Cryptic peracarids are an important component of the coral reef fauna in terms of diversity and abundance, yet they have been poorly studied. The aim of this study was to evaluate the taxonomic richness and abundance of cryptic peracarids in coral rubble in the Puerto Morelos Reef National Park, Mexico (PMRNP), and their relationship with depth.Three reef sites were selected: (1) Bonanza, (2) Bocana, and (3) Jardines. At each site six kilograms of coral rubble were collected over four sampling periods at three depths: 3 m (back-reef), 6–8 m (fore-reef), and 10–12 m (fore-reef).A total of 8,887 peracarid crustaceans belonging to 200 taxa distributed over five orders and 63 families was obtained; 70% of the taxa were identified to species and 25% to genus level. Fifty species of those collected represent new records for the Mexican Caribbean Sea. Isopoda was the most speciose order while Tanaidacea was the most abundant.Cryptic peracarid taxonomic richness and abundance were related to depth with higher values of both parameters being found in the shallow (3 m) back-reef, possibly due to a higher reef development and a greater accumulation of coral rubble produced during hurricanes. Peracarid data obtained in the present study can be used as a baseline for future monitoring programs in the PMRNP. Coral reefs are one of the most complex and productive ecosystems of the world and support one of the highest diversity of the marine realm due to its highly complex architecture . Both liCoral reefs have deteriorated in the last decades world-wide because of climate change, diseases, macroalgal overgrowth, overfishing, sedimentation, low water quality and hurricanes . DeclineThe objective of this study was to make a quantitative assessment of the taxonomic richness and assemblage composition of peracarids in coral rubble within the Puerto Morelos Reef National Park, Mexico. We also address two research questions: (i) Do taxonomic richness and abundance of cryptic peracarids vary with depth?; and (ii) do these parameters vary between reef sites?The study site is located within the Puerto Morelos Reef National Park (PMRNP), in Quintana Roo, Mexico . This maThree reef sites were selected: (1) Bonanza , (2) Bocana , and (3) Jardines  . The disin situ and immediately transported to the laboratory, where fragments were placed in buckets with fresh water to induce osmotic shock and force cryptofauna to leave the microhabitats (holes and crevices) , 6–8 m (fore-reef), and 10–12 m (fore-reef); hammer and chisel were used when the coral rubble was consolidated. One sample was collected by SCUBA divers from each depth, at each site, in four months . Samples were placed in plastic bags revices) . Consolip > 0.05). To visualize differences in the dominant taxa across sites and depths we constructed a heatmap  and a hierarchical clustering, performed with the average linkage method from a Bray–Curtis dissimilarity matrix; all taxa were used to do the hierarchical clustering but only the more abundant taxa are displayed in the heatmap (those whose relative abundance was higher than 5%). All analyses were done in R (https://github.com/rerodriguezmtz/Peracarids). This includes all underlying data and R code for all analyses.Similarities in peracarid taxonomic richness among reef sites and depths were summarized in Venn diagrams. The hypothesis that cryptic peracarid abundance varied with (1) reef site and (2) depth was tested using a 2-factor ANOVA, using the four sampling surveys as replicates. Abundance data were transformed by log10(x) prior to the statistical analysis. Homogeneity of variances was confirmed by Bartlett’s test  among sites  and depths , but the composition of the assemblage was heterogeneous and only 20 taxa were shared among all sites and depths. Venn diagrams illustrate that 85 of the 200 taxa recorded were shared by the three reef sites . The three depths also shared 85 taxa, with the shallow back-reef having the highest number of unique taxa (N = 24)  .p > 0.05; p < 0.01), with abundance being significantly lower  in the deep (10–12 m) fore-reef  than in the shallow (3 m) back-reef .The mean abundance of peracarids  was not Pseudoleptochelia sp A, Paratanais sp A, and Leptochelia dubia. Other abundant taxa were Apseudes sp A, in the shallow site of Bonanza, Cirolana parva, in the medium depth of Bonanza, and Chevalia aviculae, in the medium depth of Jardines , Tanaidacea (N = 22) and Cumacea (N = 30) recorded for the PMRNP is higher than reported for other coastal habitats in the Mexican Caribbean, and in the case of Tanaidacea it was higher than previously reported for the Caribbean Sea (N = 72) is lower and accounts for approximately one fourth of that previously reported for the Mexican Caribbean Sea and one tenth for the Caribbean Sea  to a coast that is experiencing intensive land development, as a result of the rapid growing tourism industry , and wheber 2015 . The cauCryptic peracarid crustaceans in coral rubble are diverse and abundant within the PMRNP. Taxa richness of the orders Isopoda, Tanaidacea and Cumacea was larger than previously reported for coral rubble and other costal habitat types in the Mexican Caribbean, while that of Amphipoda was lower. The most abundant order was Tanaidacea with dominant species belonging to the families Paratanaidae and Leptocheliidae. Within the reef system taxonomic richness and abundance of cryptic peracarids were higher in the shallow back-reef areas than in the reef front, where values decreased with depth. This elevated occurrence in the back-reef may result from a larger accumulation of coral rubble, which occurs during hurricanes. No significant differences in taxonomic richness and abundance of cryptic peracarids were observed between reef sites suggesting homogeneous environmental conditions for this superorder across the PMRNP. The data obtained in the present study can serve as a baseline for future monitoring programs in the PMRNP that aim to detect changes in water quality and invasive species.10.7717/peerj.3411/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.3411/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.3411/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.3411/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.3411/supp-5Supplemental Information 5Click here for additional data file.


The system is analogous to real-time PCR, but with the potential to detect small molecules, proteins, and other targets that can be recognized by a suitable aptamer. The ligand-dependent self-replication of RNA has potential applications in molecular diagnostics and biosensing that benefit from the rapid, precise, and real-time detection of various target molecules.A system was developed to detect the self-replication of an RNA enzyme in real time. The enzyme is an RNA ligase that undergoes exponential amplification at a constant temperature and can be made to operate in a ligand-dependent manner. The real-time system is based on a fluorimetric readout that directly couples the ligation event to an increase in florescence signal that can be monitored using standard instrumentation. The real-time system can also operate entirely with  Technologies based on the exponential amplification of nucleic acids are widely used in molecular diagnostics and biosensing. For example, the quantitative polymerase chain reaction (qPCR) is a sensitive and precise method for detecting target nucleic acids ,2. Due t–1, corresponding to a doubling time of 23 min [–1, corresponding to a doubling time of 5 min [The self-replicating RNA enzyme (E) is an RNA ligase that catalyzes the joining of two RNA substrates (A and B) to form another copy of itself ,6. The lof 5 min .Kd of the aptamer domain determines the degree of saturation of the aptazyme, which, as previously shown [The catalytic activity of the RNA enzyme can be made dependent on a target ligand by linking a ligand-binding aptamer domain to the catalytic center of the enzyme . The resly shown ,4, deterl-RNA [d- and l-RNA enzymes have identical biochemical properties, but only the latter have been shown to operate in the presence of biological samples such as human serum [A previous limitation of the RNA replication system was that it could not be applied to biological samples without first removing ribonucleases that would otherwise degrade the RNA. This limitation was overcome by preparing the enzyme and its substrates in the form of enantiomeric l-RNA , which ian serum . This isFor the ligand-dependent exponential amplification of RNA to be a practical method, it would be important to track the course of amplification in real time, based on the development of a fluorescent signal or other simple readout. Previous analyses were conducted after the reaction had been completed, using either polyacrylamide gel electrophoresis (PAGE) to determine the amount of ligated products or a luciferase assay that is driven by the release of inorganic pyrophosphate .d- or l-RNA, and can be made to operate in a ligand-dependent manner.In this study, a fluorimetric method was developed to monitor the amplification of the self-replicating RNA enzyme in real time. The B substrate was labeled with a fluorophore linked to the γ-phosphate of the 5′-triphosphate, and a quencher was placed at the 3′-end b. Prior N-hydroxysuccinimide (NHS) ester, followed by gel purification the conjugated product. The TMR-labeled substrate was tested in a self-replication reaction employing 19 µM of substrate A, 25 µM of TMR-B, 1.5 µM of E, and 25 mM of MgCl2 at pH 8.5 and 42 °C. The enzyme performed very poorly with the modified substrate, reaching only 7% completion after 25 h and failing to undergo exponential amplification. Similarly, poor performance was seen when other fluorophores, such as Cy5 or Alexa Fluor 488, were linked to the 5′-γ-phosphate of substrate B (data not shown).The original form of the self-replicating RNA enzyme  was testIn vitro selection was used to develop a modified form of the enzyme that can readily accept fluorophore-modified substrates. The selection strategy was based on a previous design  that req12 RNA molecules (100 pmol) was used to initiate the first round of selection. The first round employed 2 µM of TMR-B-biotin, 1 µM of E-A, and 25 mM of MgCl2, which were incubated at pH 8.5 and 42 °C for 1 h. During subsequent rounds, the selection pressure was increased by decreasing the concentration of reactants, decreasing the concentration of MgCl2, and decreasing the reaction time. By the fifth round, the reaction employed 50 nM of TMR-B-biotin, 50 nM of E-A, and 5 mM of MgCl2, which were incubated for only 15 s. The