RMRP-gene, encoding the lncRNA component of the RNase MRP complex, are the origin of cartilage-hair hypoplasia. Cartilage-hair hypoplasia is associated with severe dwarfism caused by impaired skeletal development. However, it is not clear why mutations in RMRP RNA lead to skeletal dysplasia. Since chondrogenic differentiation of the growth plate is required for development of long bones, we hypothesized that RMRP RNA plays a pivotal role in chondrogenic differentiation. Expression of Rmrp RNA and RNase MRP protein subunits was detected in the murine growth plate and during the course of chondrogenic differentiation of ATDC5 cultures, where Rmrp RNA expression was found to be correlated with chondrocyte hypertrophy. Genetic interference with Rmrp RNA expression in ATDC5 cultures caused a deregulation of chondrogenic differentiation, with a prominent impact on hypertrophy and changes in pre-rRNA processing and rRNA levels. Promoter reporter studies showed that Rmrp RNA expression responds to chondrogenic morphogens. Chondrogenic trans-differentiation of cartilage-hair hypoplasia fibroblasts was impaired with a pronounced impact on hypertrophic differentiation. Together, our data show that RMRP RNA expression is regulated during different stages of chondrogenic differentiation and indicate that RMRP RNA may play a pivotal role in chondrocyte hypertrophy, with potential consequences for CHH pathobiology.Mutations in the  RMRP long non-coding RNA (lncRNA) and several protein subunits . RNase MRP is ubiquitously present in eukaryotes and acts as an endoribonuclease which cleaves several RNA substrates. It has been reported to be involved in mitochondrial DNA replication2, cleaves pre-rRNA in the internal transcribed spacer 1 (ITS1)4, plays a role in cell cycle regulation by cleaving cyclin b2 mRNA5, and is able to cleave viperin mRNA6. In addition, it has been shown that RMRP RNA and the telomerase-associated reverse transcriptase (TERT) protein are able to form a complex that exerts RNA-dependent RNA polymerase activity, which is able to generate a double stranded RMRP RNA molecule. Via a Dicer-dependent route siRNAs are generated from this molecule, downregulating cellular RMRP levels and providing a negative feedback mechanism controlling RMRP RNA levels7. In addition, RMRP RNA is the source of at least two other short RNAs designated RMRP-S1 and RMRP-S2, which function as miRNAs and have gene-silencing activity relevant for human cartilage-hair hypoplasia (CHH)8. Recently, RMRP RNA has been shown to interact with DDX5 and play a role in RORγt-dependent TH17 biology9. RNase MRP is structurally and functionally related to the RNase P endoribonuclease, which has a unique RNA component, but shares many protein subunits with RNase MRP10. RNase P is critically involved in maturation of tRNAs by cleaving the 5′ leader of pre-tRNA and is involved in RNA polymerase I and III transcriptional activity13.RNase MRP is a small nucleolar ribonucleoprotein (snoRNP) particle, consisting of the RMRP gene are the cause of a severe form of dwarfism known as the cartilage-hair hypoplasia14 (CHH: OMIM #250250) – anauxetic dysplasia15 (AAD: OMIM #607095) spectrum of disorders16. CHH is also termed metaphyseal chondrodysplasia, McKusick-type17. To date, more than hundred individual CHH-pathogenic mutations have been identified in the RMRP gene14. These mutations are located either in the transcribed region or in the proximal promoter region, often between the TATA box and the transcription start site. Promoter mutations may result in inefficient transcription of the RMRP gene, whereas mutations in the transcribed region are thought to influence RNase MRP complex assembly, enzymatic activity, subcellular localization, substrate recognition or RNA stability14.Mutations in the 18. Longitudinal bone growth during skeletal development and lengthening of the limbs depends on endochondral ossification. Endochondral ossification is a multistage process in the growth plates of developing long bones. Growth plates are populated by highly proliferative chondrocytes, which differentiate into mineralizing hypertrophic chondrocytes that either die from apoptosis or transdifferentiate into osteoblasts21. The remaining mineralized extracellular matrix (ECM) provides a scaffold for osteoblasts and osteoclasts to adhere and remodel, setting the stage for bone apposition and thus longitudinal bone growth and limb development23. In the proliferative zone of the growth plate, Sox9 drives the expression of type II collagen (Col2a1), the main marker for this stage of chondrogenic differentiation. Runx2 and Mef2c drive the expression of type X collagen (Col10a1) during the hypertrophic phase of chondrogenic differentiation. Many skeletal abnormalities have been described that are often caused by mutations in genes involved in growth plate development24.One phenotypic hallmark of CHH is short-limbed dwarfism accompanied by abnormal growth plate development. Other symptoms include sparse thin hair, anaemia, Hirschsprung’s disease, bronchiectasis, and impaired T-cell immunity. In addition, adult patients have a predisposition to certain cancers RMRP RNA in the molecular pathology of CHH has been recognized, its role in growth plate biology remains elusive. As abnormal growth plate development is one of the radiological characteristics of CHH25, and chondrogenic differentiation in the growth plates is required for skeletal development23, we hypothesized that the RMRP lncRNA may also function as an important regulator of chondrogenic differentiation.Although the involvement of the 2PO4.H2O (Merck Millipore), 1.8 mM KH2PO4 (Merck Millipore)). Antigen retrieval was performed using 20 μg/ml proteinase K . Slides were washed extensively with demineralized water to remove proteinase K. Slides were pre-hybridized with microRNA ISH buffer . Hybridization was performed for 1 hour at 50 °C on a heating plate using 80 nM of Rmrp RNA double-digoxigenin (DIG)-labeled miRCURY LNA probe  or a double-DIG labeled scrambled LNA miRNA probe  in miRNA ISH buffer. Slides were washed three times with 5x SSC  for 10 minutes at 50 °C. Subsequently slides were blocked with blocking solution  for 30 minutes and incubated with anti-DIG-Alkaline Phosphatase (AP) Fab fragments  in blocking solution for 1 hour. Endogenous AP was blocked with Levamisole solution  and bound anti-DIG-AP Fab fragments were detected with AP substrate  in demineralized water. Slides were washed in PBS-T , counter-stained with 0.01% FastGreen (Sigma-Aldrich), dehydrated and mounted with Entellan (Merck Millipore).Decalcified knee epihyseal growth plates of 6-week-old C57BL/6 mice  were embedded in paraffin and 5 μm sections were cut. Sections were deparaffinized in a xylene/ethanol series ending in PBS , 2.7 mM KCl (Merck Millipore), 9.0 mM Na26 was used at a 1:1,000 dilution. Rabbit polyclonal anti-Rpp2527, anti-Rpp3028, anti-Rpp3828, anti-Rpp4029 and anti-hPop130 were used at a 1:100 dilution. Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (Dako EnVision) for 30 minutes at room temperature. DAB chromogen substrate (Dako) was used for detection. Sections were counterstained with haematoxylin (Dako), dehydrated and mounted with histomount . Microscopic images were acquired using a Zeiss Axioscope A1.Knee joint epiphyseal growth plates of 6-week-old C57BL/6 mice were formalin-fixed and decalcified using 0.5 M ethylenediaminetetraacetic acid , pH 7.8. After paraffin-embedding 5 μm tissue sections were cut. Sections were deparaffinized in a xylene/ethanol series ending in PBS. For antigen retrieval (COL10A1 detection) sections were treated with 0.4% hyaluronidase (Sigma-Aldrich) at 37 °C for 30 minutes. Sections for detection of RNase MRP protein subunits were incubated in hot citrate buffer (1.8 mM citric acid (Sigma-Aldrich) and 8.2 mM tri-sodium citrate (VWR Prolabo)) for 30 minutes. Endogenous peroxidase activity was inactivated using peroxidase-blocking solution . Next, slides were blocked with 10% normal sheep serum in PBS-T. Primary antibodies were incubated for 1 hour at room temperature. Rabbit polyclonal anti-COL10A131 were cultured in a humidified atmosphere at 37 °C, 5% CO2 in proliferation medium , 5% FCS , 1% antibiotic/antimycotic (Invitrogen), 1% NEAA (Invitrogen)). To induce chondrogenic differentiation, cells were plated at 6,400 cells/cm2. After 24 hours chondrogenic differentiation was initiated by changing the medium to differentiation medium (proliferation medium supplemented with 10 μg/ml insulin (Sigma-Aldrich), 10 μg/ml transferrin (Roche), 30 nM sodium selenite (Sigma-Aldrich)). Differentiation medium was refreshed every two days for the first 10 days, and each day after day 10. Chondrogenically differentiating ATDC5 cells were transfected  with 100 nM siRNA duplexes . Scrambled siRNA duplex was purchased from Eurogentec (REF: SR-CL000–005). Cells were harvested for RNA isolation on day 0, 7 and 10 during differentiation  and samples were kept at −80 °C until further analysis. In addition, ATDC5 cells were differentiated for 14 days and exposed to 5 or 50 nM PTHrP  from day 10 onwards (medium with PTHrP was refreshed on day 12 during chondrogenic differentiation). MCT cells32 were cultured in a humidified atmosphere at the permissive temperature of 32 °C, 8% CO2 in proliferation medium (DMEM/F12 (Invitrogen), 10% FCS (PAA), 1% antibiotic/antimycotic (Invitrogen), 1% NEAA (Invitrogen)). Hypertrophic differentiation was induced in the absence or presence of 50 nM PTHrP (Bachem) by switching culture conditions to 37 °C, 5% CO2 for 24 hours. Cells were harvested for RNA isolation  and samples were kept at −80 °C until further analysis. To maintain Rmrp knockdown during an extended period of time during chondrogenic differentiation, scrambled (Eurogentec SR-CL000-005) and Rmrp RNA (Table ATDC5 cellsEcoRV and HindIII (pGluc-Rmrp-prom). As a transfection control the pGL4.20[luc2/Puro]  vector was used in which the CMV promoter was cloned (pGL4.20[luc2/Puro]-CMV). Day 5-differentiating ATDC5 cells in 12 well plates were transfected with 500 ng pGluc-Rmrp-prom and 125 ng pGL4.20[luc2/Puro]-CMV using 1 μg/μl polyethylenimine  transfection reagent. DNA and PEI were complexed for 10 minutes at room temperature in DMEM/F12 (1.9 μl PEI per 625 ng construct per well) and added to the ATDC5 cells. Five hours post-transfection medium was supplemented with BMP-2 , PTHrP , TGF-β3 , dorsomorphin , (5Z)-7-Oxozeaenol , SHH , IHH , WNT5A , WNT3A , GDF-5  and bFGF . After 36 hours samples were harvested for bioluminescence detection using the Dual Luciferase reporter assay system (Promega) as described by the manufacturer and measured on a Fluostar Omega plate reader .Plasmid constructs were generated using traditional cloning methods and cloned inserts were verified by DNA sequencing. The mouse 1500 nucleotide sequence upstream of the mouse Rmrp transcription start site was amplified from ATDC5 genomic DNA and cloned into the pGluc-Basic vector (NEB) using primer-introduced restriction sites 33. Real time quantitative PCR (RT-qPCR) was performed in 96-well optical plates. For each cDNA sample a mix was prepared consisting of Mesagreen qPCR Mastermix Plus for SYBR Green (Eurogentec) and 300 nM forward and reverse oligonucleotides. Serially diluted standard curves were utilized to quantify gene expression in the samples. A Biorad CFX96 Real-Time PCR Detection System was used for amplification using the following protocol: denaturation at 95 °C for 10 minutes, followed by 50 cycles of amplification (15 seconds 95 °C and 1 