Some species of fungi can cause serious human diseases, particularly to immuno-compromised individuals. Opportunistic fungal infections are a leading cause of mortality, and present an emerging challenge that requires development of new and effective therapeutics. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of cellular protein translation machinery and can be targeted for discovery of novel anti-fungal agents.A. niger, C. albicans and C. neoformans. Hidden Markov Models were built for aaRSs and related proteins to search for homologues in these fungal genomes. Here, we provide a detailed and comprehensive annotation for 3 fungal genome aaRSs and their associated proteins. We delineate predicted localizations, subdomain architectures and prevalence of unusual motifs within these aaRSs. Several fungal aaRSs have unique domain appendages of unknown function e.g. A. niger AsxRS and C. neoformans TyrRS have additional domains that are absent from human homologs.Validation of aaRSs as potential drug targets in pathogenic microbes prompted us to investigate the genomic distribution of aaRSs within three fungi that infect humans – Detailed comparisons of fungal aaRSs with human homologs suggest key differences that could be exploited for specific drug targeting. Our cataloging and structural analyses provide a comprehensive foundation for experimentally dissecting fungal aaRSs that may enable development of new anti-fungal agents.The online version of this article (doi:10.1186/1471-2164-15-1069) contains supplementary material, which is available to authorized users. Fungal infections are emerging as a leading cause of human mortality in hospital settings –5. RecenAspergillus, Candida, and Crytococcus are the three most common genus of fungi associated with infectious diseases in humans [C. albicans. Candida is generally present on the skin and mucous membrane and does not cause infection, however invasive candidiasis occurs when Candida enters the bloodstream and spreads throughout the body [C. albicans infections [Aspergillus species causes second most common fungal infections in humans [A. fumigates is the most virulent species in this genus, and cases of pulmonary aspergillosis have been reported resulting from A. niger infections as well [Cryptococcus neoformans and Cryptococcus gattii[C. neoformans is present in soil and spreads through microscopic airborne fungal spores [C. neoformans infection increases the probability of re-hospitalization of AIDS patients who otherwise show positive response to anti-retroviral therapy [A. niger and C. albicans belong to Ascomycota phylum while C. neoformans belongs to Basidomycota phylum within the fungal kingdom. Genome sizes for A. niger, C. albicans, and C. neoformans are ~33 Mb (19 chromosomes), ~14 Mb (8 chromosomes), and ~19 Mb (14 chromosomes). Predicted number of ORFs (open reading frames) in A. niger, C. albicans, and C. neoformans are ~14000, ~6200, and ~6500. In addition to genome sequencing, transcriptomics analyses for these three fungi are also available [n humans –5. Candithe body . Immuno-fections . Aspergin humans . A. fumi as well . Cryptocs gattii. C. neofl spores . C. neof therapy , 12. A. vailable –15.Leishmania[Toxoplasma[C. albicans SerRS is the only full-length aaRS from these three fungal genomes for which crystal structure has been solved [A. niger, C. albicans and C. neoformans using profile-based Hidden Markov Models (HMM). In addition, homologs for aaRS-related proteins such as the editing domains (AlaX) and D-tyrosyl-deacylase (DTD), Gln and Asn amidotransferases, and aaRS-associated P43 protein have also been identified. Protein sequences of thus identified aaRSs have been comprehensively annotated in context of their domain organization. We discovered novel domain appendages in several fungal aaRSs that are absent from human homologs. Our results highlight key structural attributes of fungal aaRSs that could be exploited for drug targeting to combat mycoses in humans.Aminoacyl-tRNA synthetases (aaRSs) drive protein translational machinery by catalyzing the addition of amino acids to cognate tRNA , 17. Strishmania and Toxoxoplasma have alsn solved . Here, wA. niger and C. albicans were downloaded from their corresponding genome databases available at http://www.aspergillusgenome.org/ and http://www.candidagenome.org/ respectively. Similarly, ORF sequences for C. neoformans were retrieved from FungiDB [E. coli. The UniProt accession numbers for sequences in the dataset are given in supplementary Additional file Computationally translated open reading frames (ORFs) for  FungiDB . To anno FungiDB . Our dathttp://tcoffee.crg.cat/. Homology modeling for protein sequences was performed using Phyre2 server [All protein sequences from our dataset were individually used to perform BLAST searches against non-redundant databases available at NCBI. Top 1000 homologs from each BLAST search were saved for further analyses as we reasoned that these may effectively capture the evolutionary diversity at different positions within aaRSs, thus facilitating generation of robust HMMs. For each aaRS, HMMs were generated using the homologs identified from BLAST search. Sequence Alignment and Modeling Software System (SAM)  was used2 server  and PyMo2 server  was usedA. niger, 28 in C. albicans and 26 in C. neoformans  with 1524 residues and TrpRS (209919-mRNA) with 391 residues respectively , 7 C. albicans aaRSs that are present in two versions , and 5 C. neoformans aaRSs that are present in two versions  were predicted to (co-) localize in cytoplasm, mitochondria and nucleus  were observed in all the three fungal genomes  is frequently observed within mitochondria for synthesis of Gln-tRNAGln[A. niger and C. albicans.We also sought to catalog trans-editing domains within these fungal genomes. D-Tyrosyl-tRNATyr deacylase (DTD) catalyzes hydrolysis of bond between D-amino-acid and tRNA. These enzymes play an important role in evading toxicity resulting from accumulations of D-amino acids within the cell . Trans-eAla. Hence, examined , 51. In s Tables  and 3. Ts Tables  and 3. S-tRNAGln. In addi-tRNAGln. We addiIn some eukaryotes, a few aaRSs associate with each other and with auxillary proteins  to form multi-synthetase complex (MSC) , 54. It A. niger – 14 belonging to class I and 15 to class II. Among class I aaRSs – IleRS, LeuRS, GlnRS, ArgRS, and MetRS had N-terminal catalytic domain followed by anticodon binding domain (ABD) towards the C-terminal  lacks N-terminal RNA binding region that is present in human and other fungal homologs  and mitochondria (209919-mRNA). In case of CysRS, GluRS, MetRS, TrpRS and TyrRS the Pfam server could only annotate their catalytic domains within these sequences. However, based on the catalytic domain positioning it appears likely that their anti-codon binding domains lie towards the C-termini  displays an additional Brix domain at its C-terminus , BLAST searches for 194077-mRNA were performed against the A. niger transcriptome database located at the Broad Institute, USA. A highly significant hit was obtained spanning the fungal full-length protein sequence  domains. AlaRS, PheRS, GlyRS, HisRS, ProRS and ThrRS had catalytic domains at the N-termini of anti-codon binding domain , B (172989-mRNA) and F (173432-mRNA) were also identified  has an additional domain (DUF726) at its C-terminal  that were predicted to localize in cytoplasm (207470-mRNA) and nucleus (55404-mRNA) respectively. These enzymes catalyze the charging of both tRNAspartate . Asp-tRN-tRNAAsn. CuriousA. niger. Interestingly, A. niger P43 has GST C-terminal domain at the N-terminal of the tRNA binding region, which is different from the human counterpart. Indeed, GST_C domain is absent in human P43, however, it is present in P43 homolog from Toxoplasma gondii[T. gondii P43s  within the structurally conserved tRNA binding domain. Further, the N-termini of these proteins show poor homology amongst each other. Finally, GluRS (56891-mRNA), HisRS (51854-mRNA), and LeuRS (52554-mRNA) were observed to have ‘ELR’ motifs in their N-terminal regions  in a gondii  also has anti-codon binding domain at the C-terminal of catalytic domain. Further, cytoplasmic PheRS prototypically encompass α (orf19.2960) and β (orf19.2573) subunits  and orf19.198 (predicted cytoplasm). These two enzymes could act in tandem with two AsxRSs to generate Asn-tRNAAsn in an indirect manner. In addition, subunit components of Gln synthase were also identified – GatA (orf19.3956), GatB (orf19.2494) and GatF (orf19.4092)  shares ~35% identity with human mitochondrial GluRS (which is non-discriminating), raising the possibility of indirect synthesis of Gln-tRNAGln by the action of Gln synthase on Glu-tRNAGln. Standalone trans-editing domains like AlaX (orf19.5239) and DTD (orf19.297) were also identified  and TyrRS (CNBA1270 and CNBJ0260)  and functions . A reces Figure A. IntrigC. neoformans TyrRS part of the fusion protein suggests that the ‘ELR’ motif is likely to be solvent accessible  an interesting target for further investigation. In addition, we noted that ProRS (CNBB0150) also has an ‘ELR’ motif towards its N-terminal  and other two Gln synthases (subunit A (CNBD1400) and subunit B (CNBG4210)). We were not able to identify any homolog for GatF subunit. Interestingly, all these four amidotranferases were predicted to localize within the mitochondria Table . InteresAspergillus, Candida and Cryptococcus are three highly pathogenic fungi, particularly in immuno-compromised patients. Dissecting and understanding critical fungal protein machineries is therefore vital for establishing a base for launching new therapeutics against these pathogens. Within microbial proteomes, aminoacyl-tRNA synthetases have already been exploited for drug discovery [Leu in editing conformation is now an anti-fungal agent in the market [Treatment and prevention of opportunistic fungal infections in humans presents an emerging challenge. iscovery . Specifie market . Hence, A. niger, C. albicans, and C. neoformans there exist 8, 7, and 5 aaRSs that are encoded by two different set of genes, presumably for mitochondrial localization  maybe heterodimeric unlike its homologs in other two fungi where it is likely to be heterotrimeric. None of the aaRS in C. neoformans were predicted to localize within nucleus while 2 and 6 aaRSs in A. niger and C. albicans were predicted to have nuclear localization  schematic representation for genome and transcriptome sequencing analyses for VRS-Brix and (B) modelling for Brix domain from C. neoformans. Figure S3. Schematic representation for genome and transcriptome sequencing analyses for YRS-SAICAR fusion protein in C. neoformans. (DOCX 356 KB)Additional file 1: Table S1 and Table S2: Having the details of the dataset used for analyses and summary of fungal aaRSs with ELR motif, respectively.


Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity ( The use of chemically modified antisense oligonucleotides (ASOs) as potential therapeutics has received much attention in recent years . Chemica(n–1) possible stereoisomers.While the PS modification has been used to enhance drug-like properties of ASOs which function through a variety of antisense mechanisms , this moTm and RNase H activating profile while the Sp isomer shows better nuclease stability  monomers d Figure  were pree et al. . The proTM support (315 μmol/g). Fully protected nucleoside phosphoramidites were incorporated using standard solid-phase oligonucleotide synthesis conditions: i.e. 3% dichloroacetic acid in dichloromethane (DCM) for deblocking, 1 M 4,5-dicyanoimidazole with 0.1 M N-methylimidazole in acetonitrile as activator, 10% acetic anhydride in THF and 10% N-methylimidazole in THF/pyridine for capping and 0.2 M phenylacetyl disulfide in pyridine:acetonitrile 1:1 (v:v) for thiolation. DNA amidites were prepared at 0.1 M in acetonitrile:toluene 1:1 (v:v) and were incorporated using 5 min coupling time. OAP monomers were prepared at 0.2 M in acetonitrile:toluene 1:1 (v:v) and were coupled utilizing two applications of OAP, with a 6 min contact time for each pass. After conclusion of the synthesis, the 5′-dimethoxytrityl group (DMT) group was removed. The solid support was suspended in aqueous ammonia (28–30 wt.%) and heated at 55°C for 48 h. The reaction mixture was allowed to come to room temperature, and the solid support was filtered and washed with a solution of EtOH and H2O 1:1 (v:v). The washings and filtrate were combined and were concentrated under reduced pressure to ∼ 20% of the original volume. The residue obtained was dissolved in water and purified by high performance liquid chromatography (HPLC) on a strong anion exchange column . Oligonucleotides were desalted over HiPrepTM 26/10 columns   or by utilization of C18 reverse-phase cartridges . The oligonucleotides were lyophilized to give an overall isolated yield of 6–8% based on the initial loading of the solid support.ONs were prepared on an ABI 394 DNA/RNA synthesizer  or an AKTA oligopilot 10  using polystyrene-based NittoPhase unylinkerSp configuration at the 5′-most internucleotide linkage and the second peak is the group of diastereoisomers Rp configuration at the 5′-most internucleotide linkage (vide infra). Comparison of the relative ultraviolet (UV) areas of the two peaks is a direct measure of Sp/Rp ratio at the 5′-most internucleotide linkage.Oligonucleotides were characterized by ion-pair-HPLC coupled mass spectrometry (MS) analysis with Agilent 1100 MSD system. PS oligonucleotides that terminate at the 5′-end in a 4, 4′-DMT are resolved chromatographically into two peaks on the basis of the absolute configuration of the internucleotide linkage located at the 5′-end of the oligonucleotide. The first peak corresponds to the group of diastereoisomers with Tm values) were measured in quartz cuvettes (pathlength 1.0 cm) on a Cary 100 UV/visible spectrophotometer equipped with a Peltier temperature controller. Absorbance at 260 nm was measured as a function of temperature using a temperature ramp of 0.5°C/min. Tm values were determined using the hyperchromicity method incorporated into the instrument software.ASO and RNA were mixed in 1:1 ratio (4 μM duplex) in buffer containing 10 mM phosphate, 100 mM NaCl and 10 mM ethylenediaminetetraaceticacid (EDTA) at pH 7.0. The duplex was denatured at 85°C and slowly cooled to the starting temperature of the experiment (15°C). Thermal denaturation temperatures . Reduction of target mRNA was determined by quantitative reversetranscriptase-polymerase chain reaction (qRT-PCR) as previously described .Electroporation. 10× ASO was diluted to 1× by either hepatocytes or b.END cells at a density of 35,000 cells/well for hepatocyte cells or 20,000 cell/well for b.END cells. Hepatocyte cells were electroporated at 165 V and b.END cells at 115 V for a pulse length of 6 ms. Cell/oligo mixture was transferred into a 96-well tissue culture plate containing Williams E medium (Life Technologies) for hepatocytes or Dulbecco's modified Eagle's medium (Life Technologies) for b.END and 10% fetal bovine serum. Cells were incubated overnight at 37°C in 10% COescribed . The prin = 3, were maintained at a constant temperature of 23°C and were allowed standard lab diet and water. Dosing solutions were prepared in phosphate-buffered saline, sterile filtered and quantified. Mice were dosed, at the indicated dosage, twice a week for two weeks by subcutaneous injection. Mice were sacrificed 48 h after the last injection. Animals were anesthetized with isoflurane and terminal bleed was performed as previously described . Male Balb/c mice (Charles River Laboratories), aged 6–8 weeks and escribed . Immediam/z corresponding to the −3 charge state of cEt gapmer and −5 charge state of the internal standard. Peak areas from extracted ion chromatograms were determined for each analyte and normalized to the peak area of the internal standard. Concentrations were determined using a trend line established with the extracted standards and reported as μg/g liver.cEt gapmer ASOs were extracted from liver samples along with five to eight standards of each analyte prepared in control liver and analyzed by LC-MS using previously described methods ,37. Brie32P using 20 U of T4 polynucleotide kinase, 120 pmol (7000 Ci/mmol) of [γ-32P]ATP, 40 pmol RNA, 70mM Tris–HCl, 10 mM MgCl2 and 50 mM dithiothreitol, at pH 7.6. The labeling reaction was incubated at 37°C for 30 min followed by heating at 90°C for 1 min. Labeled RNA was purified using 12% denaturing polyacrylamide gel. ASO (200 nM), unlabeled 19-mer RNA (100 nM) and a small amount of 32P-labeled RNA was mixed in hybridization buffer  and heated to 90°C for 2 min. To the hybridization mixture was added 0.1 mM TCEP, 1mM MgCl2 and 40 U of RNaseOUT and incubated at 37°C for 1 h. The human RNase H1 enzyme was incubated in dilution buffer  for 1 h at rt. Enzyme solution (1/25 ml duplex solution) was added to duplex solution and incubated at 37°C. At time point's aliquots were removed and reaction quenched in loading buffer and snap-frozen on dry ice. Cleavage products were separated by 12% denaturing polyacrylamide gel and products were quantitated on a Phosphor-Imager. We have found that the absolute initial rates vary as a function of the RNaseH assay preparation; however, the relative rank ordering between experiments remains consistent.RNA was 5′-end labeled with et al. (A5) targeting scavenger receptor B1 (SR-B1), a ubiquitously expressed gene whose physiological role is related to cholesterol uptake into tissues (A10) was designed to inhibit the production of the AR—a well-validated target associated with prostate cancer  results in an Rp linkage , while addition of an Rp OAP  results in an Sp linkage  Figure To evaluate the coupling efficiency and stereoselectivity of the reported OAP method a series of 15-mers was prepared. The first 14 bases were synthesized using standard phosphoramidites and standard conditions (i.e. the first 14 bases were 2′-deoxynucleosides and the first 13 PS linkages were stereo-random), followed by a 5′-terminal nucleotide which was connected by either a diastereomeric PS linkage, an Sp linkage or an Rp linkage. The resulting 15-mers were analyzed by RP-HPLC (UV area) as described in the Materials and Methods section. The chromatograms can be found in Figure OAP monomers do not couple as efficiently as phosphoramidite monomers. A variety of conditions were tested in an attempt to improve coupling efficiency and to push the coupling reactions to completion. Choice of activators, thiolating agents and/or capping reagents did not increase the coupling efficiency, though it is interesting to note that the stereoselectivity was unaffected by these variables . By increasing the coupling time, concentration and use of multiple applications of monomer, we were able to achieve ∼ 93% coupling efficiency per incorporation. Oligomers containing phosphodiester linkages were observed as major by-products either due to incomplete manual sulfurization or oxidation of the PS bonds post-synthesis. Isolated yields of 6–8% were typical after ASO purification.A1–A8 were designed to target mRNA for the SR-B1 receptor and ASOs A9–A16 were designed to target mRNA for the AR (see Table Tm) (Table A set of four ASOs was prepared for each sequence that was tested: (i) full length PO, (ii) full length PS (stereo-random), (iii) uniform Rp-gap and (iv) uniform Sp-gap. ASOs A2, A6, A10 and A14) were found to be less thermally stable than their corresponding full PO ASOs  by ∼ 0.5°C per linkage, which is consistent with previous studies; (ii) The incorporation of Rp PS linkages in the gap  led to an increase in Tm by ∼ 0.2°C per incorporation relative to the Tm of stereo-random PS ASOs  The incorporation of Sp PS linkages in the gap  led to a decrease in Tm by ∼ 0.4°C per incorporation relative to the Tm of stereo-random PS ASOs . The net result is the following rank order for thermal stability: PO > Rp > stereo-random PS > Sp. This trend is conserved regardless of sequence, ASO length or gap size.For each set of ASOs, the following observations held true: (i) Stereo-random PS ASOs ; (ii) Incorporation of Sp linkages has a larger negative affect on Tm than incorporation of Rp linkages; and (iii) The ASOs containing a mixture of Rp- and Sp-linkages lies in between that of pure Rp and Sp gaps.To further explore the effect that multiple chiral PS linkages has on A10–A12 (10-base gap AR ASO) and A14–A16 (8-base gap AR ASOs) were tested for stability against S1 endonuclease to confirm configurational assignment. ASOs were incubated with nuclease S1 from Aspergillus oryzae (Sp-specific) at room temperature over a time course. ASOs and digestion products were analyzed by LCMS. As expected, the digestion profile showed that the all-Sp gap compounds A12 and A16 were rapidly digested (< 10 min) while the all-Rp gap compounds A11 and A15 were found to be significantly more stable than the Sp-gap ASOs (A12 and A16) and the diastereomeric PS parent ASOs (A10 and A14) .ASOs 32P. The resulting duplex was then incubated with Escherichia coli (E. coli) RNase H (20-fold excess) or human RNase H1 (50 fold excess) at 37°C. Aliquots were removed over time and cleavage products were separated by 12% denaturing polyacrylamide gel, and quantitated on a Phosphor-Imager.ASOs were prepared at 200 μM concentration and were incubated for 1 h at 37°C with complementary mRNA that had been 5′-end labeled with E. coli, the results in Figure A11 and A15) and the all-Sp gap AR ASOs (A12 and A16) were more active than their corresponding stereo-random PS parent ASOs . This observation held true for both the 10-base compounds  and all-Sp gap AR ASOs (A12 and A16) were less active than their corresponding stereo-random PS parent ASOs . In the case of the 10-base gap compounds there was very little difference in initial rates of cleavage between the all-Rp and the all-Sp gap compounds  was similar to the stereo-random parent, which were both more active than the Rp-gap isomer (A15).The results in Figure A2–A4, A6–A8, A10–A12 and A14–16) were tested for activity in cell culture in a seven-point dose response assay. ASOs were transfected into mouse primary hepatocytes (SR-B1 ASOs) or b.END cells (AR ASOs) at 20, 6.67, 2.22, 0.74, 0.25, 0.08 and 0.03 μM via electroporation. The