activity of the population was assayed after each round of selection and, by the fifth round, had reached a plateau, signifying that the process was completed.A population of enzyme variants was generated by randomizing 16 nucleotides that comprise the catalytic center c. A star2, which were incubated at pH 8.5 and 42 °C. Under these conditions, the starting enzyme has a catalytic rate of 1.4 min–1 with the unmodified substrate B and only 0.0013 min–1 with TMR-B. In contrast, the selected enzyme has a catalytic rate of 0.25 min–1 with unmodified substrate B and 0.11 min–1 with TMR-B . The selected enzyme is about 6-fold slower than the starting enzyme when reacting with the unmodified substrate. Thus, the selected enzyme has, to some extent, become specialized in reacting with the modified substrate.d-RNA form of TMR-B can be prepared by in vitro transcription and subsequent chemical modification, this is not possible for the corresponding l-RNA form, which must be prepared synthetically. The chemical synthesis of l-RNA oligonucleotides is straightforward, relying on commercially available l-RNA phosphoramidites. However, the l-RNA form of TMR-B requires installation of a 5′-triphosphate and subsequent modification of the γ-phosphate with the fluorescent label. In addition, for the real-time detection system, a quencher (Q) must be installed at the 3′-end of the substrate. Thus a synthetic scheme was devised to prepare the TMR-B-Q substrate  phosphoramidites on a CPG column derivatized with 2′-O-propargyl-uridine. Following removal of the 5′-terminal dimethoxytrityl group, but prior to further deprotection, the 5′-hydroxyl was phosphitylated using salicyl phosphorochloridite, reacted with tributylammonium pyrophosphate (TBAP) to form the cyclic trimetaphosphite, and oxidized with t-butyl peroxide to form the water-sensitive and reactive cyclic trimetaphosphate (v/v) solution of trifluoroacetic acid (TFA) in hexanes, the column was washed with acetonitrile (ACN), and the RNA was deprotected and purified using standard procedures  phosphoramidites. The d- and l-RNA substrates were prepared using 2′-TOM phosphoramidites. The ligation reaction employed the simplified form of substrate A and TMR-B-Q under the same conditions as above. Fluorescence was monitored in real time using a standard qPCR instrument, but maintaining a constant temperature of 42 °C. For both the d- and l-RNA forms of the reaction, there was a progressive increase in fluorescence, with observed rates of 0.10 and 0.13 min−1, respectively (The ectively a. The slAn aptamer that binds theophylline  was linkd-RNA form of the resulting aptazyme (Etheo) was prepared by in vitro transcription and tested in a ligation reaction employing 5 µM of the simplified form of substrate A, 2 µM of TMR-B, 6 µM of Etheo, either 0 or 5 mM of theophylline, and 25 mM of MgCl2, which were incubated at pH 8.5 and 42 °C -GTP and GTP during in vitro transcription to install a derivatizable group on the 5′-γ-phosphate. Chemical synthesis also avoids the 5′-terminal sequence heterogeneity that commonly occurs during in vitro transcription [Regardless of the sequence of the enzyme, it can be readily prepared in the form of enantiomeric cription ,18.l-RNA aptamers, continues to expand, the opportunities to combine those reagents with the real-time exponential amplification system will expand accordingly.A potential application of the fluorescent-signaling, self-replicating RNA enzyme is to monitor ligand-dependent exponential amplification in real time. The instrument used in this study was a standard qPCR device with 96-well format, although maintaining a constant reaction temperature rather than thermal cycling. The time required to reach an amplification threshold that is distinguishable from background is about 2–3 h c, which O-propargyl uridine 3′-Lcaa CPG were purchased from ChemGenes . Both T7 RNA polymerase and RNaseP M1 RNA were prepared as described previously [– reverse transcriptase and Alexa Fluor 488 NHS ester were from Thermo Fisher , and yeast poly(A) polymerase was from Affymetrix . Tributylammonium pyrophosphate, 6-(Boc-amino)-1-hexanol, TMR-NHS ester, biotin-PEG4-alkyne, NTPs, dNTPs, and theophylline were purchased from Sigma-Aldrich , γ-(6-aminohexyl)-GTP was from Jena Bioscience , 2′-azido,2′-deoxyadenosine 5′-triphosphate was from TriLink Biotechnologies , and Tide Quencher™ 3 (TQ3) azide was from AAT Bioquest .Synthetic oligonucleotides were prepared by solid-phase synthesis using an Expedite 8909 DNA/RNA synthesizer. All nucleoside phosphoramidites and 2′-eviously . Bovine E. coli RNase P M1 RNA and an external guide RNA to yield RNA with a homogeneous end and free 2′- and 3′-hydroxyls [UAAGUUGCGGUCUCACCA-3′ (region of hybridization underlined). Following RNA cleavage, the RNA was extended by a single 2′-azido,2′-deoxyadenosine residue using yeast poly(A) polymerase, then coupled to biotin-PEG4-alkyne, as described previously [The TMR-B-biotin substrate was prepared by in vitro transcription of an extra-length RNA, which was cleaved using ydroxyls ,19. The eviously .TAATACGACTCACTATAGGAAGTTGTGTCTTAATGTTACGTAAGTAACAGATGGAATTGGTTGAAGTATGAGACCGCAACTTA-3′; for Etheo, the sense strand had the sequence 5′-GGCTAATACGACTCACTATAGGAAGTTGTGTCTTAAGATACCAGCCGAAAGGCCCTTGGCAGCGATGGAATTGGTTGAAGTATGAGACCGCAACTTA-3′ .In vitro transcribed RNA enzymes were prepared by run-off transcription, as described previously ,6. DoublIn vitro transcribed substrate A containing the eight mutations present in the selected enzyme was prepared by first generating the enzyme, as described above. Then, the B-substrate portion was removed using M1 RNA and the same external guide RNA described above.d- and l-RNA enzymes and both the full-length and simplified forms of substrate A were prepared by automated solid-phase synthesis using 2′-TBDMS phosphoramidites. The RNAs were deprotected using a 1:1 mixture of aqueous ammonia and methylamine at 65 °C for 10 min, then lyophilized. Then, the 2′-TBDMS group was removed by incubation with tetrabutylammonium fluoride for 16 h. The resulting materials were mixed with an equal volume of 1 M Tris (pH 7.5) and desalted, and the RNAs were purified by PAGE.Synthetic d- and l-RNA forms of substrate B were prepared by automated solid-phase synthesis using 2′-TOM phosphoramidites. For substrates containing the TQ3 quencher, synthesis was carried out on a 2′-O-propargyl uridine CPG column. The substrates were 5′-triphosphorylated on the column, based on a modification of previous methods [t-butyl peroxide in hexanes for 15 min. The resulting material was washed with 500 µL of dry ACN and allowed to react with dry 3.5 M of 6-(Boc-amino)-1-hexanol in ACN for 72 h [Both the  methods ,14 SO4, 2 mM of Tris(3-hydroxypropyltriazolylmethyl)amine, 10 mM of sodium ascorbate, 50 mM of Na2HPO4 (pH 7.4), and 10% dimethyl sulfoxide, which was incubated at 23 °C for 2 h. The resulting materials were purified by PAGE and ethanol precipitation.The oligonucleotide was deprotected as described above, and the 5′-amine was then labeled with TMR-TAATACGACTCACTATAGGAGCGAGAACGTTGT-N7-TGTTACGTAAGTAACA-N9-GTTGAAGTATGAGACCGGAAGGTTGAAGTATGAGACCGCAACGTA . The DNA was made double-stranded by extending a primer having the sequence 5′-TACGTTGCGGTCTCATACTTCAACCTTCCGGTCTCATACTTCAACC-3′; then, 1.7 × 1013 DNA molecules (282 pmol) were used to transcribe the corresponding pool of RNAs. These were cleaved using M1 RNA and an external guide RNA having the same sequence as described above for the processing of substrate B. Following purification by PAGE, 6.0 × 1012 RNA molecules (100 pmol) were mixed with TMR-B-biotin substrate and 50 mM of EPPS (pH 8.5), heated to 70 °C for 2 min, and then cooled to 4 °C over 3 min. The mixture was then heated to 42 °C, and the ligation reaction was initiated by adding MgCl2 and then quenched by adding 50 mM of EDTA. During the first round of selection the reaction mixture contained 2 µM of TMR-B-biotin, 1 µM of E-A, and 25 mM of MgCl2, and the reaction time was 1 h. During rounds 2 and 3, the concentration of reactants was the same, and reaction time was reduced to 7.5 min in round 2 and to 30 s in round 3. During rounds 4 and 5, the reaction mixture contained 50 nM of TMR-B-biotin and 50 nM of E-A, the concentration of MgCl2 was 25 mM in round 4 and 5 mM in round 5, and the reaction time was 30 s in round 4 and 15 s in round 5.The in vitro selection procedure was based on a previously reported method . The staTAATACGACTCACTATAGGAGCGAGAACGTTGT-3′ (T7 RNA polymerase promoter sequence underlined). The amplified DNA was in vitro transcribed, and the resulting RNA was purified by PAGE and then used to initiate the next round of selection.The ligated products were captured on a streptavidin-agarose resin and washed successively with the following: 10 mL of a solution containing 100 mM of NaCl, 1 mM of EDTA, and 50 mM of Tris (pH 7.5); 20 mL of a solution containing 1 M of NaCl, 1 mM of EDTA, 0.1% Triton X-100, and 50 mM of Tris (pH 7.5); 10 mL of a solution containing 100 mM of NaCl, 1 mM of EDTA, and 50 mM of Tris (pH 7.5); and 20 mL of a solution containing 1 mM of EDTA and 10 mM of Tris (pH 7.5). The ligated RNA was reverse transcribed on the resin by extending a primer having the sequence 5′-TAAGTTGCGGTCTC-3′. The DNA (as an agarose slurry) was then PCR amplified using the same reverse primer and a forward primer having the sequence 5′-GGCtheo), and 50 mM of EPPS (pH 8.5), which were heated at 70 °C for 2 min and then cooled to 4 °C over 3 min. The mixture was then heated to 42 °C, and the reaction was initiated by adding MgCl2 to a final concentration of 25 mM. Aliquots were taken from the mixture at various times. The products were analyzed by PAGE, and imaging fluorescence with a BioRad Pharos FXPlus Molecular Imager. For the real-time assays, the reaction mixture was monitored continuously using a BioRad CFX-96 Real-Time PCR Detection System.Ligation was carried out by first mixing 5 µM of substrate A, 2 µM of substrate B , 6 µM of enzyme , which were heated to 70 °C for 2 min and then cooled to 4 °C over 3 min. The mixture was then heated to 42 °C, and the reaction was initiated by adding MgClA self-replicating RNA enzyme, which can undergo exponential amplification in a ligand-dependent manner, was modified to generate a fluorescent signal throughout the course of amplification. This modification entailed both derivatization of the leaving group with a fluorescent reporter and optimization of the enzyme to accommodate that reporter. The result is a novel way to measure the concentration of various target ligands with high sensitivity and specificity.