minute 60 °C) followed by a dissociation curve. Data were analyzed using Biorad CFX Manager Software version 3.1, based on the relative quantification of mRNA expression of the target gene normalized to a housekeeping gene . Primer sequences are depicted in Table RNA was isolated from TRIzol samples by collecting the aqueous phase after phase separation. RNA was precipitated with isopropanol  and centrifuged for 30 minutes at 20,000 × g, 4 °C. RNA pellets were washed with 80% ethanol and potential DNA contamination was removed by DNase I (Roche) treatment . After subsequent ethanol precipitation, RNA was dissolved in 15 μL DNase/RNase free water (Eurogentec). RNA quantity and purity were determined spectrophotometrically . DNA-free total RNA was reverse transcribed using standard procedures and random hexamer priming as described previouslyCells were washed two times with 0.9% NaCl and fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature. Fixed cells were washed 6 times with distilled water and air-dried. Dried fixed cells were incubated for 30 minutes at room temperature with either 0.1% (m/v) Crystal Violet (Sigma-Aldrich); 1% (m/v) Alcian Blue  in 0.1 M HCl; or 40 mM Alizarin Red S (Sigma-Aldrich), pH 4.2. Cells were washed six times with distilled water to remove unbound stain and allowed to air dry. Pictures were acquired with an Epson V370 flatbed scanner . Crystal Violet was extracted by incubation with 10% acetic acid (VWR) for 15 minutes on a plate shaker . Alcian Blue was extracted by incubation with 6 M Guanidine-HCl (Sigma-Aldrich) for 2 hours on a plate shaker (IKA). Alizarin Red S extraction was initiated by incubation with 10% acetic acid (VWR Prolabo) for 30 minutes at room temperature on a plate shaker (IKA). Cells and eluate were further collected using a cell scraper and transferred to a microcentrifuge tube. Samples were heated at 85 °C for 10 minutes and subsequently centrifuged at 20,000 × g for 15 minutes at 4 °C. Ammonium hydroxide (VWR) was added to neutralize the acetic acid until a pH of 4.4 was reached. Extracted Crystal Violet, Alcian Blue and Alizarin Red S were quantified spectrophotometrically at 590 nm, 645 nm or 405 nm (respectively) using a plate reader .2; 7.5 mM p-nitrophenyl phosphate), ALP activity was determined by measuring ALP-dependent enzymatic conversion of p-nitrophenyl phosphate to nitrophenyl phosphate by spectrophotometric analysis at 405 nM. A calibration curve containing an increasing concentration of nitrophenyl phosphate was used to determine the absolute amount of ALP-generated nitrophenyl phosphate over time. Values were normalized to total protein concentration and ALP activity expressed as µmol nitrophenyl phosphate/gram/minute.Enzymatic activity of Alkaline Phosphatase in ATDC5 cultures was determined in a colorimetric assay. Cells were lysed in lysis buffer  and homogenized on ice by means of sonication . Insoluble material was removed by centrifugation . Total protein concentration was determined with a BCA assay (Sigma-Aldrich). In flat-bottom 96 wells plates containing assay buffer  in Aggrecan-coated  wells as previously described34. Cells were directly plated in trans-differentiation medium which consisted of DMEM/F12 + Glutamax (Invitrogen), 10% FCS (PAA), 1% antibiotic/antimycotic (Invitrogen), 1% NEAA (Invitrogen) + 1% ITS , 50 μg/ml ascorbic acid 2-phosphate (Sigma-Aldrich) and 1 ng/ml human recombinant TGF-β3 (Life Technologies). Trans-differentiation medium was refreshed every other day and transdifferentiated cartilaginous nodules were harvested in TRIzol on day 3, 5 and 7 during trans-differentiation. At plating, fibroblasts were harvested for day 0 samples as well.Ethical permission for the use of dermal fibroblasts was obtained from the medical ethical Institutional Review Board of Freiburg University Hospital and methods and experimental protocols to obtain dermal fibroblasts were carried out in accordance with the Freiburg University Hospital medical ethical Institutional Review Board, according to German law. Informed consent was obtained from all subjects. Human dermal fibroblasts (passage 7–9) were hyperconfluently plated  were also positive for the expression of Rmrp RNA. Subsequently, we evaluated the spatiotemporal expression of a number of RNase MRP protein subunits RPP25, RPP30, RPP38, RPP40 and POP1  and cells were exposed to different chondrogenic mediators , a negative regulator of chondrocyte hypertrophic maturation39, reduced Rmrp promoter activity by 35%. TGFβ isoforms activate SMAD signaling via SMAD2/340. Of all TGFβ isoforms, TGFβ3 was described to be able to induce matrix mineralization41. Indeed, TGFβ3 increased Rmrp promoter activity by 40%. In agreement with the TGFβ3-induced increase of Rmrp promoter activity, exposure to (5Z)-7-Oxozeaenol, an inhibitor of TGF-β activated kinase-1 (TAK1)42, decreased Rmrp promoter activity by 22%. Exposure to BMP-2 , the main ligand that activates Patched-1-mediated GLI-signaling and which is crucial in limb bud patterning during embryogenesis46, did not significantly alter promoter activity. We thus concluded that the 1500 nucleotide sequence upstream of the Rmrp RNA transcription start site is responsive to mediators known to alter the chondrocyte phenotype. This indicates that, in chondrocytic cells, Rmrp RNA levels may be controlled by transcription factors involved in chondrocyte differentiation and hypertrophy.Our data described above suggested that RMRP RNA regulates chondrogenic differentiation, Rmrp RNA expression was targeted by RNAi. ATDC5 cells were transfected with an siRNA duplex at day -1, 2 and 5 during chondrogenic differentiation and gene expression was determined at day 0, 7 and 10 in differentiation. The siRNA duplex reduced the expression of Rmrp RNA significantly  gene expression in cultures from healthy fibroblasts, substantiating trans-differentiation into the chondrogenic lineage  of RNase MRP that are involved in the molecular mechanisms of chondrogenic differentiation has/have not been identified, we can only speculate how induction of RMRP RNA expression is associated with chondrocyte hypertrophy. It is remarkable that in CHH tissues with fast-dividing cell types  seem to be the most affected ones16, suggesting a role for RMRP RNA in cell cycle regulation. Tight control over cell mitosis and differentiation is paramount to controlling growth plate development47. The proliferative zone of the growth plate presents a high mitotic activity, while terminally differentiated chondrocytes in the hypertrophic zone most likely lack a mitotic cycle. One of the identified activities of the RNase MRP complex is endoribonucleolytic cleavage of the mRNA of cyclin b2 (Clb2). This B-type cyclin activates cyclin-dependent kinase-1 (CDK1) during M-phase and anaphase-promoting complex (APC)-dependent proteolysis of Clb2 is essential for mitotic exit48. Low Clb2 levels also keep CDK1 activity to a minimum during G0 phase via similar APC-dependent proteolysis49. It is likely that the strictly regulated terminal differentiation of hypertrophic chondrocytes is orchestrated by mitotic exit or entrance into G0 phase. As RNase MRP activity has been shown to degrade the Clb2 message it is tempting to speculate that the elevation of RNase MRP levels during chondrocyte hypertrophy serves to decrease Clb2 protein levels by increased turnover of Clb2 mRNA to inhibit activation of CDK1. Compelling evidence from recent work indeed shows the crucial involvement of CDK1 in controlling chondrocyte hypertrophic differentiation50. CDK1 expression was found to be highly expressed in proliferative chondrocytes and was greatly diminished in hypertrophic chondrocytes in differentiating ATDC5 cultures, as well as in mouse growth plates. In addition genetic interference with CDK1 expression caused absence of proliferative chondrocytes in the growth plate and a switch towards hypertrophic differentiation in ATDC5 cultures, strongly suggesting that the reduction of CDK1 levels or its decreased activity is a prerequisite for hypertrophic differentiation. We thus speculate that in a healthy growth plate the increased RMRP RNA levels in hypertrophic chondrocytes may lead to decreased Clb2 levels, thereby hampering CDK1 activity and inducing an overall mitotic arrest. Work from the same group50 shows that the proliferation-promoting and hypertrophy-suppressive action of PTHrP is, at least in part, CDK1-dependent. In agreement with this notion we observed that Rmrp RNA levels are down regulated upon PTHrP exposure, presumably via decreased Rmrp promoter transcriptional activity. This may result in elevated Clb2 levels, thereby potentially aiding in PTHrP downstream CDK1 activity. In this respect, it is important to note that knockdown of Rmrp RNA levels during ATDC5 chondrogenic differentiation increases Clb2 and Pthrp mRNA levels and deregulates chondrogenic differentiation in an overall hypertrophy-suppressing fashion. One of the earliest identified roles of RNase MRP is endoribonucleolytic maturation of 5.8 S rRNA by cleaving site A3 in the internal transcribed spacer 1 (ITS1) in yeast4. Even though there is presently no conclusive evidence showing that specifically hypertrophic chondrocytes in the growth plate display the highest protein synthetic capacity, an RMRP RNA/RNase MRP mediated contribution to the synthesis of the large ribosomal subunit in the growth plate may support the high protein synthetic activity of the growth plate to produce protein-rich cartilaginous extracellular matrix. For long an involvement of RNase MRP in the processing of ITS1 in human cells remained elusive51. However, recently it has been elegantly shown that human RMRP RNA in the RNase MRP complex indeed catalyzes the endoribonucleolytic cleavage of ITS1, thereby contributing to pre-rRNA maturation4. Indeed our RMRP RNA knockdown data in ATDC5 show accumulation of an ITS1 pre-rRNA processing intermediate as well as reduced levels of mature 5.8 S and 18 S rRNA. In concert with these findings we detected similar accumulation of an ITS1 processing intermediate in differentiating CHH cells. Chondrogenic differentiation of ATDC5 and the developing growth plate is associated with high proliferative capacity31 and increased synthesis of protein-rich ECM. It is conceivable that this alters the cellular demand for mature rRNAs for ribosome biogenesis, potentially explaining the changes in Rmrp RNA expression during chondrogenic differentiation and the phenotype observed after Rmrp RNA knockdown. Finally, RMRP RNA has been described to associate with TERT7, the reverse transcriptase that is associated with the telomerase holoenzyme. This RMRP RNA-containing macromolecular complex was demonstrated to display RNA-dependent RNA polymerase activity. This activity generates a double-stranded RMRP RNA molecule that is converted by Dicer into an siRNA that targets RMRP RNA7, as well as specific mRNAs52. Other RMRP RNA-derived siRNAs have been identified as well, but it is currently unknown whether these are also generated via a similar mechanism. The latter siRNAs, termed RMRP-S1 and RMRP-S28, seem to target genes relevant for skeletal development like SOX4, PTCH2 and BMPR2. It remains elusive whether this relates to increased RMRP RNA levels in hypertrophic chondrocytes, but it is conceivable that upregulation of RMRP RNA leads to higher production of RMRP-S1 and RMRP-S2, which in turn may change the magnitude by which RMRP-S1 and RMRP-S2 targets are influenced.The involvement of the RMRP RNA expression levels can be controlled at the