Crosstalk between SOX Genes and Long Non-Coding RNAs in Glioblastoma.

Glioblastoma (GBM) continues to be the most devastating primary brain malignancy. Despite significant advancements in understanding basic GBM biology and enormous efforts in developing new therapeutic approaches, the prognosis for most GBM patients remains poor with a median survival time of 15 months. Recently, the interplay between the SOX (SRY-related HMG-box) genes and lncRNAs (long non-coding RNAs) has become the focus of GBM research. Both classes of molecules have an aberrant expression in GBM and play essential roles in tumor initiation, progression, therapy resistance, and recurrence. In GBM, SOX and lncRNAs crosstalk through numerous functional axes, some of which are part of the complex transcriptional and epigenetic regulatory mechanisms. This review provides a systematic summary of current literature data on the complex interplay between SOX genes and lncRNAs and represents an effort to underscore the effects of SOX/lncRNA crosstalk on the malignant properties of GBM cells. Furthermore, we highlight the significance of this crosstalk in searching for new biomarkers and therapeutic approaches in GBM treatment.

Crosstalk between SOXB1 proteins and WNT/ß-catenin signaling in NT2/D1 cells.

During early vertebrate embryogenesis, the expression of SOXB1 proteins is precisely regulated by a number of different mechanisms, including Wnt/ß-catenin signaling. This is essential for controlling the balance between stemness and differentiation in embryonic stem cells. In the present study, we analyzed the molecular mechanism of LiCl action in NT2/D1 cells and examined the crosstalk between SOXB1 proteins and Wnt signaling in this model system. We have shown that LiCl increases ß-catenin level, induces its translocation to the nucleus and consequently up-regulates ß-catenin/Tcf-dependent transcription in NT2/D1 cells. Our results also suggest that LiCl treatment leads to increased expression of SOX2 and SOX3 proteins in NT2/D1 cells through activation of canonical Wnt signaling. Finally, we have detected a negative feedback loop between ß-catenin and SOX2 expression in NT2/D1 cells. Since ß-catenin and SOX2 have been linked to processes of self-renewal and pluripotency, our results have implications for future research on the maintenance of stemness and lineage commitment of embryonic stem cells.

Human embryonal carcinoma cells in serum-free conditions as an in vitro model system of neural differentiation.

Serum is generally regarded as an essential component of many eukaryotic cell culture media, despite the fact that serum composition varies greatly and may be the source of a wide range of artefacts. The objective of this study was to assess serum-free growth conditions for the human embryonal carcinoma cell line, NT2/D1. These cells greatly resemble embryonic stem cells. In the presence of retinoic acid (RA), NT2/D1 cells irreversibly differentiate along the neuronal lineage. We have previously shown that the early phases of neural induction of these cells by RA involve the up-regulation of SOX3 gene expression. Our goal was to compare RA-induced differentiation of NT2/D1 cells in serum-containing and serum-free media, by using SOX3 protein levels as a marker of differentiation. We found that NT2/D1 cells can be successfully grown under serum-free conditions, and that the presence or absence of serum does not affect the level of SOX3 protein after a 48-hour RA induction. However, six days of RA treatment resulted in a marked increase in SOX3 protein levels in serum-free media compared to serum-containing media, indicating that serum might have an inhibitory effect on the expression of this neural differentiation marker. This finding is important for both basic and translational studies that hope to exploit cell culture conditions that are free of animal-derived products.

Structural, optical, and bioimaging characterization of carbon quantum dots solvothermally synthesized from o-phenylenediamine.

Carbon quantum dots as a novel type of carbon nanomaterials have attracted the attention of many researchers because of their unique optical, antibacterial, and anticancer properties as well as their biocompatibility. In this study, for the first time, carbon quantum dots were prepared from o-phenylenediamine dissolved in toluene by a solvothermal route. Subsequently, the prepared carbon quantum dots were encapsulated into polyurethane films by a swelling-encapsulation-shrink method. Analyses of the results obtained by different characterization methods (AFM, TEM, EDS, FTIR, photoluminescence, and EPR) indicate the significant influence of the precursor on structural, chemical, and optical properties. Antibacterial and cytotoxicity tests showed that these dots did not have any antibacterial potential, because of the low extent of reactive oxygen species production, and showed low dark cytotoxicity. By investigating the cellular uptake, it was established that these dots penetrated the HeLa cells and could be used as probes for bioimaging.

