Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens.For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses.We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies. Archived human specimens, with known clinical follow-up, represent a valuable resource, particularly for retrospective molecular studies and identification of biological markers that might be useful for risk prediction of disease or prognosis Recent studies have demonstrated that nucleic acids recovered from archived specimens are suitable for a variety of downstream genetic (genomic and transcriptomic) and epigenetic analyses Protocols for genomic DNA or total RNA extractions, from FFPE specimens, have been well documented and made available as reliable commercial kits The recovery of genomic DNA and total RNA from the same specimen has the advantage of providing matched nucleic acid fractions, from the same cells, which is extremely valuable for validations as well as for integrative studies. Maximizing DNA and RNA retrieval from a single specimen might also be very useful when using tissues that are of limited availability.whole-genome cDNA-mediated Annealing, Selection, Extension and Ligation (WG-DASL) assay from Illumina, microRNAs by qRT-PCR and expression profiling, and genomic DNA by methylation assays.In this study, we sought to determine if genomic DNA and total RNA could be effectively co-extracted from archived specimens within a single reaction. Therefore we optimized a co-extraction method using TRIzol, which is the most trusted reagent for total RNA extraction from fresh tissues, because it allows DNA/RNA phase separation and recovery from fresh tissues Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from Dr. Susan Fineberg at the Montefiore Medical Center (MMC), Bronx, NY. In accordance with OHRP Guidance on research involving coded private information or biological specimens, this study did not meet the definition of human subject research as defined by 45 CFR 46.102(f), as data/specimens were not collected specifically for the proposed research project and the data/specimens received by Dr. Loudig did not contain a code derived from individual personal information. Thus, experiments using these tissue blocks did not require further monitoring from the Albert Einstein College of Medicine Institutional Review Board (IRB), which also oversees MMC. Electrophoretic analysis and methylation analyses of genomic DNA from older FFPE benign breast tissue samples  were performed with specimens obtained from Kaiser Permanente Northwest, after approval of a pilot study entitled “Gene Methylation and Oxidative Stress in the Etiology of breast Cancer” from the ethical board, which was supervised by Dr. Thomas Rohan. IRB approval for this study was obtained from the Albert Einstein College of Medicine Institutional Review Board and from the Kaiser Permanente Northwest Institutional Review Board (Portland), which waived the requirement to obtain informed consent prior to use of these specimens. The tissue blocks were cut on a standard microtome (Leica-microsystems) to generate successive 10 µm sections. Fresh mouse tissues were recovered from dead animals after they had been sacrificed and analyzed in the laboratory of Dr. Rachel Hazan at the Albert Einstein College of Medicine. The animal use protocol was reviewed and approved by the Animal Institute Committee (AIC) of Albert Einstein College of Medicine, the institution's animal care and use committee, on 11/06/08. AIC approved the protocol for a period of 3 years from the approval date. The approved Animal Welfare Assurance (A3312-01) is on file with the Office for Laboratory Animal Welfare. Albert Einstein College of Medicine has been fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) since February 22, 1983. This protocol was renewed for a period of 3 additional years on 11/06/11. The tissues were processed with TRIzol in the laboratory of Dr. Rachel Hazan.Genomic DNA from fresh tissue (mouse) and cells (human MCF10A) was extracted using phenol/Chloroform method or TRIzol, following manufacturer's instructions .et al. 2007, total FFPE-RNA was obtained from the upper aqueous phase of TRIzol, and genomic FFPE-DNA from the lower organic phase of TRIzol Using the TRIzol-based method described in Loudig DNA-mediated Annealing, Selection, Extension and Ligation (DASL) assay.For extraction of FFPE-DNA alone, we used the QIAamp DNA FFPE kit  following manufacturer's instructions, using 24 hours proteinase K (pK) digestion. For co-extraction of FFPE-DNA and -RNA we used the Qiagen AllPrep DNA/RNA FFPE kit following manufacturer's instructions. We used the RecoverAll™ Total Nucleic Acid Isolation kit  to extract FFPE-DNA or -RNA, and following manufacturers' instructions the pK digested FFPE tissue solution was separated into two halves, with one half subjected to DNase for FFPE-RNA purification, and the other half left at 55°C for 16 hours before RNase treatment and DNA purification. FFPE-RNA alone was extracted with the High-Pure RNA Paraffin kit  for analysis on the Whole-Genome cet al. 2011 Non-tumorigenic breast epithelial MCF10A cells were obtained from Dr. Paraic Kenny at the Albert Einstein College of Medicine and they were cultured in DMEM/F12 , supplemented with 5% horse serum , hydrocortisone (0.5 µg/ml), mouse epidermal growth factor , insulin (10 µg/ml), cholera toxin  at 37°C in a humidified incubator (5% CO2). Fresh and FFPE cells were prepared as described in Loudig et al. 2011 Total RNA (200 ng) from fresh and FFPE cells was subjected to high-throughput miRNA profiling  using the Illumina miRNA platform  on 12 beadchip arrays, according to manufacturer's instructions, as described in Giricz DNA-mediated Annealing, Selection, Extension and Ligation (DASL) assay on 32 beadchip arrays, following manufacturer's instructions and according to Loudig et al. 2011 mRNA expression profiling  was performed with total RNA extracted from fresh and FFPE cells (200 ng) using the Illumina Whole genome cet al. 2011 et al. 2011 MicroRNAs miR-10a, miR-196b, miR-135b, miR-32a and miR-21 were quantified from total RNA from fresh and FFPE cells using Taqman® miRNA qRT-PCR  as described in Giricz et al 2011. RNU44 and RNU6B were used as endogenous controls for data normalization as described in Giricz et al. 2009 Methylation was assayed using the procedure described by Thompson For FFPE-RNA recovered by four different methods, TRI, QDR, AMB, and Roche, gene expression profiles were measured by WG-DASL assay. Raw expression intensities of mRNAs were normalized by quantile normalization method implemented in GenomeStudio For fresh tissue, DNA and RNA can be simultaneously but separately extracted from the lower organic and the upper aqueous phase of TRIzol, respectively . This apTRIzol-based FFPE DNA/RNA co-extraction method  and tested the four different extractions with seven different archived tissues , when compared with QDR (r> = 0.929) and AMB (r> = 0.810). We observed that AMB provided FFPE-RNA where miRNAs had the lowest correlation with fresh RNA, further validating the qRT-PCR results and indicating that this method of extraction appears to be the least suited for microRNA recovery from FFPE cells.Firstly, we sought to determine if the different FFPE-DNA/RNA extraction methods might influence miRNA expression measures. Using five miRNAs with known differential expression (from low to high) in fresh MCF10A cells  assay for high-throughput expression profiling of 24,526 genes to compare matched fresh and FFPE-RNA. Following Illumina's instructions for analysis of FFPE-RNA with the WG-DASL assay, we used FFPE-RNA recovered with the high-pure RNA paraffin kit from Roche (r> = 0.881), with greater correlation using FFPE-RNA obtained with AMB (r> = 0.908) and TRI (r> = 0.895). We noted that primers used for the WG-DASL assay span 50 nucleotides, whereas primers used to quantify ESR1, CCND2 and KRT14 spanned 62, 64 and 69 nucleotides, respectively, which might account for the decrease in expression measured in FFPE-RNA, when compared with fresh RNA.Next, we sought to determine if mRNA expression might be influenced by the FFPE-RNA extraction method and compared matched fresh and FFPE-RNA recovered by TRI, QDR, and AMB. First, we performed qRT-PCR experiments on three differentially expressed genes in fresh MCF10A cells, ESR1 for low (MCF10A cells are considered ER negative cells), CCND2 for intermediate, and KRT14 for high expression  approach for co-extraction of genomic DNA and total RNA from archived specimens within a single reaction whole-genome cDNA mediated selection extension ligation (WG-DASL) assay, from Illumina, is designed to interrogate 50 nucleotide regions by RT and PCR r> = 0.908). However, for miRNA expression analysis, we observed that AMB provided the lowest correlation ratios (r> = 0.810), between fresh and FFPE-RNA, when compared to the QDR (r> = 0.929) and TRI (r> = 0.944), suggesting that different FFPE-RNA extraction methods can quantitatively and qualitatively affect the analysis of miRNAs. However, our analyses reveal that co-extraction of DNA and RNA does not affect miRNA expression profiling results demonstrating that this approach provides high quality mRNA and miRNAs for molecular analyses.For FFPE-RNA recovery, we showed that our extraction method (TRI) is