman. Normal and neoplastic stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY08-02.

Structural-functional organisation of the bone marrow hematopoietic stem cells niches

This article focuses on (1) the analysis of the structural-functional organization of bone marrow niches of the hematopoietic stem cells, (2) the role of the intercellular contact interactions and humoral regulation factors in these niches, in particular CXCL12, SCF and TGFβ, and (3) the intracellular signal pathways: Notch, Wnt and Shh. The two types of niches, switching from one into another: endosteal niches located on the endost surface at the borderline with bone marrow cavity and the vascular niches included into bone marrow parenchyma. It is emphasized that the main role in the formation of the niches of both types is ascribed to the multipotent stromal cells, which serve as a base for differentiation of the osteoblasts, spindle-shaped N-cadherin+CD45– osteoblasts (SNO-cells), nestin-expressing cells (Nes+ cells), cells with leptin receptor (Lepr+ cells), abundant producing CXCL12 reticular cells (CAR-cells) and NG2-pericytes. The endothelial cells are no less important. Also, the adipocytes, osteoclasts, macrophages and megakaryocytes, regulatory T-cells and neuronal cells are involved in the niche functioning. It is postulated that osteoblasts and CAR-cells play a crucial role in the genesis of immune system cells: common lymphoid precursors, B-lymphocytes, natural killer and dendritic cells.

Aurora-A Kinase as a Promising Therapeutic Target in Cancer.

Mammalian Aurora family of serine/threonine kinases are master regulators of mitotic progression and are frequently overexpressed in human cancers. Among the three members of the Aurora kinase family (Aurora-A, -B, and -C), Aurora-A and Aurora-B are expressed at detectable levels in somatic cells undergoing mitotic cell division. Aberrant Aurora-A kinase activity has been implicated in oncogenic transformation through the development of chromosomal instability and tumor cell heterogeneity. Recent studies also reveal a novel non-mitotic role of Aurora-A activity in promoting tumor progression through activation of epithelial–mesenchymal transition reprograming resulting in the genesis of tumor-initiating cells. Therefore, Aurora-A kinase represents an attractive target for cancer therapeutics, and the development of small molecule inhibitors of Aurora-A oncogenic activity may improve the clinical outcomes of cancer patients. In the present review, we will discuss mitotic and non-mitotic functions of Aurora-A activity in oncogenic transformation and tumor progression. We will also review the current clinical studies, evaluating small molecule inhibitors of Aurora-A activity and their efficacy in the management of cancer patients.

The aged brain: genesis and fate of residual progenitor cells in the subventricular zone

Neural stem cells (NSCs) persist in the adult mammalian brain through life. The subventricular zone (SVZ) is the largest source of stem cells in the nervous system, and continuously generates new neuronal and glial cells involved in brain regeneration. During aging, the germinal potential of the SVZ suffers a widespread decline, but the causes of this turn down are not fully understood. This review provides a compilation of the current knowledge about the age-related changes in the NSC population, as well as the fate of the newly generated cells in the aged brain. It is known that the neurogenic capacity is clearly disrupted during aging, while the production of oligodendroglial cells is not compromised. Interestingly, the human brain seems to primarily preserve the ability to produce new oligodendrocytes instead of neurons, which could be related to the development of neurological disorders. Further studies in this matter are required to improve our understanding and the current strategies for fighting neurological diseases associated with senescence.

Mechanism Analyses for Elucidating the Role of LOXL2 Silencing in Hepatocellular Carcinoma

The paper aimed to study the effect of lysyl oxidase-like 2 (LOXL2) on hepatocellular carcinoma (HCC) and explore the biological mechanisms of tumorigenicity and progression in HCC. The authors used four HCC cell lines to identify LOXL2. A lentiviral vector containing LOXL2-siRNA was constructed to silence the LOXL2 gene in SMMC-7721 cell line, and mRNA of the target gene was detected by real-time polymerase chain reaction (RT-PCR). The effect of LOXL2 silencing on the growth of SMMC-7721 cells was explored with flow cytometry profiling and BrdU labeling. Downstream genes of LOXL2 were selected by microarray and verified by Western Blotting. In the results, LOXL2 expression was significantly up-regulated in four types of HCC cell lines, therefore, SMMC-7721 cell line was selected for further exploration. When SMMC-7721 cell line was infected with LOXL2-siRNA, the expression of LOXL2 mRNA decreased. The silencing of LOXL2 resulted in the cell cycle arrest at the G1-phase, the increased apoptosis and the decreased growth of SMMC-7721 cells on the indicated days by BrdU. Moreover, the MDM2, BIRC3, CDC42, FOS and TGFBR2 genes were selected and verified to be the downstream genes of LOXL2. In conclusion, LOXL2 contributes to the genesis and progression of HCC cells and works by regulating downstream genes of LOXL2 in certain pathways. Therefore, LOXL2 may play an important role in the progression and prognosis of HCC.

