The mucus that coats the gastrointestinal tract of all mammals is a dynamic and sticky gel layer and represents the first protective barrier between the host and the hostile environment. There is, however, a lack of detailed knowledge about the mucus gel organization because of the high water content and the complexity of MUC2, the main gel-forming molecule in the intestine. Histological staining and a multilabel immunofluorescence method were used to examine mucus blankets and Muc2 in mouse colon and ileum samples fixed in Carnoy's solution, unveiling an extraordinarily complex but highly structured mucus gel organization. The inner firmly adherent mucus blanket consists of alternating layers. The thicker outer loosely adherent mucus blanket in the colon is made of alternating laminated layers and loose curl-like structures. The layers consist of Muc2 molecules with different fucosylation states and glycoforms remain unmixed in the mucus. Importantly, distinct goblet cell subpopulations throughout the ileum along the crypt-to-villus axis with an alternation of goblet cells secreting fucosylated and non-fucosylated Muc2 are observed. A better understanding of the mucus structure should contribute to improve the efficiency of DNA and drug delivery and will allow for a better understanding and treatment of inflammatory and infectious intestinal diseases. O-glycosylated extracellular portion may be released into the mucus-gel by proteolysis  axis has been observed The gastrointestinal mucus is an essential complex gel-like secretion that protects the stomach and duodenum against acidic juice, prevents tissue dehydration, lubricates the gastrointestinal tract for the movement of foodstuffs and represents the first innate line of defense against the hostile environment. Its physical properties are dictated mainly by secreted gel-forming mucin MUC2 molecules libitum. Animals were euthanized by cervical dislocation. All the experiments were conducted in agreement with guidelines and the Animal Care Committee.Animal tissues were obtained from 6-wk-old C57BL/6 mice  that were housed in microisolator cages in a specific pathogen-free animal facility and maintained on pelleted rodent chow and water ad Ulex europaeus agglutinin I (UEA1), which recognizes H type 2 epitopes  and the lectin Maackia amurensis agglutinin (MAA), which recognizes the epitope sialic acid α-2,3-galactose, conjugated directly to TRITC  were used at 25 µg/ml. Sections used for immunofluorescence experiments were incubated with Hoechst 33258 (1∶1000) to stain nuclei. Images are representative of 10 fields randomly chosen per colon and per ileum using 8 C57BL/6 mice.Because of the high water content of mucus, tissues were fixed in Carnoy's solution, which provides the most satisfactory preservation of the mucus layers in paraffin blocks Multilabel immunofluorescence analysis was performed on a Leica DM4000B or a Zeiss LSM 710 confocal microscope. Images were acquired and minimally processed by importing them into the GNU image manipulation program GIMP.Histological staining and immunofluorescence imaging of mouse biopsies using an anti-MUC2 antibody in combination with lectins offers for the first time an unprecedented glimpse into the well-organized mucus layer of the gastrointestinal tract. Using AB–PAS staining, the colonic inner mucus blanket, which is attached more consistently to the apical surface of the epithelial cells than the outer mucus blanket, consisted of a tight, multilayered structure, while the thick, weakly adherent outer mucus blanket, which is more difficult to preserve, was made of alternating laminated layers and loose curl-like structures . A methoFucosylated and sulfated oligosaccharides are abundant in the carbohydrate moieties of the mouse Muc2 Histological analysis of mouse ileum using AB–PAS showed a mix of neutral and acidic mucins in goblet cells and in the mucus, with a compact mucus blanket coating the mucosa and a loose mucus layer between villi . ImportaMucus gel is the first innate line of defense that also acts as an efficient semipermeable barrier in the digestive tract. This dynamic, sticky, insulating blanket made of many layers with unique, distinct biochemical properties is highly structured. A better understanding of mucus architecture should help to understand and prevent pathogen entry, to improve the engineering of particle-based systems to better penetrate the mucus layer for drug delivery and gene therapy and, alternatively, to permit reinforcement of its properties against pathogens and chemical substances.


Hexose sugars, such as glucose and fructose produced in plants, are ubiquitous in most organisms and are the origin of most of the organic matter found in nature. To be utilized, hexose sugars must first be phosphorylated. The central role of hexose-phosphorylating enzymes has attracted the attention of many researchers, leading to novel discoveries. Only two families of enzymes capable of phosphorylating glucose and fructose have been identified in plants; hexokinases (HXKs), and fructokinases (FRKs). Intensive investigations of these two families in numerous plant species have yielded a wealth of knowledge regarding the genes number, enzymatic characterization, intracellular localization, and developmental and physiological roles of several HXKs and FRKs. The emerging picture indicates that HXK and FRK enzymes found at specific intracellular locations play distinct roles in plant metabolism and development. Individual HXKs were shown for the first time to be dual-function enzymes – sensing sugar levels independent of their catalytic activity and controlling gene expression and major developmental pathways, as well as hormonal interactions. FRK, on the other hand, seems to play a central metabolic role in vascular tissues, controlling the amounts of sugars allocated for vascular development. While a clearer picture of the roles of these two types of enzymes is emerging, many questions remain unsolved, such as the specific tissues and types of cells in which these enzymes function, the roles of individual HXK and FRK genes, and how these enzymes interact with hormones in the regulation of developmental processes. It is anticipated that ongoing efforts will broaden our knowledge of these important plant enzymes and their potential uses in the modification of plant traits. These T) Figure  (Dennis ) Figure .In the chloroplast, ADP-G is used for starch synthesis, to store extra sugar and to form a pool of reserve carbohydrates. The starch stored in the chloroplast can be degraded into the disaccharide [α1 → 4) glucose–glucose] maltose, glucose, or G1P, but probably only glucose and maltose are exported (via specific carriers) to the cytoplasm , which are galactosyl derivatives of sucrose that contain one or more galactose moieties  or sucrose synthase (SUS), the only two families of sucrose-cleaving enzyme that have been identified in plants , and fructokinases (FRKs). HXKs and FRKs were first purified from protein extracts of sink and source tissues by ion-exchange chromatography in the early 1950s  FRK (StFRK) was the first hexose-phosphorylating gene to be isolated from a plant  , three HXK and three HXK-like (HKL) genes have been isolated from Arabidopsis  and 10 HXK genes have been isolated from rice (Oryza sativa) , tomato (four HXK and four FRK genes), potato (a single FRK gene), Solanum chacoense (a wild relative of the cultivated potato), sunflower , Sorghum bicolor, grape , rice (10 genes), and Physcomitrella and Sellaginella (three HXK genes each)  , tobacco (Nicotiana tabacum), tomato, rice, and Arabidopsis within plastid stroma  SoHXK1 are associated with mitochondria  or an N-terminal membrane anchor domain (type B)  tissues, HXK and FRK might be required during both dark and light periods. Nevertheless, temporal and spatial expression patterns of HXKs and FRKs do not necessarily conform to these theoretical expectations. It appears that HXK and FRK genes are expressed in both source and sink tissues regardless of the time of day . Another exception is the tobacco plastidic HXK2. Promoter expression analysis of the tobacco HXK2, in which GUS was used as a reporter gene, indicated that it is expressed mainly in cells of the vascular starch sheath and xylem parenchyma, guard cells and root tips  were uncoupled  mutant background  enzyme  of exogenous glucose  and ABA-insensitive (abi) mutants  mutants gin1/aba2 and gin5/abi4 does not restore the glucose sensitivity, indicating that ABA acts downstream of AtHXK1 and is required for AtHXK1-mediated glucose responses  phenotype was also displayed in constitutive ethylene biosynthesis (eto1) and constitutive ethylene signaling (ctr1) mutants  of glucose, that connection was uncoupled at low (2%) concentration of glucose in absence of nitrogen source  prevented inhibition of cotyledon greening and seedling development at high concentrations of glucose in wild type seedlings  and the 19S regulatory particle of the proteasome subunit (RPT5B) , AtHXK1 accelerates senescence, enhances the appearance of lateral buds, affects root growth, closes stomata, and controls transpiration  in the gin2 null mutant background displayed substantial leaf expansion when grown in soil in the presence of intense light. Therefore, it has been suggested that catalytically inactive AtHXK1 mutants support both growth-inhibiting and growth-promoting roles of AtHXK1 under different growth conditions  was showed to be dependent on auxin as well  HXKs. Expression of potato StHXK1 and StHXK2 and rice OsHXK5 and OsHXK6 in Arabidopsis plants lacking AtHXK1 (gin2 mutant) complemented glucose sensitivity, indicating their putative role in sugar-sensing  PtHXK1 and rice OsHXK5, OsHXK6 in plant protoplasts were also correlated with photosynthetic gene expression, suggesting that these enzymes play a sensing role as well  HXKs is not yet known, but was examined in a single study of the Arabidopsis plastidic HXK n et al.  and Theln et al.  demonstrIt has also been suggested that HXKs may prevent programmed cell death  resulted in reduced tuber yield  with regard to several physiological processes, primarily related to photosynthesis and photosynthetic tissues, have been documented for a few HXKs. However, the molecular mechanisms of these processes and the roles of the various HXKs in sink and other tissues are not yet understood. Specifically, the molecular and physiological nature of the interactions between HXKs and plant hormones remain unclear. Unlike some of the HXKs, there is no strong evidence that FRK plays any sugar-sensing role. Rather, FRK may function primarily in the regulation of sugar metabolism in sink and vascular tissues. Nevertheless, the roles of HXK and FRK may be dictated by the specific tissues and cell types in which they are found. Therefore, it is essential to explore in which tissues and types of cells the various HXK and FRK genes are expressed. Later on, tissue-specific modulated expression of the corresponding genes (using mutants or gene expression under specific promoters) may help clarify the roles of these enzymes in different tissues.AtHXK1 and the role of FRK in vascular development. Due to the central roles of HXK and FRK in plant physiology, it is very likely that the modulation of specific HXK or FRK isozymes in certain tissues may have profound effects on specific economic