cells were incubated overnight and were lysed 16 h post treatment for RNA purification and analysis  exhibited activity that was virtually identical to that of the corresponding diastereomeric PS parent ASOs . Conversely, the 10-base all-Sp gap compounds  were significantly less active. The exception to the trend was the 8-base all-Sp and all-Rp gap AR ASOs (A15 and A16), both of which exhibited activity in vitro that was very similar to that of the stereo-random parent (A14).Uniform gap ASOs  and A8  were selected for evaluation in animal experiments and were compared to the performance of the stereo-random parent ASO A6. Mice (n = 3 per group) were injected subcutaneously with 0.67, 2.0 and 4.0 mg/kg of SRB-1 ASOs A6, A7 and A8 twice a week for 2 weeks. The animals were sacrificed 48 h after the last ASO dose and the SRB-1 mRNA in liver was measured by quantitative RT–PCR. Target mRNA was normalized to total RNA levels via RiboGreen  with the Sp-gap ASO (A8) being significantly more stable  (tThe relatively low abundance of the Rp-gap ASO recovered in tissue suggests that the yet unidentified nuclease(s) that metabolize gapmer ASOs have a preference for the Rp isomer. We have shown that Sp-gap ASOs are more susceptible to degradation by S1 endonuclease while Rp-gap ASOs are more stable . There are multiple reports in the literature that indicate that the Rp PS linkages are rapidly metabolized by exonucleases such as snake venom phosphodiesterase (svPDE), however, gapmer ASOs are resistant to exonuclease degradation as reported in the literature ,46–48 anmerase 1 –52. We agure S8) . In all in vivo. Conversely, ASOs that contain long stretches of Sp linkages were found to be more stable to exonucleases but gave rise to compounds with lower target affinity and lower activity in vitro. Thus, it appears likely that a mixture of Rp and Sp is necessary to achieve a balance between good target affinity, activity and nuclease stability. The stereospecific OAP coupling method will enable us to synthesize discrete Sp/Rp gap configurations in an attempt to determine whether or not specific combinations of Sp and Rp result in ASOs that were significantly more potent (5×) than the parent ASO prepared under stereo-random conditions (i.e. ‘super isomers’).The incorporation of Rp linkages increases the affinity of ASOs to their target RNA, however ASOs with long stretches of sequential Rp linkages (10 or more) are susceptible to degradation by exonucleases A6 was selected as the parent ASO for this study. Twenty-nine isomers (A27–A55) were synthesized utilizing the bicyclic OAP monomers to control the chirality of each PS linkage within the gap. These isomers were selected from a master list of the 1024 possible isomers without bias or preconceived notion of performance based on the Rp/Sp gap configuration. The ASOs were tested for activity in cell culture in a five-point dose response assay and IC50 values were calculated in an attempt to identify trends and patterns that correlate Rp/Sp pattern/configuration with activity (Table A7) exhibited activity that was equal to that of the stereo-random PS parent ASO A6, while the activity of the 10-base Sp gap ASO (A8) was greatly decreased. It is interesting that A28, which has nine sequential Sp linkages, exhibits moderate activity. Although there is not a compound with eight sequential Sp-linkages in this sample set, there are two compounds that have a total of eight Sp linkages broken up with Rp linkages (A34 and A36), both of which have similar activity to the diastereomeric PS parent. This suggests that 9–10 sequential Sp linkages are detrimental to activity, but eight or less is tolerated. This point can be further illustrated by comparing the activity AR ASOs A12 and A16 (Table A12) was found to be inactive while the 8-base Sp AR ASO (A16) was nearly as active as the stereo-random PS 8-base Gap AR ASO (A14). With the exception of A8 and A28, the remainder of the compounds in Table A6.SR-B1 ASO ty Table . Mass spinkages A4 and A36et al.  from the stereo-random parent. This suggests that, at least in terms of in vitro activity, most of the isomers are nearly equivalent in activity except for certain limiting cases of all-Sp or at least runs of 9-Sp isomers which were less active. While it is certainly possible that among the 1024 potential isomers exists within a set of ‘super isomer(s)’ (i.e. with >5× activity in vitro), the fact that we chose our specific absorption rate set randomly suggests that this is unlikely. Unfortunately, a herculean, exhaustive screen would be required to answer this question definitively. For further discussion regarding the probability of finding a ‘super-isomer’, see Supplementary Data, Figure S10.The decreased thermal affinity of the Sp isomer seems to correlate with the rs Table , a substTm or cell culture data of our 3-10-3 motif. While human RNase H1 has both a binding domain and a catalytic domain, the bacterial RNase H has a catalytic domain only (E. coli RNase H1. The fact that the Rp isomer (A11), Sp isomer (A12) and stereo-random parent (A10) all have initial rates that are comparable for the E. coli version, but both the chirally-pure ASOs are significantly less active than the stereo-random isomer is probably reflective of this additional selectivity. It appears, however, that RNase H initial rates are not rate limiting for activity; however, it remains to be seen if there is an optimal configuration of Rp and Sp isomers that produce ASOs more active relative to the stereo-random parent or if the stereo-random parent is the optimal RNase H motif.There does not appear to be a correlation of the RNase H activity to either the ain only . As a reA13–A16) remain an outlying and interesting case. While the Tm follows the general trend of the 10-base gap (i.e. Rp>Sp), the Tm, cell culture data and RNase H results are uncorrelated. Unlike the 10-base gap, the 8-base gap ASOs are nearly identical in cell culture, while the Sp isomer is more active against RNase H . Additionally, the cleavage patterns, as determined by the gels in Figure sui generis, or if these results extend as a general pattern. One possibility, if this is indeed a general pattern, is that the conformational transmission is enhanced in the Rp isomer  over that of the Sp isomer; however, the potential structural reasons for this are unclear.In this study, the 8-base gap ASOs  was more active than either the Rp isomer (A7) or the Sp isomer (A8), despite the fact that the Rp isomer was significantly more active in vitro than the Sp isomer. However, after comparing the amount of intact ASO in liver tissue (Figure A7in vivo has yet to be identified.While initially it appeared to us, based on the e Figure , it seemThe PS linkage is not limited in its use in RNase H mediated ASOs mechanisms. The PS linkage has been utilized for enhancing metabolic stability of immunomodulatory and other oligonucleotides which function through RISC pathways. It is conceivable that chirality of the PS linkage could impact the therapeutic properties of these oligonucleotides and the role that the PS moiety plays in their binding, activity and metabolism remains to be seen. However, controlling chirality of the PS linkage in the gap region of RNase H active gapmer ASOs provides no discernible benefits for therapeutic applications relative to the mixture of stereo-random PS ASOs. Furthermore, the added complexity and cost associated with the synthesis and characterization of chiral PS ASOs will likely limit their utility for broader applications.Supplementary Data are available at NAR Online.


Escherichia coli strains is a powerful virulence factor by impairing the signaling cascade of Toll-like receptors (TLRs). Several other bacterial pathogens like Salmonella, Yersinia, Staphylococcus aureus but also non-pathogens express similar proteins. We discuss here the pathogenic potential of TcpC and its interaction with TLRs and TLR-adapter proteins on the molecular level and compare its activity with the activity of other bacterial TIR-containing proteins. Finally, we analyze and compare the structure of bacterial TIR-domains with the TIR-domains of TLRs and TLR-adapters.The TIR-containing protein C (TcpC) of uropathogenic  Escherichia coli (UPEC) strains are the leading cause of urinary tract infections. The frequent disease affects almost half of all women during their lifetime and is increasingly difficult to treat since resistance against any class of antibiotics spreads globally with alarming speed [Uropathogenic ng speed ,2,3. To i.e., double stranded RNA, single stranded RNA, double stranded DNA, with TLR3, -7 and -9, respectively. Upon recognition with their ligands, TLRs interact with up to four different adaptor molecules via cytoplasmic receptor TIR-domains, which are located at the C-terminus of TLRs and the adaptor TIR-domain containing molecules MyD88, TIR-associated protein or MyD88 Adaptor-like (TIRAP/MAL), the TIR-domain containing adaptor protein-inducing IFN-β (TRIF) and TRIF-related adaptor molecule (TRAM). Different bacterial pathogens like UPECs including the CFT073 strain; Salmonella enterica subsp. enterica Serovars Enteritidis, Dublin, Gallinarum; Yersinia spp.; Brucella spp.; Staphylococous aureus (S. aureus) MSSA476 ; and Enterococcus faecalis , but also non-pathogens such as Paracoccus denitrificans harbor genes, which encode proteins containing a TIR-domain (serU t-RNA island of CFT073 [E. coli strains the gene is only found in the phylogenetic group B2 and is strongly associated with the presence of the main pathogenicity island [Brucella TIR-containing protein B (TcpB) and the Yersinia pseudotuberculosis (Y. pseudotuberculosis) serotype I TcpYI protein are located within phe t-RNA islands [TLRs comprise a family of 10 functional proteins in humans, 12 in mice, and recognize pathogen-associated molecular patterns (PAMPs) from microorganisms . The indR-domain  [5,6,7,8y island . Similar islands . The amiSalmonella enterica subspecies enteria Serovar Enteritidis [Y. pseudotuberculosis serotype I [tirS-containing S. aureus strain MSSA476 lost more weight and demonstrated higher bacterial numbers in different organs than mice infected with the tirS-deficient MSSA476 strain [Brucella melitensis (B. melitensis) augmented the spread of B. melitensis in mice [In vitro, TcpC and TlpA increased the intracellular amount of CFT073 and Salmonella Enteritidis, respectively [in vivo and impaired host immune responses. Thus, the data support a role of bacterial Tcps to subvert immune responses of the host. However, based on their broad phylogenetic distribution, which also includes environmental microorganisms, microbial TIR-domains may additionally serve more general functions as protein–protein interaction domains [TLRs initiate a broad defense program of the innate immune system to orchestrate the elimination of the invading pathogen. Several groups demonstrated that Tcps are able to interfere with this fundamental process of host defense. Thus, TlpA increased the bacterial load in the spleen and lethality of mice infected orally with eritidis . Similarrotype I . Mice in6 strain . TcpC in in mice . In vitrectively . As the ectively . Taken t domains .B. melitensis, Yersinia pestis and E. faecalis, respectively, bound to MyD88 but also PdTLP from the non-pathogenic Paracoccus denitrificans interacted with this central adaptor molecule of the TLR-signaling cascade [et al., TcpB with TLR4 impaired the latter