Developing predictive biomarkers that can detect the tipping point before metastasis of hepatocellular carcinoma (HCC), is critical to prevent further irreversible deterioration. To discover such early-warning signals or biomarkers of pulmonary metastasis in HCC, we analyse time-series gene expression data in spontaneous pulmonary metastasis mice HCCLM3-RFP model with our dynamic network biomarker (DNB) method, and identify CALML3 as a core DNB member. All experimental results of gain-of-function and loss-of-function studies show that CALML3 could indicate metastasis initiation and act as a suppressor of metastasis. We also reveal the biological role of CALML3 in metastasis initiation at a network level, including proximal regulation and cascading influences in dysfunctional pathways. Our further experiments and clinical samples show that DNB with CALML3 reduced pulmonary metastasis in liver cancer. Actually, loss of CALML3 predicts shorter overall and relapse-free survival in postoperative HCC patients, thus providing a prognostic biomarker and therapy target in HCC. Biomarkers of the tipping point before metastasis in hepatocellular carcinoma (HCC) could help stratify patient treatment. Here, the authors study dynamic network biomarkers to identify CALM3 as a potential suppressor of metastasis, the level of which can predict overall survival and relapse-free survival in postoperative HCC. The high mortality rate results from late presentation at advanced stages, high incidence of tumour metastasis, and tumour recurrence after surgical resection2. Generally, HCC is prone to both intrahepatic and extrahepatic metastasis. Extrahepatic metastasis has been reported to occur in 13.5–42% of HCC patients4. The median survival time and 1-year survival rate of HCC patients with extrahepatic metastasis are only 4.9–7 months and 21.7%–24.9%5, respectively. The most common site of metastasis is lung7. Metastasis is a nonlinear  and dynamic process involving cancer cell motility, intravasation, transit in the blood or lymph, extravasation, and growth at a new site8. Understanding the molecular mechanisms of this irreversible HCC metastasis at a network level is of great importance, both for preventing the initiation of metastasis in early HCC patients and for developing therapeutic strategies in advanced HCC patients.Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths globally9. Various oncogenes and tumour suppressors forming networks or pathways are involved in the metastatic process. Pathway-based approaches and functional experimental studies have been adopted in identifying the dysfunction of different signalling cascades in HCC metastasis , mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), and WNT/β-catenin)10 and disease-related biomarkers. Although some of these biomarkers are effective in identifying HCC patients who are in a metastasis state, it is difficult to pinpoint the critical state or tipping point before metastasis initiation  for early diagnosis. Specifically, HCC progression can be divided into three stages: non-metastatic state, pre-metastatic state , and metastatic state . Clearly, there is a phase transition just after the pre-metastasis state that leads to a drastic (irreversible) change in phenotype12. Generally, there are significant differences between non-metastatic and metastatic states in terms of gene expression, which is why we can find molecular biomarkers to distinguish the two states. However, statically there is no clear difference between non-metastatic and pre-metastatic states, because the pre-metastasis state is really a part of the non-metastatic state. Thus, ‘‘traditional’’ molecular biomarkers fail to distinguish them or fail to identify HCC patients in the pre-metastasis state.One invariable feature of the metastatic process is deregulated gene expressions and dysfunctional interactions, which dynamically affects sequential stages of tumour cell invasion, organ tropism, and growth at distant sites17. Actually, in contrast to no statically significant difference, it has been shown that dynamically there is significant difference between non-metastatic  and pre-metastatic  states, which can be explored to develop dynamic biomarkers  for predicting the pre-metastatic  state.Recently, new high-throughput omics technologies , sophisticated animal models , loss-of-function  and gain-of-function  studies have opened the field to new strategies in oncogene and tumour suppressor discovery, in particular, for studying the pre-metastatic state and the critical transition problem from the perspectives of both network and dynamics17. Actually, the DNB model has been also recently applied to analyse other complex biological processes by many other researchers, e.g., successfully identifying the tipping points of cell fate decision14 and studying immune checkpoint blockade16. Specifically, we obtained DNB genes that not only signalled the pre-metastatic state but also were strongly related to key molecules of HCC metastasis, by analysing the three DNB statistical conditions of the critical state derived from nonlinear dynamic theory17. Compared with traditional biomarkers detecting the metastatic state based on differential expression of molecules, a major advantage of the DNB method is to identify the pre-metastatic state or critical state (tipping point) just before the irreversible transition to metastasis state in tumour progression. Furthermore, we discovered several DNB members as predictive biomarkers that were shown to play a precursor role in initiating metastasis17 in both animal models and patient samples.In this work, we adopted our mathematical method, i.e., the dynamic network biomarker (DNB) model, to identify the pre-metastatic state or tipping point by exploring dynamic and network information of omics data from both animal models and clinical samples18. The model with a transplanted HCC cell line, i.e., HCCLM3-RFP18 of high metastatic potential, is labelled with a stable fluorescent protein, and is effective in resolving and quantifying the dynamics of tumourigenesis and metastasis. By analyses of time-series whole-genome expression patterns of orthotopic liver tumour samples, based on the DNB model, we find remarkable changes at the third week after orthotopic transplant , which was consistent with the results of circulating tumour cells (CTC) detection.Specifically, we use xenografts and a spontaneous pulmonary metastasis mouse model of HCCLM3 to detect the tipping point of HCC metastasis initiation and its predictive biomarkersIn this study, we demonstrate a new way to predict the critical transition in metastasis by DNB method, and provide new insights into the molecular pathology of HCC pulmonary metastasis from the perspectives of dynamics and network. In particular, we find that CALML3, as a DNB member or dynamic biomarker, could not only quantify the early-warning signals of metastasis initiation but also effectively predict outcomes in HCC patients.19. To simulate tumour growth and metastasis in livers of HCC patients, we used the spontaneous pulmonary metastasis mouse model, HCCLM3-RFP, which involved the orthotopic transplanted human HCCLM3 cell line labelled with a stable fluorescent protein18. Due to the high metastatic potential of HCCLM3, this cell line has been used frequently in studies seeking to identify biomarkers and to examine mechanisms in the pulmonary metastasis of human HCC22. From experience in previous work18, we created a xenograft model according to the flowchart shown in Fig. The tumour microenvironment plays an essential role in tumour growth and metastasis in tumour progressionWe observed that, hepatic tumours in orthotopic xenograft HCCLM3-RFP mice grew gradually from the second week to the fifth week after orthotopic implantation in the primary liver tissue, whereas spontaneous pulmonary metastasis occurred only at the last time point (the fifth week after orthotopic implantation) Fig. . The fluP < 0.05; Supplementary Data Generally, a whole-genome expression profile is expected to reflect the instant state of tumour. Thus, to characterise state-specificity during the progression of HCC pulmonary metastasis, we identified 13,247 differentially expressed genes (DEGs) by multiple comparisons with false discovery rate (FDR) adjustment  using Mfuzz23 Fig. . Genes i17  of gene expression and the PCCs (Pearson correlation coefficients) of pair-wise genes vs. those at the original time point  at the third week after implantation Fig. . This reint Fig. . Compareint Fig. . Note th30. To more exactly identify the time point of metastasis initiation experimentally for primary tumour cells in the liver, we used flow cytometry to measure CTCs31, i.e., HCCLM3-RFP cells, in plasma from the peripheral blood of orthotopic xenograft mice at each time point. The result showed that CTCs only appeared in peripheral blood of the orthotopic xenograft mice at the fourth and fifth weeks after orthotopic implantation , and selected CALML3 as the top one for further functional study Fig. . Certain32. Compared with the high expression of CALML3 in THLE-3, CALML3 was significantly downregulated in most hepatoma cell lines, especially in cells  with relatively high metastatic potential, and moderately expressed in hepatoma cells  with low metastatic potential  and serial cell lines with medium or high metastatic potential . MHCC97L, MHCC97H and HCCLM3  were established from the same parent HCC cell line, MHCC97, at Liver Cancer Institute, Zhongshan Hospital, Fudan Universityial Fig. . Notably−/− cell lines (CALML3−/− #1 and CALML3−/− #2) was testified by genomic DNA sequencing and western blot  were established from the same parent HCC cell line and showed relatively high and low CALML3 expression respectively, we established a doxycycline-inducible CALML3 overexpression system in HCCLM3 and a CRISPR/Cas9-mediated knockout in MHCC97L cells. Treatment with 50 ng/ml doxycycline induced a time-dependent expression of CALML3 protein in HCCLM3/CALML3 cells, detectably as early as 12 h Fig. . Two CAL33. The result of cell proliferation assay showed that inducible CALML3 expression significantly inhibited cell growth in HCCLM3 cells, while knockout of endogenous CALML3 promoted cell proliferation in MHCC97L cells  of pathways that regulate key disease-associated processes during the initiation and development of complex diseases17. Thus, we analysed CALML3-associated regulations and functions (or network rewiring) to systematically explore the role of CALML3 in the metastatic process at a network level. According to the knowledge-based molecular interactions of human biology, we integrated CALML3 and its 72 neighbouring genes into a CALML3-centred network and weighted the nodes of this network according to the z-score transformed data of their real gene expression across the four time points . These enriched pathways belong to typical cancer-associated signals resulting in cell growth and division37 as well as cell adhesion and migration38, and play key roles in metastasis initiation. Here, inversing DEGs refer to those genes whose expressions changed significantly from low (high) to high (low), before and after the tipping point of metastasis . The dynamics of the functional phenotypes of CALML3 and its inversed DEGs indicated the complexity and time-dependence of dysfunctions in metastasis-related biological processes during metastasis initiation  were enriched Fig. . In partion Fig. . Most ofThe above analyses showed that the dynamics resulted proximally from the regulation or dysfunction in CALML3 during the initiation of metastasis. Furthermore, we sought to determine whether CALML3 could regulate well-known metastasis-related genes in a cascading manner. For this, we used a polymerase chain reaction (PCR) array including 84 typical cancer-associated genes  components, cell cycle, cell proliferation and apoptosis) and five