traits. For example, FRK may control the amount of sugar allocated for vascular tissues and may be used to enhance xylem and vascular development in woody plants. Similarly, HXK may be used to control transpiration in agricultural crops. We believe that these discoveries and the potential uses of these enzymes will encourage further exploration of these gene families.The study of both HXK and FRK has led to significant discoveries, such as the dual-function of the mitochondria-associated The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Longevity & Healthspan, a new BioMed Central journal, has launched a thematic series on cellular senescence and aging, a quickly evolving field critical to our understanding of the biology of aging. Aging occurs at virtually every level of complexity . Two funReplicative cellular senescence was first described in cell culture as an irreversible growth arrest triggered by the accumulation of cell divisions in human fibroblasts . It has In spite of the plethora of stimuli, either or both of two central signaling pathways leading to the activation of the p53 and tumor suppressor retinoblastoma proteins (pRB) are responsible for initiating and maintaining the senescence state. Our knowledge of the senescence phenotypes has also grown in detail, and includes the manifestations of genotoxic stress, the secretion of certain inflammatory cytokines and tissue remodeling factors, and a distinctive type of facultative heterochromatin .in vivo tumor suppression mechanism [in vivo in appreciable numbers, today it is increasingly evident that they accumulate with age as well as at sites of age-associated pathologies. Implication of cellular senescence in stem cell aging has added renewed credence for its importance in species with considerable renewable tissues. Studies in mouse models lacking p16Ink4a-positive senescent cells, as a result of p16Ink4a gene inactivation or drug-induced cell clearance, have implied a causal link between senescence and age-related functional decline of tissues and organs [Recent data have implicated cellular senescence as an important echanism . Howeverd organs . This tod organs  has raisd organs , highlig


Dynamic epigenetic regulation of neurons is emerging as a fundamental mechanism by which neurons adapt their transcriptional responses to specific developmental and environmental cues. While defects within the neural epigenome have traditionally been studied in the context of early developmental and heritable cognitive disorders, recent studies point to aberrant histone acetylation status as a key mechanism underlying acquired inappropriate alterations of genome structure and function in post-mitotic neurons during the aging process. Indeed, it is becoming increasingly evident that chromatin acetylation status can be impaired during the lifetime of neurons through mechanisms related to loss of function of histone acetyltransferase (HAT) activity. Several HATs have been shown to participate in vital neuronal functions such as regulation of neuronal plasticity and memory formation. As such, dysregulation of such HATs has been implicated in the pathogenesis associated with age-associated neurodegenerative diseases and cognitive decline. In order to counteract the loss of HAT function in neurodegenerative diseases, the current therapeutic strategies involve the use of small molecules called histone deacetylase (HDAC) inhibitors that antagonize HDAC activity and thus enhance acetylation levels. Although this strategy has displayed promising therapeutic effects, currently used HDAC inhibitors lack target specificity, raising concerns about their applicability. With rapidly evolving literature on HATs and their respective functions in mediating neuronal survival and higher order brain function such as learning and memory, modulating the function of specific HATs holds new promises as a therapeutic tool in neurodegenerative diseases. In this review, we focus on the recent progress in research regarding epigenetic histone acetylation mechanisms underlying neuronal activity and cognitive function. We discuss the current understanding of specific HDACs and HATs in neurodegenerative diseases and the future promising prospects of using specific HAT based therapeutic approaches. The human genome encodes approximately 30,000 genes—but can this relatively fixed genome explain who we are or how we behave? A wealth of accumulating evidence suggests that there is much more to genome than its linear sequence of three billion basepairs. In fact, an additional level of “instructive” information superimposed on the DNA double helix in the form of a nucleoprotein entity termed the “chromatin” defines the three dimensional structure of the genome in the cell nucleus. The core unit of the chromatin is the nucleosome, which consists of 147 bp of DNA folded around histone octomers consisting two each of the histone proteins H2A, H2B, H3, and H4 Berger, . Althougvia three major levels of epigenetic changes: (1) chemical modifications at the level of nucleotides that include DNA methylation and RNA interference (RNAi); (2) covalent post-translational modifications (PTMs) of histone proteins and incorporation of histone variants; and (3) nucleosome remodeling, referring to ATP-dependent processes that regulate the accessibility of nucleosomal DNA  and adenylyl cyclase/protein kinase A (PKA), thereby triggering a series of events that eventually converge upon ERK - HDAC 1 (histone deacetylate 1)-mSin3A repressor complex from its promoter. This suggests that DNA methylation–related chromatin remodeling may play a crucial role in activity-dependent gene regulation critical for neural plasticity ,  or long term depression (LTD), whereby the efficacy of synaptic transmission is up- or down-regulated, respectively , it is conceivable that most, if not all, chromatin modifying enzymes are targeted by signaling pathways that directly link environmental cues to gene expression , zif268 , and bdnf (brain-derived neurotrophic factor) , Alzheimer's disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), and others are multifactorial illnesses in which many as yet poorly understood pathways are affected serially and in parallel, resulting in pathologic phenotypes like cognitive decline. Recent studies have linked phenotypic as well as mechanistic features common with this array of neurodegenerative diseases to epigenetic defects Kwok, . In suppDrosophila  and histone deacetylases (HDACs) are among the best characterized chromatin modifying enzymes and represent distinct classes that, respectively, catalyze forward and reverse kinetics of lysine residue acetylation in specific histone substrates. HATs function enzymatically by transferring an acetyl group from acetyl-coenzyme A to the ∈-amino groups of histone lysine residues thereby creating a specific “histone code” for chromatin modification that in general, enhances DNA accessibility for transcription factor binding. Contrarily, HDACs attenuate transcription levels by deacetylating such lysine targets , synaptic degeneration appears to be an early event in pathogenesis with synapse loss evident in patients with early AD and mild cognitive impairment , Bdnf, Fos, and Creb. Accordingly, neuronal overexpression of HDAC2 in mice, but not that of HDAC1, decreased dendritic spine density of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells, impaired hippocampus-dependent synaptic plasticity and suppressed the expression of synaptic remodeling and plasticity genes, indicating that HDAC2 negatively regulates memory formation  and suberoylanilide hydroxamic acid (SAHA)  either individually or in combination  is an inherited genetic disorder, caused by an abnormally expanded and unstable CAG repeat (polyglutamine or polyQ expansion) within the coding region of the gene encoding the huntington (Htt) protein. One of the models for mutant huntington protein induced toxicity is based on the finding that a polyglutamine peptide encoded by the first exon of Htt (Httex1p) directly binds the acetyltransferase domains of CBP and PCAF substantia nigra pars compacta (SNc) of the ventral midbrain  is a progressive neurodegenerative disorder, characterized by degeneration and death of dopaminergic (DA) neurons in the The efficacy of restoring histone acetylation levels has also been investigated in Amylotrophic lateral sclerosis (ALS) using HDACi treatments as transcriptional dysregulation is thought to play a role in the disease pathophysiology  is the most common form of neurodegenerative disorder and dementia in the elderly. AD manifests itself on the pathological background of amyloid beta (Aβ) plaques, neurofibrillary tangles (NFTs) resulting from intraneuronal aggregates of the microtubule-associated protein, +-dependent deacetylase in a mouse model of AD reduces the production of Aβ and formation of plaques via activation of transcription of the gene encoding α-secretase. Additionally, p25/Cdk5, a kinase complex implicated in AD and other neurodegenerative disorders inhibits HDAC1 in primary rat cortical neurons, rendering these neurons susceptible to DNA damage, cell cycle reentry, and ultimately cell death , a fragment that is generated by the sequential processing of APP by β- and γ-secretases and is subsequently released into the cytoplasm , in an autophagy-dependent manner. HDAC6 has also been reported to function in the autophagic clearance of aggregated Htt proteins via retrograde transport on microtubules  by which this system impacts neuronal survival and mediates memory functions. This knowledge can then be translated to novel HAT/HDAC based therapeutic strategies for the early intervention of neurodegenerative diseases. However, a major challenge with utilization of modifiers of cellular acetylation levels is the identification of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MicroRNAs (miRNAs) are small, abundant RNA molecules that constitute part of the cell's non-coding RNA “dark matter.” In recent years, the discovery of miRNAs has revolutionised the traditional view of gene expression and our understanding of miRNA biogenesis and function has expanded. Altered expression of miRNAs is increasingly recognized as a feature of many disease states, including neurodegeneration. Here, we review the emerging role for miRNA dysfunction in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and Huntington's disease pathogenesis. We emphasize the complex nature of gene regulatory networks and the need for systematic studies, with larger sample cohorts than have so far been reported, to reveal the most important miRNA regulators in disease. Finally, miRNA diversity and their potential to target multiple pathways, offers novel clinical applications for miRNAs as biomarkers and therapeutic agents in neurodegenerative diseases. Arguably one of the most important discoveries in molecular biology in recent years has been the finding and characterization of regulatory RNAs. The majority of the human genome is transcribed, however, less than 1.5% encodes protein. A vast amount appears to be biologically active, non-coding RNAs which are often referred to as the “dark matter” of the cell Mattick, . ProgresTraditionally conditions such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS) have been considered as distinct entities, however, there is increasing evidence of