Aging and oral squamous cell carcinoma development: the role of cellular senescence.

The gradual accumulation and inadequate renewal of senescent cells over time drive organismal aging. Senescent cells undergo altered gene expression and release inflammatory mediators collectively termed the senescence-associated secretory phenotype (SASP), which significantly contributes to a spectrum of age-related disorders, including cancer. In the context of carcinogenesis, the SASP produced by senescent cells has been implicated in the promotion of epithelial cancers, including oral squamous cell carcinoma (OSCC), the most common form of oral cancer. Senescent cells within the tumor microenvironment release factors that amplify the growth and invasiveness of neighboring cancer cells. Senotherapeutics, including senolytics and senomorphics, emerge as promising modalities to target senescent cells and their associated inflammatory factors, thereby opening novel avenues for augmenting the efficacy of cancer treatments. Here, we review the general aspects of cellular senescence, focusing on the relation between senescence-related inflammation with cancer development. We also analyze the available evidence linking cellular senescence with OSCC, highlighting possible clinical applications.

Bioinformatic analysis predicts that ethanol exposure during early development causes alternative splicing alterations of genes involved in RNA post-transcriptional regulation.

Prenatal ethanol exposure is associated with neurodevelopmental defects and long-lasting cognitive deficits, which are grouped as fetal alcohol spectrum disorders (FASD). The molecular mechanisms underlying FASD are incompletely characterized. Alternative splicing, including the insertion of microexons (exons of less than 30 nucleotides in length), is highly prevalent in the nervous system. However, whether ethanol exposure can have acute or chronic deleterious effects in this process is poorly understood. In this work, we used the bioinformatic tools VAST-TOOLS, rMATS, MAJIQ, and MicroExonator to predict alternative splicing events affected by ethanol from available RNA sequencing data. Experimental protocols of ethanol exposure included human cortical tissue development, human embryoid body differentiation, and mouse development. We found common genes with predicted differential alternative splicing using distinct bioinformatic tools in different experimental designs. Notably, Gene Ontology and KEGG analysis revealed that the alternative splicing of genes related to RNA processing and protein synthesis was commonly affected in the different ethanol exposure schemes. In addition, the inclusion of microexons was also affected by ethanol. This bioinformatic analysis provides a reliable list of candidate genes whose splicing is affected by ethanol during nervous system development. Furthermore, our results suggest that ethanol particularly modifies the alternative splicing of genes related to post-transcriptional regulation, which probably affects neuronal proteome complexity and brain function.

Transcriptome analysis of the response to thyroid hormone in Xenopus neural stem and progenitor cells.

BACKGROUND: The thyroid hormones-thyroxine (T4) and 3,5,3'triiodothyronine (T3)-regulate the development of the central nervous system (CNS) in vertebrates by acting in different cell types. Although several T3 target genes have been identified in the brain, the changes in the transcriptome in response to T3 specifically in neural stem and progenitor cells (NSPCs) during the early steps of NSPCs activation and neurogenesis have not been studied in vivo. Here, we characterized the transcriptome of FACS-sorted NSPCs in response to T3 during Xenopus laevis metamorphosis. RESULTS: We identified 1252 upregulated and 726 downregulated genes after 16 hours of T3 exposure. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that T3-upregulated genes were significantly enriched in rRNA processing and maturation, protein folding, ribosome biogenesis, translation, mitochondrial function, and proteasome. These results suggest that NSPCs activation induced by T3 is characterized by an early proteome remodeling through the synthesis of the translation machinery and the degradation of proteins by the proteasome. CONCLUSION: This work provides new insights into the dynamics of activation of NPSCs in vivo in response to T3 during a critical period of neurogenesis in the metamorphosis.

Role and Regulation of Lin28 in Progenitor Cells During Central Nervous System Development.