housekeeping genes (as controls), to validate CALML3-regulated DEGs in wild-type HCCLM3 cells (HCCLM3/NC) and CALML3 overexpression HCCLM3 cells (HCCLM3/CALML3)  and RFS (P < 0.001); and that death and relapse were much more likely to occur in CALML3-negative patients than in CALML3-positive ones. The 1-, 3- and 5-year OS rates were 87.3%, 64.4% and 59.8%, respectively, in the intratumoural CALML3-positive group and 79.0%, 38.4% and 7.9%, respectively, in the intratumoural CALML3-negative group (P < 0.001). The 1-, 3- and 5-year RFS were 71.2%, 49.0% and 49.0%, respectively, in the positive group and 44.8%, 12.9% and 9.2%, respectively, in the negative group (P < 0.001)  Fig. . Clinica01) Fig. . These r17. Compared with traditional molecular biomarkers detecting the metastatic state by differential expression of molecules in a static manner, DNB showed superiority in identifying the critical or pre-metastatic state (a tipping point just before the dramatic transition17 to a metastatic state) during disease progression via interactions between molecules  in a dynamic manner. Then we determined the corresponding functional network of the DNB, which can signal the imminent transition. Another advantage of DNB method is that it is unnecessary to infer the unknown parameters, suggesting that it is a data-driven approach without requirements on parameters or even models.One major cause of high mortality of HCC is its high rate of metastasis. Thus, it is important to identify predictive biomarkers of the pre-metastatic state and understand the molecular pathology of metastasis initiation for early diagnosis or early prevention. To address this, we developed a prediction model of pulmonary metastasis, based on DNBsHere, by analysing time-series transcriptomic data based on DNB method, we identified the tipping point of metastasis initiation (the third week after orthotopic implantation) using HCCLM3-RFP nude mice xenograft model, which was consistent with the CTC analysis by flow cytometry Fig. . MoreoveCALML3 gene was located on chromosome 10 (10p15.1), encoding a 138-amino-acid residue calcium sensor protein, similar in structure to calmodulin (CaM)39. CaM was a single-chain protein composed of 148–152 conserved amino-acid residues40; it was a multifunctional protein that could form a Ca2+–CaM complex to regulate signal transduction and metabolic activities related to calcium ion in eukaryotic cells41. However, there were few studies on physiological functions of CALML3. In previous work, CALML3, as a calmodulin-like protein, could bind competitively with IQ protein on the neck of Myosin-10, which played an important role in cell migration42. There were other reports that CALML3 was highly expressed in normal differentiation of epithelial tissues, such as breast, thyroid, prostate, kidney and skin, and also significantly downregulated in breast tumours, oral cancers and skin cancers45. However, little is known about the role of CALML3 in HCC metastasis initiation.Human 46. Tet-on system that allows activation of gene expression by doxycycline in a time-dependent manner was used in gain-of-function assay47. In this study, to better elucidate the role of CALML3 as a tumour suppressor in HCC metastasis, we performed endogenous genetic tests on gain-of-function and loss-of-function, respectively. Gain-of-function model was established using an inducible system, which had a time-dependent increase effect of CALML3 expression in HCCLM3 cells with high metastatic capacity and low CALML3 expression. Loss-of-function model was conducted by CRISPR-Cas9 in MHCC97L cells with relatively lower metastatic capacity and higher CALML3 expression than HCCLM3 cells. To further verify the role of CALML3 in HCC metastasis, we also conducted CALML3 overexpression and knockdown models by lentivirus-mediated expression and RNA interference in HCCLM3 cells and HepG2 cells. We found that CALML3, as a suppressor, could significantly inhibit HCC carcinogenesis and metastasis initiation in vitro and in vivo  provides an effective means of introducing multiplexable targeted loss-of-function mutations at specific sites in the genomeivo Fig. . Moreoveivo Fig. . These r51. In this work, we compared CALML3 with existing HCC metastasis-related EMT markers, i.e., VEGF and vimentin by evaluating IHC. Receiver operating characteristic (ROC) analysis was performed to evaluate the performance of CALML3, VEGF and vimentin in distinguishing patients with HCC recurrence from patients without recurrence. CALML3 AUC  was higher than VEGF AUC  and vimentin AUC . CALML3 had a sensitivity of 70.8% and a specificity of 73.6%, higher than those of VEGF (67.8% for sensitivity and 60.4% for specificity) and vimentin (65.5% for sensitivity and 68.7% for specificity)  to evaluate the risk of metastasis and recurrence after resection. Many molecular markers such as AFP, VEGF, CK19, CD133, osteopontin, and epithelial–mesenchymal transition (EMT) markers  are also considered in the studyIn conclusion, our results suggested a new way to identify the tipping point of HCC pulmonary metastasis with its DNBs, and provided biological insights into the molecular pathology of this progression from the perspectives of dynamics and network. We also proposed CALML3 as a predictive biomarker as an early-warning indicator of the initiation of metastasis for clinical application.CALML3 (ab155130) and beta actin (ab8226) were from Abcam . Secondary antibodies conjugated with HRP were from Jackson ImmunoResearch Laboratories .2.The four human hepatoma cell lines  and an immortalised human hepatocyte liver cell (THLE-3) were obtained from the Type Culture Collection of the Chinese Academy of Sciences . MHCC97L, MHCC97H and HCCLM3 cells were obtained from the Liver Cancer Institute of Zhongshan Hospital, Fudan University. All the cell lines passed the conventional tests of cell line quality control methods . The cell lines have passed the test of DNA profiling (STR) between 2012 and 2014. Hepatoma cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . THLE-3 was maintained in BEGM from Lonza/Clonetics Corporation, Walkersville, MD 21793  plus 10% fetal bovine serum. Cells were maintained at 37 °C in a humidified incubator under 5% CO7cells in 0.2 ml phosphate buffer solution (PBS) were injected into the right upper flank of mice. Tumour sizes were recorded twice a week. Mice were sacrificed at 4 or 5 weeks post-injection; tumours were excised and weighed. Tumour volume was calculated by the formula: 0.5 × L × W2 . To examine the metastatic potential of cells, orthotopic liver tumour implantation model were conducted. A suspension of 1 × 107 cells in 0.2 ml PBS were injected into the right upper flank of mice. When the subcutaneous tumours reached 1 cm in diameter, 3 or 4 weeks later, they were removed, cut into pieces, of about 2 × 2 × 2 mm3, and implanted into the livers of another nude mice, as described previously17. To study of the whole-genome expression, 20 nude mice that underwent orthotopic tumour implantation, were divided into four groups. After the orthotopic tumour implantation, one group of mice were sacrificed at the second, third, fourth and fifth weeks for autopsy. For study the effect of inducible CALML3 expression by Tet-on technology on HCC metastasis, mice were received doxycycline injection (20 mg/kg) daily. Mice were euthanized at week 7 after tumour implantation. Orthotopic tumours and spontaneous metastasis to the lung were imaged . Tumours and lung nodules were analysed histologically. Consecutive tissue sections (110–135 sections) were made for each block of the lung, and stained with haematoxylin-eosin.Ethical approval was obtained from the Research Ethics Committee of Zhongshan Hospital. Four-week-old male athymic BALB/c nu/nu mice were obtained from Shanghai Slac Laboratory Animal Co., Ltd., China. Mice were housed in laminar-flow cabinets under specific pathogen-free conditions. Mice were kept for 5–7 days as an adaptation period before being used in experiments. To assess the tumourigenicity of cells, tumour-bearing mouse model were conducted. A suspension of 1 × 10http://www.ncbi.nlm.nih.gov/geo/) Accession Number GSE94016.To assess whole-genome expression, we individually extracted total RNAs from liver tumours of orthotopic xenograft mice using TRIZOL Reagent (Life technologies) and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies). The qualified total RNAs were further purified by RNeasy micro kit (QIAGEN) and RNase-Free DNase Set (QIAGEN). Then, total RNAs were amplified, labelled and purified by using GeneChip 3′IVT Express Kit to obtain biotin labelled cRNA (Affymetrix). Array hybridisation and wash were performed with constant rotation on the PrimeView Human Gene Expression Assay (Affymetrix). Microarrays were scanned by GeneChip Scanner 3000 (Affymetrix) and Command Console Software 3.1 (Affymetrix) with default settings. Raw data were normalized by robust multiarray analysis (RMA) algorithm, Gene Spring Software 11.0 (Agilent technologies). All microarray data were deposited in the Gene Expression Omnibus database  were isolated from fresh blood by density gradient centrifugation through Ficoll-Hypaque . Then, single-cell suspensions were assessed on a FACS Aria II flow cytometer  and data were analysed using the Flowjo software (version 7.6.1). HCCLM3-RFP cells (with red fluorescence) acted as a positive control and wild-type HCCLM3 as a negative control.9 TU/ml (transfection unit). To obtain cell lines expressing CALMAL3 only at the time when doxycycline existing, HCC cells were infected with lentiviruses expressing Tet-CALMAL3. At 48 h post-infection, 1 µg/ml of puromycin was supplemented in medium for selection.The human CALMAL3 sequence was cloned into Lenti-X Tet-one System expression vector . The recombinant lentivirus tet-CALMAL3 and the negative control lentivirus  were prepared and titered to 10http://crispr.mit.edu/). The targeted gRNA expression oligos were introduced into the PHY-701 vector ), which was modified according to letiCRISPRv2 (Addgene plasmid #52961). PHY-701-CALML3 sgRNA plasmids with pMD2.G and psPAX2 (Addgene plasmid #12259 and #12260) were co-transfected to 293T cell to make lentivirus. The recombinant sgRNA expression lentivirus were prepared and titered to 5 × 108 TU/ml (transfection unit). MHCC97L cells grown to 50% confluence in 6-well plate were added sgRNA lentivirus and selected with puromycin (1 μg/ml) for 48 h. Individual colonies were selected and genotyped by genomic DNA sequencing.The 20 nt target DNA sequence guide RNA #1 (5′- TGCATCACCACCCGCGAGCT -3′) and guide RNA #2 (5′- GAGATCGACCGGGACGGCAA -3′) were selected for generating single guide RNA (sgRNA) for depleting CALML3 using the CRISPR design tool , CALML3 cDNA fragment, and negative control particles (5′-GTAGCGCGGTGTATTATAC-3′) were purchased from Hanyin Biotechnology Company . HCCLM3 and HepG2 cells were seeded in 6-well plates at a density of 5 × 103VO4, and 1 mM NaF). Lysates were cleared by centrifugation and denatured by boiling in Laemmli buffer. Equal amounts of protein samples were loaded per well and separated on sodium dodecyl sulphate-polyacrylamide gels, and then transferred electrophoretically onto polyvinylidene fluoride membranes. Following blocking with 5% non-fat milk at room temperature for 1 hour, membranes were incubated with primary antibodies  at 4 °C overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) for 1 h at room temperature. Specific immune complexes were detected with chemiluminescence reagents .The cells were washed twice with ice-cold PBS and then lysed with a modified radio-immunoprecipitation assay buffer , 1 mM Na3 cells per well were plated in 24-well plates and incubated for 24 h. Then, the plates were transferred to Cell-IQ incubator with the cell-secure lid, and all-in-focus imaging recorded cells at 30-min intervals for 48 h. Analysis was carried out with a public-domain