clinical, pathological and genetic overlap. Neurodegenerative diseases can therefore be considered a spectrum of aetiologies culminating in a final common final pathway of neuronal cell death. The pathogenic mechanisms underlying neurodegeneration are complex, but the universal risk factor is aging and there are common themes across the disorders, including protein aggregation, neuroinflammation and mitochondrial dysfunction. There are also common challenges across these conditions including the lack of early diagnostic testing and a large proportion of patients having sporadic forms of the disease (excepting Huntington's disease). Unraveling the similarities and differences between these conditions, and understanding cell type specific vulnerability, will be key to developing new therapeutic interventions.MiRNAs are a novel class of small (18–25 nucleotides), non-coding RNA molecules predicted to post-transcriptionally regulate at least half the human transcriptome  and presenilin 2 (PSEN2), both of the latter encoding components of the γ-secretase pathway  genotype is known to have a modifying influence on the genotype and is associated with a predisposition for the disease. The vast majority of Alzheimer's disease is sporadic, with no obvious genetic component, suggesting that other mechanisms are responsible. Recent studies have demonstrated that alterations in the network of miRNAs contribute to the disease process.Less than 1% of Alzheimer's disease cases are familial, with autosomal dominant mutations described in only three genes that lead to early onset disease; Several studies have used profiling strategies to show miRNA dysregulation in Alzheimer's disease patient brain tissues [see Schonrock and Gotz  for a deDown regulation of miR-107 at an early stage of Alzheimer's disease has been observed in temporal cortex and correlated with the up regulation of BACE1 in two studies, which could impact upon Aβ production  mRNA transcripts , Forkhead Box O3a (FOXO3a), and E1A binding protein p300 (P300), which all have a role in neural apoptosis, and the acetylcholinesterase enzyme (AChE), inhibition of which is a standard treatment in Alzheimer's disease and links into the cholinergic anti-inflammatory pathway , interleukin-1 receptor-associated kinase-1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6), all associated with innate immunity and inflammatory pathways which are dysregulated in Alzheimer's disease , a key regulator of ADAM10 and therefore has the potential to impact upon Aβ metabolism  and leucine-rich repeat kinase 2 (LRRK2) for late-onset disease and Parkin (PARK2), oncogene DJ1 (DJ1) and PTEN Induced Putative Kinase 1 (PINK1) for early onset  that target components of the chaperone-mediated autophagy pathway  , transactive response DNA-binding protein (TARDBP) and fused in sarcoma (FUS)  and a larger complex of at least 17 polypeptides, including TDP-43 and FUS, with limited pri-miRNA processing activity  gene. The classical motor symptom of chorea is not present in all patients, whilst other motor features such as impaired balance or abnormal fine finger movements are more likely to interfere with the patient's quality of life. Huntington's disease patients frequently develop neuropsychiatric complications such as progressive cognitive decline, personality change and depression. Pathologically, there is severe degeneration of the corpus striatum and atrophy of several brain regions, including the caudate nucleus, putamen and globus pallidus, but also the cortex itself . REST is a transcriptional repressor that acts to silence neuronal gene expression in non-neuronal cells. In healthy neurons REST is sequestered in the cytoplasm, but in Huntington's disease there is increased nuclear translocation of REST in neurons leading to increased gene repression, which has a negative effect on survival  and saliva, at detectable levels where they are exceptionally stable and potential candidates for biomarker discovery  to miR-155 have been used to prolong survival and disease duration by 38%. miR-155 was previously identified as up regulated in SOD1 mouse and human ALS patients spinal cord tissues, in addition to patient peripheral blood cells, and is linked to altered inflammation in the disease (Butovsky et al., Effective treatment of neurodegenerative disorders will most likely require manipulation of multiple targets and biochemical pathways. The capacity of miRNAs to modify multiple targets is an attractive feature for developing therapeutic strategies in the future.Over the past two decades there has been an explosion of research focused on small non-coding RNAs, the so called “dark matter” of the cell. MiRNAs have emerged as key players in regulating gene expression and their dysregulation is common to many disease states, including neurodegeneration. The alteration of miRNA-mediated regulatory activity potentially upsets the delicate balance required for neuronal cell survival, thereby contributing to pathogenesis and disease progression Figure . In commLRRK2 in Parkinson's disease and BACE1 in Alzheimer's disease. A key feature of miRNAs is their short length, making them ideal candidates for non-biased expression profiling techniques, such as next generation sequencing. Such approaches would also address the concern of publication bias that may have affected the field to date. The challenge of unraveling complex gene regulatory networks calls for large, systematic studies of miRNAs in the CNS and continuous development of robust experimental approaches for studying miRNA function. These will need to take into account the issue of disparate CNS/brain regions with divergent cell type composition. Techniques such as laser capture microdissection and induced pluripotent stem cells, in combination with the increased availability of more sophisticated sequencing technologies, means that we can anticipate larger, non-biased, cell type enriched or specific studies of miRNAs for neurodegeneration in the near future.The studies highlighted in this review generally have small sample sizes, and results may reflect individual variability within the cohort rather than true disease specific changes in miRNA expression. In addition, many of the studies have focused on miRNA targets related to already known disease genes, such as While the clinical application of miRNAs as biomarkers and therapies in neurodegeneration is perhaps premature, the rate of discovery is promising. In an era of personalized medicine, the use of miRNA expression signatures to sub-classify neurodegenerative disease, provide markers for therapeutic effectiveness and prognosis prediction, is an attractive prospect. Despite the anticipated off-target effects which cannot be fully predicted, saturation of the miRNA biogenesis pathway and possible immune activation, miRNA-based therapy has shown promise in animal models of neurodegeneration. Considerably more groundwork is needed in terms of functional studies to characterize miRNA targets and identify the most appropriate candidates before their potential in clinic can be realized.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Taken together, these results indicate that initial myocardial responses to increased work load include alterations in the contractile properties of sarcomeres and transitory adjustment of mitochondrial bioenergetics and calcium availability. Ventricular hypertrophy is one of the major myocardial responses to pressure overload (PO). Most studies on early myocardial response focus on the days or even weeks after induction of hypertrophic stimuli. Since mechanotransduction pathways are immediately activated in hearts undergoing increased work load, it is reasonable to infer that the myocardial gene program may be regulated in the first few hours. In the present study, we monitored the expression of some genes previously described in the context of myocardial hypertrophic growth by using the Northern blot technique, to estimate the mRNA content of selected genes in rat myocardium for the periods 1, 3, 6, 12 and 48 h after PO stimuli. Results revealed an immediate switch in the expression of genes encoding alpha and beta isoforms of myosin heavy chain, and up-regulation of the cardiac isoform of alpha actin. We also detected transitory gene regulation as the increase in mitochondrial cytochrome c oxidase 1 gene expression, parallel to down-regulation of genes encoding sarco(endo)plasmic reticulum Ca Myocardial hypertrophy is associated with the high risk of cardiac mortality due to its established role in the development of cardiac failure, one of the leading causes of death world-wide [World Health Organization - 2007 Prevention of Cardiovascular Diseases: Guidelines for assessment and management of total cardiovascular risk.]. Myocardial hypertrophy is defined as an abnormal increase in heart muscle mass in response to exposure to neurohumoral factors and increased work load conditions. In both cases, the myocardium undergoes excessive growth as an adaptive response. Nevertheless, if sustained, this adaptive hypertrophy leads to dysfunctional and uncompensated hypertrophy, with heart failure as a possible outcome. For a comprehensive review of this subject, see et al, 2008). These multiple signaling pathways lead to the regulation of gene expression, among which are the so-called IEGs  and genes of the fetal program  , sodium-calcium exchanger (NCX) and cytochrome c oxidase 1 (COX1), were analyzed during a time span of 1, 3, 6, 12 and 48 h after transverse aortic constriction (TAC) in adult Wistar rats. Gene expression was studied by using Northern blot hybridization of mRNAs collected from TAC or sham-operated (control) rats to cDNA probes isolated from a commercial cDNA library . Detailed methods are presented as Supplementary Material.In this context, we report herein the transient regulation of various genes encoding essential components of cardiac cells, in the first few hours after induction of myocardial hypertrophy in rats. The expression of genes encoding alpha myosin heavy chain (α-MHC), beta myosin heavy chain (β-MHC), alpha cardiac actin (ACTC1), sarco(endo)plasmic reticulum CaAs expected for the short time span investigated here, rats that underwent the TAC procedure showed no significant increase in the left ventricular weight (LV) / body weight (BW) ratio, despite a significant increase in the systolic pressure gradient (SPG) with respect to the controls. These data are summarized in According to gene expression results and specifically LV in TAC rats, there were alterations in the mRNA levels of genes encoding components of the contractile apparatus , calcium cycling (SERCA2 and NCX), and the mitochondrial electron chain (COX1). Northern blot assays and respective densitometric readings are shown in -MHC there was a progressive down-regulation, more obvious at later periods (6 to 48 h), the β-MHC gene was progressively up-regulated after 1 h. Cardiac α-actin , was immediately up-regulated after PO, maintaining high levels of expression at all the times evaluated herein. This has never been reported before, and is contrary to skeletal alpha actin gene expression which is part of the fetal program plasmic reticulum CaThe following online material is available for this article:Detailed MethodologyThis material is made available as part of the online article from http://www.scielo.br.gmb.