signaling pathway as well [These results prompted the investigation of the TLR-Tcp interaction on the molecular level. Immuno-precipitation and pull-down assays revealed that MyD88 was the target of several Tcps. Thus, TcpC, TcpB, YpTIR and TcpF from CFT073,  cascade ,10,16,24 cascade ,21,25. S cascade , which a as well ,19,27,2815N-labeled MyD88-TIR domain in the absence or presence of TcpC, this approach revealed that the TcpC binding site of MyD88 included the amino acids M157, S244, K250, R269, F270, T272, V273, C280 and W286, which is predominantly located near CD-, DE- and EE-loops on one face of the MyD88-TIR domain [Eukaryotic TIR domains consist of five parallel β-strands, which are termed βA-E and are surrounded by alternating α-helices termed αA-E . Loops cR domain . The resR domain . These fOur efforts to elucidate the crystal structure of TcpC have failed so far; however, crystal structures of PdTLP and TcpB were reported recently ,20,21,24Although the basic arrangement of secondary structure β-strands and α-helices of bacterial and eukaryotic TIR-domains is very similar, a detailed structural comparison of the TIR-domain loop position of bacterial Tcps, TLRs and TLR adaptors revealed that these three TIR-domain containing subfamilies exhibit unique conformations of corresponding bacterial, receptor and adaptor loop positions, which may be important for function. For example, the position of the well characterized BB-loop differed substantially between the bacterial Tcps, PdTLP and TcpB, the TLR adaptor molecules, MyD88 and TIRAP, and the TLRs, TLR1, 2 and 10 . Althougin vivo and to induce urinary tract disease like kidney abscesses in mice by impairing TLR-signaling. This principle of virulence is spread among a variety of human pathogens but also non-pathogens. TcpC is secreted by UPECs and this process can be blocked by the efflux pump inhibitor phenylalanine-arginine-β-naphtylamide (PAβN) demonstrating the principle possibility to neutralize this new virulence factor. Therefore, deeper knowledge of the mechanisms involved in TcpC secretion might offer new target structures for small molecules to block this process and by that a strategy to develop pathogen-specific treatment strategies. This strategy would also reduce the risk of the development of antibiotic resistance in the human commensal flora, since most of these bacteria lack tcpC genes. By molecular mimicry bacteria interfere with their recognition by host TLRs. A substantial part of UPECs harbor TcpC in their genomes. In case of CFT073, TcpC was shown to increase bacterial burden Autoimmune diseases are characterized by inflammatory processes of non-infectious cause. It is assumed that endogenous ligands such as β-defensins or self DNA/RNA from necrotic cells trigger TLRs and by that inflammation is maintained . Thus, s


The reduction in coral cover on many contemporary tropical reefs suggests a different set of coral community assemblages will dominate future reefs. To evaluate the capacity of reef corals to persist over various time scales, we examined coral community dynamics in contemporary, fossil, and simulated future coral reef ecosystems. Based on studies between 1987 and 2012 at two locations in the Caribbean, and between 1981 and 2013 at five locations in the Indo-Pacific, we show that many coral genera declined in abundance, some showed no change in abundance, and a few coral genera increased in abundance. Whether the abundance of a genus declined, increased, or was conserved, was independent of coral family. An analysis of fossil-reef communities in the Caribbean revealed changes in numerical dominance and relative abundances of coral genera, and demonstrated that neither dominance nor taxon was associated with persistence. As coral family was a poor predictor of performance on contemporary reefs, a trait-based, dynamic, multi-patch model was developed to explore the phenotypic basis of ecological performance in a warmer future. Sensitivity analyses revealed that upon exposure to thermal stress, thermal tolerance, growth rate, and longevity were the most important predictors of coral persistence. Together, our results underscore the high variation in the rates and direction of change in coral abundances on contemporary and fossil reefs. Given this variation, it remains possible that coral reefs will be populated by a subset of the present coral fauna in a future that is warmer than the recent past. Most present-day coral reefs differ from the reefs that were first described by ecologists and explorers Porites spp. is an example of a group of corals that is faring better than others and is increasing in abundance in the Pacific and Caribbean Coral reefs in remote settings provide some of the best examples of reefs with high coral cover and intact trophic structures Considerable effort is being dedicated to elucidating the processes driving shifts in coral community structure on contemporary reefs, and characterizing the biological and ecological traits of scleractinian corals that are resistant to disturbances The goals of this study were to use long-term data from modern and fossil coral reefs to test for variation among coral genera in the rates and directions of change in abundance over time, to use these trends to consider which genera have the potential to persist as seawater warms through climate change, and to evaluate in what form these genera might assemble in the future to form coral communities. To achieve these goals, we synthesized data from extant reefs at seven locations (“case studies”) and from fossil coral reef communities in the Caribbean, and developed a mathematical model to evaluate which traits are most likely advantageous in promoting persistence of coral genera in warming oceans. We present our analyses in three parts: first, we describe the events taking place on extant reefs by examining aspects of ecological records from our case studies ; second, we use the fossil record  to gain insight into the temporal novelty of the changes affecting the community ecology of extant reefs, and whether clues to the ultimate outcomes of these changes might be found in the past; finally, we use a mathematical model to offer insight into the potential ecological fate of coral reefs under increased thermal stress .Recent efforts to describe changes in the composition of coral reef communities have typically focused on scleractinian corals and their performance relative to other functional groups such as macroalgae 2) that would individually have considerable leverage on the analysis. The GBR data were therefore collapsed into three habitats - inshore (11 reefs), mid shelf (18 reefs), and outer shelf (18 reefs) - and pooled among latitudes. For all sites, data were summarized annually as percentage cover of scleractinian corals by genus, using taxonomy as described in recent papers To support our analysis, data were gathered from nine projects in seven locations where multiple sites have been censused frequently, and together span up to 33 y  (Table S1 in −1) were used in subsequent analyses, and slopes were used regardless of their statistical significance. While the significance of any one slope can be evaluated with P values based on the ratio of the mean sum of squares explained by the regression and unexplained variance, in data compilations such as used in the present study, the least squares estimate of the slope is an unbiased estimate of the true slope that is preferable to the biased slope estimate derived by assuming non-significant slopes have a value of zero. The frequency distribution of these slopes would then be distributed more uniformally than one in which non-significant slopes were set to zero, which would create an ecologically unrealistic gap between actual zero slopes and the larger slopes that are statistically significant. Having calculated the slopes of the relationships between coral cover and time, the frequency distributions of the slopes were tested for skewness using a g2 test, and for normality using a Kolmogorov-Smirnov (K-S) test. Our objective was to understand how corals were responding to the combined effects of biotic and abiotic disturbances extending over multiple decades, and we did not intend to partition these changes to the effects of individual pulse or press disturbances. Such disturbances might reflect rapidly-acting events such as severe storms, or a large-scale predator outbreaks , or chronic effects such as rising seawater temperature or declining seawater pH. Therefore the rates of change in coral cover we report cannot be attributed to specific causal processes and cannot be used to distinguish between the response of corals to recent dramatic and local events versus long-lasting, chronic, and regional-scale events. Instead, our analyses attempted to ‘capture’ the culmination of the above-mentioned processes and events as time-averaged rates of change in cover of each coral genus.The rate of change in coral cover by genus over the duration of each study was evaluated using least-squares linear regression, with analyses separated for the Caribbean (two locations) and Indo-Pacific (five locations)  changes in abundance were independent of overall dominance in the community, meaning that abundant and rare coral genera were likely to share similar trajectories of change; and (2) the covariance between changes in absolute and relative abundance was random with regard to distributing coral genera on these axes, meaning that coral genera were equally likely to have any fate defined by all possible combinations of changes in absolute and relative abundances. These analyses were conducted to evaluate the effect of abundance on change in relative abundance , and were designed so that they could be completed for fossil data as well as ecological data from our case-study locations. Dominance was evaluated as the rank abundance by genus across the entire data set, and the relationship between abundance and success was evaluated separately for our case-history sites from the Caribbean and Indo-Pacific.strongest ecological performance by increasing cover on both absolute and relative scales; (2) M-domain corals showed moderate ecological performance by increasing in absolute cover but declining in relative cover, because other taxa increased faster still; (3) W-domain corals showed weak ecological performance by declining in absolute cover but increasing in relative cover, because other taxa declined in coral cover at a faster rate; and (4) F-domain corals showing failing ecological performance by decreasing in cover on both absolute and relative scales. The covariance between changes in absolute and relative abundance was analyzed with Pearson correlations to test for a random association between variables. Rejection of the null hypothesis would indicate a positive or negative association. Additionally, the distribution of the coral genera among the S-, M-, W- and F- domains was tested for equality using a χ2 test.The associations between change in relative abundance and rank dominance were tested using Pearson correlations, first by genus, and second by family. The two analyses were used to evaluate the extent to which the dominance–success relationships were independent of taxon. The analyses of covariance between absolute and relative abundance were used to identify genera that had increased in absolute and relative cover, and to evaluate regional variation in these characteristics. Based on