imaging software , using the Manual Tracking plug-in created by Fabrice Cordelières . All assays were performed in triplicate and repeated three times.Cell proliferation was examined using Cell Counting Kit-8  or Cell-IQ . For CCK-8, Cell proliferation was measured according to the manufacturer’s instructions. Cells were incubated with CCK-8 for 1 h with five multiples. Cell proliferation rate was assessed by measuring the absorbance at 450 nm with the Universal Micro-plate Reader. For Cell-IQ, about 1 × 106/well) in culture medium and cultured until confluent. Then the cells were treated with or without doxycycline (50 ng/ml). Then, an artificial scratch wound was drawn at the centre of the well and photographed. After 24 h of culture, the cell scratch wound was photographed again, and the migration distance was determined by the ratio of healing width at 24 h vs. the wound width at 0 hour. Each assay was carried out in triplicate and repeated three times.The migration of cells was evaluated with a wound healing scratch assay. Cells were seeded in 6-well plates (1 × 105) cells and MHCC97L (5 × 105)cells were seeded into the upper chambers, which were filled with 2% FBS-containing culture medium, with or without doxycycline (50 ng/ml). The lower chambers were filed with 10% FBS-containing culture medium complete media. After 48 h of incubation, cells that migrated through the matrigel and adhered to the lower chamber were fixed in 4% paraformaldehyde for 20 min, and stained with crystal violet. For quantification, five fields per filter were counted under a microscope. Each invasion assay was carried out in triplicate and repeated three times.The cell invasion assay was performed using 24-well matrigel invasion chambers with 8-μm pore inserts (BD Biosciences). HCCLM3 . Genes selected for this array included several classes of proteins involved in cell adhesion, ECM components, cell cycle, cell growth and proliferation, apoptosis, transcription factors, regulators and other genes related to tumour metastasis. RNA isolation, DNase treatment and RNA clean-up were performed according to the manufacturer’s protocol . The isolated RNA was reverse transcribed into cDNA using the RT2 First Strand Kit (Invitrogen). PCR was performed using the RT2 SYBR Green qPCR Master Mix (Invitrogen) on an ABI PRISM7900 instrument . Data normalization was based on correcting all Ct values for the average Ct values of several housekeeping genes present on the array. Each assay was conducted in triplicate.To investigate the molecular mechanism of CALML3 in HCC metastasis, we used the Human Tumour Metastasis RTFrom January 2009 to December 2011, curative resections were performed for HCC on 3544 patients, defined as macroscopically complete removal of the tumour, in the Liver Cancer Institute, Zhongshan Hospital, Fudan University. Of them, 100 cases were selected as the metastasis group, with pulmonary metastasis, and 170 cases as the control group, without pulmonary metastasis, from a surgical specimen database during the follow-up. None of the patients had received any preoperative anti-cancer treatment. Preoperative liver function was classified as Child-Pugh class A. The staging of HCC was determined according to the BCLC staging system. Tumour differentiation was graded according to the Edmondson grading system. Detailed clinicopathological features are provided in Supplementary Table 52. The evaluation was carried out by two independent pathologists who were unaware of the patient outcomes. Images were acquired with a Pannoramic MIDI Slide scanner Immunohistochemical staining for molecules of interest was performed on tissue microarrays (TMAs) made of formalin-fixed, paraffin wax-embedded tumour resection specimens. Two cores were taken from each representative tumour tissue  and from liver tissue adjacent to the tumour within a distance of 10 mm  to construct TMA slides . Then, two TMA sections with 270 pairs of tumours and matched peritumoural samples were constructed. Paraffin wax-embedded sections were deparaffinised in xylene and rehydrated in a decreasing ethanol series, diluted in distilled water. Following antigen retrieval with 10 mM citrate buffer, TMA sections were incubated overnight at 4 °C with a CALML3 antibody . Following 30 min incubation with secondary antibody, sections were developed in 3,3′-diaminobenzidine solution under microscopic observation and counterstained with haematoxylin. The sections were observed under a light microscope for a histological review, to examine tumour microheterogeneity in terms of antigen distribution. Five randomised microscopic views of each section at ×100 and × 400 magnification were observed and scored. The semi-quantitative scoring system was based on both staining intensity  and the percentage of positive cells 17 , satisfying the following three criteria from the observed data, then the system is near the critical state or tipping point:i) of molecules in this dominant group are increased markedly,Standard deviations (SDi) of molecules (expression levels) in this dominant group are increased significantly andPearson correlation coefficients (PCCo) are decreased significantly.Pearson correlation coefficients between molecules in this group and others , and PCCo is the average PCC of molecule-pairs between the dominant group and others . In fact, the three conditions can be expressed approximately in the following sentence: from the measured data, the appearance of a group of genes (or proteins) with strongly collective fluctuations indicates an imminent critical transition . Thus, the group members are the predictive/dynamic biomarkers for this critical  transition. When CI reaches a peak or increases markedly during the measured periods, the biological system is at the critical period or tipping point  approximately considering the all three criteria can be used as the numerical signal of the DNB method:int Fig. . The DNBmentclass2pt{minimPriority one , and all genetic information processing, cellular processes, and signalling pathways  and SPSS 19.0  was used for statistical analyses. Cumulative survival time was calculated by the Kaplan–Meier method and analysed with the log-rank test. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. ROC curve analysis was used to determine the predictive value of the parameters, and the differences in the area under the curve (AUC) were detected by SPSS. The 53 was used for canonical pathway detection. Then, we estimated enrichment significance of specific proteins in each biological process or pathway based on a hypergeometric test. Thus, significantly enriched functions were chosen by the corresponding P-value being < 0.05 after FDR correction.To uncover potential HCC-associated biological functions regulated by the identified genes or proteins, we mapped the proteins into known molecular sets with functional annotations. Here, KEGGAll data have been uploaded to GSE94016.The data that support this study are available within the article and its Supplementary Information files or available from the authors upon request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


An RNA-based fluorogenic module consists of a light-up RNA aptamer able to specifically interact with a fluorogen to form a fluorescent complex. Over the past decade, significant efforts have been devoted to the development of such modules, which now cover the whole visible spectrum, as well as to their engineering to serve in a wide range of applications. In this review, we summarize the different strategies used to develop each partner (the fluorogen and the light-up RNA aptamer) prior to giving an overview of their applications that range from live-cell RNA imaging to the set-up of high-throughput drug screening pipelines. We then conclude with a critical discussion on the current limitations of these modules and how combining in vitro selection with screening approaches may help develop even better molecules. Quoting the famous idiom: “seeing is believing”, which makes imaging and spectroscopic technologies very popular in life sciences to detect and track molecules. Among the different visualization strategies, fluorescence is the most attractive and widely used, mainly because it is safe, sensitive and it offers the possibility of analyzing multiple colors at the same time, with a long shelf life of the fluorophores when properly stored. Among the different fluorescent probes, those acquiring their fluorescence only in permissive conditions  and hereafter defined as being fluorogenic are the most useful. These probes exhibit only a weak fluorescence in non-permissive conditions, making washing steps dispensable, and permitting the monitoring of dynamic processes in time course experiments. A plethora of such fluorogenic molecules has been developed for applications as diverse as ions and small molecules sensing, pH monitoring or environment viscosity assessment ,2. MoreoAequorea victoria [The discovery of the naturally fluorescing Green Fluorescent Protein (GFP) from the jellyfish victoria  had a prvictoria ,6, makinvictoria  or the rvictoria  regions victoria  for a covictoria ) or a whvictoria ) appendevictoria .In contrast to proteins, no naturally fluorescent RNA has been discovered yet, making the development of RNA-based genetically encoded fluorescent reporters less straightforward than their protein counterparts. A first live-cell compatible strategy pioneered by Bertrand et al.  consistsbound) that should be as close as possible to 1. (ii) In its free form, the fluorogen should display the lowest possible quantum yield (Φfree) to minimize background fluorescence. Consequently, a good fluorogenic module is expected to have the highest possible fluorescence enhancement . (iii) The RNA–dye interaction should be highly specific and bio-orthogonal . (iv) Moreover, the interaction should occur with a high affinity (dissociation constant DK in the nM range or less) to make using low concentration of the dye possible and to keep fluorescence background as low as possible, which allows getting high contrast [RNA-based fluorogenic modules are made of two components: a fluorogenic dye  and a specific light-up RNA aptamer. In an ideal case, such module should feature five essential properties. (i) The module should be as bright as possible to ensure sensitive detection, ideally down to single molecule resolution. Therefore, the dye should have an elevated absorption coefficient (ε) and its interaction with the RNA should place it in a conformation and an environment maximizing its quantum yield (Φcontrast . (v) FinThe fluorogen plays a key role in the functionality of a fluorogenic module by contributing to both its brightness and its photostability. Moreover, for live-cell applications, additional parameters such as membrane permeability and the lack of toxicity of the dye should also be considered. The fluorogenicity of a dye can be obtained in various ways encompassing but not limited to charge (or proton) transfer, conformational change, isomerization or even dye aggregation . SeveralMany compounds are known to become highly fluorescent upon interaction with nucleic acids . Among tFluorogenicity can also result from intramolecular movements allowing for non-radiative relaxation of the dye upon excitation b. Such cFinally, fluorogenic dyes can be obtained by appending a quenching group to a fluorescent organic dye c. In itsAll together, these different strategies allowed designing a variety of fluorogens with fluorescence emission spanning most of the visible spectrum d and briOnce a fluorogen has been designed, the molecule can be used as target for the isolation and the optimization of a specific light-up RNA aptamer using two conceptually different approaches: (i) a selection strictly speaking during which an RNA pool is challenged to interact with the dye and only the best binders are recovered; or (ii) a screening approach