While the effects of hypoxia on gene expression have been investigated in the CNS to some extent, we currently do not know what role epigenetics plays in the transcription of many genes during such hypoxic stress. To start understanding the role of epigenetic changes during hypoxia, we investigated the long-term effect of hypoxia on gene expression and DNA methylation in hippocampal neuronal cells. Primary murine hippocampal neuronal cells were cultured for 7 days. Hypoxic stress of 1% O2, 5% CO2 for 24 hours was applied on Day 3, conditions we found to maximize cellular hypoxic stress response without inducing cell death. Cells were returned to normoxia for 4 days following the period of hypoxic stress. On Day 7, Methyl-Sensitive Cut Counting (MSCC) was used to identify a genome-wide methylation profile of the hippocampal cell lines to assess methylation changes resulting from hypoxia. RNA-Seq was also done on Day 7 to analyze changes in gene transcription. Phenotypic analysis showed that neuronal processes were significantly shorter after 1 day of hypoxia, but there was a catch-up growth of these processes after return to normoxia. Transcriptome profiling using RNA-Seq revealed 369 differentially expressed genes with 225 being upregulated, many of which form networks shown to affect CNS development and function. Importantly, the expression level of 59 genes could be correlated to the changes in DNA methylation in their promoter regions. CpG islands, in particular, had a strong tendency to remain hypomethylated long after hypoxic stress was removed. From this study, we conclude that short-term, sub-lethal hypoxia results in long-lasting changes to genome wide DNA methylation status and that some of these changes can be highly correlated with transcriptional modulation in a number of genes involved in functional pathways that have been previously implicated in neural growth and development. During early mammalian development, oxygen plays a vital role in the growth and maturation of every organ system. The central nervous system is particularly dependent on oxygen for proper development and function. Hence, a hypoxic insult during development may cause significant cognitive and behavioral impediments at a later stage in life. For example, in the United States, perinatal asphyxia has an incidence of up to 8 per 1000 live births with results ranging from mild neurological difficulties to severe neonatal encephalopathy, including cerebral palsy . This raWhile the detrimental effect of short-term hypoxia on neurological development has been phenotypically characterized, the underlying mechanisms responsible for the deleterious effects continue to be elucidated. Furthermore, although it is possible that severe hypoxia in early life can cause cell death, it is also possible that hypoxia can induce sub-lethal biochemical changes, including long-lasting alterations in gene expression. It has been shown that hypoxia activates certain genes necessary for cellular survival and adaptation in hypoxic conditions as well as genes for inducing cell injury -5. For eEpigenetic mechanisms, including DNA methylation and histone modification, result in long-term changes in gene expression . EpigeneThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of California, San Diego.The mice were anaesthetized with Isoflurane by inhalation and sacrificed. All efforts were made to minimize suffering.Time-pregnant female mice were obtained from Charles River (CD-1 strain). Dissection and culturing protocol was adapted from Fath et. al., 2008 . BrieflyThe morphology of all cultured cells was assessed with microscopy, and cell death was evaluated by trypan blue exclusion assay. Images were acquired with a Zeiss microscope with AxioCam MRm camera using the Axiovision Rel 4.5 software, which was also used to determine the dendrite length and cell area before and after hypoxia treatment.The primary neuronal cells were cultured in room air condition for 48 hours, and then the cultured cells were treated with 1% O2 for 24 hours and returned to room air condition for continuing culture for another 4 days. Cells were harvested at this time point. Genomic DNA was extracted using DNeasy Blood and Tissue Kit  to survey CpG methylation using MSCC assay. Total RNA was isolated using RNeasy Mini Kit  for RNA-Seq based transcriptome profiling.The duration and severity of hypoxia was determined prior to the experiment by exposure of different hippocampal cell cultures to varying levels of oxygen during cell incubation. After 48 hours of growing at room air condition with 21% O2 and 5% O2, the cells were transferred to hypoxia chambers and treated with 1%, 2%, or 3% O2 for 24 or 48 hours, respectively. Cell death and morphological changes were assayed immediately after each treatment with trypan blue exclusion assay and microscope imaging. In addition, cellular hypoxic response was monitored by measuring transcriptional changes of hypoxia responding genes  using quantitative real-time PCR.Sequenced reads for both RNA-Seq and MSCC assays can be accessed via the NCBI Sequence Read Archive under accession number SRP028757. E. coli DNA and included in each library generation to represent 0% methylation, 33% methylated, 67% methylated and 100% methylated CpG sites. The CpG tag libraries were sequenced using Illumina Genome Analyzer IIx and returned ~1 GB of sequence partitioned into 36-nt long reads per library. The resulting sequence files were mapped to the Mus musculus reference genome (version mm9), and the data were analyzed by creating frequency histograms showing the number of times reads identified at any given CpG site. The CpG sites with at least 30 reads for both methylation sensitive and insensitive libraries combined were included for further analyses to minimize technical variations in each sample, a list of which may be found in Genome-wide cysteine methylation profiling was carried out using methyl sensitive cut counting (MSCC) assay as previously described ,17. BrieEach RNA-seq library was generated using 200ng of rRNA-depleted RNA with the Illumina®-compatible ScriptSeq™ mRNA-Seq Library Preparation Kit  according to manufacturer’s instruction with 200-250bp insertion fragments. The quality of the libraries was checked on the Bioanalyzer using a High Sensitivity DNA Chip  and quantified using the Illumina Genome Analyzer DNA library quantification kit . Two libraries for each condition (control and hypoxia-treated) were sequenced using a 2x36 cycle paired-end run on Illumina Genome Analyzer IIx. The resulting reads were mapped using the RUM alignment package  with defThe promoter region of a gene was defined as 1500bp upstream to 500bp downstream of the gene's transcription starting site (TSS). The differentially expressed genes and the changes in CpG methylation that were identified with aforementioned filtering criteria were merged to determine the association between CpG methylation and gene expression. Further filtering was imposed to exclude all the genes with promoter regions containing less than two CpG sites measured to reduce the incidence of noise. The promoter regions of the remaining genes were profiled to determine the direction of methylation change at each site, whether positive, indicating hyper-methylation relative to control, or negative, indicating hypo-methylation relative to control.We first present the morphologic changes that occur with hypoxia; we then present the effect of hypoxia on gene expression, and finally the DNA methylation results.The hypoxic condition of 1% O2 for 24 hours was found, after a number of trials at various concentrations and durations, to induce a cellular stress response with no or very little cell death (see Methods). It was found that after 24 hours of hypoxic stress, the neuronal dendritic lengths were significantly shorter than the dendritic lengths of the control cells  . HoweverWe next investigated the effects of such short-term hypoxia at the transcriptional level. RNA-Seq revealed that 369 genes were differentially expressed (>1.5-fold change) in the hypoxic cells 4 days after hypoxic exposure was removed compared to control cells. Of these 369 genes, 225 genes were upregulated and 144 genes were downregulated .Analysis of the global methylation profile of all observed CpG sites with greater than 30 reads reveals that out of 268,479 reads , there iWhen we determined the proximity of the methylated CpG sites to the transcription start sites (TSS) of the differentially expressed genes, it was clear that long-term methylation changes due to hypoxia occur predominantly near the transcription start site . AdditioWe next determined whether the changes in DNA methylation correlated with the changes in gene expression, as expected. It is known that a decrease in DNA methylation in the promoter region is correlated with an up-regulation of the related gene, while an increase in DNA methylation induces the opposite ,26. AnalWe have made several major observations in this study. First, short-term acute sublethal hypoxia caused inhibition of process and dendritic growth of hippocampal neurons, and after return to normal oxygen conditions, cells exhibited a rapid recovery of this growth. Second, this acute hypoxic exposure caused long-term differential expression of 369 genes, of which 225 genes were up-regulated. Third, 59 of the 369 differentially expressed genes displayed modified promoter methylation consistent with the previously observed relation, namely, that decreased promoter methylation correlated with increased RNA transcription and increased promoter methylation correlated to a decrease in transcription. Finally, a whole genomic view of methylation revealed a strong tendency for CpG islands to remain hypomethylated long term after the hypoxic stress was removed. Indeed, short-term, sub-lethal hypoxia results in long-lasting gene expression changes in addition to long lasting and substantial changes to DNA methylation.Analysis of hippocampal cells exposed to hypoxia revealed blunted growth during stress but accelerated growth following return to normoxia. Previously, other studies have shown that hypoxia indeed suppresses genes involved in neuronal maturation and growth ,28 but tIn this study, hypoxia resulted in more upregulated (225) than downregulated (144) genes. Also, a greater proportion of promoter and CpG islands were hypomethylated rather than hypermethylated, several days after hypoxic exposure. These findings indicate that there is a correlation between whole genome changes in methylation and gene expression, days after sub-lethal hypoxic stress. When investigating the site of methylation in the 59 genes, it was evident that methylation as an epigenetic mechanism was strongly responsible for the observed lasting changes in gene expression. While these 59 genes fit the widely-accepted paradigm of the influence of methylation on gene expression, the majority of genes that had altered expression either did not have observed methylation changes within their promoter or displayed methylation modification that were contrary to the expected paradigm. This suggests that, while methylation plays a potential role in gene regulation, it is not the only mediator, and there is still much to be discerned regarding the role of methylation in gene expression regulation. Transcription regulatory mechanisms are diverse and influenced by various environmental and chemical conditions. Histone acetyltransferases and deacetylases have been found to have a role in HIF1-dependent transcriptional induction -37. HypoOf the 59 genes predominantly impacted by DNA methylation, 4 of them that displayed upregulation of RNA and demethylated promoter regions  were found to be involved in the Wnt signaling pathway. APC and CTNNBIP1 act as negative regulators of the canonical pathway through disruption of β-catenin nuclear localization ,41. PLCB We found that RICTOR, a subunit of the mTORC2 complex in the mTOR pathway, was found to have undergone both transcriptional up-regulation and substantial promoter de-methylation in the culture subjected to hypoxia . HoweverWe conclude that acute sublethal hypoxia has a lasting impact on DNA methylation that persists days after normoxia is restored and that this modulation of methylation status, particularly in promoter regions and CpG islands, is correlated with the expression of neuronal genes involved in networks such as Wnt and mTOR that have been linked to roles regulating growth and development. The clinical significance of hypoxia to cell viability and functionality cannot be understated, and a deeper understanding of the mechanism of how hypoxia leads to changes in gene expression through epigenetic mechanisms will shed some light on the effect of hypoxia on gene expression and thus the phenotypic consequence of a stress like hypoxia.Table S1Observed genome methylation status after filtering.A listing of CCGG sites observed with MSCC assay after being filtered for reads and percentage change as described in Methods.(XLSX)Click here for additional data file.Table S2Genes with significant expression changes.A listing of genes with significant changes in expression based on RNASeq reads processed with the RUM aligner and DESeq differential expression analysis as described in Methods.(XLSX)Click here for additional data file.


