UNC-16, through LRK-1, also enables Golgi-localization of the μ-subunit of the AP-1 complex. AP1 regulates the size but not the composition of SVP transport carriers. Additionally, UNC-16 and LRK-1 through the AP-3 complex regulates the composition but not the size of the SVP transport carrier. These early biogenesis steps are essential for dependence on the synaptic vesicle motor, UNC-104 for axonal transport. Our results show that UNC-16 and its downstream effectors, LRK-1 and the AP complexes function at the Golgi and/or post-Golgi compartments to control early steps of SV biogenesis. The UNC-16 dependent steps of exclusion, inclusion and motor recruitment are critical for polarized distribution of neuronal cargo.JIP3/UNC-16/dSYD is a MAPK-scaffolding protein with roles in protein trafficking. We show that it is present on the Golgi and is necessary for the polarized distribution of synaptic vesicle proteins (SVPs) and dendritic proteins in neurons. UNC-16 excludes Golgi enzymes from SVP transport carriers and facilitates inclusion of specific SVPs into the same transport carrier. The SVP trafficking roles of UNC-16 are mediated through LRK-1, whose localization to the Golgi is reduced in  Synaptic vesicles (SVs) have a defined composition and size at the synapse. The multiple synaptic vesicle proteins (SVPs) found on these vesicle membranes are synthesized at and trafficked out of the cell body in distinct transport carriers. However, we do not yet understand how different SVPs are sorted and trafficked to the synapse. We show that UNC-16/JIP3 plays a critical role, in a series of essential steps, to ensure proper membrane composition and size of the ensuing SVP carrier exiting the cell body. These processes are “exclusion” of resident Golgi enzymes followed by the “inclusion” of synaptic vesicle proteins in the same transport carrier. Regulation of composition and size seems to occur independently of each other and depends on two distinct AP complexes acting downstream to LRK-1. Our study further indicates that the composition of the transport carrier formed is important for the recruitment of motors and consequently for the polarized localization of SVPs. The secretory pathway in a cell involves the synthesis and trafficking of proteins through the ER-Golgi network and their subsequent targeting to different sub-cellular compartments. Generation of a defined transport carrier, with a characteristic protein and lipid composition, along the trafficking pathways is known to involve at least three steps , several occurring at the trans-Golgi network (TGN). (a) Protein sorting where the secretory cargo is segregated away from Golgi resident proteins –4. For ein vivo studies in C. elegans have shown that the AP-3 complex is required for synaptic vesicle biogenesis, potentially directly from the Golgi, and for the axonal targeting of SVPs respectively [Synaptic vesicle proteins (SVPs) are essential for neurotransmission and synaptic vesicles at the synapse are known to have a defined composition . Severalectively –23. SVPsectively ,25. ThusDrosophila, is thought to have roles in protein trafficking [C. elegans unc-16 mutants also show mis-trafficking and mis-accumulation of multiple neuronal cargo such as SVPs, dendritic receptors, lysosomes and early endosomes [C. elegans homolog of LRRK2, has been previously implicated in the polarized trafficking of SVPs and is also found at the Golgi, like UNC-16 [JIP3/UNC-16/dSYD, a JNK-signaling scaffold protein present at the Golgi in fficking –28. The ndosomes . It is ue UNC-16 ,31. Thusunc-16 and lrk-1 mutants have an altered composition and size. These mutants also show defects in polarized distribution of SVPs, which mis-localize to the dendrites [In this study, we show that the SVP transport carriers formed in endrites ,30. We sC. elegans neuronal cell bodies (unc-16 (tb109), with an early stop codon at amino acid 423, that caused mis-trafficking of the trans-membrane VAMP Synaptobrevin-1 (SNB-1) to the dendrite of the amphid sensory neurons  from SVP transport carriers, “inclusion” of specific SVPs into the same transport carrier and regulates the size of such compartments exiting the cell body.lrk-1 mutants show mis-trafficking of SNB-1 into dendrites of C. elegans chemosensory neurons, similar to phenotypes seen in unc-16 animals [To identify other genes in the UNC-16-mediated SVP trafficking pathway, we examined mutants in LRRK2/LRK-1, known to be present on the Golgi . LRRK2/L animals .lrk-1 animals, Golgi enzymes Man-II and ST occasionally exit the cell body  complex in C. elegans [Dendritic trafficking of proteins is known to require the AP-1 complex ,23,38. T elegans . Therefounc-101 animals, like in wild type TRNs, Man-II and ST are restricted to the cell body and the incidence of co-transport of SVPs RAB-3 and SNB-1 is about 40%  on the Golgi.As the phenotype of long moving compartments observed in nd lrk-1 . UNC-101ild type . The ovehe Golgi . Since ounc-16 are thought to recruit non-canonical motors [C. elegans [unc-104 mutants, the SVPs RAB-3 or SNB-1 are found to be trapped in the cell body and absent from synapses  are sorted into transport carriers and trafficked out of the cell body are not yet clearly understood. It is thought that like other membranous cargo such as secretory granules and dense core vesicles, SVPs are sorted at the Golgi and post-Golgi compartments and the transport carrier that is formed moves out of the cell body by recruiting specific motors, such as KIF1A/UNC-104 ,43–45. U elegans  as well unc-16 mutants  typically excluded from these carriers. Earlier studies have also shown that the AP-1 complex is required for trafficking of proteins into dendrites [apb-3 mutants show inclusion defects wherein the co-transport of SVPs, SNB-1 and RAB-3, is reduced . methods . L4 or 1unc-16 (tb109) allele was isolated from a behavioral suppressor screen carried out in unc-104 (e1265) worms. This was a non-clonal screen of approximately 60,000 haploid genomes. Animals were mutagenized using 50 mM EMS for 4 h. F1 and F2 progenies were screened for improved locomotion. Suppressors were further identified by improved localization of GFP::RAB-3 in PLM neuron . The tb109 allele was separated from the background mutation and mapped to chromosome III and tested for non-complementation with unc-16 (e109). The tb109 phenotypes could be rescued by the expression of Punc-16::UNC-16::GFP. Sequencing revealed that allele tb109 contains a point mutation in exon 9 leading to a stop codon at amino acid 423 .The unc-16 (tb109) and N2 were fixed for electron microscopy by high-pressure freezing (HPF) technique [Young adults of echnique . Serial echnique .Immunostaining was performed as described previously . For douSamples were prepared by mechanical homogenization using homogenization buffer ) followed by mild sonication at 4°C. Sample was then incubated with mouse monoclonal anti–Flag (1:50) (Biovision) or mouse monoclonal anti-GFP sera (1:10)  at 4°C for 4 h. The antigen-antibody complex was incubated with Protein-A agarose beads  for another 4 h. Antibodies used for Western blots to probe for UNC-16::GFP and LRK-1::Flag were rabbit anti-GFP  and mouse anti-flag  respectively.L4 or 1-day adult worms were immobilized using 30mM Sodium Azide and mounted on 2–5% agarose pad. Images were acquired on an Olympus IX71 Epifluorescence Microscope with an Andor Camera, an Olympus IX73 Epi-fluorescence microscope with Evolution Camera or the Olympus IX83 with Perkin Elmer Ultraview Spinning Disc microscope and a Hamamatsu EMCCD camera or the Olympus Fluoview FV1000 confocal laser-scanning microscope. Imaging conditions vary from experiment to experiment.L4 worms were anesthetized in 5 mM Levamisole (Sigma-Aldrich) and mounted on 2% or 5% agarose pad. Time-lapse images were acquired in Olympus IX83 with Perkin Elmer Ultraview Spinning Disc microscope and a Hamamatsu EMCCD camera. Imaging conditions vary from experiment to experiment.Image analysis was done using ImageJ software .Kymographs from a time-lapse image were made utilizing the “MultipleKymographs” ImageJ plugin. In a kymograph, straight vertical lines represent static particles whereas lines with a positive or negative slope represent moving particles. The length of the moving compartments was quantified by measuring the distance along the x-axis of the sloped line. This was done at regions not overlapping with stationary particles or other moving particles. Particles having length greater than 0.6μm was considered as longer compartments. We chose the 0.6μm cut-off for the following reasons: (i) The limit of resolution in our microscope is about 200nm leading to possible errors in length estimation in this size range, (ii) in wild type the majority of SVP carriers are between 0.4–0.6μm. Vesicles above 0.6μm in length are easy to measure accurately and very rare in wild type.Kymographs were made from identical regions of the movie in both colour channels utilizing the ImageJ plugin “MultipleKymographs”. The kymographs were then aligned and the number of sloped lines that overlapped was considered as co-migrating particles.First a threshold for an image was set using the background intensity as cut-off value and then the number of puncta were quantified. To calculate the ratio of intensity, intensity measurements of a circle with fixed area (5x5 pixels) were taken from both puncta and the background regions.Kymographs from a time-lapse image were made utilizing the “MultipleKymographs” ImageJ plugin. Number of EBP-2::GFP comets (anterograde and retrograde) were quantified from kymographs.In all graphs, data are presented as mean values ± SEM. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). Wherever possible, an independent t-test was used, or for multiple comparisons, one-way ANOVA followed by Dunett’s multiple comparison tests was done. For grouped datasets, two-way ANOVA, followed by Tukey’s multiple comparison tests was used. Differences were considered significant when P < 0.05 .S1 Fig(a) Schematic diagram of UNC-16, with reported and newly identified (tb109) lesions of unc-16 at the indicated positions. The predicted functional domains are colour-coded. (b) SNB-1::GFP is mis-localized into the dendritic compartment of amphid sensory neuron in two different alleles of unc-16 (e109 and tb109). The mis-localization is eliminated by an UNC-16 rescue construct . (c) Dendritic protein ODR-10::GFP is mis-trafficked to the axon of the AWB neuron in unc-16 (tb109) mutants. This defect in unc-16 cannot be rescued by over-expression of LRK-1 . Red arrows indicate axonal localization. (d) The fraction of EBP-2::GFP comets moving in either anterograde or retrograde directions in both axon and dendrites in wild type and unc-16 (tb109) are similar. This suggests that microtubule polarity is unchanged in unc-16 (tb109) mutants. No. of animals examined = 5 per genotype; no. of EBP-2 comets quantified ≥ 60 (e) Double immunostaining of endogenous RAB-3 and SNT-1 in the sublateral neuronal process of wild type and unc-16. Merge panel shows the degree of co-localization between the two proteins. White arrows indicate an absence of co-localization. Scale bar represents 10 μm.(TIF)Click here for additional data file.S2 Fig(a) Quantitation from dual colour imaging and kymograph analysis shows Golgi enzyme ST::GFP is co-transported in the same compartment along with synaptic vesicle marker mCherry::RAB-3 into the PLM neuronal processes of unc-16 (tb109) animals but not of lrk-1 (km17) or unc-101 (m1) animals. (b) Imaging of ST::GFP shows that the Golgi enzyme exits the cell body only to a low extent in dhc-1 (js319) animals and not at all in jnk-1 (gk7) animals. (c) Imaging of Man-II::GFP shows that it is mis-trafficked into dendrites in unc-16 and lrk-1 (km17) mutants. However, this accumulation depends on UNC-101 as indicated by unc-101(m1); unc-16 (tb109) and unc-101 (m1), lrk-1 (km17) double mutants. Red arrows indicate dendritic tip accumulation. (d) Static images of neuronal cell bodies showing expression of Man-II::mCherry (PLM), ST::GFP (PLM), RUND-1::TagRFP (tail ganglia) and eGFP::RAB6.2  is shown. The number of puncta seen within each cell body has been quantified and shown as a fraction of total number of cell bodies examined. n ≥ 50 cell bodies. Scale Bar: In kymographs, horizontal scale bar represents 5 μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10 μm.(TIF)Click here for additional data file.S3 Fig(a) Comparison between wild type and two different mutant alleles of unc-16 –tb109 and e109. (b) Comparison between wild type and two different mutant alleles of lrk-1 –km17 and km41. (c) Comparison between wild type, unc-16 (tb109) and LRK-1 overexpression in unc-16 (tb109) animals. (d) Comparison between wild type, unc-101 (m1) and LRK-1 overexpression in unc-101; unc-16 animals. In each genotype, n ≥ 200 particles.The graphs show a frequency distribution of the lengths measured of SVP transport carriers across different genotypes. (TIF)Click here for additional data file.S4 Fig(a) and (b) show immunoprecipitation from animals overexpressing (a) LRK-1::FLAG and (b) UNC-16::GFP animals, where either LRK-1::FLAG or UNC- 6::GFP was pulled down and probed for the other protein. (c) Static images of LRK-1::Venus fluorescence in sensory and pharyngeal neurons shows that LRK-1::Venus is mis-localized to dendrites in unc-16 (tb109) mutants. (d) Quantitation and comparison of punctate distribution of LRK-1::Venus in neuronal cell bodies between wild type and unc-16 (tb109) animals. n ≥ 25 cell bodies (e) Static images showing punctate distribution of UNC-16::GFP in the ventral nerve cord in wild type and lrk-1 (km17) animals. Quantitation and comparison of puncta distribution suggests that UNC-16 localization is not affected in lrk-1 mutants. n ≥ 25 cell bodies (f) Anterograde flux measurements and comparison between wild type, unc-16 (tb109), unc-104 (e1265); unc-16 (tb109), unc-116 (e2310); unc-104 (e1265); unc-16 (tb109) mutants indicates that flux in unc-16 decreases in absence of Kinesin-3 and Kinesin-1. This further suggests that the RAB-3 containing transport carriers depend on multiple motors. n ≥ 10 animals.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 MovieDual colour live imaging of the PLM neuronal process was done (3fps for 3 min at 100 X) using wild type L4-animals expressing GFP::RAB-3 and Man-II::mCherry.(AVI)Click here for additional data file.S2 Movieunc-16 (tb109) L4-animals expressing GFP::RAB-3 and Man-II::mCherry.Dual colour live imaging of the PLM neuronal process was done (3fps for 3 min at 100 X) using (AVI)Click here for additional data file.S3 MovieLive imaging of the PLM neuronal process was done (5fps for 3 min at 100 X) using wild type L4-animals expressing GFP::RAB-3 shows no large compartments.(AVI)Click here for additional data file.S4 Movieunc-16 (tb109) L4-animals expressing GFP::RAB-3 shows large compartments.Live imaging of the PLM neuronal process was done (5fps for 3 min at 100 X) using (AVI)Click here for additional data file.S5 Movieunc-16 (tb109) L4-animals expressing SNB-1::GFP shows large tubular compartment in the dendritic process.Live imaging of the ASI neuronal process was done using (AVI)Click here for additional data file.S6 Movieunc-101 (m1) L4-animals expressing GFP::RAB-3 shows large compartments.Live imaging of the PLM neuronal process was done (5fps for 3 min at 100 X) using (AVI)Click here for additional data file.S7 Movieunc-101 (m1) L4-animals expressing GFP::RAB-3 shows large compartments.Live imaging of the motor neurons was done (5fps for 3 min at 100 X) using (AVI)Click here for additional data file.


Tm) measurement by HRM. The accuracy of HRM was assessed as compared with UV melting method, and little difference between the two methods was found when the DNA Tm was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher Tm, showing that the proportion of dye should be considered for precise Tm measurement of nucleic acids. Finally, HRM method was also successfully used to measure Tms of DNA triplex, hairpin, and RNA duplex. In conclusion, HRM can be applied in the evaluation of thermal stability of nucleic acid (DNA or RNA) or secondary structural elements (even when dNTPs are present).High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping. For the aim of extending HRM for the evaluation of thermal stability of nucleic acid secondary structures on sequence dependence, we investigated effects of the dye of EvaGreen, metal ions, and impurities (such as dNTPs) on melting temperature ( Tm, data are required to be collected at narrow temperature increments of 0.02~0.2°C. Due to the high sensitivity to small variations, HRM can distinguish small differences in nucleic acid sequences, even for single nucleotide polymorphism (SNP) [High resolution melting (HRM) is a robust technique for mutation scanning, methylation, and genotyping, which possesses high sensitivity to distinguish DNA species from tiny differences in their melting temperatures based on sequence composition, length, GC content, or strand complementarity –4. To obsm (SNP) , 6. Meansm (SNP) .Tm of short DNA sequences by HRM, not to mention RNA [Tms of short DNA duplex, triplex, or hairpin and even RNA duplex. We found that Tms of DNA duplexes with various GC contents and lengths (even shorter than 10 bp) could be well measured by HRM. Effects of the dye and metal ion as well as other composites (such as deoxyribonucleoside triphosphates and free single strands) were also investigated.However, at present, HRM has been mainly applied to distinguish two DNA species longer than 60 bp in PCRs. It is unknown whether it can be applied to the high-throughput evaluation of thermal stability of short DNA or RNA sequences, which provide significant information for investigating sequence dependence or predication of secondary structures. From the viewpoint of basic data for thermodynamics, there has been no specific report on measuring tion RNA . In thisDNA and RNA oligonucleotides , 1x EvaGreen, and Mg2+ (1–100 mM) was used. For DNA triplex, DNA oligomers were prepared in a solution containing 1x EvaGreen, 2.5 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 10 mM MgCl2, and 0.5 mM spermine (pH 5.5).DNA duplex, DNA hairpin, or RNA duplex was prepared in a solution containing 10 mM phosphate buffer (pH 7.4), EvaGreen , and NaCl (0-1 M). For experiments involving MgμL) was pipetted into 96-well microtiter plates; then, the microtiter plates were transferred to a PikoReal Real-Time PCR instrument . Annealing was performed with a cooling rate of 0.1°C/s from 95°C to 10°C; then, fluorescence data were collected over a temperature range of 10–95°C in 0.1°C increments (the holding time was 2 seconds). All fluorescence data were acquired from at least three parallel tests in one plate and repeated twice.For fluorescence melting profiles, the mixed oligomer solution  was dissolved in a buffer as the cases of HRM, but with no EvaGreen. The solution was added into a cuvette with 1 mm or 10 mm path lengths. Melting profiles were obtained at 260 nm over the temperature ranges from 95°C to 10°C and then from 10°C to 95°C at a ramp rate of 1°C/min. All UV melting assays were performed for three times at least, and Tms were calculated by the first derivatives of melting curves.UV melting assays were operated by a UV-Vis Spectrophotometer . For Tm measurement by HRM, which was one of the most popular saturating dyes for HRM analysis. The detailed effects of DNA sequences, metal ions, and other factors on Tm measurement by HRM were investigated.In this study, EvaGreen was adopted as the fluorescent dye in Tm measurement by HRM, we used a series of DNA duplexes with lengths from 6 bp to 20 bp  and GC12/15 (80% GC content) were used  were shown in  GC3/15 (1 μM) showed a lowest Tm when the EvaGreen was 0.5x . When the DNA concentration was less than 1 μM, the Tm difference between two methods was less than 2.5°C. The Tm difference became greater as DNA concentration increased to 5 or 10 μM. Obviously, for a higher concentration of DNA, more EvaGreen is required, and a reasonable proportion of dye to DNA is essential for Tm measurement by HRM.Meanwhile, the DNA concentration effect on HRM Tm of nucleic acid, its accuracy is a necessary aspect to be investigated by comparison with the traditional UV melting method. Two series of 15 bp or 20 bp DNA duplexes with various GC contents  were measured by HRM and UV melting method, respectively. Some single stranded