where the light-up capacity of each molecule is analyzed and only the most fluorogenic ones are recovered.12 to 1015 sequences) in a single experiment.Historically, RNA (or DNA) aptamers are isolated using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach ,57, a seDK ~0.1–1 µM) for their cognate dyes. This limitation could be overcome by changing the elution mode. For instance, the insertion of a disulfide bond within the linker between the dye and the bead, allows for the elution of dye/RNA complexes by adding dithiothreitol [DK in the nM range, the highest affinity described so far for a fluorogenic module  allows the expression of abundant non-coding RNAs to be monitored in prokaryotes [Direct expression of light-up RNA aptamers in cells can be challenging due to their degradation by cell nucleases. Therefore, the molecules are usually expressed inserted into a second RNA such as a transfer RNA (tRNA) ,86 or a karyotes ,32,55 ankaryotes ,31,34. Mkaryotes , within karyotes  or in mekaryotes  while prkaryotes ,92,93, ykaryotes , mammalikaryotes , viruseskaryotes ,96,97 ankaryotes . The limkaryotes  or labelkaryotes  and enabkaryotes . InteresDirect insertion of light-up aptamers into target RNAs is mainly limited by the laborious genetics required for modifying the locus of each target gene. This limitation can nevertheless be overcome by using in trans-acting fluorogenic modules that are destabilized forms of the light-up aptamer ,78 Figub. In thil-homocysteine (SAH) [Its great structural flexibility enables RNA to fold into aptamers able to specifically recognize virtually any type of molecule. Moreover, if the interaction between the aptamer and its target leads to a structural remodeling or even the stabilization of RNA structure, then such aptamer can be used for the conception of an allosteric biosensor in which a sensing module is connected to a reporting module via a communication module e. Brieflne (SAH) , Thiaminne (SAH)  and neurne (SAH) . The majne (SAH) . With sune (SAH) ,106,107.Whereas both sensing and reporting modules have key roles in the proper function of allosteric fluorogenic biosensors, the communication module is probably the most critical element, as it drives the information from one aptamer to the other. Usually, a communication module is rationally designed based on thermodynamic considerations since such module would be extremely challenging to isolate using a SELEX approach. However, the recent advances in high-throughput screening technologies introduced above offer a new way of rapidly identifying optimal communication modules. For instance, a large-scale integration microfluidic device was used to systematically screen a library of 94 different Spinach-based glycine biosensor containing different communication modules while measuring their response to various glycine concentrations . TherefoThe possibility of detecting RNA via fluorogenic assays opened a whole bunch of applications in vitro. First, inserting the sequence of a light-up aptamer into that of another RNA allows monitoring in real-time the in vitro transcription of the construct ,115 and,The great ability to predict to some extent the folding of an RNA sequence makes possible the design of molecular circuits composed of two or several RNA (or DNA) elements trapped by folding to keep the circuit silent ,123. HowSplit and full-length light-up aptamers can also be used to devise in vitro procedures aimed at assisting catalytic RNAs engineering ,127, traOver the past decade, significant efforts were devoted to the development of new RNA-based fluorogenic modules leading to a toolbox of modules covering the whole visible spectrum . Many ofDK of a few nM), likely resulting from the use of very stringent wash and elution conditions. Therefore, applying the same concept to other systems could yield modules with much higher affinity. This would reduce the amount of dye in the assays and lead to a gain in sensitivity by improving the contrast of the experiment [Interestingly, even though current modules are already pretty efficient and permitted multiple questions to be answered, none of them completely fulfills all the criteria enounced in periment . Moreoveperiment  and Mangperiment . This phperiment . This prperiment . Therefoperiment . New genperiment ) to moreperiment ,52 and o


Acoustic recording has been recognized as a valuable tool for non-intrusive monitoring of the marine environment, complementing traditional visual surveys. Acoustic surveys conducted on coral ecosystems have so far been restricted to barrier reefs and to shallow depths (10–30 m). Since they may provide refuge for coral reef organisms, the monitoring of outer reef slopes and describing of the soundscapes of deeper environment could provide insights into the characteristics of different biotopes of coral ecosystems. In this study, the acoustic features of four different habitats, with different topographies and substrates, located at different depths from 10 to 100 m, were recorded during day-time on the outer reef slope of the north Coast of Moorea Island (French Polynesia). Barrier reefs appeared to be the noisiest habitats whereas the average sound levels at other habitats decreased with their distance from the reef and with increasing depth. However, sound levels were higher than expected by propagation models, supporting that these habitats possess their own sound sources. While reef sounds are known to attract marine larvae, sounds from deeper habitats may then also have a non-negligible attractive potential, coming into play before the reef itself. The existence of coral reefs at depths of more than 150 m in tropical regions has been known for decades . RecentlA soundscape is defined as the collection of all sounds that are present in a landscape, which vary over space and time . Within These studies performed in shallow waters demonstrate that acoustical differences between reef habitats are due to variations in the sonic activity of marine organisms, i.e., soniferous fishes, snapping shrimps  and the The study was carried out at the end of the warm season, from June to July 2015, along three North-orientated seaward transects characterized by increasing depths, extending from the barrier reef , to the sandy plain , the reef slope  and to the more distant reef drop-off . The barrier reef of transect 1 was located in a Marine Protected Area (MPA) while barrier reefs of transects 2 and 3 were located in non-protected areas. Transects 1 and 2 were separated by the pass of Tiahura, a coral-free area located in front of an opening in the reef crest that canalizes the water flow to the ocean . BertuccRecordings were conducted between 09:00 and 16:00. This period of time shows little variations in acoustic activity, in contrast to early morning and late afternoon where drastic changes in sound intensity and complexity may happen rapidly . RecordiAn underwater Remora acoustic recorder  connected to a HTI96-min hydrophone  was attached to a block of lead placed at the end of a 100 m rope. This minimized vibration of the rope and current-driven movement of the device. The measurements sequence consisted of recording the four different habitats of a single northward transect, i.e., BR, SP, RS, DO, in a random order. For each habitat, the recorder was suspended from the boat and lowered by an experimenter into the water until it was 5 m above the sea floor. The depth of the device (recorder and hydrophone) was determined by means of marks positioned every 5 m along the rope. Water depth was measured every 2 min with the sonar system to ensure that recordings were made within the appropriate habitat, at a constant depth, and that the hydrophone did not risk hitting the sea floor. Recordings lasted 10 min  before the recorder was pulled back on board and switched off. Completing one transect took approximately 90 min. For each transect, three replicates were obtained for each habitat type with a one week time interval between them . A totalN = 116) of power spectra were normally distributed  and were compared between the three transects and the four habitat types with two-way ANOVAs followed by Tukey’s post-hoc tests for pairwise comparisons.A 20 Hz high-pass frequency filter was applied to all recordings to eliminate very low frequencies. The start and end sections corresponding respectively to the positioning and withdrawal of the recorder were deleted. Recordings were further cleansed by visually inspecting sound spectrograms using Avisoft SASLab Pro 5.2.07 software  in order to cut-out anthropogenic sound sources and other artefactual sounds . This cleansing-step shortened some of the recordings down to 4 min. For each habitat, a set of 12 subsamples of 60 s were used, which were randomly extracted from the 3 replicates in order to produce spectra based on recordings of the same duration . The sound pressure levels measured for each 21.53 Hz frequency band  were transformed into µPa  and averaged. Averaged sound pressure levels were then converted back into dB re: 1 µPa to present the average spectrum of each habitat. The characteristics of each habitat were described on the basis of variations of the average spectral profiles. For each habitat, the root mean square (RMS) of the sound pressure level was measured on the 20 Hz–2.5 kHz frequency band using Avisoft SASLab Pro 5.2.07 software. This low frequency band is dominated by fish vocalizations . Due to α = 0.05 and carried out with R 3.1.2      . For tra < 0.05) . For tra < 0.05) . For tra < 0.05) .F2,116 = 2.80 ; P < 0.05)    . For RS, < 0.05) . Spectra < 0.05) .2 = 11.24, df = 2, P = 0.004, Tukey’s post-hoc tests for pairwise comparisons, all P-values < 0.05)   with all values below 92 dB re: 1 µPa. RS of transect 2 showed significantly lower average sound pressure level . The DO of transect 2 showed a significantly higher average sound pressure level of 95 dB re: 1µPa   . No diff < 0.05) . No diff < 0.05) .Recent studies on coral reefs have highlighted the positive relationships between sound signatures and coral cover, density of fishes or increased number of biotic sound sources in shallow waters . In thisRecordings made at the barrier reef presented higher sound pressure levels in the low frequency range despite the fact that low frequencies transmit poorly in shallow waters . The higRecorded sound pressure levels in deeper habitats highlight that sounds do not result from the sole propagation and degradation of sounds produced at the level of the noisy barrier reef, as predicted by propagation models  Table 2Table 2. Overall, as in shallow waters, differences in spectral patterns seem to exist in deeper habitats and may therefore reflect different characteristics of the habitats, i.e., physical and/or biological. Acoustic cues produced by deeper habitats may therefore also be used in the orientation of marine larvae and may come into play earlier in the recruitment of coral reefs organisms. However, the short period of time sampled in this study remains insufficient to reliably characterize the different habitats and only provides initial information on the different acoustic signatures of deeper habitats. Fish vocal activity can change drastically at dusk or dawn and be more sustained at night with specific species vocalizing in specific time windows . MonitorWe still know very little about the acoustic ecology of deeper habitats adjacent to coral reefs. It has been assumed that deeper environments were less likely to be impacted by anthropogenic activities or by global change, and therefore may provide refuge areas for shallow reef species . This hyResearch in practically unexplored depths will undoubtedly bring new knowledge and tools that will be extremely valuable for the creation of Marine Protected Areas (MPAs), watershed management plans and the development of conservation plans for coral reefs as a whole, from shallow to deep water habitats.