transcriptional level. RMRP RNA expression is driven by RNA polymerase III (RNAPIII) and the proximal RMRP RNA promoter has been studied in the past to some extent53, showing that the −84 bp promoter region is sufficient to drive RNAPIII-dependent transcription of RMRP RNA. However, to fine-tune promoter activity and to be able to respond to alternating demands of RMRP RNA (e.g. during chondrocyte hypertrophy), it is expected that additional transcription regulatory elements are present. Indeed, WNT-3A was recently found to be able to drive RMRP transcription in cancer via activation of β-catenin and YAP proteins54. Here, we showed that the 1500 base pair sequence upstream of the Rmrp RNA transcription start site is responsive to a series of chondrocyte morphogens or pathways. Several of the growth factors tested increased or decreased the transcriptional activity of the minus 1500 Rmrp promoter, indeed indicating that in chondrocytic cells transcription of Rmrp RNA may be under transcriptional control of chondrogenic cues. In agreement with their known role in chondrocyte hypertrophy45, WNT-5A, WNT-3A and BMP-2 induced Rmrp promoter activity, suggesting that increased Rmrp RNA expression in chondrocyte hypertrophy is, at least in part, transcriptionally controlled by one or more of these morphogens and their downstream pathways. Indeed, in silico prediction of putative transcription factor binding sites in the minus 1500 bp Rmrp promoter sequence suggests the presence of CREB, MEF2, TCF/LEF and SMAD binding sites  which are transcription factors acting downstream of WNT-5A, WNT-3A and BMP-2 signaling. Dorsomorphin (a BMP/SMAD1/5/8 inhibitor) exposure greatly reduced Rmrp promoter activity, further supporting BMP-mediated control of RMRP transcription. In keeping with a potential feedback mechanism it is noteworthy that BMPR2 (the type II BMP receptor) is a potential target of RMRP-S2, the RMRP RNA-derived siRNA8.Our observations that the expression of RNase MRP components is modulated in differentiating cells promoted us to investigate whether Rmrp transcription in chondrocyte hypertrophy is further substantiated by the observation that PTHrP and bFGF are able to reduce Rmrp promoter activity. PTHrP and bFGF delay chondrocyte hypertrophy/terminal differentiation55. At this point we can only speculate whether reduced Rmrp promoter activity and Rmrp expression by PTHrP and bFGF is an indirect result of a morphogen-dependent changing chondrocyte hypertrophic phenotype , or that these morphogens directly control Rmrp abundance by downstream inhibition of Rmrp promoter activity. We conclude that Rmrp transcription in chondrocytes is controlled by well-known chondrogenic signaling pathways that are associated with hypertrophic differentiation. It remains to be determined whether such transcriptional control is also present in other cell types and how this communicates with the RNAPIII transcription machinery56.Active control over RMRP RNA during chondrogenic differentiation and is especially linked to chondrocyte hypertrophic differentiation. Genetic interference with Rmrp RNA expression in ATDC5 cells and chondrogenic trans-differentiation of CHH fibroblasts indicates delayed chondrocyte hypertrophy. Growth plates of CHH patients are characterized by delayed ossification and the presence of very few hypertrophic chondrocytes25. Whether and how delayed chondrocyte hypertrophy may influence the development of the growth plate in CHH remains to be determined. Finally, considering our observations that the expression of PTHrP is induced during knockdown of Rmrp RNA and in chondrogenic differentiating CHH fibroblasts and its inhibiting action on RMRP promoter activity and RMRP RNA expression, it is interesting to realize that disrupted PTH/PTHrP signaling is deleterious for skeletal development by endochondral ossification57. Considering this it is tempting to speculate whether there are molecular connections between RMRP RNA and PTH/PTHrP in skeletal dysplasias like cartilage-hair hypoplasia, metaphyseal chondrodysplasia Jansen type (OMIM #156400)58 and Blomstrand type chondrodysplasia59, (OMIM #215045).The clinical presentation of CHH is dominated by short stature, caused by impaired skeletal development. Our data are consistent with a pivotal role for 60 and we found that the RMRP lncRNA is differentially expressed during different stages of chondrogenic differentiation and displaying a prominent association with chondrocyte hypertrophic development. Our findings shed new light on potential pathobiological mechanisms involved in the skeletal dysplasia phenotype associated with cartilage-hair hypoplasia.In conclusion, lncRNAs involved in mesenchymal cellular differentiation processes are scarcely known


Aegilops tauschii L. Here, we investigated the chloroplast RNA editing of Ae. tauschii and compared it with other wheat relatives to trace the evolution of wheat. Through bioinformatics prediction, a total of 34 C-to-U editing sites were identified, 17 of which were validated using RT-PCR product sequencing. Furthermore, 60 sites were found by the RNA-Seq read mapping approach, 24 of which agreed with the prediction and six were validated experimentally. The editing sites were biased toward tCn or nCa trinucleotides and 5′-pyrimidines, which were consistent with the flanking bases of editing sites of other seed plants. Furthermore, the editing events could result in the alteration of the secondary structures and topologies of the corresponding proteins, suggesting that RNA editing might impact the function of target genes. Finally, comparative analysis found some evolutionarily conserved editing sites in wheat and two species-specific sites were also obtained. This study is the first to report on RNA editing in Aegilops tauschii L, which not only sheds light on the evolution of wheat from the point of view of RNA editing, but also lays a foundation for further studies to identify the mechanisms of C-to-U alterations.RNA editing is an important way to convert cytidine (C) to uridine (U) at specific sites within RNA molecules at a post-transcriptional level in the chloroplasts of higher plants. Although it has been systematically studied in many plants, little is known about RNA editing in the wheat D genome donor  Th. ThAe. that tool . SNPs wehat tool . Only thhttp://www.biogem.org/tool/chou-fasman/) [http://phobius.sbc.su.se/) [CFSSP (Chou and Fasman Secondary Structure Prediction Server) (fasman/) ,36 was u.su.se/) .Ae. tauschii were compared to those of Hordeum vulgare [Lolium perenne [Oryza sativa [Saccharum officinarum [Zea mays [Triticum urartu, Aegilops speltoides, and T. turgidum, as well as T. aestivum  were put on moistened filter paper in a dish and then cultured in a growth chamber under the condition of 20 °C and a natural light/dark cycle. One-week-old seedlings were harvested for DNA and RNA isolation. Genomic DNA was extracted, as described previously [® Plant RNA Kit  and then treated with HiScriptTM Q RT SuperMix (+gDNA wiper)  to remove genomic DNA and to synthesize cDNAs, according to the manufacturer’s instructions.Seeds of eviously . Total RpetL, ndhK, rpoA, rps3, ndhD, ndhG, and rpoB were sequenced directly from both directions by Sangon Biotech Corporation . The products of ndhB, atpA, matK, ndhA, and rpoB were subsequently ligated into pMD18-T Vector  and transformed into Escherichia coli DH5α. Two positive clones were selected for each fragment to sequence (both directions for ndhB and only one direction for others) by Sangon Biotech Corporation. Both DNA and cDNA sequences were spliced using the ContigExpress tool implemented in Vector NTI suite 6.0 , and the RNA editing sites were validated by aligning cDNAs with corresponding genomic DNA sequences using the ClustalW program  [The extracted DNA and obtained cDNAs were used as PCR templates for the validation of specific RNA editing sites, namely 18 sites of six genes, selected randomly from the prediction, as well as six sites in six genes identified by RNA-seq read mapping. In total, 11 specific primers  were desstalw2/) .Ae. tauschii, all of which were C-to-U conversions. Among them, ndh genes showed the most abundant editing sites with a total of 15, followed by rpo genes with a value of 10, and ycf with two editing events. Furthermore, 30 out of 34 (88.24%) editing events were observed in the second position of the codon and the rest were in the first position.In total, 34 editing sites present in 15 chloroplast protein-coding genes  were preA. thaliana [rps16.A total of 60 sites were identified as RNA editing sites by read mapping and SNP calling, 24 of which agreed with the prediction . Among tthaliana . The abuthaliana  and mighIt has been demonstrated that the editing sites in the chloroplasts of seed plants preferred 5′-T–A-3′ flanking bases and/or a 5′-pyrimidine due to the genomic context with lower substitution rates ,47,48. TatpA-234 and rpl16-36, spike-specific psbB-414, and rps3-30 edited in all tissues. Editing events at sites rps18-448 and ndhF-1891 generated stop codons, resulting in the premature termination of transcription, which only occurred in pistils with relatively low efficiency (9%). Furthermore, editing events at ndhB-586, ndhB-611, ndhB-704, and ndhB-1481 on the sense-strand, as well as ndhB-836, ndhB-149, ndhA-1070, and ndhA-563 on the antisense-strand, were found to only occur in leaves, while rps4-305 was specifically edited in seeds.Additionally, six silent editing sites with no impact on the encoded amino acid were also detected, including pistil-specific rpoB-617 was not validated, as nucleotide C was present in both the mRNA and genomic DNA . Only omic DNA . In addiCycas taitungensis, 85 editing sites were found in 25 transcripts using a combination of prediction by CURE and experimental determination [Aegilops tauschii L. chloroplasts, we performed identification through a combination of these three methods, which provide a systematic and comprehensive RNA editing profile of the transcriptome of Aegilops tauschii L. chloroplasts.Generally, there are three methods to identify RNA editing sites, namely bioinformatics prediction and RNA-Seq, as well as molecular cloning. Molecular cloning is a common, widely used and accurate method to identify RNA editing in different species. Kugita et al.  verifiedmination . In cocomination . With thmination . Howevermination . Thus, tpetB, rpoA, and rps8 possessed the same secondary structures, before and after editing, while editing events of the remaining 12 genes could alter the composition of the secondary structures around the editing sites , which was in the proline codon CCA at the DNA level and was subsequently edited to the leucine codon CTA at the RNA level. Although the editing event at petB-662 had no impact on the hydrophobicity of the amino acids, the composition of the secondary structures or the number of transmembrane structures, the transmembrane region at codons 201 to 220 expanded to the C terminal of the protein (petB-662 contributed to maintaining the functional activity of cytochrome b6f in Ae. tauschii.In the cp genome of  residue . This ge protein A. We thundhA, before and after editing, which encoded NADH dehydrogenase subunit A. Nevertheless, a contraction in the region of codons 296 to 323 was observed and the transmembrane region at codons 335 to 353 shifted to the C terminal (ndhA-encoded peptides of other chloroplast species, as was found in maize [ndhB, with all sites edited, revealed a new transmembrane region at codons 449 to 510 (NdhB editing site III