Lin28 is a highly conserved RNA binding protein that regulates stemness whose molecular role has been widely studied in vitro. However, the regulation and the molecular role of Lin28 during the development of the vertebrate central nervous system (CNS) in vivo are not completely understood. Here, the expression and the putative role of Lin28 in the development of the mammalian CNS are reviewed in the context of recent results showing the progressive cellular and molecular changes in neural progenitor cells. Downstream genes that may play a role during CNS development and the effect of misregulated expression of Lin28 are discussed. Evidence suggests that Lin28 promotes symmetric divisions over asymmetric divisions, increasing the number of progenitors during early neurogenesis. Future quantitative analysis of Lin28 isoforms levels and stabilities together with single cell transcriptomics data, cell cycle dynamics and cell fate analysis in Lin28 gain- and loss-of-function experiments will provide a better understanding of the molecular role of Lin28 during development.

Identification of the naphthoquinone derivative inhibitors binding site in heat shock protein 90: an induced-fit docking, molecular dynamics and 3D-QSAR study.

The combination of molecular modeling methods to identify the putative binding site of inhibitors constitutes an important tool in drug discovery. In this work, we used these analyses to understand the potent inhibitory effect of naphthoquinone derivatives on heat shock protein 90 (Hsp90), one of the proteins involved in many types of cancer. Molecular docking results indicated that some favorable interactions of key amino acid residues at the binding site of Hsp90 with these quinones would be responsible for the inhibition of Hsp90 activity. Molecular docking and molecular dynamics simulation were carried out to further understand the binding modes and the interactions between the protein and these inhibitors. The main residues of the internal cavity were Val136, Phe138, Tyr139, Val150, Trp162 and Val186. The high concordance between the docking results and 3D-QSAR contour maps gives us helpful information about the environment of the binding site. Our results provide the bases for a rational modification of new molecules based in quinone scaffold, in order to design more potent Hsp90 inhibitors, which would exhibit highly potent antitumor activity.Communicated by Ramaswamy H. Sarma.

Overexpression of Lin28a delays Xenopus metamorphosis and down-regulates albumin independently of its translational regulation domain.

BACKGROUND: Lin28 regulates stem cell biology and developmental timing. At the molecular level Lin28 inhibits the biogenesis of the micro RNA let-7 and directly controls the transcription and translation of several genes. In Xenopus, Lin28 overexpression delays metamorphosis and affects the expression of genes of the thyroid hormone (TH) axis. The TH carrier albumin, synthesized by the liver, is down-regulated in limbs and tail after Lin28 overexpression. The molecular mechanisms underlying the interaction between Lin28, let-7, and the hypothalamus-pituitary-thyroid gland (HPT) axis are unknown. RESULTS: We found that precursor and mature forms of let-7 increase during Xenopus metamorphosis. In the liver, lin28b is down-regulated and albumin is up-regulated during metamorphosis. Overexpression of a truncated form of Lin28a (Lin28aΔC), which has been shown not to interact with RNA helicase A to regulate translation, delays metamorphosis, indicating that the translational regulation domain is not required to inhibit the HPT axis. Importantly, both full length Lin28a and Lin28aΔC block the increase of albumin mRNA in the liver independently of changes in TH signaling. CONCLUSIONS: These results suggest that Lin28 delays metamorphosis through regulation of let-7 and that the decrease of the TH carrier albumin is one of the early changes after Lin28 overexpression.

Molecular Properties and Evolutionary Origins of a Parvovirus-Derived Myosin Fusion Gene in Guinea Pigs.