For most sample types, the automation of RNA and DNA sample preparation workflows enables high throughput next-generation sequencing (NGS) library preparation. Greater adoption of small RNA (sRNA) sequencing has been hindered by high sample input requirements and inherent ligation side products formed during library preparation. These side products, known as adapter dimer, are very similar in size to the tagged library. Most sRNA library preparation strategies thus employ a gel purification step to isolate tagged library from adapter dimer contaminants. At very low sample inputs, adapter dimer side products dominate the reaction and limit the sensitivity of this technique. Here we address the need for improved specificity of sRNA library preparation workflows with a novel library preparation approach that uses modified adapters to suppress adapter dimer formation. This workflow allows for lower sample inputs and elimination of the gel purification step, which in turn allows for an automatable sRNA library preparation protocol. NGS is a powerful tool that yields vast amounts of sequence data and allows the analysis of many samples in parallel. Correlation with genetic databases allows identification of novel biomarkers for better diagnosis and more personalized treatment. Next-generation sequencing can be used to examine DNA or RNA for whole genome or transcriptome analysis, respectively. In addition, various techniques such as Chromatin Immunoprecipitation (ChIP), Cross-linking Immunoprecipitation (ClIP), and RNA Immunoprecipitation Sequencing (RIP-Seq) isolate protein/nucleic acid complexes and sequencing of the partitioned nucleic acids allows cataloging of molecular interactions. Transcriptome analysis can be further divided into long coding RNA (messenger RNA or mRNA), long non-coding RNA (lncRNA), or small RNA (sRNA). sRNA are < 500 nucleotides (nt) and consist of a variety of sRNA species including piwi interacting RNA (piRNA), small interfering RNA (siRNA), Y RNA, transfer RNA (tRNA), and mircroRNA (miRNA) –3. miRNALong RNA-Seq and DNA-Seq library preparation techniques have matured quickly and allow PCR free library preparation with low sample inputs and automated protocols. Inherent obstacles for small RNA-Seq (sRNA-Seq) library preparation have thus far limited sequencing of lower RNA inputs and have prevented sRNA-Seq automation. In a traditional sRNA library preparation, oligonucleotides called adapters are ligated onto both the 5΄ and 3΄ ends of the small RNA targets (library) to form a tagged library pool . These aSeveral methods have been developed to curtail adapter dimer formation. One method requires early inclusion of the reverse transcription (RT) primer, which is complementary to the 3΄ adapter. Directly after the first ligation step, the RT primer is added to create a double stranded product which cannot be ligated by T4 RNA Ligase 1 to the 5΄ adapter . Other mGel purification is a common and often necessary size selection procedure as a final step in library preparation. However, even with a laborious gel purification step that can substantially reduce the amount of adapter dimer, its presence is not completely eliminated. Minimal amounts of adapter dimer contamination will be loaded onto the flow cell along with the tagged library and again preferentially amplify to form clusters and take up valuable sequencing reads that could otherwise be occupied by tagged library. Reproducibility of gel extractions can vary tremendously in percent recovery of sample and success largely depends on the technician performing the procedure. Furthermore, with low product recovery it is possible to lose RNA sequences that have low expression levels, leading to false negative data. Gel purification significantly limits the ability to automate library preparation for sRNA thus limiting high throughput experiments. The two most common commercially available kits, TruSeq Small RNA Library Preparation Kit (Illumina) and NEBNext Small RNA Library Prep Set (New England Biolabs), recommend 100 ng total RNA input as the lowest sample amount achievable . Below 1We have developed a novel small RNA library preparation method which uses chemically modified adapters to prevent adapter dimer formation by blocking ligation of the 5΄ and 3΄ adapters to one another, while allowing for efficient tagging of adapters onto the small RNA library . ChemicaModified adapter oligonucleotides and PCR primers were synthesized at TriLink BioTechnologies, LLC. All oligonucleotides were purified by polyacrylamide gel electrophoresis (PAGE). P = 5΄ phosphate; ddC = 2΄,3΄-dideoxycytidine; rApp = adenylate; MP = methylphosphonate; 2΄OMe = 2΄-O-methyl.5΄ FAM-GTACCAGTCGCCTAGAATACT-3΄LuoDon R FAM: (P)AGTTGTCATAGTTTGATCCTCTAGTCTGGGAGTATTCTAGGCGACTGGTAddC 3΄5΄ Luo 3΄ Adapter (51nt): Original sequence taken from Luo et al. paper in bold. Primer binding site added to end (underlined) 5΄ (rApp)TGGAATTCTCGGGTGCCAAGG(ddC) 3΄Unmodified 3΄ Adapter: 5΄ GUUCAGAGUUCUACAGUCCGACGAUC 3΄Unmodified 5΄ Adapter: 5΄ (rApp)T(MP)GGAATTCTCGGGTGCCAAGG(ddC) 3΄CleanTag 3΄ Adapter: 5΄ GUUCAGAGUUCUACAGUCCGACGAU (C) 2΄OMe 3΄CleanTag 5΄ Adapter: 5´rAppA(MP)GATCGGAAGAGCACACGTCT-NH2–3´Alternate 3΄ Adapter Sequence: 5΄ GCCTTGGCACCCGAGAATTCCA 3΄RT primer: 5΄ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA 3΄Forward primer: CGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA 3΄5΄ CAAGCAGAAGACGGCATACGAGATReverse primer:  Illumina compatible barcoded reverse PCR primers:CGTGAT, Index 2: ACATCG; Index 3: GCCTAA; Index 4: TGGTCA; Index 5: CACTGT; Index 6: ATTGGC; Index 7: GATCTG; Index 8: TCAAGT; Index 9: CTGATC; Index 10: AAGCTA; Index 11: GTAGCC; Index 12: TACAAG.Index Primer set 1 for Illumina : Index 1: GGAACT; Index 15:TGACAT; Index 16:GGACGG; Index 17:CTCTAC; Index 18:GCGGAC; Index 19:TTTCAC; Index 20:GGCCAC; Index 21:CGAAAC; Index 22:CGTACG; Index 23:CCACTC; Index 24:GCTACC.Index Primer set 2 for Illumina : Index 13:TTGACT; Index 14:5' P-NNNUACGACCUGCUGCCUUNNN 3'Let7d-3p (NNN): 5' P-NNNGGUAGUAGGUUGCAUANNN 3΄Let7d-5p (NNN): 5' P-CUAUACGACCUGCUGCCUUUCU 3'Let-7d-3p RNA: 5' P-AGAGGUAGUAGGUUGCAUAGUU 3΄Let-7d-5p RNA: Initial ligation reactions were performed with higher concentrations of adapters (each ~ 1μM) and synthetic RNA (1μM) for visualization purposes. Ligation conditions for each ligase are as listed in library preparation section below unless otherwise indicated. 1X tris/borate/ethylenediaminetetraacetic acid (TBE)-Urea buffer (ThermoFisher) was added in equal volumes to ligation reactions. Samples were heated for 2 minutes at 70°C prior to loading onto a 10% TBE-Urea Novex precast gel (ThermoFisher). Gel was run at 165 volts for approximately 25 minutes and then stained for 5 minutes with SYBR Gold (ThermoFisher). Gel densitometry was performed using ImageJ to quantify product bands  followed by final extension at 72°C for 10 minutes. For RNA inputs below 1 μg the 3΄ and 5΄ adapter concentrations and PCR cycles were adjusted depending on RNA input amount: 1000 ng total RNA , 100 ng , 10 ng , 1 ng , 100 pg , 10 pg . These parameters were optimized for samples with high quality RNA.Ligation Step 1: 1X Buffer 1 (50 mM Tris(hydroxymethyl)aminomethane HCl pH 7.5, 10 mM MgClLigases: T4 RNA Ligase 2, truncated (New England Biolabs) and T4 RNA Ligase 2, truncated K227Q (New England Biolabs). Other RT enzymes: Superscript II Reverse Transcriptase (ThermoFisher).A number of commercially available RNA templates were used for these studies. FirstChoice Human Brain total RNA and Breast Adenocarcinoma (MCF-7) Total RNA are available from Life Technologies . Universal Human Reference RNA (UHR) is composed of total RNA from 10 human cell lines including adenocarcinoma (mammary gland), hepatoblastoma (liver), adenocarcinoma (cervix), embryonal carcinoma (testis), glioblastoma (brain), melanoma, liposarcoma, histiocytic lymphoma (macrophage and histocyte), lymphoblastic leukemia (T lymphoblast), and plasmacytoma/myeloma (B lymphocyte) . Human trachea total RNA is available from Agilent Technologies . T Helper Cell (CD4+) total RNA can be purchased from Miltenyi Biotec . miRXplore Universal Reference (Miltenyi Biotec #130-093-521) is a commercially available pool of 963 synthetic miRNA  at equimolar quantities of 5 fmol/μL.Libraries (crude or purified) were visualized by 4% EX agarose E-gel  run for 30 minutes and imaged with UV transilluminator.Library prep samples after PCR (crude or purified) were analyzed by 2100 (Agilent Technologies). Samples were diluted 1:10 with water and then 1 μL was loaded onto a High Sensitivity DNA chip for analysis. The 2100 Expert software was used to quantitate library peaks. For smear analysis all peaks within regions 100–300 nt were included in quantification.Size selection targeting 100–200 nucleotides was performed on small RNA libraries using Agencourt AMPure XP beads (Beckman Coulter).1X AMPure XP magnetic beads (80 μL) was added to 80 μL of final PCR product then mixed and incubated at room temperature for 10 minutes. Tubes were then placed on magnetic rack for 4 minutes to separate the beads. Supernatant containing desired product was then transferred to a new clean tube and 1.8X original PCR volume (144 μL) of beads were mixed and incubated with supernatant for 10 minutes at room temperature. Again, tubes were placed on magnetic rack for 4 minutes to separate beads and supernatant. This time, supernatant was discarded and beads were washed twice with 500 μL of 70% ethanol on magnetic rack. Beads were then air dried for 5 minutes on magnetic rack before resuspended in 17 μL nuclease free water. Beads were incubated in water for 2 minutes at room temperature before placed back on magnetic rack for elution. 15 μL of eluate was collected as purified product.Gel purification was performed on PCR samples run on 4% EX agarose E-Gels  using Zymoclean Gel DNA Recovery Kit .In most cases, experiments were planned to contain 24 samples which would be barcoded, pooled equally, and then loaded onto one lane of a flow cell. Libraries were prepared individually and barcoded with reverse primers during the PCR step which contained Illumina compatible indices #1–24. Individual library concentrations were determined by running a high sensitivity Bioanalyzer chip followed by smear analysis. Since all samples were to occupy a similar amount of space on the flow cell, we divided the final concentration of our pooled mixture, 10 nM for example, by 24 to determine that each samples final concentration should equal 0.416 nM. We then calculated the volume of each sample that would be required to meet this number based on the individual concentration. Each of the 24 samples was pooled accordingly to occupy equal space on a single lane of a flow cell.All next-generation sequencing experiments were performed on an Illumina HiSeq 2500 with single-end 100 nt reads. 