superior to the two commercial kits . Considering that RNA is highly degradable in solution and that some studies suggested that mildly degraded FFPE-RNA could still be subjected to linear amplification and conventional microarray analyses For extraction of FFPE-DNA, we selected the Qiagen QiaAmp FFPE DNA kit , as a control for yield and quality, because it has been shown to be a robust approach when compared to other methods and kits et al. [2010], in which nucleic acids released by proteinase K digestion are magnetically purified, split, and subjected to RNAse for DNA purification and DNAse for RNA purification, might be more appropriate Our analyses demonstrated that the two co-extraction methods tested (optimized TRIzol method (TRI), and Qiagen AllPrep DNA/RNA FFPE kit (QDR)) provided higher yields as well as more reliable material for molecular studies than the separate extraction method (Ambion RecoverAll™ kit (AMB)). However, advantages and disadvantages of either method should be weighted carefully. On one side, the QDR has a short pK digestion (15 minutes), and might be automated, but it might not provide the highest amounts of FFPE DNA and RNA. On the other side our optimized method (TRI) requires two digests (45 min and 48 h), use the QD kit for final purification, and is incompatible with automation (due to the use of TRIzol), but our results indicate that it provides higher genomic DNA yields when used with older archived specimens . and genIn conclusion, our study is the first to demonstrate that high-quality FFPE-DNA can be purified from the lower aqueous phase of TRIzol, without affecting optimal recovery of FFPE-RNA from the upper organic phase. We demonstrated that co-extraction of DNA and RNA from a single archived specimen is highly efficient and provides the options of direct usage or storage of material for additional or subsequent studies. Based on our experiments we advise researchers to extract genomic DNA and total RNA at the same time for effective use of archived specimens.Figure S1Co-extraction of total RNA and genomic DNA from fresh mouse tissues using TRIzol. RNA and DNA were extracted from brain, muscle, heart and liver in triplicate to determine technical reproducibility using TRIzol and following manufacturer's instructions. Based on simultaneous extractions of DNA and RNA performed using TRIzol we consistently recovered more RNA than DNA and recovery of DNA appears highly reproducible.(TIF)Click here for additional data file.Figure S2Comparison of RNA extraction between the Ambion RecoverAll™ kit (AMB) and the TRIzol-based optimized method (TRI) using archived normal and tumor human breast tissues. Total RNA was recovered from normal breast tissue (see left side of graph) and from tumor breast tissue (see right side of graph). For normal tissue (left side of graph), total RNA was extracted from two, three, and four 10 µm sections, in triplicate experiments, using either the AMB or TRI methods. For tumor tissue (left right of graph), total RNA was extracted from one, two, and three 10 µm sections, in triplicate experiments, using either the AMB or TRI methods. The average of total RNA, in micrograms, of three experiments was plotted and error bars were determined for each individual condition.(TIF)Click here for additional data file.Figure S3Electrophoretic and methylation analyses of genomic DNA recovered from older formalin-fixed paraffin-embedded benign breast disease tissue specimens. A. Analysis of 200 ng of genomic DNA recovered from 8, 13, 20, 27 and 31 year-old BBD tissue specimens using the TRIzol-based optimized method (TRI) and the Qiagen AllPrep DNA/RNA FFPE (QDR) kit. For each specimen 5× 10 µm sections were used for each method and the total amounts of genomic DNA recovered are displayed below the image of the agarose gel, showing that TRI provides at least twice the amount of DNA than QDR. The genomic DNA displays an overall degraded profile identical in both methods. B. 1% agarose gel analysis of 25  and 27  year-old breast specimens displaying significant differences in genomic DNA quality. C. and D. Methylation analyses of CpG regions of ESR1 (283 bp region) and CCND2 (261 bp region) using FFPE genomic DNA from the 25 and 27 year-old BBD tissue specimens and representative images of the PCR products on a 2% agarose gel, respectively. Lower quality genomic DNA (25 year old specimen) did not yield a PCR product indicating either absence of methylation or failed PCR reaction, possibly due to low genomic DNA quality.(TIF)Click here for additional data file.


Human choice is not free—we are bounded by a multitude of biological constraints. Yet, within the various landscapes we face, we do express choice, preference, and varying degrees of so-called willful behavior. Moreover, it appears that the capacity for choice in humans is variable. Empirical studies aimed at investigating the experience of “free will” will benefit from theoretical disciplines that constrain the language used to frame the relevant issues. The combination of game theory and computational reinforcement learning theory with empirical methods is already beginning to provide valuable insight into the biological variables underlying capacity for choice in humans and how things may go awry in individuals with brain disorders. These disciplines operate within abstract quantitative landscapes, but have successfully been applied to investigate strategic and adaptive human choice guided by formal notions of optimal behavior. Psychiatric illness is an extreme, but interesting arena for studying human capacity for choice. The experiences and behaviors of patients suggest these individuals fundamentally suffer from a diminished capacity of willful choice. Herein, I will briefly discuss recent applications of computationally guided approaches to human choice behavior and the underlying neurobiology. These approaches can be integrated into empirical investigation at multiple temporal scales of analysis including the growing body of experiments in human functional magnetic resonance imaging (fMRI), and newly emerging sub-second electrochemical and electrophysiological measurements in the human brain. These cross-disciplinary approaches hold promise for revealing the underlying neurobiological mechanisms for the variety of choice capacity in humans. What is free will?” must be addressed. Unfortunately an empirical answer to even this obvious question remains murky—narratives aimed at describing the subjective experiences associated with free will fail to pin down concrete variables for measurement and experimentation. However, discussions on the topic of free will lurk around issues related to observable choice behavior and the causes of our actions. In the extreme, no organism has complete freedom in their capacity to make choices. There are great numbers of evolved physical constraints that restrict any given organism's capacity to choose; for example, humans cannot choose to breath underwater (unassisted) no more than a fish can choose to become a doctor. Yet, we observe choice behavior all around us and this phenomenon is a very active area of research for many academic disciplines including neuroscience, economics, psychology, politics, and philosophy.A scientific perspective of “free will” must be rooted in the parts of the problem that are measureable and consistent with the wealth of empirical data. Others have discussed the need for this kind of approach and make good philosophical arguments for the progress a scientific framework promises Dennett, . But, beChoice behavior is arguably the basis for all that is interesting in humans. The impact of our choices pervades all aspects of our day-to-day existence and has long-term consequences for our planet and the life it supports. The choices individuals make are likely the basis for how we assign personality and define identity. The apparent freedom individuals possess in carrying out these decisions is a highly valued concept in many cultures. The social value of this concept and beliefs about “free will” may have evolved around the complex social development of our species. Thus, we hold others (and ourselves) accountable for our actions; and, we reward or punish individuals based on our acceptance of the choices expressed. Accountability and the choices we make in social domains assume that we possess agency and determine the course of our own actions Skyrms, . In caseMathematically explicit models have been incorporated into empirical investigations of choice behavior and are beginning to generate good hypotheses about how nervous systems deal with computations associated with various choice problems. Herein, I will briefly introduce two dominant quantitative theories that deal with choice behavior . I will discuss the impact these disciplines have already had on neurobiological investigations of human choice behavior and highlight how the integration of these disciplines is beginning to open new arenas for investigating the biological basis for choice capacity. These arenas include the diminished capacity for choice experienced by individuals with mental illness and investigation into the variation expressed by healthy decision makers. It is in the intersection of theoretical and empirical disciplines where advances in our understanding of our