Employing Gamma-Ray-Modified Carbon Quantum Dots to Combat a Wide Range of Bacteria.

Nowadays, it is a great challenge to develop new medicines for treating various infectious diseases. The treatment of these diseases is of utmost interest to further prevent the development of multi-drug resistance in different pathogens. Carbon quantum dots, as a new member of the carbon nanomaterials family, can potentially be used as a highly promising visible-light-triggered antibacterial agent. In this work, the results of antibacterial and cytotoxic activities of gamma-ray-irradiated carbon quantum dots are presented. Carbon quantum dots (CQDs) were synthesized from citric acid by a pyrolysis procedure and irradiated by gamma rays at different doses (25, 50, 100 and 200 kGy). Structure, chemical composition and optical properties were investigated by atomic force microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, UV-Vis spectrometry and photoluminescence. Structural analysis showed that CQDs have a spherical-like shape and dose-dependent average diameters and heights. Antibacterial tests showed that all irradiated dots had antibacterial activity but CQDs irradiated with dose of 100 kGy had antibacterial activity against all seven pathogen-reference bacterial strains. Gamma-ray-modified CQDs did not show any cytotoxicity toward human fetal-originated MRC-5 cells. Moreover, fluorescence microscopy showed excellent cellular uptake of CQDs irradiated with doses of 25 and 200 kGy into MRC-5 cells.

Interplay of SOX transcription factors and microRNAs in the brain under physiological and pathological conditions.

Precise tuning of gene expression, accomplished by regulatory networks of transcription factors, epigenetic modifiers, and microRNAs, is crucial for the proper neural development and function of the brain cells. The SOX transcription factors are involved in regulating diverse cellular processes during embryonic and adult neurogenesis, such as maintaining the cell stemness, cell proliferation, cell fate decisions, and terminal differentiation into neurons and glial cells. MicroRNAs represent a class of small non-coding RNAs that play important roles in the regulation of gene expression. Together with other gene regulatory factors, microRNAs regulate different processes during neurogenesis and orchestrate the spatial and temporal expression important for neurodevelopment. The emerging data point to a complex regulatory network between SOX transcription factors and microRNAs that govern distinct cellular activities in the developing and adult brain. Deregulated SOX/microRNA interplay in signaling pathways that influence the homeostasis and plasticity in the brain has been revealed in various brain pathologies, including neurodegenerative disorders, traumatic brain injury, and cancer. Therapeutic strategies that target SOX/microRNA interplay have emerged in recent years as a promising tool to target neural tissue regeneration and enhance neurorestoration. Numerous studies have confirmed complex interactions between microRNAs and SOX-specific mRNAs regulating key features of glioblastoma. Keeping in mind the crucial roles of SOX genes and microRNAs in neural development, we focus this review on SOX/microRNAs interplay in the brain during development and adulthood in physiological and pathological conditions. Special focus was made on their interplay in brain pathologies to summarize current knowledge and highlight potential future development of molecular therapies.

Current Opportunities for Targeting Dysregulated Neurodevelopmental Signaling Pathways in Glioblastoma.

Glioblastoma (GBM) is the most common and highly lethal type of brain tumor, with poor survival despite advances in understanding its complexity. After current standard therapeutic treatment, including tumor resection, radiotherapy and concomitant chemotherapy with temozolomide, the median overall survival of patients with this type of tumor is less than 15 months. Thus, there is an urgent need for new insights into GBM molecular characteristics and progress in targeted therapy in order to improve clinical outcomes. The literature data revealed that a number of different signaling pathways are dysregulated in GBM. In this review, we intended to summarize and discuss current literature data and therapeutic modalities focused on targeting dysregulated signaling pathways in GBM. A better understanding of opportunities for targeting signaling pathways that influences malignant behavior of GBM cells might open the way for the development of novel GBM-targeted therapies.

Highly Efficient Antibacterial Polymer Composites Based on Hydrophobic Riboflavin Carbon Polymerized Dots.

Development of new types of antimicrobial coatings is of utmost importance due to increasing problems with pathogen transmission from various infectious surfaces to human beings. In this study, new types of highly potent antimicrobial polyurethane composite films encapsulated by hydrophobic riboflavin-based carbon polymer dots are presented. Detailed structural, optical, antimicrobial, and cytotoxic investigations of these composites were conducted. Low-power blue light triggered the composites to eradicate Escherichia coli in 30 min, whereas the same effect toward Staphylococcus aureus was reached after 60 min. These composites also show low toxicity against MRC-5 cells. In this way, RF-CPD composites can be used for sterilization of highly touched objects in the healthcare industry.