DNA may form secondary structures ; however, we found that they did not affect the Tm measurement of the duplex by HRM . As shown in Tm difference between the two methods when Tm was higher than 40°C, although the difference became greater at the lower Tm range. In detail, for DNA duplexes with Tms higher than 70°C, their Tms by HRM were almost consistent with those by UV melting method. For Tms of 40–65°C, the Tm differences were 1-2°C. For Tms lower than 40°C, the Tm difference was within 4°C. We also found that there was a good linear relationship between Tms by the two methods  and that the root mean-squared deviation (RMSD) of corrected Tm was 0.5°C, less than that of mfold Tm to UV Tm (1.7°C).To apply HRM to measure  methods  which wa GC3/15 and GC12/15 at various concentrations of Na+ or Mg2+ by HRM , the initial fluorescence intensity was lower when Na+ was at 0.6–1.0 M. The lower Tm by HRM indicated that Na+ at a high concentration affected the binding of EvaGreen to DNA.In terms of the metal ion effect, we measured DNA duplexes+ by HRM . HRM Tm 1000 mM . The sam2+ concentration was shown in 2. Even at 10 mM MgCl2, Tm of GC3/15 by HRM was 2.7°C lower than that by UV. Similar results were also obtained for GC12/15, and the difference between two methods became even greater. These results indicated that Mg2+ competed for DNA binding with EvaGreen containing only one positive charge; then, EvaGreen dissociated at a lower temperature.The effect of MgTm measurement of DNA duplexes by UV melting method becomes difficult. Whether dNTPs can affect Tm measurement by HRM was determined. In the presence of 0.02–2.0 mM dNTPs, Tms of GC0/15~GC15/15 with various GC contents were measured, and almost no Tm change was observed at various concentration of dNTPs  by HRM. The obtained Tms of IAA-, GAI-, and IAI-loop hairpins  were much lower than those of GAA- and GIA-loop hairpins . This was in line with Moody and Bevilacqua's findings by UV melting method indicating that the first and third base of loop dominated the stability of DNA triloop hairpin [In terms of DNA hairpin, we investigated a series of triloop hairpins possessing the same loop sequence, GAA, but differing in their length of stems . We found that when the stem was 5 bp or longer, n HP-S6,  and its  hairpin , 12. TheTm of a 21-bp RNA duplex . It was interesting that EvaGreen could bind to A-form conformation of RNA duplex. Therefore, HRM could be applied in Tm analysis of short RNA duplex, or other RNA secondary structures.Considering the significance of RNA SSEs, we measured the plex RR,  by HRM. Tms of small nucleic secondary structures was investigated. Surprisingly, HRM can measure as short as 7-bp DNA duplex with Tm of about 20°C  (Supplementary Table S1). Because the work platform of 96- or 384-well plate could be used for HRM and the data could be analyzed by well-edited processing software, HRM has been used as a high-throughput approach for determining SNP, gene methylation, and mutant analysis on a large scale [Tms of thousands of DNA sequences can be obtained in hours, even using 96-well plates .In this study, whether HRM could measure out 20°C . For the hairpin . For haige scale –15. ObviTm measurement by HRM is that it is less affected by the presence of dNTPs  between HRM Tm and UV Tm (Tms (Supplementary Table S2). Therefore, HRM can be utilized as an accurate approach to evaluate the thermal stability of nucleic acids.In terms of  Figures . The difd UV Tm , which cTm values are required [Tm  and EvaGreen for binding with DNA. Accordingly, HRM using EvaGreen is not suitable for the studies of salt dependence of nucleic acid stability [+ concentration is lower than 400 mM, and Mg2+ concentration is not higher than 10 mM; thus, the accuracy of HRM is acceptable for most cases.Another important point to be considered is the proportion of dye to nucleic acid when highly accurate required , 9. Our constant , which dant  data of 15 bp and 20 bp DNA duplexes, respectively. The Tm difference between UV and HRM in Table S1 indicated that HRM measurement performed better stability and reproducibility compared with UV melting method. Moreover, the linear formula obtained from Table S1 can be used to correct the HRM Tms of 20-bp DNA duplexes (Table S2), and the error of most corrected data was within 0.9oC as compared with UV data. The HRM Tm of DNA duplex GC3/15 and GC12/15 at various strand concentrations from 0.1 to 10 μM in 1× EvaGreen was investigated .The Tm difference between HRM and prediction became greater as DNA concentration increased to 5 or 10 μM. The results demonstrated that a reasonable proportion of dye to DNA is essential for Tm measurement by HRM. The HRM melting curves of GC14/15 duplex, GC14/15 duplex with additional F or R strand, and only F strand or R strand were investigated and the results showed that the single strands did not affect the Tm measurement of the duplex by HRM . For the UV melting method, it was found that dNTPs (≥0.2 mM) resulted in a high absorbance beyond the accurate scale of UV spectrophotometers . Melting curves of a series of triloop hairpins with 3~8-bp stem and a 21-bp RNA duplex were detected by HRM , which demonstrated that HRM could be applied in Tm analysis of DNA and RNA secondary structural elements. For the melting curves of 96 samples on a 96-well plate by HRM , all the data could be obtained in 1 to 2 h. The results showed its high efficiency and high throughput.The supplementary material contains additional data used for assessing the melting temperature measurement of nucleic acid by HRM. Supplementary Tables S1 and S2 supply the detailed melting temperature (


FGF4) and deletion of 9p21.3 (CDKN2A) were found to be recurrent in all 38 superficial ESCCs analyzed. Notably amplifications of 3p26.33 (SOX2OT), 8q24.21 (MYC), 14q21.1 (FOXA1) and deletion of 3p12.1 (GBE1) were only found to be recurrent in metastaic superficial ESCCs. In conclusion, using CNAs analyses, we identify a 39-gene signature which characterizes the high risk metastatic superficial ESCCs and discover several recurrent CNAs that might be the driver alterations in metastasis among superficial ESCCs.Superficial esophageal squamous cell carcinoma (ESCC) is generally considered a subtype of less invasive ESCC. Yet a subset of these superficial ESCC would have metastasis after esophagostomy or endoscopic resection and lead to poor prognosis. The objective of this study is to determine biomarkers that can identify such subset of superficial ESCC that would have metastasis after surgery using genome wide copy number alteration (CNA) analyses. The CNAs of 38 cases of superficial ESCCs originated from radical surgery, including 19 without metastasis and 19 with metastasis within 5 years’ post-surgery, were analyzed using Affymetrix OncoScan™ FFPE Assay. A 39-gene signature was identified which characterized the subset of superficial ESCC with high risk of metastasis after surgery. In addition, recurrent CNAs of superficial ESCC were also investigated in the study. Amplification of 11q13.3 ( Esophageal squamous cell carcinoma (ESCC) is one of the most deadly tumors worldwide, with 5 year survival of only 10%. ESCC related death rates are particularly high in China, in part due to the lack of early screening tools and limited treatment options [Superficial ESCC has significant better prognosis than the advanced ESCC, and can be treated effectively by endoscopic resection, including both mucosal resection and submucosal dissection , 3. EndoCCND1), 3q26.33 (SOX2), 8q24.2 (MYC) and deletion of 3p14.2 (FHIT) have been associated with metastatic diseases [Several clinicopathological features have been associated with high metastatic risk in superficial ESCC, including invasion depth, tumor size, angiolymphatic invasion and histological differentiation –6. ESCCsdiseases . NeverthGenomic instabilities, in the form of chromosome instability (CIN), microsatellite instability (MIN) and point mutations, are characteristic for human cancers. CIN, including chromosome structural and number changes, is a major form of genomic instability . AccordiHere we reported the analyses of 38 superficial ESCCs originated from radical surgery with both metastatic and metastasis-free samples with Affymetrix OncoScan™ array. The CNAs landscape of superficial ESCC were determined, and the focal recurrent CNAs were compared between the metastasis and metastasis-free cases.p = 0.07). In addition, the comparison of GII on each chromosome in non-metastasis and metastasis groups was also performed. In non-metastasis group, chromosomes 3 (65.7%), 8 (78.2%) and 20 (59.8%) have the highest GII, whereas chromosomes 4 (29.5%), 12 (28.7%) and 21(25.2%) have lowest GII. In metastasis group, chromosomes 3 (75.2%), 8 (72.1%) and 14 (60.0%) have the highest GII, whereas chromosomes 15 (35.9%), 21 (31.8%) and 22 (38.1%) have the lowest GII. Chromosome 18 has higher GII in metastasis group than in the non-metastasis group (p = 0.03)  Figure .The average number of genome segments with CNAs in all 38 samples was 218, and the average length was 13.26 Mb . In metastasis group, the average number of segments with CNAs was 217, and the average length was 13.35 Mb . In non-metastasis group, the average number of segments with CNAs was 210, and the average length was 13.05 Mb . The distribution of size of CNAs length was shown in Figure FGF4), 3q28 (TP63), 14q21.1 (FOXA1), 8q24.21 (MYC), and deletions of 9p21.3 (CDKN2A), 22q11.23 (GSTT1), 3p13 (MITF) and 2q22.1 (LRP1B). In metastasis group, eight recurrent focal CNAs were found, including 4 amplifications and 4 deletions (FGF4), 14q21.1 (FOXA1), 3q26.33 (SOX2-OT), 8q24.21 (MYC) and deletions of 22q11.23 (GSTT1), 9p21.3 (CDKN2A). In non-metastasis group, fourteen recurrent focal CNAs were found, including 12 amplifications and 2 deletions (FGF4), 2q33.1 (PLCL1), 3q28 (TP63) and deletions of 22q11.23 (GSTT1) and 9p21.3 (CDKN2A). In conclusion, amplifications of 11q13.3 (FGF4), 8q24.21 (MYC) and deletions of 9p21.3 (CDKN2A) and 2q22.1 (LRP1B), which previously reported in ESCCs were also found in all 38 superficial ESCCs in our study. Furthermore, deletions of 3p13 (MITF) and 22q11.23 (GSTT1) were also found as CNAs targets of ESCC but were not previously