While overexpression of TINCR had opposite effect. In addition, we also found that TINCR specifically bound to EpCAM through RNA IP and RNA pull down assays. Loss of TINCR promoted hydrolysis of EpCAM and then released EpICD, subsequently, activated the Wnt/β-catenin pathway. Further studies shown that c-Myc repressed the expression of TINCR through repressing sp1 transcriptive activity, which established a positive feedback loop controlling c-Myc and TINCR expression. These findings elucidate that loss of TINCR expression promotes proliferation and metastasis in CRC and it could be considered as a potential cancer suppressor gene.Long non-coding RNAs (lncRNAs) are involved in kinds of human diseases, including colorectal cancer (CRC). TINCR, a 3.7 kb long non coding RNA, was associated with cell differentiation in keratinocyte and gastric cancer cells. However, little is known about the role of TINCR in regulation CRC progression. Here, we showed that lncRNA TINCR was associated with CRC proliferation and metastasis. TINCR was statistically downregulated in CRC tissues and metastatic CRC cell lines compared with their counterparts. TINCR was reversely correlated with CRC progression and promoted tumor cells growth, metastasis  Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females worldwide . The estLncRNAs, a subgroup of non-protein coding transcripts, ranging in length from 200 bp to tens of kilobases (kb), and accumulating evidences have shown that lncRNAs may promote the formation and progression of colorectal cancer –6. FirstTerminal differentiation-induced lncRNA (TINCR) is a 3.7 kb lncRNA which is isolated from human well differentiated somatic tissues. TINCR interacts with a range of differentiation mRNAs though a specific motif. TINCR also binds to the stau1 protein and mediates the stabilization of differentiation mRNAs , 14. TINIn this study, we demonstrated that lncRNA-TINCR which associated with tissues differentiation, was downregulated in the colorectal cancer tissues and inhibited CRC cell proliferation, invasion and metastasis. TINCR specifically bind to EpCAM, and loss of TINCR accelerating the hydrolysis of EpCAM and released EpICD, then activate the EpICD-Wnt/β-catenin signaling in CRC. We also revealed that c-Myc could repress the expression of TINCR by a sp1-dependent way. This study provides a novel mechanism for TINCR in proliferation, migration, and metastasis in CRC.p < 0.0001)  Figure  and 1B.p = 0.001), lymph metastasis (p = 0.037), and tumour node metastasis (TNM) classification (p = 0.016), while positively correlated with differentiation degree (p = 0.017). Moreover, the expression level of TINCR in the high malignant potential cell lines LoVo, RKO, and SW620 was significantly decreased compared with low malignant potential cell lines HCT116, SW480 and LS174T , while increased the proportion of cells in S phase (from 20.52% ± 0.97 to 51.97% ± 1.68). The overexpression of TINCR drove progression beyond the G2/M transition in RKO cell lines. The percentage of cells in G2/M phase in RKO/oe-TINCR (14.51% ± 1.35) was significantly higher than that in RKO/Vector cells (8.59% ± 0.87) Figure . The uprEpCAM was one of the disrupted genes when TINCR was depleted and had the highest score by transcript profiling . RNA immp = 0.005, r = −0.478, Figure As shown in Figure EpICD is one of the components of Wnt pathway. EpICD colocalized with FHL2 and β-catenin to form a nuclear protein complex, leading to gene transcription . ConsistPotential transcription factors (TFs) promoting the expression of TINCR were analyzed by bioinformatics algorithms. Sp1 was the predicted TF with one binding site on the TINCR promoter showed by the JARSPAR, and PROMO databases Figure . An obviin vitro by causing cell cycle arrest and inducing apoptosis. However, TINCR is strongly upregulated in human gastric carcinoma (GC) and promoted cell growth [Accumulating evidence indicates that the abnormal expression of lncRNAs is a frequent molecular event in human malignances including CRC , 21. In l growth . The incThe molecular mechanisms of TINCR on cell proliferation remain poorly identified. It reported that lncRNAs could act together with specific proteins when performing its functions –24. In tSp1 promotes a variety of cancer associated genes that are implicated in cell growth, differentiation, and apoptosis –33. We cTaken together, our results indicate that TINCR is a tumour suppressor that, when downregulated, may promote CRC growth and metastasis, and accelerate hydrolysis of EpCAM. With regard to the fantastic potential applications of targeted drugs for EpCAM, this finding suggests the considerable therapeutic value of TINCR for CRC.2 at 37°C according to the recommendation by ATCC.Total 44 human colorectal cancer tissues and their paired normal colorectal mucosa were obtained from Nanfang Hospital, Southern Medical University . Patients and/or their relatives approved the use of these clinical materials for this research according to the Ethics Committee of Southern Medical University. All tissues were frozen in liquid nitrogen immediately after surgical exeresis, and then stored at −80°C. The CRC cell lines used in this research were obtained from ATCC, and cultured in RMPI-1640 medium containing 10% FBS in COThe full length of TINCR, psen2, c-Myc and Sp1 were amplified by PCR using cDNA from non-tumour colorectal mucosal tissues, and cloned into pcDNA3.0 vectors respectively. The pcDNA3.0-anti-TINCR was generated with the antisense of TINCR. The TINCR promoter DNA fragment containing the wild-type (wt) or mutant (mut) putative target site of the transcription factor Sp1 were subcloned into the pGL3-basic vectors. All the PCR primers were collected in RNA isolation, qRT-PCR and western blotting were performed as previously described –36. The The cell proliferation, plate colony formation, cell migration, invasion assays and Flow Cytometry were performed as previously described .5/well) and cultured for 24 hours. Then, either pGL3-wt promoter vectors or pGL3-mut promoter vectors were co-transfected with pcDNA3.0-sp1 plasmids in HCT116 and SW480 cells using lipofectamine 3000 . The control luciferase plasmids pRL-TK was also transfected. Luciferase activity was measured 48 hours after transfection according to the Dual-luciferase Reporter Assay System .Cells were seeds in 24-wells plates . Briefly, 1 × 10promoter .6 cells were lysed by RIP lysis buffer, immunoprecipitate with EpCAM antibody or lg G antibody with protein A/G magnetic beads overnight. The immobilized magnetic beads bound complexes were collected and then extract RNA. PCR was performed with primers specific for TINCR promoter (RNA immunoprecipitation (RIP) experiments were performed according to the instruction provided by the kit . 1 × 10promoter .™ RNA 3′ End Desthiobiotinylation Kit .To synthesize TINCR and anti-TINCR transcripts, RT-PCR was conducted using forward primers containing the T7 RNA polymerase promoter sequence. PCR products were purified according to the protocol of Biomiga DNA gel/PCR Extraction Kit . TINCR and anti-TINCR were biotinylated according to the protocols of T7 RNA polymerase kit  and Pierce™ Magnetic RNA-Protein Pull-Down Kit . Briefly, labeled TINCR or anti-TINCR was bound to streptavidin magnetic beads, and then incubated the RNA with cell lysate. RNA binding protein complexes were washed and eluted. Western blotting was performed with specific antibody for EpCAM.RNA pull down assay was conducted according to the instruction of Pierce6 HCT116-NC or HCT116-shTINCR cells per animal were subcutaneously injected into the BALB/c nude mice respectively. The size of the tumors was measured by a caliper as length × width2 × 1/2 every 2 days. A total of 2 × 106 HCT116-NC or HCT116-shTINCR cells per animal were also injected into the tail veins of the mice, which were sacrificed 6 weeks after injection. The lungs and livers were removed and photographed, and visible tumors on the lungs and livers surface were counted. For histology and immunostaining experiments, subcutaneous tumours, lungs, livers, and the other visceral organs were excised, fixed in formalin, paraffin embedded in paraffin. Sections from the tumours and visceral organs were stained with hematoxylin and eosin (H & E) for general histology, or immunostained with specific antibodies (anti-Ki67 for proliferation) [Four to 5 weeks old athymic nude mice were obtained from the Central Laboratory of Animal Science, Southern Medical University. All experimental procedures involving animals were performed in accordance with animal protocols approved by the Animal Care and Use Committee of Southern Medical University. A total of 2 × 10eration) .t test were used for comparisons independent experimental groups. The spearman correlation coefficient was used to assess the degree of the linear relationship between the expression level of TINCR and EpCAM in colorectal cancer tissues. One asterisk, two asterisks, and three asterisks implied p < 0.05, p < 0.01, and p < 0.001, respectively.Statistical analysis was carried out using SPSS 19.0 to assess the differences between experimental groups. Quantitative data were presented as mean ± SD. One-way ANOVA or the independent samples


We evaluated the impact of CAP exposure on the FCV viral capsid protein and RNA employing several cultural, molecular, proteomic and morphologic characteristics techniques. In the case of long exposure (2 min) to CAP, the reactive species of CAP strongly oxidized the major domains of the viral capsid protein (VP1) leading to disintegration of a majority of viral capsids. In the case of short exposure (15 s), some of the virus particles retained their capsid structure undamaged but failed to infect the host cells in vitro. In the latter virus particles, CAP exposure led to the oxidation of specific amino acids located in functional peptide residues in the P2 subdomain of the protrusion (P) domain, the dimeric interface region of VP1 dimers, and the movable hinge region linking the S and P domains. These regions of the capsid are known to play an essential role in the attachment and entry of the virus to the host cell. These observations suggest that the oxidative effect of CAP species inactivates the virus by hindering virus attachment and entry into the host cell. Furthermore, we found that the oxidative impact of plasma species led to oxidation and damage of viral RNA once it becomes unpacked due to capsid destruction. The latter effect most likely plays a secondary role in virus inactivation since the intact FCV genome is infectious even after damage to the capsid.Possible mechanisms that lead to inactivation of feline calicivirus (FCV) by cold atmospheric-pressure plasma (CAP) generated in 99% argon-1% O The standard curve was generated from average Ct values of three measurements per dilution. At each real-time RT-qPCR measurement, a standard curve  was generated under the same conditions as the tested samples.The real-time RT-qPCR primer set [rRT-forward (ere used  as descr5’-TAC GCA TCA AGG TTT AGG CC-3’) and FCV-R5 (5’-CTC AGT ATG TGC TGG CGA AA-3’)] was used for conventional RT-PCR. The primers were designed to cover the region from nucleotide 2870 to 3850 of the viral genome with amplicon size of 980bp. Amplification reactions (25 μl reaction mixture) were performed in an Eppendorf thermocycler  under the following conditions: reverse transcription step was carried out at 50°C/30 min 95°C /15 min followed by 40 cycles of 1 