SOX transcription factors and glioma stem cells: Choosing between stemness and differentiation.

Glioblastoma (GBM) is the most common, most aggressive and deadliest brain tumor. Recently, remarkable progress has been made towards understanding the cellular and molecular biology of gliomas. GBM tumor initiation, progression and relapse as well as resistance to treatments are associated with glioma stem cells (GSCs). GSCs exhibit a high proliferation rate and self-renewal capacity and the ability to differentiate into diverse cell types, generating a range of distinct cell types within the tumor, leading to cellular heterogeneity. GBM tumors may contain different subsets of GSCs, and some of them may adopt a quiescent state that protects them against chemotherapy and radiotherapy. GSCs enriched in recurrent gliomas acquire more aggressive and therapy-resistant properties, making them more malignant, able to rapidly spread. The impact of SOX transcription factors (TFs) on brain tumors has been extensively studied in the last decade. Almost all SOX genes are expressed in GBM, and their expression levels are associated with patient prognosis and survival. Numerous SOX TFs are involved in the maintenance of the stemness of GSCs or play a role in the initiation of GSC differentiation. The fine-tuning of SOX gene expression levels controls the balance between cell stemness and differentiation. Therefore, innovative therapies targeting SOX TFs are emerging as promising tools for combatting GBM. Combatting GBM has been a demanding and challenging goal for decades. The current therapeutic strategies have not yet provided a cure for GBM and have only resulted in a slight improvement in patient survival. Novel approaches will require the fine adjustment of multimodal therapeutic strategies that simultaneously target numerous hallmarks of cancer cells to win the battle against GBM.

SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis.

The SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the Sox/SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed.

Bis-Bibenzyls from the Liverwort Pellia endiviifolia and Their Biological Activity.

Based on previous investigations where bis-bibenzyls isolated from liverworts showed various biological activities (cytotoxic, antimicrobial, and antiviral), we investigated their cytotoxic activity in several human cancer cell lines. From the methylene-chloride/methanol extract of the liverwort Pellia endiviifolia, three bis-bibenzyls of the perrottetin type were isolated, namely perrottetin E, 10'-hydroxyperrottetin E, and 10,10'-dihydroxyperrottetin E. The last two were found for the first time in this species. Their structures were resolved using 1D and 2D NMR, as well as by comparison with data in the literature. Cytotoxic activity of the isolated compounds was tested on three human leukemia cell lines, HL-60 (acute promyelocytic leukemia cells), U-937 (acute monocytic leukemia cells), and K-562 (human chronic myelogenous leukemia cells), as well as on human embryonal teratocarcinoma cell line (NT2/D1) and human glioblastoma cell lines A-172 and U-251, and compared to the previously isolated bis-bibenzyls (perrottetins) of similar structure. The isolated compounds exhibited modest activity against leukemia cells and significant activity against NT2/D1 and A-172. Overall, the most active cytotoxic compounds in this investigation were perrottetin E (1), isolated in this work from Pellia endiviifolia, and perrottetin F phenanthrene derivative (7), previously isolated from Lunularia cruciata and added for a comparison of their cytotoxic activity.

Facile Synthesis of L-Cysteine Functionalized Graphene Quantum Dots as a Bioimaging and Photosensitive Agent.

Nowadays, a larger number of aggressive and corrosive chemical reagents as well as toxic solvents are used to achieve structural modification and cleaning of the final products. These lead to the production of residual, waste chemicals, which are often reactive, cancerogenic, and toxic to the environment. This study shows a new approach to the modification of graphene quantum dots (GQDs) using gamma irradiation where the usage of reagents was avoided. We achieved the incorporation of S and N atoms in the GQD structure by selecting an aqueous solution of L-cysteine as an irradiation medium. GQDs were exposed to gamma-irradiation at doses of 25, 50 and 200 kGy. After irradiation, the optical, structural, and morphological properties, as well as the possibility of their use as an agent in bioimaging and photodynamic therapy, were studied. We measured an enhanced quantum yield of photoluminescence with the highest dose of 25 kGy (21.60%). Both S- and N-functional groups were detected in all gamma-irradiated GQDs: amino, amide, thiol, and thione. Spin trap electron paramagnetic resonance showed that GQDs irradiated with 25 kGy can generate singlet oxygen upon illumination. Bioimaging on HeLa cells showed the best visibility for cells treated with GQDs irradiated with 25 kGy, while cytotoxicity was not detected after treatment of HeLa cells with gamma-irradiated GQDs.