was reported to remain unedited in the non-photosynthetic mutants of tobacco, as well as during the etiolated stage of the seedling development of maize, whereas it was fully edited in plants with an intact photosynthetic apparatus, indicating that the editing events in ndhB of Ae. tauschii might occur with a similar developmental stage-dependent manner, suggesting an unexpected link between ndhB editing and photosynthesis and light-induced chloroplast development [There was no change in the number of transmembrane structures of terminal B. The edin maize . In addi9 to 510 C. NdhB eelopment .Ae. tauschii, H. vulgare, L. perenne, O. sativa, S. officinarum, and Z. mays, 14 sites of which were found to be fully or partially edited (ndhA-1, ndhK-1, rpl2-1 and rps14-1, and the absence of edited rpl2-1 was specific to Ae. tauschii. NdhK-1 was not edited in Ae. tauschii, and nucleotide C was present at the genomic DNA level. For the other three sites, nucleotide T already existed at the genomic DNA level. These sites had probably re-mutated to T at the DNA level to remedy detrimental genomic mutations that occurred after the water-to-land transition of land plants [ndhB-1, ndhB-5 and ndhB-8, Ae. tauschii, H. vulgare, L. perenne and O. sativa shared edited ndhB-5 and ndhB-8, whereas ndhB-1 was only edited in Triticeae crops, suggesting that patterns of RNA editing were in accordance with the phylogenetic relationship (A total of 31 conserved RNA editing sites in 15 chloroplast protein-coding genes were systematically compared in y edited , establid plants . In termtionship  and therAe. tauschii and other wheat relatives including T. urartu, Ae. speltoides and T. turgidum. In total, 35 editing sites were detected in Ae. speltoides, T. turgidum, and T. aestivum, while only 33 sites were found in T. urartu (T. urartu (atpB-35 and ndhB-737 were edited in Ae. speltoides and T. turgidum compared to T. urartu (atpB-35 and ndhB-737 in T. urartu, as well as atpB-35 in Ae. tauschii, were not edited, the encoded T in the DNA level might be a result of re-mutation to repair the deficient transcripts that arose from mutations as described previously. If we posited that editing sites remained stable when the genome progenitors evolved to common wheat, undergoing the natural cross and chromosome doubling, atpB-35 and ndhB-737 of T. turgidum might come from Ae. speltoides. As predicted, the editing event at atpB-35 was not observed in Ae. tauschii but occurred in T. aestivum (atpB-35 of T. aestivum came from T. turgidum and originated from Ae. speltoides. However, the editing event at ndhB-737 also occurred in Ae. tauschii. Thus, further study is needed to reveal the origin of ndhB-737 in common wheat.Furthermore, to trace the evolution information of polyploid wheat from the chloroplast RNA editing point of view, we investigated and compared the chloroplast RNA editing patterns between . urartu . Moreove. urartu , indicat. urartu A. As atpaestivum B,C, suggAe. tauschii chloroplast genome through bioinformatics prediction, combined with RNA-Seq mapping and molecular cloning. A total of 34 editing sites in 15 protein-coding genes were predicted by PREP-Cp, of which 23 sites in 12 genes were validated by RNA-Seq mapping and 17 sites in six genes by molecular cloning. Additionally, RNA-Seq mapping detected 60 sites, seven of which were confirmed experimentally. Ae. tauschii shared similar patterns and features of RNA editing with other land plants and editing events could result in the alteration of the secondary structures of the encoded protein, which might have an impact on biological functions. Finally, comparative analysis was performed to identify the evolutionarily conserved or species-specific sites. Two sites, including atpB-35 and ndhB-737, were found to underlie some origin and evolutionary information of common wheat. This study, for the first time, reported on RNA editing in Aegilops tauschii L. and compared the RNA editing pattern among wheat relative species, which not only shed light on the evolution of wheat from the point of view of RNA editing, but also lay the foundation for further studies to identify the mechanisms of RNA editing in wheat and beyond.Here, we identified the RNA editing sites in the


The APOBEC3 family of cytidine deaminases cause lethal hypermutation of retroviruses via deamination of newly reverse-transcribed viral DNA. Their ability to bind RNA is essential for virion infiltration and antiviral activity, yet the mechanisms of viral RNA recognition are unknown. By screening naturally occurring, polymorphic, non-human primate APOBEC3H variants for biological and crystallization properties, we obtained a 2.24-Å crystal structure of pig-tailed macaque APOBEC3H with bound RNA. Here, we report that APOBEC3H forms a dimer around a short RNA duplex and, despite the bound RNA, has potent cytidine deaminase activity. The structure reveals an unusual RNA-binding mode in which two APOBEC3H molecules at opposite ends of a seven-base-pair duplex interact extensively with both RNA strands, but form no protein–protein contacts. CLIP-seq analysis revealed that APOBEC3H preferentially binds to sequences in the viral genome predicted to contain duplexes, a property that may facilitate both virion incorporation and catalytic activity. The APOBEC3 family cytidine deaminases with antiviral activity are proteins that catalyze the deamination of newly reverse-transcribed viral DNA. Here the authors present the crystal structure of full-length pig-tailed macaque APOBEC3H with bound RNA, which reveals how the APOBEC3H dimer binds around a short RNA duplex. Primate genomes encode seven A3 proteins, of which at least three  inhibit human immunodeficiency virus (HIV-1) replication, but are antagonized by the viral Vif accessory protein in a species-dependent manner5. A3 proteins are comprised of one (A3H) or two (A3F and A3G) “Z” domains containing Zn-coordinating His and Cys amino acids that, together with a conserved Glu, form the deaminase catalytic center2. Among the three identified Z-domain clades (Z1–Z3), A3F and A3G are comprised of Z1 and Z2 domains, while A3H is unique among primate A3 proteins in possessing a single Z3 domain2.APOBEC3  antiviral proteins7. Although the mechanisms by which A3s recognize RNA are unknown, they are sufficiently non-specific that widely divergent RNA sequences are recognized, yet they exhibit sufficient selectivity to enable virion RNA binding and incorporation in the presence of a vast excess of cellular RNA9. A3 binding to RNA may also contribute to the deaminase-independent ability to inhibit HIV-1 reverse transcriptase (RT)13. In addition, A3H and the RNA-binding domains of A3F and A3G drive the formation of higher-order oligomeric complexes15. RNA binding is thought to inhibit deaminase activity, via an unknown mechanism17, and in the two-domain A3F and A3G proteins, one Z-domain binds RNA and the other catalyzes cytidine deamination2. Crystal structures obtained for the isolated catalytic domains of A3F and A3G and for an A3G RNA-binding domain reveal a common domain structure and Zn-binding site22. Nevertheless, in each case, the A3 protein was extensively truncated or engineered to improve stability or crystallization properties. The structure of an unmodified huA3C has also been obtained23, however, this protein lacks antiviral activity. Therefore, thus far, no structure is available for a naturally occurring, full-length APOBEC3 with antiviral activity, and the structural basis for RNA recognition by any APOBEC3 is unknown.RNA binding is a key feature of A3 proteins that is necessary for their encapsidation into virions and for antiviral activityHere, we present the 2.24-Å crystal structure of a full-length, natural polymorphic variant of pig-tailed macaque APOBEC3H (pgtA3H) bound to a short RNA duplex. Furthermore, we show that human A3H haplotype II (huA3H) and several pgtA3H variants retain cytidine deaminase activity in the presence of bound RNA, demonstrating that the RNA does not obstruct access to the A3H active site. CLIP-seq analysis reveals that the regions of the viral genome most frequently bound by A3H are predicted to form short duplex structures. These results illuminate important elements of RNA recognition and potential mechanisms of antiviral activity by a natural, catalytically competent pgtA3H.25. Because our initial attempts to crystallize human (hu) A3H hap II were unsuccessful, we explored natural A3H variants encoded by non-human primates, specifically pig-tailed macaques (pgt). Remarkably, analysis of pgtA3H sequences from 14 macaques revealed no less than 13 allelic variants, each of which was expressed as two spliced isoforms that included or excluded a Gln182 codon at the exon 4/5 boundary  as opposed to Glu62 , and introduction of Lys62 in variants ζ and η increased their antiviral activity  have been identified in humans, but only three  restrict HIV-1ity Fig. . In addi. ζ Fig. . The levEscherichia coli expression system26 and recombinant huA3H from an insect-cell expression system27. The complex also contained bound RNA with a length of 10–12 nt, determined by denaturing gel electrophoresis   the use of the same 40-nt DNA substrate for the in vitro deaminase assays that may not account for any subtle differences in substrate sequence preference between the A3H variants. For example, the activity of huA3H (hap II) varied by twofold with substrates differing in the +2 position relative to the reactive cytosine26. The variation in activity was not due to differences in Zn incorporation among the A3H variants. We confirmed that each A3H variant was purified with a full complement of Zn by comparing activities of samples with and without added Zn. For each variant, the deaminase activity was identical in parallel assays with and without added Zn    Fig. . At the end Fig. . The onlend Fig. . The unpend Fig. . These tend Fig. .Fig. 4Un9. Accordingly, the co-purified RNA lacks a distinct sequence. For each of the seven base pairs, electron density is continuous across the paired bases , one hydrogen bond to the nt2 2′-hydroxyl, and aromatic interactions with bases in both RNA strands  Fig. . There a8. The ten most frequently bound sites in the viral genome all contained one or two predicted RNA duplexes of seven or more nucleotides , the unusual mode of RNA binding by A3H appears to be distinct. Indeed, the 10 most frequent A3G-binding sites in HIV-1 virion RNA were significantly less likely to contain predicted, overlapping ≥7 nt duplexes than were the preferred A3H-binding sites  (8 × 105 cells per well—six-well) using 12 μL polyethylenimine (PEI)  together with 800 ng of a murine leukemia virus (MLV) GagPol expression plasmid and 100 ng of a vesicular stomatitis virus glycoprotein (VSV.G) expression plasmid to produce MLV-based retroviral stocks, which were subsequently used to transduce 293T cells. Cells stably expressing each pig-tailed A3H (pgtA3H) variant were selected and maintained in medium containing hygromycin (10 ng/mL)8.Total RNA and genomic DNA were extracted from 14 activated, IFNα-treated pig-tailed macaque peripheral blood mononuclear cell samples using TRIZOL (GibcoBRL). Thereafter, complementary DNA (cDNA) was synthesized using the SuperScript III RT kit (Invitrogen) using gene-specific primers (Supplementary Table 5 cells per well—six-well) were infected with VSV.G pseudotyped HIV-1NL4.3ΔVif at a multiplicity-of-infection (MOI) of 2. At 16 h post infection, supernatants were collected, clarified by centrifugation at 748×g, filtered through a 