Sequences derived from parvoviruses (family Parvoviridae) are relatively common in animal genomes, but the functional significance of these endogenous parvoviral element (EPV) sequences remains unclear. In this study, we used a combination of in silico and molecular biological approaches to investigate a fusion gene carried by guinea pigs (genus Cavia) that is partially derived from an EPV. This gene, named enRep-M9l, encodes a predicted polypeptide gene product comprising a partial myosin9-like (M9l) gene fused to a 3' truncated, EPV-encoded replicase. We examined the genomic and phylogenetic characteristics of the EPV locus (enRep) that encodes the viral portions of enRep-M9l, revealing that it derives from an ancient dependoparvovirus (genus Dependoparvovirus) that was incorporated into the guinea pig germ line between approximately 22 and 35 million years ago (MYA). Despite these ancient origins, the regions of the enRep locus that are expressed in the enRep-M9l gene are conserved across multiple species in the family Caviidae (guinea pigs and cavies), consistent with a potential function at the amino acid level. Using molecular biological approaches, we further demonstrated that (i) enRep-M9l mRNA is broadly transcribed in guinea pig cells, (ii) the cloned enRep-M9l transcript can express a protein of the expected size in guinea pig cells in vitro, and (iii) the expressed protein localizes to the cytosol. Our findings demonstrate that, consistent with a functional role, the enRep-M9l fusion gene is evolutionarily conserved, broadly transcribed, and capable of expressing protein.IMPORTANCE DNA from viruses has been "horizontally transferred" to mammalian genomes during evolution, but the impact of this process on mammalian biology remains poorly understood. The findings of our study indicate that a novel gene has evolved in guinea pigs through fusion of host and virus genes.

A role for Lin-28 in growth and metamorphosis in Drosophila melanogaster.

Insect metamorphosis has been a classic model to understand the role of hormones in growth and timing of developmental transitions. In addition to hormones, transitions in some species are regulated by genetic programs, such as the heterochronic gene network discovered in C. elegans. However, the functional link between hormones and heterochronic genes is not clear. The heterochronic gene lin-28 is involved in the maintenance of stem cells, growth and developmental timing in vertebrates. In this work, we used gain-of-function and loss-of-function experiments to study the role of Lin-28 in larval growth and the timing of metamorphosis of Drosophila melanogaster. During the late third instar stage, Lin-28 is mainly expressed in neurons of the central nervous system and in the intestine. Loss-of-function lin-28 mutant larvae are smaller and the larval-to-pupal transition is accelerated. This faster transition correlates with increased levels of ecdysone direct target genes such as Broad-Complex (BR-C) and Ecdysone Receptor (EcR). Overexpression of Lin-28 does not affect the timing of pupariation but most animals are not able to eclose, suggesting defects in metamorphosis. Overexpression of human Lin-28 results in delayed pupariation and the death of animals during metamorphosis. Altogether, these results suggest that Lin-28 is involved in the control of growth during larval development and in the timing and progression of metamorphosis.

The heterochronic gene Lin28 regulates amphibian metamorphosis through disturbance of thyroid hormone function.

Metamorphosis is a classic example of developmental transition, which involves important morphological and physiological changes that prepare the organism for the adult life. It has been very well established that amphibian metamorphosis is mainly controlled by Thyroid Hormone (TH). Here, we show that the heterochronic gene Lin28 is downregulated during Xenopus laevis metamorphosis. Lin28 overexpression before activation of TH signaling delays metamorphosis and inhibits the expression of TH target genes. The delay in metamorphosis is rescued by incubation with exogenous TH, indicating that Lin28 works upstream or parallel to TH. High-throughput analyses performed before any delay on metamorphosis or change in TH signaling showed that overexpression of Lin28 reduces transcript levels of several hormones secreted by the pituitary, including the Thyroid-Stimulating Hormone (TSH), and regulates the expression of proteins involved in TH transport, metabolism and signaling, showing that Lin28 disrupts TH function at different levels. Our data demonstrates that the role of Lin28 in controlling developmental transitions is evolutionary conserved and establishes a functional interaction between Lin28 and thyroid hormone function introducing a new regulatory step in perinatal development with implications for our understanding of endocrine disorders.

Conservation in the involvement of heterochronic genes and hormones during developmental transitions.