Urologic tumors continue to represent a huge fraction of cancer cases in the United States, with over 376,310 estimated new diagnoses in 2013. As with many types of tumors, urologic tumors vary greatly in their phenotype, ranging from minimally invasive to malignancies possessing great metastatic potential. The increasing need for more efficient and less invasive methods of cancer detection, as well as the ability to predict severity of the disease phenotype is readily evident—yet reliable methods remain elusive in a clinical setting today. Comprehensive panels of gene clusters are being developed toward the generation of molecular signatures in order to better diagnose urologic malignancies, and identify effective treatment strategies in the emerging era of personalized medicine. In this review, we discuss the current literature on the credibility and biomarker value of such molecular signatures in the context of clinical significance relating to the pathological aggressiveness of urologic tumors —also exploiting their predictive potential in the response to treatment. In 2013, an estimated 58,610 deaths in the United States will result from prostate, bladder and kidney cancers [The use of molecular signatures to detect and determine rate of progression in various cancers represents the latest shift in moving toward identifying initial events contributing to early stage tumors, ultimately allowing for optimizing therapeutic response and treatment outcomes. Indeed the need for biomarkers with high predictive value in tumor progression to advanced disease and requiring less invasive procedures (than characterization of tumor morphology) is now higher than ever for urologic malignancies. Since the 1960’s, scoring of urologic tumors, such as the Gleason Score of prostatic tumors based largely on morphological characteristics, has been common practice. “Primitive” as the pursuit of morphological differences might seem, and with cell morphology alone proven to be an imprecise predictor of cancer progression in the past, one must recognize that factors controlling tissue architecture, cell polarity and signaling processes such as epithelial-mesenchymal-transition (EMT), invasion and cell adherence may represent attractive options within a signature collection. EMT is now viewed as an essential early step in cancer metastasis. Significantly the switching between cell phenotypes that occurs during EMT is specified by certain microRNAs, , thus enabling expansion of the molecular platforms for the pursuit of molecular signatures, towards predicting the metastatic course of cancer progression, as well as therapeutic response. In this review, we discuss the increasingly recognized value of biomarkers in molecular signatures and their impact on identification of the three major types of urologic tumors—with a focus on projecting their clinical impact in patient diagnosis and treatment.de facto standard biomarker for prostate cancer detection. However, it is common knowledge that even in the absence of elevated PSA values, prostate cancer can be present [Prostate cancer remains the second most common cancer-related death in men, and will result in more than 29,720 estimated deaths in 2013 [ present . In an eTMPRSS2:ETS gene fusions have been the focus of intense investigations, as well as a point of contention with regards to its role in prostate cancer as an indicator for the aggressive phenotype. The transmembrane serine protease TMPRSS2 is androgen regulated, and in response to ligand exposure, fuses with members of the ETS family of transcriptional activators [ERG is the most commonly fused member of the ETS family, with a TMPRSS2:ETS fusion occurring in approximately 46% of prostate cancers, in a study of needle biopsies in patients with prostatic malignancy [TMPRSS2:ETS fusions alone as prognostic indicators [TMPRSS2:ETS fusion via interaction with the androgen receptor (AR) enables a new promise for a potential role of the gene fusions in predicting the emergence of castration resistant prostate cancer (CRPC). While primary prostate cancer has several treatment options ranging from “active surveillance” to radical prostatectomy, all showing similar and extraordinarily high patient survival rates—with 5 year survival approaching 100% [Since their identification in 2005 by the pioneering studies of Arul Chinnayan [tivators . ERG is lignancy . An exacdicators . The imping 100% . AndrogeTMPRSS2:ETS fusions and the AR is the transitory dip in expression immediately after androgen deprivation therapy [TMPRSS2:ETS expression rises once again to levels seen before ADT [TMPRSS2:ETS fusions. The potential benefit to the patient and physician alike would be two-fold: firstly, serving as a more accurate indicator of prognosis; secondly, allowing the physician to better time the introduction of second-line chemotherapeutic agents.Specifically relevant to the  therapy . Howeverfore ADT  as primaet al. after exploring a 16 gene signature in CRPC, further highlighting the vital role of persistent AR signaling in CRPC [The primary target of ADT, the androgen receptor (AR) transcriptionally regulates a series of growth response genes, the intricacies of which have challenged not only their therapeutic exploitation towards the development of effective treatment strategies in CRPC, but also the identification of biomarkers better predicting tumor aggressiveness and ultimate lethality associated with advanced disease. Recent elegant studies by Heemers and colleagues documented the role of the AR in regulation of effector genes along with the transcription factor Serum Response Factor in a 158 gene signature. The resultant androgen dependent gene signature indeed directly correlated with the presence of aggressive disease, poor outcome, biochemical recurrence, as well as demonstrating the ability to accurately distinguish malignant from benign prostate tissue . Similar in CRPC .PTEN) is another genetic aberration involved in prostate cancer, as well as many other types of cancer. PTEN acts as a tumor suppressor by encoding for a phosphatase protein product which is involved in cell cycle regulation [TMPRSS2:ERG fusion, the concurrent loss of PTEN contributes to a poor prognosis in cancer in the presence of an existing TMPRSS2:ERG fusion [PTEN has been shown to directly influence tumor progression and early recurrence in both fusion positive and fusion negative tumors [PTEN, SMAD4, Cyclin D1, and SPP1 significantly outperformed Gleason scoring using data from the Physicians’ Health Survey cohort in predicting lethal metastasis, and was further improved when combined with Gleason scoring [The loss of the Phosphatase and Tensin Homolog , has been shown to be present in a subtype of prostatic tumors not containing a S fusion . The expS fusion . Unfortuof cases .et al. found a marked correlation between number of CTCs and mortality in patients with metastatic castration resistant prostate cancer; patients with >5 CTCs per 7.5 mL blood had a median survival of 7 years compared with a median survival of 4 years for those patients having <5 CTCs per 7.5 mL blood [TMPRSS2:ERG gene fusion status in CTCs with outcomes in men with CRPC. Men in this study treated with Abiraterone Acetate who demonstrated post therapeutic levels of <5 CTCs per 7.5 mL blood benefitted from a median survival of 72 weeks longer than their cohorts with post therapeutic levels of >5 CTCs per 7.5 mL [In a novel attempt to further tailor treatment to the individual patient, methods of identifying and capturing malignant cells that arise from primary or metastatic tumor sites have been developed. The cell-capture techniques of “moving targets” are constantly improving and guided towards the detection of CTCs in peripheral blood, becoming extremely valuable in the prognosis of cancer patients—in both disease recurrence and likelihood of metastatic invasion . High-comL blood . More rer 7.5 mL .) represent a unique class of molecules recently discovered and classified as small non-coding RNAs that regulate gene expression at the post-transcriptional level in eukaryotic cells. Since their discovery, miRNAs have