0.22 μm filter, and virions were purified via ultracentrifugation through a 25% sucrose cushion. Supernatants from uninfected A3H-expressing cells were used as control for specificity of A3H incorporation into viral particles. Purified virions and cell lysates were analyzed by immunoblot using a rabbit anti-HA at a 1:1000 dilution (600-401-384 Rockland) and a mouse anti-HIV-1 p24 capsid at a 1:100 dilution  antibodies, followed by an anti-rabbit IgG conjugated to IR Dye680 and an anti-mouse IgG conjugated to IR Dye800 (LiCOR).293T cells stably expressing huA3H or pgtA3H variants (7 × 104 cell per well—96-well) were transfected using 2 μL PEI  with 50 ng of an HIV-1NL4.3ΔVif proviral plasmid, 12 ng of a VSV.G expression plasmid, and 0, 37.5, 75, or 150 ng of each A3H-3xHA expression plasmid using the LHCX plasmid as filler to maintain a constant total amount of DNA per transfection. At 48 h post transfection, supernatant was collected, frozen at −70 °C and then thawed and titrated on Helios indicator cells that constitutively express CD4 and CCR5 and also express a nanoluciferase gene under the control of the HIV-2 LTR. At 48 h post inoculation, infectivity was quantified by measuring nanoluciferase expression using the Nano-Glo Luciferase assay (Promega) and is expressed as Relative Light Units (RLU).293T cells , and catalytically inactive pgtA3Hα E56A13 were produced in E. coli using codon-optimized synthetic DNAs  protease cleavage site), including all pgtA3H variants, or pMocr38  for huA3H. All sequences were confirmed by Sanger sequencing. Plasmids phA3H001 and ppgtA3H001-003 were transformed into E. coli strain BL21(DE3) carrying the pGro7 plasmid for co-expression of chaperones GroES-GroEL (Takada)39. Transformed cells were grown in 4% glycerol terrific broth media at 37 °C until the OD600 reached 0.6–0.8. Chaperone expression was induced with l-arabinose (1 g/L) at 37 °C for 1 h, then cultures were cooled to 20 °C, induced with 200 µM IPTG, and grown overnight. Cells were collected via centrifugation and stored at −20 °C.Recombinant huA3H, three pgtA3H variants  and are included in Supplementary Table 2, 0.5 mg/mL DNaseI, 0.5 mg/mL RNase A, and 0.1 mg/mL lysozyme (lysis conditions). Under these conditions, resuspended cells from a 1-L culture were incubated on ice for at least 1 h, followed by sonication. Supernatants of centrifuged cell lysates were batch-bound to 5 mL Ni-NTA resin (Qiagen) overnight at 4 °C. The resin was washed with 5 column volumes (CVs) of buffer A followed by a 5-CV wash using buffer A with 1 M NaCl and 20 mM imidazole. Protein was eluted with 6-CV buffer A with 200 mM imidazole. Purification tags were removed by overnight incubation with TEV protease (+2 mM dithiothreitol (DTT)) in dialysis against buffer A. Tag-free protein was separated from uncleaved protein and TEV protease with a 5 mL His Trap column , and the flow-through was concentrated and incubated for 4 h with 1 mg/mL RNase A at 25 °C. Attempts to remove nucleic acid from the protein, including PEI precipitation, were unsuccessful, however with extensive RNase A treatment, a monodisperse chromatogram was achieved. Concentrated protein was loaded onto a Superdex 16/60 S200 gel filtration column and eluted in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 1 mM Tris(2-carboxyethyl)phosphine. All APOBEC3H proteins eluted as monodisperse complexes with an apparent molecular weight (MW) of 60 kDa . Peak fractions were pooled, concentrated to 10 mg/mL, and flash-frozen in liquid N2. All purified proteins, despite removal efforts, contained nucleic acid as shown by the absorbance ratio (A260 nm/A280 nm) of 1.5. Tag-free protein was used for all biochemical and crystallographic experiments. No RNase A was used in the purification of proteins for the assay data shown in Supplementary Fig. Bacterial cell pellets were resuspended in buffer A  with 2 mM MgCl41. Extracted nucleic acids from each A3H preparation were separated on a 15% urea polyacrylamide denaturing gel followed by overnight staining with Stains All (Sigma) to determine the identity of the bound nucleic acid  and its approximate size .Nucleic acid was extracted from each A3H preparation after the gel filtration step using a phenol:chloroform extraction followed by ethanol precipitation (95% ethanol for 2 h at −20 °C)42. Recombinant UDG43 was produced in E. coli from pUDG001 generated by LIC of a synthetic DNA  contained 1 µM DNA substrate, 40 nM UDG, 50 mM Tris pH 7.5, 40 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 4 nM, 20 nM or 100 nM A3H. Reactions at 37 °C were initiated by addition of A3H. At each time point, the reaction was quenched with two volumes of 0.2 M NaOH followed by heating 15 min at 70 °C to hydrolyze DNA at the deglycosylated nucleotide. After addition of three volumes of formamide loading solution, the samples were heated 10 min at 95 °C, and the products were resolved by 15% urea denaturing polyacrylamide gel electrophoresis. Gels were scanned using a Typhoon fluorescence imager, and bands corresponding to cleaved and uncleaved DNA were quantified using the ImageQuant software . To confirm that Zn was not limiting in the deaminase assay, parallel experiments were performed as described above with or without addition of 2 and 10 µM ZnCl2 to the assay buffer (2× and 10× molar excess of A3H). Activities were identical within experimental error 2, 0.1 M Tris pH 8.5). Crystals grew in 1–2 days, were collected directly from the growth solution, and were flash-cooled in liquid N2. Data were collected at the Advanced Photon Source  on GM/CA beamline 23ID-B. Anomalous pair data to 2.6 Å were recorded at the X-ray energy of peak absorption (9.667 keV) above the Zn K-edge (9.6586 keV) in 15° wedges with inverse-beam geometry. Additional data to 2.24 Å, recorded from two crystals, were collected at an X-ray energy of 12 keV. Data were indexed, integrated, and scaled using XDS44. Initial crystal screening suggested the possibility of a tetragonal space group (a ≈ b), and we retained these axial assignments throughout the structure determination, which resulted in a nonstandard primitive orthorhombic space group (P22121). Anomalous differences to 3.5 Å were used with AutoSol45 in the Phenix suite46 to determine the Zn substructure, and for initial single-wavelength anomalous phasing. Phases were refined and extended to 2.6 Å by exploiting the fourfold noncrystallographic symmetry using DM47. A 78% complete initial model of the protein was built into the 2.6-Å phase-refined map using AutoBuild, and the remainder of the model, including an RNA duplex, was built manually in coot48. Although the RNA density was consistent with a mixture for the base paired nucleotides (nt3–9) and for nt2, for simplicity, the RNA strands were modeled with unique sequences (5′-A-A-C-C-C-G-G-G-G-3′ and 5′-A-A-C-C-C-C-G-G-G-3′). The nt1 nucleotide had clear purine density and was modeled as adenine. For two of the RNA strands, weak density extended beyond nt9, and the ribose for an nt10 was included in the model. The model was refined against the 2.24-Å data using phenix.refine49. Structure validation was done with MolProbity50. In the final model, 98% of amino acids are in favored regions of the Ramachandran plot, 2% in additionally allowed regions, and none are outliers. Structure images were prepared with PyMOL51, electrostatic surface potentials calculated with APBS53, and buried surface areas calculated with PISA56.Crystals of the full-length pgtA3Hζ (10 mg/mL) were grown at 20 °C by sitting-drop vapor diffusion from a 2:1 mixture of protein stock and reservoir solution  14 h prior to collection. Thereafter, cells and virions were irradiated, lysed in NP-40 lysis buffer, and the soluble fraction was separated by centrifugation. Lysates were subsequently treated with RNase A (Fermentas) and DNaseI (Roche) and then transferred to ice. Protein G-conjugated magnetic Dynabeads coated with a mouse monoclonal anti-HA antibody were added to lysates. After binding, the beads were washed thoroughly57. Dephosphorylation was done using calf-intestinal phosphatase (NEB), and the beads were then washed and resuspended in 1 bead volume of polynucleotide kinase (PNK) buffer, [γ-32P] ATP and T4 PNK (NEB). Cold ATP was added and the phosphorylation reaction continued. The beads were then washed and resuspended in NuPAGE SDS-PAGE loading buffer to elute protein–RNA adducts. Protein–RNA adducts were separated by SDS-PAGE, transferred to nitrocellulose and detected by autoradiography. RNA was isolated by proteinase K treatment and phenol:chloroform extraction57. Sequential 3′ and 5′ adapter ligations were then performed on the isolated RNA resulting in RNA of unknown sequence that was flanked by known sequence that contained primer-binding sites for subsequent reverse transcription and PCR-amplification of the cDNA library. Sequencing of the cDNA library was performed on an Illumina HiSeq 2000 platform. The analysis pipeline used the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) to process the raw reads prior to mapping them to the HIV-1 viral genome57. Preferred A3H-binding sites in the viral genome (Peaks) were identified as the positions of the HIV-1 genome with the highest read densities. The 10–12 highest peaks were identified for each A3H protein. Thereafter, 50 nucleotides on either side of the nucleotide with the highest read density value were selected for further analysis. Potential RNA duplexes were detected using M-fold58 with default parameters. An equal number of regions with low frequencies of A3H binding  were identified as 101-nucleotide stretches in the HIV-1 genome with low read density (<200) and subjected to the same analysis as the peaks. To determine the nucleotide composition of the tips of the peaks, the nucleotides with the highest read density within a peak were selected and the base composition determined. For comparison, we identified short valleys as stretches of nucleotides with identical length to the peak tips in a proximal region of the HIV-1 genome. In each CLIP-seq experiment, approximately two to three peaks had wide tips that rendered the identification of short valleys in the vicinity of those peaks impossible, and therefore distal regions of the genome were used.CLIP-seq analysesAtomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) database under the accession code 5W3V. The pgtA3H variant sequences identified in these studies have been deposited to GenBank (accession numbers MF509624 through MF509631). Other data are available from the corresponding authors upon reasonable request.Supplementary Information