min at 94°C (denaturation); 1 min at the annealing temperature corresponding to the used primer  of sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE). The peptide bands on the gel were stained with Imperial Protein Stain . Two gel regions from each lane (an upper region ~ 50–74 kDa and a lower region ~27–43 kDa) were excised followed by in-gel trypsin digestion using a trypsin solution  [Triplicate samples of plasma-exposed and unexposed FCV were separated on 4–15% gradient (Criterionhloride] . The hydhloride] .Triplicate samples of peptides (~1 μg) were injected and analyzed by capillary liquid chromatography (LC)-mass spectrometry (MS) on an OrbitrapVelos MS system  using MS1 (survey) scan mode. The parameters of the MS analysis and LC gradient profile are included in Label-free relative quantification of analytes/peptides between control and exposed samples was done with in-RIPPER software . Brieflyi) an RNA sample (referred to as “packed RNA”) in which, the RNA was isolated from FCV after the virus was exposed to CAP for 15 s and 2 min, ii) an RNA sample (referred to as “unpacked-RNA sample”) isolated in pure form from FCV and then exposed to CAP directly for 15 s and 2 min while it was unprotected by capsid protein, and iii) a control (unexposed to plasma) RNA sample.Viral genome integration test (described below) was conducted to study the effect of CAP species on viral RNA. In this experiment we tested genome integration of three viral RNA samples, We studied the integration of FCV genome by comparing RT-PCR products of the CAP-treated unpacked viral RNA and CAP-treated packed viral RNA (RNA isolated from FCV after CAP treatment). RNA samples compared to CAP untreated RNA (control). Eleven primer pairs  coveringAliquots (100 μl each) of FCV suspended in sterilized Millipore water containing RNase inhibitor (10 U/ml), and aliquots (100 μl each) of pure RNA sample (unpacked RNA) isolated from FCV in sterilized RNase-free water containing10 U/ml RNase inhibitor were placed in 96-well plates (100 μl/well) and then exposed to plasma for 15, 30, 60, 120, 180, and 240s. Then packed RNA was extracted from CAP-exposed FCV samples. In both packed and unpacked RNA samples treated with CAP for different exposure times, One-Step RT-PCR reactions were carried out using QIAGEN® One Step RT-PCR Kit  and FCV primer set number 5 only  using Primer3 software. Amplification reactions (25 μl reaction mixture) were performed in an Eppendorf thermocycler  under the following conditions: reverse transcription at 50°C/30 min 95°C /15min, 40 cycles of 1 min at 94°C (denaturation); 1 min at the annealing temperature corresponding to the used primer (Aliquots (100 μl) of DW-suspended FCV were exposed to CAP for 2 min followed by incubation at room temperature for 2 hrs. Viral RNA was extracted and One-Step RT-PCR reactions were carried out to amplify directly the complete genome of plasma-exposed and unexposed FCV by primer walking using a set of specific primers  which wed primer , 1 min ahttps://genecodes.com) followed by BLAST analysis in GenBank database. The compatible nucleotide (nt) sequences were aligned by using the Clustal W option in MEGA 6.0  to obtain a consensus sequence. The variations in plasma-treated and untreated viral genome were analyzed by P distance option in Mega 6.0 based on  in FCV capsid protein sequence. The modifications at His202 and His354 were detected by MS/MS without quantification. The monoisotopic [M + 2H]2+ precursor peak 605.6633 m/z matched to peptides [H202HLAKQSLGPLLNPYL] and the monoisotopic precursor 768.3952 m/z matched to [H354WTDINGFAIRPFVFQANR] as seen in 1O2) species and was suggested to be a unique marker for singlet oxygen-mediated oxidative stress on proteins  (M248LQYPHVLFDARLAAIVVP(+28)PGVRP(+28)VQSTS], [M331LTHGSIPSDLIPKNSSLWIGNRFHLLKPPGS], and [M409DSVVPGIP(+28)DGWPDTTIPEKLGIGVA], respectively.The observed molecular mass of methionine residues  shifted to Met+16 Da, which was identified as methionine sulfoxide. The methionine sulfoxide (Met +16 Da) modifications were detected in the monoisotopic [M + 2H]HVLFDAR] ,  . This oxidative modification was detected in the monoisotopic M + 2H]383ITPR] . The detH383ITPR]2+ precursor peak at m/z = 809.1879, which matched with peptides [P237PGVRP242VQSTSM(+16)LQYPHVLFDARLAAIVV]. In addition, pyroglutamic acid (Gln -17 Da) was detected at Gln260 in peptide [Q260VH262PVIFSIPDLR], with monoisotopic [M + 2H]2+ peptide precursor peak at m/z = 759.4071. Pyroglutamic acid is an uncommon amino acid derivative in which the free amino group of glutamic acid or glutamine cyclizes to form a lactam. N-terminal glutamic acid and glutamine residues have been found to cyclize spontaneously forming pyroglutamate lutamate  indicatiCapsid-related functions such as virus assembly, antigenicity, and receptor interactions are mostly encoded in a single protein in the icosahedral capsid of the virus . Hence, The oxidative species of plasma not only affected the outer surface of the capsid  is a functional receptor on the host cells (CRFK) that attaches with FCV and plays a role in its entry into the host cell , 55. AlsFurther, the fJAM-A is known to engage the top of the P2 domain causing a rotation in the P dimer at the dimeric interface between the two capsid monomers . This isThe interaction of reactive oxygen and nitrogen species (RONS) with nucleic acids is a suggested mechanism of bacterial and viral inactivation by cold plasma exposure through interference with viral gene expression and viral RNA replication . It is tThe RT-PCR products from CAP-exposed packed-RNA showed agarose patterns identical to the control sample  and protrusion (P) domains as well as the dimeric interface regions of the major capsid protein (VP1) of FCV. This oxidation of the fundamental structure of FCV capsid leads to a loss of structural integrity and distortion of FCV virions. In addition, the short time exposure of virus to CAP leads to inactivation of the virus by oxidizing specific functional peptide residues located in P2 domain and NTA hinge that are known to play an important role in virus attachment and entry to the host cell. This indicates that short exposure to CAP may inactivate FCV by hindering virus binding to host cell even if it retained an intact capsid. Furthermore, the oxidative impact of plasma species leads to oxidize and damage the viral RNA once it becomes unpacked subsequent to capsid destruction. However, the former effect plays a less important role in virus inactivation since the intact FCV genome as well as other calicivirus are infectious even after uncoated from the capsid.S1 File(PDF)Click here for additional data file.S1 FigA) X-ray structure of FCV viewed along the icosahedral 2-fold axis. Location of a set of A/B and C/C dimers and icosahedral 5-fold and 3-fold axes are shown. B) Ribbon representation of the VP1 subunit structure. The NTA (green), S domain (blue), and P1 (red) and P2 (yellow) subdomains are indicated. Source: Ossiboff et al., 2+ observed precursors 605.6633 m/z matched to peptide QSLGPLLNPYLH202HLAK, PEAKS -10logP score 31.22. B) Monoisotopic [M + 2H]2+ observed precursor 768.3952 m/z matched to peptide H354WTDINGFAIRPFVFQANR PEAKS -10logP score 22.87. Theoretical b- and y-type fragment ion types matched to experimental product ion peaks are labeled (spectrum copied and pasted from PEAKS® Studio 7.0). The identified b and y ions are mapped onto the primary sequence and demonstrate modifications in the identified peptides. The peptide scores for the set of representative peptides have a false discovery rate of 0.5%.(TIF)Click here for additional data file.S1 SpreadsheetSpreadsheet A): Relative quantification of peptides between band 1 of control and 15s CAP-exposed samples. (Spreadsheet B): Relative quantification of peptides between band 2 of control and 15s CAP-exposed samples. (Spreadsheet C): Relative quantification of peptides between band 1 of control and 120 s CAP-exposed samples. (Spreadsheet D): Relative quantification of peptides between band 2 of control and 120 s CAP-exposed samples.((XLSX)Click here for additional data file.


To evaluate the incidence and associated risk factors of massive subretinal hemorrhage (SRH) in patients with retinal angiomatous proliferation (RAP).A total of 187 eyes of 135 treatment-naıve patients diagnosed with RAP were evaluated retrospectively. Clinical records including the time between the initial visit, last anti-vascular endothelial growth factor (VEGF) treatment, last stable examination, and the date of massive SRH were reviewed. Imaging findings including indocyanine green angiography (ICGA) and optical coherence tomography (OCT) were analyzed.Massive SRH developed in 18 eyes (9.6%) a median of 20 months after the initial presentation. Kaplan-Meier survival analysis revealed that the incidence  continuously increased. Among 14 eyes with discernable vascular anastomosis on baseline ICGA, 13 (92.8%) showed retinal arteriole involvement. On spectral-domain OCT imaging of the last visit prior to the massive SRH, a layered lamellar tissue complex was noted under the retinal pigment epithelium in 9 of 13 eyes, which was significantly associated with massive SRH. The average time between the last stable examination/last injection and the massive SRH was 2 and 5 months, respectively. The patients were treated with anti-VEGF, gas and recombinant tissue plasminogen activator injection; however, all except one eye had visual acuity worse than 20/1000 at the final visit.Massive SRH can occur in RAP in the course of anti-VEGF treatment, resulting in severe vision loss. A proactive dosing regimen may be more appropriate for these RAP eyes. Retinal angiomatous proliferation (RAP) is a subtype of neovascular age-related macular degeneration (nAMD) characterized by proliferation of deep retinal capillaries, with the later stages involving the progression from subretinal to choroidal neovascularization (CNV) with or without pigment epithelial detachment (PED)., 2 The pOn angiography, RAP lesions exhibit retinal-retinal anastomosis and retinal-choroidal anastomosis, which are thought to contribute to a high blood supply to the lesions. PossiblyAnother possible complication that can limit a good visual prognosis in nAMD is subretinal hemorrhage (SRH). SRH is a quite common finding in patients with nAMD and is associated with a worse visual prognosis, especially when massive subfoveal blood is present., 14 SheaWe reviewed medical records and images retrospectively of consecutive treatment-naïve patients diagnosed with RAP over a period of 10 years (between June 2005 to May 2015) at Seoul St. Mary’s Hospital , The Catholic University of Korea. This study received approval from the Seoul St. Mary’s Hospital of The Catholic University of Korea Institutional Review Board and was conducted in accordance with the Declaration of Helsinki. The institutional review board waived the need for a written consent from the participants, because of the retrospective design. Patient information was anonymized and de-identified prior to analysis.Each case was diagnosed as RAP based on characteristic features on funduscopy, fluorescein angiography (FA), indocyanine green angiography (ICGA), and optical coherence tomography (OCT), which included intraretinal hemorrhage, intraretinal vascular anastomoses, and the hyperreflective intraretinal vascular complex. Massive SRH was defined by subretinal and/or sub-RPE hemorrhages larger than a circle with a radius