258. Universal Stem Cell Gene Therapy Platform: Broadening the Transient Epigenetic Gene Therapy Arm by Using Nuclease-Null dCas9 as an Additional Drive for Efficient Epigenetic Editing

Bacterial CRISPR/Cas9 is an RNA-guided endonuclease that has been converted into a breakthrough tool for human gene targeting culminating in efficient gene repair/genomic editing and is now emerging, through its nuclease-null dCas9 double-mutant, as a popular new drive for human endogenous gene control culminating in epigenomic editing. Cas9 overwhelming strength is its easy and inexpensive customization to DNA targets since it relies on a short single-guided RNA. Therefore, dCas9 has already been successfully used as a programmable RNA-guided DNA-binding module for efficient targeting of dimeric FokI endonuclease and a powerful tripartite transcriptional activator. Fusing dCas9 to a variety of epigenetic effector domains stands thus as an attractive option to broaden the transient epigenetic gene therapy arm of our proposed 2005 Universal Stem Cell Gene Therapy Platform. Coined as a translational application of human promoter-specific siRNA-mediated transcriptional gene silencing (promoter methylation) and promoter demethylation mediated by non-coding antisense-RNA, transient epigenetic gene therapy is aimed at long-term transcriptional gene silencing/activation through the transient action of promoter/enhancer-specific siRNAs/dsRNAs or short sense/antisense RNAs/oligonucleotides (oligos). Unlike canonical RNA interference mediated by siRNAs that target cytoplasmic mRNAs for post-transcriptional gene silencing through sequence-specific mRNA cleavage or translational repression, promoter-specific small RNAs/oligos-mediated transcriptional gene silencing/activation is a long-lasting epigenetic process that involves DNA methylation/demethylation and post-translational histone modifications. Therefore, transient epigenetic gene therapy should provide a new dimension to basic gene therapeutics in which the transient action of a dCas9-Methylase/Demethylase fusion protein or of promoter/enhancer-specific small RNAs/oligos could be used for long-term up-/down-regulation of target genes. Transient epigenetic gene therapy has exciting applications for inherited diseases such as allele-specific silencing for autosomal-dominant mutants or activation of fetal genes as substitutes for mutant adult cognates. However, long-term transcriptional up-/down-regulation of target genes has many other applications since promoter/enhancer-specific small RNAs/oligos target non-coding sense/antisense RNAs including long ones, i.e. the very RNAs that appear to drive the complex digital regulation of human epigenetic processes and inherent pathologies. Therefore, our approach is discussed in terms of new therapeutic/prophylactic avenues for 1) epigenetic pathologies such as neurodevelopmental disorders originating from deleterious early life epigenetic programming (eg depression, schizophrenia), 2) therapeutic remodeling of imprinted gene expression (eg Angelman syndrome), 3) degenerative diseases and age-related disorders (eg Alzheimer's & Parkinson's diseases), 4) cancer and infectious diseases, 5) the ex vivo genesis of clinical-grade iPS cells and 6) rising stem cell conversion.

Effects of ethanol exposure in utero on Cajal-Retzius cells in the developing cortex.