reported. Amplification of 3q26.33 (SOX2-OT) was the most significantly recurrent CNA in metastasis group whereas amplification of 3q28 (TP63) was the most significantly recurrent CNA in non-metastasis group.We found 28 significantly recurrent focal CNAs, including 16 amplifications and 12 deletions in all 38 superficial ESCCs  . Hierarchical clustering was performed on the CNA profiles of these 39 genes and two groups were obtained, including metastasis and non-metastasis groups. In general, good separated performance of metastasis and non-metastasis was obtained with a small amount of misclassification (two metastasis misclassified and five non-metastasis misclassified)  were 5.14. Amplification of FGF4 happened in 68% cases in metastasis group, and 58% in non-metastasis group. FGF4 gene was located on chromosome 11q13.3, which were found to be amplified in ESCCs from previous studies. Average copy number of MRAS gene  in all cases was 2.61, and 68% cases had amplification of MRAS gene in metastasis and 84% cases had amplifications in non-metastasis group. Average copy number of ALK  in all cases was 2.25. Amplification of ALK gene happened in 26% of non-metastasis cases, and 63% of metastasis cases. Average copy number of CHEK1  in all cases was 1.91. In non-metastasis group 3 cases (16%) had amplification of CHEK1 and 3 cases (16%) had deletion of CHEK1, and in metastasis group only one case had amplification and 10 cases (53%) had deletion of CHEK1.The genomic distribution and copy numbers of these 39 genes was shown in Figure p = 7.59E-11), “Chromosomal rearrangement” (p = 8.64E-9), “Pathways in cancer” (p = 6.66E-04) and “Nucleotide-binding” (p = 0.015).The most significantly enriched pathway/functional terms were “Proto-oncogene”  to analyze genome wide CNAs of 38 superficial ESCCs and compared recurrent focal CNAs level, and the CNAs of 891 cancer genes between ESCCs with and without metastasis.FGF4, PIK3CB, MRAS, ALK, LMO2, AKT3 and CHEK1 genes were included in the 39-gene signature. Copy number of CHEK1 gene was significantly lower in metastasis than in non-metastasis groups (p = 0.017). Checkpoint kinase 1 (Chk1) which encoded by CHEK1 gene trigger cell cycle arrest upon DNA damage [CHEK1 was initially thought to be a tumor suppressor gene due to its role in checkpoint activation and cell cycle arrest, several studies documented a positive correlation between Chk1 expression and tumor grade and disease recurrence, suggesting that Chk1 may promote tumor growth [CHEK1 in ESCC should be explored further. In addition, FGF4 gene located in 11q13.3 was also recurrently amplified in superficial ESCC.The Oncoscan SNP microarray is an ideal platform to perform analyses on FFPE-derived tumor materials with increased probe density within 891 cancer genes at 50-100 kb resolution. Among the 891 cancer genes, 39 cancer genes had significantly different CNAs between the metastasis and non-metastasis cases. These genes might represent candidate biomarkers for superficial ESCC patients with high risk of metastasis after surgery. Previous studies focus on using gene expression profiles to guide prognosis prediction and diagnosis , 19. HerA damage  Althoughr growth –24. So tCCNL1 gene, and 76% (19/25) cases for PIK3CB gene. The difference of calculation of copy number alterations in these two experiments might lead to inconsistency of results.In discovery and validation experiments, there were 88.5% (23/26) cases showed the identical copy number aberrations of FGF4 and FGF19), 8q24.21 (MYC), 7q22.1 (CDK6) and deletions of 9p21.3 (CDKN2A), 3p12.3 (MITF), 2q22.1 (LRP1B), 13q14.2 (RB1) which were previously reported in all stage of ESCC [FOXA1), which has not been previously reported as associated with ESCCs, was found in 20 cases (52.6%) in our study. Forkhead box protein A1 (FOXA1), the representative member of the Forkhead-box (FOX) proteins subfamily, is a DNA-binding transcription factor. Amplification of FOXA1 gene has been found in lung cancer, esophageal adenocarcinoma, [GSTT1, GSTTP1, and GSTT2) occurred in 20 superficial ESCC samples (57.9%) in our study. Glutathione S-transferase (GST) theta 1 (GSTT1) which encoded by GSTT1 gene is a member metabolizing dimeric phase II enzymes superfamily. These enzymes play a vital role in cellular defense system by catabolism of a broad range of xenobiotics and carcinogen [GSTT1 gene deletion polymorphisms increases susceptibility to lymphoma, breast cancer, colon cancer and lung cancer [GSTT1 deletion in the etiology of ESCC should be explored further.The recurrent focal CNAs which determined by GISTIC2.0 were more likely to identify cancer-causing genes , 25. Amo of ESCC –30 were rcinoma,  estrogenrcinoma,  and anaprcinoma, . And thercinoma, , 35. Delrcinogen , 37. Mang cancer –40. So tSOX2-OT), 8q24.21 (MYC), 14q21.1 (FOXA1) and deletion of 3p12.1 (GBE1) were found as the recurrent CNAs in metastasis group only, which indicated that those CNAs might be associated with metastasis in superficial ESCC. Deletion of 3p12.1 (GBE1) has been associated with prognosis in cervical cancer [SOX2 gene embedded in the introns of SOX2 overlapping transcript (SOX2OT) gene, which encodes a long non-coding RNA (lncRNA) [SOX2 have been associated with metastasis or poor prognosis in previous studies of ESCC [SOX2OT gene on 3p26.33 was found as recurrent CNA in metastasis group. SOX2OT gene encodes the lncRNA which have been demonstrated that involved in regulation of SOX2 expression and/or other related processes. And expression of SOX2 and SOX2OT were concordant in ESCC and breast cancer [SOX2OT gene and expression in ESCC metastasis should be explored further.Amplifications of 3p26.33  . Amplifi of ESCC , 44–46. t cancer , 48. TheIn conclusion, our study constructed a 39-gene signature associated with metastasis from superficial ESCCs. The comparison of recurrent CNAs between superficial ESCCs with and without metastasis also revealed a subset of metastasis specific events. A larger set of independent samples are warranted to validate and refine this 39-gene signature from our study.Thirty eight superficial ESCC patients, at stage T1N0M0, were collected from Cancer Hospital, Chinese Academy of Medical Sciences between 2004 and 2010. The most recent follow-up visit was dated in June 2015 and the follow was conducted via telephone interview or clinical data consultation. Among the 38 cases, 19 metastasis cases have lymph node metastasis or distant metastasis within five years from radical surgery, and the 19 metastasis-free cases have no detectably metastasis within five years after radical surgery. All the patients underwent radical resection without radiotherapy or chemotherapy prior to surgery. The clinicopathological characters of 38 patients were summarized in Table Genomic DNA were extracted from formalin-fixed and paraffin-embedded (FFPE) tissues of 38 superficial ESCCs using QIAamp DNA Mini kit  according to the manufacturer's instruction. H&E sections of all cases were reviewed to manually identify areas with minimum of 85% malignant cells for microdissection.Molecular Inversion Probe (MIP) based Oncoscan Array was used to detect CNAs, loss of heterozygosity, and somatic point mutations. The experiments were performed according to the user guide of Affymetrix OncoScan™ FFPE Assay Kit . Briefly, 75 ng FFPE DNA were hybridized to MIP probes and allowed to anneal 58°C overnight (16-18h) after denaturation at 95°C for 5 min. Then each sample was split into two tubes and gap fill reaction was performed by adding dATP (A) and dTTP (T) (A/T) in one tube and dGTP (G) and dCTP (C) (G/C) to another. After removing the un-circularized MIP probes through exonuclease treatment, the cleavage enzyme was added to linearize the gap-filled circular MIP Probes. Then the circular MIP probes were amplified by first and second round PCR. The enriched product was digested by HaeIII enzyme and the 44bp fragments were hybridized to the OncoScan™ Array for 16-18h. The hybridized arrays were washed, stained using the GeneChip® Fluidics Station 450 and scanned through GeneChip® Scanner 3000 7G .CCNL1 and PIK3CB genes with the internal control HBB gene using SYBR-Green II fluorescence and Mx3005P System )  was used to validate CNAs of A, USA)) . The resof genes .The intensity (CEL) files generated by the scanner were imported into Oncoscan Console Software  and analyzed by the Affymetrix TuScan algorithm  to create segmentation to differentiate between adjacent clusters of probes and determines the CNAs. Amplification was defined as copy number which was calculated using Affymetrix TuScan algorithm > 2, and copy number < 2 was considered to be deletion. In addition, the copy number information of 891 cancer genes were obtained as Gene Report text files from the Console software.The recurrent CNAs were determined using GISTIC2.0 (Genomic Identification of Significant Targets in Cancer) with a Q-value cutoff < 0.25. And the significant recurrent focal CNAs has a 90% likelihood of containing the targeted genes.The Mann-Whitney U-test was used to detect the significantly different CNAs of 891 cancer genes between metastasis group and non-metastasis group. And then, hierarchical clustering was performed on significantly differential CNAs of cancer genes. Pheatmap function in R pheatmap package was used to draw heat maps to visualize the clustering results.p < 0.05). All the statistical analyses were performed on R (version 3.2.1). Significance were deemed as < 0.05 for all statistical analyses.Genome instability index (GII) was used to evaluate the levels of DNA copy number changes in all 38 samples. To take the variation of chromosome size into consideration, weighted GII was used. First, percentage of aberrant SNPs for each chromosome was calculated separately to obtain GII of each chromosome. For individual chromosome, numbers of SNPs with aberrant copy number were divided by numbers of all SNPs in each chromosome to get the GII of each chromosome. Then the mean percentage aberration of all 22 autosomes in each sample was calculated to generate the weighted GII , 51. PaiFunctional enrichment analyses was performed on the 39 genes using DAVID  tool. These terms achieved significant enrichment p values after adjusted with the Benjamini method.