24 samples were loaded onto one lane of a flow cell. All sequencing was outsourced to a core facility (TSRI- Scripps Research Institute) or company (GENEWIZ).PRJNA350292, SRA study number SRP092414 . Data analysis was performed using a number of different methods and in some cases outsourced, as described below. See figure and table legends for analysis method used. Initial NGS runs were outsourced to a core facility at TSRI, with data analysis and statistics performed by their in-house bioinformatician. Subsequent data sets were analyzed using Geneious version R8 from Biomatters  and the Illumina compatible CleanTag 5΄ adapter [5΄- GUU CAG AGU UCU ACA GUC CGA CGA UC(OMe)-3΄].Next, three top combinations of modified adapters were used in a full library preparation workflow on a synthetic miRNA pool (Miltenyi) and then sequenced in an NGS run to dissect out any minimal differences between the modifications . The PS Finally, we investigated whether these modifications, when in close proximity without an RNA insert (adapter dimer), would inhibit the cDNA synthesis step. This in turn would further help to suppress any residual adapter dimer if formed at the ligation step. The ability to reverse transcribe through a single MP and a single OMe modification was evident since a product was formed from a tagged library but there was no specific investigation into whether these modifications slowed down cDNA synthesis. To test the effect of the modifications on reverse transcription for the CleanTag modifications, we used a FAM labeled RT primer to track cDNA synthesis yield on various adapter dimer versions: A) unmodified; B) 3΄ adapter modified only; C) 5΄ adapter modified only; and D) both 3΄ and 5΄ modified adapters. At very high concentrations and with nothing else in the reaction to compete with, we were able to force ligation of the modified adapters to one another and conduct the downstream RT reaction. Reading through two modifications in close proximity to each other proved challenging for the reverse transcriptase enzyme as cDNA synthesis was decreased by 70% . This gaWe then introduced our top modifications onto another set of adapter sequences but interestingly, we determined that adapter dimer suppression was not as dramatic as when using the CleanTag adapter sequences . No furtIn order to determine if the chemical modifications introduced any significant changes in miRNA detection, we compared sequencing results between unmodified and modified adapters within our optimized workflow. We found that a similar population of miRNA was tagged by unmodified adapters compared to those tagged by the modified adapters . Though With strong suppression of adapter dimer using CleanTag modifications in the library preparation workflow, sequencing from much lower RNA inputs is now possible. Samples using a range of human total brain RNA inputs were sequenced to determine the limit of detection using the modified adapters. Adapters were diluted to optimized concentrations for each amount of total RNA input and PCR cycles were increased accordingly . HoweverIn addition to allowing for lower RNA inputs, removal of the adapter dimer also eliminates the need for the gel purification step. This in turn facilitates automation of the small RNA library prep process. A two-step AMPure XP bead-based purification protocol was optimized to size select product between 100–200 nucleotides. Library preparation with the CleanTag workflow results in limited adapter dimer but also a decrease in side products. This leads to a cleaner bead purified sample downstream, especially when compared to TruSeq Small RNA Library Prep Kit . While bWe further pushed the limit of detection for small RNA library prep using modified adapters down to 100 and 10 pg of human brain total RNA . A singlHerein we have demonstrated the use of oligonucleotide modifications to specifically inhibit the ligation of the 5΄ adapter to the 3΄ adapter thereby significantly reducing adapter dimer formation and improving the specificity of small RNA library preparation workflows. The specific interaction of a 2΄ OMethyl (OMe) and a methyl phosphonate (MP) modification on the adapters proved to be one of few combinations which inhibited ligation and furthermore repressed RT read through and downstream amplification. While certain other modifications initially appeared to suppress ligation, after RT-PCR, the adapter dimer ligation product could be detected and was an indication that not all modifications that suppressed ligation had the added benefit of preventing RT read through. We therefore chose to optimize the workflow around the top combination of modified adapters that suppressed adapter dimer to the greatest extent after both the ligation and reverse transcription-PCR steps. Although initial ligation yields using modified adapters were slightly lower than unmodified adapters, upon workflow optimization the benefit of reduced adapter dimer seemed to help promote RT and PCR amplification of the tagged library and diminish any lower yield effects brought on by the modifications during ligation.Interestingly, the adapter dimer suppression effect seen with our top modifications seems to depend largely on the sequence of the adapters. These modifications were later tested on several other sets of adapter sequences (data from one example shown) but the same level of adapter dimer suppression was not achieved . Simply It is known that RNA ligases have specific preferences for their substrates, thus introducing a level of bias into the small RNA library preparation workflow . It is cA limitation of current small RNA library preparation is high input requirements. Low RNA inputs often result in high adapter dimer yield as this product is preferentially formed and amplified. This makes investigation of small RNAs from precious biological samples difficult as they often give low RNA yield. We have demonstrated that small RNA sequencing from as low as 1 ng total RNA input is made possible due to suppressed adapter dimer. We were unable to produce sufficient library at such low inputs using commercially available kits, as adapter dimer was the dominant product, which emphasizes the importance of dimer suppression, even when gel purification is used. At 1 ng total RNA input we obtained quality sequencing data without significant loss of miRNA reads. At these input levels more limiting biological samples can be easily interrogated. Lower RNA inputs will expand the small RNA-Seq field in several ways. Small RNA from biofluids , FACS cells, exosomes, Clip-Seq, and FFPE samples can more easily be analyzed by next-generation sequencing with higher quality sequencing data. Examples of sRNA library preparation with CleanTag and these challenging sample types are presented elsewhere .We found that moving to lower RNA input levels also required the dilution of the adapter input in order to maintain a reduction in adapter dimer formation. Very low amounts of adapter were needed to form tagged libraries at low RNA inputs. With the reduction in RNA input, an increase in number of PCR cycles was needed to generate enough copies of tagged library for downstream sequencing.Automated high throughput sample preparation that is common for DNA-Seq or RNA-Seq has not yet been implemented for small RNA sequencing. This is largely due to the post library preparation gel clean up needed to eliminate adapter dimer. With the suppression of adapter dimer formation an automatable bead-based purification method can replace manual gel extractions. In general our workflow using modified adapters results in cleaner small RNA libraries with less side products as compared to other commercial kits. Therefore bead purification of CleanTag libraries is a sufficient clean up method for sequencing at most routine RNA inputs. While bead purified samples contain a variety of tagged species which can also be sequenced, we showed that this did not significantly detract from the number of mappable miRNA reads in the sample. Sequencing data generated from bead purified samples produced comparable number of miRNA reads to that of gel purified samples despite the presence of additional tagged species. With the elimination of adapter dimer there is more functional space on the flow cell for sequencing other important small RNA targets in addition to miRNA. miRNA are often still the dominant and shortest product in the reaction so these will preferentially amplify over any other larger molecular weight species, thus maintaining number of mappable reads for this important small RNA category.While maintaining miRNA information, bead-based methods also offers the ability to analyze all types of sRNA tagged in a library ranging in size from 100 to 200 nt, not just a specific type or length of sRNA such as miRNA that has been size selected from a gel. Different cell types or bodily fluids may contain different RNA signatures and be enriched in other types of sRNA other than miRNA. Valuable information including new small RNA biomarkers can be extracted from these sample types and a more in depth analysis can be done when data from other small RNA would have been previously excluded by gel excising the 140 nt targets exclusively. As further information is gained and various new types of sRNA are discovered, small RNA-Seq is becoming increasingly important. Now, more information can be gained from sequencing bead-purified samples and analyzing all small RNA species that are present in a given sample.It is now possible for liquid handling robots to be programmed for automated small RNA library preparation and purification to prepare samples for direct loading onto the sequencer. This will significantly decrease hands on time, human error, and increase throughput. CleanTag improves small RNA sequencing by 1) enabling sequencing of samples containing as little as 1 ng total RNA; 2) allowing for automation by the use of a bead-based purification; and 3) increasing throughput potential with an automated workflow.Samples with ultra low levels of RNA including single cell quantities (10 pg) of material can now be easily analyzed by NGS. Preliminary results reveal adequate amounts of library generated from these low inputs, a sufficient amount of quality reads generated after filtering, but an overall loss in low expressed miRNA species and an overabundance of tRNA reads. The biological relevance of this observation remains to be examined. The importance of tRNA fragments as other small RNA biomarkers is increasing, especially in breast cancer research , 36, 37.We have demonstrated that rendering adapter dimer formation negligible overcomes many of the current challenges for sRNA library preparation. There are multiple benefits from adapter dimer suppression including the feasibility of using ultra-low total RNA inputs, potential to automate the entire workflow, and elimination of a gel-extraction clean up step. These improvements significantly enhance sRNA library preparation workflow and the ability to sequence ultra low inputs now opens up sRNA-Seq to more sample types regardless of limiting material. This includes single cell samples, FACS sorted cells, FFPE, Clip-Seq, biological fluids, etc. This modified adapter technology may also be applied to other library preparation techniques such as long RNA-Seq, and cell free ssDNA-Seq in the near future, as our investigations of these applications is ongoing.S1 FigA) 4% agarose gel analysis of bead purified PCR products. Libraries were prepared with unmodified adapters and 0.7 to 70 ng of a synthetic miRNA (Let 7d-3p (NNN)). B) NGS data showing average number of mapped reads and average number of adapter dimer reads. Libraries were prepared with the recommended conditions of the TruSeq Small RNA Library Prep Kit using unmodified adapters and brain total RNA at inputs of 10, 100, and 1000 ng. Data analysis was performed using Geneious.(TIF)Click here for additional data file.S2 FigRepresentative chemical modifications screened on adapter oligonucleotides for sRNA-Seq library preparation. Nomenclature for the positions modified is shown.(TIF)Click here for additional data file.S3 FigExample of screened modifications on A) 3´ adapter Luo or B) the 5´ adapter for ligation efficiency. Red box indicates a modification in which ligation to the substrate was undetectable. Reactions were incubated with 10 U T4 RNA Ligase 1 for 1 hour at 37°C.(TIF)Click here for additional data file.S4 FigReverse transcription was performed on different modified adapter dimer substrates using a FAM-labeled RT primer. Ligation product from unmodified adapters served as the control for normalization (column A). Read through from a single modification on either the 3´ adapter (B) or 5´ adapter (C) was compared to a double modified substrate (D), both 5´ and 3´ adapters modified. RT products were run on a gel, imaged, and quantified for relative cDNA synthesis yield determination.(TIF)Click here for additional data file.S5 FigA) Library preparation using 7 ng synthetic miRNA (Let 7d-3p (NNN)) input. U = both adapters were unmodified; M = both adapter were modified with top modifications. The CleanTag adapter set was compared to an alternate adapter set with a different sequence for the 3´ adapter. The same modifications were used for the alternate set. B) The alternate adapters were also tested with 1000 ng brain total RNA input. The CleanTag library preparation workflow was used to prepare libraries.(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableComparison of data between different small RNA library preparations using human brain total RNA input between 1–100 ng. Libraries sequenced on HiSeq 2500 SR, 1x 100bp. Data analysis was performed using Geneious. Samples run in triplicate.(TIF)Click here for additional data file.S3 TableLibraries prepared with human brain total RNA at various input amounts using CleanTag library preparation kit. All samples performed in triplicate. Inputs at 1000 and 1 ng were bead purified. Inputs at 100 and 10 pg were pooled and gel purified. Libraries sequenced on HiSeq 2500 SR, 1x 100bp and data analysis was performed with Galaxy.(TIF)Click here for additional data file.S4 TableBreakdown of data from (TIF)Click here for additional data file.