capacities for choice can develop. I will conclude with a depiction of possible future avenues of basic neuroscience research in the domain of human choice capacity and how these investigations may impact psychiatric medicine and more generally our conception of “free will.”A neurobiological understanding into the mechanisms underlying willful choice behavior in humans must integrate the wide range of knowledge gained from empirical studies at multiple scales of analysis. Molecular systems build neural circuits that respond to and direct the actions of a wholly integrated organism; the behavior of these systems can be measured in a wide range of spatial and temporal scales, thus complicating the job of any integrative hypothesis. Quantitative theory has had a big impact on integrating the growing body of neurobiological data, but much work remains Abbott, . Investist”, the agent is in at time “t” ) for a given state-action pair [an action (a) taken while in state (s)]. Here “E” is the expectation of the reward “rt+1” plus the discounted (γ) state-action value function ) for the next state (st+1) given some chosen action (a′); a′ is chosen to maximize Q*, for which the policy is implied. Finally, the expected value “E{·|}” is calculated given the actual state-action pair at time t . When everything is known about the decision problem  the calculation of this problem is straightforward. But, in the real world these decision problems are faced with incomplete knowledge and the theoretical work in this domain has been aimed at different solutions to variations on this problem. This framework has been deployed on a number of biological decision-making problems including those that humans face or are pitted against in experiments aimed at understanding how humans make decisions in environments with various statistical properties  is wrong due to incomplete or inaccurate information about the decision to be made . An example of this kind of problem during willful decision making has been explored in a formal manner by Dayan et al.; here, the authors present a computational theoretical depiction of the problem faced by an agent that utilizes both reflexive and contemplative mechanisms for navigating choice spaces and show how one or the other system may misestimate the value of available options  the primary tools available to investigate the underlying neurobiology of choice were highly invasive, and thus restricted primarily to non-human decision-making experiments. fMRI has opened the door to determining physiological responses associated with willful choice. Experiments aimed at understanding human decision-making have taken advantage of the theoretical frameworks developed in game theory and computational reinforcement learning theory (Montague et al., In the context of willful choice, psychiatric disorders pose a number of interesting issues. The altered behavioral profiles of patients with psychiatric disorders compared to healthy individuals suggest an amplification of the kinds of biological determinants that naturally constrain human choice. Computational psychiatry aims to characterize psychiatric illness in objectively measureable quantitative terms. Using game theoretic paradigms to investigate altered social behavior in patients diagnosed with mental disorders (Kishida et al., The computational role of reward and valuation in adaptive decision-making has been explored at the level of individual neuron activity. In 1996 Montague et al., proposed a mathematical model of dopamine neuron activity in non-human primates . NeurosComputational reinforcement learning theory and game theory have already made a significant impact in human decision neuroscience. Not only have they framed the problem of choice in quantitative terms, but also they have begun to open new areas of research in domains where human choice is severely restricted as observed in individuals diagnosed with psychiatric disorders. I argue that continuing along these lines will be important of a neuroscience of human choice capacity, or what it means to have anything like “free will.” This would include beginning with a computational definition of “free will.” Huys and Dayan have made a positive step in this direction by addressing one aspect of what it would mean in computational terms to have “control” (Huys and Dayan, The development of theory without application to, or verification with, empirical investigation is not a scientific endeavor. Indeed, the theoretical frameworks discussed herein have guided investigations into the neurobiological substrates supporting human choice. The primary tool thus far has been fMRI, however, new developments in access to human brains and supporting clinical and investigational technology is providing an unprecedented look into the neurobiology of human choice and agency. Going forward, the biology of willful choice behavior in humans is wide open for investigation and will likely make major advances in our basic understanding of the underlying neurobiology. More practically speaking, these developments will also likely have a major impact on our understanding of human mental illness—conditions where capacity for choice becomes so restrictive that individuals suffer without apparent alternatives. What it means to have “free will” isn't very clear and this concept continues to evolve, but it is clear that humans express choice and have preferences. The quantitative exploration of these concepts has provided a clarifying foundation for theoretical and empirical developments. This same framework promises to allow us to measure human choice capacity and in this context we may come to understand and exercise the freedoms we do possess.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic diseases are a leading contributor to work disability and job loss in Europe. Recent EU policies aim to improve job retention among chronically ill employees. Disability and occupational health researchers argue that this requires a coordinated and pro-active approach at the workplace by occupational health professionals, line managers (LMs) and human resource managers (HRM). Little is known about the perspectives of LMs an HRM on what is needed to facilitate job retention among chronically ill employees. The aim of this qualitative study was to explore and compare the perspectives of Dutch LMs and HRM on this issue.Concept mapping methodology was used to elicit and map statements (ideas) from 10 LMs and 17 HRM about what is needed to ensure continued employment for chronically ill employees. Study participants were recruited through a higher education and an occupational health services organization.Participants generated 35 statements. Each group (LMs and HRM) sorted these statements into six thematic clusters. LMs and HRM identified four similar clusters: LMs and HRM must be knowledgeable about the impact of chronic disease on the employee; employees must accept responsibility for work retention; work adaptations must be implemented; and clear company policy. Thematic clusters identified only by LMs were: good manager/employee cooperation and knowledge transfer within the company. Unique clusters identified by HRM were: company culture and organizational support.There were both similarities and differences between the views of LMs and HRM on what may facilitate job retention for chronically ill employees. LMs perceived manager/employee cooperation as the most important mechanism for enabling continued employment for these employees. HRM perceived organizational policy and culture as the most important mechanism. The findings provide information about topics that occupational health researchers and planners should address in developing job retention programs for chronically ill workers. Chronic diseases are increasingly prevalent among people of working age in Europe ,2. In thIn the Netherlands, the responsibility for the provision of work disability benefits has shifted from the state to the employer in 2002 . In respafter illness or disability has occurred and aims at reducing long-term disability . The stThis study identified both similarities and differences between the perspectives of Dutch LMs and HRM on conditions that may facilitate job retention for chronically ill employees. LMs and HRM identified four similar conditions: LMs and HRM must be knowledgeable about the impact of chronic disease on the employee; employees must accept responsibility for work retention; work adaptations must be implemented; and clear company policy. Two conditions were only identified by LMs: good manager/employee cooperation and knowledge transfer within the company. Unique conditions identified by HRM were: a company culture that facilitates trust and openness and organizational support. LMs perceived manager/employee cooperation as the most important mechanism for enabling continued employment for these employees. HRM perceived organizational policy and culture as the most important mechanism. These findings provide information about topics that occupational health researchers, planners and practitioners should take into consideration when developing job retention programs for chronically ill workers.LM: Line Manager; HRM: Human Resource Manager; HR: Human Resources; OHS: Occupational Health Services.The authors declare that they have no competing interests.JH designed and conducted the study, analyzed the data and wrote the paper. HK critically reviewed several drafts of the paper. MM helped to design and conduct the study, the interpretation of the data and she critically reviewed several drafts of the paper. FvD contributed the data analysis and interpretation and the drafting of the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/11/104/prepubConcept maps. Figure s1 shows the cluster map indicating perspectives of line managers on what is needed to ensure continued employment for chronically ill employees. Figure s2 shows the cluster map indicating perspectives of HRM on what is needed to ensure continued employment for chronically ill employees.Click here for file