Functional specialization of cells and tissues in metazoans require specific gene expression patterns. Biological processes, thus, need precise temporal and spatial coordination of gene activity. Regulation of the fate of messenger RNA plays a crucial role in this context. In the present review, the current knowledge related to the role of RNA-binding proteins in the whole mRNA life-cycle is summarized. This field opens up a new angle for understanding the importance of the post-transcriptional control of gene expression in cancer cells. The emerging role of non-classic RNA-binding proteins is highlighted. The goal of this review is to encourage readers to view, through the mRNA life-cycle, novel aspects of the molecular basis of cancer and the potential to develop RNA-based therapies. Regulation of eukaryotic gene expression is crucial in pathophysiological responses to extracellular and intracellular signals in the context of homeostasis maintenance and cellular differentiation or in stress response and cell survival. It is the result of multiple, complex, and tightly regulated processes occurring in different cell compartments. Each process involves regulation-points, from pre-messenger RNA biosynthesis in the nucleus, to messenger RNA (mRNA) translation and, finally, to mRNA and protein catabolism in the cytoplasm. Regulation of gene expression at the mRNA level involves dynamic organization and reorganization of ribonucleoproteic structures. This requires many RNA-binding proteins controlling every step of the mRNA lifetime, e.g., nuclear maturation of transcripts, nuclear export, cytoplasmic transport and localization, stability and storage, translation, and degradation. The mRNA life-cycle can be separated into two phases, the first one being nuclear and transcriptional  and the other one being cytoplasmic and post-transcriptional. This division is artificial and is not supported by recent molecular basis. Thus, the whole process is now called the “mRNA life-cycle”. This term highlights the continuity between the different steps of mRNA fate. This continuity allows cells to quickly respond to various stimuli by regulating the expression of large subsets of messenger RNAs called RNA regulons.cis-elements recognized by RNA-binding proteins (RBPs) in their nucleotide sequences. These RBPs can bind either to coding or non-coding sequences. They target structural specific elements that are important for RNA fate in terms of processing, transport, localization, stability, or function. Associations between RBPs and RNAs form ribonucleoproteic particles called “RNPs” or specifically called “mRNPs” when the complex involves a mRNA . . 118]. T the CRD . ExperimUC4 mRNA . Many quUC4 mRNA . On the UC4 mRNA . TogetheCharacterization of new factors acting on mRNAs and their functional relationship with canonical RBPs, as well as those regulating or coordinating all complex stages of the whole mRNA life-cycle, is necessary to conceive new RBP-based anticancer therapies. Targeting the mRNA life-cycle appears to be a relevant strategy in developing new anti-cancer therapies. As previously described, many actors involved in the mRNA life-cycle can be targeted, such as microRNAs, RBPs, and RBP expression protein regulators. Alternative splicing can also be considered, as recently reviewed by Song et al. .An increasing number of targets, including RBPs such as HuR, hnRNPA1, IGFBP3, Sam68, DDX3, DHX9, and MBNL2, are under evaluation for the development of new cancer treatments ,22,23,24MYC gene a target for new cancer therapy [It is interesting to focus on multi-functional cancer players such as the Myc protein. Myc is well known to function as a transcription factor which is able to positively or negatively regulate the transcription of sets of genes mainly involved in proliferation, cell growth, reprogramming, and RNA biogenesis. However, Myc is also a regulator of many post-transcriptional mechanisms. It promotes cap addition for its target genes, upregulates several components of the alternative and constitutive splicing machinery, and it indirectly regulates RNA degradation by modulating the expression of AU-binding proteins (AUBPs) and components of the exosome machinery. On the other hand, Myc inhibits the NMD pathway and upregulates the transcription of ribosomal RNAs by RNA polymerase I and III, as well as enhances pre-rRNA processing and rRNA post transcriptional processing. Myc also regulates the translation of many targets through the modulation of several microRNAs’ maturation. Myc also controls the expression of the hnRNP protein family . All the therapy . MicroRNAs constitute promising elements for cancer diagnosis or cancer patient stratification. In vitro and in vivo studies have also shown that, in cancer, microRNAs such as let-7, miR-10b, miR-21, miR-34, miR-155, and miR-221 are promising targets in developing miRNA-based therapy for human malignancies ,139,140.MALAT1 lncRNA in a mouse breast cancer model [These last few years, new therapeutic tools have been developed to target the mRNA life-cycle. Synthetic RNA oligonucleotides hybridizing to specific microRNAs are, for instance, tested for kidney injury and fibrosis treatments through miR-21 targeting. miR-21 overexpression contributes to the pathogenesis of the disease through inhibition of mitochondrial biogenesis and functions . A recener model ,143,144.er model . Recent er model ,147.cis-elements. Due to difficulties for ASOs in reaching the nucleus, the CRISPR-Cas9 system could alternatively be used to delete targeted exons  or to inhibit gene expression . Engineered RNA-binding domains of splicing factors that bind to specific cis-elements, in order to modulate alternative splicing, constitute another way to achieve this purpose. This last strategy has demonstrated efficiency in increasing tumor cell apoptosis and the sensibility of the tumor cell to chemotherapy [Since RNA mis-splicing plays key roles in cancers, it should be of interest to manipulate splicing of specific RNA targets to restore normal isoforms in cancers. However, there is currently limited technology targeting RNA splicing. The most commonly used method is based on ASOs that pair with splicing sites or splicing regulatory otherapy . For insotherapy . A recenotherapy .The direct use of RNA constitutes another way to take advantage of mRNA metabolism. A recent study describes the use of a RNA-lipoplexes vaccine in mice to specifically deliver RNA-encoding tumor proteins to dendritic cells, instead of directly exposing these cells to tumor antigens. This strategy was able to prevent tumor development when the treatment was administered before the tumor cell grafts. Successful results were also observed to have a therapeutic purpose since tumors were reduced in size or disappeared when the vaccine was given to mice after tumor cell grafts . MYC gene or ASOs. Dominant negative inhibitors complexing with the Myc protein have also been developed to block its interaction with binding partners and, thus, inhibit its activity [As we previously described, Myc is a key factor in carcinogenesis. To target it, several strategies have been developed, such as small inhibitors acting as transcriptional repressors of the activity ,152. In the same way, tools targeting key RBPs in the mRNA life-cycle are of interest. For instance, strategies disrupting HuR binding to mRNA with chemical compounds are able to inhibit HuR activity, as described by Wu et al.  Figure . RNA-binding proteomes show the important place of IDRs in the ever-growing field of the RNA-interactome. The important role of IDRs in the dynamics of RNA ultrastructures, such as granules, is largely unexplored today. Nevertheless, because IDRs in granules are thought to regulate many aspects of RNA stability and translation during stress, they should be the subject of more systematic functional studies which could lead to therapeutic innovations, particularly in oncology.Until recently, the number of human RBPs, initially identified through their canonical RNA binding domains, was estimated to be around 500. Thanks to new methods of genome-wide RNA target identification (crosslinking and immunoprecipitation (CLIP) combined with high-throughput sequencing), bioinformatic tools and interactome capture coupled with mass spectrometry, this number is now above 1500, including conventional and non-conventional RBPs. Conventional RBPs binds to RNA targets by one or several canonical RNA-binding domains. Binding modes of non-conventional proteins to RNA are new and involve intrinsically disordered regions. These IDRs, through their structural flexibility, fulfill versatile roles in the constitution and remodeling of many macromolecular complexes and ultrastructures associated with mRNA throughout their life-cycle, from their biogenesis to their catabolism. IDRs also seem to be the key players during transcriptional and post-transcriptional regulation associated with all major basic processes of the cell.The continuity all along mRNA life-cycle is ensured by the RBPs which provide a quick and appropriate cell response to stimuli. The major regulatory role of RBPs, and in particular of these new unconventional RBPs, raises the question of their involvement in the development of pathological processes such as carcinogenesis a,b. They thus constitute emerging or potential therapeutic targets. Their deregulation in terms of overexpression or loss of expression during carcinogenesis is an interesting, but not sufficient element, to select them as relevant targets. To achieve this purpose, investigation of their biological role and identification of their partners through cellular and animal models is a prerequisite.


In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 , binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells.  Human cytomegalovirus (HCMV), a member of the betaherpesvirus family, retains global endemic status, with infection rates ranging from 30% in industrialized countries to more than 90% in developing ones  was replaced with the UL36 ORF. We demonstrated that UL36 replaces the function of M36 in MCMV infection. While ΔM36.MCMV showed a severe loss of fitness in macrophages and Gesellschaft für Versuchstierkunde/Society of Laboratory Animals. C57BL/6 and BALB/c mice were purchased from Janvier . The experimental procedures were approved by the responsible state office (Lower Saxony State Office of Consumer Protection and Food Safety) under permit no. 33.19-42502-04-12/0838.All animal experiments were conducted according to guidelines defined by the Federation of Laboratory Animal Science Associations and the national animal welfare body WT is pSM3fr-MCK-2fl derived from Smith strain . Viruses were propagated on M2-10B4 cells and virus stocks were prepared by purification using sucrose cushion as described previously  with supplements . IC-21 cells were maintained in RPMI-1640 medium with above stated supplements. The primary C57BL/6 mouse embryo fibroblasts (MEFs) were prepared and maintained as described earlier  at −80°C. Organs were homogenized in 5 ml of DMEM  on 100 μm-pore-size cell strainers. Organ homogenates were used for titration on C57BL/6 MEFs as described above.Six to eight week-old BALB/c and C57BL/6 mice were inoculated with 2 × 10WT and MCMVUL36 at an MOI of 1.0. The mRNA was isolated with Magnetic mRNA isolation kit (New England BioLabs) according to manufacturer's instructions. DNase I treated mRNA was used to produce the cDNA by SuperScript® II Reverse Transcriptase (Thermo Fisher Scientific) using the oligo dT primer. Reverse transcription PCR was performed with UL36Exon2-seq-F2 (5′-TTTTTTGGGATGTTGACAGGTG-3′) and UL36Exon2-seq-R3 (5′-GCCCACCATGAAGGATTTTC-3′) primers yielding a 604 bp product.NIH3T3 cells were infected with MCMV4Cl-PBS (500 μL) for 30 min at room temperature. Cells were then stained by suspending them in 100 μL permeabilization solution (eBioscience) and 20 μL antibody solution, and incubated for 1 h in darkness at room temperature. After staining, the cells were washed once and then suspended in FACS buffer. The cells were then acquired in a flow cytometer and analyzed with FlowJo software.Apoptosis was induced in HEK293 and NIH3T3 cells by exposing the cells to 100 ng/mL CD95L and 10 μg/mL cycloheximide (CHX). Active caspase-3 was stained with either FITC or PE labeled anti-active caspase-3 antibodies (BD-Clone C92-605), following CD95L induced apoptosis in HEK293 and NIH3T3, or virus induced apoptosis in murine macrophage cell lines. The protocol used for active caspase-3 staining was adopted from Cicin-Sain et al. . BrieflyFor annexin V staining the cells were harvested as described in previous paragraph. After washing, cells were suspended in 250 μL 1X Annexin V binding buffer  containing 5 μL of APC labeled Annexin V . The cells were incubated at room temperature for 30 min and acquired directly in Annexin V