corresponding to the distance between the optic disc and fovea, and extending past the major vascular arcade of the retina.,22Information regarding the patient’s medical history and types and dates of treatment were collected along with clinical details of the massive SRH. The interval between the last treatment and massive SRH, and between the last stable examination and massive SRH, were analyzed. Snellen best-corrected visual acuity (BCVA), digital color fundus photographs, and OCT images were obtained at baseline and at each follow-up visit for all patients. OCT images were obtained using a Stratus  or Cirrus OCT (Carl Zeiss Meditec) before November 2011, and then with a Spectralis OCT . FA and ICGA , with a confocal scanning laser ophthalmoscope system, were performed at baseline and when required. Although Yannuzzi and associates classified the three stages of RAP, it is often difficult to differentiate whether the serous PED is associated with subretinal neovascularization (stage 2 RAP) or CNV (stage 3 RAP). Thus, in this study, RAP was classified as early stage (RAP without PED), which was equivalent to stage 1, or as advanced stage (RAP with PED) that included stages 2 and 3.3F8 gas were intravitreally injected, followed by an anti-VEGF injection within 1 week. Patients injected with gas were instructed to remain in the prone position for as long as possible . A pars plana vitrectomy was performed when a breakthrough vitreous hemorrhage occurred.Initially, treatment-naïve RAP eyes were treated with a combination of photodynamic therapy (PDT) and bevacizumab  followed by anti-VEGF injections with as-needed dosing [pro re nata (PRN)]) or anti-VEGF monotherapy from the beginning, based on PRN dosing with or without three loading injections. Both ranibizumab  and bevacizumab were used, and selection between the two drugs was determined mainly by considering the national health insurance coverage and reimbursement schedules. All patients were followed regularly at 1–3-month intervals, depending on the lesion activity, and retreatment was given whenever evidence of persistent/recurrent fluid, involving or threatening the fovea, was detected on follow-up OCT. For the treatment of massive SRH, patients received anti-VEGF injections with or without gas/recombinant tissue plasminogen activator (rt-PA) injection, and all decisions were at the discretion of a single physician (WK Lee). When gas/rt-PA injection was performed, 0.1 mL (50 μg) of rt-PA and 0.2 mL of 100% CThe proportion of patients who developed a massive SRH at each time point was analyzed with a Kaplan–Meier survival curve model using SPSS software . The date of initial treatment visit was designated as the index date. If a patient was lost to follow-up or did not present with a massive SRH, the latest visit was considered the last visit and it was regarded as censored data in the survival analysis. Cox regression analysis was performed to investigate the risk factors for massive SRH with clinical variables, such as age, hypertension, initial BCVA, use of PDT, and other funduscopic/OCT findings.During the study period, in total, 187 eyes of 135 patients were diagnosed with RAP and 104 patients were females (77.0%). The mean age at onset was 75.1 ± 6.3  years and baseline logMAR BCVA was 0.51 ± 0.46 . The mean follow-up period for all patients was 47.6 ± 29.0  months. During the follow-up period, 39 (20.9%) eyes received a combination of PDT and bevacizumab as the initial treatment and 148 (79.1%) eyes received anti-VEGF monotherapy. Baseline characteristics of the 187 enrolled eyes are shown in Among these eyes, RAP as a cause of massive SRH was diagnosed in 18 eyes of 17 patients; thus, the overall incidence of massive SRH was 9.6%. Massive SRH appeared after a median follow-up period of 20  months. Kaplan—Meier survival analysis revealed that 2.8% of eyes had developed massive SRH within the first year of the follow-up period. Thereafter, 5.8%, 13.1%, and 21.0% suffered massive SRH within 2, 5, and 10 years, respectively, during the follow-up period . Non-masThe median age of the 17 patients with massive SRH was 71  years. Of them, 3 patients were males and 14 were females. Six (35.3%) patients were on aspirin therapy and no patient was using any other kind of anticoagulation therapy. The median follow-up duration was 59  months for these patients. Bilateral involvement of RAP was noted in 14 eyes during the follow-up period, and GA without neovascularization was diagnosed in the contralateral eyes of the other four eyes. Macular soft drusen within the major vascular arcade were seen in all eyes and reticular pseudodrusen in 15 (83.3%) eyes. Based on the baseline examinations, all 18 eyes were in the advanced stages (RAP with PED).Although retinal-retinal anastomosis or retinal-choroidal anastomosis was found in all RAP lesions, their venous or arterial origin was detectable on baseline ICGA in 158 of 187 (84.5%) eyes. In the other 29 eyes, we failed to obtain enough images of the early filling sequence and/or the image quality was too poor to identify the origin of vascular anastomosis. Among 158 eyes, 75 (47.5%) showed retinal arteriole involvement on baseline ICGA and 83 (52.5%) showed involvement of retinal venules alone. The vascular component was detectable in 14 of 18 eyes with massive SRH. Among them, 13 (92.8%) eyes showed retinal arteriole involvement on baseline ICGA and one showed involvement of retinal venules only. The rate of retinal arteriole involvement was significantly higher in eyes with massive SRH .Two patients (two eyes) received combination therapy with PDT and bevacizumab as an initial treatment, and the intervals between the last PDT and SRH were 37 and 98 months, respectively. The other 15 patients (16 eyes) were treated with anti-VEGF monotherapy. The median number of injections before the massive SRH was eight , and the median time between the last injection and the massive SRH was 5 months . In two eyes, massive SRH occurred within 1 month after the last anti-VEGF injection . The other 16 eyes showed no sign of disease activity, such as macular hemorrhage or fluid, on the most recent examination and OCT before the occurrence of the massive SRH. The average time between the last stable examination and the massive SRH was 2  months in these 16 eyes.OCT at the last visit prior to the massive SRH showed widespread fibrovascular tissue under the RPE in 17 of 18 eyes, without any intraretinal/subretinal fluid in 16 eyes. Spectralis OCT was performed at this visit in 13 eyes, and interruption of the RPE line (atrophic RPE layer) was noted within an area of widespread fibrovascular tissue in all 13 eyes. A well-organized layered lamellar band was seen within fibrovascular tissue in 9 of 13 eyes. Representative cases are shown in Figs In total, 14 eyes received intravitreal anti-VEGF with gas/rt-PA injections, and four eyes were treated with anti-VEGF injections only. Treatment decision was made at the discretion of a single physician (WK Lee) considering the height of SRH under the fovea, visual acuity at the last visit prior to the massive SRH, and whether the patient can maintain a prone position. In 10 (55.6%) eyes, massive SRH resulted in vitreous hemorrhage, and these eyes underwent pars plana vitrectomy. During the follow-up period, RPE tears (a bare choroid with elevation or scrolling of the torn RPE flap on fundus photography and OCT), which were not seen initially with massive SRH, were noted in six eyes after the absorption of massive SRH .The median BCVA was 20/100 (range: 20/200–20/32) at baseline, decreasing to 20/200 (range: 20/1000–20/40) at the last visit prior to the massive SRH. All except one patient (case 10) had vision worse than 20/1000 at the final visit after the massive SRH. The clinical characteristics of the patients and the treatment results are summarized in the Among 187 eyes of 135 patients, 169 (92.0%), 4 (2.1%), and 11 (5.9%) eyes were analyzed with Spectralis, Cirrus, and Stratus OCT, respectively, until their final visit. Cox regression analysis of 169 eyes analyzed with Spectralis OCT revealed a statistically significant association of massive SRH with the layered lamellar tissue complex on OCT . No other variable, such as age, hypertension, baseline BCVA, use of PDT, reticular pseudodrusen, or fibrovascular tissue under the RPE at final visit, showed a significant association with the occurrence of massive SRH .This is the first study to report the incidence of massive SRH in eyes with RAP lesions (9.6%). The incidence increased steadily, from 2.8% at year 1 to >10% after the 5-year period of treatment and follow-up. In the 18 eyes of our study, massive SRH occurred at the advanced stages of the disease. All except one eye showed widespread fibrovascular tissue under the RPE at the visit prior to the massive SRH, indicating disease progression despite treatment primarily with anti-VEGF drugs . The medThe proposed mechanism of SRH in PCV involves the rupture of polyps, which may occur before any treatment by an increased tension within the polyps during the natural exudative course of the disease. In contrIt is noteworthy that retinal arterioles were involved in the RAP lesion in 13 of 14 (92.8%) eyes with discernable vascular anastomosis on ICGA. A limited number of studies have investigated the nature of the vascular anastomosis found in RAP.–28 The iThe use of intravitreal injections of anti-VEGF agents has resulted in a significant improvement of visual outcomes for patients with RAP; however, the optimal dosing regimen still remains to be determined. The most widely used dosing strategy in clinical practice involves a variety of as-needed regimens and treat-and-extend regimens.,31 An adOur study had several limitations, including its retrospective nature. The initial treatment regimens differed among patients and two patients received PDT initially. However, the intervals between the last PDT and SRH were too long in these patients to suggest a direct effect of PDT on massive SRH. Since we could not collect that data from all enrolled RAP patients, the use of antiplatelet and/or anticoagulation therapy was not included in Cox regression analysis. Although antiplatelet/anticoagulation drugs could be a risk of ocular hemorrhage, the association between such drugs and SRH in nAMD has not been established in large clinical studies yet. ,35 FinalTo our knowledge, this is the first report on the incidence of massive SRH with a large RAP-only cohort, and we collected a relatively large number of cases with massive SRH which was previously considered as an uncommon complication. We performed a survival analysis using follow-up data, which ranged up to 11 years from the initial presentation. The cumulative incidence rate of massive SRH in RAP increased steadily with time and massive SRH usually occurred during injection-free intervals, in the chronic, advanced stages of RAP. We assume that layered lamellar tissue complexes within fibrovascular tissue under the RPE and involvement of retinal arterioles were contributing factors in the development of massive SRH. Because the incidence of massive SRH in RAP is not rare, and the clinical course is devastating even after many different treatments, proactive dosing of an anti-VEGF agent may be a more appropriate approach in RAP eyes at high risk. Additionally, identification of the precise mechanisms, risk factors, and management of massive SRH in RAP should be investigated in future studies.


Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5’ and 3’ termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely. Bunyaviridae family, which causes severe hemorrhagic fever with a mortality of 30% in more than thirty countries worldwide  CTP was from Perkin Elmer. T7 transcription and RNA purification kits were from Promega. RNeasy and other RNA purification kits were from Qiagen. NiNTA beads were from Gold Bio. All other chemicals were purchased from Sigma.PCR primers were from Invitrogen. All other PCR reagents were from New England Biolabs and Genscript. The radioactive  CTP as previously reported  CTP during synthesis, as mentioned above. All binding reactions were carried out in RNA binding buffer  at a constant concentration of RNA with increasing concentrations of N protein. Reaction mixtures were incubated at room temperature for 30–45 min and filtered through nitrocellulose membranes under vacuum. Filters were washed with 10 ml of RNA binding buffer and dried. The amount of RNA retained on the filter at each input concentration of N protein was measured by quantifying the radioactive signal, using a scintillation counter. The background signal from nonspecific binding of RNA to the filter was subtracted from each data point. A binding profile was generated by plotting the radioactive signal along Y-axis and N protein concentration along X-axis. The percentage of bound RNA at each input concentration of N protein was calculated using the Interaction of bacterially expressed and purified CCHFV N protein with the RNA of interest Figs  was studmax is the same parameter when the RNA is totally bound to the N protein. Double reciprocal plot  was useΔRmax under the conditions where Cp >> initial concentration of RNA  denotes the input concentration of N protein, Kd is the dissociation constant and “n” represents the Hill coefficient. If n < 1 the system exhibits the negative cooperativity, if N > 1 the system exhibits the positive cooperativity, if n = 1, the system does not have the cooperativity.The binding profiles showing sigmoidal behavior were examined for cooperativity using the Hill equation  31,32].,32.31,32We used mFold to analyze the most probable secondary structures of RNA molecules shown in Figs 32P] CTP labeled RNA and increasing concentrations of cold competitor RNA of interest. The reaction mixture was incubated at room temperature for 45 min and then filtered through nitrocellulose filters under vacuum. The bound radiolabeled RNA retained on the filter at each input concentration of the competitor RNA was measured using scintillatation counter. Percent radiolabeled RNA bound to the protein was plotted against input competitor RNA concentration.Competition experiments were performed using filter binding assay as previously reported . A constKd), the fluorescence intensity value at 334 nm at each input concentration of the ligand was recorded. The binding profile was generated by plotting the fluorescence value at 334 nm along the Y axis and ligand concentration along the X axis. The percentage of N protein bound to the RNA of interest at each input concentration of the RNA was calculated from The binding of wild type vRNA panhandle, 5’ and 3’ NCR sequences with CCHFV N protein was carried out in Shimadzu spectrofluorometer RF-5301PC as previously reported ,29. Fluomax is the same parameter when N protein is totally bound to the RNA. Double reciprocal plot  was useΔFmax is valid under the conditions where Cp>> initial concentration of N protein, only the saturating data points of the binding profile were fitted to ΔFmax. A plot of percent bound N protein versus RNA concentration was used for the calculation of apparent dissociation constant (Kd), which corresponds to the concentration of RNA required to obtain half saturation, assuming that complex formation obeys a simple bimolecular equilibrium.Since F0/F = 1 + Ksv[Q], where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively, [Q] is the concentration of the quencher, and Ksv is the Stern-Volmer quenching constant. Small aliquots (1–5 μl) of acrylamide from concentrated stock solutions (3M-10M) were added to 300 μl of N protein at a concentration of 250 nM in the binding buffer, and the tryptophan fluorescence signal at 334 nm was recorded at each input concentration of acrylamide. The total volume of added acrylamide solution was less that 10% the total volume of N protein solution in the cuvette. Similar quenching studies were carried out with N protein that was pre-incubated with the desired concentration of RNA of interest. The data points in a plot of F0/F versus [Q] fit to a single straight line, except when N protein was simultaneously bound to both the vRNA panhandle and single strand 3’ or 5’ NCR. Fitting of data points to a single straight line indicates a single species of tryptophan residues that remain equally accessible to the neutral quencher. The Stern-Volmer quenching constant (Ksv) was calculated from the slope of the straight line fitted to the data points as reported previously , where Kapp is the apparent binding constant, K0 is the intrinsic binding constant and n is the binding stoichiometry, a possible difference in reported binding affinities due to potential variation in binding stoichiometries cannot be ruled out. It is evident from luc RNA 5’ del and Gluc RNA 3’ del show a sigmoid behavior. This sigmoidal behavior was also observed with gel purified Gluc RNA 3’ del  before and after binding to the RNA of interest, as described in materials and methods. As shown in We next employed bis-ANS as probe to monitor the difference in conformational changes, if any, induced by the binding of vRNA panhandle, single strand 5’ or 3’ NCR or the simultaneous binding of both vRNA panhandle and single strand RNA. The increase in the fluorescence signal of bis-ANS observed upon association with N protein is due to the binding of probe to the hydrophobic pockets of the protein. The fluorescence signal of bis-ANS increased with similar high magnitude upon binding to the preformed complex of N protein with either 5’ or 3’ NCR . This suSince N protein bound the vRNA panhandle with high affinity, we asked whether panhandle remains double stranded after association with N protein. To rule out the possible melting of vRNA panhandle we examined the RNA dissociation activity of N protein, as previously reported . We haveluc RNA harboring the vRNA termini, suggesting that non-coding vRNA termini are sufficient for high affinity binding. Deletion of either 5’ or 3’ terminus impacted the binding, indicating that panhandle structure might be important for the high affinity binding with the N protein. Further analysis by two independent experimental approaches confirmed that N protein bound to the panhandle structure, composed of 32 nucleotides from both 5’ and 3’ termini of S-segment vRNA, with same affinity as full length vRNA (Kd ~42 nM). Further characterization demonstrated that terminal nucleotides of the panhandle play an important role and the primary sequence of the panhandle is required for high affinity binding  demonstrated a racket-shaped overall structure with distinct “head” and “stalk” domains. The structure significantly differs from other nucleoproteins reported from other negative-sense RNA viruses. We modeled the 3D structure of CCHFV N protein (strain 10200). Although, the primary sequences of these two strains are slightly different, the modeled structure of strain 10200 is very similar to the X-ray structure of strain YL04057 . The eleIn vitro binding studies have previously used bacterially expressed and purified Bunyaviridae N protein to examine its specific binding with the synthetic viral genomic RNA [Bunyaviridae N protein appears to preferentially interact with a target site near the 5’ end of the vRNA, proposing that vRNA 5’ terminus might have cis-acting encapsidation signals. However, the recent studies have proposed that nascent vRNA is co-transcriptionally encapsidated. Recently, the x-ray and cryo-EM studies of La Crosse virus have provided insights about the location of important functional sites in the Bunyaviridae RdRp [luc RNA 3’ del : A 6% acrylamide urea gel showing four RNA molecules that were purified by gel extraction using denaturing PAGE (B): Binding profiles for the interaction of N protein with RNA molecules shown in panel A.(TIF)Click here for additional data file.S2 FigA 14% acrylamide urea gel showing three radiolabled RNA molecules that were tested for N protein binding using the filter binding assay (A). Binding profiles for the interaction of N protein with the 5’ NCR (B), 3’NCR (C) and wild type panhandle (D) form panel A are shown. The binding profiles were generated by filter binding assay.(TIF)Click here for additional data file.S3 Fig(A). A 14% acrylamide urea gel showing six radiolabled RNA molecules. The sequence of these RNA molecules is shown in (TIF)Click here for additional data file.


Coronavirus HKU15 is a deltacoronavirus that was discovered in fecal samples of pigs in Hong Kong in 2012. Over the past three years, Coronavirus HKU15 has been widely detected in pigs in East/Southeast Asia and North America and has been associated with fatal outbreaks. In all such epidemiological studies, the virus was generally only detected in fecal/intestinal samples. In this molecular epidemiology study, we detected Coronavirus HKU15 in 9.6% of the nasopharyngeal samples obtained from 249 pigs in Hong Kong. Samples that tested positive were mostly collected during winter. Complete genome sequencing of the Coronavirus HKU15 in two nasopharyngeal samples revealed quasispecies in one of the samples. Two of the polymorphic sites involved indels, but the other two involved transition substitutions. Phylogenetic analysis showed that the two nasopharyngeal strains in the present study were most closely related to the strains PDCoV/CHJXNI2/2015 from Jiangxi, China, and CH/Sichuan/S27/2012 from Sichuan, China. The outbreak strains in the United States possessed highly similar genome sequences and were clustered monophyletically, whereas the Asian strains were more diverse and paraphyletic. The detection of Coronavirus HKU15 in respiratory tracts of pigs implies that in addition to enteric infections, Coronavirus HKU15 may be able to cause respiratory infections in pigs and that in addition to fecal-oral transmission, the virus could possibly spread through the respiratory route. The presence of the virus in respiratory samples provides an alternative clinical sample to confirm the diagnosis of Coronavirus HKU15 infection. Quasispecies were unprecedentedly observed in the 5′-untranslated region of coronavirus genomes. Alphacoronavirus, Betacoronavirus and Gammacoronavirus, respectively.1 In the same year, we discovered three novel CoVs in avian cloacal swabs.2 These CoVs formed a distinct novel CoV genus, named Deltacoronavirus.1 Subsequently, in a large epidemiological study, we discovered seven additional deltacoronaviruses.3 Interestingly, one of these deltacoronaviruses, which was originally named porcine CoV HKU15, was found in fecal samples of pigs in Hong Kong, and it is the only mammalian deltacoronavirus.3 In 2016, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses rectified the species name for this virus to Coronavirus HKU15.4Coronaviruses (CoVs) are found in a wide variety of animals, in which they can lead to enteric, hepatic, neurological and respiratory illnesses of differing severity. On the basis of genotypic and serological characterization, CoVs were traditionally divided into three distinct groups. In 2009, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses replaced the traditional CoV groups 1, 2 and 3 with three genera, Coronavirus HKU15 has been widely detected in pigs in East/Southeast Asia and North America and was found to be associated with fatal outbreaks.23 In these epidemiological studies, Coronavirus HKU15 was generally only detected in fecal/intestinal samples. However, some CoVs, such as transmissible gastroenteritis CoV (TGEV)/porcine respiratory CoV (PRCV) of Alphacoronavirus 1 and bovine CoV of Betacoronavirus 1, could be detected consistently in both fecal and respiratory samples.25 Therefore, we hypothesized that Coronavirus HKU15 may also be present in