Danio rerio, is an important vertebrate model system. We focused on the grin1b gene of zebrafish due to its important function in the nervous tissue as a glutamate receptor. Using a comparative sequence-based approach, we located possible RNA editing events within the grin1b transcript. Surprisingly, sequence analysis also revealed a new editing site which was not predicted by the comparative approach. We here report the discovery of two novel RNA editing events in grin1b transcripts of embryonic zebrafish. The frequency of these editing events and their locations within the grin1b transcript are also described.Discovering RNA editing sites in model organisms provides an insight into their adaptations in addition to finding potential sites for the regulation of neural activity and the basis of integrated models of metazoan editing with a variety of applications, including potential clinical treatments of neural dysregulation. The zebrafish,  Danio rerio) including one located in the GRIA2 gene [grin1b (NMDAR1.2). Adenosine-to-inosine RNA editing occurs primarily in components of neural function and synaptic transmission; adenosines within the targeted transcript are converted to inosine which is interpreted as guanosine during translation and manifests as A/G mixed signals in sequence chromatograms. Several editing sites have been reported in zebrafish ; the nomenclature of the editing sites incorporated an “E” designation to emphasize that embryonic tissue was used. All Danio research was done in accordance with institutional IACUC guidelines.RNA was extracted from wild-type  Reverse transcription was performed using Invitrogen MMLV-RT and associated components , primed with a polythymidylate (polyT) primer. Subsequent PCR was performed using Promega GoTaq  and specific primer sets  gel extraction kit instructions. MluI and BstNI, following NEB recommendations regarding temperature and inclusion of bovine serum albumin (BSA).Sequencing services were provided by SeqWright . Restriction digests were performed with NEB  enzymes  Intensity values based on ethidium bromide fluorescence were acquired using Kodak Gel Logic  software and adjustments as described in the text.grin1b coding sequence (CDS) against NCBI databases; substantial regions of sequence identity were detected only against Danio sequences. Danio grin1b CDS was then compared against EST sequences, and upon filtering though several hits we selected a sequence comparison containing several A–G mismatches  under the same conditions to verify the occurrence of these editing sites. As expected, these produced robust bands at the size predicted and confirmed A/G mixed signals corresponding to E5 and E6 in both new reactions (data not shown).grin1b transcript of zebrafish. For this purpose we amplified the genomic region of grin1b in the region of our candidates E5 and E6. Since single nucleotide polymorphisms (SNPs) can often be misinterpreted as RNA editing events, it was necessary to amplify and sequence the genomic grin1b regions to distinguish between these explanations for the mixed chromatographic signals; ADAR editing enzymes do not edit DNA. Therefore, we designed two new sets of primers for subsequent zebrafish PCR reactions using genomic DNA; these primers included regions from the introns bracketing the regions corresponding to E5 and E6, separately, and do not generate products when used in RT-PCR (data not shown). We conducted separate PCR reactions for E5 and E6.  The next step in our investigation was aimed at confirming these signals as editing sites, rather than single nucleotide polymorphisms, in the MluI (A/CGCGT) was chosen for site 5, and BstNI (CC/WGG) was chosen for site 6. Editing at site 5 creates an MluI restriction enzyme site (ACGCAT to A/CGCGT), while editing at site 6 creates a BstNI restriction site (CCAAG to CC/AGG). No editing at site 5 prevented MluI restriction and therefore gave a full-length 311 bp product; editing at site 5 produced 2 bands . No editing at site 6 produced 3 bands after BstNI digestion , while editing followed by BstNI digestion produced 4 bands . Incomplete editing at either site manifested as a mix of full-length and cut products for each restriction. We chose to use restriction digestion and densitometry to quantitate levels of RNA editing. Editing occurs within a transcript population and may result in the creation or destruction of a restriction enzyme site. The unedited and edited transcript forms were analyzed using New England Biolabs (NEB) and Accelrys DS Gene software, and restriction enzymes were chosen that could differentiate between editing and lack of editing in a transcript at either site 5 or site 6, separately. MluI digestion, one for BstNI, and one for an untreated control. The products were extracted from an agarose gel and purified using a Qiagen gel extraction kit and protocol. Presence of the extracted band was confirmed via gel electrophoresis, and 25 uL restriction digests were performed as per NEB recommendations. Restriction digests were analyzed following gel electrophoresis using a Kodak Gel Logic imaging station and Kodak software. The intensity of the diagnostic versus uncut bands  was analyzed and corrected for band size  PCRs from each of three independent, oligo dT-primed RTs  were used for these analyses: from each set of three reactions, one was used for http://www.mirbase.org/) for D. rerio miRNAs targeting the E5 and E6 region reveal that there are no known zebrafish miRNAs that bind in this area (within 100 bases 5′ of E6 and greater than 100 bases 3′ of E5), although we suggest the presence of additional unidentified novel miRNAs  could have an effect. A number of such possibilities for non-recoding edits are discussed by Morse and colleagues [ The positions edited occur within the reading frame of the gene at 3rd codon positions and do not result in amino acid substitutions. The positions of these edits indicate that they do not result in transcript recoding. This considerably complicates an analysis of the purpose of ADAR regulation at these loci; however, an intriguing possibility is that nonrecoding edits affect the binding of additional factors such as micro RNAs or positioning cues for ADARs. Searches of existing microRNA databases such as miRBase (lleagues . There are several avenues of future research that may elucidate the function of non-recoding editing at these positions. Secondary structures of these regions were predicted using the mfold program developed by Zuker  in consigrin1b ortholog in humans is the GRIN1 NMDA receptor gene. Several mutations in GRIN1 are associated with severe mental retardation. When tested in Xenopus oocyte systems, increased calcium entry occurred with one mutated form [grin1b in model vertebrates, such as Danio. The ted form  and the gria2 transcript of zebrafish [grin1b gene transcript of zebrafish. We also call attention to the fact that site E6 was detectable only by direct sequencing and not by comparative methods; many editing sites, especially those that are unique to a single species, may remain to be discovered. Moreover, by characterizing editing in terms of frequency and location we hope to contribute to the current knowledge of RNA editing with the goal of participating in the full elucidation of this intriguing molecular process. RNA, in its many forms, plays a pivotal role in cellular processes. The processes of RNA editing and splicing along with micro RNAs, RNA interference, snRNAs, ribozymes, and so forth certainly point to RNA as a fundamental conductor and orchestrator of genetic instructions. Although RNA editing has been widely observed in many model organisms, finding a specific editing site is still a daunting task. We are interested in discovery of new editing sites as well as regulation of those sites, in a variety of model organisms. The Department of Biomolecular Sciences contains faculty who work collaboratively with a variety of experimental systems, and the record of Kung and colleagues' discovery of RNA editing in the ebrafish  was a pr