PBX1 and MEIS1 up-regulate SOX3 gene expression by direct interaction with a consensus binding site within the basal promoter region.

Sox3/SOX3 [SRY (sex determining region Y)-box 3] is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. We have previously reported characterization of the SOX3 promoter and demonstrated that the general transcription factors NF-Y (nuclear factor-Y), Sp1 (specificity protein 1) and USF (upstream stimulatory factor) are involved in transcriptional regulation of SOX3 promoter activity. In the present study we provide the first evidence that the TALE (three-amino-acid loop extension) transcription factors PBX1 (pre-B-cell leukaemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue) participate in regulating human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus PBX/MEIS-binding site, which is conserved in all mammalian orthologue promoters analysed. PBX1 is present in the protein complex formed at this site with nuclear proteins from uninduced cells, whereas both PBX1 and MEIS1 proteins were detected in the complex created with extract from RA (retinoic acid)-induced NT2/D1 cells. By functional analysis we also showed that mutations of the PBX1/MEIS1-binding sites resulted in profound reduction of SOX3 promoter responsiveness to RA. Finally, we demonstrated that overexpressed PBX1 and MEIS1 increased endogenous SOX3 protein expression in both uninduced and RA-induced NT2/D1 cells. With the results of the present study, for the first time, we have established a functional link between the TALE proteins, PBX1 and MEIS1, and expression of the human SOX3 gene. This link is of particular interest since both TALE family members and members of the SOX superfamily are recognized as important developmental regulators.

Comparative analysis of SOX3 protein orthologs: Expansion of homopolymeric amino acid tracts during vertebrate evolution.

To understand more fully the structure and evolution of the SOX3 protein, we comparatively analyzed its orthologs in vertebrates. Since complex disorders are associated with human SOX3 polyalanine expansions, our investigation focused on both compositional and evolutionary analysis of various homopolymeric amino acid tracts observed in SOX3 orthologs. Our analysis revealed that the observed homopolymeric alanine, glycine, and proline tracts are mammal-specific, except for one polyglycine tract present in birds. Since it is likely that the SOX3 protein acquired additional roles in brain development in Eutheria, we might speculate that development of novel brain functions during the course of evolution was affected, at least in part, by such structural-functional changes in the SOX3 protein.

Graphene quantum dots as singlet oxygen producer or radical quencher - The matter of functionalization with urea/thiourea.

Due to their low cost and possible green synthesis, high stability and resistance to photobleaching, graphene quantum dots (GQDs) can be considered as one of the class of carbon nanomaterials which may have great potential as an agent for photosensitized oxygen activation. In such a way, GQDs can be used as a theranostic agent in photodynamic therapy. In this work pristine GQDs, GQDs irradiated with gamma rays and GQDs doped with N and N, S atoms are produced using a simple, green approach. By using different techniques (AFM, HR-TEM, SEM-EDS, FTIR, XRD, PL and UV-Vis) we investigated structural and optical properties of the new types of GQDs. We showed that GQDs functionalized with thiourea (GQDs-TU) completely lost the ability to produce singlet oxygen (1O2) upon photoexcitation while functionalization with urea (GQDs-U) improves the capability of GQDs to produce 1O2 upon the same conditions. Thus, presented GQDs modification with urea seems like a promising approach for the production of the efficient photosensitizer. On the opposite, GQDs-TU are efficient OH quencher. Due to high singlet oxygen production and low cytotoxicity below 100 µg/mL against HeLa cells, GQDs-U is a good candidate as an agent in photodynamic therapy at this concentration.

Graphene oxide size and structure pro-oxidant and antioxidant activity and photoinduced cytotoxicity relation on three cancer cell lines.