respiratory samples of pigs, which has implications for its transmission and potential role in respiratory diseases and for the use of nasopharyngeal sampling as an alternative method of identifying infected pigs. To test this hypothesis, we performed a molecular epidemiology study on nasopharyngeal samples collected from pigs in Hong Kong. Two complete genomes of the ‘respiratory’ Coronavirus HKU15 were sequenced, and comparative genomic and phylogenetic studies were performed. The implications of the presence of Coronavirus HKU15 in respiratory samples are also discussed.Over the past three years, Nasopharyngeal samples from pigs were collected in Hong Kong over a 26-month period (January 2012–February 2014). These samples were obtained from slaughterhouses and pig farms in Hong Kong with the assistance of the Veterinary Public Health Section, Food and Environmental Hygiene Department; and the Agriculture, Fisheries and Conservation Department of the Government of Hong Kong. Immediately after sample collection, each nasopharyngeal swab was submerged in viral transport medium for viral transport and maintenance.Viral RNAs were extracted from the nasopharyngeal samples of pigs by using 200 μL of inoculated viral transport medium for each sample and by utilizing the EZ1 Advanced XL system  and EZ1 Virus Mini Kit v2.0 (Qiagen) according to the manufacturer’s protocol with RNase-free water as the eluent. Reverse transcription (RT) was performed using the SuperScript III Reverse Transcriptase  according to the manufacturer’s protocol by random priming.Coronavirus HKU15 was performed by polymerase chain reaction (PCR) amplifying a 289-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene, using the specific primer pair LPW14077 (5′-ACA CAC TTG CTG TAA CCA AA-3′) and LPW14080 (5′-ATC ATT AGA GTC ACC ACG AT-3′). PCR and DNA sequencing were carried out following our previous publications with slight modification.27 Briefly, each PCR mixture contained PCR buffer , 200 μM of each deoxynucleoside triphosphate , 1 μM of each primer (Invitrogen), 1.0 U of AmpliTag Gold DNA polymerase (Applied Biosystems) and cDNA. The mixtures were subjected to 60 thermocycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min, with an initial denaturation at 95 °C for 10 min and a final extension at 72 °C for 10 min for DNA amplification using the GeneAmp PCR System 9700 automated thermal cycler (Applied Biosystems). Standard precautions were taken to avoid contamination, and no false-positive result was observed for the negative controls. PCR products were agarose gel-purified using the QIAquick Gel Extraction kit (Qiagen). Both strands of the PCR products were sequenced twice by the ABI Prism 3130xl Genetic Analyzer (Applied Biosystems) using the two PCR primers.Detection of Coronavirus HKU15 strains detected in the nasopharyngeal samples of two different pigs were sequenced following our previous publications27 with modifications. Briefly, viral RNAs were converted to cDNAs using SuperScript III reverse transcriptase with a combined random priming and oligo(dT) priming strategy. The cDNAs were PCR-amplified by primers  according to the manufacturer’s protocol, and DNA sequencing was performed as mentioned above. When ambiguous peaks were observed consistently in the electropherograms after several attempts of PCR-sequencing for certain genomic regions, cloning followed by plasmid sequencing was performed according to our previous publication,28 except the Zero Blunt TOPO PCR Cloning kit (Invitrogen) was used to resolve the sequence ambiguities. PCRs using recombinant plasmids as templates were also performed to confirm that indels at mononucleotide polymeric regions were not the result of polymerase slippage. The 5′ ends of the viral genomes were amplified and sequenced by the rapid amplification of cDNA ends (RACE) using the SMARTer RACE 5′/3′ kit  according to the manufacturer’s protocol with the reverse primers LPW15379 (5′-TGG GTA ATG TGT CCG CTG ACG GGC GGT G-3′), LPW15380 (5′-AGA AGT GGT GGA TGG TCA GAG GAA CGG T-3′) and LPW16265 (5′-GTG GCT GGT TTC CAG GTA ATC TA-3′). The sequences of the PCR products were then assembled manually to obtain the genomes of the two nasopharyngeal strains.The genomes of two  primers  that wer29 or EMBOSS Stretcher (Nucleotide Alignment);30 whereas multiple sequence alignment was performed using MUSCLE 3.8,31 where the aligned sequences were further manually inspected and edited. Tests for substitution models and phylogenetic analysis by the maximum likelihood method were performed using MEGA 6.0.6.32Pairwise alignment was performed using BioEdit 7.2.5 Coronavirus HKU15 strains were calculated based on the complete genome sequence data, utilizing the Bayesian Markov chain Monte Carlo method using BEAST 1.8.033 with the substitution model GTR +G (gamma-distributed rate variation)+I (estimated proportion of invariable sites), a strict molecular clock, and a constant coalescent. Fifty million generations were run with trees sampled every 1000th generation to yield 50 000 trees. Convergence was assessed based on the effective sampling size after a 10% burn-in using Tracer 1.6.0. The mean time to the most recent common ancestor (tMRCA) and the highest posterior density (HPD) regions at 95% were calculated. The trees, after a 10% burn-in, were summarized as a single tree using TreeAnnotator 1.8.0 by choosing the tree with the maximum sum of posterior probabilities (maximum clade credibility) and viewed using FigTree 1.4.0.Divergence times for the Coronavirus HKU15 strains were deposited into the International Nucleotide Sequence Databases with accession numbers LC216914 and LC216915.The complete genome sequences of the two nasopharyngeal Coronavirus HKU15 was positive in 24 (9.6%) of the 249 nasopharyngeal samples. The samples that tested positive were mostly collected during winter (December–March)  . DNA seqng Kong3 .Coronavirus HKU15 found in two of the positive nasopharyngeal samples (S579N and S582N). Excluding the 3′ poly(A) tail, the genomes of S579N and S582N were 25 411–25 413 and 25 397 nucleotides long, respectively. The genome organization of the two strains was the same as that of other Coronavirus HKU15 strains. The lengths of the seven open reading frames (ORFs) of the two strains S579N/S582N were 18 803/18 788, 3480, 252, 654, 285, 1029 and 603 bp, respectively. The genomes of the two strains possessed 98.9%–99.2% sequence identity to that of the representative isolate HKU15-155. Quasispecies were detected in one of the samples (S579N) at the 5′ genomic region via two independent nested PCRs targeting the 122nd–505th bases of the genome using two different primer pairs for the first round and the same primer pair for the second round of reaction. For S579N, direct sequencing of the PCR products yielded ambiguous peaks in the sequencing electropherograms, which could only be resolved after cloning  substitutions per site per year, which is approximately 1.7-fold higher than that estimated in a previous study.19 The root of the tree was September 1638 (95% HPD: June 1570–March 1698). The tMRCA of the diversity of Coronavirus HKU15 was dated to June 1991 (95% HPD: November 1987–June 1994); and the tMRCA of the Thai/Laos strains was traced back to September 2014 (95% HPD: May 2014–January 2015). The tMRCAs for the clade containing US/Korean strains was estimated to be October 2012 (95% HPD: June 2012–January 2013), which is slightly delayed compared with that estimated in a previous study.19 For the two nasopharyngeal strains characterized in this study (S579N and S582N), they were estimated to have diverged from their respective MRCAs in December 2011 (95% HPD: May 2011–July 2012) and August 2009 (95% HPD: November 2008–April 2010), respectively , mesenteric lymph node (n=2) and saliva (n=10)/oral fluid (n=73) of pigs,20 implying that Coronavirus HKU15 can cause systemic infections in occasional cases. In this study, Coronavirus HKU15 was found in 9.6% of the nasopharyngeal samples of pigs, which is similar to the 10.1% positive rate of Coronavirus HKU15 in fecal samples of pigs that we reported previously.3 Seasonal variation in the detection rate of Coronavirus HKU15 from pigs was noted, where most of the positive samples were collected in winter. This is similar to the pattern of seasonal variation in a surveillance study carried out in the United States, where the detection rate for Coronavirus HKU15 was much lower during summer.19 It has recently been confirmed that Coronavirus HKU15 is able to cause swine enteric infections by infecting gnotobiotic and conventional pigs with Coronavirus HKU15.15 The detection of Coronavirus HKU15 in respiratory tracts of pigs has the following implications. First, in addition to enteric infections, Coronavirus HKU15 may be able to cause respiratory infections in pigs. Second, in addition to fecal-oral transmission, the virus may be able to spread through the respiratory route. Third, the presence of the virus in respiratory samples provides an alternative clinical sample to confirm the diagnosis of Coronavirus HKU15 infection. Further studies will determine the full spectrum of clinical diseases and pathologies associated with Coronavirus HKU15.Coronavirus HKU15 may possess similar properties. A number of animal CoVs possess dual or multiple tissue tropisms. For example, TGEV, which is another enteropathogenic CoV that infects pigs, could also be found in the nasopharynx of pigs as PRCV, which is a deletion mutant of TGEV.25 Moreover, bovine CoV is both an enteric and a respiratory pathogen in cattle.24 Similar to TGEV/PRCV, Coronavirus HKU15 is recovered from both respiratory and gastrointestinal samples. However, unlike TGEV/PRCV, in which there is a 621–681 nucleotide deletion at the 5′ end of the spike (S) gene leading to a loss of 1–2 antigenic sites in PRCV,34 comparative genome analysis of Coronavirus HKU15 from respiratory and fecal samples did not show any obvious difference in their S proteins or other parts of their genomes. Phylogenetic analysis also did not reveal a separate clustering of fecal/intestinal and nasopharyngeal isolates . These two indels were genuine variant sites instead of being due to polymerase slippage during the amplification process because recombinant plasmid-dependent PCR–sequencing no longer resulted in sequence ambiguities in the electropherograms. Although the existence of quasispecies has been reported in CoVs, the variant sites were found in coding regions or 3′-UTR.38 In the case of severe acute respiratory syndrome-related coronavirus, all of the variant sites observed in the quasispecies were located at the S gene.35 For bovine CoV, one of the two strains with naturally occurring intra-isolate quasispecies had all seven variant sites located at ORF1a, whereas for the other strain with naturally occurring intra-isolate quasispecies, there were 85 polymorphic sites scattered across ORF1a (n=28), ORF1b (n=19), 32 kDa-non-structural protein (NSP) gene (n=6), hemagglutinin esterase (HE) gene (n=2), S gene (n=18), 4.9 kDa-NSP gene (n=2), 4.8 kDa-NSP gene (n=2), membrane (M) gene (n=2), nucleocapsid (N) gene (n=5) and 3′-UTR (n=1).36 Similar to bovine CoV, Middle East respiratory syndrome-related coronavirus also possessed all the intra-host single nucleotide variations throughout its genome except the 5′-UTR.38 In this study, all four variant sites  were present in the 5′-UTR and were not located in the leader sequence or the transcription regulatory sequence. We speculate that the existence of quasispecies in CoVs may play a role in CoV evolution, in addition to the more well-known high-recombination and mutation rates in CoV genomes.39This is also the first report of CoV quasispecies in the 5′-untranslated region (UTR). In one (S579N) of the two