Rhinolophus pusillus and Chaerephon plicata bats, which showed close genetic relationships with severe acute respiratory syndrome coronaviruses.To clarify the evolutionary relationships among betavoronaviruses that infect bats, we analyzed samples collected during 2010–2011 from 14 insectivorous bat species in China. We identified complete genomes of 2 novel betacoronaviruses in  The 2003 outbreak of severe acute respiratory syndrome (SARS) was caused by a novel betacoronavirus and rapidly spread globally, causing ≈8,000 cases and nearly 900 deaths (www.ncbi.nlm.nih.gov/blast) with parameters “-e 1e-5 -F T -b 10 -v 10.” No assembly was performed before alignment. Sequence similarity–based taxonomic assignments were conducted as described . The located reads were then used for reads-based nested PCR to identify genomic sequences. We established the complete genome sequences of 2 betacoronaviruses (Bat Rp-coronavirus/Shaanxi2011 and Bat Cp-coronavirus/Yunnan2011), which are 29,484 nt and 29,452 nt, respectively. The G+C content of Bat Rp-coronavirus/Shaanxi2011 and Bat Cp-coronavirus/Yunnan2011 is 41.6% and 40.9%, respectively.We estimated the approximate locations of those reads on the CoV genome and their relative distances on the basis of alignment results exported with MEGAN 4–MetaGenome Analyzer (www.ebi.ac.uk/Tools/psa/emboss_needle/) with default parameters. The overall nucleotide sequences between Bat Rp-coronavirus/Shaanxi2011 and Bat Cp-coronavirus/Yunnan2011 indicated 88.7% nt identity. They shared 87.4%–89.5% nt identity with SARS-CoV, 88%–89.9% nt identity with the bat SARS-like CoV (bat SARS-CoV Rm1), and 87.6%–89.6% nt identity with the civet SARS-like CoV (civet SARS-CoV SZ16). On the other hand, comparison between the betacoronavirus genomes and human betacoronavirus (HCoV-OC43) showed only 49.9%–50.4% nt overall identity, whereas the betacoronavirus genomes and HCoV-EMC showed 52.1% nt overall identity.We conducted complete genome comparison and phylogenetic analysis on the basis of polymerase and spike protein. Pairwise genome sequence alignment was conducted by using EMBOSS Needle software (www.megasoftware.net) was used to construct the phylogenetic trees on the basis of the nucleotide sequences and deduced amino acid sequences. First, we used the MUSCLE package and default parameters (www.megasoftware.net/) to construct the alignment. The best substitution model was then evaluated with the Model Selection package implemented in MEGA5. Finally, we used the maximum-likelihood method with an appropriate model to process the phylogenetic analysis with 1,000 bootstrap replicates. We constructed a phylogenetic tree based on the nucleotide sequences of the RdRp gene to show the evolutionary relationship between these 2 betacoronaviruses and other CoVs  and are close in distance to SARS-CoVs (92.9%–94.8% nt identity) and civet SARS-like CoVs (93.1%–94.8% nt identity) but that bat CoV (BtCoV-HKU9) and HCoV-OC43 are placed among the relatively distant groups . Therefore, collectively we called these betacoronaviruses and bat SARS-like CoVs the bat SARS-like cluster of CoVs. Bat Rp-coronavirus/Shaanxi2011 and Bat Cp-coronavirus/Yunnan2011 showed little genetic similarity (<66.2%–67.3% nt identity) to HCoV-EMC.The RNA-dependent RNA polymerase  is a highly conserved gene of CoVs, which is frequently used for phylogenetic comparison  that we analyzed. In contrast, Bat Rp-coronavirus/Shaanxi2011 and Bat Cp-coronavirus/Yunnan2011 showed little genetic similarity with human betacoronaviruses and HCoV-EMC. Although several CoVs are found in horseshoe bats (Rhinolophus spp.), to our knowledge, the SARS-like CoVs in R. pusillus and C. plicata bats in China have not been identified. The description presented here will further the understanding of CoVs distribution in different bat species found in human habitats and provide clues for rapid response to potential public health threats.We detected and characterized 2 novel betacoronaviruses—Bat Rp-coronavirus/Shaanxi2011 in


Previously we reported that nuclear export of both unspliced and spliced murine leukemia virus (MLV) transcripts depends on the nuclear export factor (NXF1) pathway. Although the mRNA export complex TREX, which contains Aly/REF, UAP56, and the THO complex, is involved in the NXF1-mediated nuclear export of cellular mRNAs, its contribution to the export of MLV mRNA transcripts remains poorly understood. Here, we studied the involvement of TREX components in the export of MLV transcripts. Depletion of UAP56, but not Aly/REF, reduced the level of both unspliced and spliced viral transcripts in the cytoplasm. Interestingly, depletion of THO components, including THOC5 and THOC7, affected only unspliced viral transcripts in the cytoplasm. Moreover, the RNA immunoprecipitation assay showed that only the unspliced viral transcript interacted with THOC5. These results imply that MLV requires UAP56, THOC5 and THOC7, in addition to NXF1, for nuclear export of viral transcripts. Given that naturally intronless mRNAs, but not bulk mRNAs, require THOC5 for nuclear export, it is plausible that THOC5 plays a key role in the export of unspliced MLV transcripts.  Two major classes of transport receptors are known to mediate viral RNA export: nuclear export factor 1 (NXF1) and karyopherins, also known as importins/exportins . Most reportin1) ,9,10,11.portin1) ,12. The portin1) . We haveportin1) . HoweverSuccessful RNA processing requires recruitment of multiple nuclear proteins on bulk mRNAs ,16. EukaIn this study, we examined whether TREX components are involved in MLV RNA export. Depletion of TREX components by siRNAs demonstrated that UAP56 is required for efficient nuclear export of both unspliced and spliced viral transcripts. Under a limited loss of function by targeting THO complex by siRNAs , we alsoRecruitment of NXF1 to mRNA requires TREX components in metazoans . We haveComponents of TREX complex are formed by assembly of UAP56, adaptor proteins, and THO complex, which include THOC1, THOC2, THOC3, THOC5, THOC6, and THOC7 ,43. Assopol) in the cytoplasm (env) was observed upon disruption of THOC5  in the cytoplasm , we performed RNA immunoprecipitation assay. Regions of primers targeting unspliced and spliced MLV genome are summarized in In order to examine the interaction of the viral transcripts and components of TREX complex  was used according to the manufacture’s instruction. ON-TARGETplus Non-targeting siRNA  was used for siRNA control. siRNAs targeting NXF1, UAP56, THOC1, THOC2, THOC5, THOC6, THOC7, and CHTOP were purchased from Qiagen . siRNAs targeting Aly/REF, and UIF were purchased from ThermoScientific. For Western blot analysis, 293T cells (2 × 105 cells/well) were plated in a 6 well plate 24 h prior to siRNA transfection (25 nM), and 1.0 µg of plasmid was transfected with FuGENE6  24 h after siRNA transfection, and cells were then harvested at 48 h after plasmid transfection. For cell lines chronically infected with MLV, cells were transfected with 25 nM of siRNAs, and harvested at 48 h post-transfection.293T and TE671 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37 °C with 5% COeviously . SimilarProbes were generated by using North2South Random Prime DNA Biotinylation Kit  as described previously . Two micNuclear and cytoplasmic RNA samples were extracted by using a PARIS kit  according to the manufacturer’s protocol. Total RNA was extracted by RNeasy Plus Mini Kit . The RNA concentrations were determined by a NanoDrop Spectrophotometer (Thermo Scientific) and 1.0 µg of RNA was used for cDNA synthesis . Primers for UIF were 5'-ATGAACCGGTTTGGTACCCGG-3' and 5'-CTATCCCACGGTGACAAAGCG-3', and GAPDH was amplified by 5'-GTCCATGCCATCACTGCCA-3' and 5'-TTACTCCTTGGAGGCCAT-3'. The real-time qPCR assay was performed as described previously ,49. For Protein samples were first lysed in RIPA buffer. Cell debris was centrifuged and the supernatants were mixed with Laemmli sample buffer, supplemented with β-mercapto, USAthanol. The samples were boiled at 95 °C for 5 min and subjected to 4%–15% SDS-PAGE , and transferred to a polyvinylidene diflouride membrane. Membranes were blocked in 5% milk/PBS-0.2% Tween 20 and probed with the indicated antibodies. BAT1 polyclonal antibody was purchased from Abnova. Mouse anti-Aly/REF, clone 11G5 was purchased from Millipore. Rabbit anti-NXF1 antibody was purchased from Sigma. Goat-anti MLV antibody was purchased from ATCC. Polyclonal antibodies for CIP29 and THOC5 were purchased from Pierce. Mouse HRP-1 affinity purified polyclonal antibody was purchased from R&D Systems. Polyclonal antibodies for THOC2, THOC6, THOC7, CHTOP were purchased from Proteintech Group. g for 5 min at 4 °C. Two milligrams of the supernatant and 1 µg of antibody were incubated for 2 h at 4 °C. Protein A Sepharose CL-4B beads  were added to the mixture, and incubated for 1 h at 4 °C. Beads were washed with RIP buffer for five times, followed by one wash in PBS. RNA was extracted by RNeasy Plus mini kit (Qiagen), and cDNA was synthesized (Clontech) for PCR reaction. Unspliced viral transcript, AM-MLV gag, was amplified by using primers 5'-ATGGGCCAGACTGTTACCACT-3' and 5'-TGGTCTTGGGTCCCTATAAGGC-3'. Spliced viral transcript, AM-MLV env, was amplified by using primers 5'-ATCGGGAGACCCCTGCCCAGGGACCA-3' and 5'-TCTCCAGGTTACATTAAAGACCTGATGG-3'.RIP assay was performed as described previously . BrieflyIn this study, we have studied the RNA export mechanism of MLV and found: (i) cellular factor UAP56 is involved in the nuclear export of both spliced and unspliced viral RNAs; and (ii) the recruitment of THO component is essential for the export of unspliced RNAs. Because recruitment of TREX complex is utilized by other human pathogens, such as herpesviruses, our study provides novel insight into the host-virus interaction within the nuclear export of a MLV life cycle.