the ratio of the change in absolute to relative coral cover, coral genera were separated into four domains with differing trajectories of change in cover: (1) S-domain corals showed the 2 ≥14.9 −1), and departed significantly from a normal distribution   . At leas−1  to 0.004±0.005% y−1  for the Caribbean, and from −0.439±1.056% y−1  to 0.638±0.004% y−1  for the Indo-Pacific . On a relative scale, the range of changes were -0.149±0.319% y−1  to 0.172±0.236% y−1  for the Caribbean, and ranged from −0.869±1.572% y−1  to 1.274±2.695% y−1  for the Indo-Pacific. Mean coral abundance by genus, averaged across all study locations and dates within each region, ranged from 0.002% (Mussa) to 15.538% (Orbicella) for the Caribbean, and from 0.002% (Stylocoeniella) to 7.031% (Acropora) for the Indo-Pacific. The regions were characterized by 16 and 41 coral genera respectively, with these representing 9 families in the Caribbean and 12 families in the Indo-Pacific. Project-wide mean coral covers were used to establish a ranking scheme for the abundance of coral genera and families in each location (Table S4 in P≥0.619). Analysis of these relationships at a high taxonomic level was problematic because of the limited replication of genera within each family, although this was accomplished for Meandrinidae (n = 3 genera) and Mussidae (n = 6 genera) in the Caribbean, and Acroporidae (n = 4 genera), Agariciidae (n = 3 genera), Lobophylliidae (n = 5 genera), Merulinidae (n = 12 genera), Pocilloporidae (n = 5 genera) and Poritidae (n = 4 genera) in the Indo-Pacific. For the Caribbean, the trajectories of change in relative cover did not vary between families , and for the Mussidae, they were unrelated to relative dominance in the community  , and for the Acroporidae, Lobophylliidae, Merulinidae, Pocilloporidae, and Poritidae, were unrelated to relative dominance in the community   and Indo-Pacific . Overall, there were positive relationships between the rates of change in relative and absolute cover, showing that there were more genera in the S- and F- domains than chance alone would predict. This outcome was consistent with the distribution of genera among the F-, M-, S- and W- domains, which departed significantly from an expectation of equal representations for the Caribbean  and Indo-Pacific ; in these cases, significance was a result of the relatively large number of genera in F-domain for the Caribbean and in the F- and S- domains for the Indo-Pacific. S- and F- domain corals were found in the Caribbean and in the Indo-Pacific, but ≤3 genera were categorized as M- or W-domain corals in either region .Analyses of the covariation between relative and absolute cover by genus  rather than unequivocal declines Orbicella in the US Virgin Islands and Belize and Pocillopora and Acropora in Moorea, the GBR, Kenya, Hawaii, and Taiwan, have declined rapidly, and (3) the absolute or relative covers of only a few coral genera have increased.In summary, our analyses of coral communities at seven locations describe trends that provide higher resolution details of the large-scale declines of coral cover that have been reported elsewhere pCO2 was as high as 400 ppmv Motivated by the findings from our analysis of extant coral reefs, which revealed evidence of diverse trajectories of change that was dependent on genus , but thahttp://nmita.iowa.uiowa.edu/index.htm).To describe the dynamics of coral communities on fossil reefs, we drew on records extending from 0.125 to 6.8 Ma, which encompassed the separation of the Caribbean and Indo-Pacific biogeographic regions. Samples were extracted from outcrop exposures at 70 localities through four Miocene-Pleistocene sequences: Costa Rica −1) were used as a measure of relative success or failure  were plotted against the rank of relative dominance.Late Miocene and Pliocene coral reefs supported more diverse coral assemblages than extant coral reefs in the Caribbean, and their taxonomic composition overlapped at the genus level with contemporary Indo-Pacific coral communities. Approximately 80% of the>100 coral species became extinct during the Plio-Pleistocene, and over 60% of the species that now live in the modern Caribbean originated during that period D = 0.078, n = 39, P = 0.971), ranging from −1.758% Myr−1 (Trachyphyllia) to 2.650% Myr−1 (Acropora) (−1) of the slope of abundance on time . Analysis of the relationship between changes in relative abundance and dominance rank by genus , although more abundant taxa displayed larger shifts in relative abundances (both increases and decreases). Interestingly, coral families often were represented by some genera that increased in relative abundance and others that decreased. This suggests that the relative success of coral genera is independent of family affiliation. These results collectively are consistent with other work that focused on the fossil record and past extinction events Fossil data revealed information on 39 coral genera, which showed normally distributed changes in relative abundances (cropora) . Of the In summary, our analysis of fossil communities demonstrated that coral genera responded differently to Plio-Pleistocene environmental perturbations , maturation, and dispersal Mathematical models are used regularly to explore coral dynamics under general or specific climate change scenarios . Our goal was not to synthesize the existing models or to explicitly forecast the fate of any particular coral taxon. Instead, our intent was to build a generic model of the ecological dynamics of the full suite of different possible corals under expected future environments, where we consider each coral in isolation and use global sensitivity analysis GSA  to ask wi, AiR, and iM represent the proportion cover of coral recruits, coral adults, and macroalgae in each patch i of n patches in total. Changes in each of these state variables are given by the differential equations: Ar represents coral recruitment (from reproduction by adults), δ is the proportion of dispersing larvae, a quantifies coral maturation, g represents adult coral growth, Rd and Ad represent recruit and adult coral loss of cover due to mortality and/or shrinkage, respectively; Mr is algal growth, bh is baseline algal mortality , sh is additional algal mortality from recruited herbivores (a rate with the same 1/time units as bh), ω scales the rate at which adult corals provide habitat for herbivores , and β≤Mr is the rate at which algae overgrow adult corals depending on the degree of overgrowth resistance. Note our assumption here that the relative coral versus macroalgal cover affects grazing rate, as suggested in Our model  is a staThe model is driven by stochastic thermal anomalies with frequency and intensity drawn from the GFDL 2.1 climate model for 2051–2100 For each location and each climate scenario, we simulated dynamics for 1000 different collections of randomly chosen coral parameters. After discarding 50 y of transients, we recorded the average coral cover across all patches for 20 y. We then pooled simulations across locations and ran a global sensitivity analysis GSA  for eachWe repeated the exercise for different simulation assumptions to determine the robustness of our results. In particular, we varied the number of patches, spatial variance in thermal stress, and the parameter range for coral recruitment. We investigated the recruit and adult cover sensitivity separately, and explored sensitivity with coral dynamics only . The relative rankings presented are consistent across all of these tests as well as consistent across the four locations (consolidated in the results presented here).As is inevitable with models, this model excludes more than it includes. For example, the model does not explicitly include other components of predicted future environments , although we quantify the importance of the demographic processes that other environmental changes are expected to impact. For example, a high importance value for coral growth rates would suggest that total coral cover would be strongly influenced by decreases in growth rates from ocean acidification. The model also does not account for factors such as genetic adaptation of corals or their symbionts, competition or other interspecific interactions among multiple coral species on the same reef, hydrodynamical differences in susceptibility to disturbance based on relative position in the reef, or size-dependent demographics and disturbance susceptibility beyond our two stage classes (recruits and adults). These exclusions are necessary to keep the model tractable and transparent, but provide ample scope for future work.Our GSA suggested that the most important ecological processes and biological traits favoring coral persistence  were aduOf the possible two-way combinations between fast growth, low mortality, and high thermal tolerance, a coral having both low adult mortality and high thermal tolerance is most likely given life history trade-offs between growth and longevity, and the tendency for massive coral morphology to be associated with both slow growth and greater thermal tolerance While the most influential parameters: bleaching resistance, coral growth, and adult coral mortality are intuitively important, what is perhaps more surprising is that these three parameters clearly stand apart from others that relate to processes that have previously been identified as essential to future reef persistence. In particular, our model suggests that larval dispersal and recruitment, juvenile maturation, and competition with macroalgae , while always part of our modeled dynamics, are less influential in comparison with other processes in determining projected coral abundance. These results might seem to contradict those of other models in which recruitment dynamics and coral-macroalgal interactions are important to future coral persistence With respect to recruitment, previous studies indicate that larval exchange between multiple patches of coral experiencing different stress levels affects the outcome of coral population projections With respect to coral-macroalgal competition, previous studies found coral cover was highly sensitive to competition with macroalgae, particularly where disturbances pushed coral cover close to an unstable threshold between coral-dominated and macroalgal-dominated states We simulated coral reef community dynamics using climate model output from the fourth IPCC assessment report, released in 2007. Sea surface temperature projections are available from the fifth IPCC assessment report, released in 2013. However, large increases in mean and maximum sea surface temperature in AR5 relative to AR4 caused decreases in simulated coral cover to under 10% in all model run and extirpation in>90%  than increased (n = 5) in absolute abundance, but almost equal numbers increased (n = 21) and decreased (n = 20) in the five Indo-Pacific locations. These results suggest that the future of scleractinian corals over the current century will likely include the persistence and perhaps increased abundances of some coral genera, even while others become less common. These results highlight the strong likelihood that changes in the generic-level composition of coral communities will occur in coming decades Acropora, Stylophora, and Pocillopora, and Caribbean Acropora, Orbicella, and Madracis – relatively little is known about the functional biology of taxa that are emerging as S-domain corals Click here for additional data file.