emerged as attractive and “sophisticated in their cell-uncoded behavior” molecular species that may have therapeutic targeting value as well as diagnostic potential in tumors. These short  sequences are non-coding segments of RNA regulating approximately 60% of human genes [MicroRNAs  are more often found in low grade urothelial carcinoma (LGUC) than in high grade urothelial carcinoma (HGUC). A study in 2011 found FGFR3 mutations in 84% of LGUC, whereas a mutation was present in only 17% of HGUC samples, which did reach statistical significance [FGFR3 mutations has generally been found to be associated with low tumor grade, early stage, and low recurrence rate—all leading to a more favorable prognosis [Mutations in Fibroblast Growth Factor Receptor-3  protein family, survivin plays a role in tumor development and progression by inhibiting caspase activity . Althouget al. . An extecytology . Moreovepression .p27 gene encodes for a key cell cycle regulatory protein that interacts with cyclin-dependent kinase E towards controlling entry into the cell cycle and ultimately cell proliferation. Over a decade ago, the clinical correlation between loss of p27 protein expression and reduced survival of bladder cancer patients was first documented by Del Pizzo, et al. [SKP2 gene was highly elevated, and was once again shown to degrade p27 [SKP2 carries much promise in terms of therapeutic targeting value for the future management of bladder tumors harboring a mutation impacting p27 degradation.The , et al. . Consequ, et al. ,49. Whil, et al. . More rerade p27 . InhibitILK gene expression is correlated with tumor invasiveness of bladder cancer in humans, and plays an important role in metastasis [ILK has displayed rather significant potential as a therapeutic target in the treatment of bladder cancer. Knockdown of ILK gene expression by siRNAs provides molecular targeting promise as a novel therapeutic strategy towards targeting the multiple and occasionally functionally redundant downstream signaling effectors that play significant roles in navigating cellular processes regulating EMT, inflammation, angiogenesis and anoikis. Diminishing ILK expression and the resulting functional consequences on tumorigenesis are mechanistically dictated by the following signaling pathways, inhibition of AKT survival pathway, leading to increased apoptosis of tumor cells; reduction of expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, and upregulation of nm23-H1, a potent suppressor gene of metastasis, impairing angiogenesis, vascularity and metastasis [Integrin-linked kinase (ILK) is a signaling and scaffolding protein that regulates cell survival, proliferation, migration, and angiogenesis . ILK gentastasis , predisptastasis .et al. recently reported two novel miRNAs (miR-145 and miR-200a) present and stable in urine of bladder cancer patients. In addition to being cell-free and detectable in urine, each of the miRNAs individually demonstrated potential utility in detection and prognostic value in bladder malignancies; miR-145 demonstrated significant correlation with grade, where miR-200a served as an independent predictor of recurrence [Yun currence . FurtherRenal cell carcinoma (RCC) presents a unique challenge for clinicians and investigators alike due to the vast heterogeneity in renal malignancies. As recently as 2012, there were only six FDA approved agents for treatment of RCC, mainly targeting angiogenesis and the mammalian target of rapamycin (mTOR) signaling pathways  for the Von Hippel Lindau mutations, found in 34%–57% of Clear Cell Renal Cell Carcinoma (ccRCC), cause the dramatically reduction in breakdown of HIF-1α and promote its nuclear translocation, that subsequently transcriptionally activates the master pro-angiogenic gene, VEGF [VHL gene defects leads to greater probability of metastatic recurrence coupled with lower overall survival rates when compared to renal malignancies not possessing a VHL mutation, and has long been established [The hypoxia response signaling emerges as a therapeutic target for renal cancer via its causal imposition on tumor vascularity. In response to oxygen changes in the tumor microenvironment, hypoxia inducing factor-α induces angiogenesis . Von Hipne, VEGF . Consequablished . HIF-1α ablished . The impAnhydrase IX is strongly up-regulated in tumors under hypoxic conditions, and is partially responsible for the acidification of the extracellular environment [Fitting the mold as a marker showing relative specificity for clear cell renal carcinoma (ccRCC), CAIX has been identified as an enzyme not present in normal renal tissue, but is present in as many as 97% of ccRCC tumors . Carboniironment . Acidifiironment ,63. CAIXironment . To dateWnt pathway ultimately leads to increased levels of a critical transcriptional coactivator β-catenin; accumulation of β-catenin leads to transcriptional activation of genes regulating processes involved in malignant development, including proliferation, survival, and differentiation [Wnt signaling pathway identified the functional involvement of the VHL suppressor gene with β-catenin signaling action in tumor cells [VHL gene affects β-catenin levels in the cell—linking the Wnt signaling pathway to renal malignancy [The Wnt signaling pathway is a critical regulator of embryonic development, and serves a major role in maintaining cell homeostasis throughout adulthood . Disruptntiation . Accumulntiation . Signifior cells . Under nlignancy .HIF-2α is a protein at least partially responsible for regulating the transcription of genes related to erythropoiesis and angiogenesis, glycolysis, and vasodilation . HIF-2α The existing evidence is rather contradicting, as miR-34a has been shown to be elevated in kidney cancer, while it is markedly downregulated in other human malignancies, opening the possibility of miR-34a overexpression as a specific diagnostic marker for renal cell carcinoma . In the Advancements in technology such as high throughput DNA microarrays have revolutionized the efficiency with which researchers are able to catalogue variance in genetic expression between healthy cells and particular types of tumors. However, just as important as gathering vital information is the ability to fully interpret it—which is currently the major obstacle in applying these gene expression profiles for therapeutic use. Downstream effects not yet studied or regulatory steps not yet elucidated or fully understood remain problematic in developing effective treatments tailored to a tumor’s molecular signature. Herein lies another obstacle for clinically significant biomarkers of malignancy in the context of yet-to-be-elucidated molecular pathways—ensuring that “new” markers affecting survival are not purely a product of lead-time bias, but are relevant to carcinogenesis and represent tangible therapeutic targets.Global translational research efforts continue, with a focus on the optimization of highly-efficacious therapeutic strategies that benefit patients through the development of treatments fitted to unique genetic and pathophysiological characteristics. Considering the cellular multiplicity (in the context of the microenvironment) and the diversity of the genetic factors contributing to cancer incidence such as the patient genetics and family history, exposure to various diets, lifestyles and cellular heterogeneity that characterizes a multistep, multifactorial disease call for novel treatments tailored to the molecular dynamics of individual tumors—ultimately toward optimization of outcomes in a personalized medicine approach. Identifying the cellular and molecular landscape of individual tumors  and targeting the initial molecular events triggering manifestation of tumorigenic growth would allow effective targeted therapeutic strategies and successful clinical outcomes. Collection of gene signatures, including miRNAs panels, hold great promise in enabling investigators to proceed with an accurate diagnosis and physicians to provide patients with appropriately optimized treatment strategies selectively impairing their tumors, eventually leading to cancer prevention and improved quality of life.