StBEL5, functions to activate growth, whereas two other, sequence-related StBEL’s, StBEL11 and StBEL29, function antagonistically to repress StBEL5 target genes involved in promoting tuber development. This dynamic system utilizes closely-linked phloem-mobile mRNAs to control growth in developing potato tubers. In creating a complex signaling pathway, potato has evolved a long-distance transport system that regulates underground organ development through closely-associated, full-length mRNAs that function as either activators or repressors.Included among the many signals that traffic through the sieve element system are full-length mRNAs that function to respond to the environment and to regulate development. In potato, several mRNAs that encode transcription factors from the three-amino-loop-extension (TALE) superfamily move from leaves to roots and stolons via the phloem to control growth and signal the onset of tuber formation. This RNA transport is enhanced by short-day conditions and is facilitated by RNA-binding proteins from the polypyrimidine tract-binding family of proteins. Regulation of growth is mediated by three mobile mRNAs that arise from vasculature in the leaf. One mRNA,  Aux/IAA, FT, ATC, GAI and KNOX (Arabidopsis that were mobile across an Arabidopsis/tobacco graft union. Approximately ten of these were transcription factors (TFs), including transcripts from a BEL1-like and a KNOX-type TF. In another study, using variant ecotype transcript profiling in heterografts, Thieme et al. [Arabidopsis. Many of these mobile transcripts followed the phloem-dependent sugar allocation pathway from leaves to roots, but a high number of transcripts also moved in a root-to-shoot direction. Yet, despite these valuable insights, mobile RNAs with an established function or phenotype are still rare. Some of the best examples of these latter types include StBEL5, -11, -29 [POTH1 [CmGAI of pumpkin [PFP-LeT6 from tomato [AUX/IAA [FLOWERING LOCUS T and CENTRORADIALIS [Arabidopsis.Plants have evolved a unique long-distance signaling system that utilizes cell-to-cell plasmodesmatal connections and a specialized phloem cell network. In addition to its function in the transport of sugars from source to sink organs, the phloem is an important conduit for moving signals that mediate responses to stress and processes of development . Catalogand KNOX . Using ae et al.  reported-11, -29  and POTH pumpkin , PFP-LeTm tomato  and AUX/[AUX/IAA , FLOWERIRADIALIS ,16,17 frIn addition to full-length mRNAs, small RNAs/miRNAs are also mobile through the plant’s phloem system and play important roles in both defense and development . NumerouStBEL5 mRNA are both mobile signals that originate in the leaf and under favorable conditions move down underground to the stolon tip to initiate tuber formation. We now understand that StBEL5 functions upstream to induce StSP6A activity, auto-regulates its own gene in stolons and induces numerous genes involved in the formation of the tuber [StBEL5 transcripts move through the phloem to stolon tips, the site of tuber induction. StBEL5 mRNA originates in the leaf, and its movement to stolons is induced by a short-day (SD) photoperiod. This remarkable whole-plant communication system involves light induction of transcription in the leaf, photoperiod-activated mobilization of RNA and protein signals through the phloem and SD regulation of the promoters for both StBEL5 and StSP6A in target organs growing underground in the dark [Tuberization in potato is controlled by phloem-mobile signals that arise from the leaf. Based on previous work, the three most important signals that regulate the onset of tuber formation in potato are StCDF1, StSP6A and StBEL5 ,27,28. She tuber . POTH1 ihe tuber . The foche tuber  that inthe tuber . RNA movthe dark ,31.Arabidopsis. Based on this analysis, StBEL proteins may be grouped into five main clades  promoter was employed  [StBEL5 RNA [StBEL11 and StBEL29 [StBEL5 RNA [In both cases, the key RNA-binding proteins involved were polypyrimidine tract-binding (PTB) proteins. These proteins contain four RNA recognition motifs that bind at four cytosine/uracil (CU) motifs located within 100–200-nt regions inside the UTRs of the target RNAs . In the s 3′ UTR  facilitaprotein) . PreviouBEL5 RNA . Movemen StBEL29 ,63,64, t StBEL29 . All thrBEL5 RNA . StBEL5, -11 and -29 are by far the most abundant RNAs in the StBEL family and the only ones confirmed to be phloem-mobile [StBEL5, StBEL11 and StBEL29 contain 16, 7 and 11 CU motifs, respectively (AtGAI, a confirmed mobile RNA of Arabidopsis, contains 10 CU motifs in its 3′ UTR (StBEL mRNAs, StBEL14 and -22, have three and two CU motifs, respectively, in their 3′ UTRs. Considering that the mobility of all three of these RNAs was enhanced under an SD photoperiod (The thirteen BEL RNAs of potato exhibit a wide range of RNA accumulation patterns . StBEL5,m-mobile ,31. The s 3′ UTR . These utoperiod , it is fHow does the repressive activity of StBEL11 and -29 work within the framework of tuber development? Could these mobile tuber signals mediate cell-specific control of growth in coordination with StBEL5 activity? The nascent tuber evolves from a specific cell layer within the apical meristem of the stolon and is characterized by a change in the direction of cell growth from transverse divisions and cell elongation to longitudinal cell division . The apiStBEL5, StBEL11 and StBEL29 are phloem-mobile and that this movement is enhanced by SD and may be regulated by a common mechanism. Enhanced movement and accumulation of StBEL5 RNA results in a phenotype characterized by increased earliness and tuber yields. The movement and stability of its transcript is mediated by an interaction with the RNA-binding proteins, StPTB1 and StPTB6. StBEL11 and StBEL29 regulate tuber formation, but they function in opposition to the growth-promoting features of StBEL5. They both appear to inhibit growth by targeting select genes that are involved in tuber development. Consistent with this premise, suppression of both StBEL11 and -29 specifically increases tuber yields. In creating a complex signaling pathway, potato has evolved a long-distance transport system that regulates underground organ development through full-length mRNAs that function as both activators and repressors. The three StBEL’s of the tuberization clade appear to balance tuber growth by mobilizing their mRNAs through an interaction with RNA-binding proteins. StBEL5 functions to directly activate the tuberization program and to amplify other important signals in the pathway [In summary, previous work indicates that the full-length mRNAs of


Solitary metastasis of a malignancy to the spleen is rare, particularly for gastric cancer. Only a few case reports have documented isolated splenic metastasis from early gastric cancer. We describe a case of splenic metastasis from early gastric cancer.A 60-year-old man underwent a distal gastrectomy for early gastric cancer. It infiltrated the submucosa with pathological nodal involvement . One year after the gastrectomy, an abdominal computed tomography scan showed a low-density lesion, 17 mm in diameter, at the upper pole of the spleen. Positron emission tomography/computed tomography showed focal accumulation of fluorine-18 fluorodeoxyglucose in the spleen without extrasplenic tumor dissemination or metastasis. We diagnosed splenic metastasis of gastric cancer, and performed a splenectomy. Histological examination confirmed moderately differentiated tubular adenocarcinoma and poorly differentiated adenocarcinoma (solid type) that was consistent with the features of the primary gastric cancer. The splenic tumor was pathologically and immunohistochemically diagnosed as a metastasis from the gastric carcinoma. More than 18 months after the splenectomy, the patient has had no evidence of recurrent gastric cancer.When solitary metastasis to the spleen is suspected during the postoperative follow-up of a patient with gastric cancer, a splenectomy is a potentially effective treatment. Isolated metastasis to the spleen from early gastric cancer is very rare. Once splenic metastasis from gastric cancer occurs, it is usually accompanied by multiorgan metastasis and dissemination –3. Only A 60-year-old man visited our institution because of dysphagia. He was diagnosed with early gastric carcinoma in the middle third of the stomach, based on upper gastrointestinal endoscopy and computed tomography (CT) imaging. The preoperative carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA19–9) values were within the normal ranges. He underwent a distal gastrectomy with a D1+ lymph node dissection. Pathologic histology of the resected stomach macroscopically revealed a tumor, 25 × 20 mm in diameter, with a depressed and elevated form (Type IIa/IIc)  with obvious contrast enhancement and no evidence of other metastasis. Upper gastrointestinal endoscopy findings were negative in the residual stomach. Colonoscopy revealed no abnormalities. During the postoperative follow-up, the serum CEA and CA19–9 levels were within normal limits.We finally suspected that the tumor was a solitary splenic metastasis of the gastric cancer. The patient underwent a laparotomy because his splenic metastasis was isolated and resectable. In addition, there were no other metastases.The laparotomy revealed no lymph node involvement, hepatic metastasis, or peritoneal dissemination. The tumor was in the upper pole of the spleen. The splenectomy preserved the residual stomach. The patient’s postoperative period was uneventful.The specimen was a white mass without a capsule that measured 20 × 18 mm; it was at the upper pole of the spleen Fig. . HistoloThe patient refused to receive adjuvant chemotherapy. For 18 months, he has been well and healthy without any evidence of gastric cancer recurrence.The spleen is a hypervascular organ; however, splenic malignancy or metastasis is rare, except in cases of malignant lymphoma and leukemia . The freBy contrast, isolated splenic metastasis of early stage cancer is rare. Although it is not clear why splenic metastasis rarely occurs from malignancies, several hypotheses have been proposed , 7. FirsSplenic metastases from gastric cancer are rare, regardless of their proximity anatomically. Compét et al. reviewed 93 cases of isolated splenic metastasis, and reported that the primary sites of malignancies were the colorectum (20 cases), ovary 18 cases), lung (10 cases), endometrium (9 cases), kidney (9 cases), and stomach (7 cases) [ cases, lFor the present report, searches were performed for related reports published between April 1983 and October 2016 in Japan Medical Abstracts Society Web , and from the earliest possible date to October 2016 in PubMed, using the keywords “splenic metastasis,” “gastric cancer,” and “gastric neoplasms”. To our knowledge, there have been 19 reported cases of radical treatment by splenectomy for isolated splenic metastasis from gastric cancer, which comprised 5 cases of synchronous metastasis and 14 cases of metachronous metastasis (including our case) (Table A gastric cancer can result in splenic metastasis by 3 pathways: (1) via the splenic artery, (2) via the splenic vein, and (3) via the lymphatic route . For theIn our patient, the route of metastasis to the spleen was unknown. However, we found lymph node metastases during the gastrectomy (N2). Vascular invasion in the cancer specimen was not histologically identified. In addition, the gastric cancer was early stage cancer with submucosal invasion and without the indications for liver disease. We accordingly considered the possibility that isolated splenic metastasis developed via the lymphatic route.Most isolated splenic metastases are asymptomatic. However, isolated splenic metastasis is sometimes detected when examining a patient for general fatigue, weight loss, abdominal pain, splenomegaly, anemia, or thrombocytopenia . IsolateCavanna et al. reported 160 cases of splenic tumor for which the patients underwent fine needle aspiration (FNA) for diagnosis . They deA PET/CT scan is useful for differentiating between malignant and benign tumors, and for assessing metastasis to many organs or para-aortic lymph nodes . In the Splenectomy provides the only reliable possibility for curative treatment of solitary splenic metastasis from gastric cancer . The aimThe prognosis of an isolated splenic metastasis from gastric cancer treated by splenectomy is unclear because of its rarity. All past reports , 5, 9–23Streptococcus pneumoniae, followed by Haemophilus influenzae type b and Neisseria meningitides. Preventing OPSI involves administering a pneumococcal polysaccharide vaccine and immunizing a patient against H. influenzae type b and N. meningitides. The incidence of OPSI is infrequent; however, once it occurs, the mortality rate is very high, at 50%–70% [Overwhelming postsplenectomy infection (OPSI) is a possible critical life-threatening complication. An OPSI can result in sepsis and meningitis after splenectomy, and 3.2% of patients who have undergone a splenectomy acquire an OPSI. The risk of OPSI continues throughout a patient’s life after splenectomy. The most frequent causative agent of an OPSI is  50%–70% . PreventWe presented a rare case of solitary splenic metastasis after curative resection of early gastric cancer. The metastasis was treated by splenectomy. When a solitary mass in the spleen is detected at the diagnosis of gastric cancer or during the postoperative follow-up of a patient with gastric cancer, even early stage gastric cancer, the mass may be a splenic metastasis. PET/CT scanning is useful for diagnosing splenic metastasis and for assessing metastasis to other organs or the para-aortic lymph nodes. If a solitary metastasis to the spleen without distant metastasis is suspected, a splenectomy should be considered for a curative treatment.