Photoactive materials called photosensitizers can be used for treatment of different types of cancer in combination with light source. In this paper, we have investigated pro-oxidant and antioxidant potentials of four graphene based nanomaterials (graphene oxide-GO, graphene quantum dots-GQDs, carbon quantum dots-CQDs and N-doped carbon quantum dots-N-CQDs) depending on the presence/absence of visible light source. Structural and optical properties of these materials and their potentials for reactive oxygen species generation/quenching are investigated by applying different microscopy and spectroscopy techniques (transmission electron microscopy, FTIR, UV-Vis, photoluminescence, electron paramagnetic resonance). Results show that all types of quantum dots has pro-oxidant and antioxidant potentials whereas GO demonstrated only moderate antioxidant effect. The best free radical scavenger is CQDs sample in the absence of light. CQDs are the best singlet oxygen generator under blue light irradiation as well. To check photo-cytotoxicity of these materials, photo-cytotoxic concentrations of the GO, GQDs, CQDs and N-CQDs were determined for three cellular lines: human rhabdomyosarcoma (RD), cell line derived from human cervix carcinoma Hep2c (HeLa) and fibroblast cell line from murine (L2OB). Cytotoxicity test has indicated that all samples are much less photocytotoxic than cis-diamminedichloroplatinum (cis-DPP). The production method and doping of quantum dots affect the photodynamic activity of tested samples very much.

Comparison of promoter regions of SOX3, SOX14 and SOX18 orthologs in mammals.

SOX proteins constitute a large family of diverse and well conserved transcription factors implicated in the control of various developmental processes. Previously we have cloned and characterized human SOX3, SOX14 and SOX18 genes and performed functional characterization of their promoter regions. To better understand organization and function of SOX3, SOX14 and SOX18 promoters and to determine evolutionary conserved regulatory regions, we performed comparative genomic analyses of orthologous genes promoters. Mammalian orthologs of the human SOX3, SOX14 and SOX18 genes show high sequence identity in their promoter regions, particularly within basal promoters of the respective human genes. Binding sites for transcription factors NF-Y, Sp1 and USF1, previously shown to play critical roles in transcriptional regulation of these human genes, are highly conserved in sequence and position among diverse mammalian species. Conservation of binding sites might indicate their highly significant roles in maintaining the transcriptional regulation of these genes among different species.

Does Dietary Provision of Guanidinoacetic Acid Induce Global DNA Hypomethylation in Healthy Men and Women?

BACKGROUND/AIMS: Guanidinoacetic acid (GAA) is an experimental dietary additive and has been reported to induce methyl depletion when provided by the diet. However, no study evaluated whether supplemental GAA affects DNA methylation, a critical epigenetic process for genome regulation. METHODS: In this open-label, repeated-measure interventional trial, we evaluated the impact of 12 weeks of GAA supplementation on global DNA methylation in 14 healthy participants (8 women and 6 men, age 22.2 ± 2.3 years, body mass index 24.8 ± 5.7). RESULTS: Dietary provision of GAA had no effect on global DNA methylation, with 5-methylcytosine (m5C) nonsignificantly increased by 13.4% at postadministration when averaged across participants (95% confidence interval -5.5 to 32.3; p = 0.26). Notable DNA hypomethylation (corresponding to a 5% drop in m5C) was found in 3 of 14 participants at follow-up. CONCLUSION: Global DNA methylation seems to be unaltered by dietary provision of 3 g of GAA per day for 12 weeks in healthy men and women.

Benzothiazole carbamates and amides as antiproliferative species.

A series of new benzothiazole-based carbamates and amides were synthesized and their antiproliferative activity was determined. Derivatives with profound activity were identified and further investigated for their possible mechanism of action. It was found that these compounds induce specific apoptosis, G2/M cell cycle arrest and decrease ROS level in MCF-7 human breast cancer cell line. Moreover, submicromolar antiproliferative activity of examined carbamates against NT2/D1 testicular embryonal carcinoma was shown. The most potent derivatives strongly inhibited NT2/D1 cell migration and invasiveness.

Regulation of SOX3 gene expression is driven by multiple NF-Y binding elements.

The presented results demonstrate that human SOX3 promoter possesses three CCAAT box control elements involved in the regulation of SOX3 gene expression in NT2/D1 cells. By mutational analysis we have shown that all three elements are of functional relevance for constitutive SOX3 expression. Electrophoretic mobility shift assays indicate that the active complexes at three sites involve the ubiquitously expressed CCAAT binding protein NF-Y. The involvement of NF-Y in the up-regulation of SOX3 expression in NT2/D1 cells was demonstrated in vivo by Northern and Western blot analyses. Furthermore, in co-transfection experiments we have shown that NF-Y mediates transcriptional activation of SOX3 promoter. Our data indicate that multiple CCAAT control elements are involved in the regulation of the SOX3 promoter, suggesting that NF-Y functions as a key regulator of SOX3 gene expression. Further, our results indicate that these elements can be recognized as modulators of retinoic acid induced activation of SOX3 expression.