Developmental transitions include molting in some invertebrates and the metamorphosis of insects and amphibians. While the study of Caenorhabditis elegans larval transitions was crucial to determine the genetic control of these transitions, Drosophila melanogaster and Xenopus laevis have been classic models to study the role of hormones in metamorphosis. Here we review how heterochronic genes (lin-4, let-7, lin-28, lin-41), hormones (dafachronic acid, ecdysone, thyroid hormone) and the environment regulate developmental transitions. Recent evidence suggests that some heterochronic genes also regulate transitions in higher organisms that they are controlled by hormones involved in metamorphosis. We also discuss evidence demonstrating that heterochronic genes and hormones regulate the proliferation and differentiation of embryonic and neural stem cells. We propose the hypothesis that developmental transitions are regulated by an evolutionary conserved mechanism in which heterochronic genes and hormones interact to control stem/progenitor cells proliferation, cell cycle exit, quiescence and differentiation and determine the proper timing of developmental transitions. Finally, we discuss the relevance of these studies to understand post-embryonic development, puberty and regeneration in humans.

Wnt/ß-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells.

The development of the central nervous system is known to result from two sequential events. First, an inductive event of the mesoderm on the overlying ectoderm that generates a neural plate that, after rolling into a neural tube, acts as the main source of neural progenitors. Second, the axial regionalization of the neural plate that will result in the specification of neurons with different anteroposterior identities. Although this description of the process applies with ease to amphibians and fish, it is more difficult to confirm in amniote embryos. Here, a specialized population of cells emerges at the end of gastrulation that, under the influence of Wnt and FGF signalling, expands and generates the spinal cord and the paraxial mesoderm. This population is known as the long-term neuromesodermal precursor (NMp). Here, we show that controlled increases of Wnt/ß-catenin and FGF signalling during adherent culture differentiation of mouse embryonic stem cells (mESCs) generates a population with many of the properties of the NMp. A single-cell analysis of gene expression within this population reveals signatures that are characteristic of stem cell populations. Furthermore, when this activation is triggered in three-dimensional aggregates of mESCs, the population self-organizes macroscopically and undergoes growth and axial elongation that mimics some of the features of the embryonic spinal cord and paraxial mesoderm. We use both adherent and three-dimensional cultures of mESCs to probe the establishment and maintenance of NMps and their differentiation.

A competitive protein interaction network buffers Oct4-mediated differentiation to promote pluripotency in embryonic stem cells.

Pluripotency in embryonic stem cells is maintained through the activity of a small set of transcription factors centred around Oct4 and Nanog, which control the expression of 'self-renewal' and 'differentiation' genes. Here, we combine single-cell quantitative immunofluorescence microscopy and gene expression analysis, together with theoretical modelling, to investigate how the activity of those factors is regulated. We uncover a key role for post-translational regulation in the maintenance of pluripotency, which complements the well-established transcriptional regulatory layer. Specifically, we find that the activity of a network of protein complexes involving Nanog, Oct4, Tcf3, and ß-catenin suffices to account for the behavior of ES cells under different conditions. Our results suggest that the function of the network is to buffer the transcriptional activity of Oct4, which appears to be the main determinant to exit pluripotency. The protein network explains the mechanisms underlying the gain and loss of function in different mutants, and brings us closer to a full understanding of the molecular basis of pluripotency.

A membrane-associated ß-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.

The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/ß-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of ß-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that ß-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of ß-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/ß-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that ß-catenin, but not its transcriptional activity, is central to pluripotency acting through a ß-catenin/Oct4 complex.

Transcriptomics using next generation sequencing technologies.

Next generation sequencing technologies may now be applied to the study of transcriptomics. RNA-Seq or RNA sequencing employs high-throughput sequencing of complementary DNA fragments delivering a transcriptional profile. In this chapter, we aim to provide a starting point for Xenopus researchers planning on starting an RNA-Seq transcriptomics study. We begin by providing a section on template isolation and library preparation. The next section comprises the main bioinformatics procedures that need to be performed for raw data processing, normalization, and differential gene expression. Finally, we have included a section on studying deep sequencing results in Xenopus, which offers general guidance as to what can be done in this model.