BioEssays, Hamid and Makeyev explore an interesting new idea regarding the evolutionary origins of regulated mRNA turnover pathways In this issue of The hypothesis is supported by recent results that NMD not only regulates cellular transcripts but also viral RNAs NMD and other regulated mRNA decay pathways need to correctly identify target mRNAs. Interestingly, a similar molecular recognition problem of selecting RNA molecules is faced by the innate immune system. Defence against RNA viruses often involves cellular recognition of viral RNA molecules. This triggers direct effector functions such as degradation or sequestration of viral RNAs as well as the activation of signalling pathways that induce an antiviral state


These proteins recapitulated features of ALS-linked molecules, including insoluble aggregation, formation of cytoplasmic IBs and components of RNA granules, and cytotoxicity instigation. These findings indicated that these artificial RNA-binding proteins mimicked features of ALS-linked molecules and allowed the study of mechanisms associated with gain of toxic functions related to ALS/FTD pathogenesis.Aberrant RNA-binding proteins form the core of the neurodegeneration cascade in spectrums of disease, such as amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Six ALS-related molecules, TDP-43, FUS, TAF15, EWSR1, heterogeneous nuclear (hn)RNPA1 and hnRNPA2 are RNA-binding proteins containing candidate mutations identified in ALS patients and those share several common features, including harboring an aggregation-prone prion-like domain (PrLD) containing a glycine/serine-tyrosine-glycine/serine (G/S-Y-G/S)-motif-enriched low-complexity sequence and rich in glutamine and/or asparagine. Additinally, these six molecules are components of RNA granules involved in RNA quality control and become mislocated from the nucleus to form cytoplasmic inclusion bodies (IBs) in the ALS/FTD-affected brain. To reveal the essential mechanisms involved in ALS/FTD-related cytotoxicity associated with RNA-binding proteins containing PrLDs, we designed artificial RNA-binding proteins harboring G Six ALS-related molecules, TDP-43, FUS, TAF15, EWSR1, heterogeneous nuclear (hn) RNPA1 and hnRNPA2 are RNA-binding proteins containing candidate mutations identified in ALS patients. These molecules share several common features. First, they harbor a prion-like domain (PrLD) and are aggregation-prone2, and PrLDs involving self-propagating amyloid-fibril conformations exhibit two critical features: they contain a glycine/serine-tyrosine-glycine/serine (G/S-Y-G/S)-motif-enriched low-complexity sequence (regions of protein sequences with biased amino acid composition)3 and are rich in glutamine and/or asparagine (Q/N)4. PrLDs can mediate pathologically aggregated states observed in the ALS/FTD brain as a result of aging, and/or ALS-linked mutations clustered in the PrLDs of TDP-43 and FUS and which accelerate this toxic conversion to initiate neurodegeneration.Growing evidence indicates that disturbed RNA quality control systems mediated by aberrant RNA-binding proteins, such as TDP-43 and FUS, form the core of the neurodegeneration cascade in ALS/FTD7. A low-complexity sequence in PrLDs mediates interactions between biophysical molecules and RNA granules and it drives the assembly of cytoplasmic RNA granules, with ALS-linked mutations in TDP-43, FUS, and hnRNPA211.Second, they are included as components of RNA or messenger ribonucleoprotein granules, such as stress granules (SGs), which are involved in the RNA quality control system by stabilizing and storing mRNAs and arresting translation to prevent the accumulation of misfolded proteins14. The mutation of a truncated protein lacking a NLS (R495X) was identified as being causative of juvenile ALS with rapid disease progression, indicating that impairment of FUS nucleocytoplasmic trafficking is directly linked to motor-neuron degeneration15. Given these commonalities, cytoplasmic aggregation of RNA-binding proteins could constitute a critical process in the development of the ALS/FTD pathological cascade linked to the RNA quality control system.Third, a common pathological feature of ALS/FTD is the mislocalization of candidate RNA-binding proteins from the nucleus to the cytoplasm and their subsequent formation of cytoplasmic inclusion bodies (IBs) in disease-affected regions. In FUS, ALS-associated mutations are clustered at the non-classical nuclear-localization signal (NLS) in the C-terminus, which directly impedes nuclear localization/S-Y-G/S-motif-enriched low-complexity sequence with and without enriched glutamine residues and signal sequences for nuclear import/export. To minimize superfluous functions in mammalian cells, a well-characterized plant-based RNA-recognition domain [poly(A)-binding protein (PABPs)]16 was selected as a standard RNA-recognition motif (RRM). These proteins exhibited features similar to those of ALS-linked molecules, including insoluble aggregation, formation of cytoplasmic IBs and RNA-granule components, and instigation of cytotoxicity. These findings indicated that cytoplasmic RNA-binding proteins containing PrLDs can initiate neurotoxicity independent of their specific physiological functions. We propose that these artificial RNA-binding proteins effectively replicated features of ALS-linked molecules and constitute novel experimental molecules that enable enhanced understanding of the molecular basis of ALS/FTD-related cytotoxicity.An emerging question involving these ALS/FTD-related RNA-binding proteins is whether their associated cytotoxicity is dependent upon specific physiological functions related to target RNAs or their robust and non-specific assembly with ribonucleoproteins. To address this, we generated artificial RNA-binding proteins harboring repeats of the G/S-Y-G/S motif and minimize repeats of the nucleotide sequence to avoid formation of aberrant RNA structures. Additionally, we used the well-characterized PABP RRM from Citrus sinensis, which shares 43% identity with the human PABPN116, to minimize the possibility of functional redundancy in mammalian cells. Furthermore, we added a NLS (MPKKKRKVGG) or NES (MLPPLERLTLDGG) to investigate compartment-specific cytotoxicity.To reveal cytotoxicity induced by RNA-binding proteins containing PrLDs, we generated synthetic cDNAs encoding 67 repeats of the G/S-Y-G/S motif, 50 repeats of G/S-Y-G/S + Q or G/S-Y-G/S + Q/N motifs containing the PABP RRM, signal sequences for nuclear import/export, and GFP tags  at ~73 kDa according to western blot results. Both artificial proteins were also detected within the stacking gel, indicating that both were prone to aggregation and formation of high-molecular-weight complexes. A series of deletion experiments revealed that the SYG ± Q PrLD was essential for the formation of these high-molecular-weight complexes ; Fig. n = 1)] and HeLa cells [percentage of cells with IBs: 3.8% ] ], SG markers {, HuR [percentage of HuR+ IBs: 12.5% ], Ataxin2 [percentage of Ataxin2+ IBs: 74.4% ], and SMN [percentage of SMN+ IBs: 15.4% ]}, mRNA transporters, fragile X mental retardation protein (FMRP) [percentage of FMRP+ IBs: 52.5% ], and Staufen [percentage of Staufen+ IBs: 26.8% ]. Notably, SYGQ-NES IBs recruit components of SGs, including nuclear proteins such as TDP-43 and HuR; however, they show varying staining patterns, indicating that SYGQ-NES IBs possess partial features of SGs, and possibly belong to diverse subcellular entities known as heterogeneous RNA granules6.Previous studies demonstrated that IBs involving TDP-43 and FUS in ALS/FTD-patient brain tissue recruit other RNA-binding proteins, such as components of SGs21. As shown in Fig. P < 0.05)] in 293 T cells expressing SYGQ-NES and also mutant FUS10, but not aggresomes containing mutant forms of superoxide dismutase 1. These findings strongly suggested that SYGQ-NES IBs exhibited characteristics associated with SGs.Furthermore, administration of the inhibitor of translational elongation, emetine, stabilizes polysomes and suppresses SG formation26. Here, we observed that expression of SYG/SYGQ-NES proteins significantly reduced nuclear GEMs consistent with results observed in the presence of other ALS-linked RNA-binding protein , and mutation of TDP-43 or FUS disrupt GEM assembly or stabilization27. ALS-linked RNA-binding proteins TDP-43, FUS, and Ataxin2 are SG components, and their mutation leads to the formation of aberrant SGs in vitro and in vivo18. To examine whether SYGQ-NES proteins are recruited to SGs, we performed double immunofluorescence staining in HeLa cells, which is commonly used for examining the characters of SG8 after treatment with arsenite, a well-established SG inducer (38). As shown in Fig. 11.SGs are formed as cytoplasmic foci that aggregate mRNA and RNA-binding proteins under stress conditions, such as hypoxia and heat shock. SGs function as part of the RNA quality control system by stabilizing and storing mRNAs and arresting translation to prevent the accumulation of misfolded proteins30. SYG/SYGQ-NES-expressing cells become rounded and shrink, and showed shorter neurites compared with those observed on control cells  in PrLD(s) also exhibit similar mechanisms of seeded aggregation and propagation.Second, abundant molecular and cellular evidence suggests that the misfolding of specific proteins, such as α-synuclein, β-amyloid, and tau, can act as seeds for aggregation and can be transmitted intercellularly, thereby constituting a manner of self-dissemination similar to that observed with prions47. Analyses by cross-linking and immunoprecipitation suggested that there might be thousands of RNA molecules targeting TDP-43 and FUS during the mRNA-maturation and gene-regulation processes50. A study using a conditional mouse TDP-43 gene-targeting approach in spinal cord motor neurons reported progressive development of ALS-related phenotypes51. Therefore, theories supporting a loss-of-function phenotype associated with TDP-43 proteinopathy and leading directly to RNA dysregulation and development of neurodegeneration remain plausible52. In this study, we were unable to directly examine loss-of-function phenotypes associated with ALS pathogenesis using artificial proteins.Third, affected regions in most ALS patients are characterized by TDP-43-positive IBs in the cytoplasm and also by the depletion of TDP-43 protein in the nucleus50. Therefore, FUS and TDP 43-proteinopathies possibly cause gross RNA dysregulation rather than specific mRNA disruptions in ALS/FTD development. Therefore, an RRM of PABP from Citrus sinensis, which represents an authentic ortholog of the animal PABPs, was used for these artificial proteins to avoid specific binding to RNA in mammalian cells. We speculated that the overexpressed plant-based RRM interacted widely and non-specifically with mRNAs and caused gross RNA dysregulation; however, this does not exclude the possibility that off-target tight-binding to unexpected RNAs might affect the interpretations in studies using de novo proteins. Future studies should define comprehensive artificial SYG/SYGQ-NES proteins -RNA interaction and compare it with other ALS-linked RNA binding protein interactions.Forth, analysis by cross-linking and immunoprecipitation of sequences suggested that