The vertebrate genome contains large spans of non-coding RNA, which for the most part were considered of little functional value to the organism. Recent studies have indicated that vertebrate genomes may have stored hidden secrets in this large span of non-coding RNA, which we refer to here as “Natural Antisense Transcripts (NATs).” NATs can be found in introns, exons, promoters, enhancers, intergenic sequences, and untranslated regions of the genome. They can be located in either the plus or minus DNA strand. NATs utilize several mechanisms that include DNA replication interference, chromatin remodeling, transcriptional interference, RNA masking, double-stranded RNA (dsRNA)-dependent mechanisms and translation interference to mechanistically regulate gene expression. Recently, NAT levels have been identified as dysregulated in various disease states. This review presents an overview of the current state of NAT biology and highlights the main points with specific examples. Natural antisense transcript (NAT) is defined as any RNA transcript that is complementary to an endogenous RNA transcript . Our knoMost S/AS transcript pairs are conserved in evolution and are concurrently or inversely expressed in a cell suggesting a correlative functional role. Six mechanisms widely dictate how NAT's function: (a) DNA replication interference, (b) chromatin remodeling, (c) transcriptional interference, (d) RNA masking, (e) double-stranded RNA (dsRNA)-dependent mechanisms and (f) translation interference 4. We wilE. coli plasmid ColE1. The ColE1 primer transcript (RNA II) binds to the template DNA near replication origin. The plasmid replication is dependent on the primer formation (RNA II). Hybridization of RNA II to DNA is inhibited when a second RNA molecule RNA 1 (NAT RNA) binds to RNA II, which results in the alteration of secondary structure of RNA II [DNA replication can be interfered and thereby inhibited by NATs. This was demonstrated elegantly in the f RNA II .in cis and in trans through heterochromatin formation in leukemic cells and in mouse ES cells.Chromatin remodeling is generally defined as any event that alters the nuclease sensitivity of a region of chromatin. These events can occur independently or in concert with other events, such as transcription . S/AS pain cis. This happens through 3 kinds of mechanisms: (1) occlusion in which the passage of elongating RNA polymerases (RNAPs) blocks the access to the promoter; (2) collisions between elongating RNAPs, moving in opposite directions, leading to premature termination of transcription; and (3) ‘sitting duck' interference which refers to the removal of promoter-bound complexes by the passage of RNAPs from the opposing promoter [in cis on the complementary strand of icsA, a virulence gene of Shigella flexneri. The icsA and RnaG promoters are in opposite direction and 120 bp apart. In vitro transcription from the strong RnaG promoter (aggressive) dramatically inhibits transcription from the weaker icsA promoter (sensitive) [Transcriptional interference refers to the direct negative impact of one transcriptional activity on a second transcriptional activity promoter . A non-tnsitive) .RNA duplex formation may mask critical regulatory features within transcripts, thereby inhibiting the binding of other trans-acting factors. RNA masking intervenes mRNA splicing, transport, polyadenylation, translation and degradation . Zeb2 NAX. Laevis, double stranded Slc34a/antisense Slc34a injected into the nucleus of xenopus oocytes were degraded into short RNAs of ~23 bases within 4 h [D. melanogaster [Duplex RNA can be recognized by RNAi machinery. These dsRNAs are rapidly processed into short RNA duplexes of 21 to 28 nucleotides in length, which then guide the recognition and ultimately the cleavage or translational repression of complementary single-stranded RNAs, such as messenger RNAs or viral genomic/anti-genomic RNAs. In a recent experiment in nogaster .NATs can also prevent transcripts from translating by simply interfering with the translation apparatus. Antisense PU.1 regulates PU.1 expression level via translation interference. The absolute levels of PU.1, a transcription factor required for normal hematopoiesis is critical for specifying cell fate, and, if perturbed, even modest decreases in PU.1 can lead to leukemias and lymphomas. Antisense PU.1 forms complex with three translation factors eIF4A, eEF1A and 4E-BP1. The complex stalls translation between the initiation and elongation steps .BACE1) is a crucial enzyme in Alzheimer's disease pathophysiology and antisense BACE1 (BACE1-AS) is markedly up-regulated in brain samples from Alzheimer's disease patients [Although the physiological level of NATs is very low, their levels go up dramatically in disease. For example, β-secretase-1  increases 10- to 100-fold . Interestie-1), tie-1AS lncRNA in zebrafish, mouse and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear & brain). This argues for a tie-1 gene role previously unrecognized in the brain and ear function, perhaps in the vasculature of these organs. In fact two recent studies implicate Angiopoietins, ligands for Tie's, in lymphatic vessel development in ear [tie-1 is conserved in humans and mice, this paradigm of using non-coding RNA expression pattern to speculate putative novel gene function  of well-studied genes is intriguing.Similar to tumor studies, the relevance of AS RNA levels to progression of cardiovascular disease is unknown. In a recent study, the analysis in peripheral blood mononuclear cells from 1098 patients of coronary artery disease demonstrated positive correlation of AS noncoding RNA in the INK4 locus (ANRIL) expression with atherosclerosis . Our rect in ear ,30. Sinctie-1 vs. tie-1AS lncRNA is altered in human vascular anomaly samples. The expression level of tie-1AS is much lower than that of tie-1 in human placenta, but it goes up dramatically compared to the expression level of tie-1 in hemangioma samples [tie-1 and tie-1AS in zebrafish cardiovascular mutant cloche [tie-1 in cloche embryos where the expression is quite low when compared to WT embryos. The vascular anomaly (disease) and the cloche (genetic mutant) results together suggest that when the cognate gene level (tie-1) is low, the cognate tie-1 AS levels are high suggesting an inverse relationship between the tie-1 and the AS transcript. In fact, this interpretation makes sense since we have shown the tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels [tie-1AS is a log lower than tie-1 across different embryonic stages in zebrafish.Mutations in Tie's have been previously implicated in patients with vascular malformations -35. Our  samples . We obset cloche  -positive, but not in the t-negative lymphoid cell lines, and thus appears to be dependent on the bcl-2/IgH fusion . Anotherin vitro. Since then synthetic antisense oligonucleotides have been used extensively to block target gene expression [NATs hold immense potential for future applications. We will highlight three distinct areas of biology where NATs are expected to make promising contributions. More than 30 years ago, the inhibitory effects of antisense oligonucleotides on translation were observed pression . Howeverpression . A studypression . NATs arpression . TherebyThe second example highlights the role of NATs in a clinical trial setting. In clinical trials, an anticancer agent Gemcitabine can produce cytotoxic synergism with Cisplatin when given to cancer patients. It was proposed that the nucleotide excision repair (NER) process was responsible for the synergism. NER removes cisplatin-DNA adducts by a series of steps including damage recognition, dual incision/excision, repair synthesis and ligation. ERCC1 protein is an important factor in the incision process—the rate-limiting step of the pathway. Antisense therapy targeting the ERCC1 protein was proposed as a target for this approach. As a result, NER activity was downregulated and the cytotoxic synergism between Gemcitabine and cisplatin was abolished .The third example highlights the role of NATs in tumor therapy. The bcl-2/IgH antisense transcript is presumed to be responsible for the overexpressed bcl-2, the root of oncogenicity in follicular lymphomas. Therapeutic ODNs targeting the bcl-2/IgH antisense transcript induced an early strong inhibition of cell growth and a late sudden cell death. The induction is restricted to follicular lymphomas which have t chromosomal translocation, but not effective for the cells carrying a normal bcl-2 gene, suggesting tumor selectivity. However, antisense-oriented ODN, complementary to the bcl-2/IgH mRNA is ineffective .NATs account for majority of non-coding RNAs in the vertebrate genome. Numerous mechanisms are utilized by NATs to regulate gene expression and these mechanisms span over both plant and animal kingdoms. Recently, NATs have been identified in various diseases as well and the regulation that they mediate when lost may in part be responsible for disease progression and pathogenesis. Although most studies identify dysregulation of NATs, very few studies have identified genetic mutations in them. As of now, NATs hold immense clinical value but their application is still at its infancy.