binding buffer by flow cytometer.2HPO4, 2.7 mM KCl, 1.5 mM K2HPO4, 2 mM EDTA, 10 mM NaF, 1 mM PMSF, 5 μg/ml apotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 5 μg/ml chymostatin, 1 mM Na3VO4, pH7.4). Extracts were incubated overnight at 4°C with 2 μg of anti-myc antibody  in the presence of magnetic Protein G beads (Thermo Fisher Scientific). Subsequently, precipitates were washed, denatured and eluted according to the manufacturer's instructions. Proteins were separated on a 12% polyacrylamide gel. For further analysis, the proteins were blotted onto a polyvinylidene difluoride membrane , probed with antibodies against caspase-8 , α tubulin  and UL36  and developed with ECL Select chemiluminescence reagent .For co-immunoprecipitation of endogenous caspase-8 with UL36, NIH3T3 cells were transfected with myc-tagged UL-36. Cells were harvested 48 h post transfection and lysed in TPNE buffer  route with 2 × 106 PFU). As in the BALB/c strain, ΔM36.MCMV did not show severe growth defect in the lungs of the C57BL/6 mice on day five post infection . The ΔM36.MCMV showed growth defects in all macrophage lines . Since the UL36 expression from the ΔM36.MCMV prevented apoptosis in infected murine macrophages, we tested if UL36 physically interacts with murine caspase-8. To determine this, co-immunoprecipitation assay was performed. For this purpose, NIH3T3 cells were transfected with myc-tagged UL36. Cell lysates were immunoprecipitated with anti-myc antibody coated beads and protein complexes were separated by SDS PAGE. Immunoblot analysis was performed using anti-caspase-8 and anti-UL36 antibodies. UL36 was detected in anti-myc immunoprecipitates from myc-tagged-UL36 transfected NIH3T3 cells, whereas it was not detected in control cells transfected with empty vector. Also, pro-caspase-8 was detected in the immunoprecipitates from UL36-transfected NIH3T3 murine cells but not from the controls, indicating that the protein pUL36 interacted with the murine pro-caspase-8  in the context of virus infection in vivo, because our experiments with MCMVUL36 demonstrated that UL36 functionally replaces the MCMV M36 gene, and largely rescues the growth defect shown by ΔM36.MCMV.Similar approaches were conducted to study the HCMV chemokine receptor (CKR) homologs UL33 and US28, or the HCMV protein kinase UL97. Farrell et al. developed ΔM33/UL33 and ΔM33/UL28 MCMV viruses, to replace the MCMV CKR homolog M33 with HCMV genes UL33 or UL28  infected with ΔM36.MCMV, by secreting TNFα  introduced ectopically in the MCMV genome and driven by the CMV i.e., promoter (Cicin-Sain et al., DN contains the death domain to associate with trimeric complex of death receptors, but lacks the N-terminal death effector domain required to activate caspase-8 (Chinnaiyan et al., in vivo fitness of the ΔM36.MCMV was not completely rescued by FADDDN and ΔM36.FADDDN.MCMV showed moderate attenuation in some organs (Cicin-Sain et al., DN.MCMV may either reflect differences in the molecular targets of M36 and FADDDN, or differences in expression kinetics, where FADDDN is overexpressed by an exceedingly strong ectopic promotor. In term of expression kinetics, our current model differs from the previous one, because the homologous UL36 protein was expressed via the M36 promotor, and the splice acceptor and donor sites of M36 remained intact, arguing that splice efficiency of M36 is retained. This excludes any confounding factors associated with gene expression kinetics or amount, indicating that UL36 expression levels equal to that of M36 are enough to protect the infected cell from apoptosis and confer growth fitness. MCMVUL36 completely rescued the virus growth in liver and spleen. However, the UL36 expression only partially rescued the growth in the salivary glands. The low titers in the salivary glands might be either due to growth defects or to the inability of the virus to spread to distant organs from the initial site of infection. It has been shown that the M36 gene plays an important role in the dissemination of MCMV from the initial site of infection (Cicin-Sain et al., UL36 (Figure in vivo spread and the incomplete rescue of virus replication in salivary glands. However, further experiments are required to validate this hypothesis.We have previously shown that the growth of ΔM36.MCMV can be rescued by overexpression of a dominant-negative FADD variant (FADDUL36 provides a novel tool to study the UL36 gene and demonstrates that UL36 may act as a determinant of viral fitness in the context of in vivo infection. In combination with previous studies, our data also suggest a vital role of the UL36 gene for in vivo fitness of HCMV (Skaletskaya et al., in vivo fitness and the fact that M36 and UL36 exert their anti-apoptotic function by direct binding to pro caspase-8 (Skaletskaya et al., in vivo. The system will hence allow us to validate pharmacokinetics and toxicity properties of promising lead candidates targeting UL36 in the context of MCMV infection while additionally giving insight into their antiviral efficacy in vivo. This experimental model therefore, has a strong potential to support studies of human biology and applications in clinical medicine.Notwithstanding the moderate loss of fitness in salivary glands, the recombinant MCMVMZC, BK, CP, MB, RC, and LB performed experiments. TL provided reagents. AS, SJ, IS, and LC designed the study. MZC and LC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Caribbean coral reefs have transformed into algal-dominated habitats over recent decades, but the mechanisms of change are unresolved due to a lack of quantitative ecological data before large-scale human impacts. To understand the role of reduced herbivory in recent coral declines, we produce a high-resolution 3,000 year record of reef accretion rate and herbivore (parrotfish and urchin) abundance from the analysis of sediments and fish, coral and urchin subfossils within cores from Caribbean Panama. At each site, declines in accretion rates and parrotfish abundance were initiated in the prehistorical or historical period. Statistical tests of direct cause and effect relationships using convergent cross mapping reveal that accretion rates are driven by parrotfish abundance (but not vice versa) but are not affected by total urchin abundance. These results confirm the critical role of parrotfish in maintaining coral-dominated reef habitat and the urgent need for restoration of parrotfish populations to enable reef persistence. Fishing of keystone herbivores such as parrotfish is thought to negatively impact corals. Here, Cramer and colleagues analyse the sedimentary record for the last 3,000 years from three coral reef sites in the Caribbean and show that indeed coral declines are linked to parrotfish declines. Acropora genus) are now dominated by macroalgae and low-relief corals tolerant of lower water quality (higher turbidity and nutrient) conditions34en masse the Acropora corals and the sea urchin Diadema antillarum in the early 1980s as well as coral bleaching outbreaks that became widespread in the late 1980s  of fish teeth, coral fragments and urchin spines preserved within reef sediment cores collected at three sites in Bocas del Toro, Caribbean Panama. Whereas previous studies of historical reef change in the Caribbean have had relatively poor temporal resolution due to the imprecision of radiocarbon dating for recent material42425263839At each site, declines in accretion rates and parrotfish abundance were initiated in the prehistorical or historical period. CCM analyses revealed that accretion rates were driven by parrotfish abundance (but not vice versa) but were not affected by total urchin abundance. These results confirm the critical role of parrotfish in maintaining coral-dominated reef habitat and the urgent need for management actions to maintain and restore parrotfish populations to enable reef persistence in the Caribbean.Cores were collected from three sites within the semi-enclosed lagoon Almirante Bay that span a gradient of influence from land-based runoff24230Th ages obtained from all six cores reveal that these cores span the period 1239±15 BC to 1984±3 AD. Corrected 230Th age errors (2σ) range from±3 to 15 years  within a sandy-muddy matrix of carbonate grains. The 27 ears see . Nearly se sites . Reef ac∼1925 AD . A linea∼1925 AD .The cessation of reef growth at the two fringing reefs, despite areas of active coral growth directly downslope of our coring locations and within 0.5 m of the water surface, suggests that the zones in which we cored may have transitioned from actively accreting to non-accreting environments due to changes in local hydrography related to changing sea levelPorites spp. (mainly Porites furcata), Agaricia spp., Madracis mirabilis and Acropora cervicornis, and the urchin assemblage is is composed primarily of Echinometra spp., Lytcehinus/Tripneustes spp. and Diadema antillarum . The coral assemblage is composed primarily of branching tillarum . There itillarum . Within Diadema Caribbean reefsDiadema die-off1Porites consistently dominates the coral assemblage within most cores and time periods, except for periods of dominance of staghorn coral A. cervicornis from 1000–1920 AD at Cayo Adriana, branching M. mirabilis from approximately 1500–1800 AD in both Punta Donato cores, and temporary and periodic dominance of lettuce coral Agaricia at Airport Point and Cayo Adriana , whose abundance closely tracks that of overall urchin abundance , the most important herbivores on post- Adriana . The urcbundance . TypicalLytechinus/Tripneustes has a significant positive causal effect on accretion rate, but neither Echinometra nor Diadema are causally related to accretion  positively affects reef accretion rate, reef accretion rate has no causal effect on parrotfish abundance . In contccretion . Parrotfpneustes . There ion rates . Diagnoson rates .The subfossils and sediments analysed from our reef matrix cores provide a 3,000-year continuous record of reef growth and herbivore abundance in Bocas del Toro that spans the period of intensifying human impacts to coastal marine ecosystems1819Our analyses of the causal relationship between reef herbivore abundance and accretion rates demonstrate the essential role of herbivory in coral growth and abundance. During the prehistorical and historical periods, parrotfish abundance was a positive driver of reef accretion, indicating that their positive effects on coral growth via removal of benthic macroalgae outweigh their negative effects via corallivory and bioerosion45Echinometra throughout the past three millennia) and reef accretion, likely signifying that Echinometra's positive effect on reef accretion via herbivory is of similar magnitude to their negative effect via intense coral boring activity48Lytechinus/Tripneustes group, likely indicating their positive effects on accretion via herbivory are greater than any negative effects from bioerosion50Diadema was not found to be causally related to reef accretion, likely a result of the consistent rarity of this urchin on the forereef slope zone of our coring sites. The multispatial CCM analyses reveal a positive causal effect of parrotfish abundance on total urchin abundance in general and Echinometra abundance in particular (Echinometra that may be caused by parrotfish-mediated facilitation of (1) coral-dominated habitat and/or (2) palatable benthic algal communities preferred by this urchin. Reef accretion rates are not causally affected by the relative abundance of the most common coral species, branching Porites. While accretion rates would undoubtedly be affected by changes in the relative abundance of exceptionally fast growing species such as Acropora cervicornisWe did not detect a causal relationship between overall urchin abundance . Reef accretion rates declined dramatically in the top 1 m of the Airport Point and Cayo Adriana cores and each included one age reversal (which we excluded from the age models). Therefore, age estimations near these reversals are less certain than those in other samples. Although reef accretion rates were used as a proxy of coral growth, non-coral carbonate producing organisms also contributed to sediment production. However, 83% of the weight of the undigested sediments>500 μm , is made up of coral. Accretion rates could also have been affected by the degree of bioerosion or physical erosion which could create finer sediments that might be more easily removed from the reef matrix by current or wave action. However, because accretion rates are not related to sediment grain size , accretiOur analysis of millennial-scale change in reef communities in Bocas del Toro suggests that historical fishing may have been significantly affecting Caribbean reefs for over two centuries, initiating ecosystem declines from which they have not recovered. The clear positive causal effect of parrotfish abundance on reef accretion rate