Autonomic Nervous System (ANS) function has been assessed in several muscular dystrophies. In the last two decades it has been recognized a significant relationship between ANS and cardiac mortality  and Becker (BMD) muscular dystrophies are X-linked recessive disorders due to complete or partial loss of dystrophin protein. The presence of persistent sinus tachycardia, atrial and ventricular arrhythmias, sweating and chills has suggested an ANS involvement in these disorders.Yotsukura et al. first described an impairment of ANS in 55 DMD, characterized by an increase in sympathetic activity and a reduction in parasympathetic output. These abnormalities were present early in the course of the disease, when cardiopulmonary function was still essentially normal, and became more marked with the progression of the disorder. The authors demonstrated also that, although HRV tended to decrease with the progress of clinical severity, it cannot be considered a good predictor of death in DMD  neurons, combined with the dystrophin-associated muscle damage, might lead to ganglion neuron death  and the degree of left systolic impairment  is a chronic and slowly progressive, autosomal-dominant, multisystem disease characterized by a wide spectrum of clinical findings and by marked intrafamilial and interfamilial clinical variability. There are two genetically distinct form of DM, myotonic dystrophy type 1 (DM1) and type 2 (DM2) with different clinical phenotypes. The most frequent form is DM1 and on the basis of clinical severity the disorder is divided into three groups: mild, classical, and congenital. DM1 is caused by a (CTG)n expansion in the gene encoding Dystrophia-Myotonica-Protein-Kinase (DMPK), a zinc finger protein. When transcribed into CUG-containing RNA, mutant transcripts aggregate as nuclear foci that sequester RNA-binding proteins, resulting in a spliceopathy of downstream effector proteins . DM1 may be considered as a toxic RNA disease  and high-frequency component  resulted to be lower than those in the control group  is the third most frequent form of muscular dystrophy. HRV was evaluated in 55 FSHD patients and evidenced the presence of autonomic modifications, characterized by slight increase in sympathetic activity and a progressive decrease in parasympathetic output, which became more evident with the progression of the disease  is genetically heterogeneous since it may be caused by a mutation in the STA gene encoding emerin (X-linked inheritance) or as AD trait determined by mutations in the LMNA gene encoding lamin A/C. EDMD is clinically characterized by a slow progressive muscular dystrophy, joint contractures and prominent cardiomyopathy with cardiac conduction abnormalities that might cause sudden death.[123] MIBG myocardial scintigraphy in a patient with a nonsense mutation in the STA gene. MIBG is an analog of norepinephrine and is stored in presynaptic terminals of sympathetic nerves. The scintigraphy evidenced a diffuse and severe decrease in accumulation of MIBG in the heart suggesting an abnormality in the cardiac sympathetic nerve terminals in the patient. They suggested that the abnormality on I [123] MIBG myocardial scintigraphy may predict LV dysfunction in EDMD  is caused by mutation in the gene encoding the protein dysferlin. In 1994, Tomoda et al. performed accurate autonomic studies in 2 MMD affected girls, including laser doppler flowmetry, study of the component analysis of the cardiographic R-R interval and sympathetic skin response (SSR). They demonstrated marked abnormalities such as sensitive vasoconstrictive response, a suppressed peak of low frequency components, and an absence of SSR, compared to healthy controls. Sympathetic nerve blocking produced clinical improvement  is an autosomal recessive disease caused by mutation in the fukutin gene. The study of brainstem tissue of 10 patients affected by FCMD showed a notable reduction of the catecholaminergic neurons in the reticular formation, vagal nuclei, and nucleus tractus solitarius of FCMD cases compared to controls. The Authors suggested that neuronal dysfunction in the cardiovascular and respiratory centers may lead to sudden death in FCMD (Itoh et al., In conclusion, in agreement with ENMC International Workshop (van Engelen et al.,


Brain metastases are a much-feared complication of cancer. The development of brain metastases requires a malignant cell to acquire characteristics that facilitate dissemination away from the primary site, entrance into the nervous system, and establishment in the brain. This review summarizes recent work focused on the molecular derangements leading to brain metastases and outlines areas in need of greater understanding. The dissemination and growth of tumor cells distant from their site of origin is a much-feared complication of cancer. Brain metastases are particularly devastating due to their neurocognitive impact and resistance to conventional therapy, and are often perceived as a marker of end-stage cancer and imminent death. Over 150,000 patients are diagnosed with brain metastases each year in the United States, which is an increase compared to a previously reported incidence of 17,380 three decades ago . This maMore than 100 years ago, Paget’s “seed and soil” hypothesis—which proposes that the basis of metastasis involves a favorable interaction between a cancer cell (the “seed”) and an organ microenvironment (the “soil”)—established the fundamental relationship between the tumor cell and a secondary organ to support the establishment and growth of a metastasis. The process resulting in the distant colonization of tumor cells into a secondary organ is a consequence of multiple interrelated events that is classically referred to as the “metastatic cascade.” Traits that increase a tumor cell’s potential to access and establish itself in the brain and other distant organs can be acquired during this process. Earlier models depict this process as a unidirectional sequence of events—oncogenic transformation, local invasion, intravasation, survival in the circulation, extravasation, and colonization. Though the order in which the steps occur may vary among tumor types, each step is pertinent to determining a tumor cell’s fate and can be interrupted at any time by homeostatic mechanisms –18.Before reaching the central nervous system (CNS), cells of the primary tumor first acquire mutations that lead to genetic instability, sustained proliferative signaling, evasion of growth suppressors, increased invasive potential, replicative immortality, resistance to hypoxia and cell death, and some develop stem-like capabilities. Recent work suggests tumor cells that have already spread to secondary sites can reinfiltrate and colonize the primary tumor, interact with the microenvironment, acquire new genetic signatures that breed more aggressive phenotypes, and proceed to seed and colonize distant organs again –21. ThisThe blood brain barrier (BBB) can be defined anatomically by the neurovascular unit, which consists of endothelial cells, pericytes, and astrocytes. These cells function as a highly regulated unit to control blood flow in response to neural demand at the micro-anatomic level. Since the brain does not contain lymphatics, circulating tumor cells reach the brain parenchyma only via a hematogenous route. Unlike capillary structures in the majority of the body, the cerebral vasculature is unique in that continuous tight junctions and low pinocytic activity at the capillary level restrict the entrance of most macromolecules from the blood into the CNS.in vivo to regulate the water permeability across the BBB and are implicated in mediation of astrocyte end-feet attachment to the abluminal vessel surface [The tight junctions between capillary endothelial cells are composed of intricate links between various transmembrane and cytoplasmic proteins; three integral proteins—claudin, occludin, and junctional adhesion molecule—have been identified and together they form a seal, which permits free transport of only small solutes . Pericyt surface . Astrocy surface –28.There is evidence that invading metastatic cancer cells interact with all of these cell types—endothelium, pericytes, and astrocytes—to breach the BBB and gain access to the brain parenchyma . How somin vitro experiments on human breast carcinoma cell line MDA-MB-231 have identified tumor cell β1 integrins as essential to the adhesion of tumor cells to the vascular basement membrane [in vivo with brain-tropic breast cancer cell line MDA231) may also facilitate tumor-endothelium adhesion [In the breast cell line MDA-MB-435, cells adapt to the narrow cerebral capillaries by reshaping themselves into an elongated form until just before or during extravasation. This process involved expansion of the surrounding vessel wall by amassing and extending tumor cytoplasmic projections to penetrate the blood vessel wall. Although disruption of the vessel wall occurred, no significant destruction of the vascular endothelium was identified . This memembrane . Cell-toadhesion ,34.The secretion of proteolytic enzymes , cytokines , and chemokines  have been associated with the extravasation process in other cancer cell lines, and implies variation in the manner in which tumor cells exit capillary channels . In braiin vitro [Once tumor cells enter the brain parenchyma, a number of factors are released by the tumor cells and underlying brain. In response to tumor-associated migration inhibitory factor (MIF), interleukin-8 (IL-8), and plasminogen activator inhibitor-1 (PAI-1), astrocytes release various inflammatory cytokines including IL-1β, TNF-α, and IL-6, all of which have been shown to promote tumor cell proliferation in vitro –42.Some patients develop new brain metastases while their systemic disease is responding to chemotherapy, which implies that the CNS may serve as a sanctuary site for some tumor cells. This is exemplified in patients who receive trastuzumab for HER-2 positive breast cancer and those receiving gefitinib for EGFR-mutant lung adenocarcinoma ,44. One in vitro study suggests that chemoprotection against paclitaxel is an active process exploiting the actions of activated astrocytes. In response to direct contact with human breast or lung tumor cells, murine astrocytes induce upregulation of survival genes in the cancer cell such as GSTA5, BCL2L1, and TWIST1 [The penetration of large molecule therapeutics through the BBB is one obstacle to the effective treatment of CNS tumors with systemic agents, though the process may be more active than previously thought. A recent d TWIST1 ,48. ThesA potential method of circumventing the physical barrier of the BBB is to combine a cytotoxic agent with an efflux pump inhibitor. In one experiment, paclitaxel-sensitive human melanoma cells (K1735) were implanted subcutaneously and intracerebrally into immunodeficient mice. While the subcutaneous tumors regressed after administration of paclitaxel, the intracerebral tumors did not. This resistance was overcome by combining paclitaxel with HM30181A, a P-gp inhibitor, thus facilitating drug entry into the brain . This fiThe long latency between the successful treatment of systemic disease and the development of brain macrometastases suggests that tumor cells might exist within the CNS inactive for some time. This phenomenon has been demonstrated in experimental animals by injecting tagged 231-BR human breast cancer cells expressing green fluorescent protein with micron-sized iron oxide particles, making the cells detectable on MRI. The animals were then monitored with serial brain MRIs for evidence of CNS dissemination. Cells that actively divided lost ferric signal but retained fluorescence; 94% of the original cells could not be detected on MRI, 1.6% developed into macrometastases, and 4.5% retained ferric signal but did not proliferate . The staThe mechanisms that drive tumor cell dormancy are still unknown, but current research may shed more light on this and provide a pathway for prevention or treatment of brain metastases . Three uN-terminal kinase (SAPK/JNK) or p38, these genes can induce growth arrest at metastatic sites without initiating apoptosis. These genes could be critical to maintaining dormancy for prolonged periods of time, even if tumor cells have gained access and survive within a secondary organ. Further understanding of the early changes that occur in the primary tumor and events in the target organ that trigger the expression of MSGs may provide new therapeutic targets to suppress brain metastasis growth.Genetic signatures responsible for inducing quiescence are referred to as metastasis suppressor genes (MSG). The earliest MSG was identified in 1988 through differential gene expression of cell lines derived from K-1375 melanoma ,64. The HOXB9 and LEF1 alters the interactions among tumor cell, pressures within the circulation, and brain endothelium is currently unknown.Enhanced potential for the development of CNS metastases may be identified in the primary and define future therapeutic targets. For example, overexpression of human epidermal growth factor receptor 2 (HER2/neu) is predictive of a 3-fold increase in metastases to the lungs, liver, and brain compared to HER2/neu negative breast carcinomas –69. ThisOther recently described gene expression signatures may predict outcome such as the risk of organ-specific dissemination ,74. For The pathophysiology of brain metastasis is complex and dependent upon both oncogenic processes and host organ responses. Multiple mechanisms determine the ultimate development of a brain metastasis including but not limited to brain-trophic tumor cell phenotypes, tumor cell survival in the vasculature and extravasation of those cells from the bloodstream and into a host organ, and the structure and function of the BBB. Many unanswered questions remain, including the molecular basis of brain-tropism and the signals that govern dormancy of tumor cells and their subsequent proliferation after a period of quiescence. Future work will hopefully identify therapeutic targets that will prevent brain metastases from taking hold or treat established CNS metastases.