Trinucleotide-repeat expansion diseases (TREDs) are a group of inherited human genetic disorders normally involving late-onset neurological/neurodegenerative affectation. Trinucleotide-repeat expansions occur in coding and non-coding regions of unique genes that typically result in protein and RNA toxic gain of function, respectively. In polyglutamine (polyQ) disorders caused by an expanded CAG repeat in the coding region of specific genes, neuronal dysfunction has been traditionally linked to the long polyQ stretch. However, a number of evidences suggest a detrimental role of the expanded/mutant mRNA, which may contribute to cell function impairment. In this review we describe the mechanisms of RNA-induced toxicity in TREDs with special focus in small-non-coding RNA pathogenic mechanisms and we summarize and comment on translational approaches targeting the expanded trinucleotide-repeat for disease modifying therapies. Table 1; In the human genome trinucleotide repeats (TNR) are especially abundant in intergenic regions, gene introns and untranslated regions, and translated segments of protein coding genes. Short tandem-TNR have generally less than 30 copies in the normal population . AbnormaHTT mRNA (The largest group of inherited polyglutamine (polyQ) disorders is caused by expansions of CAG repeats in the open reading frame (ORF) of unique genes, including the Huntington’s disease (HD) genes and several spinocerebellar ataxias (SCA) genes. In these diseases the predominant hypothesis has been that the expanded polyQ track confers detrimental properties to the protein, that compromise cell homeostasis. The consequences of polyQ expansion in the HTT protein have been systematically characterized, with detrimental effects in transcriptional activity, vesicle trafficking, mitochondrial function and proteasome activity . HoweverHTT mRNA .DMPK gene (50–3000 repeats) leads to neuromuscular degeneration  are associated to different pathologies such as fragile X tremor/ataxia syndrome (FXTAS) and primary ovarian failure  a CTG expansion in the 3′UTR of the neration . A CGG eure POF; . ExpansiAtaxin8_OS) with an expanded CAG has been shown in different frames, in SCA8 mouse models and in patients with SCA8 (FMR1 5′-UTR (The recent discovery of repeat associated non-ATG (RAN) translation  has chanith SCA8 . The sam1 5′-UTR . A cryptIn this review we focus on the RNA pathogenic mechanisms in TREDs. We present the existing evidences for RNA binding protein (RBP) sequestration by different expanded TNR and the linked altered biological processes. We address the possible relevance of bidirectional transcritption in TREDs loci and further discuss about the role of small non-coding RNAs in TREDs pathogenesis. Finally, we summarize the latest therapeutic strategies in TREDs, based on selective targeting of the allele with the expanded TNR.Recent findings indicate that alterations of RNA sequences can lead to abnormal RNA–protein interactions, alteration of protein translation, or RNA interference (RNAi) activation, among other anomalous processes. These altered pathways contribute to disruption of normal cell function and homeostasis, eventually leading to cell degeneration.Figure 1). Muscleblind-like splicing regulator 1 (MBNL1) and Elav-like family member 1 (CUGBP1 or CELF1) are regulators of mRNA splicing that present affinity for CUG and/or CAG repeats. In DM1, long CUG repeats lead to decreased MBNL1 activity and increased CELF1 activity in muscle cells, which results in mis-splicing events in different developmentally regulated genes including the insulin receptor (IR), the chloride channel (CLCN1) and the cardiac tropin T (TNNT2), which explain several aspects of DM1 symptomatology  pre-mRNA has been recently described in DM1 , and exon 7 in the amyloid precursor protein (APP) and exon 5 in glutamate receptor NMDAR1, have been detected in brains of DM1 patients and mouse models . MBNL1 and the RNA helicases p68 and p72 influence the activity of proteins involved in microRNA (miRNA) biogenesis . Depletion of these processors compromises the biogenesis of many miRNAs, thus triggering downstream detrimental gene expression perturbations, which likely contribute to FXTAS pathogenesis. However, this mechanism may be tissue-dependent, as small RNA profiling in peripheral blood of FXTAS patients does not reveal a general miRNA downregulation . Dicer recognizes the expanded triplet and cleaves it, producing small repeated RNAs (sCNG). In vitro approaches demonstrated that single stranded CGG-RNA constructs are cleaved by Dicer, producing short CGG-RNAs of approximately 21 nt  derived from cells expressing expanded HTT exon-1 produced neuronal death. Both the biogenesis and the toxic activity of sCAG were dependent on Dicer. Similarly, transfection of sRNAs isolated from the striatum and cortex of patients with HD induced significant neuronal toxicity. This toxic effect was prevented by oligonucleotides complementary to short CAG, strongly suggesting a detrimental effect of sCAG. Furthermore, toxicity may depend on downstream silencing effects, as HTT exon-1-derived sCAG were loaded onto Ago2 complexes and knocking-down of Ago2 prevented damage. However, the sequence/structure requirements for effective silencing of sCAG-targets remain to be resolved, since similar moderate inhibition was detected in luciferase assays performed with targets harboring a perfect sCAG-complementary CTG repeat or a CAG repeat that offers an interrupted sCAG target site. Whether the detrimental properties of sCAG include silencing effect through mRNA degradation and/or translational repression, or perturb gene expression networks through other mechanisms, remains to be determined. Interestingly, this study shows that the effect of sCAG-RNAs differed depending on the cell type, with high toxicity detected in BDNF-differentiated neuroblastoma cells. In this scenario, sCAG activity may provide a mechanism contributing to tissue selective affectation. The relevance of sCNG biogenesis and activity in other TREDs is an interesting field for future studies.The relevance of sCNG in TREDs pathogenesis has been recently addressed in HD . This stTable 1; Ataxin 8OS mRNA (Ataxin 8OS) and an anti-sense transcript resulted in a CAG expansion that was translated into a highly enriched polyQ track (Ataxin 8). Intranuclear inclusions immunopositive for anti-polyQ antibodies, which are typical from PolyQ diseases and CUG foci formation co-localizing with MNBL1 were detected in cerebellar cells of the mouse model and patients with SCA8  locus , The alttoxicity . The exitoxicity .FMR1AS) in human lymphoblastoma cells , similar to the FMR1 sense transcript. A recent study suggested that elevated expression levels of the sense and antisense expanded FMR1 involving mitochondrial dysfunction participate in parkinsonism phenotype that is associated with CGG-repeat moderate expansions  transcripts have been identified that contain the repeated CAG track . Complementary repeats can form double-stranded structures compatible with endonuclease Dicer slicing activity. This results in the formation of short repeated RNAs that are incorporated into the RISC complex, possibly driving downstream gene silencing with detrimental consequences. This mechanism has been proven in a DM1 Drosophila model, in which the toxic effect of an expanded CUG track was largely enhanced if co-expressed with a CAG expansion. The co-expression of sense and anti-sense transcripts lead to the formation of repeat-derived small interfering RNAs in a process dependent of Dicer-2 and Ago-2  show compatibility with double stranded hairpin formation  was also shown to be deleterious in transgenic mice . Mice exHTT  further These data indicate that cell failure in polyQ diseases may be the result of both an abnormal function of the protein harboring the expanded glutamine and the altered properties of the expanded-CAG RNA. The secondary structure of the CAG-repeat in each gene context and the dynamic expression and activity of RBP may provide specific pathogenic scenarios for cell dysfunction.MicroRNAs are small RNA molecules of 20–24 nucleotides that generally inhibit the expression of target mRNA, by a mechanism involving mRNA degradation, translational inhibition or a combination of the two  that carries an intronic GAA. TTC triplet repeat expansion in Friedeich ataxia  that causes spinocerebellar ataxia 1 (SCA1), when presenting a CAG expansion  is caused by a CAG/polyQ expansion in DRPLA gene/protein, respectively. miR-200b and miR-429 target to DRPLA . In anotia FRDA; . FXN mRNxpansion . The autn . RNAi using shRNA against mutant TREDs gene mRNA and protein have been successfully used in vivo. Intrastriatal adenoviral-delivery shRNA targeting mutant human HTT resulted in improved neuropathology and behavioral deficits in HD mouse models  or RNAi , targeting the expression of the mutant gene . In addition, the RNA structures of the expanded CAG that differ from those of the wild type allele (HTT mRNA degradation and LNA ASOs were shown to form stable structures with the target RNA. Although expanded RNA toxicity in HD has not been addressed, the formation of stable LNA ASOs : RNA duplexes has also the potential to block expanded CAG toxic effects in HTT mRNA.Other potential therapeutic strategies in polyQ diseases, are based on the use of several types of modified single stranded ASOs that target the CAG expansion in the mutant e allele . The lone allele , may offDMPK mRNA (DMPK expression and reduced the ribonuclear aggregates. Intramuscular administration of these ASO in a DM1 mouse model further reduced expanded RNA toxicity (Several studies have demonstrated a similar therapeutic potential of a CAG-repeat antisense or morpholino targeted to expanded-CUG in the MPK mRNA . In celltoxicity . In anottoxicity .Together, these data suggest that in TREDs, targeting the expression and activity of the expanded allele both at the RNA and protein levels is a promising therapeutic strategy.RNA toxicity is a process underlying pathogenicity in TREDs, with TNR expansions occurring in both coding and non-coding regions of specific genes. Sequestration of transcriptionally active RBP, and RBP participating in miRNA biogenesis result in direct and indirect perturbations of the coding-transcriptome, which likely contribute to cell dysfunction. Full characterization of the repertoire of RBP in different types of TNR expansions is essential to understand common detrimental pathways in etiologically diverse neurological disorders. Studies that take into consideration the gene context may provide hints to understand disease specific aspects.The biogenesis and activity of sCNG may contribute to TREDs pathogenesis. The gene silencing activity of these species likely trigger downstream detrimental effects, which may differ, depending on the cell type. This mechanism may complement the damaging activity of expanded protein and/or expanded RNA. Whether sCNG mechanisms are only related with gene silencing or present other activities and the real importance of sCNG mechanisms in each TRED remain to be determined.In each TRED, the mechanistic bases for tissue specificity, with particular affectation of selective neuronal types, remain largely unknown. The different RNA-based pathogenic processes provide a number of scenarios that may underlie this specificity. These include the dynamic expression and cellular and subcellular localization of RBP, the amount of expression of the sense and anti-sense transcripts spanning the expanded TNR in each cell context, the regulation of the biogenesis and activity of sCNG in different brain areas and/or the temporal and spatial primary and secondary perturbations of the miRNA transcriptome.The mechanistic complexity in TREDs stresses the need of additional studies to dissect the relative relevance of expanded protein- RNA-, and/or sRNAs-mechanisms in each disease. In this context, the use of modified ASOs or siRNA directed to the expanded TNR has the potential to block deleterious effects of expanded full-length RNA and derived sCNG, with concomitant inhibition of expanded protein expression.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.