Vasopressor infusion (VPI) is used to treat hypotension in an ICU. We studied compliance with blood pressure (BP) goals during VPI and whether a statistical model might be efficacious for advance warning of impending hypotension, compared with a basic hypotension threshold alert. Retrospective data were obtained from a public database. Studying adult ICU patients receiving VPI at submaximal dosages, we analyzed characteristics of sustained hypotension episodes (>15 min) and then developed a logistic regression model to predict hypotension episodes using input features related to BP trends. The model was then validated with prospective data. In the retrospective dataset, 102-of-215 ICU stays experienced >1 hypotension episode (median of 2.5 episodes per day in this subgroup). When trained with 75% of retrospective dataset, testing with the remaining 25% of the dataset showed that the model and the threshold alert detected 99.6% and 100% of the episodes, respectively, with median advance forecast times (AFT) of 12 and 0 min. In a second, prospective dataset, the model detected 100% of 26 episodes with a median AFT of 22 min. In conclusion, episodes of hypotension were common during VPI in the ICU. A logistic regression model using BP temporal trend features predicted the episodes before their onset. The rationale for treating severe hypotension with vasopressors is based in large part on classical physiology studies showing that central nervous system (CNS) auto-regulation of CNS perfusion fails in healthy animals for MAP < 65 mmHg4. Below this limit, CNS hypoperfusion and tissue ischemia occur. Indeed, associations between duration of hypotension and measures of end-organ injury have been reported in critically-ill7 and intraoperative8 patient populations.Many patients treated for circulatory shock receive hours or days of vasopressor infusion to manage persistent hypotension. Vasopressors generally act by elevating vascular tone, cardiac contractility and heart rate, and these physiological effects result in higher arterial blood pressure (BP). Administration of vasopressors to prevent mean arterial pressure (MAP) < 65 mmHg for sepsis is recommended by the consensus Surviving Sepsis guidelinesVasopressor infusion requires ongoing titration of dose to ensure that the patient’s MAP is high enough to prevent hypotension, but not too high, which can result in unnecessary cardiac demand, cardiac ectopy, and excessive vasoconstriction that impairs tissue perfusion. As a practical matter, the infusion dose must be continually re-adjusted through time, because the critically ill patient’s underlying condition is not static, leading to either an increase or decrease in the dose necessary for maintaining a target MAP.9, we reported that ICU patients frequently experience sustained episodes (>15 min) of hypotension while receiving vasopressor infusions. In a related report, Hawryluk et al. found that nearly half of BP measurements in ICU patients with acute spinal cord injuries were below the BP range that is recommended by consensus guidelines to prevent secondary injury to newly damaged spinal cord10. Therefore, the first goal of this investigation was to better understand compliance with BP management guidelines during ICU vasopressor administration.Recent capabilities in electronic data archiving for the ICU reveal that rates of compliance with BP goals may be suboptimal. In a preliminary studyThe second goal of this study was to investigate the efficacy of statistical methods for predicting when the patient’s vasopressor dosage requires a change, because computerized decision-support offers a potential means to enhance clinician practice. We sought to develop and evaluate a simple statistical model that might be applied in an ICU as a tool for preventing hypotension by providing a reliable advance warning before hypotension develops.11 or suboptimal outcomes after acute spinal cord injury10. On the other hand, if standard statistical methods are efficacious at predicting when dose changes are necessary, even before hypotension develops, then statistical models might offer a relatively practical method for improving compliance rates with BP targets and avoiding preventable end-organ injury during vasopressor therapy.Overall, if compliance with BP management guidelines during vasopressor infusion is suboptimal, that suggests that ICU patients may be suffering preventable harm. For example, failure to maintain MAP > 65 mmHg via suitable vasopressor infusion doses might be a causal factor (via CNS ischemia) to the permanent cognitive impairment reported in many survivors of septic shockWe studied an archived dataset of adult patients from a mixture of ICUs from a single institutional hospital . Prospectively, we collected a second dataset from the surgical ICU of our institution  to rigorously validate our statistical model.We studied ICU patients with vasopressor infusion doses documented in the nursing flowcharts  with concurrent heart rate (HR) and MAP data from indwelling arterial catheters. Only records from patients who survived at least 48 hours after their ICU stays were included in this study, to exclude patients who may have been comfort measures only (CMO) and therefore not receiving aggressive BP management.12. Minute-by-minute numeric data of MAP and HR originally sourced from the bedside monitor, as well as vasopressor dose information and MAP charted in the nursing documentation, were downloaded for analysis.For the Hospital 1 dataset, the MIMIC II database  was accessed through the PhysioNet websiteFor the Hospital 2 dataset, we prospectively identified patients in the surgical ICU receiving vasopressor infusions. MAP and HR were collected by the BedMaster data archiving system  while vasopressor dose information and intermittent MAP measurements were obtained by chart review of nursing documentation.For both datasets, a moving “causal” 5 min median filter was applied to the MAP data . We assumed vasopressor was infused no more than 60 min after its last documented infusion.We focused our study of dose-titration behavior of clinicians on specific data intervals. Specifically, within each patient record, data intervals were excluded from primary analysis when the interval (a) lacked either MAP or vasopressor dose data, (b) contained non-physiological MAP measurements (<5 mmHg or >300 mmHg), (c) occurred during the first 30 min of a patient’s ICU stay, when initial resuscitation was occurring, (d) occurred during maximum vasopressor infusion defined as per hospital protocols, or (e) contained two or more simultaneous vasopressor infusions. This left us with paired dose and MAP data during vasopressor mono-therapy at sub-maximum infusion doses. This dataset was deemed optimal for studying the dose-titration behavior of clinicians.The validity of the recorded MAP was confirmed by comparing it against contemporaneous non-invasive MAP values, from the nursing documentation.We also performed a secondary analysis by relaxing the exclusion criteria (c), (d), and (e), for assessing the performance of the statistical model under wider-ranging conditions.The study outcome was sustained hypotension. For each patient record, hypotensive measurements were identified (MAP < 60 mmHg) and hypertensive measurements were also identified (MAP > 100 mmHg). Next, sustained episodes of hypotension were identified, defined as 15 min or more of continuous hypotension. Episodes begin upon the first hypotensive MAP sample and terminate with any subsequent in-range MAP sample, defined as 100 mmHg ≥ MAP ≥ 60 mmHg.Patients’ ICU stays were divided into three categories: ICU stays with sustained hypotension, ICU stays with only transient (non-sustained) hypotension (<15 min), and ICU stays without any hypotension. For each category, we computed: demographic statistics of patients receiving at least 30 min of vasopressor infusion; documented reason(s) for vasopressor infusion based on the patient’s discharge summary; and descriptive statistics about vasopressor infusion and MAP trends during vasopressor infusion.For the ICU stays with at least one sustained episode of hypotension, we analyzed vasopressor dose changes before and after the onset of the episodes, to characterize clinician behavior before and after the onset of the episodes. Specifically, we analyzed the incidence of dose changes in vasopressors within 30 min prior to the onset of hypotension and after the onset of hypotension. For the Hospital 2 dataset, we also examined whether the episodes of hypotension were associated with additional doses of sedatives; other cardiovascular medications; or documented clinical event within the nursing note.Many episodes of hypotension were followed within 30 min by a subsequent episode of hypotension. Sequential episodes of sustained hypotension separated by 30 min or less were termed “episode spans.”We developed a logistic regression model for predicting sustained episodes of hypotension, using cases that account for 75% of the data from the Hospital 1 dataset. We performed analysis in the MATLAB computing environment , using the greedy backward feature selection until only statistically significant features remained (P < 0.05 significance level).We explored 54 candidate predictor features derived from the MAP trend, vasopressor trend, and HR trend data: mean, least-squares slope, and standard deviation of MAP, vasopressor dosage, and HR over time-scales of 5, 10, 20, 30, 45, and 60 min. These are features that are available at the bedside via electronic interfaces with the patient monitor and the infusion pump and do not require additional clinical information. We computed these features at 5 min time steps throughout the duration of vasopressor infusion of each subject. We normalized the candidate features before application to logistic regression.glmfit to develop the logistic regression model.There were a total of 54,276 time point observations used for training the model, after excluding time points that lacked a complete set of feature data. For each time point, we calculated whether or not it was followed by sustained hypotension within the subsequent 15 min and used it as the model output for