indicates that modern Caribbean reefs that have largely been depleted of these herbivores may be locked into an alternative stable state of macroalgal dominanceTo prevent damaging living corals, we collected cores from rubble zones on the reef slope adjacent to living coral colonies. At each site, two replicate cores 10.1 cm in diameter and ranging from 3–5.5 m in length were extracted ∼10–30 m apart at 6–7 m water depth using using a self-contained underwater breathing apparatus (SCUBA) and a combination of push-coring and vibra-coring techniques. Cores were split lengthwise, and the working half was sliced into 5 cm increments (referred to as samples throughout the paper). Subfossils were analysed from every sample from the top 1 m of each core to produce a high-resolution record of change from the most recent period, while every third sample was analysed from deeper parts of the core to produce a moderately high-resolution record from the pre-historical period. This scheme resulted in the analysis of 36–41 samples from each core. Samples were dried and weighed, then wet sieved at 2 mm, 500, 250, 104 and 63 μm fractions to facilitate sorting and counting different taxonomic groups.http://ichthyolith.ucsd.edu/.We tracked changes in the abundance and taxonomic or functional composition of reef fish, corals and urchins through time. Reef accretion rate, a measure of carbonate production by hermatypic corals as well as other carbonate-producing organisms, was computed by determining sedimentation rates based on our age models (time/5 cm increment). Fish teeth were analysed from all five sediment size fractions. To remove small carbonate grains from acid-insoluble tooth subfossils, we dissolved the three sediment size fractions <500 μm in 10% acetic acid. Parrotfish teeth (family Labridae) were identified from gross tooth morphology and with the assistance of a tooth and jaw reference collection that we developed from dissection and photography of oral and pharyngeal jaws of 287 positively identified Caribbean reef fish species that represent all Caribbean reef families and most genera. Modern fish specimens were obtained from the Fish Collection of the Smithsonian Institution's National Museum of Natural History and fish markets in Bocas del Toro and Colón, Panama. Our photographic reference collection is at Acropora cervicornis, Agaricia spp., branching Porites spp., Madracis mirabilis). Urchin spines were picked from the 0.5–2 mm fraction and separated into two typically reef-associated taxa  and one typically seagrass- or reef-associated group (Lytechinus and Tripneustes spp.) and counted and weighed. Total urchin abundance was also computed to track effects of overall urchin herbivory on reef accretion rates.Coral and urchin subfossils were assigned to species or generic level. Coral fragments from the >2 mm sediment fraction were weighed and identified to common taxa  and to facilitate complete sample-tracer homogenization. U and Th were separated using conventional anion-exchange column chemistry using Bio-Rad AG 1-X8 resin. After stripping off the matrix from the column using double-distilled 7N HNO3 as eluent, 4N HNO3 and 2%HNO3+0.03% HF mixture was used to elute U and Th into 3.5 ml pre-cleaned test tubes, respectively. After screening U and Th concentrations in the U and Th fractions using their 1:100 dilute solutions on a quadrupole ICP-MS, the U and Th separates for the samples were then re-mixed in 2% HNO3 to make ∼3 ml solution in a pre-cleaned 3.5 ml test tube. The mixed solution for each sample contained the entire Th fraction and a small percentage of the U fraction. The amount of U fraction to be added to the mixed solution was calculated based on the screening results and the MC-ICP-MS working sensitivity, aiming to achieve ∼5 volts of 238U signal. The remixed U–Th solution was injected into the MC-ICP-MS through a DSN-100 desolvation nebulizer system with an uptake rate of ∼0.1 ml min−1. U–Th isotopic measurements were performed on the MC-ICP-MS using a detector configuration to allow simultaneous measurements of both U and Th isotopes. The 230Th/238U and 234U/238U activity ratios of the samples were calculated using the decay constants given in ref. 230Th ages were calculated using the Isoplot 3.75 Program230Th ages were corrected for non-radiogenic 230Th contributions using a modelled two-component-mixing non-radiogenic 230Th/232Th value based on the equation in ref. A high-resolution chronology of reef ecosystem change was obtained by U–Th dating coral fragments within the cores using a Nu Plasma multi-collector inductively-coupled plasma mass spectrometer (MC-ICP-MS) in the Radiogenic Isotope Facility at University of Queensland, following chemical treatment procedures and MC-ICP-MS analytical protocols described in ref. 230Th ages were obtained from the top 1 m of the core and one date from approximately every 0.5 m interval below that, yielding 6–8 ages per core  from which they were picked. Because proportional abundances of living communities are generally faithfully represented in subfossil assemblagesTemporal trends in individual community components were assessed by plotting abundance values of individual samples, with trends assessed within each core and across all cores combined using non-parametric locally weighted regression (‘loess')-smoothed trendlinesTo assess the presence and direction of causal relationships between herbivore abundance and reef accretion rates and between herbivore groups, we used CCM, a technique that compares the ability of time-lagged components of one process to estimate the dynamics of another. CCM tests for significant causal relationships by recognizing that the observed values of a forcing process should be significantly better explained by observed values of a response process than expected by chance, and the accuracy of that explanation should improve with increasing time series length3536Briefly, the algorithm proceeded in five steps: (1) using cross-validation, determine the optimal embedding dimension (E) that describes the number of time steps that best predicts the dynamics of each explored process , (2) test for nonlinearity and stochastic noise to ensure that none of the processes were purely random and that stochastic noise was not so large that causal links could not be recovered. Stochasticity and nonlinearity were tested by visually assessing whether predictive power was reasonably high for short time steps and decreased with increasing prediction time, respectively38The data that support the findings of this study are available from the corresponding author upon reasonable request.How to cite this article: Cramer, K. L. et al. Prehistorical and historical declines in Caribbean coral reef accretion rates driven by loss of parrotfish. Nat. Commun.8, 14160 doi: 10.1038/ncomms14160 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary References


Measuring at ~30 nm, a fully customizable holliday junction DNA nanoconstruct, was designed to simultaneously carry three unmodified SiRNA strands for apoptosis gene knockout in cancer cells without any assistance from commercial transfection kits. In brief, a holliday junction structure was intelligently designed to present one arm with a cell targeting aptamer (AS1411) while the remaining three arms to carry different SiRNA strands by means of DNA/RNA duplex for inducing apoptosis in cancer cells. By carrying the three SiRNA strands  into triple negative breast MDA-MB-231 cancer cells, cell number had reduced by up to ~82% within 24 hours solely from one single administration of 32 picomoles. In the immunoblotting studies, up-elevation of phosphorylated p53 was observed for more than 8 hours while the three genes of interest were suppressed by nearly half by the 4-hour mark upon administration. Furthermore, we were able to demonstrate high cell selectivity of the nanoconstruct and did not exhibit usual morphological stress induced from liposomal-based transfection agents. To the best of the authors’ knowledge, this system represents the first of its kind in current literature utilizing a short and highly customizable holliday DNA junction to carry SiRNA for apoptosis studies. This is largely due to the wide choices available in innovative molecular/drug designs, ranging from nanoparticles6 to drug-antibody conjugates7. Yet not a single system can be truly beyond reproach9. Of the many novelties advocated in literature, delivering RNA interference (RNAi) remains a strong contender for treating diseases at a cellular level by means of repressing and shutting down disease-causing genetic anomalies13. While RNAi technology currently represents the forefront in gene suppression from an academic standpoint, it does suffer from some drawbacks as well. High degradation rate within the cytoplasmic environment14 as well as the requirement for high dosage16 had drawn much criticism from the scientific community. Chemically modified RNA may improve the circulation longevity15 but is highly susceptible towards immunogenic responses18. Hence, the plasmid derivatives in the form of ShRNA (short hairpin RNA) are often promoted in its place for the maintenance of interfering RNA levels19. Nevertheless, the main problem remains in the selection of the most appropriate delivery mechanism into the cell. The most common mode of deliverance is via liposomal-based technology but the issues of cytotoxicity21 as well as the lack in cell-specificity were serious enough to impede its development as a viable clinical option. While handling issues pertaining to cytotoxicity remains a tricky and daunting task, gaining high cell specificity is comparatively more straightforward. Many groups in the past had tried to covalently conjugate antibodies directly to liposomes24 but the problems of immunogenic responses towards the antibodies had plagued these hybrid systems right from the start26. This had subsequently regressed antibodies-liposome hybrids (coined “immunoliposomes”) to the role of useful in-vitro tools. In contrast, DNA aptamers, with its lower level of immunogenicity27 as well as being more economically viable compared to antibodies, are often proposed as another alternative for cell targeting.Some of the quintessential characteristics of any customizable therapeutics should be non-immunogenic, low toxicity as well as high tissue specificity31. Indeed, synergizing both aptamer and RNAi had already gained much footing in literature and reports had already shown considerable success in recent years34. Advantages of using aptamers over antibodies are that they are usually inexpensive and have a higher shelf life compared to antibodies35. They can be easily tailor-made through SELEX enrichment procedures and are more thermally stable. Compared to liposomal-based delivery36, aptamers do not require any additional preparation steps other than purification prior to administration to cells and they do not typically induce any of the cytotoxicity compared to liposomal delivery37.DNA aptamer are short strands of DNA that can readily self-hybridized with itself to present important tertiary structures. They can serve to bind to cell surface receptors and ultimately gaining entry into cell targets with high specificity38. These bioconjugated DNA/RNA nanocomplexes were then administered directly to the designated cell, and gene suppression was subsequently measured. However, the process of bioconjugation can be technically difficult in untrained hands, and RNA chimeras may suffer from potential immunogenic responses. Coupled by the fact that many of these systems only dealt with a single gene target, this may not be as efficient compared to multiple gene targets being singled out simultaneously. It was with these thoughts that we proposed this DNA/RNA nanoconstruct that was designed to simultaneously carry multiple copies of antisense RNA strands into cells. Borrowing from the concepts of the self-assembled holliday junction as demonstrated by Li et al. delivering fluorophores39 as well as complex DNA/RNA machinery by Lee et al.40, we assembled a four-armed nanoconstruct in a single pot fashion and an aptamer (AS1411) was selected to anneal towards one of the arms via a sequence specific ‘sticky’ end. The remaining three arms comprised of overhanging sequences that were complementary to its individual antisense RNA sequences and the respective RNA strands were then hybridized to these selective regions via DNA/RNA hybrid duplex.Much of the current research has reported the use of aptamers to deliver single antisense RNA (double stranded) and most often involved covalent conjugation of the ends of the DNA aptamer directly to the functional end of the RNA or as “chimeras”In short, all the strands were introduced into a single PCR tube and placed in a thermocycler and the temperature slowly reduced from 95 °C to 4 °C in order to self-assemble into its final