Characterization of small RNAs in X. tropicalis gastrulae.

Here, we report and characterize deep sequencing data and bioinformatics analysis of small RNAs from X. tropicalis gastrula. A total of 17,553,124 reads with perfect match to the genome derived from 2,616,053 unique sequences were identified. Seventy-seven percent of theses sequences were not found in previous reports from X. tropicalis oocytes and somatic tissues. Bioinformatics analyses indicate that a large fraction of the small RNAs are PIWI-interacting RNAs. Up to 23.9% of small RNAs mapped to transposable elements and 27% to genic regions. Most of the abundant transposon-derived small RNAs are found in oocyte and gastrula libraries, suggesting that transposons also need to be silenced during early embryonic development. Importantly, novel clusters of piRNAs whose expression is activated after zygotic transcription begins were identified in the genome of X. tropicalis. Additionally, miRNAs were also identified and many of them are not present in oocytes, suggesting that miRNA expression is stage-specific. To the best of our knowledge, this is the first high throughput data release and bioinformatics characterization of small RNAs during Xenopus early embryonic development.

Characterization of small RNAs in Xenopus tropicalis gastrulae.

Here, we report and characterize deep sequencing data and bioinformatics analysis of small RNAs from Xenopus tropicalis gastrula. A total of 17,553,124 reads with perfect match to the genome derived from 2,616,053 unique sequences were identified. Seventy-seven percent of theses sequences were not found in previous reports from X. tropicalis oocytes and somatic tissues. Bioinformatics analyses indicate that a large fraction of the small RNAs are PIWI-interacting RNAs. Up to 23.9% of small RNAs mapped to transposable elements and 27% to genic regions. Most of abundant transposable derived small RNAs are found in oocyte and gastrula libraries, suggesting that transposon needs to be silenced also during early development. Additionally, miRNAs were identified and many of them are not present in oocytes, suggesting that miRNA expression is stage specific. To the best of our knowledge, this is the first high throughput data release and bioinformatics characterization of small RNAs during Xenopus development.

Expression of transposable elements in neural tissues during Xenopus development.

Transposable elements comprise a large proportion of animal genomes. Transposons can have detrimental effects on genome stability but also offer positive roles for genome evolution and gene expression regulation. Proper balance of the positive and deleterious effects of transposons is crucial for cell homeostasis and requires a mechanism that tightly regulates their expression. Herein we describe the expression of DNA transposons of the Tc1/mariner superfamily during Xenopus development. Sense and antisense transcripts containing complete Tc1-2_Xt were detected in Xenopus embryos. Both transcripts were found in zygotic stages and were mainly localized in Spemann's organizer and neural tissues. In addition, the Tc1-like elements Eagle, Froggy, Jumpy, Maya, Xeminos and TXr were also expressed in zygotic stages but not oocytes in X. tropicalis. Interestingly, although Tc1-2_Xt transcripts were not detected in Xenopus laevis embryos, transcripts from other two Tc1-like elements (TXr and TXz) presented a similar temporal and spatial pattern during X. laevis development. Deep sequencing analysis of Xenopus tropicalis gastrulae showed that PIWI-interacting RNAs (piRNAs) are specifically derived from several Tc1-like elements. The localized expression of Tc1-like elements in neural tissues suggests that they could play a role during the development of the Xenopus nervous system.

SHh activity and localization is regulated by perlecan.

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.

Expression of DNA transposable elements during nervous system development: A discussion about its possible functions.

Transposable elements (retrotransposons and DNA transposons) comprise a large proportion of animal genomes, for example 20% in D. melanogaster, 36% in X. tropicalis and 45% in humans. After invading a new genome, the transposable element increases its copy number and subsequently accumulates mutations. These may eventually result in inactive copies. Until recent days transposons have been considered "junk" DNA and no clear function have been assigned for this important amount of information on genomes.

SHh activity and localization is regulated by perlecan

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.