Xanthine oxidoreductase (XOR) is involved in oxidative metabolism of purines and is a source of reactive oxygen species (ROS). As such, XOR has been implicated in oxidant-mediated injury in multiple cardiopulmonary diseases. XOR enzyme activity is regulated, in part, via a phosphorylation-dependent, post-translational mechanism, although the kinase(s) responsible for such hyperactivation are unknown.in silico approach, we identified a cyclin-dependent kinase 5 (CDK5) consensus motif adjacent to the XOR flavin adenine dinucleotide (FAD) binding domain. CDK5 is a proline-directed serine/threonine kinase historically linked to neural development and injury. We tested the hypothesis that CDK5 and its activators are mediators of hypoxia-induced hyperactivation of XOR in pulmonary microvascular endothelial cells (EC) and the intact murine lung. Using complementary molecular and pharmacologic approaches, we demonstrated that hypoxia significantly increased CDK5 activity in EC. This was coincident with increased expression of the CDK5 activators, cyclin-dependent kinase 5 activator 1 (CDK5r1 or p35/p25), and decreased expression of the CDK5 inhibitory peptide, p10. Expression of p35/p25 was necessary for XOR hyperactivation. Further, CDK5 physically associated with XOR and was necessary and sufficient for XOR phosphorylation and hyperactivation both in vitro and in vivo. XOR hyperactivation required the target threonine (T222) within the CDK5-consensus motif.Using an These results indicate that p35/CDK5-mediated phosphorylation of T222 is required for hypoxia-induced XOR hyperactivation in the lung. Recognizing the contribution of XOR to oxidative injury in cardiopulmonary disease, these observations identify p35/CDK5 as novel regulators of XOR and potential modifiers of ROS-mediated injury. Xanthine oxidoreductase (XOR) is a prototypical molybdo-flavoenzyme known for its role in purine catabolism, oxidizing hypoxanthine into xanthine and xanthine into uric acid . XOR alsIn vitro data indicates that XOR is a phosphoprotein . To . To 7]. 2+, ATP and buffer. H1 phosphorylation, a CDK5 target, was assessed by Western blotting. The phosphorylation status of H1 was thus a marker of CDK5 activity. There was a significant and time-dependent increase in CDK5 activity in hypoxic compared to normoxic EC  protein in 293A cells via transient transfection and purified it using chromatography as detailed in Methods. The hXOR was then incubated in the absence or presence of immunoprecipitated CDK5 with and without its inhibitor olomoucine with ATP and Mgine site .2+. The XOR enzymatic activity was assessed using the Amplex Red assay in the presence or absence of olomoucine. There was no demonstrable difference in the activity of T222 versus A222 XOR at baseline indicating that T222 was not obligatory for basal enzymatic activity. The activity of T222 XOR was significantly increased in the presence of CDK5  of the CDK5 consensus sequence and an alanine mutant (A222) that cannot be phosphorylated at residue 222. Both recombinant hXOR proteins were then incubated in the presence or absence of immunopurified CDK5 with ATP and Mg of CDK5 , coincid of CDK5 . In cont of CDK5 . CDK5 enin vivo, we exposed 8 week old male C57/Bl6 mice to hypoxia (10% O2) or room air for 24 hours with designated arms receiving pretreatment with olomoucine (3mg/kg) versus vehicle delivered via peritoneal injection. At the end of exposure to hypoxia, there was significant lung XOR hyperactivation compared to normoxia  , we demoCDK5 activity is controlled, in part, by binding to its regulatory subunit p35 and/or the p35-derived peptide p25, which contribute to CDK5 hyperactivation in certain disease states . RecogniAnother binding partner and potential activator of CDK5 is p39 which is homologous to p35 . Unlike in vitro [Importantly, we also demonstrate that CDK5 is sufficient to increase XOR activity and that this CDK5-mediated hyperactivation is dependent on an intact CDK5 consensus sequence, specifically threonine 222. Consistent with our prior work We identified a consensus target sequence for CDK5, a serine/threonine kinase, adjacent to the FAD binding domain within XOR. Here, we demonstrate that hypoxia-induced hyperactivation of XOR is associated with a significant increase in its immunoreactivity for phosphothreonine specific antibodies . In addiin vivo and in pulmonary EC. Hypoxia increases expression of p35/p25 in EC and these CDK5 regulators are necessary for XOR hyperactivation. Finally, threonine 222 of XOR is the critical target site for CDK5-dependent activation of XOR. The link between XOR and CDK5, as well as, their importance in EC response to hypoxia are novel findings. Thus, we demonstrate a novel role for p35-CDK5 in the regulation of XOR.In conclusion, our findings demonstrate CDK5 is sufficient and necessary for hypoxia-induced XOR activation both


Rhodococcus equi is an important pulmonary pathogen in foals and in immunocompromised individuals. Virulent R. equi strains carry an 80-90 kb virulence plasmid that expresses the virulence-associated protein A (VapA). VapA expression is regulated by temperature and pH. The LysR-type transcriptional regulator, VirR, is involved in the regulation of the vapA gene. To examine the mechanism underlying transcriptional regulation of vapA, we characterized an R. equi mutant in which another putative transcriptional regulator encoded on the virulence plasmid, VirS, was deleted.virS reduced vapA promoter activity to non-inducible levels. Complementary expression of VirS in the virS deletion mutant restored transcription at the PvapA promoter, even under non-inducing conditions (30°C and pH 8.0). In addition, VirS expression increased PvapA promoter activity in the absence of functional VirR. Further, transcription of the icgA operon containing virS was regulated by pH and temperature in the same manner as vapA.Deletion of vapA-promoter activity may be achieved by controlling VirS expression levels.This study suggests that VirS is required for VapA expression and that regulation of PThe online version of this article (doi:10.1186/s12866-014-0243-1) contains supplementary material, which is available to authorized users. Rhodococcus equi is a Gram-positive bacterium and a facultative intracellular pathogen of alveolar macrophages. Rhodococcus equi can cause bronchopneumonia in foals up to five months of age  was synthesized and cloned into pUC57at the EcoRI and HindIII sites. Next, the apramycin resistance gene cassette was excised by digestion with EcoRI and HindIII, then cloned into pBluescript II SK(+) digested with EcoRI and HindIII to create pTKR131. oriT was amplified from pEco101 by polymerase chain reaction (PCR) using primers oriT-F and oriT-R. The PCR product was digested with SpeI and EcoRI, and cloned into pTKR131 digested with SpeI and EcoRI to create pTKR144. The aphII promoter (PaphII) region was amplified using primers aph2-F and aph2-R. The amplified DNA fragment was digested with HindIII and ClaI, and cloned into pTKR144 digested with HindIII and ClaI to create pTKR159. Finally, the codA-upp cassette was excised from pORF-codA-upp  by digesting with NcoI and HindIII, and then cloned into pTKR159 digested with NcoI and HindIII to create pDelta. Primers used in this study are listed in Additional file To construct mutants with unmarked in-frame gene deletions within Streptomyces ϕC31 integrase gene was constructed to generate the integration vector for the complementation experiments  of the culture onto MM-Ac agar plates supplemented with 5-FC (100 μg/mL). Plates were incubated at 30°C for 2–3 days. Virulence plasmids were isolated from 5-FC-resistant and apramycin-sensitive mutants, and analyzed by digestion with EcoRI. Mutants that produced the expected digestion pattern were selected . The deleted region in the virS gene comprised codons 2–252. Fragments that contained ΔvirS and virRΔHTH were excised from pTKR361 and pTKR226 by EcoRI digestion and ligated to EcoRI-digested pDelta to create pTKR265 and pTKR391, respectively. pTKR265 and pTKR391 were separately electroporated into the PvapA-lacZ strain (TKR255), and the ΔvirS and virRΔHTH mutants  were selected and confirmed as described above  was produced using the primer pair virS D57A-1 and virS D57A-2. This plasmid was electroporated into TKR303. Transformants were recovered on LB agar containing 60 μg/mL apramycin.The virR ORF and promoter region was amplified using the primer pair PvirR-F and virR-R. The DNA fragment was cloned into the pGEM-T vector to create pTKR509. pTKR509 was digested with NotI and ligated to NotI-digested pINT to create pTKR528. pTKR528 was electroporated into TKR474. Transformants were recovered on LB agar containing 60 μg/mL apramycin.The fragment that contained the 600) of 0.5–0.7. Cells were washed twice with 0.9% NaCl and resuspended in 500 μL Z buffer  . To. TovirS l Figure . ComplemvapA-lacZ in the virS (Asp57Ala) mutant was comparable to that of the strain expressing wild-type VirS when they were both grown under inducing conditions  was deleted and the promoter of the icgA operon was kept intact. The virRΔHTH mutation reduced the transcription level of PvapA-lacZ to undetectable levels under non-inducing conditions  to R. equi virulence and the expression of VapA, we constructed an R. equi virS deletion strain paired with a PvapA-lacZ reporter virulence plasmid. In the current study, we demonstrate that VirS is another transcriptional regulator encoded on the virulence plasmid that is required for vapA transcription.Pneumonia-causing virulence by the bacterial pulmonary pathogen  plasmid . To examvirS reduced the transcriptional activation of the vapA promoter to non-inducible levels. Further, previous studies have demonstrated that virR is constitutively expressed and that VirS has no effect on the transcription of virR [ΔvirS mutant, and that VirR alone is not sufficient to activate the transcription of vapA in the absence of VirS. In addition, deletion of virR completely abolished vapA promoter activity. However, it has previously been demonstrated that VirR is required for VirS expression [vapA gene transcription. Indeed, VirS expression via the PaphII promoter restored expression of the PvapA-lacZ fusion plasmid in the virRΔHTH mutant, thereby supporting this hypothesis. Furthermore, when VirS was expressed via the PaphII promoter, there was no difference in transcription from the vapA promoter in the presence or absence of VirR. These results indicate that VirS can activate vapA transcription via the vapA promoter in the absence of functional VirR.With this work, we show that deletion of  of virR . Togethepression . Thus, waphII-virS fusion did not restore PvapA-lacZ expression to wild-type levels in the ΔvirS and virRΔHTH deletion mutants. Furthermore, activation of the PvapA promoter was observed under non-inducing conditions. It is possible that expression of the PaphII-virS fusion, which was present as a monocopy on the chromosome, may have resulted in lower VirS expression levels when compared with VirS expression from the virulence plasmid, as each cell harbors two or more plasmid copies [aphII promoter but not from the original promoter found on the virulence plasmid.Interestingly, chromosomal integration of the Pd copies . In addivapA promoter under non-inducing conditions; thus, pH and temperature are unlikely to be the stimuli that are responsible for VirS activation. We further observed that the Asp57Ala mutation did not affect activity of the PvapA promoter when compared with that of wild-type VirS under inducing conditions. These data suggest that this putative phosphorylation site is not necessary for function of this domain. Although most residues that are critical for canonical functions in response regulators are well conserved in VirS, Asp13, which chelates the Mg2+ necessary for aspartic acid phosphorylation, is substituted with arginine [E. coli CheY protein means that it cannot chelate Mg2+ effectively and phosphorylation of Asp57 is blocked [Myxococcus xanthus FrzS and Helicobacter pylori HP1043 retain their functional activity in the absence of phosphorylation [VirS is an orphan response regulator, and its cognate sensor is not found on the virulence plasmid . VirS caarginine -35. Subs blocked . Howeverrylation -38. ThericgA promoter is regulated by both temperature and pH, and this corresponds to regulation of the PvapA promoter. Further, expression of VirS from the PaphII promoter could induce the vapA transcription under non-inducing conditions. These results suggest that regulation of vapA by temperature and pH may be achieved by controlling VirS expression levels, and a proposed model for this regulation is presented in Figure icgA operon are likely crucial in controlling vapA gene expression via environmental stimuli in the bacterial pathogen R. equi.In the present study, we demonstrated that the PvapA, and VirS expression is regulated by both temperature and pH. We hypothesize that regulation of the PvapA promoter is influenced by VirS expression levels. Future studies are required to examine the mechanisms that regulate transcription of the virS-containing icgA operon, and how this operon contributes to R. equi virulence.VirS is required for the expression of