logistic regression. We used the MATLAB routine In addition to the above primary analysis, we also performed a secondary analysis to test whether the aforementioned primary logistic regression model was improved with additional candidate predictor features, adding parameters about the individual patient’s age, gender, vasopressor name, and type of shock pathology, i.e., presence of sepsis (yes/no), presence of hemorrhagic hypovolemia (yes/no), etc., using the chart review methodology described above.13).The first evaluation metric was model goodness-of-fit. For deciles of the dataset, we compared the model’s predicted versus the actual probabilities that any time point would be followed within 15 min by sustained hypotension. We did not apply a formal goodness-of-fit test, i.e., Hosmer-Lemeshow test, because it is not reliable for datasets larger than 50 K samples . We computed the following statistics: false alarms per 24 hours; undetected episodes of hypotension; advance forecast time prior to the onset of the episode of hypotension; and duration of the alarm episodes. These metrics were compared using the Wilcoxon Rank Sum test.de novo prospective dataset collected from Hospital 2, we repeated the comparison between the logistic regression model  and simple threshold alerts. We repeated the same evaluation methodology with the goal of evaluating the generalizability of the results from Hospital 1, i.e., whether the logistic regression model was externally valid outside of Hospital 1. We computed the same performance metrics, and again compared the alerting methods using the Wilcoxon Rank Sum test.After collecting the We sought to explore the relationship between documented dose increases versus the logistic regression model output. We identified instances where the vasopressor dose was documented to increase ≥50%, and we calculated the proportion for which the logistic regression model was predicting sustained hypotension.A total of 175 patients accounting for 215 ICU stays from Hospital 1 were identified as patients receiving at least 30 minutes of vasopressor mono-therapy at sub-maximal doses. There were 102 ICU stays (47%) with at least one sustained episode of hypotension during vasopressor infusion. These populations are characterized in Tables The majority of the episodes of hypotension appeared valid, as we found that only 5.4% of these episodes had two or more MAP measurement samples that differed by more than 10 mmHg from the documented non-invasive MAP measurements. During episodes, the difference between MAP measurements and non-invasive MAP measurements was 5.1 ± 6.6 mmHg (mean ± std. dev.).Compared to ICU stays without hypotension, ICU stays with sustained episodes of hypotension had significantly longer durations of vasopressor infusion, lower MAP during vasopressor infusion, and lower overall proportion of in-range MAP (P < 0.05 per Mann-Whitney U and Chi-squared tests).There were no additional significant differences in the characteristics in Table any documented dose increase in vasopressors. The top two panels in Fig. In Hospital 1, there were 354 episode spans . Most (92%) episode spans resolved without In Hospital 2, there were 26 episode spans. The majority of episode spans were not associated with an increase in vasopressor infusion dose  increasing likelihood of undetected episodes of hypotension; ii) decreasing false alarm rate; and iii) decreasing advance forecast time. The results of evaluating the logistic regression model in the Hospital 1 testing dataset are shown in Table The trade-off between detection sensitivity and false alarms for different thresholds for the logistic regression model output are shown in Fig. Results from prospective evaluation in Hospital 2 are also shown in Table For the primary analysis, we excluded data intervals that made it hard to interpret the clinician dose-titration behaviors. In a secondary analysis, we repeated the evaluation of the logistic regression model, this time including data intervals from the initial 30 min of a patient’s ICU stay and during maximum vasopressor dosages and during infusion of two or more vasopressors at the same time. This did not alter our findings. Specifically, there were no statistically significant differences, as compared to the numerical results shown in Table We found that gender and “presence of sepsis (yes/no)” were additional significant predictor features in the secondary logistic regression model. However, when this secondary model was tested and compared with the primary logistic regression model , there was no improved performance. Specifically, there were no statistically significant differences, as compared to the numerical results shown in Table The logistic regression model was predicting sustained hypotension during 48% of the instances when the vasopressor dose was increased ≥ 50%. Within a wider 60 min window, the logistic regression model was predicting sustained hypotension during 76% of the instances.There are three main findings of this analysis. First, ICU patients receiving vasopressor infusion commonly experienced sustained episodes of hypotension, with MAP below the limits of CNS auto-regulation often for 30 min or even longer. Classical physiological studies and retrospective analyses have suggested that such episodes cause deleterious hypoperfusion of the CNS and other end-organs. Second, most of these episodes developed in the absence of any preceding dose adjustments, and after commencing, many episodes persisted without any documented increases in vasopressor dose. This raises the possibility that many of the episodes were preventable via more vigilant clinical interventions, including vasopressor dose increases immediately upon the onset of the episodes, or dose increases before the onset . Third, a statistical model only requiring BP trend data as an input was able to predict the onset of these episodes well in advance of the onset, and with significantly fewer false alarms than simple threshold alerts. This suggests that advance warning prior to the onset of sustained hypotension is technically feasible.10.The findings in this investigation arose from two medical centers, so while the generalizability of this finding is not known, it is evident that BP goals may not always be met during vasopressor infusions. A recent report about ICU patients with acute spinal cord injury (ASCI) revealed a similar failure to maintain MAP goals: although current ASCI guidelines advise maintenance of MAP of 85–90 mmHg for the first week after ASCI, the investigators reported that MAP was below that threshold for 42% of all documented values4. Therefore, low-level sustained hypotension might cause CNS ischemic injury. Indeed, major cognitive injury is a tragic sequelae in survivors of sepsis11 and it is reasonable to speculate that sustained hypotension could be directly causing some of the cognitive injury. Clinical studies have shown a correlation between hypotension and worse ICU outcomes7 but of course in retrospective studies, it is not possible to distinguish between causal versus correlative relationships.The actual effect of these episodes of hypotension is not known, but there is ample indirect evidence that such hypotension is deleterious. In animal models it has been shown that the CNS cannot effectively autoregulate perfusion below MAP of 65 mmHg, which means that CNS ischemia is likely to occurOverall, further study of the actual consequences of sustained low-level hypotension on neurons and nephrons may be advisable, to establish the true risk of sustained hypotension during ICU care. If a causal relationship between hypotension and patient injury is established, it would behoove clinical guidelines to strengthen their guidance .Are sustained episodes of hypotension during vasopressor therapy in the ICU preventable? We found that most episodes of hypotension occurred not because the vasopressor had been excessively weaned in the time preceding the episode, nor because there had been an insufficient increase in vasopressor dose preceding the episode. Rather, most episodes of hypotension occurred in the absence of any preceding vasopressor dose change. As well, when we reviewed the nursing notes from the time of the episodes, we also did not find any other clinical events documented to explain the episode. In other words, in none of the cases that we evaluated did the patient experience sudden-onset problem such as new onset GI bleeding or new arrhythmias. Typically, for our sustained episodes of hypotension, the MAP simply drifted out of goal range.Moreover, the clinical response to sustained episodes of hypotension often lacked any vasopressor dose increase, nor consistent responses such as volume boluses. In Hospital 1, the top quartile of sustained episodes of hypotension persisted longer than 72 min  may be clinically indicated.Although the prediction of hypotension in an ICU (e.g. early warning systems) has been explored in other reportsOur analysis suggests that advance warning is quite feasible based solely on statistical associations between BP trend data and subsequent hypotension: a relatively simple logistic regression model was able to predict episodes of hypotension well before their onset. Performance was favorable both in the testing dataset from Hospital 1, and also in the dataset collected prospectively from Hospital 2. Specifically, the logistic regression model was able to detect almost all of the sustained episodes of hypotension with advance warning, and with fewer false alarms, relative to a simple threshold alert , vasopressor dose was essentially constant in a median patient in the feature extraction window. Evidently, the absolute value of the dose was not strongly associated with the likelihood of an imminent episode of hypotension.18.The fact that the statistical model only required MAP input data is a strength for real-world implementation, since electronic MAP data are accessible via serial port interface to most vital-sign monitors. By contrast, a predictive model that requires additional clinical data would likely require electronic communication with the patient’s electronic medical record (EMR), which can be challenging “False alarms” by the logistic regression model occurred when the MAP was trending towards hypotension but never actually developed sustained hypotension see Fig. . Under sTo be sure, predictive performance may further improve with additional clinical data, or with more sophisticated statistical methods. Logistic regression is a standard, simple classification technique, and we found that the logistic regression model offered consistent performance between Hospital 1 retrospective testing and Hospital 2 prospective testing .There are several limitations to this report. First, regarding the incidence of sustained hypotension during vasopressor therapy, our findings were only from two hospitals. The degree to which this problem is widespread is not known, although it has been corroborated by another study of preventable hypotension in an ICUIn conclusion, during vasopressor therapy, sustained episodes of hypotension were common and typically without associated vasopressor dose increases. Because the likelihood is that sustained hypotension causes CNS and other end-organ injury, the prevalence of this practice and its clinical consequences should be further investigated. In a prospective validation, a logistic regression model demonstrated promising test characteristics and it may offer one tool for reducing hypotension during vasopressor therapy in the ICU.