approximately 30% mRNA of the murine transcriptome interacted with TDP-43 and FUS54. As shown in Fig. http://web.expasy.org/protparam/) was high (49.22), indicating that this must be improved to ensure stable expression in future studies. However, our findings suggested that the use of de novo proteins, specifically SYG/SYGQ-NES, effectively allowed the study of mechanisms associated with gain of toxic functions related to ALS/FTD pathogenesis. These results support the acquisition of an enhanced understanding of RNA dysregulation mediated by aggregated cytoplasmic RNA-binding proteins and promote the development of new strategies for the treatment of ALS/FTD proteinopathies.Finally, most neurodegeneration-associated aggregated proteins, such as tau and TDP-43, are easily targeted by protein degradation systems and are multiply fragmented in the affected brain10.cDNAs encoding SYG/SYGQ-NES/NLS and synthetic prion-forming domain (sPFD)/control prion-forming domain (cPFD)-NES/NLS (Sequences are shown in Supplementary information) were synthesized  and subcloned into pcDNA (3.1) or pEGFP-N1  or. Wild type and mutant TDP-43 and FUS were described in previous papersHeLa human carcinoma cells, human embryonic kidney 293 cells (293 T cells), were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum. Neuro2a neuroblastoma cells were cultured in αMEM (Gibco) supplemented with 10% fetal bovine serum. Transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Neuro2a cells were differentiated where stated by incubating in DMEM supplemented with 2% (vol/vol) FBS and 20 μM retinoic acid  for 24 h.Mouse monoclonal anti-ubiquitin, HuR, and GFP were purchased from Santa Cruz Biotechnology . Mouse monoclonal anti-α tubulin was from Invitrogen. Mouse monoclonal Anti-βIII Tubulin (2G10), p62/SQSTM1, MAP2 and FMRP was obtained from Millipore . Rabbit polyclonal anti-N-terminus FUS was from Bethyl Labs . Anti-TDP-43 and G3BP antibodies were from Proteintech . Rabbit polyclonal anti-GFP antibody was from Rockland Immunochemicals Inc. . Mouse monoclonal anti-GFP antibody was from Santa Cruz Biotechnology, Inc .Mouse Anti-Ataxin-2 was purchased from BD Biosciences . Rabbit polyclonal anti-GAPDH antibodies were from Cell Signaling Technology . Propidium iodide was purchased from Dojindo . Emetine, and Arsenite was purchased from Sigma-Aldrich.Cells were briefly sonicated in cold lysis buffer . Cell lysates were separated using SDS-PAGE with 10% Tris-glycine gel, after which proteins were transferred to a polyvinylidene difluoride membrane . The membrane was incubated with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies and then visualized using enhanced chemiluminescence reagents . Protein levels were determined by densitometry using an Epson ES-2000 scanner and Image J .To examine the solubility profile of artificial protein, sequential extractions were performed. The cells were lysed in cold RIPA buffer . The cell lysates were cleared by centrifugation at 100,000 × g for 30 min at 4 °C to generate the RIPA soluble samples. To prevent carry-overs, the resulting pellets were washed with RIPA buffer twice. RIPA insoluble pellets were then extracted with urea buffer , and centrifuged at 100,000 × g for 30 min at 22 °C.At 48 h after transfection, cells were fixed with 4% paraformaldehyde on ice for 10 min and then permeabilized in 0.2% Triton X-100 at room temperature for 10 min. Post-fixation with 4% paraformaldehyde on ice for 5 min followed. After blocking of nonspecific binding, coverslips were incubated with primary antibodies and then incubated with Alexa 488 and/or Alexa 586-conjugated secondary antibodies (Invitrogen). Immunofluorescent staining was examined using a Leica TCS SP5 confocal microscope .Well-defined cytoplasmic foci of 1.0–2.5 μm diameter were counted as IBs. To detect cell death, cell were trypsinized and stained with 1 μg/ml propidium iodide . Cell sorting was performed using MoFlo XDP .29.For cytotoxicity analysis in differentiated Neuro2a cells, neurites in more than 30 transfected cells from independent experiments (n = 3) were evaluated using ImageJ, and cells containing neurites of at least two cell-body-diameter length were scored as neurite-bearing cellsStatistical analysis of the data was performed by one-way ANOVA with Fisher’s protected least squares difference test or Student’s t-test using the Statview 5.0 system .Supplementary InformationSupplementary Information


DCBCT) is currently available in the Vero4DRT image‐guided radiotherapy system. We evaluated the image quality and absorbed dose for DCBCT and compared the values with those for single‐source CBCT (SCBCT).Dual‐source cone‐beam computed tomography (HU) linearity, image contrast, and spatial resolution were evaluated using a Catphan phantom. The rotation angle for acquiring SCBCT and DCBCT images is 215° and 115°, respectively. The image uniformity was calculated using measurements obtained at the center and four peripheral positions. The HUs of seven materials inserted into the phantom were measured to evaluate HU linearity and image contrast. The Catphan phantom was scanned with a conventional CT scanner to measure the reference HU for each material. The spatial resolution was calculated using high‐resolution pattern modules. Image quality was analyzed using ImageJ software ver. 1.49. The absorbed dose was measured using a 0.6‐cm3 ionization chamber with a 16‐cm‐diameter cylindrical phantom, at the center and four peripheral positions of the phantom, and calculated using weighted cone‐beam CT dose index (CBCTDIw).Image uniformity, Hounsfield unit (SCBCT, the image uniformity of DCBCT was slightly reduced. A strong linear correlation existed between the measured HU for DCBCT and the reference HU, although the linear regression slope was different from that of the reference HU. DCBCT had poorer image contrast than did SCBCT, particularly with a high‐contrast material. There was no significant difference between the spatial resolutions of SCBCT and DCBCT. The absorbed dose for DCBCT was higher than that for SCBCT, because in DCBCT, the two x‐ray projections overlap between 45° and 70°.Compared with that of DCBCT than for SCBCT in the Vero4DRT.We found that the image quality was poorer and the absorbed dose was higher for  Each imaging unit consists of a kV X ray tube and a FPD. Miura et al.Two important issues need to be addressed when using DCBCT. First, how does the image quality of DCBCT compare with that of SCBCT? Second, does DCBCT increase the patient dose compared with SCBCT? The purpose of CBCT is to provide a volumetric image for patient positioning for radiotherapy; thus, it is important to study the dose–image quality tradeoffs. However, no information is available on the imaging performance of a commercial DCBCT.In this study, we evaluated the performance of a DCBCT, by comparing it with a SCBCT. Both were used in a Vero4DRT. The American Association of Physicists in Medicine (AAPM) Task Group (TG) 142 recommends several QA values for imaging by IGRT.22.AThe characteristics of the Vero4DRT system were published previouslyTo acquire a set of SCBCT images, a kV X‐ray source is rotated 215° clockwise (CW) or counterclockwise (CCW) , was used to assess HU linearity , LDPE (low‐density polyethylene), polystyrene, acrylic, Delrin2.B.3P¯ROI,insert is the mean pixel value in a circled ROI inside an insert and ROI, background is the mean pixel value of the background of the contrast module, respectively.We used the CTP 404 module, described above, to assess the image contrast, which was calculated using the following equation:2.B.4The high‐resolution module CTP 528 has a high‐resolution pattern of 1 through 21 line pairs per centimeter (Lp/cm). High‐contrast resolution was calculated using the method reported by Droege et al.2.B.53 ionization chamber  and a poly(methyl methacrylate) (PMMA) cylindrical phantom , 16 cm in diameter and 15 cm long with a density of 1.19 g/cm3. The midpoint of the cylindrical cavities, located at the center and four peripheral positions in the phantom, corresponded to the in‐room laser system. The ionization chamber was inserted at the center and the four peripheral holes of the cylindrical phantom, with the mechanical isocenter at the center of the phantom. Because CBCT performed with the Vero4DRT involves a partial rotation of the gantry to acquire images, the dose at each of the peripheral measurement points is different. The average absorbed dose was calculated by analogy to the weighted cone‐beam CT dose index (CBCTDIw) Dcenter is the central axis dose and Dperipheral is the average peripheral dose of the scanning phantom.Dose measurements were performed using methodology adapted from that outlined in AAPM Task Group Report No. 111.33.ATable 3.BR2) >0.9997. The measured HU values for SCBCT were closer to the reference HU values than those for DCBCT. The largest differences between the reference and the measured HU values occurred for Teflon; the difference in the HU values obtained using SCBCT (tube 1/tube 2) and DCBCT were 221.9/249.6 and 510.4 HU, respectively.Figure 3.CFigure 3.DTable 3.Ew values of SCBCT (tube 1/tube 2) and DCBCT were 54.3/59.4 and 56.4 mGy, respectively. The CBCTDIw value of DCBCT was 13.5% and 3.8% higher than those of SCBCT (tube 1/tube 2).Table 4We compared the image quality and absorbed dose for the first commercial DCBCT, in the Vero4DRT image‐guided radiotherapy system, with those for SCBCT. The CBCT of the Vero4DRT system cannot rotate a full 360°, which leads to lower image uniformity. The image uniformity of DCBCT is worse than that of SCBCT. With DCBCT, the detector may detect the photons scattered by the object, resulting in degradation of the image quality.No significant degradation in HU linearity for DCBCT was observed when compared to that for SCBCT. The HU linearity of both CBCTs differed from the reference HU. The Teflon HU value obtained via DCBCT had the largest difference with respect to the reference HU, which is attributed to cross‐scatter.Several authors proposed techniques to reduce the cross‐scatter and improve the image quality for DCBCT 16‐18, 20]. Giles et al.. Giles eA CBCT scan can be acquired over a 360° rotation at a maximum angular speed of one rotation per minute. In the Vero4DRT or other on‐board SCBCT systems, acquisition of a CBCT half‐scan using a single kV source takes approximately 30 s, which makes a breath‐hold CT scan difficult to implement. Future studies should investigate the effect of DCBCT on reducing motion artifacts. Data from a phantom may not represent the final say in image quality with respect to clinical patient data because a phantom is relatively uniform, unlike a patient. Figure 5In this study, we evaluated the image quality and absorbed dose for DCBCT by comparing them with the corresponding values for SCBCT, using the Vero4DRT. Compared with SCBCT, DCBCT had slightly reduced image uniformity. A strong linear correlation existed between the mean HU values in the ROIs obtained by DCBCT and the reference HU, although the linear regression slope was different from that of conventional CT. The image contrast with DCBCT, particularly for extreme‐contrast material, was worse than that with SCBCT, even though the absorbed dose was higher.No conflicts of interest.