Prenatal exposure to ethanol exerts teratogenic effects on the developing brain. Here, we tested the hypothesis that exposure to ethanol in utero alters the disposition of Cajal-Retzius cells that play a key role in orchestrating proliferation, migration, and laminar integration of cortical neurons in the embryonic cortex. Pregnant Ebf2-EGFP mice, harboring EGFP-fluorescent Cajal-Retzius cells, were subjected to a 2% w/w ethanol consumption regimen starting at neural tube closure and lasting throughout gestation. Genesis of Cajal-Retzius cells was assessed by means of 5-bromo-2-deoxyuridine (BrdU) immunofluorescence at embryonic day 12.5, their counts and distribution were determined between postnatal day (P)0 and P4, patch clamp electrophysiology was performed between P2 and P3 to analyze GABA-mediated synaptic activity, and open-field behavioral testing was conducted in P45-P50 adolescents. In Ebf2-EGFP embryos exposed to ethanol in utero, we found increased BrdU labeling and expanded distribution of Cajal-Retzius cells in the cortical hem, pointing to increased genesis and proliferation. Postnatally, we found an increase in Cajal-Retzius cell number in cortical layer I. In addition, they displayed altered patterning of spontaneous GABA-mediated synaptic barrages and enhanced GABA-mediated synaptic activity, suggesting enhanced GABAergic tone. These findings, together, underscore that Cajal-Retzius cells contribute to the ethanol-induced aberration of cortical development and abnormal GABAergic neurotransmission at the impactful time when intracortical circuits form.

SOX4 cooperates with neurogenin 3 to regulate endocrine pancreas formation in mouse models.

The sex-determining region Y (SRY)-related high mobility group (HMG) box (SOX) family of transcription factors is essential for normal organismal development. Despite the longstanding knowledge that many SOX family members are expressed during pancreas development, a role for many of these factors in the establishment of insulin-producing beta cell fate remains to be determined. The aim of this study is to elucidate the role of SOX4 during beta cell development. We used pancreas and endocrine progenitor mouse knockouts of Sox4 to uncover the roles of SOX4 during pancreas development. Lineage tracing and in vitro models were used to determine how SOX4 regulates beta cell formation and understand the fate of Sox4-null endocrine lineage cells. This study demonstrates a progenitor cell-autonomous role for SOX4 in regulating the genesis of beta cells and shows that it is required at multiple stages of the process. SOX4 deletion in the multipotent pancreatic progenitors resulted in impaired endocrine progenitor cell differentiation. Deletion of SOX4 later in the Neurog3-expressing cells also caused reductions in beta cells. Lineage studies showed loss of Sox4 in endocrine progenitors resulted in a block in terminal islet cell differentiation that was attributed to reduction in the production of key beta cell specification factors. These results demonstrate that SOX4 is essential for normal endocrine pancreas development both concomitant with, and downstream of, the endocrine fate decision. In conclusion, these studies position Sox4 temporally in the endocrine differentiation programme and provide a new target for improving in vitro differentiation of glucose-responsive pancreatic beta cells.

Cancer/testis antigens trigger epithelial-mesenchymal transition and genesis of cancer stem-like cells.

Malignant tumors aberrantly overexpress various embryonic genes and proto-oncogenes, including a variety of cancer-testis antigens (CTAs). CTAs belong to a class of testis-derived proteins which are only expressed in germ cells in the male testis, and the expression of CTA genes is entirely silenced in the adult somatic tissues. They are, however, aberrantly overexpressed in a variety of malignant tumor tissues. Emerging evidence shows that a number of CTAs promote epithelialmesenchymal transition (EMT) and genesis of cancer stem like cells, escalating tumorigenesis, invasion, and metastasis. The can cer-testis antigens, such as SSX, MAGE-D4B, CAGE, piwil2, and CT45A1, upregulate EMT and metastatic genes, promoting EMT and tumor dissemination. In addition, certain members of CTAs, including Piwil2, DNAJB8, CT45A1, MAGE-A, GAGE, and SPANX, are implicated in the initiation or maintenance, of cancer stem-like cells, promoting tumorigenesis and malignant progression. Clinically CTAs are closely associated with poor prognosis in cancer patients. Intriguely, CTAs are strongly immunogenic and normally restricted to the male testis after birth, however, these proteins are aberrantly overexpressed in cancer stem-like cells and in a variety of cancers, suggesting their target potential for cancer immunotherapy, as diagnostic biomarkers, and as targets for novel anticancer drug discovery. Thus, the targeting of tumorigenic CTAs is a promising strategy to eradicate cancer stem-like cells and inhibit tumorigenesis for effective cancer treatment.