The molecular mechanisms by which RNA viruses coordinate their transcriptional activities are not fully understood. For rotavirus, an important pediatric gastroenteric pathogen, transcription occurs within a double-layered particle that encloses the viral genome. To date, there remains very little structural information available for actively-transcribing rotavirus double-layered particles, which could provide new insights for antiviral development. To improve our vision of these viral assemblies, we developed a new combinatorial strategy that utilizes currently available high-resolution image processing tools. First, we employed a 3D classification routine that allowed us to sort transcriptionally-active rotavirus assemblies on the basis of their internal density. Next, we implemented an additional 3D refinement procedure using the most active class of DLPs. For comparison, the refined structures were computed in parallel by (1) enforcing icosahedral symmetry, and by (2) using no symmetry operators. Comparing the resulting structures, we were able to visualize the continuum that exists between viral capsid proteins and the viral RNA for the first time. RNA viruses are ubiquitous in nature and represent some of the most severe pathogens known to mankind . Rotaviruses (RVs) are non-enveloped, eleven-segmented, double-stranded RNA viruses that can cause life-threatening gastroenteritis in children. RVs express their genes as single-stranded, messenger RNA (mRNA) molecules early in infection through the process of transcription . RV tranThree-dimensional (3D) structures of non-transcribing RV DLPs have been determined to high resolution 3.8 Å) using cryo-electron microscopy (cryo-EM) or X-ray crystallography ,7. Not o Å using Recent cryo-EM structural assessments performed by our laboratories revealed the unexpected finding that transcriptionally-active DLPs exhibited varying degrees of disorder in their external VP6 capsids . AdditioRotavirus (strain SA11-4F) DLPs were prepared as described in our previous work . Transcr2 and 10 mM CaCl2. The Protein A solution was incubated for 1 minute on each grid and the excess solution was blotted away with filter paper. Next, 3 μl aliquots of VP6-specific guinea pig polyclonal anti sera (#53963) (0.01 mg/ml) contained in the same HEPES buffer solution were added to Protein A-decorated grids. After a 1 minute incubation step, the excess solution was gently removed using a Hamilton syringe. Finally, the transcriptionally active DLPs  were added to the antibody-decorated grids for a 2 minute incubation. Frozen-hydrated specimens were prepared by plunge-freezing the grids into liquid ethane slurry using a Gatan Cryoplunge™ 3 equipped with Gentle Blot capabilities  while employing a one-sided blotting routine for approximately 8 seconds.EM Affinity grids containing Nickel-nitrilotriacetic acid (Ni-NTA) coatings were prepared using holey carbon grids  as previously described . The Ni-6 filament and operated at an acceleration voltage of 120 kV under low-dose conditions (∼5 electrons/ Å2). Images of transcriptionally-active DLPs were recorded on a FEI Eagle 2k HS CCD camera with a pixel size of 30 μm using a defocus range from -1.0 μm to -3.0 μm at a nominal magnification of 60,000× for a final sampling of 5 Å/pixel at the specimen level.Frozen-hydrated specimens were transferred to a Gatan 626 cryo holder  and maintained under liquid nitrogen until transferred into the TEM. Specimens were imaged using a FEI Tecnai Spirit BioTwin TEM  equipped with a LaBhttp://www.image-analysis.net/EM). Prior to particle selection, the raw images were inverted, normalized and CTF-corrected using standard routines in PARTICLE. The final image stack containing 2769 particles was exported in MRC format for 3D reconstruction and refinement calculations in the RELION software package [Images of transcriptionally-active DLPs were recorded as described above and individual particles were selected from the images using automated routines in the PARTICLE software package .Improving our understanding of protein-RNA interactions can provide important new insights to dissect infection at the molecular level. The dynamic manner in which these interactions occur remains unclear, especially in the case of viral mRNA production. To expand upon our previous findings of transcriptionally-active RV DLPs, we implemented a new computing protocol to better visualize the dynamic nature of these complexes. We collected images of frozen-hydrated transcriptionally-active DLPs that were tethered to C-flat holey carbon Affinity Grids . These grids were decorated with adaptor molecules including IgG antibodies against the VP6 capsid protein (please see Methods section). EM images of the DLP specimens were acquired using a FEI Tecnai Spirit BioTwin TEM  equipped with a LaBhttp://www.image-analysis.net/EM), we inverted and normalized each image  of the DLP structure , RELION Overall, the 3D classification routine reproducibly distinguished various structures among the active particles. However, considering the fact that not all DLPs displayed perfect symmetry in the images, we investigated whether applying the icosahedral symmetry operator to the active DLPs obscured information about protein-RNA arrangements. We performed additional refinement calculations using only the particles contained within the presumed most active class , Class 3Upon examining the resulting reconstructions generated from applying icosahedral symmetry, we visualized strong internal density along the 5-fold axis in contoured sections through the EM structures . ProteinUpon examining the 3D density maps calculated from the same images of active DLPs, but without imposing icosahedral symmetry, we found very different results . The oveAs a control experiment, additional 3D structures were calculated in the C1 space group for non-transcribing particles contained within the same image stack . For theReoviridae), we found an essential commonality- a series of conformational changes must occur in the viral capsid during mRNA synthesis, capping, and egress [In comparing our results to the high-resolution structures of the transcriptionally-active subviral particle of cypo virus, a member of the same viral family as RV (i.e., d egress . It remaCollectively, the experimental results presented here complement the multi scale molecular dynamics simulations of Miao and Ortoleva . Their sAdditional work by Vlijmen and Karplus demonstrated the existence of structural variability among viral transition states using normal mode analysis while applying icosahedral symmetry . AlthougFinally, studies performed by Brooks et al. described the need to examine viral capsid reconstructions with and without enforcing symmetry elements ,18. Theiin situ TEM analysis of transcriptionally-active viral assemblies within a liquid environment may reveal additional information to delineate real-time mechanisms within these exquisite nanomachines.Based on these theoretical frameworks, and considering the continuum of density visualized in the more disordered structures presented here, it seems feasible that the movements within the DLP cores are influenced by RNA-induced mobility in cooperation with internal protein subunits. As such, the transitional movements in the VP6 capsid are likely to be asymmetric due to the natural fluctuations needed to accommodate RNA synthesis and extrusion. We expect similar movements may occur in other pathogenic RNA viruses, and that our strategies can be broadly utilized to provide new insights for structure-based drug design. Overall, we anticipate future work involving


RNA molecules have the tendency to fold into complex structures or to associate with complementary RNAs that exoribonucleases have difficulties processing or degrading. Therefore, degradosomes in bacteria and organelles as well as exosomes in eukaryotes have teamed-up with RNA helicases. Whereas bacterial degradosomes are associated with RNA helicases from the DEAD-box family, the exosomes and mitochondrial degradosome use the help of Ski2-like and Suv3 RNA helicases. All living cells encounter situations where they need to adapt gene expression to changing environmental conditions. The synthesis of new mRNAs to be used for translation and the release of sequestered or translational inactive mRNAs allow the cells to express new proteins. On the other hand, processing and degradation of RNAs not only helps to recycle essential components, but also to shut down expression of genes that are no longer required or would even be detrimental for living under a new condition. Moreover, remnants of processed or aberrant transcripts must rapidly be degraded to avoid the production of useless or even toxic peptides and proteins. Eubacteria, Archaea, and eukaryotes have developed dedicated pathways and complexes to process RNA, check the accuracy of RNAs (surveillance), and feed undesired RNA into exoribonucleases that degrade RNA in a 3′–5′ or 5′–3′ direction. In addition to the ribonucleases, these complexes often contain adaptor proteins such as Hfq (an RNA chaperone), RraA (a regulator of RNase E and DEAD-box helicases), poly(A) polymerase (to render 3′-ends accessible to the 3′–5′ PNPase), or others ,2. It isRNA helicases are large families of proteins that share up to 12 motifs involved in nucleotide-triphosphate binding, interaction with RNA, and intramolecular contacts . Accordii, the helicase will have reduced affinity for RNA and dissociate again. The binding to RNA induces a bending of the substrate that is incompatible with a double-stranded RNA and therefore induces a local, non-processive, unwinding . Th. Th59]. activity . The RNAactivity  and in tactivity . Recentlactivity . RNAs aractivity ) and PAXactivity ) complexactivity ,56.FiguIt is interesting to note that certain Archaea also possess an exosome with a similar structure to the eukaryotic complex ,67. In tin vitro reconstitution assays, it has been shown that Suv3 has a 3′–5′ helicase activity requiring a short 3′ single-stranded tail and that this helicase activity was dependent on the presence of Dss1, whereas the exonuclease showed basal activity alone that could be greatly stimulated by Suv3.RNA turnover and the degradation of RNA fragments or splicing products are also important in mitochondria and chloroplasts. Several RNA helicases from the DEAD-box protein family and Suv3, an RNA helicase forming its own family most closely related to the Ski2-like RNA helicases in eukaryotes and purple bacteria , are reqArabidopsis chloroplasts revealed an RNA-binding protein with similarity to transcription termination factor Rho, which is an RNA translocase [So far, little is known about RNA degradation and turnover in chloroplasts. These organelles contain RNase E and RNase J homologues, but so far no degradosome as such has been described. RNase E purified from nslocase  and may RNA turnover is crucial to maintain faithful gene expression and to allow cells to adapt to changing growth conditions, and in many instances, the inactivation of RNases causes strong growth defects. The structures of bacterial PNPases or eukaryotic exosomes require the insertion of single-stranded RNA into the complex, explaining the presence of RNA helicases to unwind inhibitory structures or to remove bound proteins. A crucial point in RNA turnover is the specificity that insures that a given RNA is degraded if necessary, but left alone if still useful to the cell. Although several adaptor proteins have been described for the different systems, the specificity of RNA turnover remains an important open question. Moreover, bacterial degradosomes are often associated with metabolic enzymes, but how they regulate degradosome activity is not known. In general, RNA helicases are not (very) substrate-specific; nevertheless, exchange of RNA helicases in bacterial degradosomes has been observed under different growth conditions. Future work will show why and how these RNA helicases are exchanged and function together with the RNA decay machineries.An intriguing difference in the degradation of RNAs in bacterial and eukaryotic machineries is the differences in the RNA helicases. DEAD-box proteins, used in the bacterial degradosome, are non-processive RNA helicases that unwind locally secondary structures. In case of RhlB, the helicase is bound and stimulated by RNase E, which holds it on the substrate and will provide a certain artificial processivity. The role of Mtr4, and by analogy Ski2, may be more active in the process, since it has been shown that the RNA helicase translocates on the substrate to unwind the RNA. Thus, it remains to be seen to what extent the RNA helicases contribute to the active RNA degradation and the specificity of the decay complexes.