We aimed to evaluate the immunologic nature of sentinel lymph nodes (SLNs) in gastric cancer patients and to determine whether it can predict non-SLN metastasis.Sentinel lymph node samples were collected from 64 gastric carcinoma patients who had undergone gastrectomy with SLN biopsy. One representative SLN sample was selected from each patient and was subjected to immunostaining for CD8, CD57, FOXP3, and DC-LAMP. The numbers of marker-positive cells in each sample were counted. The relationships between various immune cell densities and clinicopathologic parameters or metastasis status of SLNs and non-SLNs were sought.High FOXP3+ Treg density of the SLN was found to be significantly associated with the presence of metastasis in either SLNs or non-SLNs. DC-LAMP+ cell density of the SLN was the highest at the isolated tumours cell level, and this decreased along with an increase in tumour metastasis in either SLNs or non-SLNs. Univariate and multivariate logistic regression models revealed that high FOXP3+ Treg density of the SLN was an independently significant predictor of non-SLN metastasis.This study is the first to indicate an important role of SLNs in metastatic dissemination of gastric cancer. Our findings suggest that Tregs could be a new therapeutic target for regulating the metastasis of gastric cancer. In the treatment of gastric cancer, the standard procedure has been complete excision of both the primary tumour and the regional lymph nodes (LNs). However, metastasis to regional LNs is observed in only 2–8% and 40–50% of pT1 and pT2 gastric cancer patients, respectively  is an independent predictive factor of non-SLN metastasis in gastric cancer.We collected SLN samples from 64 patients (35 men and 29 women) with cT1-2, NO gastric cancer, for whom SLN mapping and biopsy and subsequent laparoscopic gastrectomy were performed at Seoul National University Bundang Hospital. No patient had received preoperative chemotherapy or radiotherapy. The pathologic tumour-node-metastasis stage (according to AJCC seventh edition) and histologic classification  were evaluated after the operation. Histologic findings showed that 35 patients (54.7%) had pT1a cancer; 19 (29.7%), pT1b cancer; 5 (7.8%), pT2 cancer; 4 (6.3%), pT3 cancer; and 1 (1.6%), pT4a cancer. There were 31 (48.4%) and 33 (51.6%) cases that were classified as intestinal and diffuse type of Lauren classification, respectively. The tumour was located in the lower third of the stomach in 35 cases (54.7%) and in the middle third in 29 cases (45.3%). This study was approved by the Institutional Review Board for research using human subjects at Seoul National University Bundang Hospital.μm). In 30 cases, an additional serial cut was stained for rapid immunohistochemistry (IHC) using an SLN rapid IHC kit . The SLN portions remaining after intraoperative examination were formalin fixed and paraffin embedded. All dissected non-SLNs were sliced and stained with HE. Two serial slides were obtained and stained with HE and subjected to immunostaining for CK  using an automated immunostainer  according to the manufacturer’s instructions. Nodal metastases were subdivided into three groups according to AJCC guidelines using the maximum size of tumour deposits: isolated tumours cells (ITCs), micrometastases, and macrometastases. Finally, 45 (70.3%), 4 (6.3%), 11 (17.2%), and 4 (6.3%) cases showed negativity, ITCs, micrometastases, and macrometastases in SLNs, respectively; and 49 (76.6%), 3 (4.7%), 4 (6.3%), and 8 (12.5%) showed negativity, ITCs, micrometastases, and macrometastases in non-SLNs, respectively. When only micrometastasis and macrometastasis were considered as presence of metastasis, two cases showed skip metastasis, which represent non-SLN metastasis without any SLN metastasis, and five cases displayed SLN metastasis but no further metastasis in non-SLNs.We previously determined the metastasis status of SLNs and non-SLNs with the following pathologic examination method (under submission). Briefly, 207 SLN samples from 64 patients were examined. During the operation, we sliced the samples at 2 mm intervals in the plane of the largest dimension. All pieces were frozen, sectioned, and stained with haematoxylin and eosin (HE) , FOXP3 , CD57  cell marker; 1 : 150; NK-1; Zymed), and DC-LAMP  using an automated immunostainer  according to the manufacturer’s instructions.Formalin-fixed, paraffin-embedded SLNs were sectioned into 4 http://rsb.info.nih.gov/ij). The numbers of antibody-positive immune cells in each case were counted in two representative areas at a magnification of × 100. The mean numbers of immune cells in two different areas were calculated, and their density was determined for each patient.Slides subjected to immunostaining were analysed using NIH ImageJ software with a Java-based colour deconvolution plugin  was assessed for each factor. All statistical analyses were conducted using SPSS statistics 17.0 , and P-values ⩽0.05 were considered statistically significant.Continuous variables were compared using the Mann–Whitney We identified CD8+, FOXP3+, CD57+, and DC-LAMP+ immune cells in SLNs using IHC . CD8+ ceP=0.015).The median numbers of CD8+, FOXP3+, CD57+, and DC-LAMP+ immune cells of SLNs were 2686 , 928.5 (42–2347), 592 (8–1371), and 377 (46–1040), respectively. Correlations between the numbers of the various immune cells were not statistically significant, except for the numbers of CD8+ and FOXP3+ immune cells, which were significantly correlated , and 54.7% were men. Associations between immune cell density of SLNs and clinicopathologic characteristics are summarised in P=0.001). DC-LAMP+ cell density of the SLN was highest at the ITC level and was found to decrease as tumour metastasis in SLNs increased. CD8+ or CD57+ cell density of the SLN was not associated with the metastasis status of SLNs . Similar to the comparison with metastasis status of SLNs, DC-LAMP+ cell density of the SLN was highest at the ITC level, and was found to decrease as tumour metastasis in non-SLNs increased. In non-SLNs, CD57+ cell density of the SLN was significantly higher in the cases of macrometastases than in those of micrometastases (P=0.048). No significant association was found between CD8+ cell density of the SLN and metastasis status of non-SLNs  of 1.002 (1.000–1.003) .Because the SLN is the site where naive lymphocytes initially encounter tumour-specific antigens and develop antitumour immunity  . In partPreviously, it was found that NK cell-mediated immunity has an important role in the inhibition of LN metastasis, especially in the early stage of metastasis development, and that the main factor responsible for the regression of ITCs after primary tumour resection is the immunologic activity conferred by NK cells against metastatic tumour cells in the LNs , and oneα act as immunomodulators of SLNs (Significant advances have been made in the field of cancer immunotherapy over the last decade, some of which was based on immunomodulation in SLNs to prevent or eradicate tumour metastasis in SLNs (In conclusion, we revealed that high Treg density of the SLN is associated with SLN or non-SLN metastasis and, through logistic regression models, is an independent predictor of downstream non-SLN metastasis in gastric cancer. We believe that the findings can help decide the extent of gastrectomy and lymphatic dissection to be performed, and thus offer a more individualised therapy for patients in the near future. In addition, our findings suggest that Tregs within the SLN have an important role in promoting metastatic dissemination of cancer. We expect Tregs to be a new therapeutic target for regulating the metastasis of gastric cancer.