holliday configuration as shown in Fig. et al.41. One other important consideration was the potential cleavage by cellular Ribonuclease H1 of the DNA/RNA duplex within the cell cytoplasm. To mediate this, the system was designed in such a way that the 5′ end for all three RNA strands (19 bases) was approximated less than 5 nm relative to the center of holliday junction. Molecular dynamics simulation on the cellular ribonuclease H1 interaction with DNA/RNA hybrid strand revealed that the binding process would require a surface area of 177 nm2 on site42. But it is essential that the DNA region to be free of any structured loops (such as hairpin loops etc) in order to accommodate the binding domain of the ribonuclease43, which would not be in our case due to the immediate flanking of the RNA’s 5′ end with DNA. The key recognition of the binding domain for Ribonuclease H1 to the width of the minor groove had been reported to be around 7.5 Å44 and is highly intolerant towards other dimensions. The holliday junction DNA right next to the 5′ RNA/DNA duplex antisense strand would distort and rigidize the backbone of the DNA/RNA which could offer resistance towards ribonuclease H1 cleavage. Moreover it was already well-established that structured RNA tends to cleave off significantly slower than unstructured RNA45. Hence, we postulated that the cross-like contorted nano-architecture could offer much steric hindrance towards the ribonuclease antisense and this would also help to reduce the overall processivity for the ribonuclease antisense to work on cleaving the DNA/RNA. It is important to note that if cleavage had indeed occurred for the DNA/RNA duplex, calculations from closest neighbor parameter would shown that the fragmented RNA oligomers were incapable of forming stable duplex with mRNA at 37 °C. It was therefore with these considerations that we proceeded with unmodified RNA strands rather than chemically modified species in order to reduce toxicity and facilitate for rapid clearance from the cellular system. The successful attainment of protein repression in later section of this manuscript had helped to reinforce these notions.Four strands of DNA were specially designed to host three separate antisense RNA strands while leaving a single arm for hybridization to an aptamer DNA see Fig. . We had 47. Nucleolin was highly expressed in cancer cells and they were often localized on the outer-membrane while their surface presentation was not typically found on non-cancer targets48. The rationale was that upon the complete assembly of the nanoconstruct, the aptamer would bind to the cell surface to facilitate for uptake  were mixed in equimolar concentration within a single PCR tube and were subjected to a melting temperature of 95 °C before allowing all oligomers to reassemble via complementary pairing with the slow gradual reduction of temperature to 4 °C. In order to validate the proper assembly of the full nanoconstruct, non-denaturing acrylamide gel electrophoresis was also performed  was subsequently performed to verify the sizing of the nanoconstruct. As shown in Fig. As observable in Fig. Using a holliday junction decorated with the AS1411 aptamer, a FITC tagged DNA sequence homologous to that of the antisense RNA belonging to the MDM2 was used to hybridized to the nanoconstruct and MDA-MB 231 cells were incubated for 2 hours. This was to examine if the cells were able to uptake nanoconstruct on the virtues of the AS1411 aptamer as selected. As shown in Fig. In a series of dose dependent studies, the full nanoconstruct carrying the three apoptosis inducing antisense RNA was purified from non-denaturing polyacrylamide gels and were added at 4, 8, 16 and 32 pmoles to MDA-MB 231 cells for a period of 24 hours. After the incubation period, cells were collected and the cell numbers were determined using the hemocytometer. Figure In order to further demonstrate the stringent cell-type selectiveness of this nanoconstruct, two other cell types were incubated with the apoptosis-inducing nanoconstruct at the dosage of 16 pmoles. As shown in Fig. Cell count for lipofectamine-based transfection was performed using the three single strand antisense RNA at 16 picomoles. As these antisense were unmodified, we did not envisage for any high efficiency from this delivery event and this was as shown in Fig. et al.54 by which that the maintenance of high p53 levels was deemed as a prerequisite for inducing cell apoptosis. However, this study could not be directed comparatively with that report as we had deliberately suppress three proteins in a simultaneous fashion although it is important to note that this was not the first time that this simultaneous repression of multiple protein target was attempted56. Nonetheless the general consensus was that multiple protein suppression in a concurrent fashion could indeed shortened the time-line required for apoptosis (24 hours)58 and our findings here were no different.As mentioned three antisense RNA protein were selected under special consideration that all the genes contribute towards cell death. AKT and its downstream MDM2 are proteins that regulate the expression of p53 and the suppression of these genes would upregulate phosphorylated p53 expression levels that would contribute towards cell apoptosis. On the other hand, survivin was another protein that inhibits caspase activity, thus contributing to the overall unnatural longevity of cancer cells. Hence, by suppressing all these three genes, it was in principle possible to induce rapid cell death. In Fig. Furthermore, in order to demonstrate that this nanoconstruct was easily programmable as a proof of concept, we decided to introduce three separate RNA antisense strand (8 picomoles) that would simultaneously suppressed the same metastatic Twist protein and we were able to obtain an even early repression at the 2 hour mark of up to ~77%  was purchased from Genetex, Survivin  was acquired from Novus Biologicals and AKT  was obtained from Cell Signalling. Anti-rabbit IgG, HRP-linked Antibody (#7074) was purchased from Cell Signalling as well. DMEM/F12 cell culture media, EGF (20 ng/ml), Insulin (10 ug/ml) was purchased from Invitrogen while Hydrocortisone (0.5 mg/ml) was from Sigma. 5% Horse Serum was acquired from HyClone Donor Equine Serum (#SH3007403) while Fetal Bovine Serum was purchased from Gibco2 incubator at 37 °C. MDA-MB-231 cells were cultured in DMEM-F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 90% RMPI 1640 medium, 10% fetal bovine serum and 1% penicillin-streptomycin was used to maintained AGS cells. Upon reaching 80% confluency, the cells were treated with trypsin after washing with PBS (3 times). The detached cells were centrifuged at 1000 rpm for 5 minutes. The pellets were then resuspended with growth medium at appropriate dilution, and cultured in a new 75 ml polystyrene cell culture flask.All cell cultures were incubated in 5% CO2 with DMEM and Nutrient Mixture F12 (Ham) (Invitrogen) with high glucose containing L-glutamine, pyridoxine hydrochloride, and HEPES buffer supplemented with 5% Donor Equine Serum (Hyclone), 20 ng/ml Epidermal Growth Factor, 0.5 mg/ml Hydrocortisone, 10 ug/ml Insulin, and 1% penicillin-streptomycin.The medium recipe for MCF-10A cells was adapted from Brugge’s lab. In brief, MCF-10A cells were grown at 37 °C incubator with 5% CO™ unless otherwise specified. To assemble the holliday junction nanoconstruct, the following sequences were selected to form a holliday junction with 4 overhanging ends that are complementary of the the individual RNA antisense strands and the respective aptamer sequence are also as listed below:All DNA and RNA were obtained from InvitrogenHolliday Junction 1 = 5′TTTGTGCAGCCAACCCTCCGTGTGTGTGCCATAGTGCATTGCGAGAGAGAG 3′Holliday Junction 2 = 5′ATACTATCAGATTTGTGGCTTTCCTTTGCATTCGGACTATGGCACACACAC 3′Holliday Junction 3 = 5′GCAGTGGATGAAGCCAGCCTTAAGGCCCGTGCTCACCGAATGCAAAGGAAA 3′GGGGGGGGGGGCTCTCTCTCGCAATGCTGAGCACGGGCCTTAA 3′Holliday Junction 4 = 5′59AS1411 (nucleolin) aptamer = 5′ GGTGGTGGTGGTTGTGGTGGTGGTGG CCCCCCCCCCC 3′CCCCCCCCCCC 3′60AGS cell specific aptamer = 5′ CGACCCGGCACAAACCCAGAACCATATACACGATCATTAGTCTCCTGGGCCG Three strands of antisense RNA was selected from well-cited sources in literature and was shown below:62AKT: 5′ GGAGGGUUGGCUGCACAAA 3′64MDM2: 5′ GCCACAAAUCUGAUAGUAU 3′65Survivin: 5′ GGCUGGCUUCAUCCACUGC 3′™) and 1 nmol for each strands  were collectively pooled to a PCR tube and annealing buffer  was added to bring the mixture to a final volume of 100 μl. The oligomer mixture was then mixed well before subjecting to the thermocycler  to 95 °C and held for 10 minutes. The temperature was then slowly reduced to 4 °C over 30 minutes before purification.To assembly the full nanoconstruct, all received oligomers (DNA and RNA) were firstly resolubilized in Rnase Free DEPC water (Invitrogeng for 10 mins. All purified nanoconstruct was quantified with UV-Vis Spectrophotometer (Eppendorf Biophotometer plus) at 260 and 280 nm. The “roadmap” of the nanoconstruct was as shown in Supplementary Figure The nanoconstruct was purified using an 8% native polyacrylamide gel in Tris/Borate/EDTA buffer. The gel was sectioned out according to the size of the fully assembled nanoconstruct (a lane was stained with EtBr as a guideline). The sections were crushed and transferred to Nanosep® MF centrifugal devices and spun at a 17949 × 4 in a 24-well plate and then transfected with the nanoconstruct at the nanoconstruct concentrations of 4, 8, 16 and 32 picomoles (plated out in quadruplicates) for 24 hours. The cells were washed three times with phosphate-buffered saline and resuspended with 0.05% Trypsin-EDTA for ~15 mins. After which, 10 μl of the cell suspension was loaded into a hemocytometer and counted according to the manufacturer’s protocol. Statistical analysis of differences was performed via a one-way ANOVA analysis whereby P value of ≤0.05 was taken as statistically significance.For all cell types, the cells were seeded at a density of 2 × 106 and co-incubated with the nanoconstruct. Total protein concentration was determined by bicinchoninic acid assay (Thermo Fisher). Whole cell lysates were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Milipore) using a wet transfer system (Hoefer) with a Tris-glycine buffer containing 20% methanol. The membrane was then blocked with 3% bovine albumin serum in Tris-buffered saline with 0.1% Tween-20 (TBST) for an hour room temperature and gently washed twice with TBST. The membrane was probed with antibodies against Akt , MDM2 (Genetex), phosphorlayted-p53 , and GADPH (Genetex) for ~16–18 hours at 4 °C and washed three times for ~10 mins with TBST. Afterwards, the blot was detected with HRP-linked Anti-rabbit IgG antibodies  for an hour at room temperature and followed by three washes with TBST for ~10 mins. The blot was developed using Immobilon Western Chemiluminescent HRP Substrate (Milipore).For immunoblotting, the cells were seeded at a density of 1 × 10The following staining protocol was adapted from Sir William Dunn School of Pathology. In brief, Uranyl acetate was dissolved in deionized water to prepare a 2% stain solution. The nanoconstruct was diluted by 10-folds in DEPC-treated water. Equal ratios of the stain solution and nanoconstruct were combined. The mixture loaded onto the grid for ~30 seconds and then absorbed with filter paper. Transmission electron microscopy was performed on Hitachi HT7700 platform at an acceleration voltage of 70 kV.Atomic force microscopy (AFM) images were acquired using Bruker Dimension Icon AFM system using an in-build AFM tapping mode. Scan area on the surfaces were of 0.3 μ m × 0.3 μ m and the scan speed was set at 0.5 hz with the integral and proportional gain set at automatic mode. Post image processing was performed with Gwyddion MacOS version 2.38.Particle size of the nanoconstruct was determined using a disposable plastic cuvette through the Zetasizer Nano  at room temperature using DEPC water as the solvent.6. After 2 hours, the cells were washed 3 times with PBS followed by the nominal fixation protocol with paraformaldehyde and stained with Alexa Fluor® 594 phalloidin before mounting on cover slips. The surfaces were then examined under Nikon® N-SIM super resolution microscopy using using an oil immersion objective lens CFI SR at laser wavelength of 488 nm and 561 nm for the visualization of the nanoconstruct carrying FITC and the cell membrane respectively. Image processing was done with NIS Elements Revolutionizes Imaging software .To evaluate the uptake of the nanoconstruct, the four arm holliday junctions with the AS1411 aptamer was assembled without antisense RNA but was replaced with a FITC-tagged complementary strand to holliday junction 2 (5′ GCCACAAATCTGATAGTAT 3′). The fluorescence labeled nanoconstruct (16 picomoles) was administrated to MDA-MB-231 cells at density of 1 × 10Supplementary Information