The structure of the hEAG PAS domain is presented and compared with similar segments of human and insect ion channels. ether-à-go-go channel (hEAG channel) was determined. By comparing it with the structures of the Homo sapiens EAG-related gene (hERG) channel and the Drosophila EAG-like K+ (dELK) channel and analyzing the structural features of the hEAG channel, it was identified that a hydrophobic patch on the β-sheet may mediate interaction between the PAS domain and other regions of the channel to regulate its functions.KCNH voltage-gated potassium channels play critical roles in regulating cellular functions. The channel is composed of four subunits, each of which contains six transmembrane helices forming the central pore. The cytoplasmic parts of the subunits present a Per–Arnt–Sim (PAS) domain at the N-terminus and a cyclic nucleotide-binding homology domain at the C-terminus. PAS domains are conserved from prokaryotes to eukaryotes and are involved in sensing signals and cellular responses. To better understand the functional roles of PAS domains in KCNH channels, the structure of this domain from the human  We present the structural details and discuss the implications for the functional roles of the PAS domain in the hEAG channel.In this study, we first determined the crystal structure of the PAS domain found at the N-terminus of human EAG  cells for expression.The DNA sequence encoding residues 1–146 of the N-terminus of the human EAG channel, named the N-PAS domain, was amplified from a human cDNA library by PCR. The gene was cloned into the pET-GST vector (Invitrogen), which adds a glutathione transferase (GST) tag and a PreScission protease cleavage site at the N-terminus. The reconstructed plasmids were transformed into E. coli cells were cultured until the OD600 reached ∼0.6 at 310 K and overexpression of the fusion protein was then induced using 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 298 K for 16 h in LB medium . The cells were harvested by centrifugation, resuspended in buffer A  and lysed by sonication. The lysate was centrifuged at 18 000g for 40 min and the supernatant was loaded onto a GST affinity column which had been equilibrated with buffer A. The column was washed with buffer A and eluted using 10 mM reduced glutathione. The fusion protein was cleaved with 2 mg ml−1 PreScission protease at 277 K for 16 h to remove the N-terminal GST tag. A HiTrap Q column  equilibrated with 20 mM Tris–HCl pH 7.5 was then used to remove the N-terminal GST tag. The column was eluted with an NaCl concentration gradient from 5 to 700 mM, which was applied to the column over a 75 min period, and the target protein eluted between 200 and 260 mM NaCl. Finally, the eluate was concentrated to 10 ml by ultrafiltration using a 10 kDa cutoff membrane. The eluate was then loaded onto a HiLoad 26/60 Superdex 200 size-exclusion column in the presence of 20 mM Tris–HCl pH 7.5, 200 mM NaCl, 5 mM DTT and was immediately subjected to crystallization trials. SDS–PAGE was used to determine the purity. The calculated molecular mass of the N-PAS domain is 16 933.16 Da. The eluted peak containing the N-PAS domain from the HiLoad 26/60 Superdex 200 size-exclusion column corresponded to a dimer. Samples were applied to SDS–PAGE and showed a single band at ∼17 kDa for the N-PAS domain . Finally, a UV spectrophotometer was used to determine the concentration, and the molar extinction coefficient was calculated using Vector NTI (Thermo Fisher). The molar extinction coefficient was 18 260 and one A280 unit corresponds to 0.93 mg ml−1 protein. The sample was concentrated to 20 mg ml−1 for crystallization.The in Fig. 1a. Final2.2.−1 N-PAS domain, 20 mM Tris–HCl pH 7.5, 200 mM NaCl, 5 mM DTT) was mixed with 1 µl precipitant solution  as well as Wizard I and II . For crystallization, 1 µl protein solution (20 mg mln Table 1. Differev/v) glycerol as a cryoprotectant on beamline BL17U at Shanghai Synchrotron Radiation Facility  and the figures were prepared using PyMOL (http://www.pymol.org).The structure of the N-PAS domain was determined by molecular replacement using 3.3.1.M sodium malonate pH 6.0, 20%(v/v) PEG 3350 and condition No. 88 consisting of 0.03 M citric acid pH 7.6, 0.07 M bis-tris propane pH 7.6, 20%(v/v) PEG 3350. These conditions were optimized, and after a week crystals were obtained in a condition consisting of 20%(v/v) PEG 3350, 0.1 M bis-tris propane pH 7.0. The crystals were suitable for X-ray analysis and diffracted to ∼3.5 Å resolution on beamline BL-17U1 at SSRF. The Additive Screen, Detergent Screen and Silver Bullets kits  were used for further optimization. Larger crystals were obtained after 7 d using condition No. 57 of the Detergent Screen kit: 20%(v/v) PEG 3350, 0.1 M bis-tris propane pH 7.0, 244.0 mMn-octanoylsucrose . The N-PAS domain crystals used for X-ray diffraction were flash-cooled in liquid nitrogen with a cryoprotectant consisting of 20%(v/v) PEG 3350, 0.1 M bis-tris propane pH 7.0, 25%(v/v) glycerol and diffracted to ∼1.9 Å resolution on beamline 17U at SSRF .N-PAS domain microcrystals were obtained at 291 K in condition No. 54 of PEG/Ion consisting of 0.2 se Fig. 1c. The NRF Fig. 1b.3.2.C2, with unit-cell parameters a = 213.974, b = 39.058, c = 106.802 Å, β = 118.93°. Resolution-dependent Matthews coefficient probability analysis suggested the presence of six molecules per asymmetric unit, with around 50% solvent content and a VM of 2.54 Å3 Da−1.The crystal belonged to space group 3.3.We have determined the structure of the PAS domain found at the N-terminus of hEAG. The three-dimensional model consisting of residues 28–137 was refined to 1.9 Å resolution, while the first 27 amino acids could not be detected in the electron-density map. Crystallographic statistics are shown in Table 1a). The topological order of β-strands is 2–1–5–4–3. The secondary structure from β1 to β5 is referred to as the core region of the N-PAS domain and the extensions to the N-terminus and C-terminus are referred to as flanking regions.In the crystal structure of the N-PAS domain, there are six molecules per asymmetric unit. Each molecule displays the canonical fold of a PAS domain comprising a central β-sheet with five strands labelled β1–β5. Four α-helices, α1–α4, decorate the β-sheet Fig. 2a. The tc). The crystal structure of the PAS domain from mouse EAG . The root-mean-square deviation (r.m.s.d.) of the aligned structures is 0.362 Å. The sequence alignment shows that there are two conservative differences between the hEAG PAS domain and the mEAG PAS domain, The68 and Ile80, which were not present in either structure. These results show that the PAS-domain structures of EAG channels are highly conserved.In order to investigate the evolutionary conservation of PAS domains in EAG channels, we aligned the amino-acid sequences of the human, mouse, fruit-fly and zebrafish proteins. The amino-acid sequences are highly conserved Fig. 2c. The c3.4.a). To analyze the differences in the amino-acid sequences, we performed a multiple sequence alignment of PAS domains from hEAG, mERG and dELK. The amino-acid sequences, including the core β-sheet region, have low conservation . These results indicate that while the amino-acid sequences share little similarity among KCNH channels, the secondary structures are highly conserved.To better understand the structural features of PAS domains from KCNH channels, we compared the structures of PAS domains from hEAG, mERG and dELK. We superposed these structures using their main-chain atoms. The overall structures show high similarity, apart from the N-terminal helix, which is not defined in the hEAG structure. The main differences appear in the β1–β2, α4–α5, α5–β3 and β4–β5 loop regions Fig. 3a. To anon Fig. 3b. These3.5.b). The patch is thought to mediate the interactions between the PAS domain and other channel regions or those among PAS domains . In the hERG and dELK structures, the patches are found in the same region and also mediate the intermolecular interaction  and is highly conserved in hERG and dELK. These results suggest the possibility that the functions of KCNH channels may be regulated by the interactions between this hydrophobic patch on the PAS domain and other entities. Based on the previously reported structures, the PAS domain is a monomer in hERG, while it is a dimer in dELK. It is possible that the dimerization of PAS domains may have little effect on the assembly of the channels. However, another crucial region regulating the functions of the hEAG channel, residues 1–27, at the N-terminus was not defined in the structure (Schönherr & Heinemann, 1996One of the interesting regions in the structure of the N-PAS domain of hEAG is a hydrophobic patch on the outer side of the β-sheet Fig. 4b. The p D Fig. 4c. In thDespite many years of study, the functional roles of the PAS domain in KCNH channels still remain unclear. In particular for EAG, which has been determined as a diagnosis marker or a therapeutic target, clarification of the functional roles of the PAS domain is urgently required. In our study, we determined the structure of the PAS domain at the N-terminus of hEAG. The overall structure fits the conserved fold of the domain family. Alignment with previously determined structures of PAS domains from hERG and dELK indicates that the hydrophobic patch on the outer surface of the β-sheet may mediate both the interaction between homodimers and the interaction between the PAS domain and other channel regions in order to regulate the channel function.hEAG PAS domain, 5j7ePDB reference:


To that end, we characterized the localization of exogenously expressed Ribeye A- and B- domains and the closely related protein, CtBP1, in the hair cells of transgenic zebrafish larvae. Unexpectedly, exogenously expressed Ribeye A-domain showed variable patterns of localization in hair cells; one zebrafish paralog of A-domain failed to self-associate or localize to synaptic ribbons, while the other self-assembled but sometimes failed to localize to synaptic ribbons. By contrast, Ribeye B-domain/CtBP2 was robustly localized to synaptic ribbons. Moreover, both exogenously expressed B-domain/CtBP2 and CtBP1 were preferentially localized to the basal end of ribbons adjacent to the postsynaptic density. Overexpression of B-domain/CtBP2 also appeared to affect synaptic-ribbon composition; endogenous levels of ribbon-localized Ribeye were significantly reduced as hair cells matured in B-domain/CtBP2 transgenic larvae compared to wild-type. These results reveal how exogenously expressed Ribeye domains interact with synaptic ribbons, and suggest a potential organization of elements within the ribbon body.Synaptic ribbons are presynaptic structures formed by the self-association of RIBEYE–the main structural component of ribbon synapses. RIBEYE consists of two domains: a unique N-terminal A-domain and a C-terminal B-domain that is identical to the transcription co-repressor C-terminal binding protein 2 (CtBP2). Previous studies in cell lines have shown that RIBEYE A-domain alone is sufficient to form ribbon-like aggregates and that both A- and B- domains form homo-and heterotypic interactions. As these interactions are likely the basis for synaptic-ribbon assembly and structural plasticity, we wanted to examine how zebrafish Ribeye A- and B- domains interact with synaptic ribbons  Sensory receptors of the visual, auditory, and vestibular systems contain presynaptic specializations that enable them to transmit sensory information with high fidelity and for extended periods of time Sensory receptors in different organs and species have distinct coding requirements, and synaptic ribbon architecture likely contributes to synaptic function–synaptic ribbons are found in a variety of different sizes and shapes, from generally spherical in hair-cell synapses in situ; that is, within an intracellular milieu containing all the necessary components to generate synaptic ribbons, should give important further insight into how RIBEYE self-association is regulated.RIBEYE is the main structural component of synaptic ribbons Here we characterized the intracellular localization of stably expressed exogenous Ribeye A-domain, B-domain, and the closely related protein CtBP1 within hair cells of transgenic zebrafish larvae at 3 and 5 days-post-fertilization (dpf). Our results indicate that Ribeye A-domain alone does not efficiently localize to synaptic ribbons and suggest a potential substructural organization of CtBPs within synaptic ribbons.This study was performed with the approval of the Oregon Health and Science University Institutional Animal Care and Use Committee as well as the Massachusetts Eye and Ear Infirmary Animal Care Committee, and in accordance with NIH guidelines for use of zebrafish.Transgenic lines were created and maintained in Tübingen, Tupfel Long Fin, and WIK wild-type backgrounds. Adult zebrafish strains were maintained as previously described myosin6b promoter was previously cloned into the 5′entry vector, p5E, 228 . Ribeye domains were PCR amplified from cDNA containing full-length ribeye a (NCBI Accession Number NM_001195491.1) or ribeye b (NM_001015064.1), and ctbp1 was amplified from a cDNA clone ID: 7995426 . The following primers were used to PCR amplify clones for insertion into the Tol2 Middle-entry vector : ribeye a (full length) [5′ggggacaagtttgtacaaaaaagcaggctatgttgatctccagtaagcag3′] and [5′ggggaccactttgtacaagaaagctgggtgggtatacattttgtcttgcaggcc3′]; ribeye a (A domain) [5′ggggacaagtttgtacaaaaaagcaggctatgttgatctccagtaagcag3′] and [5′ggggaccactttgtacaagaaagctgggtgacttgtgtctggtgatgc3′]; ribeye b (A domain) [5′ggggacaagtttgtacaaaaaagcaggctatgatggcagtgcgag3′] and [5′ggggaccactttgtacaagaaagctgggtgacttgtttccggtgaagc3′]; ribeye a (B-domain/ctbp2s) [5′ggggacaagtttgtacaaaaaagcaggctgccaccatgataaggcctcagatcatgaat3′] and [5′ggggaccactttgtacaagaaagctgggtgggtatacattttgtcttgcaggcc3′]; ctbp1 [5′ggggacaagtttgtacaaaaaagcaggctgccaccatggctctgatggacaaac3′] and [5′ggggaccactttgtacaagaaagctgggtg ttggtcggaagggatgtctc3′]. PCR products were confirmed by restriction digest and gel electrophoresis. Middle-entry (pMe) vectors were generated by BP reaction per the Tol2 Kit (Invitrogen Live Technologies). BP reaction products were confirmed by sequencing with M16 forward and reverse primers. Injection plasmids were created by using LR clonase enzyme (Invitrogen), and then subsequently confirmed by multiple restriction digests and gel electrophoresis. To generate transgenic fish, plasmid DNA and tol2 transposase mRNA were injected into zebrafish embryos as previously described Plasmid construction was performed using the Tol 2/Gateway kit . The hair cell specific All nucleotide and protein alignments were performed using Lasergene software (DNAStar).Danio rerio peptide sequences of the A-domains of Ribeye a  and Ribeye b , and the N-terminal region of Vglut3 . We also used clone 9E10 monoclonal and rabbit polyclonal purified antibodies (Sigma-Aldrich) to label c-Myc and K28/86 purified antibody  to label MAGUK.We used previously described 2 for 4 h (3 dpf) or 6 h (5 dpf) at 4°C. Following rinse, larvae were permeabilized with ice cold acetone and blocked with phosphate buffered saline (PBS) containing 2% goat serum, 1% bovine serum albumin (BSA), and 1% dimethyl sulfoxide (DMSO). Then they were incubated with primary antibodies diluted in PBS buffer containing 1% BSA and 1% DMSO overnight at 4°C, followed by diluted secondary antibodies coupled to Alexa 488, Alexa 647 , or DyLight 549 (Jackson ImmunoResearch).Immunohistochemistry was performed as previously described Z-stack images of whole neuromasts (spaced by 0.5 µm over 7–10 µm) were acquired as previously described Digital images were processed using ImageJ software. 3D isosurface renderings were created using Amira 3D Analysis software . Subsequent image processing for display within figures was performed using Photoshop and Illustrator software (Adobe).Quantitative image analysis was performed on raw images using Amira 3D Analysis software. To reduce background noise, a Gaussian smoothing convolution was applied to each x-y image within a stack with a kernel size of 3 and a σ value of 0.4. To quantitatively measure immunolabel intensity, a user-defined inclusive threshold was applied to isolate pixels occupied by immunolabeled Ribeye spheres or MAGUK patches. The inclusive threshold values for each label were determined using 3D isosurface renderings, with the minimum threshold value defined as the value above which the user could resolve two closely adjacent spheres or patches. Minimum threshold values were typically 80 (of 256) for Ribeye and 110 (out of 256) for MAGUK. The Material Statistics function was then used to measure the cumulative intensity of fluorescent pixels  within each individual sphere or patch. Ribbon-synapse localized Ribeye spheres were identified as such by juxtaposing MAGUK patches.ribeye b was first calculated from a cDNA standard curve, then normalized to β-actin RNA. Primers used for ribeye b transcript are as follows: forward 5′-agttgatgcgcaaaggag-3′ and reverse 5′-atggtggacacgatgactg-3.At 5 dpf, groups of 30 transgenic larvae or WT siblings were anesthetized on ice and decapitated with fine surgical scissors to separate the posterior lateral line hair cells of the tail from other ribbon synapse containing tissue in the head. Larval tail tissue was immediately placed into RNAlater (Applied Biosystems/Ambion) and total RNA was extracted using the RNAqueous 4-PCR kit (Applied Biosystems/Ambion). Reverse transcription (RT)-PCR was performed the using 5 ug total RNA and the Sprint RT Complete Oligo(dT) kit (Clontech). For qPCR, 0.2 ul cDNA in SsoFast Supermix (Bio-Rad) with appropriate primers was used for each qPCR reaction, and the reactions were run in 96-well plates using a Bio-Rad CFX96 Real-Time System. The RNA level for in situ by examining its localization in zebrafish stably expressing Ribeye A-domain in lateral-line neuromast (NM) hair cells.The A-domain of RIBEYE contains three specific interaction sites that are thought to mediate RIBEYE self-association ribeye a and ribeye bribeye a (A-domain) fused to a c-terminal 6x-myc tag and ribeye b (A-domain) fused to a c-terminal GFP tag (myosin6b promoter Zebrafish have two copies of the gene encoding Ribeye:  GFP tag . Expressribeye a (A-domain)-6xmyc and ribeye b (A-domain)-gfp, then examined their localization to synaptic ribbons. Because both of the antibodies we use to label synaptic ribbons interact with the A-domains of each paralog of Ribeye Because duplicate genes in zebrafish often recapitulate the functions of a single ancestral gene ribeye a (B-domain) fused to either 6x-myc or GFP in hair cells /CtBP2s by creating transgenic zebrafish lines expressing ir cells . Becauseir cells ; 91%, weic lines . We alsoic lines ; insets.ic lines . We alsoribeye in 5-day-old zebrafish hair cells. We therefore performed qPCR and saw no difference in the relative amount of ribeye b transcript in B-domain overexpressing larvae versus wild-type  Ribeye a (A-domain) fails to self-associate or localize to synapses, (ii) Ribeye b (A-domain) incompletely self-associates, but generally localizes to synapses, (iii) both B-domain/CtBP2s and CtBP1 localize to the nucleus, the cytosol, and synaptic ribbons, (iv) B-domain/CtBP2s disrupts endogenous Ribeye retention at synaptic ribbons in 5-day-old hair cells, and (v) CtBP1 and B-domain/CtBP2s localize to the basal end of synaptic ribbons adjacent to the postsynaptic density. Collectively, these results suggest that zebrafish Ribeye A-domains do not contain all the required elements needed to completely localize to synaptic ribbons, while Ribeye B-domain contains elements that enable effective localization to synaptic ribbons such that it may displace endogenous Ribeye in mature zebrafish hair cells.Self-association of the protein RIBEYE, which is mediated by homo- and heterotypic interactions between its two domains in vivo, which is in contrast to the robust and complete self-assembly of RIBEYE A-domain observed in cell culture. However, the two zebrafish paralogs of A-domain share only ∼50% sequence similarity both with each other and with human RIBEYE A-domain (in vivo.The incomplete self-assembly and variable association of Ribeye A-domains with synaptic ribbons suggest that multiple cellular mechanisms regulate A-domain association with itself and/or full-length Ribeye A-domain , which mBy contrast, zebrafish Ribeye B-domain, which shares a high degree of sequence similarity with mammalian B-domain, appears to associate with synaptic ribbon structures in hair cells in a similar way as previously described in heterologous expression systems; that is, it associates with full-length Ribeye The observation that exogenous B-domain/CtBP2s and CtBP1 accumulated within synaptic ribbons adjacent to postsynaptic densities may reflect the behavior of endogenous CtBP proteins in sensory hair cells. Both CtBP1 and CtBP2s have been found to localize to conventional presynapses throughout the brain While CtBP1is indisputably part of the synaptic ribbon protein complex, there exist conflicting reports as to whether CtBP2 is also a component of synaptic ribbons In conclusion, our results suggest that multiple mechanisms exist in sensory cells that regulate both Ribeye self-association and Ribeye’s interaction with CtBP1 at synaptic ribbons. Our results also raise the possibility that the organization of CtBP proteins within synaptic ribbons may be compartmentalized. It is important to note that overexpression of Ribeye subunits or CtBP1 did not grossly affect 5-day-old zebrafish hearing or balance, however we did not test for subtle defects in auditory or vestibular function. Future studies addressing whether and how an overabundance of CtBP1 or CtBP2 affect ribbon-synapse activity could give important insight into the functional organization of synaptic ribbons.