Global warming is a major threat for agriculture and food safety and in many cases the negative effects are already apparent. The current challenge of basic and applied plant science is to decipher the molecular mechanisms of heat stress response (HSR) and thermotolerance in detail and use this information to identify genotypes that will withstand unfavorable environmental conditions. Nowadays X-omics approaches complement the findings of previous targeted studies and highlight the complexity of HSR mechanisms giving information for so far unrecognized genes, proteins and metabolites as potential key players of thermotolerance. Even more, roles of epigenetic mechanisms and the involvement of small RNAs in thermotolerance are currently emerging and thus open new directions of yet unexplored areas of plant HSR. In parallel it is emerging that although the whole plant is vulnerable to heat, specific organs are particularly sensitive to elevated temperatures. This has redirected research from the vegetative to generative tissues. The sexual reproduction phase is considered as the most sensitive to heat and specifically pollen exhibits the highest sensitivity and frequently an elevation of the temperature just a few degrees above the optimum during pollen development can have detrimental effects for crop production. Compared to our knowledge on HSR of vegetative tissues, the information on pollen is still scarce. Nowadays, several techniques for high-throughput X-omics approaches provide major tools to explore the principles of pollen HSR and thermotolerance mechanisms in specific genotypes. The collection of such information will provide an excellent support for improvement of breeding programs to facilitate the development of tolerant cultivars. The review aims at describing the current knowledge of thermotolerance mechanisms and the technical advances which will foster new insights into this process. Based on the “Special Report on A2 and A1B Emissions Scenarios  are associated with most forms of stress  and the relation to the reproductive system.In addition to specific reactions, it is well known that overproduction of reactive oxygen species  thermotolerance – an inherent plant ability to survive exposure to temperatures above the optimal for growth, not preceded by acclimation to non-lethal temperature elevationsAcquired thermotolerance (adaptive) (ATT) – induced by pre-exposure to elevated but non-lethal temperatures that gives the ability to survive a subsequent severe heat stress that would be lethal in the absence of the preconditioning heat treatment. ATT is transient in nature, and enhances basal thermotolerance and heat endurance via a transition to “efficient” cellular performance when acclimatory homeostasis is reachedHeat stress response (HSR) – response to elevated temperatures impairing cell homeostasis by disturbing structural and metabolic integrity of the cellHeat Shock Proteins (HSPs) – proteins accumulated in response to elevated temperatures and function as molecular chaperones in protein folding and protectionUnfolded Protein Response – subcomponent of HSR related to protein unfolding in ER and in the cytosolCompatible solutes – low molecular weight molecules, with low inhibitory action on metabolic processes compared to other solutes, acting as osmoprotectants for the maintenance of cell volume homeostasis, but might also have chemical chaperone functionReactive oxygen species (ROS) – Reactive molecules and free radicals derived from molecular oxygen, as by-products of metabolism in mitochondria and other cellular sources with the potential to cause damage to lipids, proteins and DNA when the antioxidant capacity of the cell is exceededBox 1) due to their inherent ability to survive exposure to temperatures above the optimal for growth, but they also have the ability to acquire tolerance to otherwise lethal HS  at the cellular and molecular levels to withstand more acute and frequent heat waves. ATT can be induced by subjecting plants to a moderate level of HS , followed by recovery for a few hours. Alternatively, priming for ATT can also be induced by a slow and continuous rise in temperature as exemplified for A. thaliana  of the plant transcriptome is upregulated twofold or more . A signiin wheat , which pin wheat . The remin wheat .MBF1c, a highly conserved protein involved in different developmental and metabolic pathways in organisms ranging from yeast to humans  is required for thermotolerance  and trehalose during HS (DREB2A (dehydration-responsive element binding protein 2A) that functions upstream to HSFA3 . Each of these putative sensors is thought to activate a similar set of HSR genes leading to enhanced thermotolerance, but the relationship between the different pathways and their hierarchical order remains unclear  and a PIPK  are activated, and phosphatidic acid (PA), PIP2 (phosphatidylinositol phosphate kinase), and IP3  rapidly accumulate. For this transduction of heat-initiated signal required for PIP2 and PA accumulation, active cycling of a G protein appears necessary. This adaptation response is considered to be similar to that observed during drought stress where PLDα1 mediates ABA stomatal effects through interactions with PP2C and G protein . The redA. thaliana mutants impaired in heat sensing identified the gene ARP6 as involved in mediating responses to temperature changes  in the cytosol and the ER ) is imported into the nucleus to activate UPR target genes , and rubisco activase  in plant cells and links the HSR to NO signaling  in HSR, but also identified other proteins which play an important role for HSR.Although gene expression analyses have offered insights in understanding the mechanisms underlying HSR and thermotolerance, there is frequently poor correlation between transcript and protein levels in data from heat stressed samples, probably due to alternative splicing and/or posttranslational modifications . Organ-sS-transferase and dehydroascorbate reductase  proteins are most commonly identified to be induced during HSR and have been proposed to play role in protein degradation, protection against the effects of oxidative damage and dehydration . In addieductase , cytosoleductase  and Mn-peductase . Proteineductase . Thus, teductase . In the eductase .2 fixation upon HS, indicating potential chaperone function of EF-Tu . Heat stressed Arabidopsis plants have been found to accumulate several pyruvate- and oxaloacetate-derived amino acids, the oxaloacetate precursors fumarate and malate, some amine-containing metabolites such as β-alanine, γ-aminobutyric acid (GABA), and putrescine, as well as several carbohydrates, including sucrose, raffinose, maltose, galactinol, and myo-inositol (Box 1) and their primary function is the maintenance of cell turgor but they might also act as free-radical scavengers or chemical chaperones, by stabilizing membranes and/or proteins  and radish  in soybean improved drought and HS tolerance -pyrroline-5-carboxylate synthetase 1 gene (AtP5CS1) under the control of the HSP17.6II gene promoter had enhanced proline biosynthesis under HS conditions but a decreased thermotolerance, most likely due to higher ROS production and inhibition of ABA and ethylene biosynthesis , the ultimate enzyme of the GABA biosynthesis pathway fail to prevent the accumulation of H2O2 when plants were exposed to HS, suggesting a protective role for GABA under environmental conditions that cause oxidative stress  is the best studied. The levels of GB vary within the plant species and organs under control conditions and many plants such as oduce GB . Neverthoduce GB . The pronditions . GB alsonditions .in planta  in the process known as RNA-directed DNA methylation  and RDR2 (RNA-DEPENDENT RNA POLYMERASE 2). DCL3 (DICER-LIKE3) mediates processing of dsRNA to siRNAs, which are loaded into AGO4 (ARGONAUTE 4) effector complexes  is arising as a perspective approach to dissect mechanisms which underpin the systemic functionality on the organismic level.Finally, taking the experimental observation for manifesting the ATT as guide, where the moderate increase of the temperature has a more pronounced impact on ATT than a short term preheating, it becomes obvious that not a single drastic change but rather moderate global changes are the anticipated alterations to search for. Thus, it will be important to include the reaction of, e.g., organelles or the hormone network into the cascades of HSR to describe the global plant behavior under HS. And last, it will be important to further dissect the HSR in specifically targeted tissues, namely vegetative, and reproductive, as response mechanisms might differ considering the importance for the biological system to be aimed for the reproduction. In this context a special role for “Phaseolus vulgaris, Solanum lycopersicum, Vigna unguiculata,  Capsicum annuum) the meiotic phase of pollen development has been reported as an exceptionally thermosensitive stage of the reproductive process  and stomium). Microsporogenesis (Box 2) is initiated by meiosis of PMCs and formation of microspore tetrads, followed by microgametogenesis (Box 2) and pollen maturation which includes two subsequent mitotic divisions to form the tri-cellular pollen grain with two spermatocytes embedded in the vegetative pollen cell. Both tapetum and microspore development are essential for male fertility as documented by numerous studies on male sterile mutants  growth on the stigma, pollen tubes could not reach the ovule (Box 2) characterized by tight closure of the locules, which was shown to reduce pollen dispersal in rice and tomato  is triggered , a gene coding for a mitochondrial sHSP, LeMtHSP (TC187014), a cathepsin B-like cysteine proteinase (TC171192) and the fructose-1,6-bisphosphate aldolase gene (TC176475). Generally induced upon HS independent of the genotype are several ethylene-responsive genes in developing tomato pollen grains, including ER5 , LeHSC70/ER21 , LeJERF1 (Jasmonate and ethylene responsive factor 1) (AY044235), and LeMBF1 , as well as a gene encoding ACC synthase , has been identified as critical for thermotolerance and pollen fertility under heat as well as under drought stress (2+-permeable cation transport channels that are activated by cyclic nucleotide monophosphates (cNMPs) and deactivated by binding Ca2+/CaM  during HS . Tomato has long served as a model system for genetic studies because of a series of advantageous characteristics including a diploid, small size genome relatively to other crop species  has been established to investigate fundamental and applied aspects of thermotolerance mechanisms of tomato pollen. The aim of the network is (i) to define common protocols for the analysis of HS effects on pollen, (ii) to establish the HSR at the different levels discussed, (iii) to accompany this information by the analysis of a broad variety of heat-sensitive and heat-tolerant tomato genotypes and mutant lines, and (iv) to analyze these data in the required complexity. The combination of cell biological and molecular strategies with “-omics” technologies and bioinformatics can provide valuable information for breeding programs (Solanaceae family (To approach these central challenges, the Marie Curie Initial Training Network SPOT-ITN (programs . ISOL@ be family  will serThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.